T-Cell Defect in Diffuse Large B-Cell Lymphomas Involves Expansion of Myeloid Derived Suppressor Cells Expressing IL-10, PD-L1, and S100A12

Introduction: In diffuse large B-cell lymphoma (DLBCL), the number of circulating monocytes and neutrophils represents an independent prognostic factor. These cells include monocytic and granulocytic myeloid derived suppressor cells (M- and G-MDSCs) defined by their ability to suppress T-cell responses. MDSCs are a heterogeneous population described in inflammatory or infectious diseases as well as in numerous tumors including multiple myeloma, chronic lymphocytic leukemia and DLBCL. However, their mechanisms of action in DLBCL remain unclear. The aim of the study was to investigate the presence and mechanisms of suppression of MDSC subsets in DLBCL.

Methods: Whole blood from 56 DLBCL at diagnosis and 45 healthy donors (HD) were analyzed by flow cytometry. Quantitative Real Time PCR were analyzed on Taqman@ array microfluidic cards. Cytokine levels in plasma and culture supernatants were measured by Luminex and Elisa. T-cell proliferation after monocyte depletion or treatment with inhibitors, was analyzed by CFSE assay.

Results: By flow cytometry, we identified an expansion of G-MDSC (Lin^neg HLA-DR^neg CD33^pos CD11b^pos) and M-MDSC (CD14^pos HLA-DR^low) in DLBCL compared to HD (p<0.001). Interestingly, only M-MDSC number was correlated with the International Prognostic Index (IPI), the event-free survival (EFS), and the number of circulating T_reg. Furthermore, T-cell proliferation was restored after monocyte depletion. To identify the mechanism of suppression involved, we evaluated the gene expression of key enzymes (ARG1, IDO1, NOS2, HO-1 and PTGS2) or immunomodulatory molecules (IL10, TGF β1, CD274/PD-L1, S100A8, S100A9, S100A12) involved in MDSC biology. ARG1, IDO1, IL10, CD274/PD-L1 and S100A12 were significantly up-regulated in DLBCL (p<0.05). At the protein level arginase 1, IL-10 and S100A12 were increased in DLBCL plasma, whereas PD-L1 expression on monocyte was increased in DLBCL compared to HD (p=0.03). Arginase 1 and IDO activities where increased in DLBCL patients. In DLBCL, IL-10 and S100A12 were detected in culture supernatants from stimulated PBMC and these molecules were decreased after CD14^pos depletion (p<0.05). Finally, we defined 2 groups of DLBCL depending on M-MDSC content and analyzed the percentage of proliferating T cells after CD3/CD28 stimulation in the presence of various inhibitors or blocking antibodies (L-1MT, nor-NOHA, anti-IL-10, anti-PD-1 and anti-S100A12). In the group of patients with circulating MDSC, neutralizing IL-10, PDL-1 and S100A12 resulted in an increase of CD4 and/or CD8 T-cell proliferation.

Conclusion: In summary, we identified MDSC subsets expanded in DLBCL as well as new mechanisms of immunosuppression in DLBCL. Myeloid-dependent T-cell suppression is attributed to a release of IL-10 and S100A12 and an increase of PD-L1 expression.