Epstein-Barr virus encoded EBNA2 alters immune checkpoint PD-L1 expression by downregulating miR-34a in B cell lymphomas

Cancer cells subvert host immune surveillance by altering immune checkpoint (IC) proteins. Some Epstein-Barr virus (EBV) associated tumors have higher Programmed Cell Death Ligand, PD-L1 expression. However, it is not known how EBV alters ICs in the context of its preferred host, the B lymphocyte and in derived lymphomas. Here, we found that latency III expressing Burkitt lymphoma (BL), diffuse large B cell lymphomas (DLBCL) or their EBNA2 transfected derivatives express high PD-L1. In a DLBCL model, EBNA2 but not LMP1 is sufficient to induce PD-L1. Latency III expressing DLBCL biopsies showed high levels of PD-L1. The PD-L1 targeting oncosuppressor microRNA miR-34a was downregulated in EBNA2 transfected lymphoma cells. We identified early B cell factor 1 (EBF1) as a repressor of miR-34a transcription. Short hairpin RNA (shRNA) mediated knockdown of EBF1 was sufficient to induce miR-34a transcription, which in turn reduced PD-L1. MiR-34a reconstitution in EBNA2 transfected DLBCL reduced PD-L1 expression and increased its immunogenicity in mixed lymphocyte reactions (MLR) and in three dimensional biomimetic microfluidic chips. Given the importance of PD-L1 inhibition in immunotherapy and miR-34a dysregulation in cancers, our findings may have important implications for combinatorial immunotherapy, which include IC inhibiting antibodies and miR-34a, for EBV associated cancers.