*Purpose: The selective in vivo expansion and activation of regulatory T cells via administration of interleukin-2 (IL-2) coupled to a specific antibody against IL-2 (IL-2 cplx) was successfully used to induce tolerance towards islet allografts, however survival of fully mismatched skin grafts could not be significantly prolonged. Here we investigate whether depletion of alloreactive CD8+ T cells has a positive impact on Treg mediated skin graft survival.

*Methods: Recipient C57BL/6 mice received fully mismatched BALB/c or single MHCII mismatched BM12 skin grafts in combination with IL-2 cplx, Rapamycin and a short term treatment of anti-IL-6 mAb. To analyze the role of CD8+ T cells experimental settings devoid of alloreactive CD8+ T cells were created by using an anti-CD8 antibody or the single MHCII mismatched mouse model. To study the mechanisms of skin graft rejection, development of donor-specific antibodies, formation of T cell memory as well as graft infiltrating leucocytes were investigated.

*Results: The combination of IL-2 cplx with Rapamycin and an IL-6 neutralizing antibody leads to significantly prolonged survival of fully mismatched (MST=30.5 days) as well as single MHCII mismatched (MST=77.5 days) skin grafts. Furthermore, analysis of sera showed a significant decrease of donor reactive IgG1 (p<0.05) and IgG2a/b (p<0.01) in this settings. Interestingly, depletion of CD8+ cells, did not lead to further prolongation of skin graft survival (MST=33.5 days) but a significant increase of donor specific IgG1, which was detectable by d28 post skin graft rejection. Furthermore, deficiency of CD8+ cells resulted in a shift from Th1 to Th2 immune response and higher recipient CD4+ effector T-cell infiltration into the skin grafts by day 20 post transplantation. In addition, analysis of Tfh as well as Tfr revealed increased frequencies in mice devoid of CD8+ alloreactivity.

*Conclusions: Combined treatment with IL-2 cplx, Rapamycin and anti-IL-6 leads to significantly prolonged skin allograft survival. Particularly noteworthy, humoral response and sensitization are impeded in this setting. Depletion of CD8+ cells results in – albeit delayed – formation of donor-reactive antibodies suggesting that a CD8+ cell population is needed for sustainable prevention of sensitization.

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