B2M drives PD-1 inhibitor resistance in DLBCL through independent dual immune escape mechanisms

Abstract PD-1/PD-L1 immune checkpoint inhibitors (ICIs) demonstrate promising therapeutic potential in diffuse large B-cell lymphoma (DLBCL). However, a subset of DLBCL patients exhibits primary or acquired resistance to PD-1 blockade. This study investigated the mechanistic role of β2-microglobulin (B2M) mutations in modulating PD-L1 expression and their contribution to ICI resistance in DLBCL. The association between B2M mutations and PD-L1 expression in DLBCL patient samples was analyzed via immunohistochemistry. A B2M-knockout DLBCL cell line model was established, followed by evaluation of PD-L1 and MHC-I expression changes through qRT-PCR, western blot, and flow cytometry. T cell activation (CD69/CD25 markers) and cytotoxic activity (lactate dehydrogenase release) were assessed in PBMC co-culture experiments. Mechanistic studies employing MHC-I inhibitors and IFN-γ stimulation were conducted to dissect the regulatory relationship between MHC-I and PD-L1. Co-culture systems with PD-1 blockade was performed to validate the impact of B2M on PD-1 inhibitor resistance. The results demonstrated that B2M knockout significantly downregulated both PD-L1 mRNA and protein levels, concomitant with impaired MHC-I complex assembly and reduced TAP1/2 expression. B2M-deficient cells failed to activate CD8+ T cells, evidenced by diminished CD69+/CD25+ surface expression and reduced cytotoxic efficiency. Notably, B2M ablation abolished IFN-γ-induced PD-L1 upregulation (p < 0.05), demonstrating that intact MHC-I functionality is a prerequisite for PD-L1 expression. Further co-culture systems with PD-1 blockade validated the functional roles of MHC-I and PD-L1 in modulating PD-1 inhibitor resistance mediated by B2M. In conclusion, this work indicated that B2M mutations synergistically drive DLBCL resistance to PD-1 inhibitors through dual mechanisms-suppressing PD-L1 expression and completely disabling antigen presentation. The study highlights that combined strategies restoring MHC-I expression and targeting PD-L1 may overcome immunotherapy resistance, offering a novel direction for precision therapy in DLBCL.