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WO1990007009A1 - Novel lymphokine/cytokine genes - Google Patents

Novel lymphokine/cytokine genes Download PDF

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Publication number
WO1990007009A1
WO1990007009A1 PCT/US1989/005603 US8905603W WO9007009A1 WO 1990007009 A1 WO1990007009 A1 WO 1990007009A1 US 8905603 W US8905603 W US 8905603W WO 9007009 A1 WO9007009 A1 WO 9007009A1
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WIPO (PCT)
Prior art keywords
pat
protein
cells
human
cytokine
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PCT/US1989/005603
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French (fr)
Inventor
Ulrich Siebenlist
Peter F. Zipfel
Kathleen R. Kelley
Steven G. Irving
Monica Napolitano
Warren J. Leonard
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United States Department of Commerce
US Department of Health and Human Services
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United States Department of Commerce
US Department of Health and Human Services
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Publication of WO1990007009A1 publication Critical patent/WO1990007009A1/en
Priority to NO91912189A priority Critical patent/NO912189L/en
Priority to DK113491A priority patent/DK113491A/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to genes which are activated by mitogenic stimulation of immune cells, particularly to those encoding polypeptides similar to a family of secreted factors including lymphokines and cytokines. This invention also relates to synthesis of product ' s' of such immune activation genes by recombinant cells, and to the manufacture and use of certain novel products enabled by the identification and cloning of DNAS encoding these factors. "
  • Quiescent non-dividing T lymphocytes respond to antigen and mitogen stimulation by the activation of specific cellular " genes which are thought to control events that lead to cell proliferation and the expression of a differentiated phenotype.
  • T cells produce a large variety of factors which are important for the growth and differentiation of many cells participating in an immune reaction.
  • lymphocyte proliferation and effector functions are regulated by antigen/mitogen and lymphokine binding to cell surface receptors.
  • the bind ⁇ ing of such extracellular ligands results in a cascade of intracellular biochemical events (see references 11-44, 11-45 in Experimental Section II, below) that ultimately set in motion a genetic program for activation-related growth and expression of differentiated functions.
  • quiescent T cells undergo a series of sequentially dependent, ordered transcriptional events following acti- . vation of quiescent cells that culminates in the initi ⁇ ation of DNA synthesis after about 24 hours (II-5, II- 17).
  • Primary gene transcriptional events defined by their early kinetics and lack of dependence on prior protein synthesis, include protooncogenes (e.g., c-myc and c-fos), lymphokines (e.g. IL-2, IFN, GM-CSF), and a cell surface lymphokine receptor (IL-2 receptor) (II-6, II-9, 11-12, 11-17, 11-27, 11-36, 11-44), all of which are thought to have significant effects upon T cell proliferation and/or regulation of an immune response- It is expected that some of the immediately induced .genes play a role in; initiating and thus _.regu- lating the cascade of molecular events that.-.follow mito- genie activation.
  • protooncogenes e.g., c-myc and c-fos
  • lymphokines e.g. IL-2, IFN, GM-CSF
  • IL-2 receptor cell surface lymphokine receptor
  • Such genes are also likely targets of tumorigenic events.
  • a variety of data supports the notion that uncontrolled growth occurs when the? ⁇ mitogen inducible gene c-myc is expressed, inappro ⁇ priately (11-11, 11-26).
  • c-myc a gene that is expressed, inappro ⁇ priately (11-11, 11-26).
  • a limited number of primary induced genes in T cells have been described.
  • genes induced in activated T cells, IL-2 and IL-2 receptor for example, often have been identified on the basis of their encoded proteins.
  • the transcriptional response of T cells is likely more complex than has been reported to date (II-2, II-4, 11-15, 11-23, 11-28, 11-32, II- 41).
  • PB T cells Human peripheral blood (PB) T Cells were used in the development .of this invention, as the model system in which to study activation, for several reasons.
  • PB T cells are primary quiescent cells that have not been selected for adaption to growth in culture, and they can be polyclonally stimulated ⁇ n_ vitro.
  • T cells upon activation, express a variety of differenti- ated functions that play critical roles in the pro ⁇ gression of an immune response. For example, novel lymphokines/cytokines would be expected to be included within the family of inducible genes, since a primary function of PB T cells following activation is to secrete soluble factors that affect ultimately a number of physiological functions (II-5, II-6, II-9, 11-13).
  • CSA immunosuppressive drug cyclosporin A
  • T cell system allows one.-to dissect and contrast the * activation* requirements mediated through various distinct surface molecules that are thought to play roles in T cell development and function (see Experimental Section III, below).
  • peripheral blood T cells Treatment of human, peripheral blood T cells with the mitogenic combination of PHA (phyto- hemagglutinin) and PMA (phorbol myristate acetate) appears to mimic the effects of antigen and antigen presenting cells jLr ⁇ vivo.
  • the two agents are known to induce the expression of genes which are important for progression through the cell cycle, including the c-fos and the c-myc oncogenes, the IL-2 growth factor gene and the IL-2 receptor gene (see references IV-1, IV-2 in Experimental Section IV, below).
  • T cells activated in this manner elaborate many lymphokines/cytokines which exert their effect on a variety of cells.
  • f ctors as IL-3, IL-4, IL-5 and IL-6, GM-CSF, -IFN (gamma interferon), TNF (tumor necrosis factor) and LT (IV-3 - IV-9) and several others that are less well defined (IV-10 - IV- 13).
  • factor genes may be subject to a specific control mechanism in T cells, since the immunosuppressive drug cyclosporin A appears to abrogate their activation. As noted earlier, this drug acts during the early activation phase of T cells and interferes the production of IL-2, IL-3, and -IF (IV-14 - IV-17). The inhibitory effect is specific, since many other inducible genes such as c-fos and HSP 70 are unaffected (IV-15).
  • the present invention relates to DNA segments which encode amino acid sequences with similarities to members of a- newly emerging family of small, secreted proteins (see Figure IV-6 in Experimental Section IV) .
  • This family of structurally related factors includes platelet_ factor 4 (PF-4) (IV-18), platelet basic protein (PBP) and its processed forms ⁇ -thromboglobulin ( ⁇ ?-TG) and connective tissue activating peptide III (CTAP.
  • PF-4 platelet_ factor 4
  • PBP platelet basic protein
  • CTAP connective tissue activating peptide III
  • Ill Ill
  • IB- 10 gamma interferon inducible ⁇ protein
  • MIP macrophage inflammatory protein
  • NAF neurotrophil-activating factor
  • fibroblast ⁇ human growth related protein
  • H-gro human growth related protein
  • Inflammation and wound healing/tissue repair are very complex processes which involve many different cells. Coordination of the various cellular functions is likely to be reflected in a multitude of secreted factors needed to communicate between cells.
  • This family of genes which may have been derived from one common ancestral gene, may well serve to establish such a net- work.
  • the individual factors may possess multiple functions, as shown for several of the members of this family (see below. Experi ⁇ mental Section IV, subsection Discussion) .
  • the conserved similarities between these proteins may indicate an interaction with conserved receptor elements. As enumerated above, the members of this family of proteins display a remarkable variety of tissue specificities. The distinctions between the factors may determine unique functions required during an immune reaction depending on the cell type activated.
  • This invention contemplates the application of methods of recombinant DNA technology to fulfill such needs and to develop means for producing certain protein factors which appear to be related to inflammatory or cell; growth processes, and which could not be produced otherwise.
  • This invention also contemplates the appli ⁇ cation of the molecular mechanisms of these factors related to inflammation and healing processes.
  • the present .invention relates to a development of recombinant DNA technology, which includes production of novel lymphokine-like or cytokine-like proteins, free of other peptide factors. Novel DNA segments, RNAs, and bioassay methods are also included.
  • the present invention in particular relates, in part, to DNA segments which encode mRNAs and/or proteins having structural and/or functional characteristics of such molecules related to lymphokines/cytokines that are inducible in T cells.
  • Members of one particular group of these proteins feature hydrophobic N-terminal leader or signal peptides characteristic of secreted proteins, have predicted sizes of about 8 kD (kilodaltons) upon cleavage of the putative leader peptide, and also share some critical amino acid sequence similarity with a newly emerging family of secreted factors which have been shown to display functions associated with an inflammatory response and/or to have mitogenic activities.
  • these DNA segments are capable of being expressed in suitable host cells, thereby producing lymphokine-like or cytokine-like proteins.
  • the invention also relates to mRNAs produced as the result of trans-, cription of the sense strands of the DNA segments of. this invention.
  • This invention furthe ' r comprises novel bioassay methods for determining effects of various mitogenic agents on expression in human cells of, the mRNAs and proteins produced from the genes related to DNA segments of the invention. - - _
  • the present invention comprises DNA segments encoding lymphokine-like or cytokine-like proteins, selected from the group con ⁇ sisting of: Act-2 [i.e., an approximately full length (-0.7 kb) cDNA clone of activated human peripheral blood mononuclear cells (PBMC) mRNA, number 2, hereinafter referred to as "full-length Act-2" DNA]; human cDNA clones of activated T cell mRNAs, pAT 744 and PAT 464; and related DNA segments which can be detected by hybridization to any of the above human DNA segments, which related segments encode related lymphokine-like or cytokine-like proteins.
  • Act-2 i.e., an approximately full length (-0.7 kb) cDNA clone of activated human peripheral blood mononuclear cells (PBMC) mRNA, number 2, hereinafter referred to as "full-length Act-2" DNA
  • PBMC peripheral blood mononuclear cells
  • this invention relates to an RNA transcript of a DNA of the invention.
  • these RNA transcripts are capable of being trans ⁇ lated to produce complete molecules of the encoded lymphokine-like or cytokine-like proteins.
  • the invention in another embodiment, relates to a recombinant DNA molecule comprising a vector and a DNA of the present invention.
  • recombinant molecules include molecules comprising full-length DNA and any of the following vector DNAs: a baculovirus vector (such as derivatives of Ac-Nuclear Polyhedro ⁇ is Virus; AcNPV); a bacteriophage lambda vector (gtlO); an Ml3 bacteriophage vector; or the RNA transcription vector pGEM-1.
  • Recombinant DNAs of this invneiton also include recombinant molecules comprising clone pAT 744 DNA or clone pAT 464 DNA and any of the following vector DNAs: lambda (GtlO); and Ml3 vector; and a mammalian expression vector (such as CDM-8 or PMT2-2) capable of expressing inserted DNAs in mammalian (e.g., COS) cells.
  • GtlO lambda
  • Ml3 vector a mammalian expression vector capable of expressing inserted DNAs in mammalian (e.g., COS) cells.
  • the invention comprises a cell, preferably a-mammalian or insect cell, transformed with a DNA -of the invention.
  • the invention comprises cells, including yeast cells and bacterial cells such as those of E_. coli and _B. subtilis, transformed with DNAs of the invention.
  • the transforming DNA is capable of being expressed in the cell, thereby increasing the amount of lymphokine/cytokine-like protein encoded by this DNA, in the cell.
  • the cell transformed by the DNA of the invention secretes the protein encoded by that DNA in the (truncated) form secreted by activated T cells.
  • the invention comprises novel lymphokine/cytokine-like proteins made by expression of a DNA of the invention, or by translation of an RNA of the invention.
  • these proteins will be of a secreted form (i.e., lacking an apparent signal sequence) .
  • These protein factors can be used for f nc ⁇ tional studies, and can be purified for additional bio ⁇ chemical and functional analyses, such as qualitative and quantitative receptor binding assays.
  • the novel lymphokine/cytokine-like proteins will be pro ⁇ tein products of "unmodified” DNAs and mRNAs of the invention, or will be modified or genetically engineered protein products. As a result of engineered mutations in the DNA sequences, modified lymphokine/cytokine-like proteins will have one or more differences in amino acid sequence from the corresponding naturally occurring "wild-type" proteins.
  • This invention also comprises novel antibodies made against a peptide encoded by a DNA segment of the invention. In 'this embodiment of the invention, the antibodies will specifically bind to a lymphokine/cytokine-like protein which includes ⁇ the sequence of such peptide, preferably when that protein is in its native (biologically active) conforma ion. These antibodies can be used for detection .or purification of the protein factors. " • * • - ⁇ . -
  • This invention further comprises novel .bioas ⁇ ay methods for detecting the expression-of genes related -to
  • DNAs of the invention may be used as probes to determine steady state levels of or kinetics of induction of related mRNAs.
  • Such bioassays may be useful, for example, for identification of various classes of tumor cells or genetic defects in the inflammatory response.
  • the bioassays of this invention may also be useful for detecting activation of the immune system in vivo. Genes related to DNAs of this invention are expressed very early upon immune activation, in T cells (and also some other hematopoietic cells), and, there- fore, serve as extremely early markers for immune system activation.
  • various body fluids may be tested routinely with antibodies to protein factors of the invention, for example, to monitor whether tissues are rejected upon transplantation.
  • Fig. 1-1 shows Northern blots using a ⁇ ---F- labeled full-length CDNA probe and RNA derived from a variety of human cell types, which illustrate the speci ⁇ ficity of full-length gene expression, mainly in certain mitogenically stimulated immune cells.
  • Fig. 1-2 characterizes the temporal expression of mRNA in activation of normal T and B lymphocytes and monocytes (by means of Northern Blots), illustrating that Act-2 gene expression is both rapidly induced and termi ⁇ nated in response to mitogen.
  • Fig. 1-3 depicts the sequencing strategy, DNA sequence, and deduced .amino acid sequence of -0.7 kb
  • Fig. 1-4 presents a Kyte-Doolittle hydro- phobicity plot generated from the deduced amino acid sequence, which reveals an extremely hydrophobic N- terminus that appears to be a so-called leader or signal peptide.
  • Fig. 1-5 demonstrates translation of RNA species produced from full-length Act-2 cDNA in- cell-free systems, showing by gel electrophoresis a product with an apparent M r of approximately 11,000 similar to the calcu- lated molecular weight of 10,199 daltons, which apparent ⁇ ly translocates into microsomal membranes, as do secreted proteins.
  • Fig. 1-6 portrays results of using the full- length Act-2 cDNA to perform a genomic Southern blot of human DNA, a comparatively simple pattern of digestion most consistent with Act-2 representing a single copy or low copy number gene.
  • Fig. 1-7 directly compares the time course of expression of Act-2 with two other genes known to play roles in cell proliferation, c-fos, and c-myc, in stimu ⁇ lated lymphocytes, showing that Act-2 mRNA is induced coordinately with c-fos and its peak expression coincides with that of c-myc.
  • Table 1-1 presents results of N-terminal sequencing of Act-2 protein radiolabeled with * ⁇ 5 S- methionine or cysteine.
  • EXPERIMENTAL SECTION II Fig. II-l outlines the procedure used to iso ⁇ late genes which are induced immediately upon mitogenic stimulation of human periopheral blood T lymphocytes.
  • Fig. II-2 illustrates kinetics of mRNA induc ⁇ tion (with Northern blots and cDNA probes) of four selected inducible genes upon stimulation of • resting human blood T cells.
  • Fig. II-3 portrays diverse expression and regu ⁇ lation of selected cDNA clones in the human helper T -cell line Jurkat. _- ._ __ _. --. • __>
  • Fig. II-4 shows (Northern blot) analyses of expression of selected cDNA clones in human fibrobla ⁇ t ⁇ .
  • Table II-2 presents expres ⁇ ion analysis of T cell induced genes in the helper T cell line Jurka -and in human fibroblast ⁇ .
  • Fig. III-2 illustrate ⁇ (Northern blot) analysis of mitogen induced genes in CD28 T cells that _were stimulated with either anti-CD28 monoclonal antibody or ionomycin in the absence or pre ⁇ ence of phyto- hemagglutinin.
  • Fig. III-3 depict ⁇ (Northern blot) analy ⁇ i ⁇ of mitogen induced gene ⁇ .
  • T cell line Jurkat that were ⁇ timulated with either dioctanoyl-glycerol or ionomycin or both agent ⁇ together in the ab ⁇ ence or pre ⁇ ence of cycloheximide.
  • Table III-l summarizes mRNA analyses (Northern blot data) for mitogen-induced genes in response to agents used in Figs. III-1-3.
  • FIG. IV-1 portrays Northern Analy ⁇ i ⁇ of cDNA clone ⁇ pAT 464 and pAT 744 RNA in human peripheral blood T cell ⁇ , either un ⁇ timulated (control) cell ⁇ or cell ⁇ treated with cycloheximide or cyclo ⁇ porin.
  • Fig. IV-2 demon ⁇ trate ⁇ Northern analy ⁇ i ⁇ of pAT 464 and pAT 744 RNA in Jurkat cell ⁇ , either un ⁇ timulated (control) or cells stimulated with the agents indicated.
  • Fig. IV-3 illustrates the sequencing strategy, the nucleotide sequence, and the predicted ' amino acid sequence for pAT 464, including the open reading frame of 276 base pairs, corresonding to._92 amino acids. -• ⁇ *
  • Fig. IV-4 shows the sequencing strategy, the nucleotide sequence, and the predicted amino acid sequence for pAT 744, including the open reading frame corresponding to 92 amino acids.
  • Fig. IV-5 documents primer extension analysi ⁇ of the DNA ⁇ equence for pAT 744, using an oligonucleotide complementary to. positions 135 to 152 of the pAT 744 sequence annealed to RNA from human peripheral T cells.
  • Fig. IV-6 shows comparisons among the amino acid sequence ⁇ of proteins encoded by pAT 464, pAT 744 and a family of structurally related proteins.
  • Fig. IV-7 presents Southern blot analysi ⁇ of pAT 464 and pAT 744 gene ⁇ in human placental DNA digested with ⁇ everal restriction enzyme ⁇ .
  • Fig. IV-8 illu ⁇ trate ⁇ expre ⁇ ion of pAT 464 and pAT 744 mRNAs in promyelocytic (HL60) cell ⁇ and human tonsillar B Cells (by Northern blot methods).
  • Fig. IV-9 demonstrate ⁇ expression of appropriate secreted proteins in mammalian (COS) cells transformed with pAT 464 and pAT 744 DNAs in various expression vectors (by 35 s-cysteine labeling of proteins) .
  • Fig. IV-10 demonstrate ⁇ expre ⁇ ion of appro ⁇ priate secreted proteins in mammalian (COS) cells trans- formed with pAT 464 DNAs in variou ⁇ expre ⁇ ion vector ⁇ (by ⁇ S-cysteine labeling of proteins).
  • Fig. IV-11 shows the ues of rabbit antibody
  • Fig. IV-12 illustrates the activity of rabbit antibody (number 722) raised against the C-terminal 12 amino acids of the pAT 744 peptide to detect secreted peptide in supernatant ⁇ of COS cell culture ⁇ tran ⁇ formed with pAT 744 DNA (by Western blotting) .
  • Fig. IV-13 shows the use of rabbit antibody (number 720) raised against the C-terminal 12 amino acids of pAT 464 peptide to detect secreted peptide in super ⁇ natants of dbs " cell cultures transformed with pAT ⁇ 464 DNA.
  • Q Fig. IV-14 presents results with a second rabbit antibody (number 719) raised against the C- terminal 12 amino acid ⁇ of the pAT 464 peptide in reaction ⁇ with secreted peptide in supernatants of ' COS cell cultures transformed with pAT 464 DNA.
  • Fig. IV-15 demonstrates that mitogenically activated human peripheral blood T cells secrete pAT 464 protein (by Western blotting with anti-peptide antibody) .
  • DNA ⁇ of thi ⁇ invention include the following cDNA clone ⁇ : pAT 120; pAT 125; pAT 127; pAT
  • DNA ⁇ of thi ⁇ invention al ⁇ o include recombinant
  • a mammalian expression vector (exemplified by CDM-8 or PMT2-2) capable -of expressing inserted DNAs in CO
  • the sense strand DNA nucleotide sequence, and the predicted primary protein sequence encoded, are shown for full-length Act-2 DNA in Fig. 1-3.
  • four clones with identically sized inserts of approximately -0.7 kb were identified, all of which hybridized to the same 0.9 kb mRNA.
  • the ⁇ equencing ⁇ trategy u ⁇ ed, the re ⁇ ulting nucleotide ⁇ equence and the predicted amino acid sequence for the encoded protein are shown in figure IV-4 for' pAT 744 and Figure IV-3 for pAT 434.
  • the full-length Act-2 cDNA and pAT 744 cDNAs appear to be derived from es ⁇ entially the same gene.
  • base 131 is a T and 135 is A.
  • These differences cause predicted amino acid number 20 to be Pro and Leu, in full-length Act-2 and pAT 744, respectively, while the other base change does not alter the amino acid sequence. It is possible that such differences are due to inherited variations (i.e., polymorphism ⁇ ) in the gene ⁇ of the two different individuals from which the clones are derived.
  • pLD ⁇ 78 extends 7 nucleotide ⁇ further 5' than clone pAT 464 and sequence compari ⁇ on ⁇ how ⁇ a- difference of 5 nucleotides between both clones, none of which affect ⁇ the predicted amino acid ⁇ equence. These .differences in the nucleotide sequence may reflect polymorphisms between the genes of different donors. A* comparison of the sequences for pAT 744 and
  • the ⁇ e two gene ⁇ may have been derived from a common ance ⁇ tral gene and their encoded proteins may play functionally related roles.
  • Recombinant DNA molecules which comprise a vector and other DNAs of the invention are also within the scope of the invention.
  • Preferred recombinant molecules include molecules comprising full-length Act-2 DNA and any of the following DNAs: a bacteriophage lambda vector (gtlO); an M13 bacteriophage vector; or the RNA transcription vector pGEM-1.
  • Preferred recombinant DNAs of this invention also include recombinant molecules comprising clone pAT 744 or pAT 464 DNA and any of the following vector DNAs: lambda vector (e.g., gtlO); an M13 vector.
  • Most preferred recombinant molecules of tvhis invention include: molecules comprising full-length Act- 2 DNA and any of the following vector DNAs: a i>aculo- viru ⁇ vector (including pAc-Act'2, a plasmid derived from polyhedrin pla ⁇ mid pAc373; and a recombined baculovirus, vAc-Act2, derived from pAc-Act2 and a wild-type AcNPV strain E 2 DNA) .
  • a i>aculo- viru ⁇ vector including pAc-Act'2, a plasmid derived from polyhedrin pla ⁇ mid pAc373; and a recombined baculovirus, vAc-Act2, derived from pAc-Act2 and a wild-type AcNPV strain E 2 DNA
  • recombinant molecules are those comprising pAT 744 or pAT 464 DNA and a mammalian expres ⁇ ion vector (CDM-8 or PMT2-2) capable of expre ⁇ sing inserted DNAs in mammalian (e.g., COS) cells.
  • CDM-8 or PMT2-2 mammalian expres ⁇ ion vector
  • the full-length Act-2 human cDNA was synthe- ⁇ ized, clone and isolated as described in Experimental Section I.
  • 10° phage plaques from a HUT-102B2 cDNA library, constructed in lambda gtlO (1-14) were screened with a partial length (-0.4 kb) Act-2 cDNA insert, labeled using the random priming method (1-15).
  • the full-length (-0.7 kb) DNA sequence contains an open reading frame of 276 base pairs, corresponding to 92 amino acids. This reading frame utilizes the most 5' AUG, which generally is the one utilized in eucaryotic translation initiation.
  • the region of the first AUG has good homology with the consensus sequence determined for eucaryotic start sites (1-17).
  • the cDNA appears to extend to the 3' end of the mRNA since it contains classic AAUAAA poly- adenylation signals (1-18) and a start of a poly A tail.
  • the human cDNAs of this invention were synthe ⁇ sized, cloned and isolated as described in Experimental Section II by a process of "subtraction" cloning and hybridization using mRNA from activated T cells.
  • This strategy i ⁇ summarized in Figure II-l.
  • Human peripheral blood T cells were polyclonally activated for 4.5 hours in the presence of the mitogens phytohemagglutinin (PHA) and phorbol 12-mytri ⁇ tate 13-acetate (PMA) a ⁇ well as the protein synthe ⁇ i ⁇ inhibitor cycloheximide.
  • PHA phytohemagglutinin
  • PMA phorbol 12-mytri ⁇ tate 13-acetate
  • the ⁇ e clone ⁇ which exemplify DNA ⁇ of thi ⁇ invention, include: pAT 120; pAT 125; pAT 127; pAT 129; pAT 133; pAT 140S; pAT 140L; pAT 154; pAT 158; pAT 189; pAT 201; pAT 204; pAT 225; pAT 229; pAT 232S; pAT 232L; pAT 237; pAT 239; pAT 243; pAT 270; pAT 276; pAT 281; pAT 383; pAT 402; pAT 407; pAT 416; pAT 428; pAT 464; pAT 466; pAT 478; pAT 483; pAT 485; pAT 496; pAT 516; pAT 542; pAT 563; pAT 591; pAT 594;
  • Clones pAT 464 and pAT 744 which were originally derived from a subtracted cDNA library (see Experimental Section II), have been characterized in more detail. Near full-length cDNA clones were isolated from a cDNA library which was derived from RNA extracted from human peripheral blood T cells stimulated for 4 ⁇ 5 hr with PHA-P and PMA in the presence of cycloheximide. This cDNA library was constructed with oligo ⁇ _T priming as described (IV-29) and was subsequently cloned into- lambda gt 10 (IV-30).
  • Clone pAT 464 is 793 nucleotides long and clone pAT 744 is 659 nucleotide ⁇ long (including the Eco RI linker at 5' end).
  • Primer extension analysis for pAT 744 confirmed that the isolated clone represents an almost full length cDNA (Fig. IV-5). It can.be concluded that about 10 nucleotides of the 5' end of the true mRNA sequence are mis ⁇ ing in thi ⁇ cDNA clone.
  • the first ATGs for pAT 464 and pAT 744 are at po ⁇ itions 84 and 74, respectively. In each case an open reading frame of 92 amino acids follows. DNA templates for transcription of full-length
  • Act-2 RNA in the pGEM-1 vector were prepared as follows (see also Experimental Section I): The -0.7 kb full- length Act-2 cDNA was digested with Ava I, filled in with Klenow and dNTPs, and then dige ⁇ ted with Eco RI to liberate a nearly full length fragment (ba ⁇ e 27 through the 3' end of the in ⁇ ert) from which the artificial 5' G- C tail had been removed. This fragment was subcloned into PGEM-1 (Promega Biotec) which had been digested with Sma I and Eco RI.
  • Baculovirus vector constructs containing full- length Act-2 cDNA were prepared from the -0.7 kb Act-2 cDNA by cloning into the BamHI site of the baculovirus polyhedrin plasmid pAc373 (I-16a) to generate pAc-Act2, thereby placing the full-length Act-2 cDNA under control of the polyhedrin promoter (see also Experimental Section I) .
  • SF9 cells were cotransfected with pAc-Act2 and wild- type AcNPV strain E2 DNA.
  • a recombined baculovirus vAc- Act2 was isolated and purified by a procedure ; -.of succe ⁇ ive round ⁇ of plaque hybridization (I-16a). ⁇
  • sub ⁇ tantially pre lymphokine/cytokine-like protein ⁇ thu ⁇ produced can be employed, using well-known techniques, in diagnostic as ⁇ ay ⁇ to determine the pre ⁇ ence of receptor ⁇ for the ⁇ e protein ⁇ in variou ⁇ body fluid ⁇ and ti ⁇ ue ⁇ ample ⁇ .
  • the invention also comprise ⁇ a cell, preferably a mammalian or in ⁇ ect cell, tran ⁇ formed with a DNA of the invention, wherein the transforming DNA is capable of being expressed.
  • the cell transformed by the DNA of the invention secretes the protein encoded by that DNA in the (truncated) form secreted by activated T cells.
  • the invention also comprises novel lymphokine/cytokine-like proteins made by expres ⁇ ion of a DNA of the invention, or by tran ⁇ lation of an RNA of the invention.
  • the ⁇ e protein ⁇ will be of a secreted form (i.e. , lacking an apparent ⁇ ignal sequence) . These protein factors can be used for functional studies, and can be purified for additional biochemical and functional analyses, such as qualitative and quantitative receptor binding assays.
  • Insect cells transformed with a recombinant molecule containing full-length Act-2 DNA were prepared as described in Experimental Section I. , Briefly, the plaque purified, recombinant baculovirus, vAc-Act2, was used to infect SF9 cell ⁇ , according to standard -methods. The recombinant full-length Act-2/baculovirus vector was expressed in the SF9 cells to produce abundant Act-2 protein in the supernatant. Moreover, N-terminal sequencing of radiolabeled protein shows that the signal peptide is cleaved as predicted.
  • Mammalian cell ⁇ tran ⁇ formed with pAT 744 or pAT 464 DNA and secreting pAT 744 or pAT 464 protein are described in Experimental Section IV. Briefly, COS cells were transfected using standard DEAE-dextran methodology, with cDNA clones inserted into expres ⁇ ion sites of either CDM-8 or PMT2-T. The novel proteins were visualized by ⁇ S-cy ⁇ teine labelling of the cell ⁇ after the trans- faction of the DNA constructs using standard, well known methods. The size of the protein product ⁇ is appropriate as predicted from the cDNA sequence after the leader peptide is cleaved off.
  • This invention also comprises novel antibodies made against a peptide encoded by a DNA segment of the invention.
  • This embodiment of the invention is exempli ⁇ fied by rabbit antisera containing antibodie ⁇ which spe ⁇ cifically bind to pAT 744 or pAT 464 lymphokine/cytokine- like protein which includes the sequence of such peptide, were raised to synthetic peptides representing the C- terminal 12 amino acids of both pAT 744 and pAT 464 pro ⁇ teins, as predicted by the cDNA sequences. This was done by chemically synthesizing the peptides, linking them to carrier (KLH), and injecting the carrier plus peptides into rabbits, according to standard methods of peptide immunology.
  • KLH carrier
  • Figs. IV-11 and IV- 12 show the use in Western blot experiments of two cLif- ferent rabbit antibodies (721 and 722) rai ⁇ ed again ⁇ t the pAT 744 peptide. Similar studie ⁇ with antibodie ⁇ to pAT 464 ⁇ -peptides are presented in Figs. IV-13 and IV-14. *a_fi_s is "evident from the figures, the appropriate secreted factors are detected -by antisera from rabbits immunized with synthetic"peptides. ⁇ - - -- ⁇ "• 5"
  • This invention further comprise ⁇ novel bioassay methods for detecting the expression of genes related to DNAs of the invention.
  • DNAs of this invention were used as probes to determine steady state levels of or kinetics of induction T Of related mRNAs.
  • Method ⁇ for the ⁇ e bioa ⁇ ays of the invention, using full-length Act-2, or using pAT 744 -or pAT 464 DNAs, and standard Northern blotting techniques, are described in detail in Experimental Sections I, -or II-IV, respectively.
  • pAT 744 -or pAT 464 DNAs are described in detail in Experimental Sections I, -or II-IV, respectively.
  • Such method ⁇ may be readily applied to analy ⁇ is of gene expression for lymphokine/cytokine-like proteins, either in isolated cell ⁇ or variou ⁇ ti ⁇ ue ⁇ .
  • Such bioa ⁇ ay ⁇ may be useful, for example, for identification of various classe ⁇ of tumor cell ⁇ or genetic defect ⁇ in the inflam- matory re ⁇ ponse.
  • a novel immune activation gene denoted Act-2, has been identified by differential hybridization screen ⁇ ing of an activated T Cell library. The gene is included rapidly following T cell --activation with phytohemagglu- tinin, B cell activation with Staphylococcus aureus Cowan I, and monocyte activation With lipopolysaccharide. ' cDNA containing the full length coding region has been isolated. The deduced amino acid sequence predicts ran open reading frame of 92 amino acids, including a very hydrophobic N-terminus, which by weight matrix score is predicted to be a signal peptide.
  • INTRODUCTION T cells play a pivotal role in the regulatory and effector functions of the immune response.
  • a ⁇ erie ⁇ of biochemical event ⁇ occurs, including an increase in intracellular calcium, pho ⁇ phorylation of protein ⁇ , and increa ⁇ ed phosphoinositol turnover (see references 1-1, 1-2).
  • These event ⁇ generate signals which lead to the activation of cellular genes and the production of cellular proteins that are not expressed or are very weakly expressed by resting cells.
  • These induced pro- teins are e ⁇ sential for the proliferation and differenti ⁇ ation of T lymphocytes into effector T cells mediating . helper, suppre ⁇ sor, or cytotoxic T cell functions.
  • Thi ⁇ section de ⁇ cribe ⁇ the cDNA cloning, ⁇ equencing, and characterization of a novel gene, denoted Act-2, which was identified by differential screening of an activated normal T cell cDNA library.
  • Circulating human peripheral-blood mononuclear cells were obtained from healthy volunteers and isolated by Ficoll Hypaque (LSM, Litton Bionetics) gradient centrifugation. Cells were generally cultured at 1-2 x 10 6 cell ⁇ /ml overnight in RPMI 1640 medium containing 10% fetal bovine ⁇ erum (FBS), L- glutamine, and antibiotic ⁇ .
  • FBS fetal bovine ⁇ erum
  • PMA phorbol myri ⁇ tate acetate
  • B lymphocytes were purified by incubating PBMC .(4% non-specific e ⁇ tera ⁇ e po ⁇ itive cells) with 2-aminoethyliso-thiouronium bromide-treated sheep erythrocytes and removing the rosette-focusing cells. In some experiments monocytes were depleted by plastic adherence. Such cells were 80% surface Ig posi ⁇ tive, latex non-ingesting cells, and were cultured for 30 min to 72 h in the presence of a 1:10,000 dilution of Staphylococcus aureus Cowan I (SAC, Calbiochem-Behring Corp. ) .
  • Monocytes were prepared by elutriation of PBMCs and were 95% pure by Giem ⁇ a ⁇ tain and flow cytometry. Cell ⁇ were cultured for 8 h in the presence of 10 ug/mg lipopolysaccharide (LPS, Sigma).
  • T cell line Jurkat, HUT-102B2, Molt-4, CEM, and Hut-78
  • B cell lines Raji, S3, Nall-1, 8392, GM4672, U266, and SUDHL-6
  • myeloid line ⁇ 562 and U937
  • RPMI 1640 medium containing 10% FBS
  • the osteosarcoma cell line 5887 J.-9 were maintained in 5 DMEM containing 15% FBS.
  • Human fetal -lung fibroblast HFL-1 cells were passaged to a " density of approximately * 1 x 10° cells per 175 cm 2 tissue culture flask in 25 ml of DMEM containing 10% FBS and allowed to grow to confluence over a period of 7 days at 37°C. The cells were stimu- 0 lated by removing the depleted medium and replacing it with fresh DMEM containing 20% FBS. Early pas ⁇ age HFL-1 cells were used for all experiments. "•
  • Nitro ⁇ cellulose fibers were incubated for 3 h at 65°C in 0.1 M 25 NaH 2 P0 4 pH 6.8, 0.85 M NaCl, 1 mM EDTA, 10 x Denhardt' ⁇ solution, 0.1% SDS, 100 ug/ml salmon sperm DNA and 10 ug/ml poly(rA) (prehybridization solution).
  • the filters were then hybridized at 65°C for 48 h using fresh pre ⁇ hybridization solution containing 10% dextran sulfate and 30 the radioactive probe.
  • the filters were washed 4. times for 20 min at 65°C in 2 x SSC, 0.5% SDS and twice for 30 min at 65°C with 0.1 x SSC, 0.1% SDS at 65°C and then autoradio-graphed.
  • HUT-102B2 cDNA library constructed in lambda gtlO (I-
  • DNA sequencing.- Sequencing was performed via the dideoxy chain termination method using Ml3 phage DNA and Sequenase (U.S. Biochemical Corporation) according to the manufacturer's recommendations.
  • Genomic Southern blot 10 ug of DNA from either U937 or 428 cells was digested with indicated enzymes, analyzed on a 1% agarose gel, and transferred to Gene Screen Plus nylon membrane ⁇ (duPont) and hybridized according to the manufacturer ⁇ suggested protocol.
  • the -0.7 kB Act- 2 cDNA was cloned into the BamHI site of the baculovirus polyhedrin plasmid pAc373 (I-16a) to generate pAc-Act2, thereby placing teh Act-2 cDNA under control of the poly ⁇ hedrin promoter.
  • SF9 cells were cotransfected with pAc- Act2 and wild-type AcNPV strain E2 "DNA.
  • Act-2 mRNA expressed at low or undetectable level ⁇ in re ⁇ ting PBMC, increased rapidly and dramatically in re ⁇ pon ⁇ e to mitogen ⁇ timulation. Peak level ⁇ occurred at approxi ⁇ mately 4 h, and then rapidly declined to negligible level ⁇ by 24 h after stimulation. Thus, Act-2 expression is both rapidly induced and terminated in respon ⁇ e to PHA.
  • Act-2 represents a novel gene. Comparison of the DNA sequence and the predicted amino acid sequence using DNA and protein homology search programs did not reveal any homology with published sequences in the most recent GenBank (version 55, March 31, 1988) and NBRF (versions 16 and 34, released March 31, 1988) databases.
  • the DNA ⁇ equence contain ⁇ an open reading frame of 276 base pairs, corresponding to 92 amino acids. This reading frame utilize ⁇ the rao ⁇ t 5' AUG, which generally i ⁇ the one utilized in eucaryotic translation initiation. Further, the region of the first AUG has good homology with the consen ⁇ u ⁇ sequence determined for eucaryotic start sites (1-17).
  • the cDNA wa ⁇ utilized to perform a genomic Southern blot (Fig. 1-6). A comparatively simple pattern of digestion was obtained. The same blot, was ; hybridized with an interleukin-2 receptor cDNA (the IL-2 receptor is encoded -by a single copy gene) , and bands of similar intensity were identified (not shown). These data are most consistent with A ⁇ ct-2 representing a sihgle copy or low copy number gene.
  • Act-2 activated -PBMC cDNA, number 2
  • Act-2 encodes an open reading frame.of 92 amino acids, It contains,a very.hydrophobic N-terminus that by the von Heijne weight matrix analysi ⁇ i ⁇ ⁇ trongly predicted to be a signal peptide.
  • in vitro translation analyses could not demonstrate cleavage of the signal peptide, a recombinant Act- 2/baculovirus vector has been expressed in insect cells and found that Act-r2 protein is abundant in the super- natant.
  • N-terminal sequencing of radiolabeled protein shows that the signal peptide is cleaved as pre ⁇ dicted. It remain ⁇ an interesting possibility that Act-2 may also exist in a membrane as ⁇ ociated form.
  • antibodie ⁇ are being produced in order - to be able to directly identify the distribution of Act-2 protein pro ⁇ scored in normal activated human lymphoid cells.
  • the 3' untranslated region is also of signifi ⁇ cant interest in that it is A-T rich and contains the consensus sequence ⁇ ATTTA (1-23) and TTATTTAT (1-24) that have been identified as common sequences in a variety of proto-oncogenes and ⁇ ecreted factors, including tumor necrosis factor, lymphotoxin, IL-1 (both alpha and beta), multiple interferons, and GM-CSF.
  • the TTATTTAT con ⁇ ensus is not commonly found in mammalian mRNAs in general but is particularly prevalent in mRNAs encoding proteins related to the inflammatory response (1-24); thus its presence in Act-2 is supportive of the idea that this gene may represent a new cytokine.
  • TTATTTAT con ⁇ ensus is not commonly found in mammalian mRNAs in general but is particularly prevalent in mRNAs encoding proteins related to the inflammatory response (1-24); thus its presence in Act-2 is supportive of the idea that this gene may represent a new cyto
  • Act-2 wa ⁇ minimally or not expressed in re ⁇ ting PBMC. However, it wa ⁇ rapidly induced following activa ⁇ tion of T cell ⁇ with PHA or PMA, B cell ⁇ with SAC, or monocyte ⁇ with LPS. However, ct-2 i ⁇ not expre ⁇ ed in every actively growing cell, a ⁇ evidenced by it ⁇ non- expression in HeLa an K562 cells, and its failure to be induced in re ⁇ ponse to serum stimulation of quiescent human fibroblasts.
  • the activatable genes can be further differentiated on the basis of their kinetics of induc ⁇ tion, their response to cycloheximide as well as their sensitivity to the immunosuppressive drug cyclosporin A. It is of note that this latter drug inhibits the expression of more than ten inducible genes, suggesting a broad genetic mechanism for the action of this agent.
  • MATERIALS -AND METHODS Cell Culture.
  • Human peripheral blood T cells were obtained from healthy volunteers and, were isolated, over a Ficoll-Hypaque gradient and nylon wool columns. The resulting cell preparations were consitently more than 90% T cells, as judged by anti-CD3 staining.
  • PB T cells were cultured at a concentration of 2 x 106 cells/ ml in RPMI 1640 containing 10% fetal calf serum (FCS).
  • FCS fetal calf serum
  • PB T cells were stimulated for various periods of time with PHAP (1 yUg/ml Burroughs-Welcome Co.) and PMA (20 ng/ml) either with or without cycloheximide (10 uq/ ⁇ al) .
  • the Jurkat cell line was provided by K. Hardy.
  • Jurkat cells were maintained in RPMI 1640 supplemented with 10% FCS and 25 q/ ⁇ al gentamicin.
  • Jurkat cells were stimu ⁇ lated for various periods of time at a concentration of 5 x 105 with PHA-P (1 Uq/ l ) and PMA (25 ng/ml) with or without cyclo ⁇ porin A (1 yg/ml; Sandoz).
  • Human fibro- bla ⁇ t lines, CCD-11LU and WI38 were obtained from the American Type Culture Collection. Fibrobla ⁇ t ⁇ were grown to confluence in MEM containing 10% FCS and then main ⁇ tained in MEM with 0.25% FCS for 3 to 5 Day ⁇ . In order to reinitiate growth, the ⁇ pent medium wa ⁇ replaced by MEM supplemented with 20% FCS either with or without cycloheximide (10 ⁇ q/ l) .
  • PB * - ⁇ cells were isolated and cultured as. escribed above.
  • RNA was isolated as described (II-8) from unstimulated cell ⁇ or after stimulating the cells -for 4.5 hours with PHA-P arid PHA in the pre ⁇ ence of cycloheximide.
  • Poly A+ ?RNA wa ⁇ purified by one pas ⁇ age over an oligo-dT column (II- 3).
  • this cDNA wa ⁇ hybridized to a.cot value to 2000 mole ⁇ x ⁇ /1 with a 10 fold exce ⁇ s of poly A+- RNA from unstimulated cells.
  • the single stranded molecule ⁇ then were ⁇ eparated from the double stranded cDNA:mRNA hybrids by chromatography using a hydroxy- apatite column (11-14, 11-20). After the fir ⁇ t round of ⁇ ubtraction, 15% of the molecule ⁇ appeared in the ⁇ ingle ⁇ tranded fraction a ⁇ judged by the di ⁇ tribution of count ⁇ .
  • Probes were ⁇ upplied by the following individuals or institutions: GM-CSF, Genetics In ⁇ titute; -"/ ' -Interferon, Meloy Laboratories; c-fos. Dr. T. Curran; IL-2 receptor. Dr. W. Greene; IL-3, Genetics Institute; Met- and Leu-preproenkephalin, Dr. S. Sabol; human IL-4, ATCC; p53, Dr. D. Givol; lymphotoxin, Genentech; IL-5, Dr. K. Aria (DNAX); ornithine-decarboxy- la ⁇ e. Dr. D. Nathans; bcl-2. Dr. A. Bakhshi; IL-6, Dr. H.
  • the latter agent is known to superinduce a number of growth related genes (II-l, 11-27, 11-30, 11-34).
  • cyclo ⁇ heximide prevents mRNA induction which follows IL-2 and IL-2 receptor ⁇ ynthe ⁇ i ⁇ and interaction.
  • Thi ⁇ focuses the analysi ⁇ on the primary re ⁇ pon ⁇ e of activated ceils, defined by those genes which are inducible independent of new protein synthesis'. To date, approximately 40% of the subtracted lambda gtlO cDNA ? library has eeh screened with subtracted probes.
  • phage that hybridized to subtracted probes were subjected further to a differential serein in which ' cDNA probes synthesized from activated and resting T cell mRNA were used on duplicate filters of the phage. Finally, 528 phage clones were selected which harbored induced cDNAs, a ⁇ judged by both the subtractive and the differential screening methodolo ⁇ gies.
  • the next step was to cross- hybridize subcloned cDNA insert ⁇ with the 528 phage ⁇ .
  • 66 unique cDNA clone ⁇ were identified, the majority of which appear to repre ⁇ ent di ⁇ tinct gene ⁇ .
  • a limited number of group ⁇ may derive from different segments of the same mRNA, thus leading to an overestima- tion of individual gene ⁇ .
  • the number of unique inducible gene ⁇ will exceed 66, as 120 of the 528 phages have not hybridized to the selected cDNA insert ⁇ te ⁇ ted to date.
  • the number of isolated phage clones belonging to a given group via cros ⁇ -hybridization varied con ⁇ siderably and ranged from 1 to as many as 86 phage clones (see Table II-l legend) .
  • Forty-four of the novel inducible gene clones have been studied, further (see below). All hybridized to an inducible and, with few exceptions, single-sized message by Northern blot analyses (Table II-l) .
  • none of the cDNA inserts contained repetitive sequences, but a few appeared to be members of small multi-gene families, as determined by Southern blot analyses (Table II-l).
  • composition of the subtracted library was shown to repre ⁇ sent a typical activated T cell phenotype as determined by the enrichment for clones encoding IL-2, GM-CSF, gamma-IFN, c-myc and c-fos, the latter of which i ⁇ induced in T cells at 4.5 hours in the presence of cyclo ⁇ heximide, but to a much les ⁇ er extent than c-myc (II- 36).
  • ⁇ elected 528 phage were detected several isolates of c-myc.
  • the 528 phage include cDNAs homologous to the IL-2 receptor, the IL3 and IL-4 growth factors and Met- and Leu-preproenkephalin (11-46, 11-47, 11-49) (Table II-l).
  • IL-2 in the library that are not pre ⁇ ent in the 528 phage selected with subtracted cDNA probes resulted in part from the much stronger signal generated by nick-translated versus heterogenous cDNA probes.
  • the length of the induction period (4.5 hours) was optimal for a great number of genes, it was not optimal for those genes which are expressed with relatively delayed kinetics. Nonethe ⁇ less, the hybridization data indicate that the 528 selected phage encompass many but not all of the genes expected, and the subtracted library contains the known induced genes which have been assayed.
  • FIG. 1 In order to assess the heterogeneity of expres ⁇ ion for ' the i ⁇ olated inducible gene ⁇ , kinetic analy ⁇ es of mRNA levels for many of the genes were per ⁇ formed.
  • Figure II-2 displays typical Northern analyses for four novel inducible genes.
  • One pattern of expression exemplified by pAT 249, displays a very rapid appearance after activation of T cells by PHA and PMS, i.e., by 30 min. or less; another common pattern shown for .
  • pAT 464 is characterized by mRNA appearance only after 2-4 hours. Additional mRNA species (e.g.
  • pAT 129 and pAT 139, Figure 11-2) are induced at intermediate times.
  • the human CD4+ helper cell line Jurkat i ⁇ known to have retained the inducibility of ⁇ everal gene ⁇ , including the lymphokine ⁇ IL-2 and gamma-interferon as well as the 11-2 receptor (II-II-19, 11-37, 11-45).
  • CsA immunosuppre ⁇ ive drug cyclo ⁇ sporin A
  • Many lymphokines elaborated by activated T cells are known to be suppressed by this drug which appears to inhibit transcriptional induction (11-16, II- 37, 11-39).
  • Figure II-3 and summarized in Table II-2 four different patterns of expres ⁇ ion could be distinguished.
  • the remaining 13 genes tested were not induced in Jurkat T cells. Ten of these (e.g. pAT 133, Figure II-3) failed to hybridize to any message in Jurkat cells ⁇ as determined at a number of time points following induction (data not shown) . Possibly, these message ⁇ exist in a cell type distinct from Jurkat, such as CD8+ T cells. Alternatively, Jurkat cells are transformed and may have lost or modified these genes or the signalling machinery nece ⁇ sary to induce their mRNAs. The members of the last group of genes (3 out of 35) were con ⁇ titutively expresed in Jurkat cells (e.g., pAT 129, Figure II-3). These genes may contribute to the uncontrolled growth of this tumor line (see Discussion).
  • a Frequency grouping indicates the number of cros ⁇ - hybridizing phage clones, among the 528 clones isolated, which are detected by the subcloned insert listed: 1, one cros ⁇ -hybridizing phage clone; 2, two to five cro ⁇ - hybridizing phage clones; 3, six to 10 cross-hybridizing phage clones; 4, eleven to twenty-five cross-hybridizing phage clone ⁇ .
  • mRNA size (in nucleotides) was determined relative to the migration of the 28S and 18S rRNA on formaldehyde-agarose gels. time ( inute ⁇ ) at which the iduced mRNA species was first detected following PHA (1 /g/ml) and PMA (20 ng/ml) stimulation of peripheral blood (PB) T cells; the second number denotes the time (minutes) at which maximal steady state mRNA levels were noted.
  • a 542 al ⁇ o detected a mRNA species of 4600 nucleotides which wa ⁇ coordinately expre ⁇ ed with the predominant species of 6800 nucleotides.
  • h pAT 563 also detected mRNA species of 3600, 4100 and 8500 nucleotides,. coordinately expressed with the predominant 2400 nucleotide species.
  • Table II-2 Expression analysis of T cell induced genes in the helper T cell line Jurkat and in human fibroblasts. Experimental details are as described in figure II-3 and II-4. Y - induction upon stimulation of quiescent human fibroblasts (HF) with serum N - no expression in quiescent or serum stimulated human fibroblast ⁇ ND - not determined a - " denote ⁇ expre ⁇ sion data for the 900 nucleotide constitutively expres ⁇ ed mRNA species (see Table II-l) denote ⁇ expre ⁇ sion data for the 2000 nucleotide induced mRNA specie ⁇ ( ⁇ ee Table II-l)
  • these genes may encode other functions such as modulation of the immune system.
  • those genes that exhibit limited tis ⁇ ue di ⁇ tribution of expre ⁇ ion (Table II-2) are to be expected to play such roles in the differentiated function of activated T cell ⁇ .
  • two of the ⁇ e gene ⁇ , pAT 464 and 744 have been ⁇ equenced and di ⁇ play predicted hydrophobic leader peptide ⁇ and structural homology with known secreted proteins, sugge ⁇ ting their identity a ⁇ potential lymphokine ⁇ (Experimental Section IV) .
  • lymphokine cDNA clones encoding mouse and human IgA-enhancing factor and eosinophil colony- stimulating actor activities:- Relationship to interleukin 5. Proc. Natl. Acad. Sci. USA 84:7388-7392.
  • T cells with mitogenic monoclonal antibodies directed against the CD2 or CD3 cell surface molecules, or with PHA induced all nine genes.
  • stimulation by fully mitogenic agents regardles ⁇ of cell ⁇ urface binding specificity, corre ⁇ lates with the expression of all those genes studied here.
  • heterogenous patterns of gene expression encompassing five regulatory classes, were revealed, by the use of PMA, calcium ionophore and anti-CD28 mono ⁇ clonal antibody, agents which mediate only a sub ⁇ et of intracellular event ⁇ and thus an incomplete mitogenic signal.
  • IL-2 and two novel lymphokines represent one regulatory clas ⁇ that appear ⁇ to require unique tran ⁇ criptional activation signals relative to the other mitogen, induced genes.
  • the acti ⁇ vating agents studied here include monoclonal antibodies (mAbs) directed against CD2, CD3 or CD28 which are dis ⁇ tinct, physically non-associated T cell surface molecules, the lectin phytohemagglutinin (PHA), calcium ionophore, phorbol 12, myristate 13-acetate (PMA), and dioctanoylglycerol (DiC3).
  • mAbs monoclonal antibodies directed against CD2, CD3 or CD28 which are dis ⁇ tinct, physically non-associated T cell surface molecules, the lectin phytohemagglutinin (PHA), calcium ionophore, phorbol 12, myristate 13-acetate (PMA), and dioctanoylglycerol (DiC3).
  • T cell receptor or the associated CD3 complex of T cells initiates the activation of T cells (III-2, 111-18, III- 23).
  • PHA-mediated stimulation appears to require the presence of the antigen receptor complex or a coordinate ⁇ ly expressed molecule, as determined by ⁇ tudies with selective surface loss mutants (111-20).
  • An antigen- independent pathway of T cell activation involving the CD2 complex (E rosette receptor) has al ⁇ o been described (111-19).
  • Binding of the above mi ogens to cell surface receptors on T cells results in the production of inositol 1,4,5-tripho ⁇ phate , an increased intracellular calcium concentration ([Ca 2+ ]i), membrane translocation of protein kinase C (PKC) and sub ⁇ equent proliferation (III-6, III-9, 111-13, 111-15, 111-22, 111-25).
  • PKC protein kinase C
  • the signal delivered by CD28 seems to be funda ⁇ mentally distinct from that of CD2 or CD3 because stimu ⁇ lation by anti-CD28 leads to an elevation of cytoplasmic cyclic GMP concentration and does not cause increase ⁇ in [Ca2+]; or activation of PKC (111-12, 111-14, 111-22, 111-26).
  • IL-2 production initiated by CD28 i ⁇ re ⁇ i ⁇ tant to the effect ⁇ of cyclo ⁇ porin A in contra ⁇ t to that induced by CD3 or calcium ionophore and PMA (III-8).
  • anti-CD28 - alone doe ⁇ not ⁇ timulate proliferation of T cell ⁇ but require ⁇ the ⁇ imultaneous presence of a_comitogen, such as PJJA ( 11-7,, III-8).
  • a_comitogen such as PJJA ( 11-7,, III-8).
  • T cells also may be activated, bypassing celi surface interactions, following treatment with calcium ionophore in addition to PMA or DiC8; the latter two agents act, at least in part, by stimulating protein kinase C (111-16, 111-21).
  • Ionophore-mediated increa ⁇ e ⁇ in [Ca 2+ ] act synergistically with DiC3 or PMA to ⁇ timulate prolifera ⁇ tion and induced gene expre ⁇ ion in purified PB T cell ⁇ and ⁇ ome induced gene expre ⁇ ion, including lymphokine ⁇ , in the T cell line Jurkat (111-16).
  • PB mononuclear cell ⁇ were obtained from normal healthy donor ⁇ by leukophore ⁇ i ⁇ and PB lymphocyte ⁇ i ⁇ olated by den ⁇ ity gradient centrifuga ⁇ tion through Lymphocyte Separation Medium (Litton Bionetic ⁇ ) , followed by the removal of adherent cell ⁇ (monocyte ⁇ and B cells) by passage over a nylon wool column.
  • the resulting cell ⁇ had approximately 1% B cells and ⁇ 1% monocytes a ⁇ determined by FACS.
  • the T cell ⁇ were cultured at 2 X 10 /ml in RPMI-1640 medium contain ⁇ ing 10% heat-inactivated fetal calf serum and stimulated with one of the following agents: anti-CD3 (mAb OKT3, Ortho Diagnostics), used at 10 ng/ml; PHA-P (Burroughs- Wellcome), used at 3 ⁇ q/ ⁇ al; anti-CD2 (ref. 111-19: anti- T112 and anti-T113 ascites, obtained from E. Reinherz), each ascites was used at a 1:200 dilution. All reagents were titrated to determine the optimal concentration as a ⁇ ayed by the ability to stimulate peak proliferation of PB T cells or IL-2 production by Jurkat cells.
  • cycloheximide (Sigma Chemical Co.) was used at 10 Uq/ml .
  • the ability of each agent to stimulate pro- liferation was determined by 3 H-thymidine incorporation assayed after 4 days.
  • a representative experiment showed 1,003 cpm for culture medium alone, 184 / 567 cpm for PHA, 107,908 cpm for anti-CD2 and 1,248 cpm for anti-CD3.
  • the lacksof response to anti-CD3 in PB T cells appeared due to monocyte depletion, since linfractionated PBL from the same donor gave 127,760 cpm.
  • CD28+ cell ⁇ were purified a ⁇ previously described (III-8). The cells were cultured as outlined above and stimulated for 4 h with anti-CD28 mAb 9.3 (obtained from J. A. Ledbetter) at 100 ng/ml, iono ⁇ mycin (Calbiochem; dissolved in DMSO) at 133 nM, and PMA (Sigma Chemical Co.; dissolved in DMSO) at 0.3 ng/ml.
  • a preliminary experiment showed all the gene ⁇ to be maxi ⁇ mally induced at 4 h.
  • the PMA concentration wa ⁇ determined by ⁇ H-thymidine incorporation to maximally ⁇ ynergize with either anti-CD28 or ionomycin yet not to be it ⁇ elf mitogenic.
  • the mitogenic activity of each agent wa ⁇ assayed by 3 H-thymidine incorporation after 3 days of stimulation.
  • a representative experiment showed 121 cpm for medium alone, 488 cpm for PMA, 236 cpm for mAb 9.3 alone, 58,590 cpm for mAb 9.3 and PMA. 186 cpm for ionomycin alone, 44,760 cpm for ionomycin and PMA and 69,360 cpm for immobilized anti-CD3.
  • Jurkat cells were maintained in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum at a den ⁇ ity of 2 X 10-Yml to 8 X 10°/ml. Fre ⁇ h aliquots of cells were thawed at approximately 6 week intervals. Cells were stimulated at 4 X 10 /ml with one or more of the follow- ing agents: DiC3 (Molecular Probes. Inc., Eugene, OR; dissolved in absolute ethanol) at 100 /M, ionomycin at 500 nM, cycloheximide at lOjU/ml. The DiC3 and ionomycin - 62 -
  • RNA (10 pg per lane) was separated by electrophoresi ⁇ in formaldehyde-agarose gels, ' blotted onto Genescreen membrane filter ⁇ (NEN Re ⁇ earch Product ⁇ ) and hybridized to 32 p-labelled purified cDNA inserts prepared by nick- translation.
  • Fig. Ill-i show ⁇ that for each gene, PHA, anti- CD2 and anti-CD3 stimulation resulted in ⁇ imilar re ⁇ pon ⁇ es.
  • a hierarchy in which the relative levels of mRNA expression can be graded from a low level of induci- bility (pAT 229) to strong inducibility (pAT 464) can be seen in response to any of the three mitogen ⁇ .
  • pAT 464, Fig. III-l pAT 744, data not shown
  • two were induced to a moderate level pAT 563, Fig. III-l; pAT 120, data not shown
  • two were induced to a low level pAT 416, Fig.
  • III-l pAT 237, data not shown) and three were weakly induced (pAT 229, Fig. III-l; pAT 154 and pAT 225, data not shown) by either PHA, or anti-CD2 or anti-CD3 mAbs. Stimulation with any one of the above mitogen ⁇ in the presence of PMA elicited an elevated response, relative to PHA. anti-CD2 or anti-CD3 mAbs alone, for all of the genes (data not shown) . In addition, although the kinetics of mRNA accumulation are unique for each gene, the ⁇ e kinetics are relatively uniform for each gene irrespective of the stimulus used. "
  • Table III-l Summary of Northern blot data for mitogen- induced genes in respon ⁇ e to agents used in Figs. III-l- 3. Symbols: +, mRNA hybridization to labelled probe; -, no hybridization to labelled probe; -, no hybridization; +/-, weak hybridization; Ab, abrogation of DiC8- and ionomycinr-mediated expression; Incr, increased levels of DiC8- and ionomycin-mediated expre ⁇ ion; ND, experiment not performed.
  • the ⁇ e agent ⁇ include calcium ionophore, PKC activators or anti- CD28 mAb, none of which stimulates DNA synthe ⁇ i ⁇ in puri ⁇ fied T cell ⁇ (III-8, 111-16, 111-24 and reviewed in III- 26).
  • purified T cell ⁇ proliferated after treatment with the combination of calcium ionophore or anti-CD28 mAb and PMA (see Materials and Methods).
  • Fig. III-2 Steady state mRNA levels resulting from stimu ⁇ lation of CD28+ T cells with PMA, anti-CD28 mAb 9.3, ionomycin, PMA plus anti-CD28 mAb and PMA plus ionomycin are shown in Fig. III-2. Unlike stimulation via CD2 or CD3, which elicited graded responses from all nine mitogen induced genes, activation through CD28 clearly divided the nine novel genes into distinct groups (Fig. III-2 and Table III-l). One group, exemplified by pAT 416 (Fig. III-2), and including pAT 120, pAT 154, pAT 225 and pAT 229 (data not shown), showed no respon ⁇ e to anti- CD28 mAb.
  • III-2 pAT 563 and pAT 744, data not ⁇ hown) and for IL-2R (III-8 and data not ⁇ hown) .
  • PMA alone induced varying level ⁇ of expre ⁇ ion, gene induc ⁇ tion with ⁇ oluble or cro ⁇ -linked mAb 9.3 alone was not seen in this group.
  • IL-2 mRNA in contrast, was not induced by PMA alone, but was induced in respon ⁇ e to a synergistic combination of PMA and mAb 9.3.
  • DiC8 is a cell-permeant synthetic diacylglycerol (DG) that mimics the effect of endogenous DG by reversibly acti ⁇ vating PKC (III-5, 111-16, 111-17) .
  • DG cell-permeant synthetic diacylglycerol
  • the fir ⁇ t cla ⁇ detected i ⁇ represented by pAT 237 in Fig. III-3 and includes genes which are PKC inducible but display either varible and minimal or no synergy with a [Ca 2+ ]i signal (pAT 237, Fig. III-3: pAT 120, pAT 154, pAT 225 and pAT 416, data not shown). No response to ionomycin alone was detected for this cla ⁇ of gene ⁇ .
  • the final cla ⁇ ob ⁇ erved in these cells includes pAT 464 (data not shown) and . AT 744 as well a ⁇ IL-2 (Fig. III-3), whose induction exhibited a require-** ment for both signals as ⁇ ayed in thi ⁇ experiment.
  • pAT 464 data not shown
  • AT 744 as well a ⁇ IL-2 (Fig. III-3)
  • Fig. III-3 a ⁇ IL-2
  • cycloheximide added ⁇ imultaneou ⁇ l with the activating agent ⁇ entirely prevented the accumu ⁇ lation Of IL-2, pAT 744 (Fig. III-3) and pAT 464 mRNA (data not shown) , implying a mechanism of gene induction requiring newly ⁇ ynthesized protein.
  • cyclo ⁇ heximide treatment .enhanced mRNA levels in the other two groups (Fig. III-3 and data not shown) .
  • the apparent homogeneity of expre ⁇ ion pattern ⁇ for each gene in re ⁇ pon ⁇ e to PHA, anti-CD3 or anti-CD2 mAb ⁇ may reflect that qualitatively, a similar series of intracellular biochemical events re ⁇ ult ⁇ from activating T cells with the ⁇ e agents.
  • This common pattern of gene expres ⁇ ion in re ⁇ ponse to a variety of inducing agents can potentially be mediated at a number of levels in the signalling pathway.
  • PB T cell ⁇ are heterogeneou ⁇ with regard to population subtype ⁇ or from differences in the basal state of activation between quiescent, nontrans- formed cell ⁇ and cycling, tumor cell ⁇ .
  • Jurkat cell ⁇ may abnormally over-expre ⁇ s or under-expres ⁇ ⁇ pecific regulatory component ⁇ required for ⁇ ome gene induction event ⁇ .
  • Thi ⁇ regulatory cla ⁇ of gene ⁇ responds to signal ⁇ mediated through CD28, in addition to CD2 and CD3 in PB T cell ⁇ , exhibit ⁇ a requirement for two ⁇ ignal ⁇ in Jurkat cell ⁇ , and their induction i ⁇ completely abrogated by cycloheximide treat ⁇ ment in Jurkat cell ⁇ .
  • a requirement for protein ⁇ ynthe ⁇ i ⁇ preceding mRNA induction in Jurkat appear ⁇ to be unique to thi ⁇ cla ⁇ of gene ⁇ , relative to many other mitogen induced gene ⁇ ⁇ tudied here and elsewhere, suggesting a novel and conserved regulatory mechanism for these and most likely other lymphokines.
  • T cell activation II A new activation pathway used by a major T cell population via a disul- fide.bonded dimer of a 44 kilodalton polypeptide
  • T.Cell pro ⁇ liferation involving the CD28 pathway is as ⁇ oci- ated with cyclo ⁇ porin.re ⁇ i ⁇ tant interleukin 2 gene expression. Mol. Cell. Biol. 7:4472-4481.
  • T. cell vari ⁇ ants lacking molecules involved in T. cell acti- vation T3 T. cell receptor, T44. and Til:
  • the two gene ⁇ can be expre ⁇ ed in T cell ⁇ , B cells and the promyelocytic cell line HL60, but they are not expressed in human fibroblasts, suggesting that their expres ⁇ ion i ⁇ re ⁇ tricted to hematopoietic lineages.
  • the predicted peptides encoded by these two clones feature hydrophobic N-terminal leaders -character ⁇ istic of secreted proteins.
  • the predicted size of both proteins is about 8 kD upon cleavage of the putative leader, peptide.
  • pAT 464 and pAT 744 are similar to each other and also share some critical amino acid sequence similar- ity with a newly emerging family of ⁇ ecreted factors including connective tissue activating factor III (CTAP III), platelet factor 4 (PF4), an interferon-gamma induced factor (IP-10), macrophage inflammatory protein (MIP) and a factor chemotactic to neutrophils (3-10C, MDNCF, NAF) .
  • CTAP III connective tissue activating factor III
  • PF4 platelet factor 4
  • IP-10 interferon-gamma induced factor
  • MIP macrophage inflammatory protein
  • MDNCF macrophage inflammatory protein
  • Human peripheral blood T cell ⁇ were obtained from healthy human donors by lympho- phore ⁇ is on a enwall CS 3000 or an_ IBM Cebe 2997 -appa- ratu ⁇ . Blood enriched for lymphocyte ⁇ wa ⁇ subsequently purified over Ficoll Hypaque gradients and nylon wool columns. As judged by anti CD3 staining, the ⁇ e cell preparation ⁇ con ⁇ i ⁇ tently were compo ⁇ ed of greater than 90% T cell ⁇ .
  • Cell Stimulation Human peripheral blood T cell ⁇ , Jurkat cell ⁇ and HL60 cell ⁇ were maintained in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum.
  • Penicillin, Streptomycin and Gentamycin at a cell density of 2 x 10° cells/ml for human T cell ⁇ , or 5 x 10 5 cell ⁇ /ml for Jurkat cell ⁇ , re ⁇ pectively.
  • the cell ⁇ were treated with either one or a combination of the following agent ⁇ : Phytohemagglutinin (PHA-P; Burirough ⁇ Wellcome) at a final concentration of 1 Afg/ml, Phorbol 12-myri ⁇ tate 13-acetate (PMA) at 20 ng/ml, cycloheximide at 10 jq/ml and cyclo ⁇ porin A at 1 juq/ml .
  • PHA-P Phytohemagglutinin
  • PMA Phorbol 12-myri ⁇ tate 13-acetate
  • cycloheximide at 10 jq/ml
  • cyclo ⁇ porin A at 1 juq/ml .
  • RNA Isolation and Northern Analysi ⁇ Total cellular RNA wa ⁇ isolated by the quanidinium isothiocy- nate method as described (IV-27) and either 8 ⁇ q (periph ⁇ eral blood T cells) or 10 ⁇ q (Jurkat cells) of total cellular RNA were separated on a formaldehyde agarose gel and subsequently transferred to nitrocellulose (Schleicher and Schuell) with 10 x SSC. ,
  • cDNA insert ⁇ were nick-translated (IV-28) and used as probe ⁇ for hybridization at 42°C in 40% Formamide, 4 x SSC, 10% dextran sulfate and lx Denhardt's ⁇ olution. After hybridization for 10-18 hr, the filters were washed at a final stringency of 0.1 x SSC at 60°C
  • Clones pAT 464 and pAT 744 originally were derived from a sub- ⁇ tracted cDNA library (Experimental Section II, above). Near full-length cDNA clone ⁇ were i ⁇ olated from a cDNA library which wa ⁇ derived from RNA extracted from human peripheral blood T cell ⁇ ⁇ timulated for 4.5-hr with. PHA-P and PMA in the pre ⁇ ence of cycloheximide. Thi ⁇ cDNA library was constructed with oligo dT priming as described (IV-29) and was sub ⁇ equently cloned into lambda gt 10 (IV-30).
  • Primers for the sequencing reactions were syn- the ⁇ ized a ⁇ 17mer ⁇ on an oligonucleotide ⁇ ynthe ⁇ izer model 380A (Applied Biosystems) following the protocol of the supplier. All oligonucleotides were purified on a 20% acrylamide gel prior to use. Both the cDNA sequences- and the predicted protein ⁇ equence ⁇ were compared with- a- computer data bank (Bionet, current release).
  • oligonucleotide comple-. mentary. to -nucleotide 135-152 of the pAT 744 sequence (Fig. IV-4B) was annealed to 20 q of RNA f_eom human peripheral blood T cells which were stimulated for 4.5 ⁇ ir with Pi_A and PMA in the presence of cycloheximide. Annealing was performed in 100 mM KC1, and the reverse transcription reaction was a ⁇ described (IV-29), using reverse transcripta ⁇ e (Seikageiku) to extend the annealed primer (100 (g/ml) .
  • Hybrid ⁇ were extracted'asrith an-equal volume of phenol/chloroform and then-were precipitated in ethanol.
  • the final reaction product wa ⁇ analyzed on a 8% denaturing polyacrylamide gel.
  • a DNA ⁇ equence ladder ⁇ erved a ⁇ size markers.
  • COS cell ⁇ were transfected msing ⁇ tandard.DEAE- dextran methodology, with cDNA clones inserted-- into expression sites of either of the following vector DNAs: CDM-8 [obtained from Brian Seed at Massachusett ⁇ . General Hospital, Boston, Massachusetts, and described in B. Seed, Nature 329, 840-842 (1987)]; or PMT2-T [obtained from Genetics In ⁇ titute, Cambridge, Ma ⁇ achu ⁇ ett ⁇ ; a related earlier ver ⁇ ion of thi ⁇ vector ha ⁇ been de ⁇ cribed in Yu-Chung Yang, et al.. Cell _47_, 3-10 (1986)].
  • CDM-8 obtained from Brian Seed at Massachusett ⁇ . General Hospital, Boston, Massachusetts, and described in B. Seed, Nature 329, 840-842 (1987)
  • PMT2-T obtained from Genetics In ⁇ titute, Cambridge, Ma ⁇ achu ⁇ ett ⁇ ; a related earlier ver ⁇ ion of thi ⁇ vector
  • pAT 464 and pAT 744 Two genes from this collection of cDNAs, named pAT 464 and pAT 744, were noted to ⁇ hare regulatory char- acteri ⁇ tic ⁇ with the IL-2 lymphokine gene (Experimental Section III). Like IL-2, the ⁇ e genes required absolutely the combined action of signal ⁇ initiated by ionomycin and PMA for optimal expression in highly purified T cells. In addition, mRNAs for pAT 464 and pAT 744 were induced by 2 hours and maintained at high levels for at least 24 hours, kinetics different from those seen with the major ⁇ ity of the isolated genes (Experimental Section II).
  • Nucleotide Sequence of pAT 464 and pAT 744 The nucleotide sequence of near full-length cDNA clone ⁇ for pAT 464 and pAT 744 wa ⁇ determined (see Materials and Methods). The sequencing strategies used, the resulting nucleotide sequences and the predicted amino acid ⁇ for the encoded protein ⁇ are ⁇ hown in Figure ⁇ rv-3 and IV-4 for pAT 464 and pAT 744, re ⁇ pectively.
  • Clone pAT 464 is 793 nucleotides long and clone pAT 744 is 659 nucleotides long (including the Eco RI linker at the 5' end). Both cDNA sizes are in good agreement with the estimated mRNA sizes of 850 nucleotides (pAT 464) and 750 nucleotides (pAT 744), since the mRNAs are likely to include a much longer poly A tail.
  • Primer extension analysis for pAT 744 confirmed that the isolated clone represents an almost full length cDNA (Fig. IV-5).
  • U ⁇ ing an oligonucleotide complementary to nucleotide ⁇ 135-152 (Fig. IV-4) of the pAT 744 ⁇ equence a ⁇ a primer for reverse transcriptase resulted in a fragment of about 155 nucleotides in size (138 nucleotides of synthesized cDNA and 17 nucleotides of primer) .
  • the first ATGs for pAT 464 and pAT 7.44 are at positions 84 and 74, respectively.
  • the ⁇ e open read- ing frame ⁇ are the longes to be found : for the two clone ⁇ .
  • the nucleotide sequences surrounding the predicted start sites for translation for both clones display a good match with the consensus sequences derived from known translation initiation site ⁇ (IV-36).
  • the 3' untranslated region ⁇ of the two cloned gene ⁇ contain several elements which may serve regulatory functions.
  • the ATTTA motif (underlined in »igure ⁇ : IV-3 and IV-4) that Jaa ⁇ been a ⁇ ociated with the instability of lymphokine mes ⁇ age ⁇ (IV-39) appear ⁇ four time ⁇ in clone pAT 464 and twice in clone pAT 744.
  • pAT J44. and 464 contain the octamer motif L TTATTTAT (a ⁇ ub ⁇ et of the ATTTA motif ⁇ ), which i ⁇ present in the 3' untran ⁇ lated region ⁇ of a number of distinct mRNAs induced during an inflammatory response ' (IV-40 ⁇ .
  • the ⁇ e two gene ⁇ may have been derived from a common ancestral gene and. their encoded proteins " may play functionally related roles•; The higher amino acid similarity sugge ⁇ t ⁇ a con ⁇ ervation of function and further ⁇ trengthen ⁇ the encoded protein ⁇ tructure ⁇ predicted from the open reading frame ⁇ in the cDNA clone ⁇ (see also comparison with other protein ⁇ below) .
  • the pre ⁇ umed ⁇ ecreted part ⁇ of the two pro- tein ⁇ exhibit a high degree of ⁇ imilarity, wherea ⁇ their N-terminal leader peptide ⁇ have little in common except their hydrophobic nature.
  • the hydrophilicity plot ⁇ of the two protein ⁇ are virtually superimpo ⁇ able which affirms further their structural relatedness (data not shown).
  • the primary structures of the proteins encoded by pAT 464 and 744 exhibit a striking similarity to a number of other proteins, ⁇ o e of which have been detected a ⁇ ⁇ ecreted proteins.
  • ⁇ o e of proteins have been detected a ⁇ ⁇ ecreted proteins.
  • Several members of this family of proteins have been shown to exhibit functions assiciated with an inflammatory response and with mitogenesis (see Discus ⁇ ion).
  • thi ⁇ similarity is based on four almost identically spaced cysteine residues and one proline residue, present in all proteins of this family (Fig. IV-6B).
  • These highly conserved amino acids are likely to play an important role in the structure of the proteins (see Discussion). In addition to this conservation, many other amino acids are shared between various members of this family.
  • genomic DNA wa ⁇ probed with the ⁇ e two gene ⁇ Human placenta DNA was digested with various restriction enzymes and subjected to a Southern analysi ⁇ with radioactive probe ⁇ derived from the cDNA in ⁇ ert ⁇ for the two gene ⁇ .
  • pAT 464 and 744 do not readily cros ⁇ -hybridize with each other, in ⁇ pite of their remarkable ⁇ imilarity.
  • pAT 464 and 744 are not expres ⁇ ed in quiescent or serum stimulated normal human fibroblast ⁇ . Here hematopoietic cell ⁇ other than T cell ⁇ were analyzed for expression.
  • Promyelocytic HL60 cells can be differentiated terminally into macrophages or into granulocyte ⁇ . Neither pAT 464 nor pAT 744 was expres ⁇ ed in undifferentiated HL60 cells but both were induced upon induction of differentiation into macrophages with PMA (Fig. IV-8A) . In contrast, using DMSO to differentiate HL60 cells into granulocytes did not result in any indue- tion of their mRNA ⁇ .
  • pAT 464 and pAT 744 may be expre ⁇ ed by a number of dif ⁇ ferent hematopoietic cell ⁇ upon being activated in ⁇ ome manner, including viral transformations.
  • Antibodies to predicted amino acid sequences Rabbit antisera were raised to synthetic peptide ⁇ repre- ⁇ enting the C-terminal 12 amino acids of both pAT 464 and pAT 744, as predicted by the cDNA sequences. This was done by chemically synthesizing the peptides, linking them to carrier (KLH), and injecting the carrier plu ⁇ peptide ⁇ into rabbit ⁇ , according to ⁇ tandard method ⁇ of peptide immunology.
  • Figs. IV-11A and B show the use in We ⁇ tern blot experiment ⁇ of two different rabbit anti- bodie ⁇ (721 and 722) rai ⁇ ed again ⁇ t the pAT 744 peptide; Figs.
  • IV-12A and IV-B similarly illustrate two antibodies (719 and 720) to the pAT 464 peptide.
  • supernatants of COS cells transfected with various expres ⁇ ion vector con ⁇ truct ⁇ were analyzed with the anti- ⁇ era.
  • ⁇ the appropriate secreted factors are detected by antisera from rabbits immunized with synthetic peptides, but not by serum sam- ple ⁇ taken from the ⁇ ame rabbit ⁇ before immunization with peptide ⁇ .
  • Fig. IV-13 shows a Western blot of supernatant from human T cells activated in culture with PHA/PMA u ⁇ ing anti-pAT 464 antibody. The antibody detects a secreted product of appropriate size. Similar results were obtained with anti-pAT 744 antibody (data not shown). Therefore, human T cells produce not only an abundant amount of pAT 464 and pAT 744 mRNA upon mitogenic stimulation (see above), but also the secreted forms of the protein factors predicted from the cDNA sequences of these clones.
  • pAT 464 and ' 744 are very homologou ⁇ to each other, e ⁇ pecially in their pre ⁇ umed ⁇ ecreted port__6f ⁇ s-_, Thi ⁇ ⁇ ugge ⁇ t ⁇ con ⁇ ervation of function. Both gene pro-*- ducts also share important amino acid residues with a family of proteins, -whose functions are not oicL only-par ⁇ tially understood; some of these protein ⁇ have been detected a ⁇ secreted factors (see below). Although pAT 464 and pAT 744 do not possess significant amino acid homology with several well-defined immunoregulatory fac- tors like IL-2 or gamma-IFN, they are likely to ⁇ hare regulatory element ⁇ with the ⁇ e gene ⁇ .
  • pAT 464 and pAT 744 require two inducing signals for optimal expres ⁇ ion (e.g., PHA and PMA), they appear after 2 hour ⁇ of ⁇ timulation of T cell ⁇ , they are very sensitive to the inhibitory effects of the immuno ⁇ uppre ⁇ - ⁇ ive drug cyclo ⁇ porin A and their activation i ⁇ sup- pre ⁇ ed completely in Jurkat T Cell ⁇ in the pre ⁇ ence of cycloheximide.
  • the primary amino acid ⁇ tructure ⁇ of the-'vpro- tein ⁇ encoded by pAT 464 and 744 exhibit ⁇ ignificant homology with a newly emerging family of ⁇ ecreted fac ⁇ tors.
  • the homology is ba ⁇ ed primarily on the positions of four cysteine re ⁇ idue ⁇ ( ⁇ ee al ⁇ o IV-23, IV-42, IV-43) and one proline re ⁇ idue (boxed in Fig. IV-6B).
  • the ⁇ e amino acid ⁇ are found in all the protein ⁇ ⁇ hown here and the peptides were aligned accordingly in Figure IV-6B.
  • the cysteines are likely to provide a common structure.
  • cysteine ⁇ For one of the members of this family, ?-thromboglobulin (a processed form of PBP, see below), these cysteine ⁇ have been shown to form di ⁇ ulfide bonds (IV-44). Start ⁇ ing from the amino terminus, cysteine ⁇ 1 and 3 and cy ⁇ - teines 2 and 4 form ⁇ uch bonds.
  • Trp Val Gin WVQ
  • WV WV
  • Fig. IV-6B the amino acids Trp Val Gin (WVQ) (or WV) (boxed in Fig. IV-6B) are found in eight of the factors, ⁇ ix re ⁇ idue ⁇ down ⁇ tream from the la ⁇ t of the four conserved cysteines.
  • the genes listed in Figure IV- 6B can be subdivided into two principal groups: The first two cysteines are positioned in one of two way ⁇ , either next to each other as Cys (C) or separated by one amino acid as C X C.
  • IV-23) or monocyte-derived neutrophil chemotactic factor (MDNCF) (IV-43) or neutrophil activating factor (NAF) (IV-25) is a human factor which has chemotactic activity for granulocytes in vitro (IV-24, IV-45, IV-46),- induce ⁇ rapid granulocyto ⁇ is jua vitro or with systemic injections in vivo and causes skin reactions upon local injection (IV-46, IV-47).
  • IP-10 (IV-21) is an interferon induced -protein factor which is expre ⁇ ed during delayed-type hyper ⁇ en ⁇ i- tivity reactions incited by a form of tuberculin or by - lFN (IV-49). It can " be expressed in several cell types, including endothelial cells, monocytes, fibroblast ⁇ and keratinocyte ⁇ (IV-49, IV-50).
  • Platelet factor 4 i ⁇ relea ⁇ ed from the o-s-granule ⁇ in platelet ⁇ during injury and ha ⁇ chemotactic activity for monocyte ⁇ , neutrophils and fibroblasts (IV- 51, IV-52). Additional activities are the inhibition of collagenase (IV-53) and an immunoregulatory role (IV-54). Platelets al ⁇ o release the platelet basic pro ⁇ tein (PBP) ⁇ -thromboglobulin (A-TG) and connective tis ⁇ ue activating peptide III (CTAP III) (also known as low- affinity platelet factor-4).
  • PBP platelet basic pro ⁇ tein
  • A-TG ⁇ -thromboglobulin
  • CTAP III connective tis ⁇ ue activating peptide III
  • CTAP III and _>-TG represent succe ⁇ ive N-terminally proce ⁇ ed form ⁇ of PBB, a ⁇ indicated in Figure IV-6B.
  • _?-TG appear ⁇ to be chemotactic for fibroblasts (IV- 51). The remaining factors li ⁇ ted were all cloned on the ba ⁇ i ⁇ of being inducible or differentially expre ⁇ ed.
  • RANTES (IV-42) and TCA-3 (IV-57) are T cell derived gene ⁇ for which no function i ⁇ known yet.
  • the human H-Gro gene, the mou ⁇ e JE gene and the chicken 9E3 gene are all expressed by fibroblasts.
  • the JE gene is induced by various agents in 3T3 cell ⁇ , including serum and IL-1 (IV-58).
  • the 9E3 gene is acti ⁇ vated by transformation with the Rous Sarcoma Viru ⁇ and i ⁇ suppressed in growth-restricted cells (IV-59).
  • the H- Gro gene is the human counterpart of the hamster Gro gene, whose constitutive expres ⁇ ion correlate ⁇ with tumorigenic variants of the nontumorigenic Chinese ham ⁇ ster fibroblastic cell ⁇ (CHEF) (IV-26). Gro is tran- siently induced by ⁇ erum in the nontumorigenic CHEF cells. The expre ⁇ ion of thi ⁇ gene i ⁇ apparently not sufficient for induction of the phenotypes associated with the tumorigenic state.
  • the expression data for the genes discussed above is consi ⁇ tent with a role during inflammation and/or mitogene ⁇ i ⁇ a ⁇ ociated with tissue repair. This is deduced from the various functions demonstrated, from the agents inducing these genes and from the cell types involved. Therefore, pAT 464 and pAT 744 are expected to exercise similar functions, in particular because the pAT 464 gene appears to represent the human homolog of the mouse MIP gene. Inflammation and wound healing/tissue repair are very complex proce ⁇ es which involve many different cells. Coordination of the various cellular functions is likely to be reflected in a multitude of secreted factors needed to communicate between cells. This family of genes, which may have been derived from one common ancestral gene, may well serve to establish such a network.
  • the individual factors may possess multiple functions, as shown for several of the members of this family (see above) .
  • the conserved similarities between these pro ⁇ teins may indicate an interaction with conserved receptor elements.
  • the members of this family of protein ⁇ di ⁇ play a remarkable variety of ti ⁇ ue specificities.
  • pAT 464 and pAT 744 are expres ⁇ ed in T cell ⁇ and other hematopoietic cell ⁇ , but not in fibro- blasts.
  • the distinction ⁇ between the factor ⁇ may deter-: mine unique function ⁇ required during an immune reaction depending on the cell type activated.
  • CDM-8 or PMT2-2 mammalian expres ⁇ ion vector
  • this section has illu ⁇ trated novel antibodie ⁇ of thi ⁇ invention, i ⁇ ade again ⁇ t a peptide encoded by a * DNA segment of the invention and able to specifically bind to pAT 744 or pAT 464 lymphokine/cytokine-like protein which includes the sequence of such peptide.
  • MDNCF monocyte-derived neutrophil chemotactic factor
  • the carboxyi- terminal tridecapeptide of platelet factor 4 is a potent chemotactic agent for monocytes. Bio- chem. Biophys. Res. Comm. 107:130 IV-53. Hit-Rarper, J., H. Wohl, and E. Harper. 1978. Platelet Factor 4: An Inhibitor of Collagen- ase. Science 199:991

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Abstract

Discoveries are disclosed that show particular aspects of recombinant DNA technology can be used successfully to produce hitherto unknown human lymphokine/cytokine-like proteins free of other lymphokines or cytokines. These proteins can be produced from DNA segments in cells or cell-free systems in various functional forms. These forms variously enable biochemical and functional studies of these novel proteins as well as production of antibodies. Means are described for determining the level of activation of genes for the lymphokine/cytokine-like proteins, for example, by measuring mRNA during mitogenic activation of isolated T cells or by measuring antigen secreted in extracellular or body fluids.

Description

NOVEL LYMPHOKINE/CYTOKINE GENES
FIELD OF THE INVENTION
The present invention relates to genes which are activated by mitogenic stimulation of immune cells, particularly to those encoding polypeptides similar to a family of secreted factors including lymphokines and cytokines. This invention also relates to synthesis of product's' of such immune activation genes by recombinant cells, and to the manufacture and use of certain novel products enabled by the identification and cloning of DNAS encoding these factors. "
BACKGROUND OF THE INVENTION Quiescent non-dividing T lymphocytes respond to antigen and mitogen stimulation by the activation of specific cellular" genes which are thought to control events that lead to cell proliferation and the expression of a differentiated phenotype. As part of the activation response, T cells produce a large variety of factors which are important for the growth and differentiation of many cells participating in an immune reaction.
More specifically, lymphocyte proliferation and effector functions are regulated by antigen/mitogen and lymphokine binding to cell surface receptors. The bind¬ ing of such extracellular ligands results in a cascade of intracellular biochemical events (see references 11-44, 11-45 in Experimental Section II, below) that ultimately set in motion a genetic program for activation-related growth and expression of differentiated functions. Thus, quiescent T cells undergo a series of sequentially dependent, ordered transcriptional events following acti- . vation of quiescent cells that culminates in the initi¬ ation of DNA synthesis after about 24 hours (II-5, II- 17). Primary gene transcriptional events, defined by their early kinetics and lack of dependence on prior protein synthesis, include protooncogenes (e.g., c-myc and c-fos), lymphokines (e.g. IL-2, IFN, GM-CSF), and a cell surface lymphokine receptor (IL-2 receptor) (II-6, II-9, 11-12, 11-17, 11-27, 11-36, 11-44), all of which are thought to have significant effects upon T cell proliferation and/or regulation of an immune response- It is expected that some of the immediately induced .genes play a role in; initiating and thus _.regu- lating the cascade of molecular events that.-.follow mito- genie activation. Such genes are also likely targets of tumorigenic events. For example, a variety of data supports the notion that uncontrolled growth occurs when the?ιmitogen inducible gene c-myc is expressed, inappro¬ priately (11-11, 11-26). At present, a limited number of primary induced genes in T cells have been described. In the past, genes induced in activated T cells, IL-2 and IL-2 receptor for example, often have been identified on the basis of their encoded proteins. However, the transcriptional response of T cells is likely more complex than has been reported to date (II-2, II-4, 11-15, 11-23, 11-28, 11-32, II- 41). Therefore, there was a need to characterize thoroughly the initial response of T cells to activation by cloning a maximal number of induced genes. Thus, an unbiased approach was required to clone novel, mi ogen- induced genes based upon the property of differential expression.
Human peripheral blood (PB) T Cells were used in the development .of this invention, as the model system in which to study activation, for several reasons. First, PB T cells are primary quiescent cells that have not been selected for adaption to growth in culture, and they can be polyclonally stimulated ^n_ vitro. Second, upon activation, T cells express a variety of differenti- ated functions that play critical roles in the pro¬ gression of an immune response. For example, novel lymphokines/cytokines would be expected to be included within the family of inducible genes, since a primary function of PB T cells following activation is to secrete soluble factors that affect ultimately a number of physiological functions (II-5, II-6, II-9, 11-13). Third, an important aspect in investigating the genetic response to activation is to identify mechanisms of gene regulation. Use of the immunosuppressive drug cyclosporin A (CSA) that acts upon T cells provides a tool for initially dissecting regulatory categories of induced genes. CSA appears to inhibit one or a few early
T cell activation pathways while leaving others intact
(11-16, 11-24, 11-29). Finally, the T cell system allows one.-to dissect and contrast the* activation* requirements mediated through various distinct surface molecules that are thought to play roles in T cell development and function (see Experimental Section III, below).
Treatment of human, peripheral blood T cells with the mitogenic combination of PHA (phyto- hemagglutinin) and PMA (phorbol myristate acetate) appears to mimic the effects of antigen and antigen presenting cells jLr^ vivo. The two agents are known to induce the expression of genes which are important for progression through the cell cycle, including the c-fos and the c-myc oncogenes, the IL-2 growth factor gene and the IL-2 receptor gene (see references IV-1, IV-2 in Experimental Section IV, below). In addition to IL-2, T cells activated in this manner elaborate many lymphokines/cytokines which exert their effect on a variety of cells. These include such f ctors as IL-3, IL-4, IL-5 and IL-6, GM-CSF, -IFN (gamma interferon), TNF (tumor necrosis factor) and LT (IV-3 - IV-9) and several others that are less well defined (IV-10 - IV- 13). Several such factor genes may be subject to a specific control mechanism in T cells, since the immunosuppressive drug cyclosporin A appears to abrogate their activation. As noted earlier, this drug acts during the early activation phase of T cells and interferes the production of IL-2, IL-3, and -IF (IV-14 - IV-17). The inhibitory effect is specific, since many other inducible genes such as c-fos and HSP 70 are unaffected (IV-15).
The present invention relates to DNA segments which encode amino acid sequences with similarities to members of a- newly emerging family of small, secreted proteins (see Figure IV-6 in Experimental Section IV) . This family of structurally related factors includes platelet_ factor 4 (PF-4) (IV-18), platelet basic protein (PBP) and its processed forms ^-thromboglobulin (^?-TG) and connective tissue activating peptide III (CTAP. Ill) (IV-19, IV-20), a gamma interferon inducible ^protein (IB- 10) (IV-21), a macrophage inflammatory protein (MIP) (IV- 22), a factor chemotactic to neutrophils [3-10C, MDNCF (monocyte-derived neutrophil chemotactic factor), NAF (neutrophil-activating factor)] (IV-23, IVτ-24,- IV-25), and a factor produced by fibroblastε, human growth related protein (H-gro) (IV-26).* To the extent to which this has been determined, members of this family appear to mediate certain inflammatory responses and/or display mitogenic activities.
Inflammation and wound healing/tissue repair are very complex processes which involve many different cells. Coordination of the various cellular functions is likely to be reflected in a multitude of secreted factors needed to communicate between cells. This family of genes, which may have been derived from one common ancestral gene, may well serve to establish such a net- work. To mediate inflammation and repair, the individual factors may possess multiple functions, as shown for several of the members of this family (see below. Experi¬ mental Section IV, subsection Discussion) . The conserved similarities between these proteins may indicate an interaction with conserved receptor elements. As enumerated above, the members of this family of proteins display a remarkable variety of tissue specificities. The distinctions between the factors may determine unique functions required during an immune reaction depending on the cell type activated.
In light of the complexities of inflammation and the processes of wound healing and tissue repair, which may occur in association with or following in lam¬ mation, it is apparent that there has been a need for methods and compositions and bioassays which would pro¬ vide* an improved knowledge and analysis of mechanisms of inflammation, and, ultimately, a . need -. for novel diagnostics and therapies based on the factors involved therein.
This invention contemplates the application of methods of recombinant DNA technology to fulfill such needs and to develop means for producing certain protein factors which appear to be related to inflammatory or cell; growth processes, and which could not be produced otherwise. This invention also contemplates the appli¬ cation of the molecular mechanisms of these factors related to inflammation and healing processes.
SUMMARY OF THE INVENTION The present .invention relates to a development of recombinant DNA technology, which includes production of novel lymphokine-like or cytokine-like proteins, free of other peptide factors. Novel DNA segments, RNAs, and bioassay methods are also included.
The present invention in particular relates, in part, to DNA segments which encode mRNAs and/or proteins having structural and/or functional characteristics of such molecules related to lymphokines/cytokines that are inducible in T cells. Members of one particular group of these proteins feature hydrophobic N-terminal leader or signal peptides characteristic of secreted proteins, have predicted sizes of about 8 kD (kilodaltons) upon cleavage of the putative leader peptide, and also share some critical amino acid sequence similarity with a newly emerging family of secreted factors which have been shown to display functions associated with an inflammatory response and/or to have mitogenic activities. In the practice of one embodiment of this invention, these DNA segments are capable of being expressed in suitable host cells, thereby producing lymphokine-like or cytokine-like proteins. The invention also relates to mRNAs produced as the result of trans-, cription of the sense strands of the DNA segments of. this invention. This invention furthe'r comprises novel bioassay methods for determining effects of various mitogenic agents on expression in human cells of, the mRNAs and proteins produced from the genes related to DNA segments of the invention. - - _
In a principal embodiment, the present invention comprises DNA segments encoding lymphokine-like or cytokine-like proteins, selected from the group con¬ sisting of: Act-2 [i.e., an approximately full length (-0.7 kb) cDNA clone of activated human peripheral blood mononuclear cells (PBMC) mRNA, number 2, hereinafter referred to as "full-length Act-2" DNA]; human cDNA clones of activated T cell mRNAs, pAT 744 and PAT 464; and related DNA segments which can be detected by hybridization to any of the above human DNA segments, which related segments encode related lymphokine-like or cytokine-like proteins.
In another embodiment, this invention relates to an RNA transcript of a DNA of the invention. Prefer¬ ably, these RNA transcripts are capable of being trans¬ lated to produce complete molecules of the encoded lymphokine-like or cytokine-like proteins.
In another embodiment, the invention relates to a recombinant DNA molecule comprising a vector and a DNA of the present invention. Theses recombinant molecules include molecules comprising full-length DNA and any of the following vector DNAs: a baculovirus vector (such as derivatives of Ac-Nuclear Polyhedroεis Virus; AcNPV); a bacteriophage lambda vector (gtlO); an Ml3 bacteriophage vector; or the RNA transcription vector pGEM-1. Recombinant DNAs of this invneiton also include recombinant molecules comprising clone pAT 744 DNA or clone pAT 464 DNA and any of the following vector DNAs: lambda (GtlO); and Ml3 vector; and a mammalian expression vector (such as CDM-8 or PMT2-2) capable of expressing inserted DNAs in mammalian (e.g., COS) cells.
In still another embodiment, the invention comprises a cell, preferably a-mammalian or insect cell, transformed with a DNA -of the invention. .Further, the invention comprises cells, including yeast cells and bacterial cells such as those of E_. coli and _B. subtilis, transformed with DNAs of the invention. According to another embodiment of the invention, the transforming DNA is capable of being expressed in the cell, thereby increasing the amount of lymphokine/cytokine-like protein encoded by this DNA, in the cell. In a most preferred embodiment of this aspect of the invention, the cell transformed by the DNA of the invention secretes the protein encoded by that DNA in the (truncated) form secreted by activated T cells.
Still further, the invention comprises novel lymphokine/cytokine-like proteins made by expression of a DNA of the invention, or by translation of an RNA of the invention. Preferably, these proteins will be of a secreted form (i.e., lacking an apparent signal sequence) . These protein factors can be used for f nc¬ tional studies, and can be purified for additional bio¬ chemical and functional analyses, such as qualitative and quantitative receptor binding assays.
According to this embodiment of the invention, the novel lymphokine/cytokine-like proteins will be pro¬ tein products of "unmodified" DNAs and mRNAs of the invention, or will be modified or genetically engineered protein products. As a result of engineered mutations in the DNA sequences, modified lymphokine/cytokine-like proteins will have one or more differences in amino acid sequence from the corresponding naturally occurring "wild-type" proteins. This invention also comprises novel antibodies made against a peptide encoded by a DNA segment of the invention. In 'this embodiment of the invention, the antibodies will specifically bind to a lymphokine/cytokine-like protein which includes Λthe sequence of such peptide, preferably when that protein is in its native (biologically active) conforma ion. These antibodies can be used for detection .or purification of the protein factors. " •*•-■. -
This invention further comprises novel .bioasεay methods for detecting the expression-of genes related -to
DNAs of the invention. According to one such embodiment, DNAs of this invention may be used as probes to determine steady state levels of or kinetics of induction of related mRNAs. Such bioassays may be useful, for example, for identification of various classes of tumor cells or genetic defects in the inflammatory response. The bioassays of this invention may also be useful for detecting activation of the immune system in vivo. Genes related to DNAs of this invention are expressed very early upon immune activation, in T cells (and also some other hematopoietic cells), and, there- fore, serve as extremely early markers for immune system activation. Thus, according to this aspect of this invention, various body fluids may be tested routinely with antibodies to protein factors of the invention, for example, to monitor whether tissues are rejected upon transplantation.
BRIEF DESCRIPTION OF THE DRAWINGS EXPERIMENTAL SECTION I
Fig. 1-1 shows Northern blots using a ~---F- labeled full-length CDNA probe and RNA derived from a variety of human cell types, which illustrate the speci¬ ficity of full-length gene expression, mainly in certain mitogenically stimulated immune cells.
Fig. 1-2 characterizes the temporal expression of mRNA in activation of normal T and B lymphocytes and monocytes (by means of Northern Blots), illustrating that Act-2 gene expression is both rapidly induced and termi¬ nated in response to mitogen. Fig. 1-3 depicts the sequencing strategy, DNA sequence, and deduced .amino acid sequence of -0.7 kb
(kilobases) cDNA clones of Act-2 mRNA, including the open reading frame of 276 base pairs, corresponding to 92 amino acids. .-- •-«
Fig. 1-4 presents a Kyte-Doolittle hydro- phobicity plot generated from the deduced amino acid sequence, which reveals an extremely hydrophobic N- terminus that appears to be a so-called leader or signal peptide.
Fig. 1-5 demonstrates translation of RNA species produced from full-length Act-2 cDNA in- cell-free systems, showing by gel electrophoresis a product with an apparent Mr of approximately 11,000 similar to the calcu- lated molecular weight of 10,199 daltons, which apparent¬ ly translocates into microsomal membranes, as do secreted proteins.
Fig. 1-6 portrays results of using the full- length Act-2 cDNA to perform a genomic Southern blot of human DNA, a comparatively simple pattern of digestion most consistent with Act-2 representing a single copy or low copy number gene.
Fig. 1-7 directly compares the time course of expression of Act-2 with two other genes known to play roles in cell proliferation, c-fos, and c-myc, in stimu¬ lated lymphocytes, showing that Act-2 mRNA is induced coordinately with c-fos and its peak expression coincides with that of c-myc.
Table 1-1 presents results of N-terminal sequencing of Act-2 protein radiolabeled with *^5S- methionine or cysteine.
EXPERIMENTAL SECTION II Fig. II-l outlines the procedure used to iso¬ late genes which are induced immediately upon mitogenic stimulation of human periopheral blood T lymphocytes.
Fig. II-2 illustrates kinetics of mRNA induc¬ tion (with Northern blots and cDNA probes) of four selected inducible genes upon stimulation of • resting human blood T cells.
- Fig. II-3 portrays diverse expression and regu¬ lation of selected cDNA clones in the human helper T -cell line Jurkat. _- ._ __ _. --. • __>
Fig. II-4 shows (Northern blot) analyses of expression of selected cDNA clones in human fibroblaεtε.
-- Table- ''■ 1.1-1 -=■ summarizes characteristics ~- of various mitogen-induced genes of T cells, identified by cDNA cloning studies. -
Table II-2 presents expresεion analysis of T cell induced genes in the helper T cell line Jurka -and in human fibroblastε.
EXPERIMENTAL SECTION III Fig. III-l εhowε (Northern blot) -analyεiε of mitogen induced geneε in peripheral blood T cellε using selected cDNA clones as probes.
Fig. III-2 illustrateε (Northern blot) analysis of mitogen induced genes in CD28 T cells that _were stimulated with either anti-CD28 monoclonal antibody or ionomycin in the absence or preεence of phyto- hemagglutinin.
Fig. III-3 depictε (Northern blot) analyεiε of mitogen induced geneε. in the T cell line Jurkat that were εtimulated with either dioctanoyl-glycerol or ionomycin or both agentε together in the abεence or preεence of cycloheximide.
Table III-l summarizes mRNA analyses (Northern blot data) for mitogen-induced genes in response to agents used in Figs. III-1-3.
EXPERIMENTAL SECTION IV Fig. IV-1 portrays Northern Analyεiε of cDNA cloneε pAT 464 and pAT 744 RNA in human peripheral blood T cellε, either unεtimulated (control) cellε or cellε treated with cycloheximide or cycloεporin.
Fig. IV-2 demonεtrateε Northern analyεiε of pAT 464 and pAT 744 RNA in Jurkat cellε, either unεtimulated (control) or cells stimulated with the agents indicated.
Fig. IV-3 illustrates the sequencing strategy, the nucleotide sequence, and the predicted 'amino acid sequence for pAT 464, including the open reading frame of 276 base pairs, corresonding to._92 amino acids. -•■*
. Fig. IV-4 shows the sequencing strategy, the nucleotide sequence, and the predicted amino acid sequence for pAT 744, including the open reading frame corresponding to 92 amino acids. Fig. IV-5 documents primer extension analysiε of the DNA εequence for pAT 744, using an oligonucleotide complementary to. positions 135 to 152 of the pAT 744 sequence annealed to RNA from human peripheral T cells.
Fig. IV-6 shows comparisons among the amino acid sequenceε of proteins encoded by pAT 464, pAT 744 and a family of structurally related proteins.
Fig. IV-7 presents Southern blot analysiε of pAT 464 and pAT 744 geneε in human placental DNA digested with εeveral restriction enzymeε. Fig. IV-8 illuεtrateε expreεεion of pAT 464 and pAT 744 mRNAs in promyelocytic (HL60) cellε and human tonsillar B Cells (by Northern blot methods).
Fig. IV-9 demonstrateε expression of appropriate secreted proteins in mammalian (COS) cells transformed with pAT 464 and pAT 744 DNAs in various expression vectors (by 35s-cysteine labeling of proteins) .
Fig. IV-10 demonstrateε expreεεion of appro¬ priate secreted proteins in mammalian (COS) cells trans- formed with pAT 464 DNAs in variouε expreεεion vectorε (by ^S-cysteine labeling of proteins).
Fig. IV-11 shows the ues of rabbit antibody
(number 721) raised against the C-terminal 12 amino acids of the pAT 744 peptide to detect secreted peptide in supernatants of COS cell cultures tranεformed with pAT
744 DNA (by Western blotting methods).
Fig. IV-12 illustrates the activity of rabbit antibody (number 722) raised against the C-terminal 12 amino acids of the pAT 744 peptide to detect secreted peptide in supernatantε of COS cell cultureε tranεformed with pAT 744 DNA (by Western blotting) . c " Fig. IV-13 shows the use of rabbit antibody (number 720) raised against the C-terminal 12 amino acids of pAT 464 peptide to detect secreted peptide in super¬ natants of dbs "cell cultures transformed with pAT^ 464 DNA.
,Q Fig. IV-14 presents results with a second rabbit antibody (number 719) raised against the C- terminal 12 amino acidε of the pAT 464 peptide in reactionε with secreted peptide in supernatants of ' COS cell cultures transformed with pAT 464 DNA.
15 Fig. IV-15. demonstrates that mitogenically activated human peripheral blood T cells secrete pAT 464 protein (by Western blotting with anti-peptide antibody) . DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The DNAs of this invention are exemplified by
20 DNAs referred to herein as: full-length Act-2 [i.e.", an -0.7 kb clone of activated peripheral blood mononuclear cells (PBMC) cDNA, number 2]; clones of pAT 744 or pAT 464 DNA; and related DNA segmentε which can be detected by hybridization to these DNA segments.
25 Other DNAε of thiε invention include the following cDNA cloneε: pAT 120; pAT 125; pAT 127; pAT
129; pAT 133; pAT 139; pAT 140S; pAT 140L; pAT 154; pAT
158; pAT 189; pAT 201; pAT 204; pAT 225; pAT 229; pAT
. 232S; pAT 232L; pAT 237; pAT 239; pAT 243; pAT 270; pAT
30 276; pAT 281; pAT 383; pAT 402; pAT 407; pAT 416; pAT 428; pAT 466; pAT 478; pAT 483; pAT 485; pAT 496; pAT 516; pAT 542; pAT 563; pAT 591; pAT 594; pAT 603; pAT 607; pAT 620; and pAT 730.
DNAε of thiε invention alεo include recombinant
35 moleculeε compriεing the following: full-length Act-2 DNA and a baculovirus vector DNA (exemplified by plasmid pAc-Act2, and a recombinant baculovirus, vAc-Act2); full- length Act-2 DNA or pAT 744 or pAT 464 DNA and a bac¬ teriophage lambda gtlO vector; full-length Act-2 DNA or pAT 744 or pAT 464 DNA and an M13 bacteriophage (exempli¬ fied by M13 mpl9); full-length Act-2 DNA and the -RNA transcription vector pGEM-1; and pAT 744 or pAT 464 DNA and a mammalian expression vector (exemplified by CDM-8 or PMT2-2) capable -of expressing inserted DNAs in COS cells. •-*
The sense strand DNA nucleotide sequence, and the predicted primary protein sequence encoded, are shown for full-length Act-2 DNA in Fig. 1-3. As described in Experimental Section I, four clones with identically sized inserts of approximately -0.7 kb were identified, all of which hybridized to the same 0.9 kb mRNA. The εequencing εtrategy uεed, the reεulting nucleotide εequence and the predicted amino acid sequence for the encoded protein are shown in figure IV-4 for' pAT 744 and Figure IV-3 for pAT 434.
. These DNAs, full-length Act-2 (-0.7 kb), and pAT 744 and pAT 464 clones, are most preferred DNAs of this invention.
The full-length Act-2 cDNA and pAT 744 cDNAs appear to be derived from esεentially the same gene.
More specifically, viεual inspection of the two cDNA sequences revealed only two base sequence differences within the protein coding region, namely: in full-length
Act-2 (see Fig. 1-3) base number 167 is a C, and 171 is
G, while in the corresponding pAT 744 sequence (Fig. IV-
4), base 131 is a T and 135 is A. These differences cause predicted amino acid number 20 to be Pro and Leu, in full-length Act-2 and pAT 744, respectively, while the other base change does not alter the amino acid sequence. It is possible that such differences are due to inherited variations (i.e., polymorphismε) in the geneε of the two different individuals from which the clones are derived.
Computer searches with using Bionet (most current release) indicated that a clone highly similar to pAT 464 was isolated previously (TV-41_) from_stimul^ed human tonsillar lymphocytes and waε called pLD~78. Clone. pLD 78 extends 7 nucleotideε further 5' than clone pAT 464 and sequence compariεon εhowε a- difference of 5 nucleotides between both clones, none of which affectε the predicted amino acid εequence. These .differences in the nucleotide sequence may reflect polymorphisms between the genes of different donors. A* comparison of the sequences for pAT 744 and
464 indicated a striking similarity on the nucleotide level (45%) and an even higher homology on the..amino acid level (56%, Fig. IV-6A) . In addition to common regula¬ tory characteristicε, theεe two geneε may have been derived from a common anceεtral gene and their encoded proteins may play functionally related roles.
Southern blot analyseε of genomic human DNAε uεing probeε derived from full-length Act-2, pAT 744 or pAT 464 cloneε (Figε. 1-16, IV-7 and IV-6, respectively) show comparatively εimple patternε most consiεtent with full-length Act-2/pAT 744 and pAT 464 repreεenting εingle copy or low copy number genes. Apparently, pAT 464 and 744 do not readily croεε-hybridize with each other, in spite of their remarkable similarity. Thuε, by εo using the DNAs and RNAs of the invention in hybridization methodε, eεpecially the moεt preferred DNAε listed herein, those skilled in the art, without undue experimentation, can screen genomic or cDNA libraries to find other lymphokine/cytokine-like DNAs which fall within the scope of thiε invention.
Recombinant DNA molecules which comprise a vector and other DNAs of the invention are also within the scope of the invention. Preferred recombinant molecules include molecules comprising full-length Act-2 DNA and any of the following DNAs: a bacteriophage lambda vector (gtlO); an M13 bacteriophage vector; or the RNA transcription vector pGEM-1. Preferred recombinant DNAs of this invention also include recombinant molecules comprising clone pAT 744 or pAT 464 DNA and any of the following vector DNAs: lambda vector (e.g., gtlO); an M13 vector. Most preferred recombinant molecules of tvhis invention include: molecules comprising full-length Act- 2 DNA and any of the following vector DNAs: a i>aculo- viruε vector (including pAc-Act'2, a plasmid derived from polyhedrin plaεmid pAc373; and a recombined baculovirus, vAc-Act2, derived from pAc-Act2 and a wild-type AcNPV strain E2 DNA) .
Other most preferred recombinant molecules are those comprising pAT 744 or pAT 464 DNA and a mammalian expresεion vector (CDM-8 or PMT2-2) capable of expreεsing inserted DNAs in mammalian (e.g., COS) cells.
The full-length Act-2 human cDNA was synthe- εized, clone and isolated as described in Experimental Section I. To identify a full length cDNA, 10° phage plaques from a HUT-102B2 cDNA library, constructed in lambda gtlO (1-14), were screened with a partial length (-0.4 kb) Act-2 cDNA insert, labeled using the random priming method (1-15). The full-length (-0.7 kb) DNA sequence contains an open reading frame of 276 base pairs, corresponding to 92 amino acids. This reading frame utilizes the most 5' AUG, which generally is the one utilized in eucaryotic translation initiation. Further, the region of the first AUG has good homology with the consensus sequence determined for eucaryotic start sites (1-17). The cDNA appears to extend to the 3' end of the mRNA since it contains classic AAUAAA poly- adenylation signals (1-18) and a start of a poly A tail.
The human cDNAs of this invention were synthe¬ sized, cloned and isolated as described in Experimental Section II by a process of "subtraction" cloning and hybridization using mRNA from activated T cells. This strategy iε summarized in Figure II-l. Human peripheral blood T cells were polyclonally activated for 4.5 hours in the presence of the mitogens phytohemagglutinin (PHA) and phorbol 12-mytriεtate 13-acetate (PMA) aε well as the protein syntheεiε inhibitor cycloheximide.- Thiε method , focuses the analysis on the primary response of_ activated cells, defined by those__genes which are inducibl inde¬ pendent of new protein synthesiε. To date,= approximately 40% of the subtracted lambda gtlO cDNA library has been screened with subtracted probes. - Purified phage that hybridized to subtracted probes were εubjected further to a differential εcreen in which cDNA probeε cyntheεized from activated and reεting T cell mRNA were uεed on duplicate filterε of the phage. Finally, 528 phage cloneε were selected which harbored induced cDNAs, aε judged by both the εubtractive and the differential screening methodologies. Aε deεcribed in Experimental Section II, further analyεeε have identified 66 unique cDN cloneε, the majority of which appear to repreεent diεtinct geneε (εee Table II-l, legend). Forty-four of the novel inducible gene cloneε have been εtudied further (Experimental Section II). Theεe cloneε, which exemplify DNAε of thiε invention, include: pAT 120; pAT 125; pAT 127; pAT 129; pAT 133; pAT 140S; pAT 140L; pAT 154; pAT 158; pAT 189; pAT 201; pAT 204; pAT 225; pAT 229; pAT 232S; pAT 232L; pAT 237; pAT 239; pAT 243; pAT 270; pAT 276; pAT 281; pAT 383; pAT 402; pAT 407; pAT 416; pAT 428; pAT 464; pAT 466; pAT 478; pAT 483; pAT 485; pAT 496; pAT 516; pAT 542; pAT 563; pAT 591; pAT 594; pAT 603; pAT 607; pAT 620; pAT 730 and pAT 744.
The expression of nine novel mitogen induced genes, in response to various T cell activating agents, was examined to evaluate the diversity of pathwayε which regulate such genes (Experimental Section III). These nine genes include pAT 237; pAT 563; pAT 229; pAT 120; pAT 154; pAT 225; pAT 416; which are preferred DNAs of this invention, and pAT 744 and pAT 464 which are most preferred DNAs of this invention.
Clones pAT 464 and pAT 744, which were originally derived from a subtracted cDNA library (see Experimental Section II), have been characterized in more detail. Near full-length cDNA clones were isolated from a cDNA library which was derived from RNA extracted from human peripheral blood T cells stimulated for 4^5 hr with PHA-P and PMA in the presence of cycloheximide. This cDNA library was constructed with oligo ό_T priming as described (IV-29) and was subsequently cloned into- lambda gt 10 (IV-30). Clone pAT 464 is 793 nucleotides long and clone pAT 744 is 659 nucleotideε long (including the Eco RI linker at 5' end). Primer extension analysis for pAT 744 confirmed that the isolated clone represents an almost full length cDNA (Fig. IV-5). It can.be concluded that about 10 nucleotides of the 5' end of the true mRNA sequence are misεing in thiε cDNA clone. The first ATGs for pAT 464 and pAT 744 are at poεitions 84 and 74, respectively. In each case an open reading frame of 92 amino acids follows. DNA templates for transcription of full-length
Act-2 RNA in the pGEM-1 vector were prepared as follows (see also Experimental Section I): The -0.7 kb full- length Act-2 cDNA was digested with Ava I, filled in with Klenow and dNTPs, and then digeεted with Eco RI to liberate a nearly full length fragment (baεe 27 through the 3' end of the inεert) from which the artificial 5' G- C tail had been removed. This fragment was subcloned into PGEM-1 (Promega Biotec) which had been digested with Sma I and Eco RI. Baculovirus vector constructs containing full- length Act-2 cDNA were prepared from the -0.7 kb Act-2 cDNA by cloning into the BamHI site of the baculovirus polyhedrin plasmid pAc373 (I-16a) to generate pAc-Act2, thereby placing the full-length Act-2 cDNA under control of the polyhedrin promoter (see also Experimental Section I) . SF9 cells were cotransfected with pAc-Act2 and wild- type AcNPV strain E2 DNA. A recombined baculovirus vAc- Act2 was isolated and purified by a procedure ;-.of succeεεive roundε of plaque hybridization (I-16a). ~
Construction of recombinant DNAs containing pAT. 464.- or pAT 744 DNA and __a mammalian expresεion -vector (exemplified by CDM-8 orJ,PMT2-2), capable of expressing inserted DNAs in COS cellε, are described in Experiment"..! Section IV. Recombinants contained either of the follow¬ ing vector DNas: --. GDM-8 [obtained from Brian Seed *at Masεachusettε General Hospital, Boston, Massachusetts, and described in B. Seed, Nature 329, 840-842 (1987)]; or PMT2-T [obtained from Genetics Institute, Cambridge, Massachusettε^ a related earlier version of this -vector has been described in Yu-Chung Yang, et al., Call 47, ^3- 10 (1986).~ DNAs and sense strand RNAs of thiε invention can be employed, in conjunction with protein production methods of this invention, to make large quantities 'of substantially pure lymphokine/cytokine-like proteins. * In addition, subεtantially pre lymphokine/cytokine-like proteinε thuε produced can be employed, using well-known techniques, in diagnostic asεayε to determine the preεence of receptorε for theεe proteinε in variouε body fluidε and tiεεue εampleε.
Thuε, the invention also compriseε a cell, preferably a mammalian or inεect cell, tranεformed with a DNA of the invention, wherein the transforming DNA is capable of being expressed. In a most preferred embodi¬ ment of thiε aspect of the invention, the cell transformed by the DNA of the invention secretes the protein encoded by that DNA in the (truncated) form secreted by activated T cells. The invention also comprises novel lymphokine/cytokine-like proteins made by expresεion of a DNA of the invention, or by tranεlation of an RNA of the invention. Preferably, theεe proteinε will be of a secreted form (i.e. , lacking an apparent εignal sequence) . These protein factors can be used for functional studies, and can be purified for additional biochemical and functional analyses, such as qualitative and quantitative receptor binding assays.
Insect cells transformed with a recombinant molecule containing full-length Act-2 DNA were prepared as described in Experimental Section I. , Briefly, the plaque purified, recombinant baculovirus, vAc-Act2, was used to infect SF9 cellε, according to standard -methods. The recombinant full-length Act-2/baculovirus vector was expressed in the SF9 cells to produce abundant Act-2 protein in the supernatant. Moreover, N-terminal sequencing of radiolabeled protein shows that the signal peptide is cleaved as predicted.
Mammalian cellε tranεformed with pAT 744 or pAT 464 DNA and secreting pAT 744 or pAT 464 protein are described in Experimental Section IV. Briefly, COS cells were transfected using standard DEAE-dextran methodology, with cDNA clones inserted into expresεion sites of either CDM-8 or PMT2-T. The novel proteins were visualized by ^^S-cyεteine labelling of the cellε after the trans- faction of the DNA constructs using standard, well known methods. The size of the protein productε is appropriate as predicted from the cDNA sequence after the leader peptide is cleaved off.
This invention also comprises novel antibodies made against a peptide encoded by a DNA segment of the invention. This embodiment of the invention is exempli¬ fied by rabbit antisera containing antibodieε which spe¬ cifically bind to pAT 744 or pAT 464 lymphokine/cytokine- like protein which includes the sequence of such peptide, were raised to synthetic peptides representing the C- terminal 12 amino acids of both pAT 744 and pAT 464 pro¬ teins, as predicted by the cDNA sequences. This was done by chemically synthesizing the peptides, linking them to carrier (KLH), and injecting the carrier plus peptides into rabbits, according to standard methods of peptide immunology.
These antibodies can be used for detection or purification of the protein factors. Figs. IV-11 and IV- 12 show the use in Western blot experiments of two cLif- ferent rabbit antibodies (721 and 722) raiεed againεt the pAT 744 peptide. Similar studieε with antibodieε to pAT 464χ-peptides are presented in Figs. IV-13 and IV-14. *a_fi_s is "evident from the figures, the appropriate secreted factors are detected -by antisera from rabbits immunized with synthetic"peptides. ~ - - -- λ "• 5"
This invention further compriseε novel bioassay methods for detecting the expression of genes related to DNAs of the invention. In some exemplary embodiments, DNAs of this invention were used as probes to determine steady state levels of or kinetics of induction TOf related mRNAs. Methodε for theεe bioaεεays of the invention, using full-length Act-2, or using pAT 744 -or pAT 464 DNAs, and standard Northern blotting techniques, are described in detail in Experimental Sections I, -or II-IV, respectively. One skilled in the art will recognize . that without, undue experimentation, such methodε may be readily applied to analyεis of gene expression for lymphokine/cytokine-like proteins, either in isolated cellε or variouε tiεεueε. Such bioaεεayε may be useful, for example, for identification of various classeε of tumor cellε or genetic defectε in the inflam- matory reεponse.
Without further elaboration, it is believed that one of ordinary εkill in the art, using the preceding description, and following the methods of the Experimental Sections below, can utilize the present invention to its fullest extent. The material disclosed in the Experimental Sections, unless otherwise indicated, is disclosed for illustrative purposes and therefore should not be construed as being limitive in any way of the appended claims.
EXPERIMENTAL SECTION I
IDENTIFICATION, CLONING, AND CHARACTERIZATION OF A NOVEL IMMUNE ACTIVATION GENE A novel immune activation gene, denoted Act-2, has been identified by differential hybridization screen¬ ing of an activated T Cell library. The gene is included rapidly following T cell --activation with phytohemagglu- tinin, B cell activation with Staphylococcus aureus Cowan I, and monocyte activation With lipopolysaccharide. ' cDNA containing the full length coding region has been isolated. The deduced amino acid sequence predicts ran open reading frame of 92 amino acids, including a very hydrophobic N-terminus, which by weight matrix score is predicted to be a signal peptide. Using a baculovirus expression system, it has been proven that .this gene encodes a secreted product. It is, therefore, possible that Act-2 represents a new cytokine. Another cDNA clone derived from.esεentially the same gene aε Act-2 has also been isolated and charac¬ terized (εee Experimental Sectionε II, III and IV, below). Thiε clone is called pAT 744.
INTRODUCTION T cells play a pivotal role in the regulatory and effector functions of the immune response. Upon stimulation by antigens or mitogenic lectins, a εerieε of biochemical eventε occurs, including an increase in intracellular calcium, phoεphorylation of proteinε, and increaεed phosphoinositol turnover (see references 1-1, 1-2). These eventε generate signals which lead to the activation of cellular genes and the production of cellular proteins that are not expressed or are very weakly expressed by resting cells. These induced pro- teins are eεsential for the proliferation and differenti¬ ation of T lymphocytes into effector T cells mediating . helper, suppreεsor, or cytotoxic T cell functions.
There is a need to characterize new genes in T lymphocytes that are induced in response to mitogen stimulation, with the goal of eventually identifying their functional gene products and characterizing their roles in mediating T cell function and the immune response. One succeεsful approach to identifying genes i activated by mitogenic εtimuli iε to uεe the technique -of -: differential hybridization (1-3 - 1-8). The εucceεε -of thiε method depends on the finding that although there is extensive overlap"between genes expressed in resting and activated cells, each state is characterized by a small number of high abundance mRNAs that encode proteinε characteristic of the particular activation sta.e.
Thiε section deεcribeε the cDNA cloning, εequencing, and characterization of a novel gene, denoted Act-2, which was identified by differential screening of an activated normal T cell cDNA library.
-MATERIALS AND METHODS Cell culture. Circulating human peripheral-blood mononuclear cells (PBMC) were obtained from healthy volunteers and isolated by Ficoll Hypaque (LSM, Litton Bionetics) gradient centrifugation. Cells were generally cultured at 1-2 x 106 cellε/ml overnight in RPMI 1640 medium containing 10% fetal bovine εerum (FBS), L- glutamine, and antibioticε. Phytohemagglutinin (E-PHA, Burroughε-Wellcome) and phorbol myriεtate acetate (PMA, Sigma) were used at .5 ug/ml and 50 ng/ml, respectively, unless otherwise indicated. B lymphocytes were purified by incubating PBMC .(4% non-specific eεteraεe poεitive cells) with 2-aminoethyliso-thiouronium bromide-treated sheep erythrocytes and removing the rosette-focusing cells. In some experiments monocytes were depleted by plastic adherence. Such cells were 80% surface Ig posi¬ tive, latex non-ingesting cells, and were cultured for 30 min to 72 h in the presence of a 1:10,000 dilution of Staphylococcus aureus Cowan I (SAC, Calbiochem-Behring Corp. ) . Monocytes were prepared by elutriation of PBMCs and were 95% pure by Giemεa εtain and flow cytometry. Cellε were cultured for 8 h in the presence of 10 ug/mg lipopolysaccharide (LPS, Sigma).
Cell lines. T cell line (Jurkat, HUT-102B2, Molt-4, CEM, and Hut-78), B cell lines (Raji, S3, Nall-1, 8392, GM4672, U266, and SUDHL-6), and myeloid lineε ( 562 and U937) were maintained in RPMI 1640 medium containing 10% FBS. Fibroblaεt cell lineε 4312 and-4429 (1-9), and the osteosarcoma cell line 5887 (J.-9 were maintained in 5 DMEM containing 15% FBS. Human fetal -lung fibroblast HFL-1 cells were passaged to a" density of approximately*1 x 10° cells per 175 cm2 tissue culture flask in 25 ml of DMEM containing 10% FBS and allowed to grow to confluence over a period of 7 days at 37°C. The cells were stimu- 0 lated by removing the depleted medium and replacing it with fresh DMEM containing 20% FBS. Early pasεage HFL-1 cells were used for all experiments. "•
Preparation of a cDNA library from activated human PBMC. PMBC were activated with PHA and PMA as pre- 15 viously deεcribed (1-10), total cellular RNA extracted (1-11), and mRNA iεolated by oligo (dT) cellulose chroma¬ tography. Double stranded cDNA was prepared using a modification of the procedure of Okayama and Berg (1-10, 1-12). 20 Screening of the cDNA library. The library was screened in duplicate by the procedure of Grunstein and Hogness (1-13) with 32P-labeled first strand cDNA probes derived from either induced or uninduced PBMC. Nitro¬ cellulose fibers were incubated for 3 h at 65°C in 0.1 M 25 NaH2P04 pH 6.8, 0.85 M NaCl, 1 mM EDTA, 10 x Denhardt'ε solution, 0.1% SDS, 100 ug/ml salmon sperm DNA and 10 ug/ml poly(rA) (prehybridization solution). The filters were then hybridized at 65°C for 48 h using fresh pre¬ hybridization solution containing 10% dextran sulfate and 30 the radioactive probe. The filters were washed 4. times for 20 min at 65°C in 2 x SSC, 0.5% SDS and twice for 30 min at 65°C with 0.1 x SSC, 0.1% SDS at 65°C and then autoradio-graphed.
Screening the HUT-102B2 cDNA library. To
" 5 identify a full length cDNA, 10° phage plaques from a
HUT-102B2 cDNA library, constructed in lambda gtlO (I-
14), were screened with a partial length Act-2 cDNA insert, labeled using the random priming method (1-15). Phage DNA was bound to nitrocellulose and hybridized overnight at 42°C in 50% formamide, 5 x Denhardt'ε solu¬ tion, 5 x SSPE, .1% SDS and 100 Λig/ml salmon sper DNA. Filters were washed 3 times for_.^20 min in 2 x SSC, .^% SDS, then twice for 30 min at 56°C in .1 x SSC, .1% SDS, and autoradiographed..
DNA sequencing.-. Sequencing was performed via the dideoxy chain termination method using Ml3 phage DNA and Sequenase (U.S. Biochemical Corporation) according to the manufacturer's recommendations.
Northern blot analysis.. Either total cellular RNA (10 uq) or poly A+ RNA (4-5 flq) was electrophoreεed on formaldehyde gels, transferred to nitrocellulose filters (Schleicher and Schuell), and hybridized to 32P- labeled Act-2 cDNA.
Genomic Southern blot. 10 ug of DNA from either U937 or 428 cells was digested with indicated enzymes, analyzed on a 1% agarose gel, and transferred to Gene Screen Plus nylon membraneε (duPont) and hybridized according to the manufacturer ε suggested protocol.
In vitro transcription and cell free tranε- lation. The -0.7 kb Act-2 cDNA was digested with Ava I, filled in with Klenow and dNTPs, and then digeεted with Eco RI to liberate a nearly full length fragment (base 27 through the 3' end of the inεert) from which the arti¬ ficial 5' G-C tail had been removed. Thiε fragment waε εubcloned into pGEM-1 (Promega Biotec) which had been digeεted with Sma I and Eco RI. RNA waε transcribed from both strandε uεing SP6 or T7 promoterε, according to the instructionε provided by Promega Biotec. Reactions were terminated by digesting the DNA template with RNase free DNaεe I, and the RNA purified by phenol/chloroform extraction and ethanol precipitation. RNA waε tranεlated in vitro uεing rabbit reticulocyte and wheat germ lyεateε (either from Promega Biotec or Amerεham) uεing J-'S- cysteine (>600 Ci/mmol, Amerεham) . Some tranεlation reactions were performed in the presence* of canine pan¬ creatic microsomal membranes. Translation products were analyzed on 15% SDS polyac ylamide gels.
Expression of the Act-2 cDNA. The -0.7 kB Act- 2 cDNA was cloned into the BamHI site of the baculovirus polyhedrin plasmid pAc373 (I-16a) to generate pAc-Act2, thereby placing teh Act-2 cDNA under control of the poly¬ hedrin promoter. SF9 cells were cotransfected with pAc- Act2 and wild-type AcNPV strain E2 "DNA. A recombined baculoviruε, vAc-Act2, waε iεolated and purified by a procedure of εuccessive roundε of plaque hybridization (I-16a). When plaque purified recombinant viruε waε obtained, SF9 cells were infected and biosynthetically labeled with 3^S-labeled cyεteine or methionine. Radio- labeled Act-2 protein which was secreted into the cell supernatant was isolated on SDS gels, eluted, and εub- jected to sequential Edman degradation on an automated protein sequencer (Applied Biosystemε Model 477) uεing the ATZ program εuppied by the manufacturer. Tractions were counted by liquid scintillation.
RESULTS Identification of an Act-2 cDNA. 10,000 bac¬ terial clones from the activated PBMC cDNA library were screened for the presence of specifically induced cDNA sequences. Colonies were screened in duplicate on nitro¬ cellulose filterε uεing first strand cDNA probes derived from RNA from activated or resting PBMC. Approximately 1% of the colonies hybridized only to. the induced probe. Act-2 was a cDNA clone of approximately 0.4 kb that hybridized selectively and strongly only to the induced probe. This finding was confirmed by a Northern blot of RNA from unstimulated PBMC, PBMC activated for 16 h with either PMA or PHA, and HUT-102B2 cells, an HTLV-I transformed T cell line (Fig. I-1A) . There was no detectable signal in resting PBMC, but a band of approxi¬ mately 0.9 kb was detected in PBMC activated with either PMA or PHA. A leεε intenεe signal was present in the lane representing HUT-102B2 mRNAs.
Cell specificity. To determine the range*, of cell types expresεing the Act-2 gene, a variety of .T cell/ B cell, and nonlymphoid cell lineε were examined-, using Northern blot analysiε (Fig. I-1B) . As- already shown, Act-2 was expressed '-in- HUT-102B2 cells (lane 1), but not in MOLT-4 or CEM T cells (not shown) . Act-2 specific mRNA was similarly not detected in HeLa o K562 cells (lanes 2, 3), nor in the SV40 transformed fibro- blaεt cell lineε 4312 and 4429, nor in the oεteoεarcoma cell line 5887 (data not εhown) . Although Act-2 mRNA waε minimally detectable in Raji (lane 4), GM54672 (lane 5.), SB (lane 8), and 8392 (lane 9) EBV infected B cell lineε (this can be best appreciated on the original autoradio- grams), its expression could not be detected in Nall-1 (pre-B cells, lane 7), SUDHL-6 (lane 10), or in plasma- cytoma cell lineε U266 (lane 11) or 8662 (lane 6). Intereεtingly, Jurkat T cellε when induced with both PHA and PMA (Fig. I-1C, left), but not with either agent separately, expresε Act-2, aε do PMA-εtimulated U937 cellε (derived from a human histiocytic lymphoma but retaining monocyte-like characteristicε, Fig. I-1C, right) .
To further characterize the temporal expreεεion of Act-2 mRNA in normal lymphocyte activation, a ti e- course of Act-2 mRNA expreεεion waε performed in freεhly iεolated PBMC activated with PHA (Fig. I-2A) . Act-2 mRNA, expressed at low or undetectable levelε in reεting PBMC, increased rapidly and dramatically in reεponεe to mitogen εtimulation. Peak levelε occurred at approxi¬ mately 4 h, and then rapidly declined to negligible levelε by 24 h after stimulation. Thus, Act-2 expression is both rapidly induced and terminated in responεe to PHA. An analogouε examination of B cells stimulated with S_:_ aureus Cowan I (SAC, Fig. I-2B) also indicated a rapid induction and disappearance of Act-2 mRNA. Also examined were resting monocytes and monocytes stimulated for 8 h with lipopolysaccharide (LPS) for the expression of Act-2 (Fig. I-2C) . Act-2 mRNA was absent in the unstimulated cellε but waε- readily detected in monocytes induced with LPS. The observation that the Act-2 gene was expressed in several different normal cell types exposed to mitogenic stimuli raised the possibility that Act-2 expression was induced in all normal cells follow¬ ing activation to traverse the cell cycle. In view of parallels made between the proliferative response of resting lymphocytes to mitogens and of quieεcent fibro- blasts to growth factors regarding c-myc expresεion (I- 16), Act-2 expreεsion in quiescent and εerum stimulated primary cultures of normal human fetal lung (HFL-1) fibroblasts was examined. In contrast to c-myc, however, serum was uanble to induce expression of Act-2 mRNA (data not shown) .
Identification and sequencing of a full-length Act-2 cDNA. Because of the size disparity between the length of the cDNA and the band detected on Northern blots, were next proceeded to isolate a longer cDNA. Using the 0.4 kb Act-2 cDNA insert as a probe, 10° lambda gtlO plaques were from an amplified HUT-102B2 cDNA library were screened (1-14). Four clones with identi- cally sized inserts of approximately -0.7 kb were identi¬ fied, all of which hybridized to the same 0.9 kb mRNA identified by the 0.4 kb cDNA. The sequencing strategy, DNA sequence, and deduced amino acid sequence are shown in Fig. 1-3. The salient features of the sequences can be summarized as follows: (1) Act-2 represents a novel gene. Comparison of the DNA sequence and the predicted amino acid sequence using DNA and protein homology search programs did not reveal any homology with published sequences in the most recent GenBank (version 55, March 31, 1988) and NBRF (versions 16 and 34, released March 31, 1988) databases. (2) The DNA εequence containε an open reading frame of 276 base pairs, corresponding to 92 amino acids. This reading frame utilizeε the raoεt 5' AUG, which generally iε the one utilized in eucaryotic translation initiation. Further, the region of the first AUG has good homology with the consenεuε sequence determined for eucaryotic start sites (1-17). 3)- The cDNA appears to extend to the 3' end of the mRNA since it contains classic AAUAAA polyadenylation signalε (1-18) and a start of a poly A- tail. ^The 3' untranslated region also contains A-T rich domains (boxed, εee discussion). (4) There are no potential N-linked glycoεylation sites (Asn- -Ser/Thr) . (5) Six cyεteine reεidueε are preεent; thuε, it is posεible that intra- or extra- molecular diεulfide bondε are formed. (6) Examination of a Kyte- Doolittle Hydrophobicity plot (Fig. 1-4) generated from the deduced amino acid εequence revealε an extremely hydrophobic N-terminuε. Uεing the von Heijne weight matrix analysis, this appears to be a signal peptide
(calculated .score of 11*3), with .cleavage predicted between Ser 23 and Ala 24 (1-19), a cleavage site also used in the cytokines IL-2 (1-20), IL-3 (1-21), GM-CSF (1-22) .
To demonstrate that Act-2 encodes a functional mRNA, the cDNA was next subcloned into pGEM-1 and both sense and anti-senεe εtrandε were tranεcribed utilizing the SP6 and T7 promoterε. The reεulting RNA εpecieε were translated in wheat germ and reticulocyte lystateε (Fig. 1-5). The sense (lane B) but hot the antisenεe (lane A) strand resulted in a detectable product, which migrated with an apparent Mw of approximatel 11,000, similar to the calculated molecular weight of 10,199 daltonε. Unfortunately, in reticulocyte lysate translations, the artifact associated with globin comigrated with the Act-2 primary translation product (not shown), and thus the wheat germ lysate system gave clearer results. In order to evaluate cleavage of the putative signal peptide and translocation into the lumen of the endoplasmic reti- culum, next experiments were performed using canine microsome membranes. Microεomal membraneε are often more efficient in the reticulocyte lyεate system. Thus, such cotranslational translocation/cleavage experiments were performed uεing a reticulocyte lysate system, followed by pelleting the membranes at.31,000.x g, and washing.them in 10 mM tris, .15 NaCl, .2 M sncrose, pH 7.5. The membranes were then boiled in sample buffer and analyzed on SDS gels. In this way, the contaminating globin could be removed from the membrane fraction. The primary translation product was associated with the membrane fraction (lane C), suggesting that the N-terminus functions like a signal peptide. Based on the sequence, a change of 2391 daltons was expected, if the putative signal peptide were cleaved; however, no significant change was noted, suggesting that the putative signal peptide might not be cleaved. Treatment of the membrane aεεociated band εeen in lane C with proteinaεe K in the preεence or absence.of Triton X-100 resulted in much more complete degradation in the presence of detergent (not shown) . These data indicate that the protein associates with membranes but cleavage of the signal peptide could not be demonstrated.
In order to resolve the isεueε aε to whether the full-length Act-2 cDNA encoded a secreted product and whether the signal peptide was cleaved, the -0.7 kB clone of Act-2 was expreεsed as a recombinant baculovirus in SF9 cells (manuscript in preparation). Cells were bio- synthetically labeled with ""S-methionine or cysteine, supernatant recovered, subjected to electrophoresis on an SDS gel, and for each label, the specific Act-2 protein band excised. The material was eluted in water and sequenced, proving that the putative signal peptide is cleaved (see Table 1-1). The detection of a methionine at cycle 3 and cysteines at cycles 11 and 12 confirm that cleavage occurs between ser-23 and ala-24 of the primary tranεlation product (εee Fig. 1-3).
Next, the cDNA waε utilized to perform a genomic Southern blot (Fig. 1-6). A comparatively simple pattern of digestion was obtained. The same blot, was ; hybridized with an interleukin-2 receptor cDNA (the IL-2 receptor is encoded -by a single copy gene) , and bands of similar intensity were identified (not shown). These data are most consistent with Aτct-2 representing a sihgle copy or low copy number gene.
_■-- * Finally, the time coύrεe- of expression of Act- 2, c-foε, and c-myc in PBMC εtimulated with PHA or PHA pluε PMA, were directly compared and it waε found that Act-2 mRNA iε induced coordinately with c-fos and its peak expression coincides with that of c-myc (Fig. 1-7). Cycle No. Expt. 1 Expt. 2
-* --S-methionine , cpm ^S-cvεteine, cpm
80 77 76 123 201 360
389 432 406 478 2766
4651 1986 916
Figure imgf000032_0001
lts of N-terminal sequencing of Act-2 protein radiolabeled with ^S-methionine or cysteine
The high carryover (in cycle 4 for the methionine at cycle 3 [see Expt. 1] and in cycles 13 and 14 from the cysteines at positions 11 and 12 [see Expt. 2]) were expected due to the presence of prolines at poεitions 2, 7, and 8 of the mature protein (see Figure 1-4) which are inefficiently cleaved in the ^sequential. Edman degradation.
DISCUSSION This study-identified and characterized*a novel activation gene, denoted Act-2 (activated -PBMC cDNA, number 2) in a cDNA library- prepared from mRNA from normal activated PBMC. Act-2 encodes an open reading frame.of 92 amino acids, It contains,a very.hydrophobic N-terminus that by the von Heijne weight matrix analysiε iε εtrongly predicted to be a signal peptide. Although in vitro translation analyses could not demonstrate cleavage of the signal peptide, a recombinant Act- 2/baculovirus vector has been expressed in insect cells and found that Act-r2 protein is abundant in the super- natant. Moreover, N-terminal sequencing of radiolabeled protein shows that the signal peptide is cleaved as pre¬ dicted. It remainε an interesting possibility that Act-2 may also exist in a membrane asεociated form. Currently, antibodieε are being produced in order - to be able to directly identify the distribution of Act-2 protein pro¬ duced in normal activated human lymphoid cells.
The 3' untranslated region is also of signifi¬ cant interest in that it is A-T rich and contains the consensus sequenceε ATTTA (1-23) and TTATTTAT (1-24) that have been identified as common sequences in a variety of proto-oncogenes and εecreted factors, including tumor necrosis factor, lymphotoxin, IL-1 (both alpha and beta), multiple interferons, and GM-CSF. The TTATTTAT conεensus is not commonly found in mammalian mRNAs in general but is particularly prevalent in mRNAs encoding proteins related to the inflammatory response (1-24); thus its presence in Act-2 is supportive of the idea that this gene may represent a new cytokine. These sequences also correlate with relative instability of mRNA (1-23), con- sistent with the rapid decline from peak levels of induced mRNA.
This gene is especially interesting in view of itε rapid time course of activation. Human resting PBMC represent normal cellε in a physiological quieεcent state (Go) j[I-25, .1-26). Upon - activation with antigen or- mitogen, there is cellular enlargement, increase in RNA content, transcription of new genes, and synthesiε of new proteins. The cells are thereby rendered receptive to further signals, such as IL-2 (1-27) that promote cellular proliferation. The rapid induction of Act-2 expression in response to mitogenic εtimuli and its coordinate expresεion with c-foε and c-myc are conεiεtent with the poεεibility that Act-2 could play an early and potentially important role in cell growth and prolifera¬ tion.
Act-2 waε minimally or not expressed in reεting PBMC. However, it waε rapidly induced following activa¬ tion of T cellε with PHA or PMA, B cellε with SAC, or monocyteε with LPS. However, ct-2 iε not expreεεed in every actively growing cell, aε evidenced by itε non- expression in HeLa an K562 cells, and its failure to be induced in reεponse to serum stimulation of quiescent human fibroblasts.
The experiments described in this section illustrate some of the principal embodiments of this invention and demonstrate that the full-length Act-2 cDNA can be expresεed in inεect cellε tranεformed with thiε DNA, and the properly proceεεed product iε εecreted from these transformed cells.
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EXPERIMENTAL SECTION II
COMPLEXITY OF
THE PRIMARY TRANSCRIPTIONAL RESPONSE
TO MITOGENIC ACTIVATION OF HUMAN T CELLS Thiε section describes the isolation and characterization of more than sixty novel cDNA clones, which comprise part of the immediate tranεcriptional reεponεe of reεting human peripheral blood T cellε following mitogen activation- Thiε primary response is highly complex, both in terms of the absolute number of inducible genes as wells as the diversity of their regu- lation. Although a majority of the genes expressed in activated T cells are shared with the activation response of normal human fibroblasts, a significant number are more restricted in their tissue specificity and thus likely encode or affect the differentiated functions of activated T cells. The activatable genes can be further differentiated on the basis of their kinetics of induc¬ tion, their response to cycloheximide as well as their sensitivity to the immunosuppressive drug cyclosporin A. It is of note that this latter drug inhibits the expression of more than ten inducible genes, suggesting a broad genetic mechanism for the action of this agent.
MATERIALS -AND METHODS Cell Culture. Human peripheral blood T cells were obtained from healthy volunteers and, were isolated, over a Ficoll-Hypaque gradient and nylon wool columns. The resulting cell preparations were consitently more than 90% T cells, as judged by anti-CD3 staining. PB T cells were cultured at a concentration of 2 x 106 cells/ ml in RPMI 1640 containing 10% fetal calf serum (FCS). PB T cells were stimulated for various periods of time with PHAP (1 yUg/ml Burroughs-Welcome Co.) and PMA (20 ng/ml) either with or without cycloheximide (10 uq/τal) . The Jurkat cell line was provided by K. Hardy. Jurkat cells were maintained in RPMI 1640 supplemented with 10% FCS and 25 q/τal gentamicin. Jurkat cells were stimu¬ lated for various periods of time at a concentration of 5 x 105 with PHA-P (1 Uq/ l ) and PMA (25 ng/ml) with or without cycloεporin A (1 yg/ml; Sandoz). Human fibro- blaεt lines, CCD-11LU and WI38, were obtained from the American Type Culture Collection. Fibroblaεtε were grown to confluence in MEM containing 10% FCS and then main¬ tained in MEM with 0.25% FCS for 3 to 5 Dayε. In order to reinitiate growth, the εpent medium waε replaced by MEM supplemented with 20% FCS either with or without cycloheximide (10 μq/ l) .
-Subtraction cloning and hybridization. PB *-τ cells were isolated and cultured as. escribed above. RNA was isolated as described (II-8) from unstimulated cellε or after stimulating the cells -for 4.5 hours with PHA-P arid PHA in the preεence of cycloheximide. Poly A+ ?RNA waε purified by one pasεage over an oligo-dT column (II- 3). cDNA waε εyntheεized utilizing oligo-dT priming (18) from 20 μ q poly A+ RNA from activated T cellε. After hydrolysis of the RNA, this cDNA waε hybridized to a.cot value to 2000 moleε x ε/1 with a 10 fold exceεs of poly A+- RNA from unstimulated cells. The single stranded moleculeε then were εeparated from the double stranded cDNA:mRNA hybrids by chromatography using a hydroxy- apatite column (11-14, 11-20). After the firεt round of εubtraction, 15% of the moleculeε appeared in the εingle εtranded fraction aε judged by the diεtribution of countε. Thiε fraction waε again hybridized to a 10 fold exceεε of mRNA from unεtimulated cellε, and this second round of subtraction yielded about 90% single stranded material. Following size fractionation on a Sepharose CL-6B column, cDNA moleculeε which were larger than 400 nucleotideε in εize were uεed aε templated by DNA Polymeraεe I for second strand synthesis. Double- stranded DNA was εubsequently cloned into lambda gt 10 according to standard procedures (11-12). A library with a base of 45,000 individual clones was obtained. About 40% of the subtracted cDNA library was screened with a subtracted cDNA probe synthesized as described above, except that the probe was labeled to a high specific activity (5 x 108 cpm/ug) and was II-subtracted only once. Following tertiary screening with subtracted probeε and plaque purification, differential screening was performed. Duplicate filters were hybridized to a cDNA probe prepared from activated cell mRNA and a cDNA probe derived from unstimulated cell mRNA. 523 differen¬ tially hybridizing clones were obtained.
Northern analyseε♦ Total cellular RNA was extracted with guanidine isothiocyanate and purified by centrifugation through 5.7 M CsCl (II-8), , separated by electrophoresiε in formaldehyde-agaroεe gels, blotted onto Genescreen membrane filters (NEN Research Products), and hybridized to "*2P-labeled cDNA insertε. Quantitative loading of RNA was determined by hybridization to & β -2 microglobulin probe (data not shown).
Probes. Probes were εupplied by the following individuals or institutions: GM-CSF, Genetics Inεtitute; -"/'-Interferon, Meloy Laboratories; c-fos. Dr. T. Curran; IL-2 receptor. Dr. W. Greene; IL-3, Genetics Institute; Met- and Leu-preproenkephalin, Dr. S. Sabol; human IL-4, ATCC; p53, Dr. D. Givol; lymphotoxin, Genentech; IL-5, Dr. K. Aria (DNAX); ornithine-decarboxy- laεe. Dr. D. Nathans; bcl-2. Dr. A. Bakhshi; IL-6, Dr. H. Goldstein; c-myb, Dr. F. Mushinski; HSP 70 and unwinding ATPase, Dr. R. Morimoto. Dr. M. Sporn provided a nick- tranεlated TGF-beta probe.
RESULTS Subtractive cloning of inducible genes. To clone immediately inducible genes in mitogen activated T cells, this work employed the method of subtractive cDNA cloning, followed by subtractive probe hybrid!zationε, aε these techniques afford the greateεt sensitivity in detecting even those genes whose mature mRNAs appear at low levels upon activation (11-14, 11-20). This strategy is summarized in Figure II-l. Human peripheral blood T cells were polyclonally activated for 4.5 hours in the presence of the mitogens phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) aε well as the protein synthesis inhibitor cycloheximide. The latter agent is known to superinduce a number of growth related genes (II-l, 11-27, 11-30, 11-34). In addition, cyclo¬ heximide prevents mRNA induction which follows IL-2 and IL-2 receptor εyntheεiε and interaction. Thiε focuses the analysiε on the primary reεponεe of activated ceils, defined by those genes which are inducible independent of new protein synthesis'. To date, approximately 40% of the subtracted lambda gtlO cDNA? library has eeh screened with subtracted probes. Purified phage that hybridized to subtracted probes were subjected further to a differential serein in which'cDNA probes synthesized from activated and resting T cell mRNA were used on duplicate filters of the phage. Finally, 528 phage clones were selected which harbored induced cDNAs, aε judged by both the subtractive and the differential screening methodolo¬ gies.
To determine the number of distinct genes among these 523 phage clones, the next step was to cross- hybridize subcloned cDNA insertε with the 528 phageε. In thiε way, 66 unique cDNA cloneε were identified, the majority of which appear to repreεent diεtinct geneε. A limited number of groupε may derive from different segments of the same mRNA, thus leading to an overestima- tion of individual geneε. However, the number of unique inducible geneε will exceed 66, as 120 of the 528 phages have not hybridized to the selected cDNA insertε teεted to date. The number of isolated phage clones belonging to a given group via crosε-hybridization varied con¬ siderably and ranged from 1 to as many as 86 phage clones (see Table II-l legend) . Forty-four of the novel inducible gene clones have been studied, further (see below). All hybridized to an inducible and, with few exceptions, single-sized message by Northern blot analyses (Table II-l) . Furthermore, none of the cDNA inserts contained repetitive sequences, but a few appeared to be members of small multi-gene families, as determined by Southern blot analyses (Table II-l). In order to asseεε the extent to which the εubtracted library and the 528 selected phage clones might represent previously described induced genes, both were subjected to crosε-hybridization analyεeε with many of the geneε known to be inducible in T cells. The composition of the subtracted library was shown to repre¬ sent a typical activated T cell phenotype as determined by the enrichment for clones encoding IL-2, GM-CSF, gamma-IFN, c-myc and c-fos, the latter of which iε induced in T cells at 4.5 hours in the presence of cyclo¬ heximide, but to a much lesεer extent than c-myc (II- 36). Among the εelected 528 phage were detected several isolates of c-myc. In addition, the 528 phage include cDNAs homologous to the IL-2 receptor, the IL3 and IL-4 growth factors and Met- and Leu-preproenkephalin (11-46, 11-47, 11-49) (Table II-l). Among the 528 isolated phage, analyses ruled out the presence of IL-2, GM-CSF, -IFN, c-fos, p53, ornithine-decarboxylase, bcl-2, lumphotoxin, TGF-beta, c-myb, the interleukinε 5 and 6, heat-shock gene 70, and the unwinding ATPase (II-6, II-9, 11-13, 11-21, 11-25, 11-35, 11-36, 11-38, 11-48), con¬ firming that many novel genes have been isolated. The detection of known genes have been isolated. The detec¬ tion of known genes (e.g. IL-2) in the library that are not preεent in the 528 phage selected with subtracted cDNA probes resulted in part from the much stronger signal generated by nick-translated versus heterogenous cDNA probes. In addition, although the length of the induction period (4.5 hours) was optimal for a great number of genes, it was not optimal for those genes which are expressed with relatively delayed kinetics. Nonethe¬ less, the hybridization data indicate that the 528 selected phage encompass many but not all of the genes expected, and the subtracted library contains the known induced genes which have been assayed.
Regulation of induced genes and sensitivity to cyclosporin A. In order to assess the heterogeneity of expresεion for' the iεolated inducible geneε, kinetic analyεes of mRNA levels for many of the genes were per¬ formed. Figure II-2 displays typical Northern analyses for four novel inducible genes. One pattern of expression, exemplified by pAT 249, displays a very rapid appearance after activation of T cells by PHA and PMS, i.e., by 30 min. or less; another common pattern shown for. pAT 464 is characterized by mRNA appearance only after 2-4 hours. Additional mRNA species (e.g. pAT 129 and pAT 139, Figure 11-2) are induced at intermediate times. _ The observed kinetic differences with regard to both the onset and duration of expresεion, aε εummarized in Table II-l, suggesst a highly diverεe regulation of the primary reεponεe. Approximately 70% of the genes (see Table II-l), were superinduced by cycloheximide and the remaining ones are not or only minimally affected. The uεe of cycloheximide waε critical for the isolation of the rapidly induced genes as many are expreεsed transiently and are detectable at 4.5 hours only in the presence of the protein syntheεiε inhibitor (e.g., pAT 129 and pAT 249) . Cycloheximide alone did. not cauεe the expression of these genes (Fig. II-2), suggesting that many may be transcriptionally regulated. The detailed requirements for the expression of nine newly iεolated geneε are addreεεed in the accompanying paper (Experimental Section III, below).
The human CD4+ helper cell line Jurkat iε known to have retained the inducibility of εeveral geneε, including the lymphokineε IL-2 and gamma-interferon as well as the 11-2 receptor (II-II-19, 11-37, 11-45). We tested a number of the isolated genes for their expresεion and regulation in this tumor line, as well as their sensitivity to the immunosuppreεεive drug cyclo¬ sporin A (CsA) . Many lymphokines elaborated by activated T cells are known to be suppressed by this drug which appears to inhibit transcriptional induction (11-16, II- 37, 11-39). As shown in Figure II-3 and summarized in Table II-2, four different patterns of expresεion could be distinguished. Of the 35 newly cloned genes tested, the expression of 22 was induced in Jurkat cells after treatment with PHA and PMA. In 11 of these, induction was essentially unaffected by CsA (e.g. pAT 602, Figure II-3). However, the induction of a εurprisingly large number of genes, 11 but of "22, was suppressed by CsA J(e.g., pAT 464, Figure II-3). This implies that CsA affects a step during activation which is common to a fai-rly large group of genes (posεibly including many lymphokines/cytokines), and this further suggests that a distinct activation path iε required for thiε sat of geneε. The remaining 13 genes tested were not induced in Jurkat T cells. Ten of these (e.g. pAT 133, Figure II-3) failed to hybridize to any message in Jurkat cells Λas determined at a number of time points following induction (data not shown) . Possibly, these messageε exist in a cell type distinct from Jurkat, such as CD8+ T cells. Alternatively, Jurkat cells are transformed and may have lost or modified these genes or the signalling machinery neceεsary to induce their mRNAs. The members of the last group of genes (3 out of 35) were conεtitutively expresed in Jurkat cells (e.g., pAT 129, Figure II-3). These genes may contribute to the uncontrolled growth of this tumor line (see Discussion).
Table II-l.
Figure imgf000044_0001
Figure imgf000045_0001
a Frequency grouping indicates the number of crosε- hybridizing phage clones, among the 528 clones isolated, which are detected by the subcloned insert listed: 1, one crosε-hybridizing phage clone; 2, two to five croεε- hybridizing phage clones; 3, six to 10 cross-hybridizing phage clones; 4, eleven to twenty-five cross-hybridizing phage cloneε. Among the total 66 different clones that have been isolated to date, the frequency distribution is 1=22 clones; 2=17 clones; 3=16 cloneε; 4=6 clones; 5=5 clones. mRNA size (in nucleotides) was determined relative to the migration of the 28S and 18S rRNA on formaldehyde-agarose gels. time ( inuteε) at which the iduced mRNA species was first detected following PHA (1 /g/ml) and PMA (20 ng/ml) stimulation of peripheral blood (PB) T cells; the second number denotes the time (minutes) at which maximal steady state mRNA levels were noted. * in this column indicates that the mRNA could only be detected when cycloheximide (10 ^yg/rnl) waε included in the PHA/PMA stimulation; - indicates gene induction in reεponε.e to the combination of PHA, PMA and cycloheximide, although extenεive kinetic studies-were not performed. _c effect of cycloheximide on PHA/PMA induced εteady εtate mRNA levels in PB T cells, relative to levels obtained with PHA/PMA alone: S, superinduced; NE, no or marginal -effect _of cycloheximide; - effect, not determined. α The number of bands detected on a Southern blot of human thymuε DNA digeεted with either Bam Hi, Eco RI or Sεt I: S, fewer than four bandε in all three digests; F, four or more bands in. at least wo digests; M, five or more bands in all three digestε; -, genomic organization not determined. e pAT 383 alεα detected a conεtitutively expressed mRNA species of 900 nucleotides which was unaffected by cycloheximide. pAT 516 also detected a conεtitutively expreεεed mRNA of 1700 nucleotideε. 9" A 542 alεo detected a mRNA species of 4600 nucleotides which waε coordinately expreεεed with the predominant species of 6800 nucleotides. h pAT 563 also detected mRNA species of 3600, 4100 and 8500 nucleotides,. coordinately expressed with the predominant 2400 nucleotide species.
Table II-2. Expression analysis of T cell induced genes in the helper T cell line Jurkat and in human fibroblasts. Experimental details are as described in figure II-3 and II-4. Y - induction upon stimulation of quiescent human fibroblasts (HF) with serum N - no expression in quiescent or serum stimulated human fibroblastε ND - not determined a - " denoteε expreεsion data for the 900 nucleotide constitutively expresεed mRNA species (see Table II-l) denoteε expreεsion data for the 2000 nucleotide induced mRNA specieε (εee Table II-l)
10
15
Figure imgf000048_0001
Tiεεue-specificity of genes induced in T cells. An important question in the initial characteri¬ zation of mitogen-induced genetic responsiveness is the tissue specificity of the transcriptional activation pro- cess.- Do cells derived from different tissues exhibit a largely shared activation responεe or are they very dis- εimilar, given that the receptors for mitogenic agents are generally tissue-specific? Activation of the normal human lung fibroblast cells I38 and CCD-11 U (11-12) was studied. These cells enter quiescence following growth to confluence and subsequent incubation in 0.25% serum, and they can be stimulated to re-enter the cell cycle in the presence of high concentrations of fetal calf serum which contains multiple growth promoting activities. An analysis of three representative induced genes is shown in Figure 4, in which quiescent fibroblastε were stimu¬ lated with 20% serum for varying amounts of time in the presence or absence of cycloheximide. The kinetics of expression for individual mitogen-induced genes are rela- tively εimilar in T cellε and fibroblasts (compare Figure II-4 and Table II-l). Interestingly, the induction of mRNA in the preεence of cycloheximide alone occurs frequently for a variety of genes in fibroblasts but not in T cells (compare Figures II-2 and II-4). Such a result suggests that human lung fibroblastε, εuch as thoεe utilized here, contain a background of activated cellε, or alternatively, the mechanisms for suppressing gene expresεion during quiescence may differ in the two cell types. As summarized in Table II-2, approximately 80% of the novel T cell induced genes analyzed here could be detected in fibroblasts by Northern analyses. Thus, although initial responses appear to be largely similar in completely distinct cell types, some of the genes are more restricted in their tissue specificty and thus are likely to encode or effect differentiated functions of lymphocytes or T cells. To date, all four genes which display a restriction in tissue specificity are inhibited in their induction by 'CsA. interestingly, 'some of the induced genes expresεed by both tiεεues are εuppreεεed by CsA in T cells (see Table 11-2 , providing model systemε to address the tissue-specificity of the action of CsA. ~ l' ar " ΪHSCUSSION" ■*"-*-" ** . ■ - - As demonstrated in this section, the primary transcriptiShal responεe to the activation of T cellε iε very complex, involving a' large number of novel geneε with very distinct patterns of regulation and expresεion. In addition to functionε involving pro- greεεion through the cell cycle and commitment to pro¬ liferation, these genes may encode other functions such as modulation of the immune system. Specifically, those genes that exhibit limited tisεue diεtribution of expreεεion (Table II-2) are to be expected to play such roles in the differentiated function of activated T cellε. Indeed, two of theεe geneε, pAT 464 and 744, have been εequenced and diεplay predicted hydrophobic leader peptideε and structural homology with known secreted proteins, suggeεting their identity aε potential lymphokineε (Experimental Section IV) .
Beyond the limited number of geneε iεolated previouεly on the baεiε of being induced in various T cell preparations, cell lines or cloneε (II-2, II-4, II- 15, 11-23, 11-28, 11-32, 11-41), more extenεive reεearch haε been conducted on the εerum induced activation pro- cess in mouse 3T3 fibroblastε. Previouε studies in which immediately activatable genes were cloned yielded a number much smaller than discovered here (11-10, II- 30). However, a recent study utilizing the mouεe 3T3 fibroblast system concludeε that more than 70 geneε are induced, in good agreement with our studies on human peripheral blood T cells (II-l). Aε shown here, a majority of induced genes are expreεεed εimilarly in both human fibroblaεtε and ly phocyteε. Such an obεervation εuggests that the complexity of the immediate early tranεcriptional response to mitogens does not result primarily from the induction of differentiated func¬ tions. Rather, a ubiquitously-expressed activation gene more likely plays a role in a conserved aspect of cellular metabolism such aε those events ,that rprime a cell for DNA synthesis. Particularly intriguing among those induced genes isolated to date from fibroblasts are several which may encode DNA binding proteins (II-7, II- 33, 11-40, 11-43), supporting the hypothesis that some early induced genes may serve a pleiotropic regulatory role. Two such genes which are expressed in fibroblasts and lymphocytes, called Egr-1 (or NGFI-AA) and Krox20, predict primary amino acid structures containing three zinc finger binding domains, which have been implicated in binding DNA (II-7, 11-33, 11-42, 11-43).
In addition to the large number of genes induced, the variety of kinetic patterns obεerved among the induced genes following mitogenic stimulation of PB T cells suggeεt conεiderable diverεity in their regula- tion. One common pattern of expression (Table II-l) diεplayε a rapid and transient appearance analogous to c- foε (11-34) and to many genes induced in fibroblasts (II- 1, 11-31). As one poεεibility, εuch geneε may play roleε important in initiating a GO to Gl transition within the cell cycle, and their continued preεence may not neces¬ sarily be required for further progression through G. Additional kinetic patterns diεplaying later onset and/or εuεtained expreεsion were observed for other genes (Fig. II-2 and Table II-l). Further evidence that points to diverse regu¬ latory groups among the genes studies here is the variable effects of CSA upon gene induction. CSA appears to interfere with induced gene expression prior to or at the initiation of transcription (11-16). Therefore, we anticipate that CSA inhibitable genes share a common activation component required for induction which may be reflected in shared regulatory motifs for these genes. In addition, the broad effect of thiε drug on an unex¬ pectedly large proportion of geneε leadε one_ to believe < that itε immunoεuppreεεive action may be due to more than the suppression of only-a small number- of genes, such as IL-2. « x- ■= -_. Sr
. A common characteristic observed among 70 per¬ cent of the induced genes studied was the superinduction of message levels by cycloheximide. In additiony in the presence of cycloheximide many translently-expreεsed genes showed increasingly εuεtained expreεεion kineticε. Thuε, moεt induced geneε appear to be εubject to regulatory mechaniεmε that are -mediated by labile proteinε, a εyεtem that provideε maximum flexibility for the modulation of meεεage levelε -within the cell. Evidence haε been preεented in fibroblaεtε for cyclo¬ heximide inhibiting both tranεcriptional εhut-off and increaεing mRNA stability, εuggeεting-the involvement of labile tranεcriptional repressorε and degrading enzymes in the regulation of induced gene expresεion (II-l, II- 31).
A εubεet of the geneε deεcribed here will play a role in programmed cell growth. Note the conεtitutive expreεεion in the Jurkat tumor cellε of three geneε which are normally expresεed only after mitogenic activation (Figure II-3 and Table II-2). The expreεεion of εuch genes may contribute to the uncontrolled growth of this tumor-derived cell line.
Thus this section illustrates the isolation of cDNA clones of this invention which have functional and structural charateristics of mRNAs induced early in T cell activation, similar to properties of some known lymphokine/cytokine protein genes.
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1986. Cloning of two genes that are spe¬ cifically expressed in activated cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA 83:1448-1452. 11-33. Milbrandt, G. 1987. A nerve-growth factor- induced gene encodes a posεible transcriptional regulating factor. Science 238:797-799. 11-34. Muller, R. , R. Bravo, J. Burckhardt, and T. Curran. 1984. Induction of c-fos. gene and protein by growth factors precedes activation of c-myc. Nature 312: 716-720. -. ---- 11-35. Mustelin, , T., H. Poso, S.P*. Lapinjoki, g . Gynther, and L.C. Andersεon. 1987. .Growth signal transduction: Rapid activation of covalently bound ornithine decarboxylase during phosphatidylinositol breakdown. Cell 49:171- 176.
11-36. Reed, J.C, J.D. Alpers, P.C Nowell, and R.G. Hoover. 1986. Sequential expression of .proto- oncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. Proc. Natl. Acad. Sci. USA 83:3982-3986.
11-37. Reed, J.C, A.H. Abidi, J.D. Alpers, R.G. Hoover, R.J. Robb, and P.C. Nowell. 1986. Effect of cylosporin A and dexamethasone on interleukin 2 receptor gene expreεεion. J. Immunol. 137:150-154.
11-38. Reed, J.C, Y. Tεuji oto, J.D. Alperε, CM. Crose, and P.C. Nowell. 1987. Regulation of bcl-2 proto-oncogene expresεion during normal human lymphocyte proliferation. Science 236:1295-1299.
11-39. Reem, G.H., L.A. Cook, and J. Vilcek. 1983. Gamma interferon syntheεis by human thymocyteε and T lumphocytes inhibited by cyclosporin A. Science 221:63-65. 11-40. Ryder, K., L.F. Lau, and D. Nathans. A gene activated by growth factors is related to the oncogene v-jun. Proc. Natl. Acad. Sci. USA
85:1487-1491.
11-41. Schmid, J., and C Weismann. 1987. Induction of mRNA for a serine protease and a beta- thromboglobulin-like protein in mitogen- εtimulated human leukocytes. J. Immunol. 139:250-256. -42. Sukhatme, V.P., S. Kartha, F.G. Toback, R_.C Taub, R.G. Hoover, and C Tεai Morriε. 1987. A novel early growth response gene rapidly induced by fibroblast, epithelial cell and lymphocyte mitogens. Oncogene Res." 1:343-355. "' "" -43. Sukhatme, S.P., X. - Cao, L.C Chang, CH.- Tsai- " f Morris, D. StamenkOvich, P.CP. Ferreira*, D.aR. Cohen, S.A. Edwards, T.B. Showε, T. Curran, M.M. Le Beau, and E.D. Adamson. 1988. A zinc finger-encoding gene coregulated with c-fos during growth .and> differentiation, :-and after cellular depolarization. Cell. 53:37-43. -44. Weisε, A., R. Shields, M. Newton, B. Manger, J. Imboden. 1987. Ligand-receptor interactions required for commitment to the activation of the interleukin 2 gene. J. Immunol. 138:2169-2176. -45. Weisε, A., and J.B. Imboden. 1987. Cell surface molecules and early events involved in human T lymphocyte activation. Adv. Immunol.
41:1-38. -46. Yang, Y.C, A.B. Ciarletta, P.A. Temple, M.P. Chung, S. Kovacic, J.S. Witek-Giannotti , A.C. Leary, R.. Kriz, R.E. Donahue, G.G. Wong, and S.C Clark. 1986. Human IL-3 (Multi-CSF): identification by expreεεion cloning a novel hematopoietic growth factor related to murine IL-3. Cell 47:3-10. -47. Yokota, T., T. Otsuka, T. Moεmann, J. Banchereau, T. DeFrance, D. Blanchard, J.E. De
Vrieε, F. Lee, and K.I. Aral. 1986. Iεolation and characterization of a human interleukin cDNA clone, homologouε to mouεe B-cell stimulatory factor 1, that expresεeε B-cell-and T-cell εtimulating activitieε. Proc. Natl. Acad. Sci.
USA. 83:5894-5898. -48. Yokota, T., R.L. Coffman, H. Hagiwara, D.M. Rennick, Y. Takebe, K. Yokota, L. Gemmell, B. Shrader, G. Yang, P. Meyerson, J. LuK, P. Hoy, J. Pene, F. Briere, H. Spits, J. Banchereau, T. , j.÷E. De ΫVries, F.D. Lee, N. Arai "and K.I. Arai. 1986. Isolation and characterization of lymphokine cDNA clones encoding mouse and human IgA-enhancing factor and eosinophil colony- stimulating actor activities:- Relationship to interleukin 5. Proc. Natl. Acad. Sci. USA 84:7388-7392.
11-49. Zurawski, G. , M. Benedik, B.J. Kamb, J. S.
Abrams, S.M. Zurawεki, and Frank D. Lee.
1986. Activation of mouse T helper cellε induceε abundant preproenkephalin mRNA synthesis. Science 232:772-775.
EXPERIMENTAL SECTION III MITOGEN INDUCED GENES ARE SUBJECT TO MULTIPLE PATHWAYS OF REGULATION IN THE INITIAL STAGES OF T CELL ACTIVATION The delivery of a mitogenic signal to T cells via any one of several cell surface moleculeε elicitε a variety of intracellular responses, some or all of which regulate subsequent gene expression events. The expression of nine novel mitogen induced genes, in responεe to variouε T cell activating agentε, waε examined to evaluate the diversity of pathways which regulate such genes. The relative contribution of dis¬ tinct secondary signals, individually or together, to mitogen stimulated gene induction and the capability of individual genes to respond to the sometimes divergent signals generated from different cell surface structures is addressed here. The activation of T cells with mitogenic monoclonal antibodies directed against the CD2 or CD3 cell surface molecules, or with PHA, induced all nine genes. Thus, stimulation by fully mitogenic agents regardlesε of cell εurface binding specificity, corre¬ lates with the expression of all those genes studied here. However, heterogenous patterns of gene expression, encompassing five regulatory classes, were revealed, by the use of PMA, calcium ionophore and anti-CD28 mono¬ clonal antibody, agents which mediate only a subεet of intracellular eventε and thus an incomplete mitogenic signal. IL-2 and two novel lymphokines represent one regulatory clasε that appearε to require unique tranε¬ criptional activation signals relative to the other mitogen, induced genes. As demonεtrated in the previouε Experimental Section, the immediate transcriptional responεe of T cells to mitogenic stimulation is quite complex, involving numerous genes beyond those which have been previously deεcribed. Furthermore, the discrimina- tion of εeveral regulatory phenotypes among these nine genes suggeεtε that a multiplicity of εignalling pathwayε extendε from the cell εurface to the level of tranε- cription.
INTRODUCTION The previouε Experimental Section deεcribed the iεolation of greater than εixty diεtinct, novel cDNA cloneε derived from immediate early mRNA εpecieε that are induced following the mitogenic activation of quieεcent human peripheral blood (PB) T cellε by PHA and PMA. Those data suggest that the initial biochemical responεe to a stimulus delivered to T cells iε highly complex, involving a large number of directly induced geneε. It iε expected that diεtinct pathwayε mediated by charac- teriεtic εecondary signals induce groups of genes within this large family. Indeed, an important question, which is addressed here, concerns the homogeneity or hetero¬ geneity of induced gene responses elicited by those stimuli which lead to proliferation. Genes that play an essential role in cell cycle progreεεion are expected to be uniformly expressed during any mitogenic responεe, regardless of initiating agent. In addition, it is important to define potential regulatory clasεeε of induced geneε in order to ultimately elucidate regulatory motifε characteriεtic for a given class. The present study concerns detailed charac¬ terization of the activation requirements of nine novel mitogen induced genes by utilizing various mitogenic and comitogenic agents that are known to effect.-distinct secondary signals. These nine genes were selected for further study from the larger group described in the previous section, because their expreεεion waε limited to lymphoid cellε or alternatively, they displayed interest¬ ing regulatory properties εuch aε inhibition of induction by the immunoεuppressive drug cyclosporin A. The acti¬ vating agents studied here include monoclonal antibodies (mAbs) directed against CD2, CD3 or CD28 which are dis¬ tinct, physically non-associated T cell surface molecules, the lectin phytohemagglutinin (PHA), calcium ionophore, phorbol 12, myristate 13-acetate (PMA), and dioctanoylglycerol (DiC3).
The binding of antigen or mAb to the T cell receptor or the associated CD3 complex of T cells initiates the activation of T cells (III-2, 111-18, III- 23). PHA-mediated stimulation appears to require the presence of the antigen receptor complex or a coordinate¬ ly expressed molecule, as determined by εtudies with selective surface loss mutants (111-20). An antigen- independent pathway of T cell activation involving the CD2 complex (E rosette receptor) has alεo been described (111-19). Binding of the above mi ogens to cell surface receptors on T cells results in the production of inositol 1,4,5-triphoεphate , an increased intracellular calcium concentration ([Ca2+]i), membrane translocation of protein kinase C (PKC) and subεequent proliferation (III-6, III-9, 111-13, 111-15, 111-22, 111-25). In con¬ trast, the signal delivered by CD28 seems to be funda¬ mentally distinct from that of CD2 or CD3 because stimu¬ lation by anti-CD28 leads to an elevation of cytoplasmic cyclic GMP concentration and does not cause increaseε in [Ca2+]; or activation of PKC (111-12, 111-14, 111-22, 111-26). Furthermore, the IL-2 production initiated by CD28 iε reεiεtant to the effectε of cycloεporin A in contraεt to that induced by CD3 or calcium ionophore and PMA (III-8). In addition, anti-CD28 - alone doeε not εtimulate proliferation of T cellε but requireε the εimultaneous presence of a_comitogen, such as PJJA ( 11-7,, III-8). .T cells also may be activated, bypassing celi surface interactions, following treatment with calcium ionophore in addition to PMA or DiC8; the latter two agents act, at least in part, by stimulating protein kinase C (111-16, 111-21).
Ionophore-mediated increaεeε in [Ca2+] act synergistically with DiC3 or PMA to εtimulate prolifera¬ tion and induced gene expreεεion in purified PB T cellε and εome induced gene expreεεion, including lymphokineε, in the T cell line Jurkat (111-16).
MATERIALS AND METHODS Purification, culture and activation of peripheral blood T cellε. PB mononuclear cellε were obtained from normal healthy donorε by leukophoreεiε and PB lymphocyteε iεolated by denεity gradient centrifuga¬ tion through Lymphocyte Separation Medium (Litton Bioneticε) , followed by the removal of adherent cellε (monocyteε and B cells) by passage over a nylon wool column. The resulting cellε had approximately 1% B cells and <1% monocytes aε determined by FACS. The T cellε were cultured at 2 X 10 /ml in RPMI-1640 medium contain¬ ing 10% heat-inactivated fetal calf serum and stimulated with one of the following agents: anti-CD3 (mAb OKT3, Ortho Diagnostics), used at 10 ng/ml; PHA-P (Burroughs- Wellcome), used at 3 μq/τal; anti-CD2 (ref. 111-19: anti- T112 and anti-T113 ascites, obtained from E. Reinherz), each ascites was used at a 1:200 dilution. All reagents were titrated to determine the optimal concentration as aεεayed by the ability to stimulate peak proliferation of PB T cells or IL-2 production by Jurkat cells. When included, cycloheximide (Sigma Chemical Co.) was used at 10 Uq/ml . The ability of each agent to stimulate pro- liferation was determined by 3H-thymidine incorporation assayed after 4 days. A representative experiment showed 1,003 cpm for culture medium alone, 184/567 cpm for PHA, 107,908 cpm for anti-CD2 and 1,248 cpm for anti-CD3. The lacksof response to anti-CD3 in PB T cells .appeared due to monocyte depletion, since linfractionated PBL from the same donor gave 127,760 cpm.
Culture and activation of CD28+ PB T cells. Approximately 80 percent of PB T cellε express CD28+, and therefore, to ensure a maximal response by responding CD28+ cells and to eliminate the potential interaction of CD28+ and CD28+ populations, CD28+ cellε were purified aε previously described (III-8). The cells were cultured as outlined above and stimulated for 4 h with anti-CD28 mAb 9.3 (obtained from J. A. Ledbetter) at 100 ng/ml, iono¬ mycin (Calbiochem; dissolved in DMSO) at 133 nM, and PMA (Sigma Chemical Co.; dissolved in DMSO) at 0.3 ng/ml. A preliminary experiment showed all the geneε to be maxi¬ mally induced at 4 h. The PMA concentration waε determined by ^H-thymidine incorporation to maximally εynergize with either anti-CD28 or ionomycin yet not to be itεelf mitogenic. The mitogenic activity of each agent waε assayed by 3H-thymidine incorporation after 3 days of stimulation. A representative experiment showed 121 cpm for medium alone, 488 cpm for PMA, 236 cpm for mAb 9.3 alone, 58,590 cpm for mAb 9.3 and PMA. 186 cpm for ionomycin alone, 44,760 cpm for ionomycin and PMA and 69,360 cpm for immobilized anti-CD3.
Culture and activation of Jurkat cells. Jurkat cells were maintained in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum at a denεity of 2 X 10-Yml to 8 X 10°/ml. Freεh aliquots of cells were thawed at approximately 6 week intervals. Cells were stimulated at 4 X 10 /ml with one or more of the follow- ing agents: DiC3 (Molecular Probes. Inc., Eugene, OR; dissolved in absolute ethanol) at 100 /M, ionomycin at 500 nM, cycloheximide at lOjU/ml. The DiC3 and ionomycin - 62 -
concentrations were determined by IL-2 production*, . asεayed on the IL-2 dependent T cell line GTLL, to rmaci- mally synergize with each other although neither r: used alone, resulted in-any IL-2 production. «- ■*• Northern (RNA blot) analysiε. Following culture for the indicated"" i e points, total' cellular JtøϊA waε extracted with quanidine-thiocyanate and purified by
"centrifugation through a -cushion of 5.'7M -GsCl •(1ΪI-3) .
RNA (10 pg per lane) was separated by electrophoresiε in formaldehyde-agarose gels, ' blotted onto Genescreen membrane filterε (NEN Reεearch Productε) and hybridized to 32p-labelled purified cDNA inserts prepared by nick- translation.
RESULTS In initial experiments, the expresεion of nine novel mitogen induced geneε was examined in PB nylon wool non-adherent T cells. The cells were stimulated for varying amountε of time with: (i) 0KT3, a mitogenic mono¬ clonal antibody which bindε to a non-polymorphic chain" of the CD3 complex (1); (ii) a mitogenic combination of two mAbε directed againεt the CD2 molecule (111-19); and (iii) PHA; and εteady εtate mRNA levelε of the nine geneε were determined.
Fig. Ill-i εhowε that for each gene, PHA, anti- CD2 and anti-CD3 stimulation resulted in εimilar reεponεes. A hierarchy in which the relative levels of mRNA expression can be graded from a low level of induci- bility (pAT 229) to strong inducibility (pAT 464) can be seen in response to any of the three mitogenε. Of the nine genes examined, two were strongly induced (pAT 464, Fig. III-l: pAT 744, data not shown), two were induced to a moderate level (pAT 563, Fig. III-l; pAT 120, data not shown), two were induced to a low level (pAT 416, Fig. III-l: pAT 237, data not shown) and three were weakly induced (pAT 229, Fig. III-l; pAT 154 and pAT 225, data not shown) by either PHA, or anti-CD2 or anti-CD3 mAbs. Stimulation with any one of the above mitogenε in the presence of PMA elicited an elevated response, relative to PHA. anti-CD2 or anti-CD3 mAbs alone, for all of the genes (data not shown) . In addition, although the kinetics of mRNA accumulation are unique for each gene, theεe kinetics are relatively uniform for each gene irrespective of the stimulus used. "
Cells were stimulated in the absence or presence of cycloheximide, to determine whether the interruption of protein synthesis would affect the expression of genes; previous studies have shown that the mRNA levels of many induced genes are elevated under these conditionε (III-4, 111-10, III-ll). The relative effects of cycloheximide addition together with the mitogenic agent were consistent among the various stimuli for each gene examined, even though the magnitude of the cycloheximide related modulation of mRNA levels differed among the genes from virtually undetectble to greater than ten-fold.
The above resultε (εummarized in Table III-l) show that these novel genes are induced by mitogenic agents other than those used to prepare RNA for the library (Experimental Section II). Importantly, stimu¬ lation via mAb binding to one chain within the antigen binding receptor (CD3) induces expression of the novel genes studied here. Thuε, when T cellε are activated through either the CD2 or CD3 cell surface receptors or with PHA, each gene in this panel of induced genes demon¬ strates a consistent kinetic pattern and level of expression irrespective of the agent used to generate these signals.
Figure imgf000066_0001
10
20
25
Figure imgf000066_0003
Figure imgf000066_0002
Table III-l. Summary of Northern blot data for mitogen- induced genes in responεe to agents used in Figs. III-l- 3. Symbols: +, mRNA hybridization to labelled probe; -, no hybridization to labelled probe; -, no hybridization; +/-, weak hybridization; Ab, abrogation of DiC8- and ionomycinr-mediated expression; Incr, increased levels of DiC8- and ionomycin-mediated expreεεion; ND, experiment not performed.
Deεpite the binding to different cell εurface receptorε, each of the above mitogens has been shown to generate similar immediate intracellular responses, part or all of which may be required for the activation of the nine genes examined in Fig. III-l. As the conditions used in this experiment, however, did not result in pro- liferation for one of the three stimuli used (anti-CD3 mAb: see Materials and Methodε), the expreεεion of theεe nine geneε iε not εufficient to initiate DNA εynthesis.
To further ask whether the induction require¬ ments of the nine genes could be grouped into families, highly purified T cells were stimulated with comitogenic agents that generate distinct biochemical εignalε. Theεe agentε include calcium ionophore, PKC activators or anti- CD28 mAb, none of which stimulates DNA syntheεiε in puri¬ fied T cellε (III-8, 111-16, 111-24 and reviewed in III- 26). In contraεt, purified T cellε proliferated after treatment with the combination of calcium ionophore or anti-CD28 mAb and PMA (see Materials and Methods).
Steady state mRNA levels resulting from stimu¬ lation of CD28+ T cells with PMA, anti-CD28 mAb 9.3, ionomycin, PMA plus anti-CD28 mAb and PMA plus ionomycin are shown in Fig. III-2. Unlike stimulation via CD2 or CD3, which elicited graded responses from all nine mitogen induced genes, activation through CD28 clearly divided the nine novel genes into distinct groups (Fig. III-2 and Table III-l). One group, exemplified by pAT 416 (Fig. III-2), and including pAT 120, pAT 154, pAT 225 and pAT 229 (data not shown), showed no responεe to anti- CD28 mAb. This group of genes displayed variable' responsivenesε to PMA alone but in no caεe was potenti- ation of the PMA signal by anti-CD28 mAb observed._ Stimulation of cells with control mAbs, binding to CD4, CD8 or CD45^ did not affect steady 'state mRNA levels. However, steady state mRNA levels observed with PHA treatment alone" were elevated by costimulation with PHA and PMA (data not shown). In the*~secbnd dasε of genes anti-CD28 responsiveness was observed as a εynergiεm between mAb 9.3 and PMA for four novel genes (pAT 237 and pAT 464, Fig. III-2: pAT 563 and pAT 744, data not εhown) and for IL-2R (III-8 and data not εhown) . Although PMA alone induced varying levelε of expreεεion, gene induc¬ tion with εoluble or croεε-linked mAb 9.3 alone was not seen in this group. IL-2 mRNA, in contrast, was not induced by PMA alone, but was induced in responεe to a synergistic combination of PMA and mAb 9.3. All nine mitogen induced geneε reεponded to immobilized anti-CD3 mAb (data not εhown), demonεtrating that the gene expreεεion patternε in the CD28+ PB T cellε are comparable to the leεε rigorouεly purified PB T cellε uεed in Fig. III-l.
Theεe reεults extend the data presented in Fig. III-l and demonstrate that by using an alternative means of activating PB T cells, nine mitogen induced geneε which all responded to PHA, anti-CD2 or anti-CD3 mediated signals could be clearly divided into a group that responded to CD28 derived signals (in the presence of PMA) and a group that did not respond to such signalε (εee Table III-l). For thoεe genes that responded to mAb 9.3 treatment, either signals common to CD28, CD3, and CD2-mediated activation may regulate some of these geneε or mAb 9.3 deliverε a novel signal which can induce expresεion of εome geneε by an alternative mechaniεm. To compare the effectε of the comitogenε PMA and mAb 9.3 to another comitogenic agent, Ca 9+ ionophore, cells were treated with ionomycin in the absence or preεence of PMA. Interestingly, three out of four of the geneε for which εynergy between mAb 9.3 and . PMA waε obεerved diεplayed strong synergy between ionomycin and PMA (pAT 237, pAT 464, Fig. III-2; pAT 74r4, data not shown), and mAb 9.3 -nonresponεive geneε are likewise unaffected by ionomycin. mAb 9.3 does not appear to affect [Ca2+Ji (111-24). This together with the obser¬ vation that pAT 563 is regulated differently by anti-CD28 and ionomycin in the presence of PMA (Table III-l) suggeεtε that the mechaniεms by which these two partially mitogenic agents synergize with PMA are likely to be different. - . •
To further define regulatory families of mitogen induced genes, the separate and combined effects of ionomycin and DiC8-mediated PKC activation upon gene induction in the T cell line Jurkat were examined, DiC8 is a cell-permeant synthetic diacylglycerol (DG) that mimics the effect of endogenous DG by reversibly acti¬ vating PKC (III-5, 111-16, 111-17) . Previous studies have shown that eight of the nine mitogen induced genes analyzed above in resting T cells were inducible . in Jurkat with the combined addition of PHA and PMA; pAT 563 is constitutively expressed at a low level (data not shown) . Treatment of Jurkat cells with ionomycin and/or DiC8 in the presence or absence of cycloheximide, showed that three regulatory clasεeε of geneε can be defined. The firεt claεε detected iε represented by pAT 237 in Fig. III-3 and includes genes which are PKC inducible but display either varible and minimal or no synergy with a [Ca2+]i signal (pAT 237, Fig. III-3: pAT 120, pAT 154, pAT 225 and pAT 416, data not shown). No response to ionomycin alone was detected for this claεε of geneε. All of theεe geneε were εimilarly induced in PB T cellε by PMA.mediated PKC activation (Fig. 111-2 and data not shown), and the patterns of gene expresεion in reεponεe to PMA in Jurkat were identical to thoεe preεented here for DiC8 (data not εhown). A second class, consiεting of pAT 229, iε PKC inducible and able to be potentiated by a: [Ca2+]i εignal. In this regard pAT 229 resembles the-IL-; 2R (data not shown), yet it differs from the IL-2R in that pAT 229 iε induced by increaεed [Ca2+Ji levelε alonel (Fig. III-3). ■= . * ._. -. ..
The final claεε obεerved in these cells includes pAT 464 (data not shown) and . AT 744 as well aε IL-2 (Fig. III-3), whose induction exhibited a require-** ment for both signals asεayed in thiε experiment. In addition, the reεponεeε of the variouε geneε in Jurkat cellε to the inducing agentε in the presence of cyclo¬ heximide demonstrated an .additional correlation which discriminated IL-2, pAT 464 and pAT 744 from the other genes. Specifically, cycloheximide added εimultaneouεl with the activating agentε entirely prevented the accumu¬ lation Of IL-2, pAT 744 (Fig. III-3) and pAT 464 mRNA (data not shown) , implying a mechanism of gene induction requiring newly εynthesized protein. In contrast, cyclo¬ heximide treatment .enhanced mRNA levels in the other two groups (Fig. III-3 and data not shown) .
DISCUSSION The effects of different εignalling pathwayε with regard to their ability to induce a panel of nine novel mitogen induced genes are summarized in Table III- 1. A majority of the genes εtudied here εhowed εome degree of responsiveneεε to PKG activation alone. How¬ ever, thiε signal is insufficient for maximal responεive- neεε for any of the geneε analyzed, since PHA and anti- CD3 or anti-CD2 mAbs εubstantially enhance the effect of PMA for all the genes in PB T cells (data not shown), while anti-CD28 and ionomycin (Fig. III-2 and data not shown) synergize with PMA for a subset of these genes in PB T cells.
The apparent homogeneity of expreεεion patternε for each gene in reεponεe to PHA, anti-CD3 or anti-CD2 mAbε may reflect that qualitatively, a similar series of intracellular biochemical events reεultε from activating T cells with theεe agents. This common pattern of gene expresεion in reεponse to a variety of inducing agents can potentially be mediated at a number of levels in the signalling pathway. *• At the level of signal generation " either a similar constellation of intracellular messengers is generated or, alternatively, convergence of diverse signalling molecules occurs at a point proximal to their effects on- the transcriptional or post- transcriptional "regulation of these genes. Distinct regulatory motifs controlling these genes may be responsive to divergent signalε with all generating a positive effect on tranεcription.
Deεpite the apparent uniformity of PB T cell responses to stimulation with PHA or anti-CD3 and anti- CD2 mAbs, regulatory distinctions for induced genes were unmasked by probing the system with ionomycin, which is expected to generate a subset of signals relative to those above (111-24) and with anti-CD28 mAb, which appears to generate a distinct secondary signal(s) (III- 8, 111-12, 111-14, 111-22, 111-26). Class V genes (Table III-l), those that are ionomycin and anti-CD28 non- responsive, are novel relative to the majority of mitogen-induced genes previously described in T cells (III-8). Further heterogeneity was obεerved by analyzing the effects of membrane-bypasε stimuli in the Jurkat cell line. The fact that five regulatory classeε could be diεcerned among thiε relatively small group of nine mitogen-induced genes demonεtrates the multiplicity of regulatory mechanisms acting at this stage of T cell mitogenesis.
Although the requirements for gene induction are generally parallel in resting T and Jurkat cells, differences were noted in the regulation of certain genes. Specifically, simultaneous cycloheximide addition abrogated the mitogen induction of pAT 464, pAT 744 and IL-2 in Jurkat (Fig. III-3 and data not shown) yet slightly elevated mRNA levels for these genes in PB T cellε (Fig. III-l and data not shown). Secondly, iono--,-. mycin synergized with PMA to elevate pAT 237 mRNA level-T¬ in PB T cellε, but not in Jurkat cellε, whereaε pAT 225 did not reεpond to ionomycin in PB T cellε (data not shown) but did εo in. Jurkat. Theεe diεparitieε may εtem
-r -=_" ess? _u- r* ,_._**•_ _ __ _ '*•? from the fact that PB T cellε are heterogeneouε with regard to population subtypeε or from differences in the basal state of activation between quiescent, nontrans- formed cellε and cycling, tumor cellε. Alternatively, Jurkat cellε may abnormally over-expreεs or under-expresε εpecific regulatory componentε required for εome gene induction eventε.
Thiε work haε demonstrated a striking correla¬ tion in the regulation of pAT 464 and pAT 744 with that of IL-2. Indeed, the DNA sequence of pAT 464 and pAT 744 yield a derived amino acid sequence showing a leader peptide and conεervation of amino acidε at εpecific posi¬ tions that are charcteristic of one claεε of lymphokineε (P. F. Zipfel et al., in preparation). Thiε regulatory claεε of geneε responds to signalε mediated through CD28, in addition to CD2 and CD3 in PB T cellε, exhibitε a requirement for two εignalε in Jurkat cellε, and their induction iε completely abrogated by cycloheximide treat¬ ment in Jurkat cellε. A requirement for protein εyntheεiε preceding mRNA induction in Jurkat appearε to be unique to thiε claεε of geneε, relative to many other mitogen induced geneε εtudied here and elsewhere, suggesting a novel and conserved regulatory mechanism for these and most likely other lymphokines. This section demonstrates the utility of DNAs of this invention to dissect the mechanisms of genetic activation underlying mitogenic stimulation of T cells, and therefore, illustrates embodimentε of the invention comprising bioassays for induction of genes related to early events in inflammation and/or growth and repair.
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78:1805.1808.
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Acad. Sci. USA 84:1654-1658. 11-21. Nishizuka, Y. 1984. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308:693-697. 11-22. Pantaleo. G., D. Olive, A. Poggi. W.J. Kozumbo, L. Moretta, and A. Moretta. 1987. Transmem- brane εignalling via the Til-dependent pathway of human T cell activation. Evidence for the involvement of 1,2.diacylglycerol and inoεitol '" phosphates. Eur. J. Immunol. 17:55-60.
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111-24. Truneh, A., F. Albert, P. Golstein, and A-M. Schmitt. Verhulεt. 1985. Early εtepε of lympho¬ cyte activation bypaεεed by synergy between cal¬ cium ionophoreε and phorbol eεter. Nature 313:318. 320.
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1984. Role of T3 εurface moleculeε in human T- cell activation: T3-dependent activation reεultε in an increaεe in cytoplaεmic free calcium. Proc. Natl. Acad. Sci. USA 81:4169-4173.
111-26. Weiεs, A., and J.B. Imboden. 1987. Cell surface molecules and early events involved in human T lymphocyte activation. Adv. Immunol. 41:1.38. EXPERIMENTAL SECTION IV MITOGENIC ACTIVATION OF HUMAN T CELLS INDUCES TWO CLOSELY RELATED GENES WHICH SHARE STRUCTURAL SIMILARITIES WITH A NEW FAMILY OF SECRETED FACTORS
About 60 novel cDNA cloneε whoεe corresponding mRNAs are induced by mitogenic activation in human peripheral blood T cells were isolated previously (see Experimental Section II) . Here are described the primary structure and regulation of two such clones, pAT 464 and pAT 744, which encode new lymphokines/cytokines. Similar to IL-2, both genes require the synergy of agents such as PHA and PMA for optimal expression, and, in addition, the induction of both is εensitive to the immunosuppreεεive drug cycloεporin A. The two geneε can be expreεεed in T cellε, B cells and the promyelocytic cell line HL60, but they are not expressed in human fibroblasts, suggesting that their expresεion iε reεtricted to hematopoietic lineages. The predicted peptides encoded by these two clones feature hydrophobic N-terminal leaders -character¬ istic of secreted proteins. The predicted size of both proteins is about 8 kD upon cleavage of the putative leader, peptide. .Mammalian (COS) cells transformed with pAT 464 and pAT 744 DNAs secreted novel proteins of the εize expected from the cDNA εequenceε after the leader peptide iε cleaved off. Rabbit antiεera raiεed to the predicted C-terminal amino acidε of both pAT 464 and pAT 744 reacted with the products secreted from the trans¬ formed COS cells and with products of the same size secreted by mitogenically-activated human peripheral blood T cells. pAT 464 and pAT 744 are similar to each other and also share some critical amino acid sequence similar- ity with a newly emerging family of εecreted factors including connective tissue activating factor III (CTAP III), platelet factor 4 (PF4), an interferon-gamma induced factor (IP-10), macrophage inflammatory protein (MIP) and a factor chemotactic to neutrophils (3-10C, MDNCF, NAF) . Some of theεe factorε have been εhown to diεplay functionε aεεociated with an inflammatory response and/or have mitogenic activities. Collectively, the data presented here suggest that pAT 464 and pAT 744 encode novel lymphokineε/cytokineε which play roleε dur¬ ing an immune reεponεe similar to those enacted by these structurally related factors.
A cDNA clone derived from the esεentially the same gene as pAT 744 was characterized in Experimental Section I, above. That clone is called Act-2.
INTRODUCTION There is a need to analyze the genetic program initiated upon activation of resting T cells. To thiε end, more than 60 geneε that are induced in human periph- eral blood T cellε by mitogenε were iεolated (Experi¬ mental Section II) . In order to εelect_those geneε. from among thiε collection which might encode novel lympho- kines, the iεolated genes were examined for regulation characteristicε„ exhibited by known,., genes;, that encode factors. Here are described the.primary- structure of two such cDNA clones, pAT 464 and pAT 744, whose genes share many regulatory properties with the_IL-2 gene, "including total sensitivity to cyclosporin A. Their nucleotide sequences predict hydrophobic leader peptideε character- iεtic of εecreted proteins. Amino acid εequence εimilar- itieε indicate that both geneε are memberε of a newly emerging family of εmall εecured proteinε which appear to mediate certain inflammatory reεponεeε and/or diεplay mitogenic activitieε.
MATERIALS AND METHODS T Cell Iεolation. Human peripheral blood T cellε were obtained from healthy human donors by lympho- phoreεis on a enwall CS 3000 or an_ IBM Cebe 2997 -appa- ratuε. Blood enriched for lymphocyteε waε subsequently purified over Ficoll Hypaque gradients and nylon wool columns. As judged by anti CD3 staining, theεe cell preparationε conεiεtently were compoεed of greater than 90% T cellε. Cell Stimulation. Human peripheral blood T cellε, Jurkat cellε and HL60 cellε were maintained in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum. Penicillin, Streptomycin and Gentamycin at a cell density of 2 x 10° cells/ml for human T cellε, or 5 x 105 cellε/ml for Jurkat cellε, reεpectively. The cellε were treated with either one or a combination of the following agentε: Phytohemagglutinin (PHA-P; Buriroughε Wellcome) at a final concentration of 1 Afg/ml, Phorbol 12-myriεtate 13-acetate (PMA) at 20 ng/ml, cycloheximide at 10 jq/ml and cycloεporin A at 1 juq/ml . Aliquotε were taken at different timeε, the cellε were waεhed in RPMI 1640, and the total cellular RNA waε iεolated. RNA Isolation and Northern Analysiε. Total cellular RNA waε isolated by the quanidinium isothiocy- nate method as described (IV-27) and either 8 βq (periph¬ eral blood T cells) or 10 μq (Jurkat cells) of total cellular RNA were separated on a formaldehyde agarose gel and subsequently transferred to nitrocellulose (Schleicher and Schuell) with 10 x SSC. ,
Nick-translation and Hybridization. cDNA insertε were nick-translated (IV-28) and used as probeε for hybridization at 42°C in 40% Formamide, 4 x SSC, 10% dextran sulfate and lx Denhardt's εolution. After hybridization for 10-18 hr, the filters were washed at a final stringency of 0.1 x SSC at 60°C
Isolation of Full Length cDNA Clones. Clones pAT 464 and pAT 744 originally were derived from a sub- εtracted cDNA library (Experimental Section II, above). Near full-length cDNA cloneε were iεolated from a cDNA library which waε derived from RNA extracted from human peripheral blood T cellε εtimulated for 4.5-hr with. PHA-P and PMA in the preεence of cycloheximide. Thiε cDNA library was constructed with oligo dT priming as described (IV-29) and was subεequently cloned into lambda gt 10 (IV-30).
Southern Hybridization. Human placental DNA (10 Jjq ) waε digeεted with Eco RI, Bam HI or Sεt I, elec- trophoreεed in a .8% agaroεe gel, and tranεferred onto a nitrocellulose filter. After hybridization with a nick- translated cDNA insert, filters were washed at a final stringency of 0.1 x SSC at 55°C. Sequence Analysis of cDNA Clones. The longest cDNA inserts were cloned into Ml3 RF19 and sequenced by the dideoxy chain termination method (IV-31) using Sequenase (USB), dATP p£- S] and various oligonucleotide primers. Primers for the sequencing reactions were syn- theεized aε 17merε on an oligonucleotide εyntheεizer model 380A (Applied Biosystems) following the protocol of the supplier. All oligonucleotides were purified on a 20% acrylamide gel prior to use. Both the cDNA sequences- and the predicted protein εequenceε were compared with- a- computer data bank (Bionet, current release)..
Primer Extension. An oligonucleotide comple-. mentary. to -nucleotide 135-152 of the pAT 744 sequence (Fig. IV-4B) was annealed to 20 q of RNA f_eom human peripheral blood T cells which were stimulated for 4.5 ϊir with Pi_A and PMA in the presence of cycloheximide. Annealing was performed in 100 mM KC1, and the reverse transcription reaction was aε described (IV-29), using reverse transcriptaεe (Seikageiku) to extend the annealed primer (100 (g/ml) . Hybridε were extracted'asrith an-equal volume of phenol/chloroform and then-were precipitated in ethanol. The final reaction product waε analyzed on a 8% denaturing polyacrylamide gel. A DNA εequence ladder εerved aε size markers.
Expreεεion of cDNA cloneε in Mammalian (COS) cellε. COS cellε were transfected msing εtandard.DEAE- dextran methodology, with cDNA clones inserted-- into expression sites of either of the following vector DNAs: CDM-8 [obtained from Brian Seed at Massachusettε . General Hospital, Boston, Massachusetts, and described in B. Seed, Nature 329, 840-842 (1987)]; or PMT2-T [obtained from Genetics Inεtitute, Cambridge, Maεεachuεettε; a related earlier verεion of thiε vector haε been deεcribed in Yu-Chung Yang, et al.. Cell _47_, 3-10 (1986)].
RESULTS Isolation and Regulation of Two cDNA Clones in - T Lymphocytes. Previously (see Experimental Section II) an approach was described to isolate genes which are induced immediately upon mitogenic stimulation of human peripheral blood T cells. In exceεε of 60 geneε have been isolated from a subεtracted cDNA library highly enriched for induced εequenceε. Following εtimulation of T cellε, many but not all of theεe geneε were induced rapidly and transiently within 30 min. to 1 hr and their level of induction was increased in the presence of the protein syntheεis inhibitor cycloheximide (see Experi¬ mental Section II, this specification, above). The expresεion and regulation of nine of the novel mitogen- induce genes was studied in more detail and revealed different regulatory ^classes, as these genes exhibited distinct responses to a variety of T cell specific acti¬ vating agents (see Experimental Section III, above).
Two genes from this collection of cDNAs, named pAT 464 and pAT 744, were noted to εhare regulatory char- acteriεticε with the IL-2 lymphokine gene (Experimental Section III). Like IL-2, theεe genes required absolutely the combined action of signalε initiated by ionomycin and PMA for optimal expression in highly purified T cells. In addition, mRNAs for pAT 464 and pAT 744 were induced by 2 hours and maintained at high levels for at least 24 hours, kinetics different from those seen with the major¬ ity of the isolated genes (Experimental Section II).
Given these regulatory featureε, the two genes were characterized further. Treatment of human periph- eral blood T cell preparations with both PHA and PMA yielded high levels of mesεageε for both pAT 464 and pAT 744 (Fig. IV-1). PHA alone induced these genes, but to a much lesser degree, and PMA alone had little or no effect (Fig. IV-1). Thus, even with incompletely purified T cell preparations, two signals are required for optimal expresεion. The immunosuppresεive drug cycloεporin A entirely inhibited the induction of pAT 464 and 744 by PHA and PMA in blood T cells (Fig. IV-1). A similarly dramatic effect of this drug is known to occur with the IL-2 gene (IV-32). Also shown in Figure IV-1, the pres¬ ence of the protein synthesiε inhibitor cycloheximide did not change the induced levels appreciably during activa¬ tion.
To examine further the similarity of expression between the two cDNA clones and the IL-2 gene, their regulation was analyzed in the CD4+ helper T cell line Jurkat. The requirements for induction of IL-2 are well characterized in thiε tumor line (IV-32, IV-33). As shown in Figure IV-the kinetics of induction and .the activation signals needed for pAT 464 and 744 paralleled that seen in peripheral blood T cells. First, the mes- sages for both genes appeared at about 2 hours following stimulation with PHA and PMA and their levels were main¬ tained for at least 24 hours. Second, both PHA and PMA were required for optimal εtimulation "of the two cDNA cloneε, εince PMA alone did not induce theεe geneε and PHA alone induced them to a εmall degree. Third, cyclo¬ εporin A totally abrogated the expreεεion of pAT 464 and 744 during the 24 hour εtimulation period analyzed .here.
Unlike what waε observed in peripheral blood T cells, cycloheximide inclusion during activation with PHA and PMA caused a marked inhibition of expresεion. Thiε waε the caεe alεo when cycloheximide waε added together with two different activation agentε, ionomycin and the εynthetic diacylglycerol DiC3 (Experimental Section III). Aε the IL-2 gene iε εubject to preciεely the εame inhibition by cycloheximide in Jurkat cellε (IV-32), these data demonstrate the similarities in the regulation of IL-2, pAT 464 and pAT 744. The reason why cyclohexi¬ mide inhibits the mesεage induction of theεe geneε in Jurkat cellε but not in peripheral blood T cellε are not known. A protein induced early after Jurkat cell activa¬ tion appearε to be required for the binding of a nuclear factor to the IL-2 regulatory region (IV-34, IV-35).
Nucleotide Sequence of pAT 464 and pAT 744. The nucleotide sequence of near full-length cDNA cloneε for pAT 464 and pAT 744 waε determined (see Materials and Methods). The sequencing strategies used, the resulting nucleotide sequences and the predicted amino acidε for the encoded proteinε are εhown in Figureε rv-3 and IV-4 for pAT 464 and pAT 744, reεpectively. Clone pAT 464 is 793 nucleotides long and clone pAT 744 is 659 nucleotides long (including the Eco RI linker at the 5' end). Both cDNA sizes are in good agreement with the estimated mRNA sizes of 850 nucleotides (pAT 464) and 750 nucleotides (pAT 744), since the mRNAs are likely to include a much longer poly A tail.
Primer extension analysis for pAT 744 confirmed that the isolated clone represents an almost full length cDNA (Fig. IV-5). Uεing an oligonucleotide complementary to nucleotideε 135-152 (Fig. IV-4) of the pAT 744 εequence aε a primer for reverse transcriptase (see Materials and Methods) resulted in a fragment of about 155 nucleotides in size (138 nucleotides of synthesized cDNA and 17 nucleotides of primer) . Because the first 7 nucleotideε at the 5' end of the sequenced clone for pAT 744 are derived from an Eco RI linker, it can be concluded that about 10 nucleotides of the 5' end Of the true mRNA sequence are missing in this cDNA clone.
The first ATGs for pAT 464 and pAT 7.44 are at positions 84 and 74, respectively. In each caεe and open reading frame of 92 amino acid followε. Theεe open read- ing frameε are the longes to be found: for the two cloneε. For pAT 744 εeveral in-frame εtop codons precede the first ATG. The nucleotide sequences surrounding the predicted start sites for translation for both clones display a good match with the consensus sequences derived from known translation initiation siteε (IV-36). These data strongly suggest that both cDNAs encode peptideε of 92 amino acidε.
A hydrophilicity analysis of the predicted peptides for both pAT 464 and 744 revealed hydrophobic N- terminal regions, typical for signal peptides of secreted proteins. According to the criteria for signal peptide cleavage (IV-37, IV-38) the first 23 amino acidε of the leader sequenceε would be cleaved off in both proteinε, reεulting in mature εecreted proteinε of 69 amino acidε in εize. No potential .M-linked glycosylation siteε are evident in either peptide.
The 3' untranslated regionε of the two cloned geneε contain several elements which may serve regulatory functions. The ATTTA motif (underlined in »igureε : IV-3 and IV-4) that Jaaε been aεεociated with the instability of lymphokine mesεageε (IV-39) appearε four timeε in clone pAT 464 and twice in clone pAT 744. In addition, pAT J44. and 464 contain the octamer motif L TTATTTAT (a εubεet of the ATTTA motifε), which iε present in the 3' untranεlated regionε of a number of distinct mRNAs induced during an inflammatory response' (IV-40^. ' This element is located at position 527 for pAT 464 and at position 526 for pAT"744. Both cDNA clones possess a poly A addition signal (boxed in Figureε IV-3, IV-4) which precedeε a poly A εtretch.
A compariεon of the εequenceε for pAT 744 and 464 indicated a εtriking εimilarity on the nucleotide level (45%) and an even higher homology on the amino acid level (56%, Fig. IV-6A) . In addition to common regula¬ tory characteriεticε, theεe two geneε may have been derived from a common ancestral gene and. their encoded proteins"may play functionally related roles•; The higher amino acid similarity suggeεtε a conεervation of function and further εtrengthenε the encoded protein εtructureε predicted from the open reading frameε in the cDNA cloneε (see also comparison with other proteinε below) . In particular, the preεumed εecreted partε of the two pro- teinε exhibit a high degree of εimilarity, whereaε their N-terminal leader peptideε have little in common except their hydrophobic nature. The hydrophilicity plotε of the two proteinε are virtually superimpoεable which affirms further their structural relatedness (data not shown).
The primary structures of the proteins encoded by pAT 464 and 744 exhibit a striking similarity to a number of other proteins, εo e of which have been detected aε εecreted proteins. Several members of this family of proteins have been shown to exhibit functions assiciated with an inflammatory response and with mitogenesis (see Discusεion). In particular, thiε similarity is based on four almost identically spaced cysteine residues and one proline residue, present in all proteins of this family (Fig. IV-6B). These highly conserved amino acids are likely to play an important role in the structure of the proteins (see Discussion). In addition to this conservation, many other amino acids are shared between various members of this family.
In order to determine whether pAT 464 and pAT 744 themselveε are emberε of a family of even more highly related geneε, genomic DNA waε probed with theεe two geneε. Human placenta DNA was digested with various restriction enzymes and subjected to a Southern analysiε with radioactive probeε derived from the cDNA inεertε for the two geneε. The data in Figure IV-7 revealed a limited number of hybridizing fragmentε with diεtinct patterns for the two probes. Apparently, pAT 464 and 744 do not readily crosε-hybridize with each other, in εpite of their remarkable εimilarity. Thiε had been noted previouεly in εcreening the cDNA phages isolated from the subtractive library with plasmid derived cDNA probes made to pAT 464 and 744. It is also concluded that the two clones contain no repetitive elements, and that they do not belong to a large family of closely related sequences. It is not now posεible to determine whether the genes are single copy genes or belong to small fami¬ lies of highly similar εequenceε.
Tissue and Cell Type Specificity of Expres¬ sion. As determined previously, pAT 464 and 744 are not expresεed in quiescent or serum stimulated normal human fibroblastε. Here hematopoietic cellε other than T cellε were analyzed for expression. Promyelocytic HL60 cells can be differentiated terminally into macrophages or into granulocyteε. Neither pAT 464 nor pAT 744 was expresεed in undifferentiated HL60 cells but both were induced upon induction of differentiation into macrophages with PMA (Fig. IV-8A) . In contrast, using DMSO to differentiate HL60 cells into granulocytes did not result in any indue- tion of their mRNAε. -Intereεtingly, PMA did not induce ■ RNA for pAT 464 and pAT 744 in peripheral blood T cellε * or in Jurkat cellε. Therefore, the induction of theεe genes in HL60 cells must be explained by the particular differentiated state of these cells. _ pAT .464 ,and_.__744 were.. induced also in human B cells. In unstimulated small human tonsillar B cellε no message could be detected for either gene, but mRNA for pAT _744 was induced upon 4 hours of stimulation with SAC (Fig. IV- 8B), while mRNA for pAT 464 waε clearly detectable only with SAC and cycloheximide (data not shown) . Further¬ more, several EBV tranεformed B cellε lineε conεtitu- tively synthesized mRNA for these genes, as did HTLV I transformed antigen-specific T cell clones. Therefore, pAT 464 and pAT 744 may be expreεεed by a number of dif¬ ferent hematopoietic cellε upon being activated in εome manner, including viral transformations.
Expresεion of εecreted proteinε in COS cellε. COS cells transfected with various expression vectors encoding pAT 464 or pAT 744 cDNAs were found to secrete protein products, aε predicted from the DNA εequence (εee. Figε. IV-9 and IV-10). Theεe proteinε are made and εecreted from COS cellε only when the appropriate expreε¬ εion vector conεtructε are tranεfected, not when negative control conεtructε are uεed. The novel proteinε were visualized by 35S-cysteine labelling of the cellε after the transfection of the DNA constructs using standard, well known methods. The size of the protein products is
- appropriate as predicted from the cDNA sequence after the leader peptide is cleaved off.
Antibodies to predicted amino acid sequences. Rabbit antisera were raised to synthetic peptideε repre- εenting the C-terminal 12 amino acids of both pAT 464 and pAT 744, as predicted by the cDNA sequences. This was done by chemically synthesizing the peptides, linking them to carrier (KLH), and injecting the carrier pluε peptideε into rabbitε, according to εtandard methodε of peptide immunology. Figs. IV-11A and B show the use in Weεtern blot experimentε of two different rabbit anti- bodieε (721 and 722) raiεed againεt the pAT 744 peptide; Figs. IV-12A and IV-B similarly illustrate two antibodies (719 and 720) to the pAT 464 peptide. Inr all cases, supernatants of COS cells transfected with various expresεion vector conεtructε were analyzed with the anti- εera. . As is evident from the figures, τthe appropriate secreted factors are detected by antisera from rabbits immunized with synthetic peptides, but not by serum sam- pleε taken from the εame rabbitε before immunization with peptideε. Therefore, theεe reεultε demonεtrate that εynthetic peptides derived from the predicted protein sequenceε of both pAT 464 and pAT 744 do raise antibodies to the factors secreted by COS cells transformed with appropriate cDNA clones.
Secretion of factors from human peripheral blood T cells activated by mitogenε. Fig. IV-13 εhows a Western blot of supernatant from human T cells activated in culture with PHA/PMA uεing anti-pAT 464 antibody. The antibody detects a secreted product of appropriate size. Similar results were obtained with anti-pAT 744 antibody (data not shown). Therefore, human T cells produce not only an abundant amount of pAT 464 and pAT 744 mRNA upon mitogenic stimulation (see above), but also the secreted forms of the protein factors predicted from the cDNA sequences of these clones.
DISCUSSION Previously, a large number of genes inducible in T cells upon mitogenic activation were isolated (see Experimental Section II, above). This section describes the regulation and the nucleotide sequence of two induc¬ ible genes, clones pAT 464 (or pLD 78, see Results) and pAT 744. These two genes appear to encode new lympho- kines/cytokines. The nucleotide sequences for each cDNA clone predict encoded proteins of 92 amino acids, with N- terminal hydrophobic leader peptides common. to secreted proteinε. pAT 464 and '744 are very homologouε to each other, eεpecially in their preεumed εecreted port__6fιs-_,, Thiε εuggeεtε conεervation of function. Both gene pro-*- ducts also share important amino acid residues with a family of proteins, -whose functions are not oicL only-par¬ tially understood; some of these proteinε have been detected aε secreted factors (see below). Although pAT 464 and pAT 744 do not possess significant amino acid homology with several well-defined immunoregulatory fac- tors like IL-2 or gamma-IFN, they are likely to εhare regulatory elementε with theεe geneε. Like theεe lympho- kineε, pAT 464 and pAT 744 require two inducing signals for optimal expresεion (e.g., PHA and PMA), they appear after 2 hourε of εtimulation of T cellε, they are very sensitive to the inhibitory effects of the immunoεuppreε- εive drug cycloεporin A and their activation iε sup- preεεed completely in Jurkat T Cellε in the preεence of cycloheximide.
The primary amino acid εtructureε of the-'vpro- teinε encoded by pAT 464 and 744 exhibit εignificant homology with a newly emerging family of εecreted fac¬ tors. The homology is baεed primarily on the positions of four cysteine reεidueε (εee alεo IV-23, IV-42, IV-43) and one proline reεidue (boxed in Fig. IV-6B). Theεe amino acidε are found in all the proteinε εhown here and the peptides were aligned accordingly in Figure IV-6B. The cysteines are likely to provide a common structure. For one of the members of this family, ?-thromboglobulin (a processed form of PBP, see below), these cysteineε have been shown to form diεulfide bonds (IV-44). Start¬ ing from the amino terminus, cysteineε 1 and 3 and cyε- teines 2 and 4 form εuch bonds.
In addition to the completely conserved amino acids, many other reεidueε are εhared between εeveral of the genes. For example, the amino acids Trp Val Gin (WVQ) (or WV) (boxed in Fig. IV-6B) are found in eight of the factors, εix reεidueε downεtream from the laεt of the four conserved cysteines. The genes listed in Figure IV- 6B can be subdivided into two principal groups: The first two cysteines are positioned in one of two wayε, either next to each other as Cys (C) or separated by one amino acid as C X C. Consistent with this division, several positions display clear amino acid preferences distinct to one or the other group:' To list a few, in the 'C C group, the 7th residue following the conserved* proline (proline boxed in Fig. IV-6B) is alwayε a Tyr (Y) , the 9th reεidue preceding the fourth cysteine is an Phe (F) and the residue directly following this laεt conserved cysteine is an Ala (A). In the 'C X C group, the third and sixth residues following the conserved proline are lie (I) or Leu (L) and L or Val (V), respectively, and the residue directly following the fourth cysteine is always an L. At many other poεitionε, however, different εortε are poεεible, clouding the distinction between these two groups.
The homologies between pAT 464, pAT 744 and all additional proteins listed in Figure IV-6B may indicate some conservation of function. Several of these factors have been shown to be expressed during or even cause an inflammatory responεe and/or to exhibit functions consiε- tent with a role during inflammation. Some of the pro- teins also act mitogenically, possibly contributing to wound-healing and tissue repair, processes which may occur in association with or following inflammation. 3- 10C (IV-23) or monocyte-derived neutrophil chemotactic factor (MDNCF) (IV-43) or neutrophil activating factor (NAF) (IV-25) is a human factor which has chemotactic activity for granulocytes in vitro (IV-24, IV-45, IV-46),- induceε rapid granulocytoεis jua vitro or with systemic injections in vivo and causes skin reactions upon local injection (IV-46, IV-47). Inducible expresεion haε been demonεtrated in monocyteε/macrophageε by agentε εuch aε LPS, IL-1 or TNF (IV-24, IV-46) or Con A (IV-46) and in T cells by staphylococcal enterotoxin (IV-23). Macrophage inflammatory protein (MIP) iε a factor εyntheεized. by: mouse macrophages upon LPS stimulation (IV-22, IV-48). It possesεeε neutrophil chemotactic activity, cauεes some- hydrogen peroxide production and leadε to localized inflammation when,.administered subcutaneously (IV-22, IV- 48). - ..-
IP-10 (IV-21) is an interferon induced -protein factor which is expreεεed during delayed-type hyperεenεi- tivity reactions incited by a form of tuberculin or by - lFN (IV-49). It can "be expressed in several cell types, including endothelial cells, monocytes, fibroblastε and keratinocyteε (IV-49, IV-50).
Platelet factor 4 (PF-4) iε releaεed from the o-s-granuleε in plateletε during injury and haε chemotactic activity for monocyteε, neutrophils and fibroblasts (IV- 51, IV-52). Additional activities are the inhibition of collagenase (IV-53) and an immunoregulatory role (IV-54). Platelets alεo release the platelet basic pro¬ tein (PBP) ^-thromboglobulin (A-TG) and connective tisεue activating peptide III (CTAP III) (also known as low- affinity platelet factor-4). The latter two, CTAP III and _>-TG, represent succeεεive N-terminally proceεεed formε of PBB, aε indicated in Figure IV-6B. CTAP III haε been shown to act mitogenically for human dermal fibro- blasts (IV-55) and human connective tisεue cellε and to εtimulate glycolyεiε, glycoεaminoglycan εynthesis, cAMP levels and release of plasminogen activator (IV-20, IV- 56). _?-TG appearε to be chemotactic for fibroblasts (IV- 51). The remaining factors liεted were all cloned on the baεiε of being inducible or differentially expreεεed. RANTES (IV-42) and TCA-3 (IV-57) are T cell derived geneε for which no function iε known yet. The expreεεion of both iε induced by antigen and mitogenε, with the mouεe TCA-3 mRNA appearing within an hour of activation (IV-57) and the human RANTES mRNA within a day (IV-42). The human H-Gro gene, the mouεe JE gene and the chicken 9E3 gene are all expressed by fibroblasts. The JE gene is induced by various agents in 3T3 cellε, including serum and IL-1 (IV-58). The 9E3 gene is acti¬ vated by transformation with the Rous Sarcoma Viruε and iε suppressed in growth-restricted cells (IV-59). The H- Gro gene is the human counterpart of the hamster Gro gene, whose constitutive expresεion correlateε with tumorigenic variants of the nontumorigenic Chinese ham¬ ster fibroblastic cellε (CHEF) (IV-26). Gro is tran- siently induced by εerum in the nontumorigenic CHEF cells. The expreεεion of thiε gene iε apparently not sufficient for induction of the phenotypes associated with the tumorigenic state.
The expression data for the genes discussed above is consiεtent with a role during inflammation and/or mitogeneεiε aεεociated with tissue repair. This is deduced from the various functions demonstrated, from the agents inducing these genes and from the cell types involved. Therefore, pAT 464 and pAT 744 are expected to exercise similar functions, in particular because the pAT 464 gene appears to represent the human homolog of the mouse MIP gene. Inflammation and wound healing/tissue repair are very complex proceεεes which involve many different cells. Coordination of the various cellular functions is likely to be reflected in a multitude of secreted factors needed to communicate between cells. This family of genes, which may have been derived from one common ancestral gene, may well serve to establish such a network. To mediate inflammation and repair, the individual factors may possess multiple functions, as shown for several of the members of this family (see above) . The conserved similarities between these pro¬ teins may indicate an interaction with conserved receptor elements. Aε enumerated above, the members of this family of proteinε diεplay a remarkable variety of tiεεue specificities. pAT 464 and pAT 744 are expresεed in T cellε and other hematopoietic cellε, but not in fibro- blasts. The distinctionε between the factorε may deter-: mine unique functionε required during an immune reaction depending on the cell type activated.
Thiε section haε described several of the most preferred recombinant molecules of the present"invention, namely those comprising pAT 744 or pAT 464 DNA and a mammalian expresεion vector (CDM-8 or PMT2-2) capable of expressing inserted DNAs in mammalian (e.g.~, COS) cells. It also documents preferred cells transformed by these DNAs, and secretion by such cells of appropriately- sized protein products as predicted from the cDNA sequence after the leader peptide is cleaved off. Fur¬ ther, this section has illuεtrated novel antibodieε of thiε invention, iήade againεt a peptide encoded by a*DNA segment of the invention and able to specifically bind to pAT 744 or pAT 464 lymphokine/cytokine-like protein which includes the sequence of such peptide.
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Figure imgf000095_0001
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IV-57. Burd, P.R., G.J. Freeman, S.D. Wilson, M.
Berman, R. DeKruyff, P.R. Billings, and M.E.
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IV-58. Rollins, B.J., E.D. Morrison, and CD. Stiles.
1988. Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytoline-like properties.
Proc. Natl. Acad. Sci. USA 85:3738
IV-59. Sugano, S., M.Y. Stoeckle, and R. Hanafusa. 1987. Transformation by Rous sarcoma virus; induces a novel gene with homology to a mito¬ genic platelet protein. Cell 49:321 For purposes of completing the background: description and present disclosure, each of the published articles, patents and patent applications heretofore identified in this specification are hereby incorporated by reference into the specification.
The foregoing invention haε been deεcribed in some detail for purposeε of clarity and underεtanding.
It will also be obvious that various combinations in form and detail can be. made without departing from the scope of the invention.

Claims

WHAT IS CLAIMED IS:
1. A DNA segment encoding a human lympho¬ kine/cytokine-like protein, wherein said protein is selected from the group consiεting of translation pro- ducts of a full-length clone of Act*2 cDNA and clones of pAT 744 cDNA. #
2. A recombinant DNA molecule comprising a DNA segment according to claim 1 and a vector.
3. A culture of cells transformed with said recombinant DNA molecule according to claim 2.
4. A method of producing a human lympho¬ kine/cytokine-like protein comprising., culturing said cells according to claim 3 under conditions such that said protein is produced and isolating llaid protein.
5. A method of producing ,-_a human lympho¬ kine/cytokine-like protein comprising culturing said cells according to claim 4, wherein said protein is secreted from said cell.
6. A human lymphokine/cytokine-like protein capable of being produced by the method according to claim 4.
7. A human lymphokine/cytokine-like protein capable of being produced by the method according to claim 5.
8. An antibody induced by a peptide comprising amino acid sequence of a human lymphokine/cytokine-like protein, wherein said protein iε selected from the group consisting of the translation products from the near full length clone of Act-2 cDNA and clones of pAT 744 cDNA.
9. A bioassay compriεing the steps of: i) subjecting T cells to conditions that, produce mitogenic activation; ii) isolating mRNA from said T cells; and iii) determining the level of mitogenic activation of a gene for a lymphokine/cytokine-like pro¬ tein by hybridization to a DNA probe, wherein said probe comprises DNA sequences included in DNA segments accord- ing to claim 1.
10. A bioaεεay compriεing the εtepε of: i) obtaining fluidε in which T cellε haverbeen incubated; and ii) determining the level of a human lym¬ phokine/cytokine-like protein by reaction with an anti¬ body according to claim 8.
11. A DNA segment encoding a human lympho¬ kine/cytokine-like protein, wherein said protein iε the tranεlation product of a clone of pAT 744 cDNA.
12. A recombinant DNA molecule compriεing a DNA segment according to claim 11 and a vector.
13. A culture of cellε tranεformed with εaid recombinant DNA molecule according to claim 12.
14. A method of producing a human lympho¬ kine/cytokine-like protein comprising culturing said celiε according to claim 13 under conditionε εuch that said protein is produced and isolating said protein.
15. A method of producting a human lympho- kine/cytokine-like protein comprising culturing said cells according to claim 14, wherein said protein is secreted from said cell.
16. A human lymphokine/cytokine-like protein capable of being produced by the method according to claim 14.
17. A human lymphokine/cytokine-like protein capable of being produced by the method according to claim 15.
18. An antibody induced by a peptide comprising amino acid sequence of a human lymphokine/cytokine-like protein, wherein said protein is selected from the trans¬ lation products from a clone of pAT 744 cDNA.
19. A DNA segment encoding a human lympho¬ kine/cytokine-like protein, wherein said protein is selected from the group consisting of translation pro¬ ducts of any of the following cDNA clones: pAT 120; pAT 125; pAT 127; pAT 129; pAT 133; pAT 139; pAT 140S; pAT 140L; pAT 154; pAT 158; pAT 189; pAT 201; pAT 204; pAT
225; pAT 229; pAT 232S; pAT 232L; pAT 237; pAT 239; pAT
243; pAT 270; pAT 276; pAT 281; pAT 383; pAT 402; pAT
407; pAT 416; pAT 428; pAT 466; pAT 478; pAT 483; pAT 485; pAT 496; pAT 516; pAT 542; pAT 563; pAT 591; pAT 594; pAT 603; pAT 607; pAT 620; and pAT 730.
20. A recombinant DNA molecule comprising a DNA segment according to claim 19 and a vector.
21. A culture of cells transformed with said recombinant DNA molecule according to claim 20.
22. A method of producing a human lymphokine/cytokine-like protein comprising culturing said cells according to claim 21 under conditions such that said protein is produced and isolating said protein.
23. A method of producing a human lymphokine/cytokine-like protein comprising culturing said cellε according to claim 22, wherein said protein is εecreted from said cell.
24. A human lymphokine/cytokine-like protein capable of being produced by the method according to claim 22.
25. A human lymphokine/cytokine-like protein capable of being produced by the method according to claim 23.
26. An antibody induced by a peptide comprising amino acid sequence of a human lymphokine/cytokine-like protein, wherein said protein is selected from the group consisting of the translation products of any of the following CDNA clones: pAT 120; pAT 125; pAT 127 pAT 129; pAT 133; pAT 139; pAT 140S; pAT 140L; pAT 154 pAT 158; pAT 189; pAT 201; pAT 204; pAT 225; pAT 229 pAT 232S; pAT 232L; pAT 237; pAT 239; pAT 243; pAT 270 pAT 276; pAT 281; pAT 383; pAT 402; pAT 407; pAT 416 pAT 428; pAT 466; pAT 478; pAT 483; pAT 485; pAT 496 pAT 516; pAT 542; pAT 563; pAT 591; pAT 594; pAT 603 pAT 607; pAT 620; and pAT 730.
27. A bioasεay comprising the steps of; i) subjecting T cellε to conditionε that produce mitogenic activation; ii) iεolating mRNA from εaid T cellε; and iii) determining the level of mitogenic activation of a gene for a lymphokine/cytokine-like protein by hybridization to a DNA probe, wherein said probe comprises DNA sequenceε included in DNA εegmentε according to claim 19.
28. A bioasεay compriεing the εtepε of: i) obtaining fluidε in which T cellε have been incubated; and ii) determining the level of a human lymphokine/cytokine-like protein by reaction with an antibody according to claim 26.
PCT/US1989/005603 1988-12-16 1989-12-15 Novel lymphokine/cytokine genes Ceased WO1990007009A1 (en)

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EP0506574A1 (en) * 1991-03-29 1992-09-30 Sanofi Protein having cytokin type activity, recombinant DNA coding for this protein, transformed cells and microorganisms
FR2685919A1 (en) * 1992-01-08 1993-07-09 Sanofi Elf Protein with cytokine-type activity, recombinant DNA encoding this protein, transformed animal cells
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US6623942B2 (en) 1994-03-08 2003-09-23 Human Genome Sciences, Inc. Macrophage inflammatory protein-4 (MIP-4) polynucleotides
CN115028739A (en) * 2017-08-03 2022-09-09 泰加生物工艺学公司 Methods and compositions for treating melanoma

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Journal of Immunology; Volume 142, Number 5 issued March 1989; ZIPFEL et al. "Mitogenicativation of Human T Cells induces two closely related genes share structural similarities with a new family of secreted factors" pages 1582-1590 see sequences on page 1585. *
Procedings National Acadamy Sciences; Volume 85 issued December 1988; LIPES et al. "Identification, cloning, and charaterization of an immune activation gene" pages 9704-9708 see sequence page 9706. *
See also references of EP0449956A4 *

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EP0544743A4 (en) * 1990-08-13 1994-05-18 Us Health Lymphokine 154
WO1992005198A1 (en) * 1990-09-14 1992-04-02 Chiron Corporation EXPRESSION OF MACROPHAGE INDUCIBLE PROTEINS (MIPs) IN YEAST CELLS
US5814484A (en) * 1990-09-14 1998-09-29 Tekamp-Olson; Patricia Expression of macrophage inducible proteins (MIPS) in yeast cells
US5652123A (en) * 1991-03-29 1997-07-29 Elf Sanofi Protein having interleukin 13 activity, recombinant DNA coding for this protein, transformed cells and microorganisms
EP0506574A1 (en) * 1991-03-29 1992-09-30 Sanofi Protein having cytokin type activity, recombinant DNA coding for this protein, transformed cells and microorganisms
WO1992017586A1 (en) * 1991-03-29 1992-10-15 Elf Sanofi Protein having a cytokinin-type activity, recombinant dna coding for this protein, transformed cells and micro-organisms
FR2685919A1 (en) * 1992-01-08 1993-07-09 Sanofi Elf Protein with cytokine-type activity, recombinant DNA encoding this protein, transformed animal cells
US6495129B1 (en) 1994-03-08 2002-12-17 Human Genome Sciences, Inc. Methods of inhibiting hematopoietic stem cells using human myeloid progenitor inhibitory factor-1 (MPIF-1) (Ckbeta-8/MIP-3)
US6623942B2 (en) 1994-03-08 2003-09-23 Human Genome Sciences, Inc. Macrophage inflammatory protein-4 (MIP-4) polynucleotides
WO1996039521A1 (en) * 1995-06-06 1996-12-12 Human Genome Sciences, Inc. Human chemokine beta-13
AU713267B2 (en) * 1995-06-06 1999-11-25 Human Genome Sciences, Inc. Human chemokine beta-13
WO1997029125A1 (en) * 1996-02-09 1997-08-14 Schering Corporation Mammalian dendritic cell chemokine reagents
US6015883A (en) * 1996-04-17 2000-01-18 Incyte Pharmaceuticals, Inc. Rantes homolog from prostate
CN115028739A (en) * 2017-08-03 2022-09-09 泰加生物工艺学公司 Methods and compositions for treating melanoma

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