CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) play a central role in the maintenance of immune tolerance and T cell homeostasis. It is known that Tregs promote transplant tolerance and prevent tissue damage through multiple mechanisms including the production of IL-10 and TGF-beta. Although initial clinical trials using Tregs were promising, strategies employed to isolate and expand Tregs, however have been a limiting factor. Here, we investigated in the context of transplantation, a novel immunosuppressive approach applying NAD⁺-and discovered novel immunosuppressive properties that were CD4⁺CD25⁺Foxp3⁺ Tregs independent.

To assess NAD⁺ immunosuppressive properties we used a fully MHC II mismatch skin transplant mouse model. DBA/2 skin was engrafted; onto C57BL/6 mice treated with either PBS or NAD⁺ (30mg/day i.p.). Allograft survival and immune responses were assessed. In addition, CD4^-/- and IL-10^-/- deficient mice were used to demonstrate NAD⁺ mechanisms of action.

NAD⁺ treatment promoted a dramatic improvement of allograft survival (18 days vs 9 p<0.01) in parallel to a dramatic decline of CD4⁺CD25⁺Foxp3⁺ T cells suggesting that allograft survival was CD4⁺CD25⁺Foxp3⁺ T cell independent. Furthermore, our data indicated a significant increase of Th1/Th17 CD4⁺ T cells that are known to be two pro-inflammatory subsets. In vitro experiments indicated that in presence of NAD⁺, CD4⁺CD25^LowFoxp3⁺ were able to differentiate into Th17 cells with a change in their transcriptional and cytokine signature profile. The results indicated an augmented expression of IL-17A and RORγt and a Th17 cell proliferation in absence of IL-23 that was mediated via P2XR and STAT3. Of particular relevance and unraveling a novel mechanism, NAD⁺ promoted allograft survival by skewing Th1 pro-inflammatory cells towards immunosuppressive CD 4⁺IFN-gamma⁺IL-10⁺ regulatory type 1 cells. More importantly, CD4^-/- and IL-10^-/- mice that were treated with NAD⁺ did not exhibit a prolonged allograft survival, suggesting that NAD⁺ immunosuppressive properties were mediated via CD4⁺ T cells and IL-10 cytokine.

Collectively, our study unravels a novel immunosuppressive therapy with intriguing and novel mechanisms with reduced side effects observed when compared to current immunosuppressive therapies that may benefit transplanted patients and beyond.

CITATION INFORMATION: Elkhal A, Rodriguez Cetina Biefer H, Uehara H, Quante M, Seyda M, Schuitenmaker J, Tullius S, Elkhal A. NAD⁺ Promotes Allograft Survival Independently of CD4⁺CD25⁺Foxp3⁺ Regulatory T Cells. Am J Transplant. 2016;16 (suppl 3).

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