IL-10 Contributes to the Development of Immunosuppressive CD14+HLA-DR^low/- monocytes in B-Cell Non-Hodgkin’s Lymphoma

Background - The phenotype and biological role of monocytes in B-cell non-Hodgkin lymphoma (NHL) is not fully understood, however, an increased absolute monocyte count in the peripheral blood of lymphoma patients is associated with a poor prognosis. We have previously reported that monocytes from patients with relapsed B-cell NHL displayed an immunosuppressive CD14⁺HLA-DR^low/- phenotype that correlated with a poor prognosis. However, the underlying mechanism by which CD14⁺HLA-DR^low/- monocytes develop in patients with B-cell NHL is unknown. The goal of this study was to determine whether cytokines are responsible for the increased number and phenotype of CD14⁺HLA-DR^low/-monocytes present in lymphoma patients.

Methods – Whole blood from patients with newly diagnosed, untreated B-cell NHL (n=20) and healthy donors (n=20) was stained with a panel of antibodies and analyzed by 10-color flow cytometry. Cytokine levels in serum and culture supernatants were measured by Luminex and ELISA, respectively. Polarization of monocytes from healthy donors was performed by incubating CD14⁺ cells with specific cytokines or supernatants from B-cell NHL cell lines for 24 hours. Activation and proliferation of CD4⁺T cells cocultured with IL-10-pretreated monocytes were measured by flow cytometry.

Results – By flow cytometry, we observed that the absolute number of peripheral monocytes was increased in newly-diagnosed lymphoma patients compared to healthy donors. A significant proportion of these monocytes displayed an immunosuppressive CD14⁺HLA-DR^low/- phenotype and the numbers of CD14⁺HLA-DR^low/- cells were significantly higher in lymphoma patients than in healthy donors. To identify the underlying mechanism by which CD14⁺HLA-DR^low/- monocytes develop thereby leading to increased numbers of CD14⁺HLA-DR^low/- monocytes in B-cell NHL, we tested a panel of cytokines and found that IL-10 played a key role in the process. Firstly, treatment of monocytes with IL-10 in vitro resulted in the generation of CD14⁺HLA-DR^low/- cells. Second, monocytes co-cultured with IL-10 producing lymphoma B-cells, or treated with supernatants from lymphoma cell cultures, developed a CD14⁺HLA-DR^low/- phenotype. Thirdly, IL-10 levels were increased in the serum of DLBCL (p<0.0001) and FL patients (p=0.010) compared to healthy controls, and serum IL-10 levels correlated with increased numbers of peripheral monocytes (p=0.02). Finally, IL-10-pretreated CD14⁺HLA-DR^low/- monocytes were significantly immunosuppressive and inhibited the proliferation and activation of CD4⁺T cells in co-culture assays.

Conclusions- Taken together, our results suggest that IL-10 signaling contributes to the increased numbers of CD14⁺HLA-DR^low/- monocytes in B-cell NHL. Strategies to inhibit IL-10 production may therefore have therapeutic potential in B-cell NHL.