Soluble PD-1 Ligands Regulate T-Cell Function in Waldenstrom's Macroglobulinemia

While immune checkpoint molecules contribute to the malignant development of several cancers, including certain types of lymphomas, their potential significance in malignant progression of Waldenstrom's Macroglobulinemia (WM), a low-grade B-cell lymphoma, has not fully been described. The main characteristic of WM is infiltration of lymphoplasmacytic cells in the bone marrow (BM) and increased monoclonal IgM production by malignant cells within BM microenvironment. While next generation sequencing analysis has identified mutations in the MYD88 gene as the driver of WM development, studies by our group have also highlighted a role for the BM microenvironment in pathophysiology of WM and shown that elevated levels of certain cytokines in the BM, such as IL-6 and IL-21, promote both cell proliferation and IgM secretion in WM.

In this study, we investigated the impact of cytokines on the immune checkpoint receptor Programmed Death-1 (PD-1) and its ligands PD-L1 and PD-L2 and evaluated how these molecules contribute to malignant progression in WM.

Using RT-PCR analysis we have shown that treatment of WM cell lines with IL-21 and IL-6 significantly induce PD-L1 and PD-L2 gene expression, implying that elevated levels of these cytokines within the BM microenvironment could be associated with increased PD-L1 and PD-L2 expression by malignant cells in WM patients. We therefore performed RT-PCR analysis on cells isolated from patients' BM and confirmed increased PD-L1 and PD-L2 expression by both WM and BM cells when compared normal BM cells. In contrast, flow cytometry analysis showed minimal expression of these ligands on the surface of the WM cells, with no significant difference between WM and normal BM cells, indicating that there might be a regulatory mechanism controlling surface expression of these ligands. Further analysis using ELISA and Western Blotting showed that PD-L1 and PD-L2 are found as secreted forms in both BM plasma and peripheral blood serum of the WM patients and their level is significantly higher in WM as compared to normal subjects. Interestingly, in vitro treatment of the WM cell lines by IL-21 and IL-6 increased secreted PD-L1 and PD-L2 in the culture media of the cells, providing an additional line of evidence for elevated PD-L1 and PD-L2 levels in WM patients. Subsequent analysis showed that the secreted form of the PD-L1 and PD-L2 binds to T-cells and induces signals that reduce T-cell proliferation and cell cycle progression, the effects that are associated with a reduction in p-ERK and cyclin A levels.

In summary, our data identifies that secreted form of PD-1 ligands is elevated in WM and play an important role in regulating T-cell function. Given the capability of secreted form to be bioactive at distant sites, soluble PD-1 ligands may have potential to promote disease progression.