*Purpose: Costimulation blockade-based regimens are a promising strategy to promote transplant tolerance. However, their efficacy is affected by multiple factors, many of which remain unknown. Recent reports have highlighted the unexpected capacity of passenger donor lymphocytes (CD4 T cells in particular) to directly fuel the recipient’s anti-graft response. In this study we tested if T lymphocytes contained in the donor specific transfusion (DST) inoculum, component of a very effective CoB regimen, could limit the regulation of skin transplant rejection.

*Methods: Full mismatch skin transplants were performed from Balb/c into C57BL/6 mice and animals received DST (10⁷ splenocytes; on POD 0) and anti-CD154 mAb (MR-1; on POD 0, 7 14). To study the role of donor T cells, DST inocula were depleted in either total T cells, CD8, CD4 or more specific T cell subpopulations by negative-selection/cell-sorting. IgG Donor Specific Antibodies (DSA) were determined by flow cytometry, and the strength of the recipient direct and indirect alloresponse was measured via ELISpot.

*Results: DST+MR-1 has a profound protective effect on mouse skin allotransplantation. However, it does not induce long term survival (MST=68). Interestingly, when donor T cells were depleted from the splenocytes used as DST, transplant survival was significantly increased (MST=103). Unexpectedly, the specific removal of CD4 T cells did not improve survival (MST=66). Concordantly, the addition of CD8-CD44hi memory T cells to T-depleted DST restored the limited survival of full DST (MST=71) and ELISPot and flow cytometry results showed a correlation with the generation of a greater anti-donor specific memory response in post-rejection analyzed spleen samples. Surprisingly, the specific removal of donor CD8 T cells in the DST inoculum induced a survival even greater than that achieved with total T cell depletion (MST=148). The use of CD4-CD44hi memory T cells with T-depleted DST (inoculum still depleted in CD8 T cells), matched the survival of T-depleted DST (MST=113) and was not associated to any increment in anti-donor memory response in post-rejection analysis. In ongoing experiments, we are aiming to determine what correlation exists between the presence/absence/type of donor T lymphocytes and the levels of donor specific antibodies, or variations in the strength of the direct and indirect alloresponses.

*Conclusions: Overall, these data reveal a novel and important opposing role for donor passenger lymphocytes in the modulation of recipient’s alloimmunity by DST+MR1, with donor CD4 T cells being beneficial, and memory-CD8 possessing a deleterious role. Identification of the specific mechanisms through which this effect is exerted will be pivotal for the optimization of clinically effective tolerogenic therapies.

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