A Strong B-cell Response Is Part of the Immune Landscape in Human High-Grade Serous Ovarian Metastases

Abstract

Purpose: In high-grade serous ovarian cancer (HGSOC), higher densities of both B cells and the CD8⁺ T-cell infiltrate were associated with a better prognosis. However, the precise role of B cells in the antitumor response remains unknown. As peritoneal metastases are often responsible for relapse, our aim was to characterize the role of B cells in the antitumor immune response in HGSOC metastases.

Experimental design: Unmatched pre and post-chemotherapy HGSOC metastases were studied. B-cell localization was assessed by immunostaining. Their cytokines and chemokines were measured by a multiplex assay, and their phenotype was assessed by flow cytometry. Further in vitro and in vivo assays highlighted the role of B cells and plasma cell IgGs in the development of cytotoxic responses and dendritic cell activation.

Results: B cells mainly infiltrated lymphoid structures in the stroma of HGSOC metastases. There was a strong B-cell memory response directed at a restricted repertoire of antigens and production of tumor-specific IgGs by plasma cells. These responses were enhanced by chemotherapy. Interestingly, transcript levels of CD20 correlated with markers of immune cytolytic responses and immune complexes with tumor-derived IgGs stimulated the expression of the costimulatory molecule CD86 on antigen-presenting cells. A positive role for B cells in the antitumor response was also supported by B-cell depletion in a syngeneic mouse model of peritoneal metastasis.

Conclusions: Our data showed that B cells infiltrating HGSOC omental metastases support the development of an antitumor response. Clin Cancer Res; 23(1); 250-62. ©2016 AACR.

Figure 1

B cells in HGSOC are found in tertiary lymphoid structures. A, Immunohistochemistry pictures showing two representative sections from paraffin-embedded HGSOC omentum stained for CD20. Scale bar, 200 μm. B and C, Log₂ density of CD20⁺B cells in HGSOC omental metastases. B, Comparison of CD20⁺ B cell densities in tumor versus stromal areas (n = 33; ***, P < 0.001; Mann-Whitney test). C, Comparison of CD20⁺ B-cell densities in the stroma of unmatched HGSOC metastases obtained before (n = 10) and after chemotherapy treatment (n = 31). D-F, Characterization of the B-cell-rich lymphoid structures by single-color immunohistochemistry, immunofluorescence, and double-color immunohistochemistry. D, Identification of CD20⁺ B cells, CD4⁺, and CD8⁺ T cells within TLS. Scale bars, 50 μm. E, Presence of MECA79+ high endothelial venules in TLS. Scale bars, 20 mm. F, Germinal centers of TLS contain follicular DCs (FDC) and Ki67⁺ high proliferating cells surrounded by CD45RO⁺ memory cells. Scale bars, 50 μm.

Figure 2

Memory B-cell response in HGSOC metastases. A and B, Flow cytometry analysis of the B subpopulations in HGSOC omentum (n = 25) showing differential expression of CD27 and IgM within the CD20⁺ cells. Representative plots (A) and percentages (B) of the different B-cell subpopulations: IgM⁺ memory (CD27⁺IgM⁺), class-switched memory or IgM⁻ memory (CD27⁺IgM⁻), CD27⁻ atypical memory (CD27⁻IgM⁻) and naive (CD27⁻IgM⁺) B cells. An example of IgD expression in the different subpopulations is shown. C and D, Proportion of CD86⁺PD1⁻ (C) and PD1⁺CD86⁻ (D) cells among the B-cell subsets described above (n = 11; *, P < 0.05; **, P < 0.01; ***, P < 0.001; Mann-Whitney test and Student t test). E and F, BCR sequencing of B cells from HGSOC omentum (O; n = 7) and blood (B; n = 1) compared with healthy peripheral B cells (ctrl; n = 7). E, Diversity analysis of the B-cell repertoire. Error bars, 95% confidence interval (38). F, Analysis of hypersomatic mutations. IgH mutation rates for each sample (top). Tumor and nontumor samples were binned and IgHV mutation rates were compared (bottom).

Figure 3

Role of B cells in the recruitment of DCs in HGSOC metastases. A-D, Cytokine/chemokine secretion of HGSOC omental B cells (see Supplementary Methods). A, Correlation heatmap showing how the levels of the 24 cytokines and chemokines measured in the supernatant of HGSOC B cells (n = 4–10) vary with each other. Columns and rows represent individual cytokines and chemokines colored to indicate Pearson's correlation coefficient (r). B-D, Log₂ concentrations of CXCL8, CCL2, and IL6 in the supernatant of unstimulated healthy peripheral B cells (n = 2); PMA/ionomycin- (PMA/Iono) treated healthy peripheral B cells (n = 5–7); unstimulated and PMA/ionomycin-treated HGSOC omental B cells (n = 9–10; *, P < 0.05; ***, P < 0.001, Student t test). E, Flow cytometry analysis of CXCR1 (n = 4) and CXCR2 (n = 3) expression on CD19⁺ B cells; CD3⁺ T cells, CD11b⁺⁺CD14⁺CD11c⁻ macrophages; CD11b⁺⁺CD14⁻CD11c⁻ granulocytes and CD11b⁺⁺ CD14⁻CD11c⁺ DCs from HGSOC omentum. F, Representative immunostainings and quantification of DC-LAMP⁺ DCs and MPO⁺ neutrophils in CD20⁺-rich lymphoid structures in HGSOC omental metastases (n = 7). Arrows indicate MPO⁺ cells. Scale bar, 100 μm; ***, P < 0.001, Student t test. G, Correlation analysis between the log₂ densities of DC-LAMP⁺ DCs and CD20⁺ B cells in a cohort of 16 HGSOC patients (2 pre-chemotherapy and 14 post-chemotherapy; P < 0.05; Pearson correlation test).

Figure 4

B-cell signature is enriched in diseased omentum and linked to cytotoxic response. A-D, Heatmap and boxplots showing an increase in expression of B-cell signature genes in stage3/4 pre- (n = 4) and post-chemotherapy [post-chemotherapy diseased (post dis),n = 7; post-chemotherapy good responders (post good),n = 5)] vs. stage 1/2 (n = 6) non-involved HGSOC omentum. *, P < 0.05; **, P < 0.01; Student t test. E, Positive correlation between the geometrical mean of levels of expression of granzymeA (GZMA) and perforin (PRF1) versus CD20 (MS4A1) in HGSOC stage 3/4 omentum. F, CD20 depletion increased tumor progression in the ID8 mouse model. Left, tumor progression in anti-CD20- (n = 5) and IgG control- (n = 4) treated mice assessed by luciferase activity monitoring (P = 0.03; Two-way ANOVA on log₁₀ luminescence values). Top right, Bcells (CD45⁺ CD19⁺) and plasma cells (CD45⁺ CD138⁺) are depleted in the ascitic fluid of αCD20-treated ID8 mice vs. IgG controls. Bottom right, CD107 expression by ascitic CD8⁺ T cells in αCD20 and IgG control-treated ID8 mice (**, P < 0.001; Student t test).

Figure 5

Plasma cells are present in HGSOC omenta and IgGs deposited mainly in stromal areas but also on tumor cells and macrophages. A and B, Presence of CD38^{+ +}CD19^+/−CD20_CD27⁺plasma cells in HGSOC omentum (n =14; 11). C, Paraffin sections of omental metastases were fluorescently stained for CD20, CD68, or Cytokeratin 7 (CK7) (red) in combination with IgG (green). Arrows indicate colocalization of the CD68 and CK7 markers with IgG. Scale bar, 20 μm. D, Protein extracts isolated from frozen stage 3/4 pre-chemotherapy, post-chemotherapy diseased, post-chemotherapy with no/low residual disease and stage 1/2 noninvolved omentum were tested for the presence of human IgG1, IgG2, IgG3, and IgG4 by ELISA; * P < 0.05, Mann–Whitney. E, Tumor-derived IgGs ICs increase the expression of CD86 on DCs in vitro. IgGs were isolated from two tumor lysates using Agarose beads coupled to protein A/G and pooled. Monocyte-derived DCs (n =14; 7) from healthy volunteers were then treated with tumor IgGs alone or tumor IgGs preincubated with protein extracts from HGSOC tumor cells. IgGs from healthy plasma were used as a control. CD86 expression on DCs was assessed after 24-hour treatment.