Extended Figure 5. The development of homeostatic eTregs is not dependent on PD-1, and eTregs are limited by PD-1.

(A-F) Splenocytes from naïve 8 week-old female C57BL/6 mice or total PD-1^−/− mice were isolated and analyzed via high-parameter flow cytometry. (A) Pre-gated CD4⁺ T cells gated on Foxp3+ events (Treg cells) depicting an enrichment of Treg cells at homeostasis in PD-1^−/− age matched hosts (n = 3/group two-tailed unpaired student’s t-test, ** = p = 0.0037, 4 experimental replicates). (B) Comparative flow plots of Treg cells between C57BL/6 and PD-1^−/− hosts with gating on activated Treg cells in cell cycle (CD11a^hi Ki67⁺), demonstrating an increase in Tregs cells undergoing proliferation at homeostasis in PD-1^−/− hosts (n = 3/group two-tailed unpaired student’s t-test, ** = p = 0.0044, 4 experimental replicates). (C) Treg cell staining of ICOS and CTLA-4, depicting the proportion and number of eTreg-associated (ICOS⁺ CTLA-4^hi) Treg cells is increased in PD-1^−/− mice (n = 3/group two-tailed unpaired student’s t-test, ** = p = 0.0022, 4 experimental replicates), while (D) demonstrates this enhancement is specific to the eTreg compartment (BCL-2^low, CD25^low), as the non-eTreg compartment (BCL-2^hi, CD25^hi) is consistent in number when compared to C57BL/6 mice (n = 5/group, 2-way ANOVA with Sidak’s multiple comparisons test, *** = p = 0.0001, 3 experimental replicates). Splenocytes from isotype and anti-PD-L1 treated groups were also stimulated and stained for IL-10 and analyzed via flow cytometry. (E) Flow plots of Treg cells from C57BL/6 and PD-1^−/− hosts gated on CD11a^hi IL-10⁺ events, depicting an increase in the proportion and number of IL-10⁺ Treg cells in PD-1−/− hosts (n = 3/group two-tailed unpaired student’s t-test, ** = p = 0.0011, 3 experimental replicates). (F) Splenic cDC2 subsets were identified via flow cytometry (CD3⁻, B220⁻, CD19⁻, NK1.1⁻, Ly6G⁻, CD64⁻, CD11c⁺, MHC-II⁺, SIRPα⁺), and gated on CD80⁺ events based on an FMO (n = 4/group, two-tailed unpaired student’s t test, * = p = 0.0122, 2 experimental replicates). All data presented are means +/− SEM and show individual data points.