Background: Recently, our group has succeeded in significantly prolonging allograft survival in a murine model of skin transplantation using an approach based on selective in vivo expansion and activation of Tregs by interleukin-2 (IL-2) coupled to a specific antibody against IL-2 (IL-2cplx). In the course of this project, we aimed to investigate the effect of alloreactive CD8+ cells in Treg-mediated skin graft survival.
Methods: Recipient C57BL/6 mice received IL-2cplx, Rapamycin and a short-term treatment of anti-IL-6 mAb in addition to fully mismatched BALB/c or single MHCII mismatched BM12 (no CD8 T cell alloreactivity) skin grafts. Indicated experimental groups were treated with different anti-CD8 mAbs, depleting either all CD8 populations (anti-CD8a) or CD8 T cells (antiCD8b). To further dissect the mechanisms of skin graft rejection in this model we assessed development of donor-specific antibodies, in vitro T cell alloreactivity as well as graft infiltrating leucocyte.
Results: Combined treatment of IL-2cplx with Rapamycin and anti-IL-6 mAb prolonged the survival of single MHCII mismatched skin grafts to a median survival time of 77.5 days compared with 30.5 days for fully mismatched skin grafts and additionally resulted in prevention of recipient sensitization for the latter. Of note, depletion of CD8+ cells did not further prolong skin graft survival and, in stark contrast to non-depleted recipients, donor-specific IgG1 was detectable early after rejection. Furthermore, mice depleted of CD8+ cells demonstrated an increase in donor-responsive Th2 cells and a higher number of recipient CD4+ effector T cells infiltrating the skin grafts by POD20. In addition, T follicular helper (Tfh) and T follicular regulatory (Tfr) cell levels were increased in the spleens of mice lacking CD8+ alloreactivity compared with non-depleted/fully mismatched recipients.
Results of experiments investigating the impact of CD8+ T cells vs CD8+ non-T cells on Treg mediated skin graft survival are still pending.
Conclusion: Depletion of all CD8+ cell subsets shows no beneficial effect on skin allograft survival and results in - albeit delayed - donor-specific antibody formation suggesting that a CD8+ cell population plays an important role in the sustained prevention of recipient sensitization. In addition, we propose promoted pro-inflammatory processes as a result of CD8+ cell depletion based on elevated migration of recipient CD4+ effector cells into the skin grafts and additional increase of donor-responsive Th2 cells in this model.