TRANSPLANT SPECIFIC TOLERANCE AFTER TRANSFER OF ALLOPEPTIDE SPECIFIC TH2-, BUT NOT OF TH1 CELL CLONES, IS ASSOCIATED WITH GATA-3 EXPRESSION IN THE GRAFT AND REGULATION OF THE IMMUNE RESPONSE (2004)

P937 Aims: We have recently published the characteristics and functions of Th1/Th2 clones generated from allografts of rejecting (Th1 type) and tolerant (Th2 type) animals respectively. T cell clones were established by stimulating graft infiltrating cells of acutely rejected (WF→LEW recipients) and tolerant (CTLA4-Ig treated WF→LEW recipients) kidneys with donor-derived immunodominant class II MHC allopeptide (RT1.Duβ20-44) presented by recipient antigen-presenting cells (indirect allorecognition). In this study we analyzed the in vivo functional/regulatory capacities of these T cell clones in an established WF→LEW kidney transplant model. Methods: Recipients were treated with low dose cyclosporine (CsA, 1mg/kg day 0-3 and 5mg/kg day 4-7, s.c.) and injected with Th1 or Th2 cell clones (30x106 i.p., day 0). Grafts from all recipient groups (n=6-8/Gp) were examined histologically. Proliferation assays were performed and cytokine expression (IFN-γ, IL-10) was analyzed by ELISPOT. Activated CD4+CD25+ T lymphocytes and T cell signalling (signal 1 and 2) were assessed using double immunostaining. Gene expression was measured with RNAse protection assay and Real Time PCR. Results: Unmodified LEW recipients acutely rejected WF renal allografts (graft survival: 6.3±0.8 days, n=8). Short-term CsA therapy (Gp3) resulted in prolongation of graft survival (121.7±34.6 days, n=8) but all grafts were ultimately rejected with evidence of progressive changes of chronic allograft nephropathy (CAN) not apparent in the syngeneic controls which remained virtually normal. Adoptive transfer of Th2 cell clones (Gp2) prevented allograft rejection (>250 days, n=8) and induced tolerance proven by acceptance of donor specific skin (>60 days, n=6) in long-term surviving recipients. Urine protein excretion from these animals remained virtually at baseline. Long-term surviving recipients developed only minor morphological changes of chronic vasculopathy but no evidence of glomerular sclerosis or fibrosis. Enrichment of regulatory T cells producing Th2 type cytokines (IL-10) was observed in spleens and lymph nodes of these animals (ELISPOT) and immunohistologically in the target organ. In contrast, animals injected with the Th1 cell clones chronically rejected their allografts in an accelerated manner (65.0±20.4 days vs CsA controls 121.7±34.6 days, p<0.0001, n=8/group). Double immunostaining in grafts without CAN (Gp2) showed in contrast to Gp1 with CAN activated T cells (CD4+CD25+) and downregulated signalling (TCR/MHC class II and CTLA-4/B7-1/B7-2/CD28). The transcription factor GATA-3, important in Th2 cytokine gene expression and/or Th2 differentiation, and the Th2 marker, T1/ST2 was detectable in Gp2 allografts (not seen in Gp1 and 3). Conclusions: These results are the first description of the functions of Th1 and Th2 alloreactive T cell clones generated via the indirect pathway of allorecognition in an allogeneic transplant setting and provide a causal link between Th1 cells and development of chronic rejection and Th2 cells and regulation of alloimmune responses in vivo.