*Purpose: In a clinical trial, patients who underwent kidney transplantation and were subsequently stable off tacrolimus immunosuppression exhibited increased Gm2a transcript in PBMC relative to patients who experienced rejection. While Gm2a, a lipid transfer protein that stimulates the enzymatic processing of gangliosides, is well known to contribute to sphingolipid metabolism in neurologic disorders, its role in the immune system has not been well-investigated. In a mouse model, we found that mice lacking Gm2a exhibit accelerated allograft rejection and exhibited enhanced alloreactive CD8⁺ T cell responses compared to WT mice. However, the role of Gm2a in memory T cell responses is unknown. Because memory T cells can play a significant role in mediating breakthrough rejection in the presence of immunosuppression, here we endeavored to determine the impact of Gm2a deficiency on naïve vs. memory CD8⁺ T cells.

*Methods: An in vitro culture system in which splenocytes from WT and Gm2a^-/- mice that express the OT-I T cell receptor specific for OVA_257-264 were stimulated for 4 days with OVA_257-264 peptide to generate effector/memory CD8⁺ T cells was established. On day 4, WT and Gm2a^-/- effector/ memory CD8⁺ T cells were restimulated for 4 hours again with OVA_257-264 peptide. In addition, naïve WT and Gm2a^-/- OT-I T cells were also stimulated for 4 hours with OVA_257-264 peptide. The expression of T cell activation markers on both naïve and memory WT vs. Gm2a^-/- T cells was assessed via flow cytometry.

*Results: Results indicated that Gm2a^-/- memory CD8⁺ T cells exhibited significantly increased expression of CD44 (p < 0.0001) and CD25 (p < 0.0001) relative to WT memory CD8⁺ T cells following a short in vitro restimulation. These differences were more pronounced on memory CD8⁺ T cells relative to naïve CD8⁺ T cells. In naïve cells stimulated with a range of 1 pM to 100 µM OVA_257-264, we found that a marker of early activation (CD69) began increasing at lower OVA_257-264 concentrations in Gm2a^-/- vs. WT cells. These results demonstrate that Gm2a deficiency impacts memory CD8⁺ T cells in addition to naïve CD8⁺ T cells.

*Conclusions: These results show that Gm2a^-/- memory CD8⁺ T cells express significantly more of the high affinity IL-2R relative to WT memory CD8⁺ T cells, a finding which may underlie their increased proliferation and accumulation in vivo. Overall, these findings suggest that increasing Gm2a activity could be a potential immunotherapeutic strategy to modulate memory CD8⁺ T cell alloreactivity during transplantation.

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