Figure 6. Flow cytometry of Tregs in adoptive transfer experiments show an increased activation state of endogenous Tregs in F8S-SE Treg treated mice.

Endogenous CD45.1⁺ Tregs in HemA mice receiving CD45.2⁺ F8S-SE and Naive-NSE Tregs as described in Figure 4 were analyzed over time. Naïve CD45.1⁺ HemA mice were used as controls, (A) Representative dot plots of CD4⁺CD25⁺Foxp3⁺ lymphocytes from peripheral blood. Tregs were first gated via CD25 and Foxp3 expression as described in Figure 5. Endogenous CD4⁺CD25⁺Foxp3⁺ Tregs were gated on CD45.1 expression (top row). CTLA-4 expression was measured to compare activation states of CD45.1⁺ gated endogenous Tregs (bottom row). (B) The percentage of total Tregs relative to total CD4⁺ cells was compared across all groups. (C) Percentage of activated Tregs endogenous to the recipient mice (CD45.1⁺) was compared across all groups (p<0.00001). The experiments have been repeated at least once without significant variance between experiments. p-values over time were calculated using repeated measures ANOVA.