Supplementary Figure 5: Enhanced de novo Foxp3⁺ cell induction from _ICT_N cells utilizes IFN-I pathways but does not involve IL-10, PDL-1 or IDO.

(a) DR cells from mice treated with pIC, LPS, or gardiquimod were cultured 5 days with Ova-pulsed APCs with or without TGFβ, and intracellular Foxp3 was assessed. Bar graph depicts mean plus SEM of triplicate values of fold difference in % Foxp3⁺ relative to average control % Foxp3⁺ in PBS (ctr) DR cells. (b) Total spleen and lymph node cells were harvested from DR mice and incubated with either PBS, 3μg of polyI:C alone (pIC) or with blocking anti-IFNaR antibody (pIC + aIFNaR Ab), or recombinant IFNβ (IFNβ). After 48 hours, naïve CD4⁺ T cells were purified by MACS and cultured with Ova-pulsed APCs and TGFβ, and 3 days later stained for intracellular Foxp3. Experimental schematic shown (left). Bar graph depicts mean plus SEM of triplicate values of fold difference in % Foxp3⁺ relative to average control % Foxp3⁺ in PBS (ctr) DR cells. Data are representative of two independent experiments, n=2 mice per group. (c) WT and Ifnar^KO DR cells were cultured for 5 days in non-skewing conditions, then stimulated with PMA+Ionomycin and assessed for IFNγ production by intracellular cytokine staining. Bar graph depicts % IFNγ⁺ DR cells, mean plus SEM for triplicate values. Data are representative of two independent experiments, n=2 mice per group. (d) mRNA expression of Ifnar1 and Ifnar2 from purified DR cells from control and polyI:C-treated donors. Bar graphs depict fold change in expression of indicated mRNA from polyI:C-treated cells vs controls (set to 1) after normalization to Gapdh mRNA. Mean plus SEM of fold change ratios from three independent experiments, n=3 mice per group. (e) DR cells were rested overnight, then stimulated for 30 minutes with recombinant IFNβ and stained for intracellular phospho-STAT1. PBS added for 30 minutes as negative control. FACS histogram representative of two independent experiments. (f-g) DR cells from ctr or pIC-treated mice were cultured with Ova-peptide-pulsed APC and TGFβ in the presence of (f) rat IgG2a isotype control, anti-IL-10, or anti-PDL1 neutralizing antibodies or (g) NaOH vehicle control or 1-MT, and intracellular Foxp3 was assessed. (h-i) WT or Ifnar^KO DR cells were transferred into WT or Ifnar^KO Balb/c recipients, and host mice were treated with PBS (ctr) or pIC. Data are representative of two independent experiments, n=2 mice per group. (h) Expression of Ly6C on donor DR cells directly ex vivo. (i) DR cells were cultured with Ova-pulsed APCs in non-skewing conditions and at day 5 cultures were stimulated with PMA+Ionomycin and intracellular IFNγ was assessed. Mean plus SEM of fold change ratios from three independent experiments, n=3 mice per group. P-values by student’s two-tailed t-test, *p<0.05, **p<0.01.