TGF-β Is Selectively Expressed on Lymphoma B Cells and Regulates the Differentiation of Intratumoral T Cells in B-Cell Non-Hodgkin Lymphoma (NHL)

Abstract 1586

Transformation growth factor (TGF-β) is a highly pleiotropic cytokine critical to a variety of cellular events such as cell differentiation and apoptosis. TGF-β is synthesized as a prepro-TGF-β precursor and secreted after being processed in Golgi apparatus as a latent form that non-covalently combines both TGF-β and latency-associated protein (LAP). Our previous work in B-cell NHL has shown that the intratumoral T cell composition results in the establishment of a profoundly inhibitory tumor microenvironment. However, the underlying mechanism is only partially understood. In this study, using patient specimens and lymphoma cell lines, we evaluated the role of TGF-β in the tumor microenvironment and determined the effect of TGF-β on the generation of intratumoral T_H1, T_H17 and T_reg cells in B-cell NHL. First, we determined expression of TGF-β and found that a latent form of TGF-β was specifically expressed on the surface of CD19⁺ B cells, but not on other types of cells from B-cell lymphoma biopsy specimens. By screening cell lines, we found that latent TGF-β was also expressed on the surface of lymphoma cell lines, confirming the finding. Second, we tested whether surface expression by lymphoma cells led to the secretion of TGF-β in culture medium. Using an ELISA assay, we detected variable levels of latent TGF-β in the culture medium of primary malignant B cells (median 100 pg/ml per million cells, range: undetectable −229 pg/ml, n=7). Similarly, lymphoma cell lines secreted variable amounts of TGF-β from undetectable to 200 pg/ml per million cells. Next, we determined the effect of TGF-b on intratumoral T cell proliferation and differentiation. As expected, exogenous addition of TGF-β inhibited the proliferation of T cells. Notably, the proliferation of intratumoral T cells was significantly reduced when co-cultured with lymphoma cells bearing an active form of TGF-β compared to that with lymphoma cells without TGF-β. Using flow cytometry, we showed that the addition of exogenous TGF-β enhanced Foxp3 expression in activated CD4⁺, CD4⁺CD45RA⁺ or CD4⁺CD45RO⁺ intratumoral T cells, suggesting that TGF-β promotes the generation of T_reg cells in tumor microenvironment. In contrast, TGF-β suppressed the expression of IFN-γ in activated CD4⁺ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4⁺ cells, indicating that TGF-β suppresses the generation of T_H1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4⁺ T cells; however, TGF-β, in the presence of IL-6 and IL-23, up-regulated IL-17 expression in CD4⁺ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results suggest that TGF-β controls the generation of T_H1, T_H17 and T_reg cells contributing to the imbalance of effector T_H cells and inhibitory T_reg cells in the tumor microenvironment of B-cell NHL. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell differentiation and the host immune response.