Memory T cell (Tm) homeostasis is poorly understood and is a critical challenge to controlling transplant rejection. At present there are no effective clinical strategies to control Tm and contradictory reports exist as to their control by regulatory T cell (Treg). We previously found that TCRΑΒ⁺CD3⁺CD4^–CD8^–NK1.1^– (double-negative, DN)-Treg could effectively suppress CD4⁺ and CD8⁺ effector T cells and thus mitigate transplant rejection. We utilized this model to test whether DN-Tregs could similarly suppress Tm. Tm was generated by immunizing mice with allogeneic spleen cells. Interestingly DN-Tregs suppressed CD8⁺ Tm by >53% but had no effect on CD4⁺ Tm cells. As well DN-Tregs express very high levels of granzyme B (GzmB), suggesting a potential control mechanism and indeed no suppression was observed using perforin null DN-Tregs. In BALB/c (H-2d) to B6-Rag1^-/- mice (H-2b) skin allograft transplantation, DN-Tregs suppressed CD8⁺CD44^high Tm mediated rejection and significantly prolonged graft survival over CD8⁺ Tm cell transfer alone (63.0±4.7 days, vs. 21.5±1.7 days, p<0.05). In contrast, co-transfer of DN-Tregs had no effect on CD4⁺CD44^high Tm mediated rejection (25.3±1.4 days, vs. 20.0±0.7 days, p>0.05). DN-Tregs co-transferred with CD8⁺ Tm had significantly reduced total CD44^high CD8⁺ Tm (10.9×10⁴ vs. 27.9×10⁴), CD44^high CD62L^high central Tm (6.2×10⁴ vs. 13.1×10⁴), and CD44^high CD62L^low effector Tm (4.7×10⁴ vs. 14.8×10⁴) in spleens (n=3, p<0.05). There was no effect with co-transferred CD4⁺ Tm. CD4⁺ Tm cells express higher levels of GzmB inhibitory Serpin Protease Inhibitor 6 (SPI-6) mRNA (7.7-fold) and have higher intracellular expression of SPI-6 protein than CD8⁺ Tm (5.2-fold) (p<0.05, n=3), suggesting CD4⁺ Tm may escape DN-Treg control by resistance to GzmB. We show for the first time, that DN-Treg can control CD8⁺ Tm and suggest that DN-Treg along with targeting SPI-6 may be useful to limit the expansion of Tm in transplantation.

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