Gregg A. Hadley, Ph.D. Division of Transplantation Department of Surgery

1. Effector cells and tubulitis in the renal allograft. Gregg A. Hadley, Ph.D. Division of Transplantation Department of Surgery University of Maryland Medical School

2. Cytokines MØ activation Activation Differentiation Cytokines DC licensing Gran/Prf Fas/FasL Cytokines Chemokines Allograft destruction CD8+ CD8 CD4+ MHC II MHC I Cytokines Cognate help CD8 Effectors Plasma Cell Antibody B cell + Complement Cascade Activation Differentiation Destruction of graft endothelium Host Adaptive Immunity to Renal Allografts Renal Allograft

3. Interaction of CD8 effectors with graft epithelial compartments • Attack of the renal tubular epithelium by graft infiltrating CD8 effector populations is a cardinal feature of clinical renal allograft rejection. • Current knowledge of CD8 effector properties is derived from studies of leukocyte:leukocyte interactions • Consequently, the mechanisms underlying T cell/epithelial interactions remain poorly defined

4. 60 70 60 50 50 40 40 30 30 20 20 10 10 0 0 80:1 40:1 20:1 10:1 5:1 80:1 40:1 20:1 10:1 5:1 Generation of kidney-restricted CTL Responder Spleen Cells Allogeneic REC Stimulators Allogeneic SC Stimulators TARGETS REC LC Percent Lysis E:T Ratio

5. anti-REC anti-SC Events CD103 CD103 defines a major subset of CD8 effectors generated against renal epithelial cells

6. CD103 Background • Integrin family heterodimer (formerly aE7) • Ligand (E-cadherin) is uniquely expressed by cells comprising epithelial layers • Expressed by >95% of intraepithelial lymphocytes but poorly expressed by peripheral T cells (Brenner et al., Kilshaw et al., Lefrancois et al. ...) • Previous studies have focused on the potential role of CD103 as a homing receptor that targets IEL to the gut mucosa • Role of CD103 in adaptive immune responses remains unclear • CD103 promotes lysis of REC targets by in vitro generated CTL populations: Hadley et al., J Immunol, 1997; Rostapshova et al., Eur. J. Immunol, 1998.

7. Is CD103 expression by CD8 effector populations relevant to clinical renal allograft rejection?

8. FACS analyses of transplant nephrectomy specimens #1 #2 #3 CD8 Quad% Gated UL 20.48 UR 34.80 LL 43.64 LR 1.07 Quad% Gated UL 25.41 UR 36.79 LL 35.87 LR 1.93 Quad % Gated UL 22.46 UR 23.92 LL 49.91 LR 3.72 CD103

9. Robertson et al., Transplantation 71: 306-13, 2001 Wong et al., Transplantation 75: 505-14, 2003

10. Does a causal relationship exist between CD103+CD8+ effectors and destruction of graft epithelial compartments?

11. CD103-deficient mice • Mice with targeted disruption of the CD103 gene (Itgae, chromosome 11) on the BALB/c background • Comparison of allograft rejection by CD103-/- mice vs. wild type mice provides a definitive means of documenting the contribution of CD103 to the rejection process

12. Pancreatic islet transplant model 1) compared to mouse vascularized organ transplants, islet transplants are relatively easy to perform and thus amenable to experimentation 2) islets are specialized epithelial cells known to express high levels of the CD103 ligand, E-cadherin, and should be susceptible to destruction by CD103+CD8+ effectors 3) rejection of islet allografts transplanted into streptozotocin treated (diabetic) mice can be objectively assessed by serial measurement of blood glucose levels 4) islets can be transplanted under the renal capsule which allows recovery and analysis of graft infiltrating lymphocytes

13. 35.28 9.63 51.99 3.10 CD103+CD8+effectors are present at the graft site 55 Gated CD8+ T cells CD8 Events 18.1+3.0% CD103+ (n=4) 0 100 101 102 103 104 CD103 CD103

14. 100 80 60 40 20 CD103-/- (n=17) Wild type (n=13) 0 CD103 knockout hosts are deficient in the capacity to reject pancreatic islet allografts Graft survival (%) 0 20 40 60 80 100 Time (Days)

15. 600 500 400 wild type CD8 cells 300 CD103 -/- CD8 cells Wild type Non-CD8 control 200 100 0 0 5 10 15 20 25 Time (days) Adoptive transfer of CD8+ cells into CD103-/- hosts with long-term islet allografts Transferred CD8 cells: Blood Glucose (mg/dL)

16. Islet allograft in wildtype host at time of rejection

17. Islet allograft in CD103-/- host at day 14 post-Tx

18. Conclusion CD103 is required for destruction of graft epithelial compartments by CD8 cells, and appears to critically function at the level of intragraft homing of CD8 effectors

19. Do CD8+CD103+ effectors play a significant role in rejection of vascularized renal allografts?

20. BN (RT1n) LEW (RT1l) Classical acute rejection model 10 8 6 Serum creatinine (mg/dL) 4 2 Gated CD8+ GIL 0 0 2 4 6 8 10 Time (Days)

21. BN (RT1n) LEW (RT1l) 10 8 6 4 2 0 Serum creatinine (mg/dL) 0 20 50 10 30 40 Time (Days) Delayed rejection model CsA 5 mg/kg/day for 10 days Gated CD8+ GIL CsA

22. Two-color Immunostaining Red = E-cadherin Brown = CD103

23. Antibody blocking studies ´ CsA OX62 3.5mg IP POD 8 KTX day12 day28

24. Anti-CD103 monoclonal antibody, OX-62, blocks movement of CD8 cells into tubules 100 P<0.01 n=4 80 60 Percent tubules with infiltrating CD8 cells 40 20 0 OX-62 IgG1 Control

25. Conclusion CD103 promotes entry and/or retention of CD8 effectors in the graft renal tubules, consistent with a key role for CD103+CD8+ effectors in development of CAN.

26. What controls CD103 expression by graft infiltrating CD8 effectors?

27. 80 60 Percent CD103+ 40 no TGF (day 1) 20 + TGF (day 1) 0 + TGF (day 2) Wildtype (n = 3) DNRII (n=4) Events CD103 (Log10 Fluorescence) TGF-beta controls CD103 expression by allospecific CD8 effectors In vitro In vivo

28. Graft Epithelium TGF-b “Non-specific” inflammatory cells CD8 effectors Working Model IFN-g Graft Site

29. Acknowledgements Hadley Lab Elena Rostapshova Ye Feng Rongwen Yuan Riham El-Asady Transplant Pathology Cinthia Drachenberg