Analysis of the Treg cell response post LCMV infection in IL-10 reporter (10BiT Thy1.1) mice. (a) Quantification of the number of Tfr cells, non-Tfr Treg cells, preTfh cells, Tfh cells, and GC B cells at days 0, 5, 8, and 12 following infection. Populations are defined as follows: Tfr cells. CD4⁺Ly6C⁻ PSGL1^loCXCR5^hiPD1^hiFoxp3⁺; non-Tfr Treg cells, CD4⁺CXCR5^{int− lo}PD1^{int− lo}Foxp3⁺; preTfh cells, CD4⁺CD44^hiLy6C⁻ PSGL1^loCXCR5^intPD1^intFoxp3⁻; Tfh cells, CD4⁺CD44^hiLy6C⁻ PSGL1^loCXCR5^hiPD1^hiFoxp3⁻; and GC B cells, B220⁺IgD^loGL7⁺CD95⁺. (b) Quantification of the ratio of Tfr cells to Tfh cells or GC B cells at days 0, 5, 8 and 12 following infection. (c) Representative plot of IL-10 expression (assessed as Thy-1.1) by Tfr cells (left) and Non-Tfr Treg cells (middle) from mice as described in a. Right, frequency of Thy1.1⁺ cells in Tfr cells and Non-Tfr Treg cells at days 0, 5, 8, and 12 following LCMV Armstrong infection. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. (*, p < 0.05; **, p < 0.01, ***, p < 0.001). Data are from 2 experiments representative of 4 experiments with 3–6 mice per time point post LCMV Armstrong infection.

Analysis of the B cell response in Il10^f/f and Il10^f/f Foxp3-Cre mice 15 days following LCMV infection. (a) Representative plots of the B cell responses in Il10^f/f or Il10^f/fFoxp3-Cre mice (top). Bottom: frequency and number of cells as indicated by the gates shown above. (b) Representative confocal images of the GC from Il10^f/f or Il10^f/f Foxp3-Cre mice. Right, GC sizes as measured by imageJ. The sections were taken from four mice of each genotype. Scale bar=100um. (c) Frequency of plasmablasts and memory B cells defined by the expression of surface markers (left). Right, absolute numbers of plasma cells and memory B cells. (d) ELISA quantification of LCMV-specific IgG2a and IgG1 antibody levels, indicated by arbitrary unit (a.u.). All the analyses of the GC response were performed 15 days post acute LCMV Armstrong infection. (e) ELISPOT quantification of the number of LCMV-specific IgG2a and IgG1 memory B cells. All the analyses of the GC response were performed 60 days post acute LCMV Armstrong infection. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. (*, p < 0.05; **, p < 0.01). Data for panels a-c are from one experiment representative of 3 experiments with at 3–5 mice per group carried out at 15 days and 4 experiments with 3–5 mice per group carried out 12 days following LCMV Armstrong infection. Data for panel d are pooled from 2 experiments carried out at days 12 or 15 post infection. Data for panel e are pooled from 2 experiments carried out day 60 post infection with 5–7 mice per group.

(a) Analysis of the B cell response day 15 post acute LCMV-Armstrong infection in 50:50 Il10^f/f: Bcl6^f/fFoxp3-Cre or Il10^f/fFoxp3-Cre: Bcl6^f/fFoxp3-Cre mixed BMCs. Schematic for experiment (left). Data are pooled from 2 experiments with 4–5 mice per group carried out 15 days following LCMV Armstrong infection (right). (b) Analysis of the B cell response at day 15 post acute LCMV-Armstrong infection in 50:50 Il10^f/f: SAP^−/− or Il10^f/fFoxp3-Cre: SAP^−/− mixed BMCs. Schematic for experiment (left). Data are from one experiment representative of 2 experiments with 4–9 mice per group carried out 15 days following LCMV Armstrong infection (right). Statistical analyses were performed using the unpaired two-tailed Student’s t-test. (*, p < 0.05; **, p < 0.01).

(a) GC B cells and dendritic cells but not Tfh cells are responsive to IL-10. Analysis of IL-10 responsiveness in GC B cells, dendritic cells (CD11c^hiMHCII⁺), and Tfh cells. Splenocytes isolated from mice at day 12 post LCMV infection were stimulated for 30 minutes with IL-10 and phospo-STAT3 levels determined. Data are from one experiment representative of 3 experiments with 4 mice per group. (b) Analysis of the B cell response in Il10rα ^f/f, Il10rα^f/fCd19-Cre, Il10rα^f/fCd11c-Cre, and Il10rα^f/fCd4-Cre mice 15 days following LCMV infection. Top: Representative plots of B cell response. Bottom: quantification of B cell response shown above. Statistical analyses were performed using the unpaired two-tailed Student’s t-test (*, p < 0.05; **, p < 0.01). Data are from one experiment representative of 2–3 experiments with 3–7 mice per group carried out 15 days following LCMV Armstrong infection. Mice for the control Il10rα^f/f group were pooled from littermate controls produced from the independent crosses used to generate the experimental groups.

(a) RNA-seq analysis of select differentially expressed genes among mRNA isolated from GC B cells pooled from Il10^f/f and Il10^f/fFoxp3-Cre mice 12 days after infection with LCMV Armstrong presented as expression (log2) in Il10^f/f Foxp3-Cre cells relative to that in Il10^f/f cells (key below; columns indicate paired replicates). (b) GSEA of light zone and dark zone signatures in GC B cells, based on published gene sets (, ). A positive ES signifies enrichment in the Il10^f/f Foxp3-Cre sample relative to the Il10^f/f condition of a given gene set; i.e., more highly expressed. (c) Normalized enrichment score (NES) for select pathways identified from the Reactome Pathway Database (R), Kegg Pathway Database (K) and published gene sets (G), where dot size represents p-values adjusted by the family-wise error rate (FWER). All gene sets attain significant enrichment (FDR<.0.001), with the exception of the dark zone gene set (FDR=0.5). Data are from three independent experiments with three mice per group pooled for each sample. (d) Analysis of the light zone and dark zone GC B cell response in Il10^f/f and Il10^f/f Foxp3-Cre mice 15 days post LCMV infection. Representative plots (left) of GC B cells as gated in . The numbers of the outlined area indicate dark zone (top left) and light zone (bottom right) based on the expression of surface markers CXCR4 and CD86. Middle, frequency of GC B cells from light zone gate defined by CXCR4⁺CD86⁻ and dark zone gate defined by CXCR4⁻ CD86⁺ from Il10^f/f or Il10</i>^f/f Foxp3-Cre mice. Right, expression of CXCR4 in GC B cells. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. (*, p < 0.05). Data are from one experiment representative of 2 experiments with at least 4 mice per group carried out 15 days following LCMV Armstrong infection.

(a) Ingenuity Pathway Analysis (IPA) canonical pathway analysis. The number on the x-axis indicates the negative log p-values adjusted by the Benjamini-Hochberg (B–H) procedure, which is the significance of the probability that the genes in the dataset described in Figure 6 are associated with the canonical pathway listed. The orange line represents the threshold of significance for a B–H multiple testing correction p-value of 0.05. (b) IPA upstream regulator analysis. The top upstream regulators that are predicted to be responsible for the gene expression changes observed in the Il10^f/fFoxp3-Cre sample relative to the Il10^f/f sample are shown. A negative activation z-score indicates that the upstream regulator is predicted to be inhibited in the Il10^f/f Foxp3-Cre sample relative to the Il10^f/f sample and a positive score indicates that the upstream regulator is predicted to be activated. (c) Expression of FOXO1 in GC B cells from Il10^f/f or Il10^f/f Foxp3-Cre mice 15 days after infection with LCMV. Data are from one experiment representative of 2 experiments with at least 4 mice per group carried out 15 days following LCMV Armstrong infection. (d) Assessment of the percentage of nuclear translocated FOXO1 following IL-10 stimulation in IgD^lo B cells 5 days post LCMV infection as determined by Amnis Imagestream. Statistical analyses were performed using the paired two-tailed Student’s t-test (*, p < 0.05; **, p < 0.01). Data are pooled from 3 experiments with 4 mice per group carried out 5 days following LCMV Armstrong infection. (e) Assessment of the number of mutations in the CDR region (left) and selection strength (right) in both the CDR region and framework region of a large number of the heavy chain V_H genes of GC B cells from Il10^f/f or Il10^f/f Foxp3-Cre mice 15 days after infection with LCMV. Data are pooled from 2 experiments with 3–4 mice per group carried out 15 days following LCMV Armstrong infection.