Background: Our research group recently achieved substantial advancements in extending allograft survival in a murine model of allogenic skin transplantation through the targeted proliferation and activation of Tregs in vivo utilizing interleukin-2 (IL-2) conjugated with a specific antibody against IL-2 (IL-2cplx). The present study aims to elucidate the impact of CD8+ cells on IL-2cplx-mediated skin allograft survival.
Methods: Recipient C57BL/6 mice were administered IL-2cplx, rapamycin, and a transient regimen of anti-IL-6 mAb concomitant with fully mismatched BALB/c skin grafts. Distinct cohorts received different anti-CD8 monoclonal antibodies (mAbs), encompassing the depletion of all CD8+ populations (anti-CD8a) or exclusive targeting of CD8+ T cells (anti-CD8b). To dissect the underlying mechanisms of allograft rejection in this model, we investigated donor-specific antibody (DSA) formation, changes in leucocyte subsets of lymphatic organs, in vitro T cell alloreactivity, and the composition of graft-infiltrating leukocytes.
Results: The combined therapy with IL-2cplx, rapamycin and anti-IL-6 mAb significantly extended the survival time of fully mismatched skin grafts. Notably, IL-2cplx-based intervention prevented humoral rejection and the formation of IgG DSA, whereas targeted elimination of CD8a (as opposed to CD8b) failed to further prolong skin graft survival and significantly impaired humoral tolerance. Furthermore, CD8a depletion led to an increase in donor-reactive Th2 cells and recipient CD4+ effector T cells infiltrating the graft on postoperative day 20 (POD20). In addition, histopathological examination indicated enhanced rejection on POD14 in animals subjected to CD8a depletion, in accordance with the Banff criteria. Of note, investigations of cell populations spared by CD8b but depleted with CD8a unveiled a notable augmentation of CD8+ CD205+ dendritic cells (DC) in both IL-2cplx protocol-treated and CD8b-ablated animals, a cell subset recognized for its capability to induce peripheral Foxp3+ regulatory T cells in vivo.
Conclusion: IL-2cplx therapy induces humoral tolerance, impeding the emergence of IgG DSA, whereas depletion of pan-CD8 cells does not confer additional durability to skin graft survival but induces the generation of donor-specific antibodies. Furthermore, CD8a depletion appears to precipitate proinflammatory cascades, evidenced by heightened migration of recipient CD4+ effector cells into the skin grafts and elevated levels of donor-responsive Th2 cells. Conversely, CD8b depletion fails to restore humoral alloreactivity, underscoring the indispensable role of CD8+ non-T cells in maintaining recipient desensitization. The elevated presence of CD8+ CD205+ DC, a regulatory DC subset known for their capacity to induce peripheral Tregs, represents a potential explanation for the observed suppression of IgG DSA formation in mice treated with IL-2cplx therapy.