Modulation of PD-1 and PD-L1 expression on peripheral blood T-cells by novel rotavirus variants.

2658 Background: Promising oncolytic viruses are typically evaluated based on their direct cytotoxic effects on cancer cells. However, the therapeutic benefit of virotherapy may also stem from its immunomodulatory actions, particularly through influencing immune checkpoint molecules like PD-1 and PD-L1. This study aimed to investigate the effect of unclassified apathogenic rotavirus strains RVK100 and RVK228 on the expression of PD-1 and PD-L1 on T-cells derived from the peripheral blood of patients with breast cancer. Methods: Mononuclear cells of peripheral blood were isolated on a ficoll gradient, cultured in RPMI 1640 (Gibco, USA) without serum at 37 °C, 5.0% CO2 in 4 variants of the experiment: 1) negative control without viruses; 2) positive control of activation with the addition of PHA; 3) experience with the addition of 107 particles per 1 ml of the RVK100 strain; 4) experience with the addition of 107 particles per 1 ml of the RVK100 strain RVK228. After 24 and 72 hours of cultivation, the expression of PD-1 (CD279) and PD-L1 (CD274) was determined on T cells by flow cytometry. The study used antibodies conjugated with fluorochromes: anti-CD4 (PE), anti-CD8 (APC Cy7), anti-CD279 (FITC), anti-CD274 (PerCP-Cy5–5) (Becton Dickinson, USA). Results: After 24 hours, we observed increases in PD-1 expression on CD4+ (PHA—40.5%, RVK100—42.3%, RVK228—37.5%; vs. control—18.1%) and CD8+ cells (PHA—41.7%, RVK100—46.4%, RVK228—42.6%; vs. control—27.7%). Similarly, PD-L1 expression rose on CD4+ cells (RVK100—67.0%, RVK228—58.6%, PHA—75.1%; vs. control—44.8%) and CD8+ cells (RVK100—63.4%, RVK228—58.4%, PHA—52.8%; vs. control—46.2%). At 72 hours, PD-1 levels decreased significantly in CD4+ cells exposed to RVK100 (from 42.3% to 21.6%) and CD8+ cells (from 46.4% to 17.4%). Conversely, PD-L1 expression increased across all groups on CD8+ cells, reaching 67–79%, while minimal change occurred on CD4+ cells except for a minor decline in the RVK100 group. Conclusions: Both strains, like the non-specific T-mitogen PHA, caused the stimulation of the expression of immune checkpoint receptors PD-1 and PD-L1 on T-helpers and CTL after 24 hours of cultivation. After 72 hours of cultivation, RVK100, unlike RVK228, was revealed ability to reduce the expression of PD-1 on these cells.