*Purpose: Costimulation blockade targeting the CD28 pathway provides improved long-term transplant survival without the nephrotoxicity of calcineurin inhibitors. However, treatment with the CTLA-4Ig fusion protein belatacept is associated with increased rates of acute rejection that may be due in part to lack of CTLA-4 coinhibition. Thus, directly targeting CD28 while leaving CTLA-4 intact may prove beneficial. Fc-silent non-crosslinking CD28 antagonizing domain antibodies (dAb) are currently in clinical trials for renal transplantation. Given the current standard of care in renal transplantation at most US centers, it is likely that thymoglobulin induction therapy could be used in patients treated with CD28 antagonists. As such, we sought to investigate the impact of T cell depletion (TCD) on T cell phenotype following homeostatic reconstitution in a murine model of skin transplantation treated with anti-CD28dAb.

*Methods: We performed skin grafts from BALB/c donors to C56BL/6 recipients. Recipients were treated with or without 0.2mg anti-CD4 and 10ug anti-CD8 one day prior to transplant and with or without 100ug anti-CD28dAb on days 0, 2, 4, 6, and weekly thereafter. Mice were euthanized six weeks post-transplant and lymphoid cells were analyzed by flow cytometry.

*Results: Results demonstrated that anti-CD28dAb delayed the reconstitution of CD4⁺ T cells up to day 21 compared to TCD alone while CD8⁺ T cell reconstitution kinetics were unaffected. Mice treated with TCD+anti-CD28dAb exhibited significantly improved skin graft survival compared to TCD alone (p=0.03) and anti-CD28dAb alone (p=0.005), both of which were also significantly improved compared to no treatment (p=0.004, p=0.009). In addition, we observed reduced CD69 expression on CD4⁺ and CD8⁺ T cells in the spleen (p=0.0002, p=0.008) and lymph node (p<0.0001, p<0.0001) from mice that received TCD+anti-CD28dAb compared to TCD alone. In the kidney, the frequencies of tissue-resident CD69⁺CD103⁺CD4⁺ T cells were reduced in TCD+CD28dAb compared to TCD alone (p=0.04). We also observed an increased frequency of CD8⁺Foxp3⁺ T cells in the blood and kidney of mice given TCD+anti-CD28dAb compared to TCD alone (p=0.003, p=0.002).

*Conclusions: These data demonstrate that CD28 signaling impacts the differentiation of both of CD4⁺ and CD8⁺ T cells during homeostatic reconstitution following lymphodepletion, resulting in a favorable shift towards fewer tissue-resident memory T cells and more Foxp3⁺ CD8⁺ T cells, a profile that may underpin the observed prolongation in allograft survival.

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