Fig 2: IFN? signaling promotes chromatin accessibility and poises B cells to undergo IL-21 dependent differentiation.(a–b) Cell division and total cell recovery in day 3 Be.0, Be.IFN?, Be.IL2 and Be.?2 cultures generated with HD BN cells. (c–k) Chromatin accessibility analysis using ATAC-seq data from day 3 Be.0, Be.IFN?, Be.IL2 and Be.?2 cell. Heatmap (c) showing 15,917 differentially accessible regions (DAR) based on FDR < 0.05. Chromatin accessibility plots and histograms for T-bet (d), STAT5 (e), NF-?B p65 (f), NF-?B REL (g), IRF4 (h) and BLIMP1 (i). Plots report reads per million (rpm) in the 100 bp surrounding the transcription factor binding motifs and histograms show accessibility at the indicated motif and for the indicated surrounding sequence. Genome plots showing chromatin accessibility for the PRMD1 (j) and IL21R (k) loci. DAR are shown and consensus T-bet, IRF4 and STAT5 binding motifs within DAR are indicated. DAR are aligned with previously reported T-bet binding sites in GM12878 cells (assessed by ChIP, ENCODE Project Consortium, 2012) and with ATAC-seq data derived from B cell subsets purified from SLE patients (Jenks et al., 2018). Data reported in rpm. (l–m) Early IFN? signals control IL-21R expression levels. Representative flow plots showing IL-21R expression in day 3 (l) and day 6 (m) Be.0, Be.IFN?, Be.IL2 and Be.?2 cells. See Figure 7—figure supplement 1 for representative flow plots showing cell division profile. See Supplementary file 3 for ATAC-seq data. See Supplementary file 4 for enrichment of TF binding motifs P values. ATAC-seq analysis was performed on 3 independent experimental samples/group over 2 experiments. Flow cytometry plots depicting IL-21R expression are representative of =2 experiments. Box plots (d–i) show 1st and 3rd quartile range (box) and upper and lower range (whisker) of 2 samples/group.

Fig 3: HIV exposure modulated transcriptional regulation of mucosal T cells.Mucosal immune cells isolated from the colorectal tissue biopsies by enzymatic digestion were exposed to HIV-1BaL (250 ng/mL), either free (F-HIV), complement-opsonized (C-HIV), or virions opsonized by a cocktail of complement and antibodies (CI-HIV) or mock-treated, by spinning the cultures. The effect HIV exposure had on mucosal T cells phenotype was assessed after 4 days. (A–D) The immune cells were stained with CD3, CD4, CD8, Tbet, EOMES, PD-1, LAG3, TIM3, and CD160 mAbs. (A) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed Tbet+CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160. viSNE plot of PD-1+Tbet+CD4+ was constructed on CD4 T cells, data presented as dot plots with colored channels. Data from a donor shows CD4 T cells, subjected to tSNE algorithm, which provides cells with a unique coordinate according to its expression of PD-1+T-bet+ parameters, displayed on a two-dimensional plot (tSNE1 versus tSNE2). The heat gradient (blue to red) indicates expression level of Tbet+PD-1+ on CD4 following exposure to different conditions of viruses. (B) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed Tbet+EOMES+ CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160 were assessed by flow cytometry. Flow cytometry Zebra plots for Tbet+EOMES+ CD4+ T cells expressing PD-1. (C) Percentage of Tbet+CD8+ T cells expressing PD-1, TIM3, LAG3 or CD160. viSNE plot of PD-1+Tbet+CD8+ was constructed on CD8 T cells, and data are presented as dot plots with colored channels. Data from a donor shows CD8 T cells, subjected to tSNE algorithm, which provides cells with a unique coordinate according to its expression of PD-1+T-bet+ parameters, displayed on a two-dimensional plot (tSNE1 versus tSNE2). The heat gradient (blue to red) indicates expression level of Tbet+PD-1+ on CD8 following exposure to different conditions of viruses. (D) Percentage of unexposed, F-HIV, C-HIV, or CI-HIV exposed Tbet+EOMES+ CD4+ T cells and percentage of Tbet+EOMES+ CD4+ T cells expressing PD-1, TIM3, LAG3 or CD160 were assessed by flow cytometry. Flow cytometry Zebra plots for Tbet+EOMES+ CD8+ T cells expressing PD-1 (N = 12–15). Statistical significance was tested using repeated measures of ANOVA followed by Tukey's posttest. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM.

Fig 4: IFN? is required for development of T-bethi BDN cells and regulates ASC formation and recovery.(a) Ingenuity Pathway Analysis (IPA) to identify predicted upstream direct and indirect regulators of the HD BDN Be1 transcriptome. IPA performed using the 427 DEG (BDN Be1 over BDN Be2; FDR < 0.05) identified in the RNA-seq analysis described in Figure 3b. The predicted activation state (z-score of BDN Be1 over BDN Be2) of each regulator/signaling pathway is shown as bar color (orange, activated; blue, inhibited) with predicted upstream regulators sorted in order of significance (overlap P value). Regulators with an overlap P-value<0.00001 are shown. (b–d) IPA-identified stimuli induce development of T-bethiIRF4int BDN pre-ASC-like cells from HD BN cells. Cartoon (b) depicting in vitro stimulation conditions to activate purified HD BN cells with cytokines (IL-2, BAFF, IL-21, IFN?), anti-Ig and R848 for 6 days. Phenotypic characterization of day 6 activated cells showing expression of IRF4 and T-bet (c) and other markers (d) on the IgDnegCD27neg BDN subset. (e–h) Cartoon (e) depicting HD BN cells activated with anti-Ig +cytokine cocktail (IFN?, IL-2, IL-21, BAFF) and R848 (ALL) or activated with individual stimuli (as indicated) removed from the cultures. Representative flow plots showing T-bet and IRF4 expression (f–g) by day 3 B cells in each culture. Enumeration of CD38hiCD27+ ASCs (h) in day 6 ‘ALL’ cultures. (i–j) Transient BCR activation is required for ASC development. Cartoon (i) depicting activation of HD BN cells for 3 days with R848, cytokines (IFN?, IL-2, IL-21, BAFF) ±anti-Ig (Step 1). Cells were then washed and recultured for an additional 3 days with the same stimuli ± anti-Ig (Step 2). Enumeration of CD38hiCD27+ ASCs (j) on day 6 in cultures that were not exposed to anti-Ig during Steps 1 and 2 (-,-); were exposed to anti-Ig throughout Steps 1 and 2 (+,+); were exposed to anti-Ig only in Step 1 (+,-); or were exposed to anti-Ig only in Step 2 (-,+). (k–l) IFN?, R848 and IL-21 are required for ASC development. Cartoon (k) showing HD BN cells activated with anti-Ig + cytokine cocktail (IFN?, IL-2, IL-21, BAFF) and R848 for 3 days (Step 1) and then cultured for an additional 3 days (Step 2) with cytokine cocktail and R848. Alternatively, individual stimuli (as indicated) were excluded from the cultures for all 6 days. Enumeration of day 6 CD38hiCD27+ ASCs (l). See Figure 4—figure supplement 1 for % ASCs and number of total cells recovered in cultures lacking individual stimuli. RNA-seq IPA analysis was performed on n = 3 samples/subset derived from 3 independent paired co-culture experiments. Data in (c–l) are representative of =3 experiments. The recovery of ASCs in (j, l) are shown as the mean ±SD of cultures containing purified BN cells from 3 independent healthy donors. Statistical analyses (j, l) were performed using one-way ANOVA with Tukey’s multiple comparison test. P values *<0.05, **<0.01.

Fig 5: Recognition of viable aortic Th17 and Th17.1 cells. (A) Representative dot plots showing the gating strategy for aortic CD3+CD4+RE+CD200+ T cells. (B) mRNA levels of indicated transcription factors in total CD4+ T cells (CD4), RE+200+ T cells, and RE- T cells. Un: under detection threshold. (C) Representative dot plots showing the gating strategy for R3+R4- cells, R3+R4+ cells, and R3-R4+ cells in aortic RE+200+ T cells. (D) Representative histograms showing ROR?t and T-bet proteins in R3+R4- cells, R3+R4+ cells, and R3-R4+ cells. (E) The mean fluorescence intensities (MFI) of ROR?t and T-bet in the subsets. N = 3 or 4. *: p < .05. **: p < .01. ***: p < .001.