Human B Cells Mediate Innate Anti-Cancer Cytotoxicity Through Concurrent Engagement of Multiple TNF Superfamily Ligands

Abstract

The essential innate immunity effector cells, natural killer and dendritic cells, express multiple plasma membrane-associated tumor necrosis factor (TNF) superfamily (TNFSF) ligands that, through simultaneous and synergistic engagement, mediate anti-cancer cytotoxicity. Here, we report that circulating B cells, mediators of adaptive humoral immunity, also mediate this innate anti-cancer immune mechanism. We show that resting human B cells isolated from peripheral blood induce apoptosis of, and efficiently kill a large variety of leukemia and solid tumor cell types. Single-cell RNA sequencing analyses indicate, and flow cytometry data confirm that B cells from circulation express transmembrane TNF, Fas ligand (FasL), lymphotoxin (LT) α1β2 and TNF-related apoptosis-inducing ligand (TRAIL). The cytotoxic activity can be inhibited by individual and, especially, combined blockade of the four transmembrane TNFSF ligands. B cells from tumor-bearing head and neck squamous cell carcinoma patients express lower levels of TNFSF ligands and are less cytotoxic than those isolated from healthy individuals. In conclusion, we demonstrate that B cells have the innate capacity to mediate anti-cancer cytotoxicity through concurrent activity of multiple plasma membrane-associated TNFSF ligands, that this mechanism is deficient in cancer patients and that it may be part of a general cancer immunosurveillance mechanism.

Keywords: B cells; TNF superfamily ligands; cancer; cytotoxic; head and neck squamous cell carcinoma; innate immunity; single-cell RNA sequencing (scRNAseq).

Figure 1

Resting B cells isolated from peripheral blood induce apoptosis in a broad panel of different types of cancer cells. (A) Resting healthy donor B cells were compared to iDCs for their ability to induce apoptosis in cancer cells. MACS-purified human peripheral blood resting B cells (B cells) and in vitro-generated iDCs were co-incubated with PCI-13 target cells at the indicated E:T ratios and tested for their cytotoxic activity using 1-h ³H-thymidine release, 24-h MTT and 24-h ⁵¹Cr release assays. Each test was performed in quadruplicates and mean percent cytotoxicity values ± standard deviations (STDEV) are shown. Data are representative of three independent experiments. (B) The susceptibility of normal and cancer cell lines to cytotoxic activity of resting B cells was tested using 1-h ³H-thymidine release assay (non-adherent leukemia and healthy donor T cell blast cells) and 24-h MTT assays (adherent cell monolayers). Data are presented as LU₂₀/10⁷ effector cells based on the mean % cytotoxicity values of quadruplicate tests obtained at four different E:T ratios. HNSCC cell lines labeled with “A” or “B” originated from primary and metastatic lesions, respectively. The cytotoxicity against various targets was tested 1, 2 or, in the case of PCI-13, 11 times. The presented data are from representative experiments. NORMAL, normal cell lines; LEUKEMIA, leukemia cell lines; HNSCC, squamous cell carcinoma of head and neck cell lines; BREAST, breast carcinoma cell lines; MELAN, melanoma cell lines; COL, colorectal carcinoma cell lines; NSCLC, non-small cell lung carcinoma cell lines; SCLC, small cell lung carcinoma cell lines; GC, gastric carcinoma cell line, RC, renal carcinoma cell line; OVAR, ovarian carcinoma cell lines; GLIOMA, glioma cell lines. (C) Paired primary tumor (PCI-4A) and lymph node metastasis (PCI-4B) cancer cells obtained from an HNSCC patient were tested in parallel for their susceptibility to killing by resting B cells using 24-h MTT assay. Mean % cytotoxicity values and STDEV obtained from quadruplicate tests and at four E:T ratios (0.1:1, 0.3:1, 1:1 and 3:1) are plotted. Two-way ANOVA was used to calculate the differences between groups. Data are representative of two independent experiments. (D) Resting B cells isolated from 11 healthy blood donors were tested for their cytotoxic activity against PCI-13 target cells using the 24-h MTT assay. Data are presented as LU₂₀/10⁷ B cells based on the mean % cytotoxicity values of quadruplicate tests obtained at four E:T ratios.

Figure 2

The tumor killing ability of resting B cells is inhibited by TNF, FasL, TRAIL and/or LT-α1β2 blockades but not by cellular fixation with paraformaldehyde. (A) Healthy donor PBL were stained for flow cytometry per Materials and Methods. Lymphocytes were gated based on their FSC vs SSC profile and single-cell status and, subsequently, evaluated for CD3 and CD19 expression. CD19⁺CD3^- B cells were analyzed for surface TNF, LT-α, LT-β, FasL and TRAIL expression levels. Representative data from one of the four healthy donors evaluated are shown. Empty histograms represent IgG controls, and filled histograms represent the TNFSF ligand staining. (B) TNFSF receptor-Fc fragment fusion proteins were used to block healthy donor B cell cytotoxicity of PCI-13 cells at 1:1 E:T ratio. Where indicated, effector cells were preincubated with 10 μg/mL of the individual or combined (combination) TNFSF receptor-Fc fusion proteins specific for TNF (TNFR1:Fc), FasL (Fas : Fc), TRAIL (TRAILR : Fc) and LT-α1β2 (LTβR:Fc) for 1h at 37°C. Controls included IgG1 isotype and IL-4R:Fc fusion protein controls. Following preincubation with antagonistic agents, the cytotoxic activity of B cells was assessed using the 3-h MTT assay. Data represent the mean % cytotoxicity of 6 replicates ± STDEV, and statistical significance was calculated using the one-way ANOVA. Calculated p-values are listed in the summary table below the figure. A representative of 3 experiments is shown. (C) Resting healthy donor B cells fixed with 1% paraformaldehyde were compared to their viable counterparts for their ability to kill PCI-13 cells. The cytotoxic activity of B cells was assessed using the 24-h MTT assay at 3:1 E:T ratio. Data represent the mean % cytotoxicity of 4 replicates ± STDEV, and statistical significance was calculated using the two-tailed Student’s t-test. A representative of 2 experiments is shown.

Figure 3

HNSCC patient circulating B cells display decreased levels of TNFSF ligand expression and tumoricidal activity. (A) Using our published scRNAseq datasets (20), we performed an analysis of healthy donor (HD; n = 6) and HNSCC patient (HNC; n = 29) PBL using the clustering strategy depicted in Supplementary Figure 4 . Donor-specific relative expression levels of and fraction of B cells expressing TNFSF ligand mRNA are presented using box plots with medians, quartiles, and ranges (healthy donor PBL, HD - PBL; HNC patient PBL, HNC – PBL). (B) Resting B cells obtained from peripheral blood of 4 healthy donors (HD-PBL) and 5 HNSCC patients (HNC-PBL) were examined for the expression of cell surface TNFSF ligand proteins using multi-color flow cytometry with the specific antibodies for cell markers (B cell CD19; T cell CD3) and for TNFSF ligands (TNF, LT-α, LT-β, FasL, and TRAIL) as described in Supplementary Figure 5 . To avoid possible batch effects, all donor samples were simultaneously stained and analyzed using the same procedure. Data represent donor-specific MFI values for each TNFSF ligand adjusted for respective IgG controls. Intersecting lines represent median MFI values of each donor cohort. p-values calculated using the one-tailed Student’s t- test are indicated in (A, B). (C, D) MACS-purified circulating resting B cells were obtained from 4 healthy donors (HD) and 4 tumor-bearing untreated HNSCC patients (HNC), added to the monolayers of (C) PCI-4a and (D) PCI-13 HNSCC target cells at indicated E:T ratios and in quadruplicates, and comparatively tested for cytotoxic activity using the 24-h MTT assay. Data represent mean % cytotoxicity ± SEM of 4 healthy donors and 4 HNSCC patients. Individual donor-specific B cell cytotoxicity is shown in Supplementary Figure 6 . Statistical significance was determined using the 2-way ANOVA test.

Figure 4

Differential TNFSF ligand and GZMB expression in different B cell subsets in blood and HNSCC tumors. (A) B cells identified in healthy donor and HNSCC patient blood and HNSCC tumors were extracted, subclustered and six different B cell differentiation states were identified using the curated list of genes listed in Supplementary Figure 4B . Dot-plots showing (B) TNFSF ligand and (C) GZMB expression by different B cell subsets. Color bars indicate normalized gene expression and dot sizes indicate the fraction of cells in a group expressing the particular gene.