*Purpose: In recently published studies we discovered that the inhibitory receptor FcγRIIB regulates CD8⁺ T cell alloimmunity in a cell-autonomous fashion, and that increased expression of FCGR2B was associated with freedom from rejection following tacrolimus withdrawal in the CTOT-09 clinical trial. These observations led us to query specific pathways that are upregulated in FcγRIIB⁺ T cells that promote allograft survival.

*Methods: We cross-referenced the list of ~1009 differentially expressed genes (DEG) in FcγRIIB⁺ vs. Fc RIIB^– CD8⁺ murine T cells with the list of transcripts that were differentially expressed at baseline (before Tacrolimus withdrawal) in patients who were stable vs. rejected off immunosuppression in the CTOT-09 cohort. In subsequent mechanistic studies, Gm2a^-/- mice and WT littermate controls received BALB/c skin grafts and alloimmune responses were assessed via flow cytometry.

*Results: Analysis of Affymetrix gene array data revealed that 3 genes upregulated in CTOT-09 stable patients (Gm2a, Cpa3 and Skap2) were also differentially expressed in RNA-Seq datasets comparing transcript expression in FcγRIIB⁺ T cells vs. FcγRIIB^– CD8⁺ T cells. To determine whether these differences were associated with the CD8⁺ cellular immune response to transplantation, CellCODE deconvolution analysis was performed to identify the cell lineages associated with the differential expression between stable and rejectors. These data revealed that the difference in Gm2a, an essential cofactor associated with sphingolipid processing, demonstrated the strongest association with CD8⁺ T cells. To determine if Gm2a plays a mechanistic role in suppressing alloimmune responses in vivo, we assessed the magnitude and functionality of alloreactive T cell responses in WT vs Gm2a^-/- mice. While frequencies of CD44^hi cells in naïve WT vs. Gm2a^-/- mice were not different, analysis of splenocytes at day 10 post-transplant revealed a significant increase in the absolute number of CD44^hi activated effectors in both the CD4⁺ and CD8⁺ T cell compartments in Gm2a^-/- animals relative to WT littermate controls. Moreover, Gm2a^-/- mice also demonstrated increased frequencies of TNF-expressing CD8⁺ T cells, decreased frequencies of IL-2-expressing CD8⁺ T cells, and fewer NKT cells relative to WT controls.

*Conclusions: Taken together, our results from human renal transplant patients and experimental mouse models suggest that Gm2a may be a critical, as-yet unrecognized, immunologic mediator of transplant tolerance.

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