Fig 2: 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 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: 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).