TSAd is an SH2 domain containing intracellular adapter molecule that functions to elicit T cell receptor-mediated signals and cytokine production. TSAd knockout (KO) T cells are reported to secrete reduced quantities of IL-2, IL-4 and IFNγ in response to mitogen, and TSAd KO mice develop autoimmune disease after 6-12 months. However, little is known about the mechanism and function of TSAd, and whether its biological activity can be exploited to promote alloimmune tolerance.

In these studies, we transplanted MHC class II mismatched B6.C-H-2^bm12 (BM12) hearts into C57BL/6 wildtype (WT) or TSAd KO recipients. While allograft survival was >50 days in WT recipients, unexpectedly allograft rejection was markedly accelerated in KO recipients (mean survival time [MST] 22 days, P<0.01). On day 14 post-transplantation, there was a notable increase in CD4⁺CD44^hiCD62L^lo effector T cells (Teff) but a similar number of CD4⁺Foxp3⁺ Tregs in KO vs. WT recipients. In vivo-BrdU labeling confirmed an increased expansion of Teffs in KO recipients, and Treg function appeared to be reduced in in vitro suppression assays. We also treated fully MHC mismatched Balb/c into C57BL/6 WT or KO recipients with anti-CD40L to inhibit Teff expansion and to evaluate Treg-mediated graft survival. While survival was >30 days in WT recipients, all grafts in KO mice failed by day 21 post-transplant (MST 19, P<0.01). To determine if TSAd regulates Foxp3 activity, we evaluated the methylation status of all CpG motifs within the TSDR of the Foxp3 locus of FACS-sorted CD4⁺Foxp3⁺ Tregs. Pyrosequencing demonstrated that KO Tregs have higher levels of methylation in CpG regions 9-14. In addition, using ELISpot, we find that KO Tregs produce high levels of IFNγ suggesting an effector phenotype. To assess Treg function in vivo, we transferred (day 2 post-transplant) WT or KO Tregs into C57BL/6 Rag2γc KO recipients of fully MHC mismatched Balb/c allografts. Recipients were challenged by adoptive transfer (on day 18) of alloprimed WT Teffs. Both WT and KO Tregs prolonged allograft survival compared to challenge with Teffs alone (MST 34, P<0.05). However, WT Tregs were significantly more efficient than KO Tregs (P<0.05) to prolong survival.

Collectively, these new findings indicate that TSAd provides cell-intrinsic signals that regulate Foxp3 activity, effector cytokine production and Treg function post-transplantation.

CITATION INFORMATION: Wedel J, Stack M, Seto T, Sheehan M, Liu K, Flynn E, Briscoe D. Novel Cell Intrinsic Role of the Adaptor Protein TSAd in Maintaining Alloimmune T Regulatory Cell Function. Am J Transplant. 2017;17 (suppl 3).

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