*Purpose: T cell Specific Adaptor Protein (TSAd, encoded by the SH2D2A gene) is an intracellular adapter molecule that binds Lck and functions to elicit T cell receptor-mediated signaling and cytokine production in CD4⁺ T effector cells. Genetic polymorphisms in the SH2D2A gene in humans have been associated with autoimmunity and, in ongoing studies, we find that disruption of TSAd in mice is associated with intrinsic defects within CD4⁺Foxp3⁺ T regulatory cell (Treg) activity and resistance to costimulatory blockade post-transplantation. However, the mechanism of function of TSAd in immunoregulation is currently unknown. In these studies, we hypothesized that TSAd serves as an intrinsic adaptor to shuffle Lck towards mitochondria to facilitate mitochondrial function and oxidative phosphorylation in CD4⁺ Tregs.

*Methods: We purified CD4⁺CD25^high Tregs cells from Sh2d2a^-/- mice (TSAd KO on the C57BL/6 background) for all studies and compared responses in TSAd KO to wild type (WT) cells. The subcellular expression of TSAd and Lck was evaluated by Western blot. Activation-induced changes in mitochondrial membrane potential was assessed by JC-1 staining, and production of mitochondrial reactive oxygen species (ROS) by CM-H₂DCFDA staining using flow cytometry. We also assessed mitochondrial mass by electron microscopy, by qPCR of mitochondrial vs. nuclear DNA, and by flow cytometry using MitoTracker Green.

*Results: We find that TSAd and Lck are expressed in the cytosol, but only Lck is expressed in the mitochondria of WT CD4⁺CD25^high Tregs. Notably, however, we observed that Lck is absent on mitochondria in TSAd KO Treg cells. Since Lck is reported to be critical for mitochondrial homeostasis in T cells, we postulated that this observation is of great significance. We treated WT and TSAd KO Tregs with anti-CD3 (1 µg/ml for 30 min) and found that the activation/depolarization of mitochondria was markedly absent in KO vs. WT Tregs (P<0.05). Furthermore, mitochondria from KO Tregs were found to lack ROS production (P<0.05) following stimulation confirming a major effect of TSAd on mitochondrial activation. Finally, we evaluated mitochondrial morphology by electron microscopy, which was normal in TSAd KO Tregs, but there was a dramatic increase in the number of mitochondria per cell (P<0.0001). This increase was confirmed by MitoTracker Green uptake as well as by qPCR of mitochondrial DNA copies/nucleus (P<0.05 and P<0.0001 respectively).

*Conclusions: Thus, we identify TSAd as a novel and critical cell intrinsic molecule that regulates mitochondrial function and homeostasis in CD4⁺ Tregs. Our findings are likely of great significance to the understanding of alloimmune tolerance as Treg metabolism is both necessary and sufficient for effective immunoregulation post-transplantation.

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