Fig 2: [212Pb]VMT01 enhances tumor infiltrating lymphocytes in B16-F10 melanoma. (A) Lymphocytes were gated to exclude non-lymphocyte populations based on forward and side scatter (FSC and SSC) and stained for live/dead discriminator, CD45, CD19, CD3, CD4, and CD8a; (B) FACS analysis of CD45, CD19, CD3, CD4, and CD8a lymphocytes in control for control (n = 5) vs. 212Pb α-therapy (n = 4) treated B16-F10 tumor. Statistical analysis: n.s. non-significant; * p < 0.05; ** p < 0.01.

Fig 3: CD169 Reduces FVC Spread to Erythroblasts in the Red PulpFluorescence-activated cell sorting (FACS) plots showing the gating strategy and graphs depicting the numbers of FVC-infected cells, erythroblasts, B cells, and ratios of infected erythroblasts and B cells in splenocytes of B6 and CD169-/- mice (n = 4) 5 days after s.c. (A–E) or r.o. (F–J) administration (2,500 SFFU). Erythroblasts (CD71+ Ter119+ CD19-), B cells (CD19+), and FVC-infected cells (Glycogag+) were identified using the indicated fluorophore conjugates. p values derived from non-parametric Mann-Whitney test; mean values denoted by horizontal line.

Fig 4: IL-35 drives imbalance of plasma and Bregs in cancer(A) Quantification of tumor weights from WT, BEBi3-/-, and Bp35-/- mice orthotopically injected with KPC cells and collected at 3 weeks post injection. Control WT mice represent both BEBi3+/- and BWT control bone marrow chimera groups.(B and C) (B) Frequency of intra-tumoral naive B cells (percentage of CD45), and (C) CD21hiCD1dhiCD5+ Breg cells (percentage of CD19) and total B cells (percentage of CD45) from Figure 3A as determined by flow cytometry.(D) Representative flow cytometry plots and (E) quantification of frequency of IL-10, and IL-35 expressing Breg cells from Figure 3A.(F and G) (F) Representative flow cytometry plots and (G) quantification of CD19loCD138hi intra-tumoral plasma cells from (A).(H and I) (H) Quantification of IgG and (I) IgM levels in tumors of WT, BEBi3-/-, and Bp35-/- mice 3 weeks post orthotopic injection with KPC cells.(J) Representative antibody-dependent cellular cytotoxicity (ADCC) assay for determination of antibody mediated killing of tumor cells (target cells). Target 1, non-cancerous pancreatic cells; target 2, tumor cells (KPC 4662); ADCC, effector cells + target cells + source of antibodies (serum samples) from tumor-bearing WT, BEBi3-/-, and Bp35-/- mice. RLU, relative luminescence units.(K) Schematic of treatment schedule. Representative flow cytometry plots of intra-tumoral CD19loCD138hi plasma cells from bortezomib (BZ) or control PBS-treated WT and BEBi3-/- mice 3 weeks post orthotopic injection with KPC cells.(L) Quantification of intra-tumoral CD19loCD138hi plasma cells from (K).(M) Quantification of pancreatic tumor weights from (K). Error bars indicate SEM; p values in (A)–(I) were calculated using one-way ANOVA. p values in (J) were calculated using Student’s t test, two tailed, unpaired. p values in (L) and (M) were calculated using two-way ANOVA. NS, non-significant; *p < 0.05; **p < 0.005; ***p < 0.001. 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).