Fig 2: Increased accumulation of B cell subsets in human PDAC(A) Representative immunofluorescence staining for CD20, CD138, and Ebi3 in samples of normal adjacent (left) and human PDAC. CD20+Ebi3+ B cells are indicated by arrowheads. Scale bars, 50 µm.(B) Quantification of the frequency of CD20+ cells in human PDAC (N = 56) and adjacent normal tissues (N = 23).(C) Quantification of the frequency of CD138+ cells in human PDAC and adjacent normal tissues.(D) Quantification of the frequency of CD20+Ebi3+ cells in human PDAC and adjacent normal tissues.(E) Paired analysis of intra-tumoral CD20+Ebi3+ Bregs and CD138+ plasma cells.(F) Association with survival via Cox proportional hazards (PH) regression analysis of plasma cell signature in TCGA PAAD dataset.(G) Correlation of cancer Breg signature with plasma cell signature in PAAD (TCGA). For immunofluorescence quantification, three fields per sample were counted. Error bars indicate SEM, p values were calculated using Student’s t test (unpaired, two tailed); NS, not significant; **p < 0.01; ***p < 0.001.

Fig 3: IL-35 drives STAT3-Pax5 co-recruitment in tumor-educated B cells(A) Mouse Pax5 gene promoter with the STAT-binding consensus sites.(B) Mouse Bcl6 gene promoter with the STAT and Pax5 binding consensus sites.(C) ChIP analysis for enrichment of pSTAT1 and pSTAT3 on Pax5 promoter in naive B cells (treated with aCD40/LPS/rIL-35 for 72 h) from WT or oKPC mice.(D and E) (D) ChIP analysis for enrichment of pSTAT3 and (E) Pax5 at Bcl6 promoter from naive B cells isolated and treated as in (C).(F) Representative flow cytometry plot of Duolink proximity ligation assay (PLA) between pSTAT3 and Pax5 in naive B cells treated with LPS/aCD40/cytokine (green) or LPS/aCD40 alone.(G) Representative flow cytometry plot of PLA between pSTAT3 and Pax5 in intra-tumoral Breg (green) and Bcon (black) cells.(H) ChIP-re-ChIP analysis to detect STAT3-Pax5 complex (STAT3:S3, Pax5:P5) at Bcl6 promoter in rIL-35-treated naive B cells isolated from WT and oKPC mice.(I–K) (I) ChIP-re-ChIP analysis to detect STAT3-Pax5 complex (STAT3:S3, Pax5:P5) at promoters of p35, (J) EBi3, and (K) Cd1d genes in rIL-35-treated naive B cells isolated from WT or oKPC mice. 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. Data represent triplicates within the same experiment and are representative of three independent experiments.

Fig 4: 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.

Fig 5: 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).