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

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

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

Responder Spleen Cells Allogeneic REC Stimulators Allogeneic SC Stimulators 40:180:120:110:15: :180:120:110:15:1 Percent Lysis E:T Ratio REC LC TARGETS Generation of kidney-restricted CTL

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

CD103 Background Integrin family heterodimer (formerly  E  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.

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

Quad % Gated UL UR LL LR 1.93 #2 Quad % Gated UL UR LL LR 3.72 #3 CD103 CD8 Quad % Gated UL UR LL LR 1.07 #1 FACS analyses of transplant nephrectomy specimens

Robertson et al., Transplantation 71: , 2001 Wong et al., Transplantation 75: , 2003

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

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

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 Pancreatic islet transplant model

CD103 + CD8 + effectors are present at the graft site CD103 CD % CD103+ (n=4) CD103 Events Gated CD8 + T cells

CD103 knockout hosts are deficient in the capacity to reject pancreatic islet allografts Graft survival (%) Time (Days) CD103 -/- (n=17) Wild type (n=13)

wild type CD8 cells CD103 -/- CD8 cells Wild type Non-CD8 control Adoptive transfer of CD8+ cells into CD103 -/- hosts with long-term islet allografts Transferred CD8 cells: Blood Glucose (mg/dL)

Islet allograft in wildtype host at time of rejection

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

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

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

Classical acute rejection model Time (Days) Gated CD8+ GIL BN (RT1 n )LEW (RT1 l ) Serum creatinine (mg/dL)

Delayed rejection model BN (RT1 n )LEW (RT1 l ) CsA 5 mg/kg/day for 10 days Gated CD8+ GIL Time (Days) Serum creatinine (mg/dL) CsA

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

Antibody blocking studies CsA day12 OX62 3.5mgIP POD  8 day28KTX

Percent tubules with infiltrating CD8 cells IgG 1 Control OX-62 P<0.01 n=4 Anti-CD103 monoclonal antibody, OX-62, blocks movement of CD8 cells into tubules

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.

What controls CD103 expression by graft infiltrating CD8 effectors?

CD103 (Log 10 Fluorescence) Events no TGF (day 1) + TGF (day 1) + TGF (day 2) TGF-beta controls CD103 expression by allospecific CD8 effectors In vitro Percent CD Wildtype (n = 3) DNRII (n=4) In vivo

Graft Epithelium Working Model TGF-  CD8 effectors IFN-  “Non-specific” inflammatory cells Graft Site

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