TWO DISCRETE WAVES OF T CELL ACTIVATION FOLLOWING TRANSPLANTATION OF A SKIN ALLOGRAFT.

Abstract

P1010 Aims: To visualize the response of alloreactive CD8+ T cells during the rejection of a skin allograft. Methods: 1x105 BM3 H2Kb-reactive TCR-transgenic CD8+ T cells (Tg-TCR+ T cells) were CFSE labeled and adoptively transferred into syngeneic CBARAG−/− mice one day before transplantation of a H2Kb+ skin allograft. Mice that received T cells but no skin graft or a skin graft in the absence of T cells served as controls. The activation and proliferation of the alloreactive T cells was followed in the axillary lymph nodes (draining (dLN) and contra-lateral (cLN)), mesenteric lymph nodes (MLN) and spleen 5, 10 and 15 days after transplantation. Skin allografts were harvested at the same timepoints for real-time PCR gene expression analysis and immunohistochemistry. Results: CBARAG−/− mice that had received Tg-TCR+ T cells acutely rejected B10 skin allografts (MST=23 days; n=6) whereas allografts transplanted onto unreconstituted CBARAG−/− recipients were accepted indefinitely (MST>100 days; n=4). 5 days after transplantation, Tg-TCR+ CD8+ T cells were found to have proliferated and expanded specifically in the dLN (the level of CFSE demonstrated on average 6 rounds of division). In contrast, 10 days after transplantation the CFSE+ T cells had the same level of fluorescent of CFSE in the dLN as those in the cLN, MLN and spleen, indicating that there was no priming of naive T cells at this time. However, by day 15 a further wave of T cell activation had taken place resulting in a dramatic expansion of T cells in the dLN (10 fold) and once again the level of CFSE on CFSE+ T cells was substantially reduced (30%+/−17% compared to CFSE+ T cells in the other lymphoid organs or untransplanted mice; n=3). CD3 mRNA expression and staining for CD8+ cells by immunohistochemistry confirmed that the alloreactive CD8+ T cells had infiltrated the skin allograft following the initial wave of T cell activation and were further increased following the second wave of priming. The infiltration of CD8+ T cells correlated with a profound increase in the mRNA expression of a number of proinflammatory molecules such as IFNγ (499 fold), Perforin (366 fold), RANTES/CCL5 (26 fold) and Lptn/XCL1 (102 fold) in rejecting skin grafts compared with expression levels in skin grafts transplanted onto unreconstituted CBARAG−/− mice (2-3 mice / group). Conclusions: Upon transplantation of an allogeneic skin graft we found that T cell priming was initiated around day 5 which was followed by a second wave of priming that occured between 10 and 15 days after transplantation. To our knowledge this is the first report demonstrating the existence of two waves of CD8+ T cell priming following skin transplantation.