Purpose: Head and Neck Squamous Cell Carcinoma (HNSCC) strongly affects the immune system resulting in profound immune defects. These are associated with increased metastasis and recurrent disease. Standard treatment options include among others various regimens of chemotherapy. However, besides the anti-neoplastic effects of chemotherapy, adverse immune modulations are described. Recently, we demonstrated that regulatory B cells (Breg) play a significant role in the production of extracellular adenosine (ADO) using ectonucleotidases CD39 and CD73, which is known to be highly immunosuppressive. Here, we tested the influence of chemotherapy on the production of ADO by Breg.
Experimental Design: Mononuclear cells were isolated from peripheral blood collected from 30 healthy donors and from patients with HNSCC before (n=30) and after chemotherapy (n=30). The number, frequency and phenotype of Breg was determined by multicolor flow cytometry. Next, B cells were isolated from healthy donors by CD19+ magnetic beads, stimulated (CD40L, IL-4, hemagglutinin) and incubated for 7 days with physiologic concentrations of various chemotherapeutic drugs (cisplatin, methotrexate, paclitaxel, 5-fluorouracil, cetuximab) for 7 days. Effects on Breg proliferation were determined by CFSE proliferation assay. Production of extracellular ADO before and after chemotherapy was determined by flow cytometry, ATP luminescence assay as well as highly sensitive mass spectrometry measuring 5'AMP, ADO and inosine. Long-term effects were described analyzing Breg from patients who had received chemotherapy several years ago.
Results: Breg were numerically diminished in untreated HNSCC patients. However, subsequently to a strong numerical decrease of CD4+ T cells during chemotherapy, the frequency of B cells was increased. Chemotherapeutic drugs had variable effects in vitro and in vivo: While cisplatin, paclitaxel and 5-fluorouracil decreased expression of CD39, it increased under methotrexate. Importantly, hydrolysis of ATP to exogenous ADO correlated strongly with CD39 expression levels (p < 0.05). CD73 expression levels were increased by all drugs, except for cetuximab, which showed no effect on phenotype or function.
Conclusion: Highly suppressive Breg increase in frequency after chemotherapy and could be responsible for suppression of antitumor immune responses and recurrent disease in HNSCC. B cell function in terms of ADO production and their capacity to suppress CD4+ T cells is compromised by various chemotherapeutic drugs in vitro and in vivo amplifying therapeutic benefits.
Citation Format: Andreas Ziebart, Ulrich Huber, Silke Schwarz, Simon Laban, Thomas K. Hoffmann, Patrick Schuler. The influence of chemotherapy on adenosine-producing B cells in patients with head and neck cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B50.
- ©2015 American Association for Cancer Research.