Figure 1: Treg cell heterogeneity at homeostasis and eTreg association with PD-1.

Splenocytes from naïve 8-week old female C57BL/6 mice were analyzed via high-parameter flow cytometry for the following figures. (A) Flow cytometry plots depicting PD-1 expression amongst CD4⁺ Foxp3⁻ T cells (Tconv), in comparison CD4⁺ Foxp3⁺ T cells (Treg) (n = 5/group two-tailed unpaired student’s t-test, **** = p < 0.0001, 6 experimental replicates). (B) UMAP heatmap plot of PD-1 expression amongst Treg cells from (A), with the subsequent X-shift identified subpopulations and delineated PD-1 subsets subdivided into PD-1⁻ (black), PD-1^low (blue), and PD-1^hi (red) regions within the UMAP (see Supplemental Figure 2 for description). (C-F) Heatmap analysis of the above UMAP demonstrating median fluorescence of stained protein expression, with additional simple overlays of positive cells within the UMAP. (C) Expression of activation-associated proteins CD69, CD11a, in addition to an overlay of Nur77⁺ cells within the UMAP. (D) Expression of proliferation-associated Ki67 and an overlay of cMyc⁺ cells within the UMAP. (E) Expression of Treg-associated Helios, and survival-associated proteins CD25, and BCL-2. (F) Expression of Treg effector-associated proteins: ICOS, CTLA-4, PD-L1, CD27, CD43, CD73, and KLRG1. (G) Flow cytometry plot of Treg cells depicting PD-1 and IL-10 expression. (H-K) Flow cytometry plots depicting proportional Treg cell changes with increasing PD-1 expression, based on PD-1⁻, PD-1^low, and PD-1^hi subsetting (as shown in Supplemental Figure 2). (H) Depictions of proportional enrichment of Nur77⁺ Helios⁺ Treg cells (n = 5/group, 1-way ANOVA with Tukey’s multiple comparisons test, **** = p < 0.0001, 4 experimental replicates), in addition to (I) increases in activation-associated CD69⁺ CD11a⁺ Treg cells with increases in PD-1 expression (n = 5/group, 1-way ANOVA with Tukey’s multiple comparisons test, **** = p < 0.0001, 6 experimental replicates). (J) Flow cytometry plots depicting changes in effector-Treg associated proteins ICOS and CTLA-4 (n = 5/group, 1-way ANOVA with Tukey’s multiple comparisons test, **** = p < 0.0001, 5 experimental replicates), with subsequent plots (K) demonstrating an enrichment of CD25⁻ BCL-2^low Treg cells, both with increasing PD-1 expression (n = 5/group, 1-way ANOVA with Tukey’s multiple comparisons test **** = p < 0.0001, 4 experimental replicates). All data presented are means +/− SEM and show individual data points.