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WO2018201282A1 - Pd-1h en tant que cible dans la modulation de la taille d'un pool de lymphocytes t régulateurs inductibles - Google Patents

Pd-1h en tant que cible dans la modulation de la taille d'un pool de lymphocytes t régulateurs inductibles Download PDF

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WO2018201282A1
WO2018201282A1 PCT/CN2017/082735 CN2017082735W WO2018201282A1 WO 2018201282 A1 WO2018201282 A1 WO 2018201282A1 CN 2017082735 W CN2017082735 W CN 2017082735W WO 2018201282 A1 WO2018201282 A1 WO 2018201282A1
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cells
itreg
mice
foxp3
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Lieping Chen
Qi Wang
Jianwei He
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Sun Yat Sen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • the present invention is related to a method for modulating pool size of inducible regulatory T cells in a subject.
  • the present invention further relates to a method for treating T cell immunity mediated diseases in a subject.
  • the invention also concerns use of a PD-1H agonist or antagonist in the preparation of a pharmaceutical compositions for modulating a pool size of inducible regulatory T cells in a subject.
  • the invention also concerns use of a PD-1H agonist or antagonist in the preparation of a pharmaceutical compositions for treating T cell immunity mediated diseases in a subject.
  • Treg Regulatory T cell
  • Treg is a subset of CD4 + T cells with broad functions from maintenance of self-tolerance to regulation of magnitude of immune responses.
  • Treg is not terminally differentiated and can be converted to other CD4 + T cell subsets including Th1 and Th17 during inflammation.
  • the transcription factor Foxp3 plays an essential role in the establishment of a functional and committed regulatory T cell lineage.
  • Foxp3 + Treg cells can be divided into the thymus-derived natural Treg cells (nTreg) and the inducible Treg cells (iTreg) by TGF- ⁇ , which regulate the differentiation of iTreg cells and stabilization of thymus-derived nTreg.
  • iTreg cells In the periphery, the differentiation of iTreg cells is largely driven by the microenvironment. For example, inflammatory cytokines IFN- ⁇ and IL-4 inhibit TGF- ⁇ -induced iTreg cells, while IL-6 directs Th17 cell differentiation in the presence of TGF- ⁇ .
  • the plasticity of Treg cells may thus determine the direction of an ongoing immune response and control inflammation as shown in several mouse models including models of colitis, acute graft versus host diseases (GVHD) , and asthma.
  • GVHD acute graft versus host diseases
  • PD-1H (also called Gi24, Dies1, B7-H5, VISTA and DD1) is a cell surface immunoglobulin superfamily molecule with immune modulatory functions in addition to its myriad of roles regulating the differentiation of osteoblast, adipocyte, and embryonic stem cells and cell apoptosis.
  • PD-1H is constitutively expressed on hematopoietic cells, such as T cells, NK cells, monocytes, NK cells and DCs, but not on B cells.
  • PD-1H deficiency Unlike CTLA-4 knockout (KO) mice that rapidly develop lymphoproliferative phenotypes and fatal systemic autoimmune diseases, PD-1H deficiency has a much more mild phenotype: young PD-1H KO mice have normal numbers of T cells, NK cells, B cells, macrophages, and monocytes while older mice experience spontaneous T cell activation, and increased levels of memory cells and larger spleen size were observed when mice aged. Furthermore, PD-1H deficient mice were more susceptible to acute inflammation and immune response to antigens as shown in accelerated Con A-induced acute hepatitis and GVHD.
  • PD-1H has been shown to function on professional antigen-presenting cells (APCs) and T cells as either a ligand or a receptor, respectively, in several in vitro and in vivo studies. Consistent with these findings, agonistic mAb to PD-1H have proven to be immune inhibitors for various types of immune responses to antigens, whereas antagonistic mAb were shown to be immune stimulators. Although the counter-receptor (s) of PD-1H have yet to be identified, a recent study indicated that PD-1H/DD1 could mediate its effect via a hemophilic interaction.
  • PD-1H is constitutively expressed on Treg and several subsequent studies implicate its role in the regulation of Treg functions.
  • PD-1HIg fusion protein promoted the induction of Foxp3 + iTreg in the presence of TGF- ⁇ in both mice and human CD4 + T cells in vitro.
  • Infusion of a PD-1H mAb in the B16-OVA tumor model reduced the differentiation of tumor antigen-specific iTreg cells. This result was interpreted as a blockade of the PD-1H interaction with its putative counter-receptor by this mAb.
  • PD-1H agonist mAb MH5A was shown to promote TGF- ⁇ induced Treg cells in vitro, and infusion of MH5A suppressed progression of GVHD in mouse models, accompanied by expansion of iTreg. While these data suggest a possible role of PD-1H in Treg induction and function, it has yet to be elucidated whether PD-1H has a direct effect on Treg cells. More importantly, the mechanisms underlying the modulatory effect of Treg cells by PD-1H are unknown.
  • PD-1H deficient iTreg could also rapidly convert to CD4 + T helper 1 or T helper 17 cells in inflammatory environment. These results indicate that PD-1H is required for maintenance of iTreg pool size by promoting its differentiation and preventing its conversion to other CD4 + T cell subsets. These findings may have important implications for manipulating Tregs to control inflammation.
  • a method for modulating a pool size of inducible regulatory T cells in a subject comprises administering to the subject in need thereof a therapeutically effective amount of a PD-1H agonist or a PD-1H antagonist.
  • a PD-1H agonist or a PD-1H antagonist in the preparation of a pharmaceutical composition for modulating a pool size of inducible regulatory T cells in a subject.
  • a method for treating T cell immunity mediated diseases in a subject comprises administering to the subject in need thereof a therapeutically effective amount of a PD-1H agonist or a PD-1H antagonist.
  • a PD-1H agonist or a PD-1H antagonist in the preparation of a pharmaceutical composition for treating T cell immunity mediated diseases in a subject.
  • the administration of PD-1H agonist causes increased level of inducible regulatory T cells in the subject. In some embodiments of the invention, the administration of PD-1H antagonist causes decreased level of inducible regulatory T cells in the subject. In some embodiments of the invention, the administration is carried out intravenously.
  • the PD-1H agonist is a monoclonal antibody against PD-1H.
  • the PD-1H antagonist is an antisense oligomer selected from dsRNA, siRNA and shRNA directed to PD-1H encoding polynucleotides. In some embodiments of the invention, the subject is a human being.
  • the T cell immunity mediated diseases include inflammation, autoimmune diseases and cancer.
  • the present invention demonstrates that PD-1H inhibits the conversion of iTreg cells into Th1 and Th17 cells in an inflammatory environment, at least partially due to its role in the maintenance of Foxp3 expression and an iTreg phenotype. These findings have important implications in the regulation of Treg growth and function. Meanwhile, PD-1H inhibits activation of T cells to limit initiation of T cell-mediated immune responses, as previously shown, it promotes growth and conversion of iTreg during immune responses. In addition to regulating early stage T cell activation, PD-1H appears to participate in the regulation of T cell tolerance by regulating Treg pool size. Thus the PD-1H pathway may represent a promising target to control and manipulate T cell-mediated immunity in inflammation, autoimmune disease and cancer.
  • FIG. 1 Effect of PD-1H in de novo generation of Foxp3 + iTreg cells.
  • A T cells purified from WT OT-II or PD-1H KO OT-II mice were first labeled with 5 ⁇ M CFSE and subsequently transferred i.v. to B6 mice at 2x10 6 /mouse. Mice were fed with 1.5%OVA in the drinking water 24 hours later for 5 days. Foxp3 frequency on the gated CD4 + CFSE + V ⁇ 5.1/5.2 TCR + was analyzed by flow cytometry in the representative mice.
  • FIG. 1 Effect of PD-1H on the conversion and function of iTreg.
  • A PD-1H deficiency in the natural development of iTreg in gut-associated lymphoid organs.
  • the percentages of CD25 + Foxp3 + Treg cells in the mesenteric lymph nodes (mLN) , payer’s patch (PP) and lamina intestinal (LP) were determined by cell surface CD25 and intracellular Foxp3 expression with specific antibodies in flow cytometry.
  • C In vitro induction of iTreg. CD4 + CD25 - CD62Lhi T cells from the WT and PD-1H KO mice were stimulated with anti-CD3/CD28 in the presence or absence of 5 ng/ml TGF- ⁇ for 3-5 days. The frequency of CD25 + Foxp3 + cells was determined by intracellular staining.
  • CD8 + T cells or CD4 + T cells were purified from B6 mice, labeled with CFSE and co-cultured with sorted GFP + iTreg cells in the presence of anti-CD3 at the indicated Treg/Teff cells ratio.
  • the decrease of CSFE upon inclusion of iTreg cells was determined by comparison with the wells without the addition of iTreg cells. Data showed are representative of at least 3 independent experiments. Teff only: T cells without anti-CD3 stimulation; Control: Teff cells with anti-CD3 without the inclusion of iTreg cells.
  • FIG. 3 Effect of the cytokine milieu on the PD-1H-mediated defects on the conversion of iTreg cells.
  • A Cytokine profile in the induction of iTreg cells in the absence of PD-1H. T cells were induced to iTreg in vitro as described above and the culture supernatants were collected at day 4 to determine cytokine levels by mouse Th1/TH2/Th17 CBA kits.
  • B Neutralizing mAb to IFN- ⁇ and IL4 were added to the culture at the beginning of the culture to induce iTreg cells from WT or PD-1H KO T cells in vitro. CD25 + Foxp3 + cells were assessed 3-5 days after the culture. The presented results were from a pair of mice.
  • C The histogram graphic presentation of the data from (B) are from a group of 5 mice. Data shown are representative of at least 3 independent experiments.
  • FIG. 4 Effect of PD-1H on the stability of iTreg cells in the EAE model.
  • CD45.2 + Foxp3 (GFP + ) iTreg cells from WT or KO were obtained by cell sorting after induction in vitro.
  • the control mice were inoculated with PBS. EAE disease progression and severity was monitored as clinical score (see Methods) .
  • the data shown represent 1 of 3 experiments with similar results and disease phenotypes.
  • B, C The spleen and draining LN (dLN) cells at Day 13 of EAE induction were gated on CD45.2 + CD4 + and analyzed for Foxp3 (GFP + ) iTreg cells.
  • D Absolute numbers of CD45.2 + Foxp3 + in the spleen and dLN from the EAE mice were counted.
  • E The spleen and dLN cells from EAE mice at day 13 were re-stimulated ex vivo using PMA/Ionomycin/BFA for 4 hours.
  • FIG. 5 PD-1H promotes the commitment of iTreg cells.
  • A The transferred iTreg cells from each group (gate on CD45.2 + CD4 + ) in the EAE model were analyzed on Day 13. The spleen and dLN cells were intracellularly stained with pSTAT3 or pSTAT5 with specific mAb.
  • B Same as A but phosphorylation of STAT3 and STAT5 on transferred iTreg cells is displayed as plotted value.
  • C The DNA methylation status of the CNS2 region was determined by bisulfite sequencing. Each line represents one clone (one DNA strand) ; open cycle, un-methylated cites; filled circles, methylated cites.
  • FIG. 6 PD-1H affects de novo differentiation of iTreg cells.
  • A WT and KO OT-II T cells (CD25 - T cells) were transferred into host mice separately. The transferred OT-II T cells were labeled with CFSE before transferred. This figure showed here is the dilution of CFSE and Foxp3 induction.
  • B WT (CD45.1/CD45.2) OT-II T cells and KO (CD45.2) OT-II naive T cells were mixed at ratio 1: 1 and co-transferred into host mice (CD45.1) . After orally feeding the host mice with 1.5%OVA, the mLN and PP were analyzed and the frequency of Foxp3 were determined by intracellular staining.
  • C Absolut number of Foxp3 + T cells in the indicate organs were counted.
  • FIG. 7 PD-1H regulates differentiation of iTreg cells in a lymphopenic environment.
  • B Absolute numbers of CD25 + Foxp3 + T cells in the indicated organs from the Rag1 KO mice were counted.
  • C Cytokine production of transferred CD4 + T cells were analyzed. The cells from indicated organs were in vitro activated for 4 hours in the presence of PMA/Inomycin/BFA and then analyzed by intracellular staining. Data showed were representative of 2 independent experiments.
  • FIG. 8 PD-1H plays a redundant role on the generation and suppressive function of nTreg cells.
  • FIG. 9 determines pool size of Treg cells in bone marrow chimeric mice.
  • A Total number of 10 million mixed bone marrow cells from CD45.1 WT mice and CD45.2 mice were transfer into sub-lethal irradiated mouse (CD45.1/CD45.2) . Mice were analyzed 10 weeks after reconstruction. Gating strategy of WT and KO T cells was showed.
  • B Foxp3 frequency in the indicated organs was determined by intracellular staining.
  • C Foxp3 frequency in the spleen were summarized, and absolute number of Foxp3 + cell and Foxp3 - cells were showed. Data shown are one of two independent experiments.
  • FIG. 10 PD-1H agonist mAb slightly promotes the differentiation of iTreg cells.
  • T cells were sorted from the WT Foxp3 (GFP) mice and subsequently stimulated with pre-coated anti-CD3 with either control mouse IgG or mam82 in the presence of TGF- ⁇ for 4 days.
  • CD25 + Foxp3 (GFP + ) iTreg cells were analyzed by flow cytometry.
  • B Induction of iTreg cells at different time points were showed. The results shown are from 3 individual experiments. Data shown are representative of at least 3 independent experiments.
  • FIG. 11 Analysis of CD4 + Th1 and Th17 cells in the recipients upon transferring with WT or PD-1H KO iTreg cells in the EAE model.
  • A T cells from WT or PD-1H KO Foxp3 (GFP) mice were differentiated into iTreg cells in vitro as described above. IFN- ⁇ and IL-4 neutralizing mAb were added to the cultures to promote the generation of iTreg. Foxp3 (GFP + ) cells were sorted and the purity was assessed by FACS.
  • B, C The recipient CD45.1 + CD4 + T cells in the EAE model upon the transfer of iTreg cells were re-stimulated ex vivo by PMA/Ionomycin/BFA.
  • the IFN- ⁇ + or IL-17 + cells of recipient CD4 + T cells were determined by intracellular staining gated on CD45.2-CD4 + T cells.
  • the data from a representative pair of mice were shown in (B) and the plot graphic presentation of data in shown in a group of 4 or 5 mice (C) . Data shown were representative of at least 3 independent experiments.
  • FIG. 12 PD-1H deficient iTreg cells fails to retain their suppressive function and Foxp3 expression.
  • the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable.
  • “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal, ” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
  • the subject herein is preferably a human.
  • phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g., for detection, for a diagnostic procedure and/or for treatment.
  • the term "agonist” refers to any agent that increases the level and/or activity of PD-1H.
  • the term “agonist” refers to an agent which increases the expression and/or activity of the PD-1H by at least 10%or more, e.g. by 10%or more, 50%or more, 100%or more, 200%or more, 500%or more, or 1000%or more.
  • Non-limiting examples of agonists of PD-1H can include PD-1H polypeptides or agonist fragments thereof and nucleic acids encoding a PD-1H polypeptide.
  • an antagonist refers to any agent that decreases the level and/or activity of PD-1H.
  • An antagonist is a compound that competes with a specific protein, a ligand for example, on binding to another protein, a receptor for example. Such binding usually, induces a specific biological response or action that is blocked by the competing antagonist.
  • the present invention employs, among others, antisense oligomer and similar species for use in modulating the function or effect of nucleic acid molecules encoding PD-1H. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding PD-1H.
  • target nucleic acid and “nucleic acid molecule encoding PD-1H” have been used for convenience to encompass DNA encoding PD-1H, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA.
  • the hybridization of an oligomer of this invention with its target nucleic acid is generally referred to as "antisense” .
  • antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
  • the antisense oligomer is selected from a DNA oligonucleotide, an RNA oligonucleotide (e.g., micro RNAs) , and a chimeric oligonucleotide.
  • the antisense oligomer is selected from dsRNA, siRNA, and shRNA.
  • An aspect of the disclosure provides a method for modulating a pool size of inducible regulatory T cells in a subject.
  • the method comprises administering to the subject in need thereof a therapeutically effective amount of a PD-1H agonist or a PD-1H antagonist, or a pharmaceutical composition comprising the PD-1H agonist or the PD-1H antagonist.
  • the disclosure provides the PD-1H agonist or the PD-1H antagonist as described above for use in a method for modulating a pool size of inducible regulatory T cells in a subject.
  • the PD-1H agonist, the PD-1H antagonist or the pharmaceutical composition is administered parenterally, e.g. intravenously, intramuscularly, percutaneously or intracutaneously.
  • the administration of a PD-1H agonist causes an increased level of inducible regulatory T cells in the subject, for example expansion of the iTreg cells.
  • the administration of a PD-1H antagonist causes a decreased level of inducible regulatory T cells in the subject.
  • the decreased level of iTreg cells is accompanied by enhanced production of Th1 and/or Th17 cells.
  • a method for treating or alleviating diseases involved with T cell immunity comprises administering to the subject in need thereof a therapeutically effective amount of a PD-1H agonist or a PD-1H antagonist, or a pharmaceutical composition comprising the PD-1H agonist or the PD-1H antagonist.
  • a PD-1H agonist or a PD-1H antagonist or a pharmaceutical composition comprising the PD-1H agonist or the PD-1H antagonist.
  • An aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of PD-1H agonist or PD-1H antagonist, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is useful for modulating a pool size of inducible regulatory T cells in a subject.
  • the PD-1H agonist or PD-1H antagonist may be prepared in a suitable pharmaceutically acceptable carrier or excipient.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • aqueous composition that contains a protein as an active ingredient is well understood in the art.
  • injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • mice Eight-week-old C57BL/6 (B6) mice were purchased from Sun Yat-sen University Animal Supply Center. PD-1H-KO mice in B6 background were described previously. The transgenic strain CD45.1, OT-II, Foxp3 (GFP) , Rag1 KO were all purchased from The Jackson Laboratory. The littermate mice of PD-1H KO and wild type (control mice) were generated from PD-1H heterozygotes and maintained in the same conditions. Foxp3 (GFP) mice and OT-II mice were backcrossed to the PD-1H KO mice respectively to generate PD-1H deficient Foxp3 (GFP) reporter mice and PD-1H deficient OT-II mice.
  • B6 The transgenic strain CD45.1, OT-II, Foxp3 (GFP) , Rag1 KO were all purchased from The Jackson Laboratory. The littermate mice of PD-1H KO and wild type (control mice) were generated from PD-1H heterozygotes and maintained in the same conditions. Foxp3 (
  • mice were maintained in a specific pathogen-free facility, and all animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of Sun Yat-sen University (Guangdong, China) .
  • MH5A hamster anti-mouse PD-1H
  • anti-hamster-IgG-PE eBioscience
  • hamster IgG eBioscience
  • All other fluorescently labeled antibodies including CD4, CD25, CD44, CD69, Foxp3, TCR V ⁇ 5.1/5.2, p-STAT3, p-STAT5, CTLA-4, Lag-3, GITR, IOCS, CD45.1 and CD45.2 were purchased from eBioscience and BD Pharmingen.
  • the anti-IFN- ⁇ (clone XMG1.2) , anti-IL-4 (clone 11B11) , anti-IL-6 (clone MP5–20F3) neutralizing Abs were purchased from R&D System.
  • the intracellular staining for Foxp3 and other intracellular cytokines were performed according to BD’s Cytofix/Cytoperm kit manual.
  • Cytokine analysis was performed using the mouse Th1/Th2/Th17 CBA kits (BD Bioscience) .
  • the mouse pan-T isolation kit, CD8 + T cell isolation kit, CD4 + T cell isolation kit, CD25 micro beads kit, and the CD4 + T cell isolation kits were purchased from Miltenyi Biotec (Cambridge, MA) .
  • Flow cytometry analysis was performed using a BD FACSVerse (BD Biosciences) and data analyzed using FlowJo software (Tree Star) .
  • CD4 + CD25 - CD44loCD62hi T cells were isolated using the CD4 + T cells isolation kit (Miltenyi Biotec) .
  • CD4 + CD25 - Foxp3 (GFP-) T cells and CD4 + CD25 + Foxp3 (GFP + ) nTreg were sorted by FACSAria (BD Biosciences) after the purification using the CD4 + T cell isolation kit.
  • the CD4 + T cells were first enriched using the CD4 + T cells isolation kit, and CD25 + T cell were subsequently depleted using the CD25 microbead kit (Miltenyi Biotec) to obtain the CD4 + CD25 - T cells.
  • the purity of the cells sorted using this method was typically more than 95%.
  • CD4 + CD25 - Foxp3 (GFP-) T cells were stimulated in vitro with plate-bound anti-CD3 (clone 2C11 at 2 ⁇ g/ml, eBioscience) plus soluble anti-CD28 (1 ⁇ g/ml) in the presence or absence of recombinant TGF- ⁇ (5 ng/ml, R&D Systems) and IL-2 (5 ng/ml, PeproTech) for 3-5 days. Conversion of Foxp3 + Treg cells were then analyzed by flow cytometry based on the expression of GFP or intracellular staining for Foxp3.
  • the cells were cultured in the plate coated PD-1H agonist mam82 (10 ⁇ g/ml) after plate-coated with anti-CD3 (1 ⁇ g/ml) .
  • the mAb to IL-4, IL-6, and IFN- ⁇ at 10 ⁇ g/ml were added to the wells at the beginning of the cultures.
  • the cultured supernatants were collected at the indicated time points for the cytokine analysis.
  • Treg cells suppressive function assay was performed as previous described. Briefly, the CD8 + T cells (in some experiment using the CD4 + T cells) were first labeled with 1 ⁇ M CFSE (Life Technologies) and subsequently co-cultured at 1x10 5 in the presence of 1x10 5 mitomycin C-treated syngeneic spleen cells as feeder cells. The cultures were added with or without Treg cells at indicated ratios in U-bottomed 96-well plates for 72 h, and subsequently added anti-mouse CD3 (1 ⁇ g/mL) for stimulation. The proliferations of T cells were assayed by CFSE dilution. In some experiments, soluble mouse control IgG or PD-1H agonist mam82 (10 ⁇ g/ml) were added to the cultures. In these cases, feeder cells were PD-1H KO spleen cells.
  • CD4 + CD25 - T cells were isolated from OT-II mice (or PD-1HKO OT-II mice) as described before and labeled with 5 ⁇ M CFSE before adoptive transfer.
  • 2x10 6 cells were injected i.v. into WT B6 mice.
  • the drinking water in the cages was replaced with 1.5%OVA solution (grade V; Sigma-Aldrich) 24 hours later for 5 consecutive days.
  • the mesenteric LN and PP were collected and the TCR-specific Foxp3 + Treg cells were determined using intracellular staining for Foxp3 by flow cytometry.
  • Bone marrow chimeras Bone marrow chimeras. Bone marrows from the tibia and femurs of WT (CD45.1) and PD-1H KO mice (CD45.2) were mixed at ratio 1: 1 and a total 1x10 7 cells were transferred into sub-lethal irradiated (6Gy) congenic WT mice (CD45.1/CD45.2) . The indicated organs were analyzed 10 week later after reconstruction. The frequency of Foxp3 + cells were determined by intracellular staining.
  • EAE disease model The experimental autoimmune encephalomyelitis (EAE) model was performed as previous described. Briefly, 7-8wk old female mice were immunized s.c. with 200 ⁇ g MOG35-55 (Life Technologies) in complete Freund’s adjuvant (Difco) . Mice were given 400 ng pertussis toxin (List Biological Labs) in 500 ⁇ l PBS i.p. on day 0 and 2 post-immunization. For adoptive transfer, 1x10 6 WT Foxp3 (GFP + ) iTreg cells or PD-1HKO Foxp3 (GFP + ) iTreg cells were injected i.v. before the immunization on day 0.
  • mice The same volume of PBS (saline) was injected i.v. into the WT mice that served as the control.
  • the spleen and dLN of each group were collected, and cell suspensions were re-stimulated with PMA/Ionomycin/BD GolgiPlug for an additional 4 hours.
  • the level of IFN- ⁇ and IL-17 was assessed using intracellular staining and analyzed by FACS (BD Cytofix/Cytoperm kit) .
  • CD4 + CD45RB hi T cells (CD45.1, 4x10 5 ) were co-injected via i.p. with or without 2x10 5 iTreg cells (CD45.2) into Rag1 KO mice.
  • Mice were weighed once a week, after 10 weeks, the colon tissue were collected and histological stained with H&E. Spleen and mLN cells were collected and ex vivo activated using PMA/Inomycin/BFA for 4 hours and then analyzed the cytokine production.
  • the Foxp3 (GFP + ) iTreg cells were sorted, and genomic DNA was purified with DNeasy Blood &Tissue Kit (Qiagen) .
  • the bisulfite conversion of DNA was performed using EZ DNA Methylation-Gold Kit (ZYMO research) .
  • the CNS2 region of Foxp3 enhancer was amplified with the primer set as previously described, and T/Acloned into pMD18-T vector (Clonetech) .
  • Ten inserted plasmids from each group were purified and sequenced and the methylation results were analyzed by BiQ Analyzer 2.0.
  • PD-1H is required for de novo induction of Treg cells
  • OT-II TCR transgenic mice were backcrossed to PD-1H KO mice to generate a new KO OT-II strain.
  • CD4 + T cells purified from the spleen cells of WT OT-II or the KO OT-II mice were depleted of CD25 + T cells (to avoid nTreg contamination) and subsequently transferred into WT B6 mice. Mice were then fed with 1.5%OVA in their drinking water for 5 consecutive days (Fig. 1A) .
  • Foxp3 + V 5.1/5.2 + OT-II cells could be detected in the gut-related lymphoid organs including mesenteric lymph nodes (mLN) and Peyer’s patch (PP) while less cells could be detected in the spleen, peripheral LN, and lamina propria (LP) (data not shown) , indicating a Treg response in the gut-related lymphoid organs to OVA.
  • Foxp3 + V 5.1/5.2 + OT-II cells were significantly decreased in the mLN and PP than WT OT-II (Fig.
  • CD4 + T cells from WT (CD45.1) and KO (CD45.2) mice were mixed at ratio 1:1, and transferred into Rag1 KO mice.
  • the percentage and absolute number of CD25 + Foxp3 + T cells in the indicated organs were analyzed 3 weeks later (Fig. 7) .
  • PD-1H is required for expansion, but not generation and function, of iTreg cells
  • PD-1H KO mice display normal numbers of nTreg cells in the thymus, spleen, and lymph nodes.
  • the phenotype and suppressive function of nTreg cells in PD-1H KO mice was also comparable to those in WT littermates (Fig. 8) .
  • iTreg cells are generated mainly in the gut under either a steady state or inflammation we next examined whether the lack of PD-1H affects iTreg generation and function. Although a similar proportion of Foxp3 + Treg cells in the mLN and PP has been found in both PD-1H KO mice and WT littermates, Foxp3 + cells in the LP of PD-1H KO mice were significantly lower, although the absolute number of Foxp3 + cells was unchanged in the absence of PD-1H (Fig. 2A and 2B) .
  • CD4 + CD25 - CD62L hi cells were purified from WT and PD-1H KO spleen cells, and stimulated with anti-CD3/CD28 in the presence of TGF- ⁇ .
  • the frequency of Foxp3 + iTreg cells that were differentiated from WT naive T cells were significantly higher than that from the KO naive T cells (Fig. 2C) .
  • iTreg cells from WT or PD-1H KO Foxp3 (GFP) mice were induced in vitro as described above and purified by sorting.
  • the Teff were generated by anti-CD3 stimulation of purified CD4 + CD25 - CD62L hi T cells.
  • the WT or KO iTreg cells were co-cultured with effector cells at the indicated Treg/Teff ratios in the presence of irradiated spleen cells, as antigen-presenting cells plus soluble anti-CD3 for 3 days.
  • the Teff were CFSE-labeled before the co-culture and the decreased CFSE dilutions were used as an indication of Treg suppression.
  • iTreg cells from both WT and KO mice suppressed proliferation of Teff with comparable activity. Consistent with these findings, we did not find significant differences in the expression of activation marker CD25, GITR, Lag-3, CTLA-4, ICOS, or PD-1 in WT vs. KO iTreg (Fig. 2F) . Therefore, PD-1H does not affect the suppressive function of iTreg despite its effect on the differentiation of iTreg.
  • T cells were sorted from WT Foxp3 (GFP) knock-in mice and subsequently stimulated with anti-CD3/CD28 in the presence of TGF- ⁇ .
  • Induction of GFP + CD25 + iTreg cells was measured upon the stimulation.
  • Inclusion of mam82 slightly increased Foxp3 + CD25 + iTreg expansion albeit the effect appeared moderate (Fig. 10A and 10B) .
  • This moderate effect may be due to a rapid loss of cell surface PD-1H on T cells in vitro.
  • PD-1H regulates iTreg differentiation via cytokines
  • cytokines may modulate the differentiation of iTreg cells in the absence of PD-1H.
  • purified CD4 + T cells were first stimulated with anti-CD3/CD28 and the cultured supernatants were collected for cytokine detection.
  • activated KO CD4 + T cells produced higher levels of IFN- ⁇ and IL-17 compared with WT CD4 + T cells (Fig. 3A) .
  • TGF- ⁇ In the presence of TGF- ⁇ , production of these cytokines was further increased, while there was no change in TNF- ⁇ (data not shown) .
  • PD-1H KO CD4 + T cells produced more IL-4 with or without TGF- ⁇ (Fig. 3A) . Because these cytokines are produced by different subsets of CD4 + T cells, our findings support PD-1H as a pan inhibitor of T helper cells.
  • Neutralizing mAb to IL-4 and/or IFN- ⁇ were added to the cultures to exploit the effect of these cytokines in the induction of iTreg cells. As showed, neutralizing either IL-4 or IFN- ⁇ partially restored differentiation of PD-1H KO iTreg cells compared to the WT control, while inclusion of both mAbs restored the majority of the activity. As the controls, IFN- ⁇ /IL-4 neutralizing mAbs, either used alone or in combination, could also enhance iTreg differentiation (Fig. 3B and 3C) . These findings indicate that impaired iTreg differentiation in the absence of PD-1H is due, at least in part, to altered cytokine production by T cells.
  • Loss of PD-1H facilitates the conversion of iTreg to Th17 in an inflammatory environment
  • PD-1H has a direct effect on already differentiated iTreg cells.
  • a murine experimental autoimmune encephalomyelitis (EAE) model was performed to test the stability of Foxp3 + iTreg cells. Briefly, WT or PD-1H KO Foxp3 (GFP + ) iTreg cells were generated as described above and sorted by flow cytometry based on GFP positivity (>97%Foxp3 + , Fig. 11A) .
  • CD45.2 + iTreg cells at a concentration of 1 x 10 6 /mouse were transferred intravenously into CD45.1 + B6 mice before the immunization, where the number of transferred WT iTreg is not sufficient to prevent EAE progression. Mice were then immunized with myelin basic protein to induce EAE as previously described.
  • the transfer of WT iTreg cells slightly delayed the onset of disease compared with the control.
  • STAT5 activation drives Treg lineage commitment whereas STAT3 is inhibitory for Foxp3 expression and promotes Th17 cell response.
  • PD-1H KO iTreg cells up to 47%p-STAT3 expressed significantly higher levels of phosphorylated STAT-3 (p-STAT3) than the WT iTreg cells in the spleen and dLN of EAE mice.
  • comparable levels of p-STAT5 were found in both WT and PD-1H KO iTreg cells (Fig. 5A and 5B) , implicating a role for STAT3 but not STAT5 in PD-1H function.
  • PD-1H is a critical cell surface signaling molecule that controls the pool size of inducible Treg via two different mechanisms.
  • PD-1H is required for the generation of iTreg from T cells. This effect is largely mediated via suppression of inflammatory cytokine such as IFN- ⁇ , IL-4, and IL-17.
  • the pool size of iTreg that responds to environmental stimulation decreases.
  • PD-1H does not affect the suppressive function of iTreg on a per cell level, the decreased pool size of iTreg may eventually affect the overall suppressive function of Treg during immune responses.
  • PD-1H signaling prevents the conversion of iTreg to Th1 and Th17 in an inflammatory environment.
  • PD-1H is essential for the generation of iTreg from T cells, the suppressive function of both nTreg and iTreg is not affected. Especially, PD-1H is not required for the generation and suppressive function of nTreg cells, which are thymus-derived and selected by self-antigens (Fig. 8) . This result may partially explain why spontaneous autoimmune diseases or lymphoproliferative symptoms were not observed in young PD-1H KO mice. Nevertheless, spontaneously generated iTreg cells in mice were clearly affected (Fig.
  • Treg is not terminally differentiated and it has been shown that iTreg can be converted into Th1 or Th17 subsets by specific cytokine or inflammatory environment. This plasticity of Treg cells makes it difficult to develop therapeutic strategies during chronic inflammation.
  • This invention demonstrates that PD-1H is required for the stability of Foxp3, a hallmark of Treg lineage, and maintains the phenotype of Treg. Loss of PD-1H led to a rapid decrease of iTreg in various in vitro and in vivo systems and models.
  • the invention supports that PD-1H inhibits the conversion of iTreg cells into Th1 and Th17 cells in an inflammatory environment, at least partially due to its role in the maintenance of Foxp3 expression and an iTreg phenotype.
  • PD-1H inhibits activation of T cells to limit initiation of T cell-mediated immune responses, as previously shown, it promotes growth and conversion of iTreg during immune responses.
  • PD-1H appears to participate in the regulation of T cell tolerance by regulating Treg pool size.
  • the PD-1H pathway may represent a promising target to control and manipulate T cell-mediated immunity in inflammation, autoimmune disease and cancer.

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Abstract

L'invention concerne des procédés de modulation de la taille d'un pool de lymphocytes T régulateurs inductibles ou des méthodes de traitement de maladies à médiation par l'immunité impliquant les lymphocytes T chez un sujet, comprenant l'administration au sujet en ayant besoin d'une quantité thérapeutiquement efficace d'un agoniste de PD-1H ou d'un antagoniste de PD-1H. L'invention concerne également des utilisations d'un agoniste de PD-1H ou d'un antagoniste de PD-1H dans la préparation d'une composition pharmaceutique pour la modulation de la taille d'un pool de lymphocytes T régulateurs inductibles ou pour le traitement de maladies à médiation par l'immunité impliquant les lymphocytes T chez un sujet.
PCT/CN2017/082735 2017-05-02 2017-05-02 Pd-1h en tant que cible dans la modulation de la taille d'un pool de lymphocytes t régulateurs inductibles Ceased WO2018201282A1 (fr)

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Citations (2)

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CN103119054A (zh) * 2010-03-26 2013-05-22 达特茅斯大学理事会 Vista调节性t细胞介体蛋白、vista结合剂及其用途
WO2016090347A1 (fr) * 2014-12-05 2016-06-09 Immunext, Inc. Identification de vsig8 en tant que récepteur putatif de vista et son utilisation pour produire des modulateurs de vista/vsig8

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103119054A (zh) * 2010-03-26 2013-05-22 达特茅斯大学理事会 Vista调节性t细胞介体蛋白、vista结合剂及其用途
WO2016090347A1 (fr) * 2014-12-05 2016-06-09 Immunext, Inc. Identification de vsig8 en tant que récepteur putatif de vista et son utilisation pour produire des modulateurs de vista/vsig8

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