Figure 5. Adoptive Transfer of WT Tregs does not rescue GrB producing Spi6^−/− Tregs from cell death.

(A) Representative example of flow cytometry gating on splenocytes derived from irradiated BALB/c mice reconstituted with T cells derived from WT (H-2Kb⁺thy1.2⁺) or Spi6^−/− (H-2Kb⁺thy1.2⁺) mice and co-injected with WT (H-2Kb⁺thy1.1⁺) Tregs at 1/1 ratio. Control mice only received T cells without Tregs. Mice were sacrificed at day 10 post-adoptive transfer. Gating on Thy1.2 expression permits differentiation between induced Tregs (thy1.2⁺) and transferred WT Tregs (thy1.1⁺). A1 is a representative example of FACS staining of recipient splenocytes gated on H-2Kb⁺ and CD4⁺ (donor CD4⁺ T cells). A2 and A3 are representative examples of FACS staining of recipient splenocytes gated on H-2Kb⁺, CD4⁺, thy1.2⁺ and FoxP3⁺ (induced Tregs from donor CD4⁺T cells). A2 represents a mouse recipient of thy1.2⁺ T cells only. A3 represents a recipient mouse that received thy1.2⁺T cells in addition to WT thy1.1⁺Tregs. (B) Graph represents the percentage of apoptosis as measured by Annexin expression in induced Tregs from Spi6^−/− T-cells, with or without co-transferred WT Tregs. Transfer of WT Tregs did not rescue induced GrB⁺ Tregs from accelerated cell death. (n=4-6 mice/group)