Fig 2: 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 3: Tumor-educated naive B cells are primed for dysfunction(A) Intracellular levels of EBi3 and p35 in naive B cells from WT and orthotopic KPC (oKPC) mice treated with LPS/aCD40 ± indicated cytokines for 72 h as determined by flow cytometry.(B) Intracellular levels of Pax5 in naive B cells from WT and oKPC mice treated as in (A) were determined by flow cytometry.(C) Intracellular levels of Pax5 and BCL6 in LPS/aCD40/rIL-35 treated naive B cells isolated from WT and oKPC mice as determined by flow cytometry.(D–G) (D) Expression of Ebi3 and p35, (E) CD1d and IL-10, (F) Pax5 and Bcl6, and (G) pSTAT1 and pSTAT3 in naive B cells from WT and spontaneous KPC (sKPC) mice treated as in (A) were determined by flow cytometry.(H–J) (H) Fold change in expression of P35, EBI3, IL10; (I) BCL6 and PAX5; and (J) PRDM1 and XBP1 from naive B cells isolated from spleens of PDAC patients and activated with aCD40/LPS ± rIL-35.(K) Heatmap of top 30 differentially expressed chromatin and DNA modifiers, fold expression is indicated.(L–N) (L) Relative H3K27 methylation, (M) HDAC1, and (N) H3K27 acetylation enrichment as determined by ChIP assay at p35, EBi3, and Cd1d gene promoters. Naive B cells isolated from WT (blue) and oKPC (red) mice were treated with aCD40/LPS and rIL-35 for 72 h then used for downstream ChIP analysis.(O and P) (O) Relative H3K27 acetylation enrichment as determined by ChIP assay at EBI3, and (P) P35 gene promoters from Breg and Bcon cells isolated from spleens of PDAC patients. Data are representative of three independent experiments. Error bars indicate SEM. NS, non-significant, *p < 0.05, **p < 0.01, ***p < 0.005 (Student’s t test, two tailed, unpaired).

Fig 4: BCL6 inhibition in tumor-educated naive B cells restrains tumor growth(A) Experimental schematic used to test in vivo effect of Bcl6 inhibition (Bcl6i) in B cells on pancreatic tumor growth. Naive B cells from oKPC mice were treated with aCD40/LPS/IL-35 ± Bcl6i and transferred into µMT mice, followed by orthotopic injection of KPC cells.(B) Quantification of tumor weights from µMT mice as in (A).(C–E) (C) Absolute number of intra-tumoral B cells, (D) Breg cells, and (E) plasma cells in mice from (B).(F) Quantification of IgG and IgM levels in tumors from mice in (B).(G) Correlation between intra-tumoral Breg cells and IgG levels from mice in (B).(H) Fold change in expression of p35, EBi3, and IL-10 as determined by qPCR in intra-tumoral Breg cells isolated from mice in (B).(I) Frequency of intra-tumoral T effector (CD45+CD4+CD25-) and cytotoxic T cells (CD45+CD8+) in mice from (B).(J) Quantification of tumor weight from µMT mice 3 weeks post orthotopic adoptive transfer of pre-treated B cells and intra-pancreatic injections with KPC cells as in (A) ± treatment with aPD-1 on days 7, 9, and 11.(K) Frequency of intra-tumoral cytotoxic T cells (CD45+CD8+) in mice from (J).(L) Quantification of tumor weight from µMT mice 3 weeks post orthotopic adoptive transfer of pre-treated B cells and intra-pancreatic injections with KPC cells as in (J) ± depletion of CD8 T cells. Error bars indicate SEM; p values were calculated using two-tailed, unpaired Student’s t test. NS, non-significant, *p < 0.05, **p < 0.005, ***p < 0.001. Data are representative of three independent experiments. Experiments using CD8+ depletion were performed with 7- to 8-week-old µMT mice with at least six mice per group in duplicate.

Fig 5: IL-35-STAT3 axis deregulates naive B cells in pancreatic cancer(A) Representative flow cytometry histogram plot of intracellular IL10, p35, EBi3, and surface CD1d in naive B cells isolated from oKPC mice and treated as indicated.(B) Quantification of IL10, p35, EBi3, and CD1d in naive B cells treated as in (A), with STAT1 inhibitor (left) and STAT3 inhibitor (right).(C) Quantification of Pax5 in naive B cells treated as in (A).(D) Quantification of Bcl6 in naive B cells treated as in (A).(E) ChIP analysis for enrichment of Pax5 at p35, EBi3, and Cd1d gene promoters. Naive B cells isolated from oKPC mice were treated with aCD40/LPS and ± STAT3 inhibitor for 72 h followed by ChIP analysis.(F) ChIP analysis for enrichment of H3K27ac at p35, EBi3, and Cd1d gene promoters. Naive B cells were isolated and treated as in (E).(G) Experimental schematic used to investigate in vivo effect of STAT1 (STAT1i) and STAT3 (STAT3i) inhibition in B cells on pancreatic tumor growth.(H) Quantification of tumor weights from mice in (G).(I) Absolute number of intra-tumoral B cells from mice in (H).(J) Absolute number of intra-tumoral Breg cells from mice in (H).(K) Absolute number of intra-tumoral plasma cells from mice in (H). Error bars indicate SEM; p values were calculated using two-tailed; unpaired, Student’s t test. p values in (H) to (K) were calculated using one-way ANOVA. NS, non-significant, *p < 0.05, **p < 0.005. Data are representative of three independent experiments.