Fig 2: The responses of human B cells to VC vary among individuals.(A) Stimulated with CpG (ODN) anti-CD40 and IL-21; (B) Stimulated with SAC and IL-21. (Corresponding to Figure 1F and 1G).

Fig 3: IL-35 drives imbalance of plasma and Bregs in cancer(A) Quantification of tumor weights from WT, BEBi3-/-, and Bp35-/- mice orthotopically injected with KPC cells and collected at 3 weeks post injection. Control WT mice represent both BEBi3+/- and BWT control bone marrow chimera groups.(B and C) (B) Frequency of intra-tumoral naive B cells (percentage of CD45), and (C) CD21hiCD1dhiCD5+ Breg cells (percentage of CD19) and total B cells (percentage of CD45) from Figure 3A as determined by flow cytometry.(D) Representative flow cytometry plots and (E) quantification of frequency of IL-10, and IL-35 expressing Breg cells from Figure 3A.(F and G) (F) Representative flow cytometry plots and (G) quantification of CD19loCD138hi intra-tumoral plasma cells from (A).(H and I) (H) Quantification of IgG and (I) IgM levels in tumors of WT, BEBi3-/-, and Bp35-/- mice 3 weeks post orthotopic injection with KPC cells.(J) Representative antibody-dependent cellular cytotoxicity (ADCC) assay for determination of antibody mediated killing of tumor cells (target cells). Target 1, non-cancerous pancreatic cells; target 2, tumor cells (KPC 4662); ADCC, effector cells + target cells + source of antibodies (serum samples) from tumor-bearing WT, BEBi3-/-, and Bp35-/- mice. RLU, relative luminescence units.(K) Schematic of treatment schedule. Representative flow cytometry plots of intra-tumoral CD19loCD138hi plasma cells from bortezomib (BZ) or control PBS-treated WT and BEBi3-/- mice 3 weeks post orthotopic injection with KPC cells.(L) Quantification of intra-tumoral CD19loCD138hi plasma cells from (K).(M) Quantification of pancreatic tumor weights from (K). Error bars indicate SEM; p values in (A)–(I) were calculated using one-way ANOVA. p values in (J) were calculated using Student’s t test, two tailed, unpaired. p values in (L) and (M) were calculated using two-way ANOVA. NS, non-significant; *p < 0.05; **p < 0.005; ***p < 0.001. Data are representative of three independent experiments.

Fig 4: Vitamin C (VC) promotes the generation of bona fide plasma cells.(A, B) Expression analysis of lineage transcription factors (TFs). B cells were cultured as indicated for 7 days, and the expression of transcription factors was analyzed by intracellular staining and fluorescence-activated cell sorting (FACS) (n = 6). Representative FACS plots (A) and summaries (B) are shown. Note that BLIMP1 and IRF4 are important TF plasma cell differentiation. (C) Antibody secretion by VC-induced plasma cells. Culture supernatants were collected after day 7, and the secreted antibodies with indicated isotypes (IgM, IgG1, IgE) were analyzed by ELISA (n = 6). All data are from at least two independent experiments. Mean ± SEM is shown for bar charts, and the statistical significance was determined by unpaired Student’s t-test. ***p<0.001.