Fig 2: Transcriptional analyses of vTreg53 Treg cells stimulated by Fat1562 in vivo.(A–C) CD45.2+ vTreg53 Treg cells were transferred i.v. into CD45.1+ B6 mice. 6 weeks later, the recipient mice were immunized with CFA alone or CFA/Fat1562 s.c., and one week later, boosted with IFA alone or IFA/Fat1562 i.p. CD45.2+ clonotype+ Treg cells were sorted from Spl/LNs or VAT of the recipient mice one week later for RNA-Seq. (A) Volcano plot comparing gene expression of transferred clonotype+ Treg cells in the VAT and Spl/LNs of recipient mice immunized with adjuvant alone (left) or with adjuvant/Fat1562 (right). VAT-Treg signature genes are highlighted in red (induced) or blue (repressed). The number of genes from each signature preferentially expressed by one or the other population are shown at the top. (B) GSEA analysis of top KEGG pathways (p<0.05) that are enriched in cells activated by adjuvant/Fat1562 compared with adjuvant alone in VAT (left) or Spl/LN (right). NES, normalized enrichment score. FDR, false discovery rate. (C) Normalized reads of Pparg transcript in CD45.2+ clonotype+ Treg cells sorted from mice immunized with adjuvant alone or adjuvant/Fat1562. Data are mean ± SD. (D) Volcano plot comparing gene expression of transferred clonotype+ Treg cells in mice immunized with adjuvant/Fat1562 and adjuvant alone in the VAT (left) and spleen (right). Representative transcripts are highlighted.

Fig 3: Immunization with Fat1562 expands transferred vTreg53 Treg cells and suppresses HFD-induced VAT inflammation.(A) Scheme of the transfer and immunization protocol. (B) Frequencies of CD45.2+ cells in Treg cells (n = 7). (C) Frequencies of clonotype+ cells in transferred CD45.2+ Treg cells (n = 7). (D) Number of clonotype+ CD45.2+ Treg cells in the spleen (n = 7). (E) Number of clonotype+ CD45.2+ Treg cells in the VAT (n = 7). (F) Number of total Treg cells in the spleen (n = 7). (G) Number of total Treg cells in VAT (n = 7). (H) Numbers of eosinophils and CD11chi inflammatory macrophages in VAT (n = 7). Data are mean ± SD. Data shown are representative of at least two independent experiments.

Fig 4: Type I IFNs Act on DCs to Suppress Development of Antigen-Specific CD4+ T Cells(A) A 90:10 mix of B6.CD45.1/CD45.2 and Ifnar1-/- (CD45.2) BM cells was engrafted into lethally irradiated CD45.1 recipients.(B) Following 10–12 weeks of engraftment, chimeric mice were infected with L. donovani, and 14 days later, spleen and liver mononuclear cells were isolated to assess the activation status of control B6 (CD45.1/CD45.2) and Ifnar1-/- (CD45.2) CD4+ T cells from the same mouse using the gating strategy shown.(C) The frequency of splenic and hepatic CD4+ T cells recently activated by parasite antigen (CD11ahi/CD49dhi), Th1 cells, and Tr1 cells (gated as shown in Figure S2) was measured by flow cytometry, as indicated.(D) Ifnar1?DC mice generated by crossing Cd11c-Cre mice with Ifnar1-floxed animals were infected with L. donovani, and 14 days later, Th1 cell number and frequency as well as parasite burden in the spleen and liver were measured, as indicated.n = 4–10 mice per group. Experiments were conducted 3 times. Mean ± SEM (C and D), *p < 0.05; significance assessed by Mann-Whitney test.

Fig 5: Late-stage Treg cell depletion enhances the emergence of invasive OSCC(A) Experimental setup. 6- to 8-week-old Foxp3WT/y and Foxp3DTR/y littermates were exposed to 4-NQO drinking water for 20 weeks. During the 17th week of 4-NQO exposure, all mice were treated intraperitoneally every other day with diphtheria toxin (DT) for 1 week. Mice were sacrificed either 3 weeks later, at the 20-week endpoint, for histopathology analysis and IHC staining (B–F), or after the 1-week DT treatment, at 17-week endpoint, for flow cytometry analysis (G–I).(B) Tumor incidence at 20-week endpoint, where each mouse was scored once based on most severe histology grade observed. Summary plot of pooled data shows the percentage of mice scored as indicated histology grade. Each bar represents the cumulative data for mice of the indicated genotype. n = 18 mice (Foxp3WT/y); n = 24 mice (Foxp3DTR/y).(C) Tumor burden at 20-week endpoint. Summary plot of pooled data shows the percentage of tongue perimeter defined as the indicated histology grade. Each symbol represents an individual tongue from a single mouse of the indicated genotype. Median is indicated. n = 18 mice (Foxp3WT/y); n = 24 mice (Foxp3DTR/y).(D) Representative CD3 IHC image of SCC region of tongue isolated from mice of the indicated genotype, at 20-week endpoint. Scale bar represents 100 µm.(E) Summary plot of pooled data from CD3 IHC density analysis at 20-week endpoint, showing the number of CD3+ cells per mm2 for each lesion. Each symbol represents an individual lesion. Median is indicated. n = 3–110 lesions from 18–24 mice per group.(F) Summary plot of pooled data from Foxp3 IHC density analysis at 20-week endpoint, showing the number of Foxp3+ cells per mm2 for each lesion. Each symbol represents an individual lesion. Median is indicated. n = 3–110 lesions from 18–24 mice per group.(G) Representative flow cytometric analysis of T cells isolated from whole tongues of 4-NQO-treated mice of indicated genotype at 17-week endpoint, after 1 week of DT treatment. Left plots depict TCRß versus TCR?d expression by CD3+ T cells, middle plots depict CD4 versus CD8a expression by aßT cells, and left plots depict CD4 versus Foxp3 expression by CD4+ T cells. The frequency of cells within the indicated gates is denoted.(H and I) Summary plots of pooled data from (G), showing the frequency of TCRß+ T cells among CD45+ cells (H) or number of TCRß+, CD8+, or CD4+ Foxp3neg T cells per mg of tissue (I), isolated from whole tongues of 4-NQO-treated mice of indicated genotype. Each symbol represents an individual mouse. Mean is indicated. n = 8 mice (Foxp3WT/y); n = 9 mice (Foxp3DTR/y).Data are pooled from multiple independent experiments. Fisher’s test (B); two-tailed nonparametric Mann Whitney test (C, E, F, H, and I). *p < 0.0.5, **p < 0.01, and ***p < 0.001. See also Figure S3.