Flow cytometry analysis of PD1 expression on rat blood CD3<sup>+</sup> lymphocytes stimulated by CD3 antibodies

Abstract

The role of the PD-1/PD-L signaling pathway in the regulation of the immune response is currently the focus of research. Numerous studies have shown the key role of PD-1 molecules in the regulation of autoimmune, antitumor and antiviral reactions. The culture of rat and mice lymphocytes, as well as animal experimental models of immunopathologies are widely used in research. However, rat lymphocytes are almost not used to study the PD-1/PD-L pathway. There is no data on PD-1 expression or methods of its induction on rat lymphocytes. In human T-lymphocyte culture, PD-1 expression can be induced by NIB1412 anti-CD3 antibodies coated in the well of culture plates. In this study, we investigated the effect of G4.18 anti-CD3 antibody on PD-1 expression in peripheral blood lymphocyte of intact Wistar rats in vitro. According to some literature data, G4.18 anti-CD3 antibodies in immobilized form can activate isolated rat T cells, and according to others, inhibit allogeneic reactions in mixed lymphocyte culture and block cytotoxicity of cells obtained from rats with a developed graft rejection reaction. We found that incubation of rat blood lymphocytes with G4.18 anti-CD3 antibodies immobilized on plastic leads to a change in cell morphology and induction of PD-1 on CD3+ lymphocytes. After incubation with anti-CD3 antibodies, the proportion of PD-1+ lymphocytes among CD3+ lymphocytes was 12.056.04%, which is significantly higher than the proportion of such cells before incubation and during incubation in a cultural medium, which amounted to 2.602.62% and 4.595.81%, respectively. In the dot-plot graphs showing the distribution of cells according to the parameters of forward scatter and side scatter, PD-1+CD3+ lymphocytes induced by anti-CD3 antibodies are localized in the region of relatively lower forward scatter and greater side scatter. Perhaps these cells belong to apoptotic cells.