Impact of cryopreservation on immune cell subset frequencies in human peripheral blood mononuclear cells following assessment of monoclonal antibodies to CD39 and P2X7.

Abstract

Peripheral blood mononuclear cells (PBMCs) are commonly cryopreserved for immune cell profiling analyses, like flow cytometry. Cryopreservation of PBMCs may affect the recovery of immune cell subsets or the presence of cell-surface molecules, potentially skewing results compared to freshly isolated PBMCs. The ectonucleotidase CD39 promotes immunosuppression through the hydrolysis of extracellular nucleotides such as adenosine 5'-triphosphate (ATP) to limit P2X7 activation and downstream immune responses. Because of this, CD39 and P2X7 are emerging as potential biomarkers in cancer and other diseases. Using flow cytometry, this study demonstrated that an anti-human CD39 monoclonal antibody (mAb) (clone TU66) bound human embryonic kidney (HEK) 293 cells transfected with human CD39, but not mock-transfected HEK293 cells. In contrast, an anti-human P2X7 mAb (clone 1058613) bound similarly to HEK293 cells transfected with human P2X7 and non-transfected HEK293 cells. Using flow cytometry, this study then assessed the impact of cryopreservation on the population frequencies of immune cell subsets including CD39⁺ cells in human PBMC samples. These analyses revealed that cryopreservation had a minor, yet statistically significant impact on the population frequencies of CD4⁺, CD8⁺ and CD4^-CD8^- T cells. Cryopreservation also had a significant impact on the frequencies of CD39⁺CD19⁺ B cells and CD39⁺ non-classical monocytes. Overall, this study informs researchers and biobank workers about the need to consider the impact of cryopreservation when interpreting data from stored samples.