2. The research progress of CD4+CD25+regulatory T cell The mechanism it participates in tumor immunity

3. Team members: 李彩娥 林冰钦 金自慧 侯 毅 陈成玉 程 卫

4. History This kind of cells was first found by Mukherji in 1986.

5. The cells were first named by Sakaguchi in 1995

6. A special CD4+ T cell Treg is a special CD4+ T cell Most CD4+ T cells belong to Th1 or Th2 But 5%--10% of CD4+T cells belong to neither one which is called CD4+ CD25+ regulator T cells

7. T reg : Suppress the outgrowth of potentially pathogenic self-reactive T cells (conventional T cells) MHC II TCR Adapted from: Wood, K.J. and Sakaguchi, S. (2003) Nat Rev Immunol 3, 199- 210 Conventional T cell

8. Increased Treg’s Increase in Solid Tumors TumorTregSiteNRef NSCLCCD4+CD25+TIL11Woo OvarianCD4+CD25+Ascites9Woo BreastCD4+CD25+TIL, LN35Liyanage PancreasCD4+CD25+TIL, LN30Liyanage MelanomaCD4+CD25+Met LN12Viguier JI 2004 GI (gastric, colon, pancreas, esoph, liver) CD4+CD25 hi PBMC149Sasada GI (gastric, esoph) CD4+CD25+PBMC, TIL30Ichihara GI (gastric, esoph) CD4+CD25+PBMC114Kono K OvarianCD4+CD25+Ascites104Curiel Head and NeckCD4+CD25 hi %PBMC24Schaefer HepatocellularCD4+CD25 hi PBMC84Ormandy OvarianFoxP3 mRNATumor Bx99Wolf In most cases Treg suppressive function is confirmed in a subset of pts. Correlations between stage, prognosis, Tx in some studies – e.g. gastric

9. Source of normal Treg

10. 1. As a function-specific T cell subpopulation, Treg developed from thymus directly.

11. CD3 ‾ CD4 + CD8 ‾ TCR ‾ CD3 ‾ CD4 ‾ CD8 + TCR ‾ CD3 LOW CD4 ‾ CD8 + TCR  LOW CD3 + CD4 + CD8 + TCR  + CD3 + CD4 + CD8 ‾ TCR  + CD3 + CD4 ‾ CD8 + TCR  + CD3 + CD4 + TCR  + CD3 + CD8 + TCR  + CD3 + CD4 + CD25+T cell CD3 + CD4 ‾ CD25+ T CD3 + CD25+ + T CD3 ‾ CD4 ‾/ LOW CD8 ‾ TCR ‾ 被膜 被膜下区 皮质 骨髓 髓质 胸腺小叶

12. Source of normal Treg 2. Treg developed from peripheral lymph organ.

13. Induced Treg

14. Character. CD25+ is symbol of activation. FOXP3 is the specific mark.

15. Molecular Marker Function CD4 + -Binds MHC class II -Expressed on T h cells CD25 + -Expressed on most T reg cells CD3 + -Ensures expression of TCR GITR-Negative signaling -Required for survival CTLA-4-Binds CD80/CD86 -Antagonizes effects of CD28 to CD80/86 CCR7-Mediates migration of T reg cells back to lymph nodes Foxp3-Required for differentiation and function of T reg cells

16. NOTE! There are no known cell surface molecules that uniquely distinguish the CD4 + T reg cells from conventional activated CD4 + T cells! CD25 CTLA-4 GITR CD4 + CD25 + Regulatory T cell

17. CD4 + CD25 + Tregs In vivo Maintain immune tolerance Inhibit autoimmunity prevent transplant rejection Interfere with anti-cancer immunity Potential in immune deficiency 40 30 20 10 0 3 H uptake (x10 -3 ) CD25 – CD25 + CD25 – + CD25 + In vitro 5-10% of CD4 + T cells Anergic to TCR stimulation Suppress T cell proliferation CD4 CD25 Foxp3 FACS CD25 Foxp3 MicroscopyCo-culture 10%>90%

18. T reg CD4 CD25 CTLA-4 GITR The T reg cell phenotype CD4 Co-receptor for TCR recognition of MHC II/Ag CD25 IL-2R  IL-2R component, confers high affinity binding to IL-2R  Key T R growth factor CTLA-4 cytotoxic T lymphocyte Ag-4 Binds to B7s (CD80/86) on APC, acts as co-stimulatory molecule for T R (blocking CTLA-4 inhibits T R ) GITR glucocorticoid induced TNF related protein Ligation inhibits TR function (agonist inhibit T R, blocking augments T R ) FoxP3 Forkhead/winged-helix TF critical for T R activity and development Unlike surface markers / receptors, T E do not express FoxP3 Foxp3

19. Molecular mechanism of CD4+CD25+ inhibiting tumor immunity Two classes on its effect wayTwo classes on its effect way Ⅱ.Inhibition through cell-cell contact Ⅰ.Inhibition dependents on cytokines Act on effector T cells

20. DC is an important cell in CD4+CD25+Foxp3+regulatary inhibition The former include inhibition induced by TGF-β and IL- 10 signal pathway the latter is mainlyredu ced by CTLA-4.

24. ＊ CD25 and IL-2 signal pathway CD25 and IL-2 signal pathway ＊ TGFβand IL-10 signal path TGFβand IL-10 signal path ＊ Cell adhesion inhibition associated with CTLA-4 Cell adhesion inhibition associated with CTLA-4 ＊ Inhibit expression of DC Inhibit expression of DC

25. TUMOR

26. APC CD4 + CD8 + IL-2, IFN-γ ‘Conventional’ T cells

27. APC CD4 + CD8 + IL-2, IFN-γ ‘Conventional’ T cells

28. APC CD4 + CD8 + IL-2, IFN-γ ‘Conventional’ T cells CD4 + T reg CD8 + T reg

29. APC CD4 + CD8 + IL-2, IFN-γ ‘Conventional’ T cells CD4 + T reg CD8 + T reg

30. APC CD4 + CD8 + IL-2, IFN-γ ‘Conventional’ T cells CD4 + T reg CD8 + T reg TUMOR

31. APC CD4 + CD8 + IL-2, IFN-γ ‘Conventional’ T cells CD8 + T reg TUMOR CCL22 migration CD4 + T reg CCR4

32. APC CD4 + CD8 + IL-2, IFN-γ ‘Conventional’ T cells CD8 + T reg TUMOR CCL22 migration CD4 + T reg CCR4

33. That is all. Thank you!

34. CD25 and IL-2 signal pathway CD25, main antigen on surface of Treg cell, α-chain of IL-2R.CD25, main antigen on surface of Treg cell, α-chain of IL-2R. IL -2 is mainly secreted by effector T cell.Its competition is the main mechanism of inhibition IL-2R on effector T cell:dimer on Treg:trimerIL -2 is mainly secreted by effector T cell.Its competition is the main mechanism of inhibition IL-2R on effector T cell:dimer on Treg:trimer

35. Secretion of IL-2 can increase expression of IL-2R on Treg cells while decrease it on effector Tcells.

36. TGFβand IL-10 signal pathway TGF-βacts to inhibit reaction and proliferation of lymphocyts, and inhibit the activation of Mφ.TGF-βacts to inhibit reaction and proliferation of lymphocyts, and inhibit the activation of Mφ. Effcctor:CD8+T cell ＆ NK cell Effcctor:CD8+T cell ＆ NK cell

37. phosphorate TGF- β R + TGF- β initiate segment kinase domain activate SMAD gene expresson

38. Inhibition of Treg cell on NK cell are performed by NKG-2D to reduce its toxic activity directly.

39. IL-10 participates in Treg cell inhibition by regulating co- stimulatory molecules on APC. Antigen cytokine CD40 CD80/CD86 IL-10

40. Cell adhesion inhibition associated by CTLA-4 Activation of naïve T cells :Activation of naïve T cells : Peptide-MHC + TCR Peptide-MHC + TCR B7 + CD28 B7 + CD28

41. B7 CD28 CD28 CTLA-4 stimulate inhibit APC

42. CTLA-4 has a much higher avidity for binding B7 family members than that does CD28 and are expressed more widely in the body. Binding of CTLA-4 to B7 on DC expression of IDO(indoleamine 2,3-dioxygenase ) on DC, Tregs activated by IDO markedly upregulated programmed cell death 1 ligand 1 (PD-L1) and PD-L2 expression on target DCs, and the ability of Tregs to suppress target T cell proliferation can be abrogated by antibodies against the programmed cell death 1/PD-L (PD-1/PD-L) pathway.

44. Inhibit expression of DC NF-KbNF-Kb (CD40,CD80\CD86,IL-12,TNF-a,CCL5) (CD40,CD80\CD86,IL-12,TNF-a,CCL5) Cell-cell contactCell-cell contact cytokine: TGF-β, IL -10cytokine: TGF-β, IL -10

45. DCs may induce T cell tolerance to tumors. Tumor cells Tolerogenic DC Lack of inflammation Tumor-induced Treg infection inflammation effector T cells IL-10, VEGF IL-10R IL-10 Tumor cells STAT3 Down regulation of co-stimulation