The ectonucleotidases CD39 and CD73 are present on immune cells and play important roles in cancer progression by suppressing antitumour immunity. As such, CD39 and CD73 on peripheral blood mononuclear cells (PBMCs) are emerging as potential biomarkers to predict disease outcomes and treatment responses in cancer patients. This study aimed to examine T and B cells, including CD39 and CD73 expressing subsets, by flow cytometry in PBMCs from 28 patients with head and neck squamous cell carcinoma (HNSCC) and to assess the correlation with the treatment modality, human papillomavirus (HPV) status, and relapse-free survival (RFS). The PBMCs were examined pre-, mid-, and post-radiotherapy with concurrent cisplatin chemotherapy or anti-epidermal growth factor receptor antibody (cetuximab) therapy. Combination radiotherapy caused changes to T and B cell populations, including CD39 and CD73 expressing subsets, but no such differences were observed between concurrent chemotherapy and cetuximab. Pretreatment PBMCs from HPV⁺ patients contained increased proportions of CD39⁻CD73⁻CD4⁺ T cells and reduced proportions of CD39^−/+CD73⁺CD4⁺ T cells compared to the equivalent cells from HPV⁻ patients. Notably, the pretreatment CD4⁺:CD8⁺ T cell ratios and CD39⁺CD73⁺CD19⁺ B cell proportions below the respective cohort medians corresponded with an improved RFS. Collectively, this study supports the notion that CD39 and CD73 may contribute to disease outcomes in HNSCC patients and may assist as biomarkers, either alone or as part of immune signatures, in HNSCC. Further studies of CD39 and CD73 on PBMCs from larger cohorts of HNSCC patients are warranted.

P2X7 is an extracellular adenosine 5′-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca²⁺ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1β release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca²⁺ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y₁, P2Y₂ and P2Y₄. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.

P2X7 is an adenosine 5′-triphosphate (ATP)-gated ion channel. CD39 (ectonucleotidase triphosphate diphosphohydrolase-1) hydrolyses ATP to reduce its extracellular concentration to limit P2X7 activation. Both P2X7 and CD39 are present on leukocytes. Ficoll-Paque density gradient centrifugation is widely used to isolate human peripheral blood mononuclear cells (PBMCs), yet the extent to which other density gradient centrifugation methods influence the expression of cell surface molecules remains unclear. Using mass cytometry, this study compared the proportions of mononuclear leukocyte subsets and the relative amount of cell surface P2X7 and CD39 detected on these cells in paired, bar-coded cryopreserved human PBMC samples isolated by Ficoll-Paque or SepMate Tube density gradient centrifugation. Both techniques yielded similar proportions of mononuclear leukocyte subsets. P2X7 and CD39 were detected across all cell subsets, with the relative amount of P2X7 or CD39 comparable between separation methods. Relatively minor but statistically significant differences were observed for some populations. P2X7 expression was higher on CD3⁺, CD4⁺, and conventional CD4⁺ T cells, and naïve B cells, and lower on myeloid dendritic cells, while CD39 expression was lower on regulatory T cells in SepMate Tube samples compared to Ficoll-Paque samples. Overall, Ficoll-Paque and SepMate Tube density gradient centrifugation yield comparable results within PBMC samples, supporting the use of either method in studies examining immune phenotypes including the purinergic molecules P2X7 and CD39.