Adenosine-producing regulatory B cells in head and neck cancer

Abstract

Background: Multiple mechanisms of immunosuppression have been identified in the tumor microenvironment including regulatory B cells (B_reg). Recently, we have shown that B_reg suppress T cell function by production of adenosine (ADO). However, the autocrine effect of ADO on B cells and the role of B_reg in head and neck cancer remains unclear.

Methods: Blood (n = 42) and tumor tissue (n = 39) of head and neck cancer patients and healthy donors (n = 60) were analyzed by FACS. The effect of ADO on phenotype, intracellular signaling pathways, Ca²⁺ influx and ADO production was analyzed in B_reg and effector B cells (B_eff) by FACS, luminescence and mass spectrometry. The blockage of the ADO receptor A_2A was analyzed in a murine head and neck cancer model.

Results: ADO-producing B_reg were found in tumor tissue and peripheral blood. ADO inhibited the intracellular Bruton's tyrosine kinase (BTK) and Ca²⁺ influx only in B_eff. The inhibition of BTK by ibrutinib mimicked the effect of ADO, and ibrutinib reduced the production of ADO by downregulation of CD39 in vitro. The inhibition of ADO receptor A_2A significantly reduced tumor mass and increased B cell infiltration, in vivo.

Conclusion: Our data demonstrate the presence of a novel ADO-producing B_reg population within the tumor microenvironment in mice and humans. A new model is proposed on how ADO-producing B_reg can influence the function of B_eff cells in healthy donors and cancer patients. Thus, the modulation of the ADO pathway in B cells may serve as a therapeutic approach for cancer patients.

Keywords: ADORA2A; Adenosine; BTK; Head and neck cancer; Regulatory B cells.

Fig. 1

Human CD19⁺ B cells and their BTK phosphorylation in response to ADO. a The expression of the ectonucleotidases CD39 and CD73 measured by FACS in PBMC of healthy donors (n = 60), PBMC of tumor patients (n = 42) and in TIL of HNSCC patients (n = 39). Data are shown as means ± SD. b RNA levels for the four adenosine receptors (ADOR) in isolated peripheral B cells of healthy donors (n = 10) and tumor patients (n = 5). c Human B cells were stimulated with anti-µ-F(ab’)₂ and stained for anti-p-BTK (pY223). The experiment was performed with gated B cells (n = 3) and isolated B cells in PBMC (n = 5). d Pre-incubation with ADO induced a decreased BTK phosphorylation in a concentration-dependent manner after stimulation with anti-µ-F(ab’)₂ (n = 3). e Incubation with ADO decreased the ability of B cells to phosphorylate BTK in a time-dependent manner. Results are expressed as percentage of phosphorylated BTK shown as mean ± SD (n = 10). f The ability of ADO to decrease BTK phosphorylation in peripheral B cells is comparable in B cells of healthy donors (n = 10) and HNSCC patients (n = 7). Results are expressed as percentage of phosphorylated BTK. The five-minute value was set to 100%. All data are shown as mean ± SD. not significant (ns); p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****)

Fig. 2

Phosphorylation of BTK in B cell subsets. a Unstimulated CD73^neg B_eff cells showed a significantly higher phosphorylation of BTK than CD73⁺ B_reg two hours after isolation and staining. After pre-treatment with ADO for 2 h, BTK phosphorylation was reduced in CD73neg B_eff cells. Data shown as median with interquartile range; paired t-test; n = 5. b Isolated human B cells were first stained for CD73 and then incubated with ADO for two hours. Phosphorylation was measured after stimulation with anti-µ-F(ab’)₂. Incubation with ADO reduced BTK phosphorylation in CD73^neg B_eff cells. Analyzed with a two-way ANOVA; n = 5. c Expression of IgM and CD73 on CD19⁺ B cells in the density plot of one representative patient. d Frequency of IgM⁺ cells in the CD73^neg B_eff and CD73⁺ B_reg subset

Fig. 3

Calcium influx and B cell responses to treatment with the BTK inhibitor ibrutinib. a Isolated B cells were treated overnight with ADO and/or ibrutinib, stained with Indo-1 and stimulated with anti-µ-F(ab’)₂ for direct Ca²⁺ measurement. The overlay plot of the kinetics shows control B cells in black, ADO-treated B cells in light gray and ibrutinib-treated B cells in dark gray of one representative experiment. The results were calculated by the following ratio: intensity indo-1 violet/indo-1 blue. b The Ca²⁺ influx was significantly reduced by ibrutinib and by the combination of ibrutinib and ADO. AUC = area under the curve as mean ± SD analyzed with one-way ANOVA; median with interquartile range; n = 9. c The data suggest that peripheral B cells of tumor patients (n = 3) show significantly decreased Ca²⁺ influx after ADO treatment as compared to B cells of healthy donors (n = 9) analyzed with a two-way ANOVA; median with interquartile range. d The MFI of CD39 and CD73 on B cells was measured by FACS before and after treatment with ibrutinib or DMSO as a control. Ibrutinib induced a significant decrease in CD39 expression. Data were analyzed with a two-way ANOVA; median with interquartile range. e B cells were stimulated with CD40L, IL-4 and hemagglutinin and treated with ibrutinib for 6 days. Remaining ATP as measured by luminescence was significantly higher in ibrutinib-treated B cells as compared to control B cells (n = 7). Analyzed by two-way ANOVA; (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001. f Adenosine concentrations measured by mass spectrometry for B cells of one donor stimulated for 6 days with CD40L, IL-4 and hemagglutinin and treated with ibrutinib. ATP was added at a concentration of 20uM

Fig. 4

Cell populations in the peripheral blood of HNSCC-bearing mice. a Leukocytes were determined using animal blood counter (n = 57). b–c Populations of blood lymphocytes and B cells were analyzed by flow cytometry (n = 43/47). d–e The expression profile for CD39/CD73 and IgM/IgD was determined in peripheral B cells. Cell numbers per µl blood were calculated based on WBC. Blood of control and tumor-bearing mice, which were either left untreated or treated with the ADORA_2A antagonist SCH-58261, was analyzed at the tumor induction and after 21 days (n = 43/54)

Fig. 5

Tumor growth and TIL in HNSCC-bearing mice. HNSCC mice were either left untreated or treated with the ADORA_2A antagonist (SCH-58261). a At d21 after tumor induction, tumors were harvested for determination of CD19⁺ B cell frequency (n = 20). Infiltrating tumor cells were further characterized for b CD39 and CD73 expression (n = 22), as well as c IgM and IgD expression (n = 22). d–e Mice, which were treated with SCH-58261, showed a decrease in tumor weight and in the absolute number of tumor-infiltrating lymphocytes (n = 22/21). f However, the number of tumor-infiltrating B cells per g tumor was decreased (n = 21) and g the number of TIL per g tumor was stable (n = 21)

Fig. 6

Adenosine affects the B cell receptor pathway. In B effector cells, binding of the antigen α-µ-F(ab’)₂ to the BCR induces Syk and PIP3 activation supported by PI3K signaling. PIP3 recruits BTK, inducing auto-phosphorylation. The activated BTK activates PLCγ2 and IP3, binding to the endoplasmatic reticulum (ER), which secrets Ca²⁺. On B_reg cells, extracellular ADO is produced by hydrolysis of ATP by the ectonucleotidases CD39 and CD73. ADO binds to different ADO receptors, downregulating the auto-phosphorylation of BTK and the Ca²⁺ influx in CD73^neg B cells. In B_reg cells, no BTK phosphorylation was found upon binding of the antigen α-µ-F(ab’)₂