Figure 4. IFNγ is required for development of T-bet^hi B_DN cells and regulates ASC formation and recovery.

(a) Ingenuity Pathway Analysis (IPA) to identify predicted upstream direct and indirect regulators of the HD B_DN Be1 transcriptome. IPA performed using the 427 DEG (B_DN Be1 over B_DN Be2; FDR < 0.05) identified in the RNA-seq analysis described in Figure 3b. The predicted activation state (z-score of B_DN Be1 over B_DN 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-bet^hiIRF4^int B_DN pre-ASC-like cells from HD B_N cells. Cartoon (b) depicting in vitro stimulation conditions to activate purified HD B_N cells with cytokines (IL-2, BAFF, IL-21, IFNγ), 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 IgD^negCD27^neg B_DN subset. (e–h) Cartoon (e) depicting HD B_N cells activated with anti-Ig +cytokine cocktail (IFNγ, 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 CD38^hiCD27⁺ ASCs (h) in day 6 ‘ALL’ cultures. (i–j) Transient BCR activation is required for ASC development. Cartoon (i) depicting activation of HD B_N cells for 3 days with R848, cytokines (IFNγ, 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 CD38^hiCD27⁺ 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) IFNγ, R848 and IL-21 are required for ASC development. Cartoon (k) showing HD B_N cells activated with anti-Ig + cytokine cocktail (IFNγ, 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 CD38^hiCD27⁺ 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 B_N 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.

Figure 4—figure supplement 1. IFNγ, R848 and IL-21 play distinct roles in facilitating the development and recovery of ASCs in in vitro cultures.

HD B_N cells were activated as described in Figure 4 and analyzed on day 6. Data shown include representative flow panels depicting % CD38^hiCD27⁺ ASCs recovered (a) quantitation of % ASCs recovered across multiple cultures (b) and total cells recovered in the cultures (c). The frequency of ASCs (b) and cell recovery (c) are shown as the mean ±SD of cultures containing purified B_N cells from 3 independent healthy donors. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparison test. P values *<0.05, **<0.01, ***<0.001, ****<0.0001. The number of total ASCs recovered is shown in Figure 4l.