Fig 2: IL21R expression levels decline as B cells differentiate into ASCs.BN cells were activated for 3 days with anti-lg, R848, IFNY and IL-2, then washed and cultured for an additional 3 days with IL-21 and R848 (Be.Y2 cells). ASCs (CD27hiCD38hi and non-ASCs were identified in the Be.Y2 cultures (a) and IL21R expression levels were evaluated in each subset and are reported as histograms (b).

Fig 3: ASC development from BN precursors is enhanced in Th1 containing co-cultures.Cartoon (a) depicting day 6 paired co-cultures containing Th1 (Be1 co-cultures) or Th2 (Be2 co-cultures) effectors generated from the same HD, BN cells from a second allogeneic HD and exogenous IL-21 and IL-2. Flow cytometric analysis showing T-bet expression (b) on gated HD B cells (non-ASCs) from Be1 and Be2 co-cultures. Phenotyping (c) of day 6 B cell-gated Be1 cells showing T-bet expression in combination with other surface markers. (d–g) ASC development in HD day 6 paired Be1 and Be2 co-cultures showing representative flow plots (d) and frequencies (e) of CD38hiCD27+ ASCs in CD19+/lo-gated B lineage cells. Frequencies of IgM+ (f) or IgG+ (g) ASCs in day 6 paired Be1 and Be2 co-cultures. See Figure 2—figure supplement 1 for BN isolation strategy and characterization of polarized Th1 and Th2 effectors. See Figure 2—figure supplement 2 for gating strategy to identify IgG+ and IgM+ ASCs. See Figure 2—figure supplement 3 for proliferation analysis of B cells in paired day 6 HD Be1 and Be2 co-cultures. Analyses in (b–c) are from representative co-cultures (n > 30). Experiments (e–g) performed on 15 (e), 8 (f) or 6 (g) independent paired Be1 and Be2 co-cultures. Statistical analyses were performed using a non-parametric Wilcoxon paired t test (e) or paired Student’s t test (f–g). P values *<0.05, **<0.01, ****<0.0001.

Fig 4: Temporally distinct regulation of T-bethiIRF4int pre-ASC and ASC development by IFN?, R848 and IL-21.Cartoon (a) depicting stimulation of CTV-labeled HD BN cells for 3 days with anti-Ig, R848, IL-21 and IFN? (Step 1). Cells were washed and re-cultured for 3 days with R848, IFN?, and IL-21 (Step 2, +,+ condition) or individual stimuli were included in Step 1 only (+,- condition) or in Step 2 only (-,+ condition). Cells from day 6 cultures containing IFN? (b–e), R848 (f–i) or IL-21 (j–m) in Step 1, Step 2 or both steps were analyzed to determine ASC frequencies (b, f, j), ASC recovery (c, g, k), cell division (d, h, l) and total cell recovery (e, i, m). Summary of data (n) showing that ASC development and recovery from T-bethiIRF4int BDN pre-ASCs requires early IFN?, R848 and BCR ‘priming’ signals and late R848 and IL-21 proliferation and differentiation signals. See Figure 5—figure supplement 1 for representative flow cytometry plots from each culture showing T-bethiIRF4int BDN cells on day 3, CD38hiCD27+ ASCs on day 6 and CTV dilution on day 6. Data are representative of =3 experiments. The percentage of cells in each division, the frequency of ASCs and cell recovery (total and ASCs) are shown as the mean ±SD of cultures containing purified BN cells from 3 independent healthy donors. All statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparison test. P values *<0.05, **<0.01, ***<0.001, ****<0.0001.

Fig 5: ASC formation is augmented and accelerated in Bel co-cultures.(a-o) Proliferation and ASC formation were measured in 5 independent (Examples 1-5) day 5-6 paired Bel and 3e2 Go-cultures that contained purified CTV-IabeIed BN cells and allogenic Thl or Th2 cells plus IL-2 and IL-21 , proliferation analysis (a, d, g, j, m) Of B lineage cells in the day 5-6 co-cultures showing the frequency Of CD19•n0 cells in each cell division. Analysis Of ASC formation in day 5-6 Go-cultures (b, e, h, k, n) showing the frequency of ASCs within the CD19•'1a gated cells. ASCs defined as CD1910CD3BhilRF4hi (b, k), CD19ZCD3BhiCD27• (e) or CD1910CD38hi (h, n). proliferation analysis (c, f, i, l, o) the ASCs and non-ASCs in each cell division. Data are shown as the proportion of cells within each division that are ASCs.