Tumor-B-cell interactions promote isotype switching to an immunosuppressive IgG4 antibody response through upregulation of IL-10 in triple negative breast cancers

MDA-MB-231 cells obtained from American Type Culture Collection were maintained in RPMI-1640 medium (ThermoFisher Scientific, #11875) + 10% FBS (ThermoFisher Scientific, #10437028) and 1% antibiotic/antimycotic (ThermoFisher Scientific, #15240). SUM159 cells were obtained from Asterand (Detroit, MI) and cultured in Ham’s F12 medium (ThermoFisher Scientific, #11765) + 10% FBS, 1 × antibiotic/antimycotic, 2 μg/ml insulin (ThermoFisher Scientific, #12585) and 100 ng/ml hydrocortisone (Sigma-Aldrich, #H0135). Primary human peripheral B lymphocytes and EBV transformed B cells were grown in RPMI-1640 + CellXVivo Human B Cell Expansion Kit as described by manufacturer (R&D Systems, #CDK005), L-glutamine (ThermoFisher Scientific, #25030), 2-mercaptoethanol (ThermoFisher Scientific, #21985), Insulin, Penicillin–Streptomycin (ThermoFisher Scientific, #15070063), and 10% FBS. EBV-transformed human peripheral B cells were grown in RPMI + 10% FBS and 1 × antibiotic/antimycotic and treated with CellXVivo Human B cell expansion kit for experimental analysis.

Peripheral human blood was obtained with consent from women with TNBC prior to surgical resection or treatment and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque^® (GE Healthcare, #95021). B lymphocytes were isolated using B Cell Isolation Kit II, human (Miltenyi Biotech, #130-091-151) according to the manufacturer’s instructions. For co-cultures, B cells and tumor cells were separated with a 0.4 μm 6-well cell culture insert with B cells in the upper chamber. Cells were cultured for 6 days, with media changed on day 3. Following co-culture, B cells were prepared for flow cytometry analysis by staining with an IgG4 antibody for 30 min in the dark (#9200-09, mouse anti-human IgG4 PE-conjugated, 1:50, SouthernBiotech). Cells were washed, resuspended in 300 μl PBS + 10% FBS, and filtered through a 35-um filter cap in a 5 ml FACS tubes to create a single cell suspension. Samples were run on the BD FACSAria II (BD Biosciences) at the Christiana Care’s Helen F. Graham Cancer Center and Research Institute core facility. For IL-10 blocking studies, anti-human IL-10 (clone JES3-97D, Biolegend) was added at a concentration of 1ug/ml, daily during co-culture. Flow cytometry experiments were repeated at least three times with each tumor cell line.

Tumor cell lines were cultured with and without B cells for 24 h as described above and RNA was isolated. Real-time PCR was performed as previously described [36]. Samples were run on an Applied Biosystems’ 7500 Fast Real-Time PCR System with Power SYBR Green PCR Master Mix (Life Technologies), using the primers described in Table 1. All experiments were performed in at least four experimental replicates in each cell line.

For the IL-10 ELISA, tumor cells were co-cultured with B cells for 24-h. Control cells were incubated with media only. To ensure detection of tumor cell IL-10, B lymphocytes and media were removed, and fresh media was added back to wells for a 24-h period. Supernatants were then collected, spun down to remove cell debris, and analyzed using the Quantikine IL-10 Elisa kit (R&D systems) according to manufacturer’s instructions. For the IgG4 Elisa, EBV B cells were activated and co-cultured with or without TNBC cell lines for 5 days as previously described. B cells were then removed and allowed to expand for two weeks. Cells were then plated for 24 h and supernatant was captured. Supernatants were analyzed using the Human IgG4 Elisa kit (Invitrogen). Absorbance was read at 450 nm on the Infinite^® 200 PRO NanoQuant (Tecan Life Sciences). Experiments were performed in duplicate in each cell line.

Breast cancer tissue specimens were obtained from the Helen F. Graham Cancer Center and Research Institute (HFGCCRI) biorepository under a protocol approved by the institutional review board. Tissue was obtained from surgical resection from women who were pathologically diagnosed with TNBC and consented to the use of their tissues for research. A pathological diagnosis of TNBC is defined as less than 1% of tumor cell expression of the hormone receptors estrogen and progesterone and a negative HER finding by IHC (0 or 1 +) or negative FISH. Tissue blocks were prepared by formalin-fixation and paraffin-embedding. Tissues were constructed into 4 × 5 tissue microarrays (TMA) with a 5 mm core size and cut as serial sections. All patients underwent adjuvant radiation and chemotherapy after surgical resection.

Serial paraffin-embedded slides were deparaffinized and rehydrated, and heat antigen epitope retrieval was performed for 16 h at 60 °C. Slides were stained using the DAB Substrate kit (Abcam, ab64238) for IL-10 and CD20 and the Mouse and Rabbit Specific HRP/AEC (ABC) Detection IHC Kit (Abcam, ab93705) for IL-4 and IgG4, following the manufacturer’s instructions. Primary antibodies were incubated overnight at 4 °C with a rabbit monoclonal antibody to IgG4 (Abcam, ab109493, 1:1000), a rabbit polyclonal antibody to IL-4 (Abcam, ab9622, 1:100), and mouse monoclonal antibodies to CD20 (Abcam, ab9475, 1:100) and IL-10 (Santa Cruz, sc-8438, 1:50). Slides were counterstained with Harris hematoxylin for 10 min. Images were captured using a Zeiss Axio microscope using a 10X objective. CD20 was scored by overlaying a 20 × 20 grid of 1 mm squares and calculating the percent of tissue-containing squares which contained 10 + lymphocytes. IgG4 was scored by counting the total number of IgG4 + B cells. For IL-10 and IL-4 staining, segmentation of tumor cells and measurement of integrated density was performed using Zen Blue. The median intensity or percentage was used to determine low vs high samples. For CD20, low, intermediate and high density was determined using the mean ± one standard deviation. Control staining was performed with an isotype matched antibody (Santa Cruz, sc-2025, sc-2027) using the same staining conditions. No staining was observed. (Additional file 1: Fig. S1).

Statistical analysis was done in graph pad PRISM 8 and IBM27 SPSS. Relationships among scored slides was done using Spearman’s correlation analysis. Flow cytometry and real time quantitative PCR data was analyzed using student’s T-test. Survival and recurrence were analyzed using Kaplan–Meier curves and Mantel Log-Rank tests. Multi-variate analysis was carried out using Cox-proportional hazards tests.

Chronic inflammation is a hallmark of TNBC [11]. TIB cells have been associated with chronic inflammatory signatures [15‐18, 37] and have been shown to secrete several inflammatory cytokines in breast cancer [38]. While previous studies have shown that tumor cells are capable of activating inflammatory signaling cascades in B cells [35, 38], the effects of this interaction on tumor cells are less clear. We therefore sought to determine the effects of B cells on tumor cells in co-culture studies with primary peripheral B cells isolated from the blood of TNBC patients. We observed increases in gene expression of several inflammatory cytokines in TNBC cells co-cultured with B cells (Fig. 1a). Of particular interest was upregulation of the TH2 type cytokines IL-10 and IL-4 in triple negative cells, which have been shown to drive IgG4 expression and are associated with immunosuppressive responses [22, 39]. To confirm these findings, we conducted a 24-h co-culture of primary human peripheral B cells isolated from women with TNBC and the TNBC cell lines MDA-MB-231 and SUM159. Real-time PCR was used to analyze changes in gene expression of IL-10 and IL-4. We found significant increases in IL-10 gene expression in both cell lines (Fig. 1b). An average 7.5-fold increase was observed in SUM159 and a 7.4-fold increase was observed in MDA-MB-231 cells co-cultured with B cells compared to controls. Increases in gene expression of IL-4 was also observed in both lines, 3.4-fold increase for SUM159 and 2.1-fold increase for MDA-MB-231, but this increase was only significant in the SUM159 cell line. To determine if this gene expression correlated to an increase in protein expression, we performed an ELISA specific for human IL-10. To determine tumor-specific production of IL-10, TNBC cell lines were co-cultured for 24 h with B cells, followed by a 24 h culture without B cells, after which media was removed and analyzed for the presence of IL-10 protein. As shown in Fig. 1c, there was a significant increase in secreted IL-10 protein in tumor cells co-cultured with B cells. These findings indicate that cross-talk between tumor cells and B cells promotes inflammatory signaling in tumor cells that may be associated with immunosuppression.

IL-10 expression is often present at sites of chronic inflammation and promotes immunosuppression of humoral responses through induction of isotype switching to IgG4. Therefore, we sought to determine if TNBC could alter the isotype profile of B cells. We performed an in vitro assay to determine if IgG4 class switching is differentially induced upon activation of B cells co-cultured with TNBC cell lines compared to controls. Co -culture of both MDA-MB-231 cells and SUM159 with primary peripheral B cells isolated from TNBC patients resulted in significant increases of IgG4 + cells, suggesting increased IgG4 class switching in response to TNBC cell co-culture (Fig. 2a and b). To confirm IgG4 class switching, an IgG4 specific ELISA assay was performed in EBV-transformed B cells co-cultured with tumor cells. Significantly higher levels of IgG4 were observed in B cell supernatants two weeks after co-culture with TNBC cell lines (Fig. 2c). Likewise, EBV B cells were shown to undergo in vitro isotype switching similar to peripheral B cells upon co-culture with TNBC cell lines in flow cytometry experiments (Fig. 2d and e). To examine the role of IL-10 in class switching, we examined class switching in EBV-transformed B cells upon co-culture with TNBC with and without the addition of an IL-10 blocking antibody. As shown in Fig. 2d and e, the addition of the anti-IL10 antibody significantly blocked IgG4 class switching in co-cultured cells. These findings indicate that TNBC can induce IgG4 class switching in B cells in an IL-10 dependent manner.

To investigate the presence of IgG4 and inflammatory cytokines in TNBC, we sought to evaluate protein expression by immunostaining tumor specimens from 152 triple negative tumors. Table 2 provides a summary of clinical data and patient characteristics. In order to quantify IgG4 + B cell and total B cell infiltration in TNBC tissues, serial sections of tissue microarrays were stained by immunohistochemistry for the pan-B cell marker CD20, IgG4, IL-10 and IL-4 (Fig. 3). The majority of tumors contained CD20 + B cells. Only 5 section (3.3%) scored negative for CD20 + B cells. The average density of B cells was 30.1%. A low density of B cells was defined to be those tumors less than one standard deviation from the mean, while high density was defined as a B cell density more than one standard deviation above the mean. Twenty tumors (13.3%) had low density, 93 (62%) had intermediate density and 27 (18%) had high density. IgG4 + cells were more often found in tumors with low or intermediate B cells (Fig. 4, p = 0.002). Tumor cell IL-10 staining was detected in 68 (45%) patients with high expression observed in 42 patients. Tumor cell IL-4 staining was detected in 102 tumors (68%), with high expression observed in 75 patients.

Prior studies indicate that IL-4 and IL-10 polarize IgG4 class switching [30], and that IgG4 + B cells are increased in advanced cancer [28], and associate with poor patient survival [29, 30] and increased recurrence [34]. Therefore, we compared immunohistochemically stained markers and clinicopathological characteristics using Spearman’s correlation analysis (Table 3). We observed no correlation of CD20 + B cell infiltration and stage or grade. Interestingly, a weak negative correlation was found between IgG4 and CD20 + B cells density (r = − 0.190, p = 0.020) as well as stage and B cell density (− 0.199, p = 0.021) confirming previous findings that a high B cell density is associated with better prognosis [38]. A correlation was also observed between IgG4 staining and tumor expressed IL-10 (r = 0.0.488, p = 0.000), and IL-4 (r = 0.262, p = 0.001), as well as between IL-10 and tumor stage (IL-10 r = 0.254, p = 0.016). Interestingly, IL-10 expression was significantly higher in blacks than non-hispanic whites (r =  − 0.176; p = 0.031).

To assess the effects of IgG4 + B cells on patient outcomes, Kaplan-Meyer analysis was performed. Consistent with previous studies, high B cell density was found to have significantly better RFS (p = 0.033) and OS (p = 0.010) than intermediate or low B cell density (Fig. 5a). Patients with high IgG4 + cells had significantly worse RFS (p = 0.000) and OS (p = 0.000) (Fig. 5b). Furthermore, high tumor expression of IL-10 and IL-4 resulted in lower recurrence free survival (p = 0.000 for IL-10; p = 0.021 for IL-4) (Fig. 5c and d). While high IL-10 expression resulted in worse OS (= 0.000), high expression of IL-4 had no significant effect on OS (p = 0.137). In a multi-variant analysis, IgG4 positivity (p = 0.001; HR = 3.366; 95% CI 1.607–7.050), tumor IL-10 (p = 0.000; HR = 7.195; 95% CI 2.418–21.411) and tumor stage and tumor stage (p = 0.000; HR = 1.442; 95% CI 1.0247–1.667) were independently associated with a shorter RFS. However, only tumor stage (p = 0.000; HR = 1.329; 95% CI 1.151–1.534) and IL-10 (p = 0.005; HR = 4.1401; 95% CI 1.538–10.934) were independently associated with a shorter OS (Table 4).