Extended Data Fig. 9: Primary T. gondii infection depletes eTreg cell populations in both C57BL/6 and hemizygous Foxp3^cre x PD-1^wt/flox mice, while eTreg cells in homozygous Foxp3^cre x PD-1^flox/flox hosts are spared.

(a-e) 8 week-old male C57BL/6 (n = 5), Foxp3^cre x PD-1^wt/flox (n = 6), and Foxp3^cre x PD-1^flox/flox (n = 5) mice were IP infected with 20 cysts of T. gondii (ME49 strain), at day 10 of infection splenocytes were harvested from each group and analyzed via high parameter flow cytometry. (a) Flow plots of bulk CD4⁺ T cells from each infected group and were gated on Foxp3⁺ events (Treg cells), depicting similar Treg depletion in C57BL/6 and hemizygous (PD-1^wt/flox) groups, with increased Treg preservation in the homozygous (PD-1^flox/flox) hosts (1-way ANOVA with Tukey’s multiple comparisons test * = p = 0.0227, ** = p = 0.0050, 2 experimental replicates). (b) Flow plots of splenic Treg cells depicting an increase in eTreg-associated ICOS⁺ CTLA-4^hi cells in the Foxp3^cre x PD-1^flox/flox group, but not the C57BL/6 or Foxp3^cre x PD-1^wt/flox cohorts (1-way ANOVA with Tukey’s multiple comparisons test, ** = p < 0.01, 2 experimental replicates). (c) Flow plots depicting enhancement to the eTreg associated BCL-2^low CD25^low compartment in Foxp3^cre x PD-1^flox/flox mice only, while the non-eTreg compartment (BCL-2^hi, CD25^hi) was consistent in number across all three groups (2-way ANOVA with Tukey’s multiple comparisons test *** = p = 0.0002, **** = p < 0.0001, 2 experimental replicates). Splenocytes from all three groups were also stimulated and stained for IL-10. (d) Flow plots of Treg cells and their expression of IL-10 vs CD11a. There is no significant change between C57BL/6 and hemizygous groups, however homozygous mice have a significant increase in the number and proportion of IL-10⁺ Treg cells (1-way ANOVA with Tukey’s multiple comparisons test, ** = p = 0.0011, *** = p = 0.0004). Splenocytes were permeabilized exvivo and stained for the downstream TCR-activation protein Nur77 and analyzed via flow cytometry. (e) Treg cell plots from the three respective groups depicting no significant differences in Nur77⁺ Treg cells between C57BL/6 and hemizygous groups, while Treg cells from homozygous hosts (Foxp3^cre x PD-1^flox/flox) have an increased proportion and number of Nur77⁺ Treg cells compared to the other two groups during infection (1-way ANOVA with Tukey’s multiple comparisons test *** = p < 0.001). All data presented are means + /- SEM and show individual data points.

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