PRETREATMENT WITH ANTI-CD4 PLUS ALLOANTIGEN RESULTS IN THE INDUCTION OF A DISTINCT PATTERN OF CHEMOKINE EXPRESSION FOLLOWING CARDIAC ALLOGRAFT TRANSPLANTATION.

Abstract

P1126 Aims: To compare the intragraft chemokine and chemokine receptor gene expression during rejection or acceptance of a fully MHC mismatched cardiac allograft. Methods: C57BL/10 (H2b) cardiac allografts were transplanted into either untreated CBA (H2k) recipient mice (MST=8 days) or CBA mice that had been pre-treated with donor blood (DST) and anti-CD4 monoclonal antibody (pretreated mice), 28 days before transplantation (MST>100 days). Cardiac allografts transplanted into CBA RAG−/− recipients (lacking T or B cells) were used as controls. Cardiac allografts were analysed 1, 2, 5, 8, 10 and 100 days after transplantation. Real-time PCR analysis was used to determine the relative mRNA expression of CCL1, CCL3, CCL4, CCL5, CXCL2, CXCL5, CXCL9, CXCL10, XCL1, CCR1, CCR5, CCR8, CXCR2 and CD3. Results: Analysis of cardiac allografts from pre-treated CBA mice revealed a higher level of expression of most chemokines and chemokine receptors 2 days after transplantation, compared to allografts transplanted into untreated CBA recipients. In particular, expression of XCL1, CXCL9 and CXCL10 was dramatically increased by 276, 166 and 186 fold, respectively. The higher expression of most genes correlated with an increase in CD3 expression indicating that T cells had infiltrated cardiac allografts earlier and in greater numbers in pre-treated mice compared to naïve recipients. 5 days after transplantation the difference in gene expression between grafts from pre-treated and naive recipients persisted but was not as marked; for example, XCL1 and CXCL10 were increased by only 7 and 9 fold on day 5, compared to 276 and 186 fold on day 2 post-transplant. As expected, chemokine and chemokine receptor expression in cardiac allografts transplanted into CBA RAG−/− mice was dramatically reduced compared to that observed in CBA mice with an intact immune system suggesting that infiltrating allo-reactive T cells were the main mediators of chemokine production during allograft rejection and acceptance. Next, we analysed chemokine and chemokine receptor gene expression in accepted cardiac allografts 100 days after transplantation into pre-treated mice. Expression levels in cardiac allografts from pre-treated recipients were significantly elevated compared that found in allografts from CBA RAG−/− recipients 100 days after transplantation, for example, CCL5 (93 fold), XCL1 (39 fold) and CXCL10 (88 fold). Conclusions: This study demonstrates that pre-treatment with donor alloantigen in combination with anti-CD4 mAb results in earlier and more pronounced intragraft chemokine and chemokine receptor expression compared to that present in allografts transplanted into untreated, naive recipients. We therefore propose that under conditions leading to long-term graft survival, T cells that have seen alloantigen during the pre-treatment period are recruited to the graft prior to the priming and infiltration of naïve alloreactive T cells. The earlier and increased expression of chemokines in the grafts of pre-treated recipients may be important for the recruitment of regulatory T cells (known to be generated by this pre-treatment protocol) to the graft where they are able to suppress the T cell mediated effector mechanisms that normally result in acute rejection.