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Immunity April ， 2009

调 节 性 T 细 胞 研 究 进 展 高晓明 北京大学医学部 2009.12.11 苏州. CD4 + CD25 + Foxp3 + Tregs. Immunity April ， 2009. Other types of regulatory T cells. Tr1: Producing IL-10 after Ag triggering NKT: Secret IL-4 after activation

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Immunity April ， 2009

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  1. 调 节 性 T 细 胞 研 究 进 展高晓明北京大学医学部 2009.12.11 苏州

  2. CD4+CD25+Foxp3+ Tregs Immunity April，2009

  3. Other types of regulatory T cells • Tr1: Producing IL-10 after Ag triggering • NKT: Secret IL-4 after activation • Th3: Produce TGF-b after Ag triggering • Tact (Anergic T cells) • CD8+gd-T cells • CD4-CD8- T cells

  4. 1 Treg 细胞的表型 2 nTreg 和 iTreg Treg的免疫调节作用机制 3 Treg的临床应用前景 4

  5. Treg 细胞的表型 1

  6. Treg 细胞的表型 CD25 其他标志 分子 Foxp3 Tregs TGF-β

  7. Before depletion After depletion 5-10% Anti-CD25-PE Anti-CD4-FITC Treg与CD25 经典实验 一

  8. Treg与CD25 经典实验 二 Normal mouse CD4+ T cells eliminated CD25+ cells Autoimmune diseases Thyroid Stomach Salivary gland Langerhans islets Adrenal glands Ovaries Oestes T cell-deficient mouse

  9. CD4+CD25+ Tregs在外周淋巴细胞中的比例 Th 9 Th 17

  10. 由Foxp3基因突变或缺失导致的CD4+CD25+ Treg细胞缺陷 Foxp3 Treg与Foxp3 性联免疫失调综合征 （IPEX）患者 Scurfy 小鼠 Foxp3基因 敲除小鼠

  11. IPEX • Immune deficiency/dysregulation • Polyendocrinopathy • Enteropathy (Often have Ab against gut epithelium) • X-linked inheritance Patients have mutations in FOXP3 gene. They suffer from inflammatory diseases similar to that seen in mice deficient in CD4+CD25+ Treg cells.

  12. IPEX – Outside (Clinical Findings) • First described in 1982 by Powell et al. as a syndrome of diarrhea, polyendocrinopathy, and fatal infection in infancy. • Neonatal onset diabetes mellitus • Hypothyroidism • Enteritis (diarrhea/villous atrophy) • Hemolytic anemia & thrombocytopenia. • Dermatitis • Dermatitis (eczema) • Death by 1-2 years of age

  13. Skin Disease in IPEX

  14. IPEX – Inside (Autopsy Findings) • Lymph nodes: Follicular hyperplasia • Thyroid: Lymphocytic infiltrates • Lungs: Consolidation/Inflammation • Thymus: Atrophy • Pancreas: islets of Langerhans absent with lymphocytic infiltrates • Intestine: villous atrophy withlyphocytic infiltrates • Liver: Cholangitis • Spleen: enlarged (inflammatory diseases similar to that seen in mice deficient in CD4+CD25+ nTreg cells).

  15. IPEX Normal FOXP3 CD25

  16. Zn - finger Zip FKH (DNA binding) ATG 1 2 3 4 5 6 7 8 9 10 11 PolyA A38S C565T splicing C565T splicing R347P AgG C424Y L76 frameshift +4AgG del251E R397W R397W R397L delGA(TGA)1317-1318 delT(TGA)13115-1316 I363V F371C A384T A384T A384T A384T FOXP3 Mutations

  17. DC DC AutoreactiveT lymphocyte AutoreactiveT lymphocyte MHC TCR MHC TCR High affinity MHC MHC CD4 TCR InhibitionNo autoimmunity Expansion.Autoimmunity FOX P3 CD25 FOX P3 nTregs Non functional regulatory T cell IPEX syndrome-FOXP3 deficiency FOXP3 deficiency

  18. NLS FOXP-3 is the master control gene for Treg development, encodes a novel forkhead/winged helix transcription factor N C LEUCINE ZIPPER PROLINE-RICH DOMAIN ZINC-FINGER FORKHEAD DOMAIN

  19. What does FOXP3 do? 1 – Plays an essential role in the development and function of CD4+CD25+ Tregs, Foxp-3 expression in CD4+ thymocytes is detected 3 days after birth 2 – May function as a transcriptional repressor of cytokine promoters 3 – Foxp3 is highly enriched in CD4+CD25+ Tregs in thymus as well as in periphery 4 – Targeted disruption of Foxp3 gene prevents CD4+CD25+ nTreg development and mice develop lethal inflammatory disease (Scurfy)

  20. Signaling Pathways, Transcription Factors, and Regulatory DNA Elements that Control Foxp3 Expression. (Josefowicz et al., Immunity, 2009)

  21. Treg与TGF-β CD4+CD25+ Tregs高表达TGF-β，特别是膜结合型TGF-β Nakamura et al., JEM, 2001

  22. Treg与TGF-β 动物模型中TGF-β对Treg功能的影响 SCID mouse SCID+colitis mouse Transfer TGF-β1 缺陷的CD4+CD25+ Treg Wild-type CD4+CD25+ T cells

  23. Treg与TGF-β TGF-β 缺陷的CD4+CD25+ Treg在体内不能保护小鼠 发生自身免疫病性结肠炎 wild-type CD4+CD25+ T cells TGF-β1 缺陷的CD4+CD25+ Treg Nakamura et al., JI, 2004

  24. Treg与TGF-β TGF-β 与CD4+CD25+ Treg的诱导产生 TGF-β and TCR costimulation induces Foxp3 expression in CD4+CD25- naive responder T cells. Chen et al., JEM, 2003

  25. Treg与TGF-β The Cytokine Milieu Determines CD4+ T Cell Differentiation and Conversion Zhou et al., Immunity, 2009

  26. 肿瘤坏死因子受体超家族 （TNFRSF）GITR，OX40（CD134）等 1 3 Nueropilin-1 小鼠 CCR5；人 CCR4，CCR8 5 Treg的其他标志分子 OX40 costimulation can abrogate Foxp3+ regulatory T cell-mediated suppression of antitumor immunity. （Int J Cancer， 2009） CTLA-4 control over Foxp3+ regulatory T cell function. CTLA-4 2 Neuropilin-1 expression on regulatory T cells enhances their interactions with dendritic cells during antigen recognition. Neuropilin-1 is not a marker of human Foxp3+ Treg. Isolation of functional human regulatory T cells (Treg) from the peripheral blood based on the CD39 expression. （J Immunol Methods， 2009） 4 CD39

  27. 小结 Treg的基因表达有其特殊性： 信号转导相关基因（COS，SOCS-1，SOCS-2及SLAP130）； 分泌性分子（IL-10，IL17，IL-35，MIP-1α，MIP-1β及ETA-1等）； 细胞表面分子（CD2，OX40，CD25，CD122，Ly-6，Thy-1及GITR等）。 虽然这些分子中的一些或在一般活化态T细胞表达，或在Foxp3-的无能T细胞表达，但可能正是这样的基因组合保障了Treg的存活和低反应状态 ，且是其发挥免疫学功能的分子基础。

  28. nTreg 和 iTreg 2 In thymus IL-2 Specific antigen stimulation Foxp-3 expression

  29. Bone marrow Thymus Deletion CD25+ CD4+ Treg CD4+ CD4+ Effector Naïve cells Periphery B Mj Self antigen

  30. IL-2 is required for development of CD4+CD25+ nTregs, anti-IL-2 treatment early after birth leads to autoimmune diseases in adults • Expansion of nTresgs in thymus is likely autoantigen-driven

  31. Treg Treg Treg Treg CD4+CD25+ nTreg的产生依赖对抗原的特异识别 OVA-TCR-tg 内源性TCRα链大量表达 不表达内源性TCRα链 OVA-TCR-tg Rag-/- 产生OVA特异性CD25 nTregs 无CD25 nTregs

  32. High affinity interaction with MHC/self peptide is required Positive selection Probability of selection Negative selection CD25+CD4+ nTregs Avidity of TCR-MHC-peptide

  33. Naturally occurring Tregs: • CD4+CD25+Foxp3+ Most Foxp3+ thymic Treg (tTreg) cells differentiate from Foxp3-negative CD4+ SP thymocytes.

  34. Induction ofTregs 1: 胸腺 CD4+ TGF-b induces the expression of Foxp3 in CD4+CD25- T cells; Retroviral transduction of Foxp3 renders CD4+CD25- T cells suppressive IL-10 Treatment of CD4+CD25- T cells makes them suppressive Foxp3+Treg Foxp3- Foxp3+nTreg Foxp3- IL-10 TGF-β Foxp3+iTreg Tr1 TE 外周

  35. Induction ofTregs 2: Treg-iTreg（Thsup） CD25 Treg cells convey a suppressive activity to conventional CD4 Th cells. （Jonuleit et al., JEM, 2002)

  36. Induction ofTregs 2: Treg-iTreg Model of T effector cell regulation. Treg-iTreg

  37. The Cytokine Milieu Determines CD4+ T Cell Differentiation and Conversion Epigenetic Status of Lineage- Specific Transcriptional Regulator Gene Loci in CD4+ T Cells IL-1beta IL-2 The plasticity of T cells Zhou et al., Immunity, 2009

  38. 小结 nTreg 与 iTreg 的异同 Lafaille et al., Immunity, 2009

  39. 3 Treg的免疫调节作用机制

  40. Treg 细胞的两个功能特征 Treg 2. 免疫抑制性 （suppressive） 1. 无能态 （Anergic）

  41. 1. 免疫无能性（Anergic） 对高浓度IL-2 的单独刺激,固相包被或可溶性抗CD3 单抗,以及抗CD3 单抗、抗CD28 单抗的联合作用呈无应答状态, 也不分泌IL-2 。 当经TCR 介导信号刺激并有高浓度外源IL-2 存在的情况下, CD4+CD25+ Treg细胞可活化并增殖,但其增殖程度较CD4+CD25- T 细胞弱很多。

  42. 抑制靶细胞IL-2基因表达 2. 免疫抑制性（suppressive） 抑制作用的 接触依赖性 Treg 抑制作用 机制 对APC的抑制作用 介导靶细胞溶解

  43. CD80/86 APC Treg抑制靶细胞IL-2基因表达 GITR LAG3 IL-10, TGF-b • CD4+25+ Foxp3+ CTLA4 mTGFb TE TGF-bR MHC II IL-2

  44. 抑制靶细胞IL-2基因表达 抑制作用的 接触依赖性 2. 免疫抑制性（suppressive） Treg 抑制作用 机制 对APC的抑制作用 介导靶细胞溶解

  45. Galectin-1与Treg 细胞抑制作用的接触依赖性 Detection of galectin-1 by confocal microscopy. Galectin-1 is required for maximal regulatory T cell function. Garin et al., Blood, 2007

  46. Treg与靶细胞的接触诱导抑制性细胞因子的分泌(eg. IL-35) Treg-restricted expression of Ebi3 and Il12a. Epstein-Barr-virus-induced gene 3 (Ebi3, which encodes IL-27beta) and interleukin-12 alpha (Il12a, which encodes IL-12alpha/p35) are highly expressed by mouse Foxp3+ (forkhead box P3) T(reg) cells but not by resting or activated effector CD4+ T (T(eff)) cells, and that an Ebi3-IL-12alpha heterodimer is constitutively secreted by T(reg) but not T(eff) cells. Collison et al., Nature, 2007

  47. IL-35 suppresses Teff cell proliferation.

  48. 抑制靶细胞IL-2基因表达 介导靶细胞溶解 2. 免疫抑制性（suppressive） 抑制作用的 接触依赖性 Treg 抑制作用 机制 对APC的抑制作用

  49. Proposed Model for the Role of Granzyme B and Perforin in Treg Cell-Mediated Suppression of Antitumor NK and CD8+ T Cells Cao et al., Immunity, 2007

  50. 抑制靶细胞IL-2基因表达 对APC的抑制作用 介导靶细胞溶解 2. 免疫抑制性（suppressive） 抑制作用的 接触依赖性 Treg 抑制作用 机制

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