1. Peripheral Control of Alloreactive B cells Anita Chong Section of Transplantation Department of Surgery

2. Chong Lab YiJin LiDengping Yin LianLi Ma Jamie Kim JiKun Shen Anncy Varghese Jingwen Ding Tongmin Wang Audrea Troutman Nextran Gerry Byrne Hui Xu U Colorado Roberta Pelanda

3. Alloreactive B cells and Long-term Allograft Survival Alloantibodies mediate acute and chronic rejection Alloantibody-alloantigen complexes induce maturation of APC B cells can activate alloreactive T cells

4. Mechanisms of B cell tolerance Calame, Nat Immunol 2001:1103 Antigen CD40L

5. Nature Immunology 2, 1126 - 1132 (2001) B cells and professional APCs recruit regulatory T cells via CCL4 Richard S. Bystry 1, Varuna Aluvihare 2, Katie A. Welch 1, Marinos Kallikourdis 1 & Alexander G. Betz 1 Using gene expression profiling, we show here that activation of B cells and professional antigen-presenting cells (APCs) induces the expression of common chemokines. Among these, CCL4 was the most potent chemoattractant of a CD4 + CD25 + T cell population, which is a characteristic phenotype of regulatory T cells. Depletion of either regulatory T cells or CCL4 resulted in a deregulated humoral response, which culminated in the production of autoantibodies. This suggested that the recruitment of regulatory T cells to B cells and APCs by CCL4 plays a central role in the normal initiation of T cell and humoral responses, and failure to do this leads to autoimmune activation.

6. Two-Phase model of T cell-dependent allo-specific B cell activation Modified from Baumgarth 2000 Immunol Rev. Secreted IgM

7. Extrinsic control of alloreactive B cells Inhibiting the development of effector T helper cells Inhibiting effective T-B cell interaction

8. Tolerance Model Day 0 C57BL/6 Day 60 + C57BL/6 Anti-CD40L (1 mg MR1/mouse; D-7, 0, 7) + DST (B/6) C57BL/6 (H-2 b ) into BALB/c (H-2 d ) mice

9. Anti-CD40L + DST induces allograft tolerance TreatmentGraft Survival None8 (x7), 9 + Anti-CD40L > 160 (x11) + 2nd heart >60 (x5), >100 (x3)

10. Tolerance can be dominantly transferred Tolerant mice reject third-party C3H hearts, exhibit linked tolerance to F1 (B/6 x C3H) hearts. A role of CD25 + T cells Allograft tolerance is associated with inhibited alloantibody production Features of Allograft Tolerance

11. Peripheral B cell Tolerance m Donor-specific (B/6) heart + 3.83 BCR-Ki B cells B/c recipients of B/6 heart Day 60-80 Day 0: DST + MR1 Naïve

12. 3.83 BCR-Ki B cells recognize K b antigens m H2-K(b) H2-K(d) Tetramer CD19

13. m Peripheral B cell Tolerance: 3.83 Cell Transfer + B/6 heart Days Post-B Cell Transfer 0 0.25 0.5 0.75 1 1.25 O.D. 05101520 Anti-Id-IgM Naïve - No TxpTolRej

14. Peripheral B cell Tolerance: Lack of T cell help m Third-party C3H heart + 3.83 BCR-Ki B cells Day 60-80 Day 0: DST + MR1 Naive

15. 3.83 BCR-Ki B cells have dual alloreactivity m H2-K(b) H2-K(d) H2-K(k) Tetramer CD19

16. Peripheral B cell Tolerance: Lack of T cell help m Third-party C3H heart + 3.83 BCR-Ki B cells Day 60-80 Day 0: DST + MR1 Naive

17. m Days Post-B cell Transfer Peripheral B cell Tolerance: Inhibition of T cell help

18. m B/6 heart + 3.83 BCR-Ki B cells + Anti-CD40 Day 60-80 Day 0: DST + MR1 Naive

19. m Control of B cell Responses by limiting CD40-CD40L signals:

20. Peripheral B cell Tolerance: Regulation of T cell help m F1 (B/6 x C3H) heart + 3.83 BCR-Ki B cells Day 60-80 Day 0 DST + MR1 Naive

21. m Linked Regulation of T cell Helper Responses Days Post-B Cell Transfer -0.25 0 0.25 0.5 0.75 1 O.D. 05101520 Anti-Id IgM TolNaive

22. Cells Maintaining Peripheral B cell Tolerance m Spleen Cells from: i.Naïve ii.Tolerant (d60-120) iii.CD25-Depl Tolerant Scid recipients of B/6 heart

23. m Peripheral B cell Tolerance: Role of CD25 Cells

24. Peripheral suppression of alloantibody production: 1.Inhibiting the generation of T cell help 2.Dependent on CD25+ cells

25. m Regulation of T cell help C3H- T H cell Recpt. APC F1 antigens B/6xC3H 3.83 B cell B/6- T Tol/Reg C3H- T H cell No T cell Help T cell Zone B cell Zone B/6- T Tol/Reg Phase 1 Phase 2

26. Anti-carbohydrate Antibodies in Allograft transplantation Naturally occurring and elicited anti-A or anti-B antibodies Donor-specific B-cell tolerance after ABO-incompatible infant heart transplantation (Fan et al. 2004) Can peripheral mechanisms control anti-carbohydrate antibody responses?

27. Structure of Gal  1,3Gal and Blood Group A and B antigens CH 2 O H R OHOH O O O O O NHCOCH 3 OHOH OHOH OHOH OHOH OHOH CH 2 O H R OHOH O O O O O NHCOCH 3 OHOH OHOH OHOH OHOH O OHOH OHOH OHOH C3HC3H O CH 2 O H R OHOH O O O O O NHCOCH 3 OHOH OHOH OHOH O OHOH OHOH OHOH C3HC3H O Gal  1,3Gal-ß1,4GlcNAc Blood Group B Gal  1,3(Fuc  1,2)Gal Blood Group A GalNAc  1,3(Fuc  1,2)Gal

28. Tolerance Model Day 0 Gal + BALB/c Heart + Intact Active Bone Day 60 Gal + BALB/c Heart Anti-CD40L (1 mg MR1/mouse; D 0, 7, 14) C57BL/6-Gal -/- recipients tolerant to BALB/c-Gal +/+ grafts

29. IAB and anti-CD40L therapy induces allograft tolerance TreatmentMean None 7 d Anti-CD40L + IAB - 1st BALB/c heart> 130 d Anti-CD40L + IAB - 2nd BALB/c heart > 60 d

30. Features of IAB/Anti-CD40L-induced tolerance Tolerance can be dominantly transferred Tolerant mice reject third-party C3H hearts, exhibit linked tolerance to F1 (B/c x C3H) hearts. A role of CD25 + T cells Allograft tolerance is associated with suppressed alloantibody production

31. Anti-Gal IgM production in IAB/Anti-CD40L-induced tolerant mice 0 0.25 0.5 0.75 1 1.25 O.D. 0306090 Days Post-Transplant Anti-Gal IgMb None MR1 + IAB

32. Peripheral B cell Tolerance m Donor-specific (B/c) heart Or Third-Party C3H heart + Anti-Gal BCR-Tg B cells B/6 GT -/- recipient of B/c heart Day 60-80 Day 0: IAB + MR1 Naïve

33. IgMa + CD43 + CD21 + Madcam-1 IgMa

34. Anti-Gal IgMa production following challenge with BALB/c hearts Days Post-B cell Transfer Naive Tolerant

35. Anti-Gal IgMa production following challenge with C3H hearts Days Post-B cell Transfer Naive Tolerant

36. Conclusion Anti-Gal cell responses are inhibited even in the presence of effector T helper cells

37. m Regulation of B cell responses C3H- T H cell Recpt. APC Balb/c-Gal antigens T cell Help B/c- T Tol/Reg C3H-Gal antigens Recpt. APC Gal-B cell B/c- T Tol/Reg C3H- T H cell T cell Zone B cell Zone Phase 1 Phase 2

38. Extrinsic control of alloreactive B cells Inhibiting the generation of effector T helper cells Inhibiting the development of effector B cells

40. Histology of Spleen B/6 C3H F1 FDC-M1 B220

41. Peripheral B cell Tolerance: Effect of TI-B cell stimulation m Donor-specific (B/c) heart + Anti-Gal BCR-Tg B cells + CpG B/6 GT -/- recipient of B/c heart Day 60-80 Day 0: IAB + MR1 Naïve

42. Inhibition of Anti-Gal IgMa production is reversed by CpG