*Purpose: Transplant recipients experience lifelong immunosuppression, yet studies have shown that a subset of patients may be able to retain stable graft function even with significant immunosuppression minimization. Identifying the immunologic characteristics of patients who maintain long-term stable graft function with minimal immunosuppression would both provide potential biomarkers for stratification of patient risk for immunosuppression minimization, and illuminate novel targets for therapeutic intervention to promote allograft tolerance. Here, we identify one such factor, an enzyme which alters the threshold of TCR signaling.

*Methods: PBMC from the CTOT-09 study, in which renal transplants with stable graft function and no DSA at 6 months post-transplant were weaned from tacrolimus, were analyzed by Affymetrix gene array. WT and Gm2a^-/- global or CD8⁺ T cell-specific conditional knockout mice received allogeneic skin grafts and were monitored for survival or sacrificed post-transplant to assess alloimmune responses.

*Results: Analysis of differentially expressed genes (DEG) within PBMC from stable vs. rejecting patients in the CTOT-09 study demonstrated that upregulation of Gm2a transcript was associated with stability off tacrolimus. CellCODE bioinformatic deconvolution analysis revealed that this upregulation was most tightly associated with CD8⁺ T cells (F-statistic 1.515 at least 2.5 fold higher than for the other cell subsets) Murine experiments to determine if Gm2a expression causally regulates alloimmunity indicated that Gm2a^-/- mice exhibit significantly accelerated allograft rejection relative to WT counterparts (median graft survival 22.0 days Gm2a^-/- vs 29.0 days WT, p=0.0108). Subsequent studies using a CD8⁺ T cell-specific Gm2a conditional knockout revealed that Gm2a^-/- CD8⁺ T cells possess a competitive advantage over WT CD8⁺ T cells (p=.0079 at 10 days post OVA-expressing skin graft) in a co-adoptive transfer model. To investigate why Gm2a^-/- CD8⁺ T cells exhibit increased accumulation following allostimuation, the ability of Gm2a^-/- CD8⁺ T cells to respond to cognate antigen in vitro was assessed. Results indicated that Gm2a^-/- CD8⁺ T cells were activated by both lower doses of cognate peptide (p<0.001 for CD69 at 10nM N4) and by low affinity TCR ligands (p=0.0073 for CD69 at 10nM Q4, p=0.0073 for CD69 at 10nM T4). This increased responsiveness was underpinned by a change in the composition of membrane-associated lipid rafts in Gm2a^-/- vs. WT CD8⁺ T cells.

*Conclusions: Taken together, these results demonstrate that Gm2a functions to increase the threshold for activation of CD8⁺ T cells by altering the composition of sphingolipids/lipid rafts in the plasma membrane. These findings illuminate a novel regulatory mechanism controlling CD8⁺ T cell allorecognition, and suggest that patients in whom Gm2a is upregulated may possess a reduced spectrum of CD8⁺ alloreactivity.

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