Fig 2: Perforin-Deficient Animals Have Enhanced GC Expansion and Somatic Mutation Rates Comparable to NK-Cell-Depleted Animals(A) Total number of GC TFH (CD4+CD44hiCXCR5+ PD-1hi) cells in the spleen of C57BL/6 (WT) or perforin-deficient (Prf1-/-) control-treated (mIgG2a) animals day 12 post-NP-KLH immunization.(B) Total number of NP+IgG1+ GC B (CD19+B220+ Fas+GL-7+) cells. Data were analyzed via Stu-dent’s t test; n = 3–8 mice/group.(C) Total number of mutations in control or a-NK1.1-treated WT or Prf1-/-animals day 12 post-immunization. Data were analyzed via Kruskal-Wallis with multiple testing correction; n = 5–6 mice/group.(D) Proportion of unique VH186.2 sequences from (C) bearing W33L mutation. Experiments were repeated 2 or 3 times.

Fig 3: Transcriptional profiling identifies disease-specific naive B cell states(A) Volcano plot of differentially expressed genes (red, upregulated; blue, downregulated) with adjusted p (padj) <0.05 in naive B cells from (Figure S3A).(B) Bar plots of the top enriched GO biological processes in healthy and tumor-educated naive B cells.(C) Bar plots of the top enriched GO biological processes in healthy and tumor-educated naive BEbi3-/- cells.(D) Loading plot overlayed on principal-component analysis (PCA) as derived by differentially expressed gene (DEG) analysis between healthy wild-type (WT), healthy B cell-specific Ebi3 KO (BEbi3-/-), tumor-educated WT (WT_tum), and tumor-educated B cell-specific Ebi3 KO (BEbi3-/-_tum) naive B cells.(E) Heatmap of selected differentially expressed genes in naive B cells from healthy or tumor-bearing WT and BEBi3-/- mice. The standardization of log expression was performed by row.(F) Quantification of intracellular pSTAT3 and Pax5 as determined by flow cytometry in splenic Breg cells isolated from WT mice and activated with aCD40/LPS (Sti, blue) and aCD40/LPS + IL-35 (red) for 48 h.(G) Quantification of intracellular pSTAT3 (left), Pax5 (middle), and BCL6 (right) analyzed by flow cytometry from intra-tumoral Bcon and Breg.(H) Relative levels of indicated gene expression in each group, as determined by RNA-seq of peripheral CD19+CD24hiCD38hi Breg or CD19+CD24loCD38lo B conventiona (Bcon) cells from PDAC patients.(I) Quantification of IL-35 expression from Breg and Bcon cells isolated from spleens of PDAC patients (n = 25).(J) The mean fluorescence intensity (MFI) of the intracellular levels of pSTAT3 (left) and Pax5 (right) in Breg and Bcon cells isolated as in (H). Data are representative of three independent experiments. Error bars indicate SEM. NS, non-significant; *p < 0.05; **p < 0.005 (Student’s t test, two tailed, unpaired).

Fig 4: 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.