1. B cells and allograft injury: more than you think Geetha Chalasani, MD Departments of Medicine (Renal-Electrolyte) and Immunology Thomas E. Starzl Transplantation Institute University of Pittsburgh School of Medicine VA Pittsburgh Healthcare System AKI Symposium – October 24, 2013

2. Deceased donor grafts Long term allograft survival remains suboptimal Chronic attrition Chronic allograft rejection Alloantibodies Persistent effector and memory T cells Adapted from USRDS 2009 and OPTN/SRTR Annual Report 2009 Lamb et al, Am J Transplant 2011; 11: 450-462 Lodhi et al, Am J Transplant 2011; 11: 1226-1235

3. Antibodies Cytokines Antigen presentation Formation of tertiary lymphoid tissues B cell B cells express TLRs and respond to innate signals Proliferate and differentiate in an antigen specific manner B cells can modulate immune responses by various mechanisms Chemokines Costimulation What is the role of antibody-independent B cell functions in alloimmunity?

4. Chronic rejection? Wt >100 days B6 BALB/c (H2d) CTLA4Ig and MR1  MT (has no B cells and antibodies) or  s-/-xAID-/- (has B cells but no antibodies) or wt (has B cells and antibodies) Heart transplant Can B cells mediate chronic rejection independent of antibodies?

5. Chronic rejection with intimal hyperplasia is not dependent upon antibodies

6. B cells are sufficient and antibodies are not necessary for chronic allograft vasculopathy B cells + + - + Antibodies + - - -  s-/-xAID-/- B cells into  MT

7. B cells + + - Antibodies + - - B cells are sufficient for chronic rejection also in an alternate model not requiring immunosuppression Bm12

8. Antibody and complement deposits in allografts

9. Alloreactive T cell responses are diminished in the absence of B cells

10. Graft vascular infiltration by T cells is diminished in the absence of B cells

11. Lymphoid + + - architecture Cognate + - - B cells Non-cognate + + - B cells Cognate and non-cognate B cell functions contribute to chronic rejection

12. Cognate role of B cells are antigen presenting cells in chronic rejection  MT B6 12 weeks Chimera  MT + wt or  MT + MHC (1 & 2) ko Heart allografts Chronic rejection? Only B cells lack expression of MHC I and II in  MT+MHCko chimeras so that antigen presentation specifically by B cells and not by other APCs would be impaired

13. Expression of MHC on B cells and non-B cells in bone marrow chimeras

14.  MT+wt  MT+MHCko  MT CD4 MOMA B220 CR1/2 MOMA B220 B cells + B-APC + ---- +-+- Splenic architecture in bone marrow chimeras

15. Chronic rejection is attenuated in the absence of antigen presentation by B cells B cells + B-APC + ---- +-+-

16. Alloreactive T cell responses are diminished in the absence of antigen presentation by B cells

17. Graft vascular infiltration by T cells is decreased in the absence of antigen presentation by B cells

18. Summary B cells are sufficient and antibodies are not necessary for chronic rejection Antibody-independent functions of B cells drive chronic rejection by supporting alloreactive T cell responses

19. B cells ≠ antibodies B cells >> antibodies

20. Acknowledgements Renee Ippolito Ke Jiang Bala Ramaswami Tripti Singh Yue-Harn Ng Qiang Zeng Khalefathullah Sheriff Parmjeet Randhawa Frances Lund University of Alabama AHA Physician-Scientist Fellowship Award (Ng), AST-BMS Fellowship Award (Sheriff), Starzl Transplantation Fellowship Award (Jiang), ASN Fellowship Award (Singh), Junior DOM Scholar Award, ROTRF and NIH-NIAID Thank You!

23. Preemptive CD20+ B cell depletion attenuates cardiac allograft vasculopathy in cyclosporine-treated monkeys Kelishadi SS, Azimzadeh AM, Zhang T, Stoddard T, Welty E, Avon C, Higuchi M, Laaris A, Cheng XF, McMahon C, Pierson RN 3 rd. Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, USA. J Clin Invest. 2010 Apr 1;120(4):1275-84 Less T cell infiltration Decreased C4D Decreased CAV despite antibodies in one recipient

24. It’s not all black and white ---- B-Cell–Depleting Induction Therapy and Acute Cellular Rejection Clatworthy MR, Watson CJE, Plotnek G, Bardsley V, Chaudhry AN, Bradley A, Smith KGC. University of Cambridge School of Clinical Medicine, Cambridge CB2 2QQ, United Kingdom N Engl J Med 2009; 360:2683-2685 Cytokine storm? Breg depletion? ‘Naïve’ recipients? Timing and context of B cell depletion should be considered carefully