Panel A contains violin plots. The markers include Sell, Il7r, Itgae, Cxcr6, Tbx21, Tigit, Lag3, Pdcd1, Tnfrsf9, Icos, Ccr8, and Il2ra. Each subpopulation is color-coded for easy identification. Panel B shows flow cytometry plots defining the gating strategy for naïve, suppressive, memory-like, and recently activated/T follicular regulatory-like subpopulations of islet Foxp3-positive regulatory T cells. The naïve population is defined as CD62L-positive. The suppressive population is defined as CD62L-negative CD103-positive and/or CXCR6-positive. The memory-like population is defined as CD62L-negative CD103-positive CXCR6-positive CD127-positive. The recently activated/T follicular regulatory-like population is defined as CD62L-negative CD103-positive CXCR6-positive CD127-negative. It also displays the expression patterns of T-bet, TIGIT, LAG3, Programmed Cell Death Protein 1, Inducible T-cell Costimulator, C-C Motif Chemokine Receptor 8, and CD25 in these subpopulations. Panel C presents representative flow cytometry plots showing CD137 expression in the four subpopulations of islet Foxp3-positive regulatory T cells. Panel D summarizes the results from two experiments, showing the percentage of CD137-positive cells in each subpopulation. Panel E shows the frequencies of Foxp3-positive regulatory T cells among CD4 T cells in the islets of different mouse genotypes. Panel F displays the frequencies of suppressive, naïve, memory-like, and recently activated/T follicular regulatory-like subpopulations of Foxp3-positive regulatory T cells in the islets of different mouse genotypes.
Subset analysis of islet Foxp3 + Tregs. (A) Violin plots showing the expression levels of select markers in the suppressive, naïve, memory-like, and recently activated/Tfr-like Foxp3+ Treg clusters shown in Fig. 5 A. (B) Gating strategy defining naïve (CD62L+), suppressive (CD62L− CD103+ and/or CXCR6+), memory-like (CD62L−CD103−CXCR6−CD127+), and recently activated/Tfr-like (CD62L−CD103−CXCR6−CD127−) subpopulations of islet Foxp3+ Tregs in WT NOD mice and their expression patterns of T-bet, TIGIT, LAG3, PD-1, ICOS, CCR8, and CD25. (C and D) CD137 expression in suppressive, naïve, memory-like, and recently activated/Tfr-like subpopulations of islet Foxp3+ Tregs in WT NOD mice. (C) Representative flow cytometry plots showing CD137 expression in the four subpopulations of islet Foxp3+ Tregs. (D) Summarized results from two experiments. **P < 0.005; ****P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparisons test. ns: not significant. (E) Frequencies of Foxp3+ Tregs among CD4 T cells in the islets of Cre+-Tnfrsf9+/+, Cre+-Tnfrsf9E7fl/fl, and Cre+-Tnfrsf9fl/fl mice. Combined results from more than two experiments are shown. ***P < 0.0005; ****P < 0.0001 by an unpaired t test. (F) Frequencies of suppressive, naïve, memory-like, and recently activated/Tfr-like subpopulations of Foxp3+ Tregs in the islets of Cre+-Tnfrsf9+/+, Cre+-Tnfrsf9E7fl/fl, and Cre+-Tnfrsf9fl/fl mice. Combined results from more than two experiments are shown. *P < 0.05 by an unpaired t test. ns: not significant.