Identification of B Cell Subpopulations with Pro- and Anti-Tumorigenic Properties in an Immunocompetent Mouse Model of Head and Neck Squamous Cell Carcinoma

Abstract

Due to their high developmental diversity and different regulatory and functional roles, B cell subpopulations can promote or inhibit tumor growth. An orthotopic murine HNSCC model was applied to investigate the B cell composition and function in HNSCCs. Using flow cytometry approaches, cells from the spleen, lymph nodes and tumors were analyzed. Additionally, immunoglobulin (Ig) levels post-tumor induction were tracked via enzyme-linked immunosorbent assays (ELISA). Following tumor induction, GCs, as well as increasing numbers of GL7⁺CD95⁺ GC B cells in the spleen and tumor tissues, were detected. In parallel, we observed CD39⁺CD73⁺ B cells in tumors and spleens of tumor-bearing mice. Notably, CD39⁺CD73⁺ expression was primarily detected on MZ B cells and to a lesser extent on follicular (FO) and non-follicular, newly formed (NF) B cells, supposing an immunosuppressive function of MZ B cells in the TME. Parallel to increased MZ B cell numbers in secondary lymphoid organs (SLOs) as well as in the tumor tissue, IgM antibody (Ab) levels rose continuously. In contrast, IgG1, IgG2, and IgG3 levels increased at later time points. Understanding the complex interactions between B cell subsets and the TME could lead to new strategies for enhancing the treatment and prognosis of HNSCC patients.

Keywords: B cells; HNSCC; adenosine; germinal centers; immunocompetent mouse model.

Figure A1

Gating strategy for the detection of B cell subpopulation in the spleen, lymph nodes and tumor tissue.

Figure 1

Increased B cell number in the spleen, draining cervical lymph nodes and tumor tissue during tumor progression in orthotopic HNSCC. (A) An absolute number of B220⁺ B cells in spleen tissue (A) in cervical lymph nodes (B) on days 0, 7, 14, and 21 was assessed by flow cytometry comparing the control group (blue point) and a group of tumor-bearing mice (green point). (C) The absolute number of B220⁺ B cells in tumor tissue during observation time on days 14 and 21. Each point represents data from a single animal. Data in the graphs are shown as means ± SD (n ≥ 3 mice per control group and n ≥ 4 mice per group for tumor-bearing mice; differences in group size due to discontinuation ahead of schedule). Data are merged from at least two independent experiments. p-values were determined using a two-tailed Student’s t-test or Mann-Whitney-U test. Marked p-values can be considered statistically significant, * p < 0.05, ** p < 0.01.

Figure 2

Increased B cell activation, GC formation and plasmablast generation within the time course of tumor formation in spleen, lymph nodes and tumor tissue. (A–C) Absolute number of B220⁺GL7⁺CD95⁺ GC B cells (A), B220⁺CD69⁺ activated B-cells (B) and B220⁺CD138⁺ plasmablast B cells (C) in spleen on days 0, 7, 14, and day 21 assessed by flow cytometry comparing control group (blue point) and group of tumor-bearing mice (green point). (D) An absolute number of B220⁺GL7⁺CD95⁺ GC B cells in cervical lymph nodes during observation time on days 0, 7, 14, and 21 was assessed by flow cytometry comparing the control group (blue point) and a group of tumor-bearing mice (green point). (E,F) An absolute number of B220⁺GL7⁺CD95⁺ GC B cells (E) and B220⁺CD138⁺ plasmablasts (F) in tumor tissue during observation time on days 14 and 21. Each point represents data from a single mouse. Data in the graphs are shown as means ± SD (n = 3 mice per control group; n = 4 for tumor-bearing mice groups, differences in group size due to discontinuation ahead of schedule). Data are merged from at least two independent experiments. p-values were determined using a two-tailed Student’s t-test or Mann-Whitney-U test. Marked p-values can be considered statistically significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Tumor-induced germinal center (GC) reaction correlates with the appearance of switched immunoglobulins (Igs) in the serum of tumor-bearing mice of orthotopic head and neck squamous cell carcinoma (HNSCC). A statistical representation of the concentration of IgG1—antibody (Ab) (A), IgG2a—Ab (B), IgG2b—Ab (C) and IgG3—Ab (D) on days 0, 7, 14, and 21 assessed by Enzyme-Linked Immunosorbent Assay (ELISA) comparing control group (blue point) and a group of tumor-bearing mice (green point). Data in the graphs are shown as means ± SD (n = 6 mice per control group, n = 23 for tumor-bearing mice groups, differences in group size due to death due to tumor progress). Data are merged from at least two independent experiments. p-values were determined using a two-tailed Student’s t-test or Mann-Whitney-U test. Marked p-values can be considered statistically significant, * p < 0.05, *** p < 0.001.

Figure 4

Increased MZ B cell population in spleen, draining lymph nodes and tumor tissue during tumor progression is paralleled with increased levels of serum IgM in tumor-bearing mice of orthotopic head and neck squamous cell carcinoma (HNSCC). An absolute number of newly formed B cells (NF), follicular B cells (FO) and MZ B cells in spleen tissue (A), in cervical lymph nodes (B) on days 0, 7, 14, and 21 assessed by flow cytometry comparing control group (blue point) and group of tumor-bearing mice (green point). (C) The absolute number of B220⁺ B cells in tumor tissue during observation time on days 14 and 21. (D) The concentration of IgM on days 0, 7, 14, and day 21 was assessed by ELISA, comparing the control group (blue point) and the group of tumor-bearing mice (green point). Each point represents data from a single animal. Data in the graphs are shown as means ± SD (n ≥ 3 mice per control group; n ≥ 3 for tumor-bearing mice groups, differences in group size due to discontinuation ahead of schedule). Data are merged from at least two independent experiments. p-values were determined using a two-tailed Student’s t-test or Mann-Whitney-U test. Marked p-values can be considered statistically significant, * p < 0.05, ** p < 0.01 and **** p < 0.0001.

Figure 5

Increased B cell-mediated potential of Ado generation during tumorigenesis in orthotopic HNSCC. (A) An absolute number of splenic or (B) cervical lymph node derived B220⁺CD39⁺CD73⁺ B cells, able to generate Ado. (C) The absolute number of splenic CD39⁺CD73⁺ NF, FO, MZ B cells on days 0, 7, 14, and 21 were assessed by flow cytometry comparing the control group (blue, yellow, white points) and a group of tumor-bearing mice (green, red, black points). (D) Absolute number of B220⁺CD39⁺CD73⁺ B cells, able to generate Ado, (E) GC B cells, able to generate Ado (B220⁺CD39⁺CD73⁺CD95⁺GL7⁺⁾ and (F) CD39⁺CD73⁺ NF, FO, MZ B in tumor tissue during observation time on day 14 and day 21. Each point represents data from a single animal. Data in the graphs are shown as means ± SD (n ≥ 3 mice per control group; n ≥ 4 for tumor-bearing mice groups, differences in group size due to discontinuation ahead of schedule). Data are merged from at least two independent experiments. p-values were determined using a two-tailed Student’s t-test or Mann-Whitney-U test. Marked p-values can be considered statistically significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.