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WO1997008303A1 - Animaux transgeniques exprimant des transgenes de recepteurs de cellules t diabetogenes - Google Patents

Animaux transgeniques exprimant des transgenes de recepteurs de cellules t diabetogenes Download PDF

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WO1997008303A1
WO1997008303A1 PCT/CA1996/000581 CA9600581W WO9708303A1 WO 1997008303 A1 WO1997008303 A1 WO 1997008303A1 CA 9600581 W CA9600581 W CA 9600581W WO 9708303 A1 WO9708303 A1 WO 9708303A1
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transgenic
cells
mice
cell
tcr
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Pere Santamaria
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University Technologies International Inc
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0312—Animal model for Alzheimer's disease

Definitions

  • This invention relates generally to transgenic animal models useful in testing agents for inhibition of CD8+ and CD4+ T-cell destruction of pancreatic beta cells.
  • it relates to transgenic mice which have a T-cell receptor a and/or ⁇ chain (TCR ⁇ ) genes derived from beta cell cytotoxic CD8 + and/or CD4+ cells in their genome.
  • TCR ⁇ T-cell receptor a and/or ⁇ chain
  • the background of the present invention relates both to biological organisms which have been genetically transformed and to the mechanisms of diabetogenesis.
  • Transgenic animals are those animals which carry a gene which has been introduced into the germline of the animal or its ancestor.
  • IDDM Insulin-dependent diabetes mellitus
  • NOD non-obese diabetic mice
  • T-cells are abundant among insulins cells at IDDM onset, and IDDM recurs in diabetic patients treated with pancreas isografts or HLA- identical allografts.
  • NOD mice most insulitis cells are also T cells and IDDM does not develop in athymic- or iminunodeficient (scid -NOD mice.
  • IDDM can be prevented with anti-T cell reagents and can be transferred to immunodeficient or young NOD mice by T cells from diabetic mice. T cells are essential, but not sufficient; macrophages and/or dendritic cells also play an important role in IDDM.
  • CD4+ vs. CD8+ T cells as effectors of beta cell destruction in IDDM.
  • TCR T-cell receptor
  • splenic CD4+ T cells from pre-diabetic NOD mice can transfer insulitis, but not IDDM, to scid-NOO mice, and MHC cla$s-I/CD8+ T cell- deficient NOD mice do not develop IDDM.
  • cytotoxic CD8+ T cells CTL
  • H-2K d -restricted beta cell-specific CD8+ CTL are regularly present in islets of diabetic NOD mice, can transfer IDDM into irradiated NOD mice if co-injected widi insulitogenic CD4+ T cells, and can kill beta cells of diabetes- resistant mice in vivo.
  • CD4+ T cell clones these adoptive CTL transfer experiments have not determined if, and to what extent, beta cell-specific CD8+ T cells effect beta cell damage in spontaneous IDDM.
  • transgenic mice Previous investigations have generated strains of transgenic mice to investigate diabetogenesis or organ-specific autoimmunity in general. Expression of different transgenic antigens in islet beta cells of transgenic mice and/or TCR genes encoding receptors specific for these transgenic auto-antigens has resulted in several different outcomes (see references 31-43, below). Some of these transgenic models developed diabetes (see references 31 and 32, below) whereas others developed a non-immunologically-mediated form of hyperglycemia, possibly due to over-expression of the transgene (see references 33-34, below).
  • the T cells bearing TCRs capable of recognizing the transgenic neo-antigen were tolerized (see references 35-37, below), or ignored the transgenic autoantigen (see references 38-43, below).
  • autoreactive transgenic neo-antigen
  • the mechanisms that regulate the differentiation and function of thymocytes and peripheral T cells vary depending on the molecular nature of the antigen/TCR system studied (see references 44-46, below), as well as on the site of expression, amount, and timing of transgene expression during development (see references 44-50, below).
  • TCR-transgenic mice In transgenic mice, the TCR studied is also important, as TCR-transgenic mice generated with transgenes encoding TCR heterodimers with same specificity but different affinity show different susceptibility to autoimmunity and tolerance induction (see references 46. below). Since neither the antigens nor the autoreactive T cells that were studied in diese models are involved in spontaneous IDDM, and die genetic backgrounds in which these genes were expressed actually provide diabetes- resistance rather than susceptibility, it is difficult to determine which, if any, of die outcomes that were observed is IDDM-relevant. Thus, whether T cells recognizing disease-relevant non-transgenic beta cell autoantigens in NOD mice cause autoimmunity or undergo tolerance or ignorance in diabetes-resistant mice is unknown.
  • transgenic NOD mice expressing the TCR ⁇ and TCR/? rearrangements of a diabetogenic beta- cell specific CD4+ T cell clone (see reference 51, below).
  • expression of these TCR transgenes did not accelerate die onset of clinical diabetes.
  • diat tiiere is a need for an animal model to study d e independent patiiogenic effects of beta cell-specific CD4+ and CD8+ cells, and to develop/test tiierapies that interfere with the development and activation of tiiese cells in vivo.
  • mice carrying TCR a and ⁇ genes from a diabetogenic CD4+ T cell clone isolated from an NOD mouse Previous investigators have generated strains of mice carrying TCR a and ⁇ genes from a diabetogenic CD4+ T cell clone isolated from an NOD mouse.
  • the present inventor has unexpectedly found d at the use of TCR ⁇ and/or ⁇ transgenes from CD4+ or CD8+ T cell clones isolated from the pancreatic islets of acutely diabetic NOD mice produces a useful transgenic animal model for diabetes, where the onset of the spontaneous diabetes is drastically accelerated.
  • the present invention provides transgenic animals which have cytotoxic
  • TCR-transgenic models of the present invention are unique in that: a) the target autoantigens are non-transgenic; b) the TCR specificities are disease-relevant as they were derived from cells involved in spontaneous diabetogenesis in non-transgenic NOD mice; and c) the T cells expressing the TCR transgenes cause a very early-onset destruction of beta cells that occurs in diabetes-prone, but not diabetes-resistant genetic backgrounds.
  • the invention provides a transgenic non-human mammal useful as a model for diabetes having incorporated into its genome a T-cell receptor beta chain gene derived from beta cell cytotoxic CD8+ and/or CD4+ cells.
  • the invention provides a transgenic non-human mammal useful as a model for diabetes having incorporated into its genome T- cell receptor alpha and beta chain genes from beta cell cytotoxic CD8+ or CD4+ T cells.
  • the invention provides a method for making an animal model useful for the study of diabetes by incorporating a T cell receptor oc and/or ⁇ chain gene from a beta cell cytotoxic CD8+ or a beta cell cytotoxic CD4+ cell into the genome of a non-human mammal.
  • the invention provides a method for studying die effect of agents on diabetes comprising administering said agent to a transgenic non-human mammal having incorporated into its genome a T-cell receptor and/or ⁇ chain gene from a beta cell cytotoxic CD8+ or a beta cell cytotoxic CD4+ cell, and monitoring the development of diabetes in said transgenic non ⁇ human mammal.
  • Figure 1 Generation of transgenic constructs used in the present invention.
  • FIG. 2 CD4, CD8 and V08.1/8.2 profiles of thymocytes (A) and lymph node cells (B) from transgenic mice generated wim the TCR ⁇ chain gene derived from a CD8+ T cell clone (NY8.3) and their non-transgenic littermates.
  • Upper panels show CD4 vs. CD8 dot plots of cell suspensions stained with anti-CD8-PE. anti-V/38.1/8.2-FITC, and anti-CD4-biotin plus Streptavidin- PerCP.
  • the lower panels show the V ⁇ 8.1/8.2 fluorescence histograms of each T cell subset after electromc gating. Numbers indicate die average percentage of cells (upper panels) or the average number of V/38.1/8.2+ cells (lower panels) in each subset. DP, double positive cells; DN, double negative cells.
  • Figure 3 Proliferation of CD4+ T cell-depleted splenocytes from NY8.3 TCRjS-transgenic NOD mice in response to immobilized anti-CD3 and anti- V 38.1/8.2 mAbs.
  • FIG. 4 Beta cell-specific T cells in TCR/3-transgenic NOD mice.
  • Splenocytes from transgenic mice were either cultured with islets for 4 days, expanded widi rIL-2 for 10 days, and restimulated wim islets and rIL-2 (A, left), or depleted of CD4+ T cells, stimulated widi plate-bound anti-CD3 mAb for 4 days, and expanded with rIL-2 for 10 days (A, right). Both T cell populations were analyzed by flow cytometry, and tested for cytotoxic activity against MIN6N8a and L929-Kd cells using 51 Cr-release assays.
  • Figure 5 Limiting dilution analyses of beta cell specific precursor cytotoxic CD8+ T-cells (cytotoxic lymphocyte (CTL)) and islet-reactive CD4+ T-cells in NY8.3 TCR ⁇ transgenic and non-transgenic mice.
  • FIG. 7 Insulitis, islet-infiltrating T cells and beta cell destruction in pre- diabetic NY8.3 TCR ⁇ -transgenic and non-transgenic NOD mice.
  • E and F Progression of activation of islet-associated CD4-I- and CD8+ T cells in transgenic and non-transgenic female mice.
  • Islets from individual transgenic and non-transgenic mice were purified, mechanically disrupted into single cell suspensions and analyzed by tiiree-color flow cytometry using anti- CD4 FITC or anti-CD4-biotin, plus anti-CD8-PE and one of the following mAbs: anti-CD44-FITC; anti-CD25-FITC; anti-CD69-biotin; anti-CD62L- biotin. Average results from 4-5 transgenic and non-transgenic NOD mice are shown.
  • FIG. 8 Beta cell cytotoxic activity of islet-derived CD8+ T-cells from NY8.3 TCR ⁇ -transgenic NOD mice.
  • Purified islets from acutely diabetic TCR ?-transgenic NOD mice were cultured in the presence of rIL-2 for 5 d, depleted of CD4+ T cells by negative selection witii GK1.5-coated immunobeads, stimulated for 7 d wid irradiated NOD islets and rIL-2, analyzed by diree-color flow cytometry using anti-CD8-PE, anti-CD4-biotin and anti-VjS81J8.2-FITC (A), and tested for bet cell cytotoxic activity (B), serine esterase content (C) and target cell-induced cytotoxic granule exocytosis (D).
  • Figure 8A shows the FACS-transgenic profile of a representative islet- derived T cell line.
  • Figure 8B shows average results (x ⁇ S.E.) of experiments with 4 different islet-derived CD8+ T cell lines (Pl, P3, P6 and 2910).
  • Figure 8C shows the total serine esterase content of islet-derived CD8+
  • the values represent the difference between the absorbance at 412 nm of non-lysed vs. lysed T cells.
  • Figure 8D shows the islet cell-induced % release of serine esterase activity from anodier islet-derived CD8+ T cell line from transgenic NOD mice.
  • FIG 9 Endogenous TCR ⁇ repertoire of islet-derived (A) and peripheral (B) CD8+ T cells from diabetic NY8.3 TCR ⁇ -transgenic NOD mice.
  • Germline encoded Ja sequences are from Koop et al. (Genomics 13:1209 (1992)), or labelled as J ⁇ ...x.
  • N is the ratio between the copy number of each cDNA and die number of cDNAs sequenced.
  • N-te ⁇ ninal residues/nucleotides homologous to those of the TCR ⁇ chain of the TCRjS-transgene donor (CTL NY8.3) are underlined.
  • Va/Ja TCR ⁇ -variable/joining genes
  • N the number of copies/number of TCR cDNAs sequenced
  • Na sequences encoded by N- terminal addition of nucleotides
  • CDR3 complementary dete ⁇ nining region 3.
  • TCR ⁇ -CDR3 sequences that are homologous or identical to tiiose of the CTL clone 8.3 are underlined in the figure.
  • FIG. 10 CD4, CD8, and V/S8.1/8.2 profiles of thymocytes (A) and splenocytes (B) from RAG-2 + + and RAG-2 '- NY8.3 TCR ⁇ j3-transgenic mice. See Fig 1 for details. Note tiiat thymocyte development is skewed towards the CD8+ T cell subset in transgenic NOD mice expressing the TCR rearrangements of NY8.3.
  • Figure 11 The peripheral frequency of beta cell-specific CD8+ T cells in NY8.3 TCR ⁇ /3-transgenic vs. TCR ⁇ -transgenic NOD mice. See Fig 5 for details.
  • Figure 12 The proliferation of bulk splenic CD8+ T cells from single- and double- transgenic NY8.3 mice in response to stimulation with NOD islets, in the presence (right) and absence (left) of exogenous rIL-2.
  • FIG. 14 Phenotype of islet-derived T cells from acutely diabetic non- transgenic (A) and NY8.3 TCR ⁇ 3-transgenic (B) NOD mice. Pancreatic islets were cultured in rIL-2 and growing cells analyzed by FACS within 4 days. Note diat most T cells from transgenic mice are CD8+ and V38.1 +.
  • Figure 15 CD4, CD8 and V311 profiles of thymocytes (A) and splenocytes (B) from transgenic NOD mice generated with the TCR ⁇ and/or TCRjS chain genes derived from a CD4+ T cell clone (NY4.1). See Fig. 2 for details.
  • NOD-NON-TG non-transgenic NOD mice
  • NOD-TCR8-TG TCR 3 transgenic NOD mice
  • NOD-TCR ⁇ /3-TG TCR ⁇ jS transgenic NOD mice.
  • Figure 16 Proliferation of bulk splenic CD4+ T cells from non-transgenic (NON-NOD-TG) and double transgenic NY4.1 mice (NOD-TG) in response to stimulation widi NOD islets, in the absence of rIL-2.
  • FIG. 17 Cumulative incidence of IDDM in NY4.1 TCR-transgenic (TG) and non-transgenic (NON-TG) NOD mice. Data correspond to 13 female transgenic mice and 13 male transgenic mice. See Fig. 6 for further details.
  • FIG 18 Phenotype of islet-derived T cells from acutely diabetic non- transgenic (NOD-NON-TG) (A) and NY4.1 TCR ⁇ 3 transgenic NOD mice (B). See Fig. 14 for further details. Note diat most T cells derived from transgenic mice are CD4+ and V ⁇ ll + .
  • Figure 19 Cytolytic activity and antigen-induced serine esterase release by islet-derived CD8+ T cells from diabetic NY8.3 TCR ⁇ -transgenic NOD mice.
  • Figure 19A without PMA and ionomycin
  • Figure 19B following PMA and ionomycin activation (6h).
  • Figure 20 Cytolytic activity of islet-derived CD4+ T cells from NY4.1 TCR- transgenic NOD mice.
  • Left-hand graph cytolysis of islet cells from NOD mice in the presence (NOD/PMA/I) and absence (NOD i.e.) of PMA and ionomycin, and of islet cells from C57BL/6 mice in the presence (B6/PMA/I) and absence (B6 i.e.) of PMA and ionomycin.
  • Right-hand graph cytolysis of L1210-Fas + (L-Fas+/PMA I) and L1210-Fas- (L-Fas-/PMA/I) in die presence of PMA and ionomycin.
  • Beta cell-specific, H-2K d -restricted TCR-transgenic NOD mice (NY8.3). Islet-derived CD8+ CTL from diabetic NOD mice use highly homologous TCR ⁇ -antigen binding site (CDR3) sequences. Prior to the present invention, diis suggested recognition of an immunodominant, autoantigen/H-2K d complex on beta cells. The present inventor has shown that tiiese beta cell- specific CD8+ T cells are major effectors of beta cell damage in spontaneous IDDM by generating transgenic NOD mice with the TCR rearrangements of a representative CTL clone.
  • TCR 3 rearrangement of this clonotype in NOD mice causes a 10-fold increase in the precursor frequency of beta cell-specific CTL, and accelerates the onset of IDDM (by 5 wk in females and by 7 wk in males).
  • IDDM onset is preceded by an accelerated recruitment of CD8+ CTL using endogenously-derived TCR ⁇ chains identical to that of the clonotype donating die TCR3 transgene to islets.
  • Co-expression of the TCR ⁇ and TCR/3 rearrangements of this CTL clone in NOD mice accelerates the onset of IDDM even further, with most mice becoming diabetic between 17 and 44 days of life.
  • H-2K d -restricted beta cell-specific TCR-transgenic NOD mice do not indicate diat beta cell damage in IDDM is exclusively mediated by CD8+ T cells, or that beta cell-specific CD4+ T cells do not or cannot kill beta cells in IDDM.
  • diat splenic CD4+ T cells from diabetic NOD mice can transfer IDDM to scid-NOD mice indicates tiiat some splenic CD4+ T cell specificities are capable of destroying beta cells in vivo following adoptive T cell transfer; however, prior to the present invention is was not known whedier these cells are also capable of destroying beta cells in spontaneous IDDM.
  • the present inventor has generated transgenic NOD mice with die TCR rearrangements of an H-2I-A g7 -restricted beta cell-specific CD4+ T cell clone isolated from islets of a diabetic NOD mouse. IDDM onset in these mice is also accelerated, with most mice developing IDDM between 25 and 43 days of life. Comparison of these results widi tiiose obtained in me H- 2I-A g7 -restricted TCR ⁇ /3-transgenic NOD mice of Katz et al., which did not develop early-onset IDDM, suggest that not all beta cell-specific CD4+ T cell clonotypes that are diabetogenic in adoptive T cell-transfer experiments are capable of effecting beta cell damage in spontaneous IDDM.
  • the present invention provides transgenic animals which are uniquely suited for use as a mammalian model of autoimmune disorders, including autoimmune diabetes.
  • die transgenic animals of the present invention are transgenic mice with beta-cell specific TCR that are naturally found in T cells, preferably CD4+ or CD8+ T cells, which may be isolated from the pancreatic islets of diabetic NOD mice.
  • the transgenic animals of the present invention may have TCR which are transgenic in both the ⁇ and ⁇ chains of the receptor (TCR ⁇ 3), or in only the ⁇ chain (TCRjS ).
  • the present invention tiius provides TCR0 transgenic animals.
  • die TCR 3 transgenic animals are transgenic mice.
  • Expression of a TCR 3 transgene in the TCR ⁇ transgenic animals of the present invention accelerates the development of IDDM, in a manner which is correlated wid an increase in the peripheral frequency of precursor CTLs, and wid d e recruitment into islets of CTL using TCR ⁇ amino acid sequences which are at least homologous, and preferably identical to that of die CTL clone which donated d e TCRjS transgene.
  • the present invention also provides TCR ⁇ j ⁇ transgenic animals.
  • d e TCR ⁇ /3 transgenic animals are transgenic mice.
  • Such TCR ⁇ transgenic mice may be generated wid beta cell-specific TCR of a CD4+ T cell clone, or with beta cell-specific TCR of a CD8+ T cell clone.
  • d e TCR of a CD4+ T cell clone is a H- 2I-A g7 -restricted beta cell specific TCR (V311-JjS2.4/V ⁇ n.3-J ⁇ 33) of a CD4+ T cell clone isolated from the pancreatic islets of a diabetic NOD mouse.
  • d e TCR of a CD8+ T cell clone is a H-2K d restricted beta cell specific TCR (V08.1-J02.4/V ⁇ n.1-J ⁇ 34). Expression of the TCR a ⁇ transgene in the transgenic animals of die present invention further accelerates the development of IDDM.
  • the present invention also provides a method for producing TCR transgenic mice which develop early-onset diabetes.
  • tiiis method comprises selecting genes of beta cell specific CD8+ or CD4+ T-cell clones derived from the pancreatic islets of diabetic NOD mice.
  • d e clones are NY8.3 (H-2K d -restricted) and NY4.1 (H-2I-A g7 -restricted).
  • the TCR genes are amplified, preferably by polymerase chain reaction (PCR).
  • the PCR will use primers carrying L-V or J-C intron sequences and convenient restriction sites at their 5' ends.
  • PCR products are then subcloned into a plasmid vector; in a preferred embodiment, me PCR products are pBluescript- SK+.
  • the plasmid vector is then sequenced and inserted into TCR ⁇ and TCR ⁇ shuttle vectors. These shuttle vectors preferably carry endogenous TCRjS or TCR ⁇ enhancers, respectively, and may further comprise 5' regulatory sequences. At this stage, prokaryotic sequences are preferably removed.
  • die constructs are incorporated into the genome of an animal; in a preferred embodiment, die constructs are microinjected into fertilized eggs which are then implanted into the uteri of pseudopregnant females. In the case of mice, mouse (SJLxB ⁇ ) F2 eggs are preferred. Founder animals are then crossbred for several generations, to generate TCR-transgenic animals in the desired genetic background.
  • the present invention also provides a method for evaluating agents which may be effective in arresting the development of IDDM.
  • an agent to be evaluated is administered to d e transgenic TCR3 or TCR ⁇ transgenic mice of the present invention.
  • the agent to be evaluated may be administered by any route known to the skilled artisan; preferably the agent to be evaluated is administered orally, parenterally, or intranasally.
  • administration of test agent and control compound begins perinatally; in this embodiment d e percentage of mice which develop diabetes in the control versus the test group indicates die effectiveness of the test agent in preventing diabetogenesis.
  • test and control compounds are administered to die transgenic mice of the present invention after development of diabetes. If the test agent reversed or alleviated symptoms of diabetes, that would indicate that the agent was useful for treating diabetes.
  • the transgenic animals of the present invention provide a particularly useful test model due to die fact that they develop IDDM at an early age, which provides economic benefits for drug testing using this' model. They are also a useful test model to evaluate die effectiveness of pharmacological or genetic agents in arresting the development of beta cell destruction diat is effected by CD4+ or CD8+ T cells, respectively.
  • transgenic NOD mice with the TCR ⁇ and/or TCR 3 rearrangements of an H-2K d -restricted beta cell-specific CTL clone (NY8.3) or of an H-2I-A s7 -restricted beta cell-specific CD4+ T cell clone (NY4.1), which were derived from islets of diabetic NOD mice.
  • H-2K d -restricted beta cell-specific CTL clone NY8.3
  • H-2I-A s7 -restricted beta cell-specific CD4+ T cell clone NY4.1
  • T-cell clones NY8.3 (H-2K d -restricted) and NY4.1 (H-2I- A g7 ) were chosen because: a) d is clonotype was found to infiltrate pancreatic islets in 3 different NOD mice and uses a TCR ⁇ -CD3 sequence homologous to of most islet-derived CTL from non-transgenic NOD mice (see B, below), suggesting immunodominance; b) in addition to die functional TCR ⁇ (V ⁇ n.l-J ⁇ 34) and TCR/3 (V08.1-D 32.1-102.4) cDNAs, out- of-frame TCR ⁇ and TCR/3 transcripts from the second loci were also identified, tiius ruling out expression of additional TCR chains; and c) tiiis clonotype was diabetogenic in vivo (see C, below).
  • B. NY8.3 uses a TCRa-CDR3 sequence that is homologous to that of most islet- derived CTL.
  • TCR ⁇ and TCR 3 cDNA sequences generated by anchor-PCR from CTL lines and clones derived from pancreatic islets of 10 different NOD mice. These CTL were oligoclonal but did not show skewed V ⁇ , V3, J ⁇ or I ⁇ gene usage when compared to CD8+ splenocytes.
  • NY4.1 H-2I-A s7 -restricted
  • PCR polymerase chain reaction
  • TCR ⁇ and TCR/3 shuttle vectors carrying endogenous TCRjS or TCR ⁇ enhancers, respectively, and 5' regulatory sequences (3A9/? and PRE53 ⁇ /AN6 ⁇ , provided by Dr. M Davis (Stanford Univ.) and S. Hedrick (UCSD)) ( Figure 1).
  • TCR/3 and/or TCR ⁇ were microinjected togetiier (TCR ⁇ +jS) or alone (TCRjS) into fertilized mouse (SJLxB6) F2 eggs, which were implanted into the uteri of pseudopregnant females.
  • Example 2 CD4, CD8 and VS8.1/8.2 Profiles of Thymocytes and Lymph Node Cells from NY8.3 TCR3 Transgenic Mice Thymocytes (Figure 2A) and lymph node cells ( Figure 2B) from NY8.3
  • TCR J transgenic mice and non-transgenic littermates were stained wim anti-CD4-biotin, anti-CD8-phycoerythrin (PE) and anti- V ?8.1/8.2-fluorescein isothiocyanate (FITC) monoclonal antibodies followed by Streptavidin-PerCP. Samples were analyzed using fluorescence activated cell sorting (FACS) with a FACScan.
  • FACS fluorescence activated cell sorting
  • the upper panels of the Figures show dot plots of CD4 versus CD8.
  • the lower panels show the fluorescence histograms for V/38.1/8.2 of each thymocyte (Figure 2A) and lymph node T-cell (Figure 2B) subset. Numbers correspond to the average percentage of cells expressing the corresponding markers, and demonstrate that >85% of single positive tiiymocytes and peripheral T-cells from TCR/3 transgenic mice expressed the V ⁇ S ⁇ specificity, compared to ⁇ 18% of non-transgenic mice, even though the absolute and relative numbers of the different d ymocyte and peripheral T-cell subsets and
  • Example 3 Proliferation of CD4+ T-cell Depleted Splenocytes from NY8.3 TCR3-Transgenic NOD Mice in Response to
  • CD4-depleted splenocytes (7 x IO 4 ) from transgenic mice or non- transgenic littermates were incubated for 72 hours (h) in EUSA-grade 96 well plates which were precoated widi serial dilutions of die anti- V 38.1/8.2 monoclonal antibody (mAb) KJ16, which had been ammonium sulfate cut from ascites fluid, and affinity purified anti-CD3 mAb 2C11.
  • the cultures were pulsed witii 1 ⁇ Ci of 3 H-thymidine for 18 h before harvesting. 3 H-thymidine incorporation was assessed by scintillation counting.
  • T cells from transgenic mice proliferated much more efficiently than those from non-transgenic mice in response to anti-
  • transgenic TCR/3 chain could confer beta cell- specificity and diat such specificity was predominantly expressed on CD8+ T cells.
  • Splenocytes from transgenic mice were either: (a) depleted of CD4+ T-cells by two rounds of negative selection with anti-CD4 mAb GK1.5 and goat anti-rat IgG-coated magnetic beads and grown in wells coated widi anti-CD3 mAb for 4 days (d), followed by treatment with recombinant IL-2 (rIL-2) for 10 d; or
  • Proliferating cells were analyzed by FACS, using anti-CD8-PE and anti- V38.1/8.2-FITC, and tested for islet cytotoxicity in 51 Cr-release assays using NOD mouse-derived MIN6N8a insulinoma cells or H-2K d -transfected L929 fibroblasts (L929-Kd) as target cells.
  • die transgenic TCR/3 chain when expressed on CD8+ T cells, die transgenic TCR/3 chain can combine widi endogenous TCR ⁇ chains to form beta cell-specific TCRs on CD8+ T cells.
  • the data further show that tiiese cells can be induced to proliferate and differentiate into beta cell-cytotoxic effectors in vitro by stimulation with NOD islets.
  • CD8 + T-cell depleted splenocytes from transgenic and non-transgenic mice using anti-CD8 mAb 53-6.7 and goat anti- rat IgG-coated magnetic beads.
  • TCR/3-transgenic NOD mice have a selective increase in the peripheral frequency of beta cell-specific precursor CTL, but not an increase in the absolute or relative number of peripheral CD8 + T cells at the expense of CD4+ T cells, or in the frequency of islet- reactive CD4+ T cells.
  • IDDM transgenic and non-transgenic mice for development of IDDM. IDDM was monitored by measuring urine glucose twice per week. Mice were considered diabetic after two consecutive 3+ glucose readings.
  • the results in Figure 6 demonstrate the onset of diabetes was accelerated in transgenic mice as compared to non-transgenic littermates or NOD/Lt mice.
  • the average age for onset of diabetes in transgenic mice was significantly lower than in non-transgenic mice (12.6 +. 1.7 weeks of age vs. 17.1 +_ 3.9 weeks of age for females (p ⁇ 0.0001) and 15.6 ⁇ 4.7 weeks of age vs. 23.1 ⁇ 4.0 weeks of age for males (p ⁇ 0.0002)).
  • the cumulative incidence of IDDM by 32 wk was similar in transgenic and non-transgenic NOD mice (85.7% vs. 87.3% for females and 50% vs. 51.3% for males).
  • IDDM in non-transgenic female littermates and NOD/Lt females only affected 27.8-31.7% of mice by 15 weeks of age ( ⁇ 0.0002).
  • IDDM also started earlier in transgenics than in non- transgenics (11 weeks of age vs. 15 weeks of age); by 17 weeks of age, it already affected 33.3% of transgenic, but only 3-5% of non-transgenic and NOD/Lt mice, respectively (p ⁇ 0.003).
  • Example 7 Insulitis, Islet-infiltration by CD4+ and CD8+ T cells and
  • FIG. 7A Faster progression of insulitis in transgenic mice was attributable to faster recruitment of CD8+ T cells (95.8 ⁇ 42.3 vs. 28.7 ⁇ 8.2 cells/islet, p ⁇ 0.01), but not of CD4+ T cells (244 ⁇ 64.7 vs. 197 ⁇ 137 cells/islet) to inflamed islets (Fig. 7B, C), and resulted in an earlier onset and faster progression of beta cell depletion, as determined by comparing the pancreatic insulin content of transgenic vs.
  • non-transgenic mice p ⁇ 0.01, Fig. 7D.
  • Faster progression of beta cell depletion in transgenic mice was not a result of accelerated activation of islet-infiltration CD4+ T cells by local CD8+ T cells, or vice versa, since no differences were noted between d e percentage of islet- associated CD4+ or CD8+ T cells expressing CD25, CD44, CD49d, CD62L and CD69 in transgenic vs. non-transgenic mice (Fig. 7E, F).
  • Example 8 Cytotoxicity of Islet-Derived CD8+ T-Cells from NY8.3 TCR/5-Transgenic NOD Mice
  • TCR ⁇ -transgenic NOD mice had differentiated into ⁇ - TL
  • cytotoxic activity of CD8+ T cell lines isolated from the pancreatic islets of 5 different acutely diabetic TCR J-transgenic NOD mice As shown in Fig. 8, islet-derived CD8+ T cells from diabetic mice were >98% V/38+ and displayed specific cytotoxic activity against beta cells, but not against irrelevant targets (L929-Kd in Fig. 8).
  • Example 9 Endogenous T-Cell Receptor Alpha Chain Rearrangements of Islet-Derived CTL Lines from NY8.3 TCRjS-Transgenic NOD Mice
  • transgenic mice of the present invention we have found that there is a selective increase in the precursor frequency of beta cell-specific CTL in transgenic NOD mice. This led to accelerated onset of IDDM, widiout increasing its incidence or accelerating the onset or progression of insulitis, owing to accumulation of CTL within islets using TCR ⁇ chains identical to tiiat of the CTL clone which donated die TCR/3 transgene.
  • transgenic NOD mice expressing the TCR ⁇ and TCRjS rearrangements of NY8.3 thymocyte development is skewed towards the CD8+ T cell subset (Fig. 10, left). Both TCR transgenes are expressed appropriately because most single-positive tiiymocytes and peripheral T cells are Vj ⁇ 8+, and RAG-2 " ' " TCR ⁇ /3-transgenic NOD mice (unable to rearrange endogenous TCR genes) contain abundant CD8+, but no CD4+ T cells (Fig. 10, right).
  • RAG + TCR ⁇ /3-transgenic NOD mice most splenic CD8+ T cells are beta cell-specific; they secrete as much as 1,000 pg/ml of TNF- ⁇ in
  • CD8-r T cells (1X10 -4 ) were cultured with NOD-derived insulinoma cells (MIN6N8a) or H-2k d - transfected L929 cells (L929-Kd) (1 x IO 4 ) for 24h.
  • the concentration of TNF ⁇ in the supernatants was determined with a bioassay using WEHI clone 14 cells as indicators.
  • TCR ⁇ / 3-transgenic NOD mice develop IDDM much earlier than TCR/5- transgenic and non-transgenic NOD mice; the incidence of diabetes in N3-N4 mice is 79% for females (11/14) and 33% for males (3/9), with an average age at onset of IDDM of 39 ⁇ 4.2 days (Fig. 13).
  • the peripheral beta cell-specific CD8+ T cells of diabetic transgenic mice do not show signs of activation, as determined by FACS analysis with mAbs specific for activation and memory markers (CD25, CD44, CD62L and CD69), suggesting that they do not undergo activation in the periphery (data not shown).
  • Example 11 H-2I-A ⁇ 7 -Restricted TCR-Transgenic NOD Mice (NY4.1)
  • NNK4.1 transgenic NOD mice expressing the TCR rearrangements of the beta cell-specific CD4+ T cell clone NY4.1, most single-positive thymocytes and peripheral T cells, which proliferate in response to NOD islet cell antigen in vitro in the absence of exogenously added rIL-2 (Fig. 16), express the transgene-encoded V311 element (Fig. 15 A and B).
  • H-2I-A g7 -restricted beta cell-specific TCR in transgenic NOD mice also accelerates the onset of IDDM without increasing the overall incidence of the disease.
  • the incidence of diabetes in N3-N5 transgenic mice is 77% in females (10/13) and 54% in males (7/13).
  • the age at onset of diabetes is 38 ⁇ 9 days in females and 47 + 20 days in males (Fig 17).
  • the CD4+ T cells of diabetic transgenic mice do not appear to undergo activation in the periphery (data not shown).
  • While the islet mononuclear infiltrates of diabetic transgenic mice contain abundant CD4+ and CD8+ T cells, most T cells recovered from islets cultured in ⁇ EL-2 are CD4+ (VS11 +) (Fig. 18), as compared to 50% in non-transgenic NOD mice or ⁇ 4% in H-2K d -restricted TCR ⁇ -transgenic NOD mice (Fig. 14 and 18).
  • the islet-derived CD4+ T cells of these mice are diabetogenic because tiiey can transfer diabetes into scid- NOD mice (1/1 T cell-transferred mouse has become diabetic with all islet- infiltrating T cells being CD4+; data not shown).
  • Example 12 Cytolytic Activity of Transgenic CD8+ and CD4+ T Cells from NY8.3 and NY4.1 TCR ⁇ /3-Transgenic NOD Mice, Respectively
  • RAG-2-def ⁇ cient H-2I-A s7 -restricted TCR-transgenic NOD mice which only have CD4+ V311+ T cells in the periphery develop diabetes with an incidence and an age at onset of diabetes similar to those observed in RAG-2-positive mice (incidence of 60% and age at onset of 40 ⁇ 18 days for females and incidence of 40% and age at onset of 39 ⁇ 15 days for males).
  • islet-infiltrating T cells of tiiese mice are CD4+.
  • RAG-2-deficient NOD mice expressing the H-2K d -restricted TCR have also been generated. All peripheral T cells of these mice are CD8 +V ⁇ $.1 + , as expected (Fig. 10, right).
  • One female smdied has developed diabetes, albeit significantly later than in RAG-2-positive mice (15 weeks), with virtually all islet-infiltrating T cells being CD8+.
  • Another cohort of mice are now being followed for diabetes development. It appears that these mice do not develop diabetes at an early age. Altogether, the data suggests that differentiation can occur in the absence of endogenous T or B lymphocytes but progresses much faster in their presence.

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Abstract

L'invention concerne en règle générale les modèles d'animaux transgéniques que l'on utilise pour tester l'aptitude de certains agents à inhiber la destruction par les cellules T CD4 + et/ou CD8 des cellules bêta des îlots de Langerhans du pancréas. Elle concerne en particulier des souris transgéniques ayant dans leur génomes des gènes à chaîne α et/ou β de récepteurs de cellules T, dérivés de cellules T CD4 + ou CD8 + cytotoxiques pour les cellules bêta. On décrit par ailleurs des procédés de production d'animaux transgéniques à récepteurs des cellules T développant un diabète de type juvénile, ainsi que des procédés relatifs à l'évaluation d'agents pour la prévention et le traitement du diabète sucré diabetes mellitus insulino-dépendant.
PCT/CA1996/000581 1995-08-30 1996-08-29 Animaux transgeniques exprimant des transgenes de recepteurs de cellules t diabetogenes Ceased WO1997008303A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046734A1 (fr) * 1997-04-17 1998-10-22 Japan Science And Technology Corporation Souris transgenique exprimant la cd4 et la fusine (cxcr4) humaines
WO1999016867A1 (fr) * 1997-10-01 1999-04-08 Isis Innovation Limited Modele transgenique comprenant des chaines tcr alpha/beta

Citations (6)

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Publication number Priority date Publication date Assignee Title
WO1991019816A1 (fr) * 1990-06-20 1991-12-26 The Board Of Trustees Of The Leland Stanford Junior University Identification de sous-population de cellules et utilisation de rcp modifiee pour amplifier des intermediaires d'expression
WO1994023760A1 (fr) * 1993-04-14 1994-10-27 The United States Of America As Represented By The Secretary Of The Navy Modele d'animal transgenique pour maladies autoimmunes
WO1995021623A1 (fr) * 1994-02-14 1995-08-17 University Of Vermont Peptides et applications contre le diabete
WO1995032285A2 (fr) * 1994-05-19 1995-11-30 Institut National De La Sante Et De La Recherche Medicale Souris arthritiques transgeniques
EP0712930A2 (fr) * 1994-11-21 1996-05-22 Jeongsun Seo Animal transgénique non humain modèle pour le diabète
WO1996021028A2 (fr) * 1995-01-03 1996-07-11 Procept, Inc. Recepteurs de lymphocytes t heterodimeres solubles et leurs anticorps

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019816A1 (fr) * 1990-06-20 1991-12-26 The Board Of Trustees Of The Leland Stanford Junior University Identification de sous-population de cellules et utilisation de rcp modifiee pour amplifier des intermediaires d'expression
WO1994023760A1 (fr) * 1993-04-14 1994-10-27 The United States Of America As Represented By The Secretary Of The Navy Modele d'animal transgenique pour maladies autoimmunes
WO1995021623A1 (fr) * 1994-02-14 1995-08-17 University Of Vermont Peptides et applications contre le diabete
WO1995032285A2 (fr) * 1994-05-19 1995-11-30 Institut National De La Sante Et De La Recherche Medicale Souris arthritiques transgeniques
EP0712930A2 (fr) * 1994-11-21 1996-05-22 Jeongsun Seo Animal transgénique non humain modèle pour le diabète
WO1996021028A2 (fr) * 1995-01-03 1996-07-11 Procept, Inc. Recepteurs de lymphocytes t heterodimeres solubles et leurs anticorps

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Title
SANTAMARIA, P. ET AL.: "Beta-cell-cytotoxic CD8+ T cells from nonobese diabetic mice use highly homologous T cell receptor alpha-chain CDR3 sequences", JOURNAL OF IMMUNOLOGY, vol. 154, no. 5, 1 March 1995 (1995-03-01), BALTIMORE US, pages 2494 - 2503, XP000604922 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046734A1 (fr) * 1997-04-17 1998-10-22 Japan Science And Technology Corporation Souris transgenique exprimant la cd4 et la fusine (cxcr4) humaines
US6255555B1 (en) 1997-04-17 2001-07-03 Japan Science And Technology Corporation Transgenic mouse expressing human fusin and human CD4
WO1999016867A1 (fr) * 1997-10-01 1999-04-08 Isis Innovation Limited Modele transgenique comprenant des chaines tcr alpha/beta

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