Abstract PD-1/L1 and CTLA-4 blockade immunotherapies have been approved for 13 types of cancers and are being studied in diffuse large B-cell lymphoma (DLBCL), the most common aggressive B-cell lymphoma. However, whether both PD-1 and CTLA-4 checkpoints are active and clinically significant in DLBCL is unknown. Whether PD-1 ligands expressed by tumor cells or by the microenvironment of DLBCL are critical for the PD-1 immune checkpoint is unclear. We performed immunophenotypic profiling for 405 patients with de novo DLBCL using a MultiOmyx immunofluorescence platform and simultaneously quantitated expression/coexpression of 13 immune markers to identify prognostic determinants. In both training and validation cohorts, results demonstrated a central role of the tumor immune microenvironment, and when its functionality was impaired by deficiency in tumor-infiltrating T cells and/or natural killer cells, high PD-1 expression (but not CTLA-4) on CD8+ T cells, or PD-L1 expression on T cells and macrophages, patients had significantly poorer survival after rituximab–CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) immunochemotherapy. In contrast, tumor-cell PD-L2 expression was associated with superior survival, as well as PD-L1+CD20+ cells proximal (indicates interaction) to PD-1+CD8+ T cells in patients with low PD-1+ percentage of CD8+ T cells. Gene-expression profiling results suggested the reversibility of T-cell exhaustion in PD-1+/PD-L1+ patients with unfavorable prognosis and implication of LILRA/B, IDO1, CHI3L1, and SOD2 upregulation in the microenvironment dysfunction with PD-L1 expression. This study comprehensively characterized the DLBCL immune landscape, deciphered the differential roles of various checkpoint components in rituximab–CHOP resistance in DLBCL patients, and suggests targets for PD-1/PD-L1 blockade and combination immunotherapies.