Fig 2: Functional analysis of ICOS+CD38+ CD4+ and CD8+ T cells and response to SARS-CoV-2 Spike antigen.PBMCs collected from study participants (n = 10) pre-vaccination (day 0), day 28 post-vaccination (7 days post-boost), and day 105 post-vaccination were examined for ICOS, CD38, and other markers by fluorescence flow cytometry individually in one setting to avoid batch variation. A CD4+ T cells were gated from lymphocytes and defined as ICOS+CD38+ or ICOSloCD38lo. Percentages of CD4+ICOS+CD38+ T cells were quantified and data from each participant is connected by lines. B Mean fluorescence intensity (MFI) of CCR7 within CD4+ICOS+CD38+, CD8+ICOS+CD38+, CD4+ICOSloCD38lo, or CD8+ICOSloCD38lo T cells. C, D PBMCs were stimulated with PMA/ionomycin and examined for cytokine production. T cells defined as ICOS+CD38+ (red) or ICOSloCD38lo (blue) were examined for C IFN-? or D TNF. Representative samples on the left and individual participants are shown on right. E Day 28 PBMC samples were stimulated with activation beads or T cells were sorted based on CD38 and ICOS expression. Sorted T cells were labeled with CellTrace Violet (CTV) to track and stimulated with T-cell (CD3)-depleted autologous PBMCs ± SARS-CoV-2 Spike peptide pool (+ peptide, blue and red). Sorted T-cell populations were analyzed after 48 h for IL-2, TNF, and Granzyme b in F. BCL6 expression in sorted T-cell populations, stimulated as in E. G Tfh markers CXCR5 and PD-1 compared on day 0 and 28 samples. H Unstimulated days 0 and 28 ICOS+CD38+ and ICOSloCD38lo T cells were analyzed for BCL6 expression (FMO; fluorescence-minus-one control for BCL6). I CXCR5 MFI within BCL6+ T cells. FMO control for BCL6 in ICOSloCD38lo T cells is shown by the blue dotted line, and by the red dotted line for ICOS+CD38+ T cells. B Significance was determined by paired two-way ANOVA mixed-effects model. E, F Repeated measures one-way ANOVA. C, D, G–I Two-way ANOVA with Sidak’s multiple comparisons test. Representative samples are shown and each data point represents an individual participant. All error bars ± SD. ns = not significant, *p < 0.5, **p < 0.005, ***p < 0.0005, ****p < 0.00005.

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: 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 5: 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.