免疫调节 （ Immunoregulation ）

1. 免疫调节 （Immunoregulation） 鲁林荣 浙江大学免疫学研究所 Lu.Linrong@gmail.com

2. Immunoregulation The ability of the immune system tosense and regulate its own responses: 1. To determine the type and scale of a response; 2. To ensure Fast Response upon exogenous antigen stimulation; 3. To switch off the response properly and restore the immune system to a resting state when the response to a given antigen is no long required.

3. Immunoregulation Immune Response and Regulation: Including Positive & Negative regulation. Positive regulation ensures fast response. While negative regulation after the clearance of infection is very essential for restore the immune homeostasis. Immunoregulationand intervention: Upon the understanding of immunoregulation mechanisms, we can deliberately enhance or take away one of the control to enhance or block an immune response. Immunoregulation and Disease: Since immune regulation is achieved at many different levels by different mechanisms, any defect in this system will cause an immune disorder.

4. Immune regulation happens at different stages of an immune response • During Innate Immune Response • TLR mediated inflammatory response • Cytokine signaling • During adaptive immune response • By Inhibitory receptors • By Tregs • Other Immune regulation mechanisms • Regulation by antibody • by the idiotype and anti-idiotype network

5. Immune Response

6. Toll-like receptors and recognition of pathogens K. Takeda & S. Akira, Cell. Microbiol. 5: 143-53, 2003 diacyl- triacyl-lipopeptide 酰基脂肽；flagellin:鞭毛蛋白; LPS:脂多糖 (Lipopolysaccharides)

7. Toll-like receptor signaling pathways MyD88 pathway and TRIF pathway; Transcription factors Cytokineexpression

8. Innate response is critical for an effective immune response. Excessive response lead to endotoxic shock (sepsis)： Sepsis（败血症） is a serious medical condition that is characterized by a whole-body inflammatory state (called a systemic inflammatory response syndrome or SIRS) and the presence of a known or suspected infection.The body may develop this inflammatory response to microbes in the blood, urine, lungs, skin, or other tissues. A lay term for sepsis is blood poisoning. The Key event for innate immune response: Recognition of microbe patterns by pattern recognition receptors including TLR.

9. Regulation of TLR4 signaling Innate TLR signaling : IRAK-M: interleukin-1-receptor associated kinase M SOCS1: suppressor of cytokine signaling 1. MyD88s: myeloid differentiation primary-response protein 88 short SIGIRR: single immunoglobulin IL-1R-related molecule Inflammatory cytokines

10. TLR4 signaling is regulated by membrane trafficking Rab10 Biosynthesis Replenishment Endocytosis Degradation Recycle Rab10 promote trafficking onto cell surface. Wang D et al in Lu lab 2010 Rab7b promote endocytosis and degradation Cao X 2007

11. Regulation by signaling pathways (cytokine receptors signaling)

12. Regulation by signaling pathways (SOCS family)

14. Virus-infected cell produces interferon to act on neighboring cells Infected cell makes interferon, uninfected cells respond to interferon and become refractory to viral growth (Antiviral State) Interferon-a Virus-infected cell

15. Production of interferon by infected cells

16. Summary of the first part: Regulation of TLR signaling The role of SOCS in cytokine signaling (inducible, ubiqutin ligases) Amplifciation of IFN signaling in antiviral response.

17. Regulation by Inhibitory receptors 1. Immune cells are activated by Ligand binding to their activating receptors； 2. Activation of Immune cells is mediated by Protein Phosphorylation； 3. Phosphorylation is mediated by protein kinases; Dephosporylation is catalyzed by protein phosphotases.

18. Regulation by Inhibitory receptors 1). Activating receptor: ITAM (immunoreceptor tyrosine-based activation motif) Motifs：YxxL/V Recruit : kinases, adaptor proteins Induce activation signal 2). Inhibitory receptor: ITIM (immunoreceptor tyrosine-based inhibitory motif) Motifs：I/Vx YxxL Recruit Protein phosphatases Transduce inhibitory singnal Two type of receptors

19. Regulation by Inhibitory receptors Activating receptors mediate cellular activation

20. Regulation by Inhibitory receptors • B cell, mast cell:FcRIIB，PIRB • Cross-linking immune complex or anti-idiotype antibody with BCR • T cell: CTLA-4, PD-1 • CTLA-4 and PD-1 transmit signals that inhibit lymphocyte activation, providing a pathway of classical feedback inhibition. • NK, CTL: KIR，Ly49 (type I), CD94/NKG2A (type II)

21. Inhibitory receptors Inhibitory receptors on B cell Once the ITIM of FcRIIB is phosphorylated, SH2-containing SHIP are recruited, which influences PI3K and ERKpathways. PIRB（paired immunoglobulin-like receptor B ） ITIMs recruites SHP1，which dephosphorylates various protein-tyrosine kinases, including SYK and Bruton's tyrosine kinase (BTK).

22. Inhibitory receptors Inhibitory receptors for T cell. CTLA-4 Y201 YVKM Y208

23. Inhibitory receptors Before After activation activation • Dynamic regulations of T cell activation by CD28 and CTLA4: • CD28 and CTLA-4 has the same ligand B7. • Resting T cell only express CD28, which is an activating coreceptor provides 2nd signaling to ensure T cell activation. • Activated T cells start to express CTLA-4 on their surface, because CTLA-4 has 100 folder higher affinity to B7, CTLA-4 activation become dominant which inhibit the TCR signaling.

24. CD80 CD80 CTLA-4 Coinhibition CD80 CD86 PD-L1 CD80 Coinhibition PD-1 CD80 PD-L2 B7 - H4 (B7S1, B7x) B7H4 Coinhibition ? CD80 HVEM Coinhibition CD80 BTLA B7H3 ? Coinhibition ? CD80 Other inhibitory receptors on T cells 《科学》杂志社今年生命医学领域十大进展

25. Inhibitory receptors on NK cells

27. Inhibitory receptors on NK cells Immune surveillance for ‘ Missing Self’ by NK cells Existence of inhibitory receptors for MHC class I spare normal cells from NK cell attack which ensures NK cells preferentially kill cells that have lost MHC class I. 1. Viral-infected cells or tumor cells tends to reduce their surface expression of MHC I to avoid CD8 cell attacks. 2. NK cells thus Provides protection against cells escaping T cell recognition. 3. NK cells will also kill foreign cells with mismatched MHC class I molecule. Karre et al. Nature 319:675, 1986

29. Summary of Part II: Definition of Inhibitory receptors ITIM /ITAM (how do they work) Typical inhibitory receptors Story of CTLA4 NK cell function regulated by inhibitory receptors (KIR in human, Ly49 in mouse NKG family in both)

30. Immunoregulation by Tregs CD4+FoxP3+ Tregs CD8+ Tregs Other regulatory cells

31. CD4+FoxP3+ Tregs • Summary of CD4+ FoxP3+ Treg cells • 1) Natural Treg: constitute approximately 10% of CD4+ T cells in spleen and lymph nodes of normal mice, 10~30% of CD4+T cells in peripheral blood of human. • Naturally anergic and suppressive, appear to be produced by the normal thymus as a functionally distinct subpopulation of T cells. • 2) Surface markers • CD25 (IL-2Rα), IL-2Rβ, CTLA-4, GITR, CD44high, CD45RB, CD5high, ICAM-1high, LFA-1high, partly CD62L. • The expression pattern of some accessory molecules is in part similar to ‘primed ’, ‘activated’, ‘effector’, or ‘memory’ T cells. • 3) Inducible Treg（iTreg）-- that differ in terms of their development, specificity, mechanism of action and dependence on T-cell receptor and co-stimulatory signaling.

32. CD4+FoxP3+ Tregs CD25+ Activated T cells can suppress autoimmune diseases CD25-PE Sakaguchi et al. 155 (3): 1151. (1995)

33. Before depletion After depletion 5-10% Anti-CD25-PE Anti-CD4-FITC Another classic experiment

34. CD4+FoxP3+ Tregs FoxP3+ is the transcription factor that program the development of Tregs Shimon Sakaguchi Kyoto University，RIKEN Nature Immunol 4:330-336. Alexander Y. Rudensky University of Washington

35. 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.

36. 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(新生儿糖尿病) • Hypothyroidism (甲状腺功能减退) • Enteritis (diarrhea/villous atrophy)(肠炎) • Hemolytic anemia & thrombocytopenia. • Dermatitis • Dermatitis (eczema，湿疹) • Death by 1-2 years of age

37. IPEX Normal FOXP3 CD25 IPEX

38. 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

39. 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

40. 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

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

42. 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 • IL2 or IL2R deficiency lead to autoimmune diseases

43. 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

44. 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

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

46. CD4+FoxP3+ Tregs Mechanisms used by Tregs

47. Combined effects of Tregs

48. Part I • Tregs 与各种疾病 Tregs in Clinic Part II • Treg过继疗法 Part III • Tregs临床治疗的前景

49. Treg与自身免疫病 系统性红斑狼疮病人（SLE） 免疫治疗 从病人体内分离出部分 Treg，经过体外活化和 扩增，再输回病人体内， 向病人回输后可以改善病情 • 缺少Treg的小鼠会出现以产生大量抗双链DNA抗体为特点的SLE症状； • SLE病人的Treg数量较健康人明显减少，缺乏正常免疫调节活性，并且更容易发生由Fas介导的凋亡 。 Construction and characterization of a novel DNA vaccine that is potent antigen-specific tolerizing therapy for experimental arthritis by increasing CD4+CD25+Treg cells and inducing Th1 to Th2 shift in both cells and cytokines. （Vaccine，2009）

50. Clinical Applications of Human Treg Cells in Allo-HSCT Riley et al., Immunity, 2009