Analysis of CD20 and PD-L1 levels on small extracellular vesicles (sEV) produced by DLBCL cells and EBV-transformed B cells, and potential role in T cell inhibition

Increasing evidence supports a role for small extracellular vesicles (sEV, including exosomes) in Diffuse Large B-cell lymphoma (DLBCL) progression and resistance to treatment. CD20 and PD-L1 are found on DLBCL-derived sEV, but little is known about their patient-level heterogeneity. Moreover, the capacity of PD-L1⁺ sEV to modulate T cells needs to be clarified. Herein we analyzed sEV produced by human DLBCL cell lines and EBV-transformed B cell-lymphoblastoid cell lines (LCLs), a model allowing autologous T cell co-cultures. We determined CD20 and PD-L1 levels on plasma sEV from patient samples vs healthy volunteers (HV). sEV functional relevance was also investigated on CD4⁺ and CD8⁺ T cells. sEV derived from all cell lines showed an enrichment of CD20 and a high glycosylated PD-L1 expression when compared to cell lysates. High PD-L1 expression on LCL-derived sEV was associated with higher CD4⁺ and CD8⁺ T cell apoptosis. In patients, plasma sEV concentration was higher vs HV. Compared to sEV-CD20 level that seemed higher in patients, PD-L1 level in sEV was not different from those of HV. A high glycosylated PD-L1 level was shown in sEV from both patients and HV plasma samples, that was associated with the same inhibiting effect on activated T cells. We conclude that sEV derived from EBV-transformed B cells realize an immunosuppressive role that involved cell-cell interaction and probably at least PD-L1. Furthermore, our findings suggest the potential of circulating sEV as a source of biomarkers in DLBCL, notably to have information on immunotherapeutic target levels of parental tumor cells.