Fig 2: CRISPR/CAS9-based deletion of accessible, promoter-connected genomic regions harboring SLE variants influences TFH gene expression.a, CRISPR–CAS9 targeting of the 136 bp OCR near the TREH gene harboring the rs527619 (r2 = 0.51) and rs71041848 (r2 = 0.44) SLE proxy SNPs and captured interacting with the CXCR5 promoter leads to increased CXCR5 expression (blue histogram) by Jurkat cells compared to cells transduced with a CTRL-gRNA+CAS9 (pink histogram). b CRISPR–CAS9 targeting of the 213 bp OCR containing the rs4385425 SLE proxy (r2 = 0.99) to rs11185603 and captured interacting with the IKZF1 promoter leads to increased IKZF1 (Ikaros) expression (blue histogram) by Jurkat cells compared to cells transduced with a CTRL-gRNA+CAS9 (pink histogram). c CRISPR–CAS9 targeting of the LPP SLE proxies rs34631447 (r2 = 1.0) and rs79044630 (r2 = 0.8) captured interacting with the BCL6 promoter abrogates IFNg-induced BCL-6 expression (orange histogram) compared to cells transduced with a CTRL-gRNA+CAS9 (blue histogram). The red histogram shows BCL-6 expression by unstimulated Jurkat cells. Graphs in a–c depict biological replicate (circles) and mean (bars) fluorescence intensities (upper panels) and biological replicate (circles) and mean (bars) percent positive cells (lower panels) ±SEM (error bars) for CXCR5, Ikaros, and BCL-6 in control gRNA-tranduced vs. targeted cells. All data are representative of three independent experiments. See Supplementary Fig. 7 for design and validation of CRISPR/CAS9-mediated deletion and mutation. Source data are provided as a Source Data file.

Fig 3: A large increase in LMP1 caused by IL4 suppressed CHOP-induced cell death(A) Farage cells were treated with 1 µg/mL CHOP alone or in combination with IL4 (50 ng/mL) or an anti-SLAMF1 antibody (500 ng/mL) for 2 days. The drug-treated cells were lysed, which was followed by SDS-PAGE and western blotting. The following antibodies were used: anti-SLAMF1, phospho-STAT3 (Tyr705), phospho-STAT6 (Tyr641), phospho-NF-?B p65 (Ser536), PARP, BCL-XL, MCL1, BCL6, Bcl-2, LMP1, TP53, and GAPDH. (B) Farage cells were treated with 0.1 or 1 µg/mL CHOP alone or in combination with IL4 (50 ng/mL). The drug-treated Farage cells were washed and incubated with an Fc blocker. To stain IRF4 and BCL6, the cells were fixed and permeabilized. Flow cytometry was then performed after incubating the cells with PE-conjugated anti-IRF4 or APC-conjugated anti-BCL6.

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).

Fig 5: 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.