NANOSECOND PULSED ELECTRIC FIELD MODULATES IMMUNOPHENOTYPE OF LYMPHOCYTES

Abstract

Introduction: Nanosecond pulsed electric fields (nsPEFs) have been shown to have anticancer effects, but little is known about the mechanisms modulating the immune landscape. According to the applied parameters (voltage, pulse duration, pulse delivery frequency, and a number of pulses), it is possible to achieve a specific effect modulating the immunophenotype of cancer cells. This study aims to identify changes in the expression profile of antigens defining lymphocyte maturity and function. Materials and Methods: We treated the Jurkat cell line with PEF. We tested different parameters of electroporation (2.5, 5, 7.5, 10, and 12.5 kV/cm, 100, 200 and 100 ns pulses, 25, 100, 250, and 500 pulses, 1, 10 and Hz). For the detection of cell permeabilization, Yo-Pro-1 and flow cytometry were employed. Cell viability was analyzed after 24h by MTT Assay. ATP levels in culture were measured using Cell-Titer-Glo® Assay after 24 hours. The expression of cell markers was determined using flow cytometry. Results: The obtained results confirmed that electroporation permeabilizes the cell membrane and modulates the immunophenotype of Jurkat cells. Moreover, we observed that the ATP release increases when higher electric fields are applied. Application of 10kHz-100Hz and 25 pulses of nsPEF induces an increase in the expression of CD154 costimulatory molecule, which regulates the immune response by priming T cells and activating immune cells. Simultaneously, we increase the expression of the early activation marker CD69, indicating the ability to activate lymphocytes. The application of the 10kHz parameter upregulated the expression of antigens involved in the recruitment of inflammatory cells (CD183), controlling homeostasis of peripheral T lymphocytes (CD127) and regulating platelet adhesion (CD61). Importantly, we thus noted there was no alteration in the expression of CD95, which can mediate the induction of apoptosis in cancer cells. On the contrary, significant CD7 subexpression was observed when 10Hz nsPEF was applied. In general, the cells have not undergone cell death with lower electric field parameters but have modified their immunophenotype. Based on our studies, we propose a mechanism in which the cells: (1) permeabilize the cell membrane, (2) increase the expression of lymphocyte-activation antigens, and (3) downregulate the expression of CD7. Conclusions: Concluding, we can state that nsPEF can be a promising approach in immunotherapy via enhancing the presentation of lymphocyte-activation antigens. Also, we proved that application of electric field treatment could modulate the expression profile of lymphocytes while avoiding cell death. Encore Abstract - previously submitted to regional or national meetings (up to < 1’000 attendees) The research was funded by: This research was founded by the Polish National Centre of Science of DAINA 2 (2020/38/L/NZ7/00342; PI: J. Kulbacka), and the Research Council of Lithuania grant (Nr. S-LL-21-4, PI: V. Novickij). Keywords: Diagnostic and Prognostic Biomarkers, Basic and Translational Science - Other No conflicts of interests pertinent to the abstract.