Expression of PD-1 and its Ligands on Plasma Cells and T Cells of Multiple Myeloma Patient Samples
MetadataShow full item record
Multiple Myeloma (MM) is the second most common hematological malignancy and is an incurable disease. Treatments with hematopoietic stem cell transplants, chemotherapeutics and immunotherapeutics have been able to give the patients a better progression free survival (PFS) with longer overall survival; however relapse of the disease is seen often in cases of MM patients. Multidrug resistance is also frequently observed in the disease which illustrates the need for the development of targeted therapies which would target certain antigens on the cancer cells thereby inhibiting cancer cell survival and growth. Programmed cell death receptor (PD-1), Programmed cell death receptor ligand-1 (PD-L1) and Programmed cell death receptor ligand-1 (PD-L2) have been observed to play key roles in modulating the T cell activity in the tumor microenvironment of various cancers. The interaction of the ligands PD-L1 or PD-L2 on tumor cells with the receptor PD-1 on T cells has been shown to inhibit T cell activation, proliferation and function leading them to an anergic state and have therefore been studied as a model for the treatment of cancer. Studying the expression of PD-1, PD-L1 and PD-L2 on plasma cells and T cells from samples of MM patients therefore may help in the advancement towards targeted therapy. Although it has been demonstrated that plasma cells from bone marrow (BM) of patients with MM express PD-L1 and that the expression is upregulated by stimulation with interferon-gamma (IFN-gamma), similar investigations for PD-1 and PD-L2 have not been made on plasma cells from MM patients. Also T cells have been observed to express PD-1 when in an activated or exhausted state, however expression of PD-L1 and modulation of PD-1 and PD-L1 on T cells from MM patient samples have not been investigated. Therefore the goal of this study was to investigate the expression of PD-1, PD-L1 and PD-L2 concomitantly on T cells and plasma cells from BM samples from MM patients. The secondary aim of this study was to determine whether IFN-gamma; upregulated the expression of PD-1, PD-L1 and PD-L2 on plasma cells and T cells from BM of MM patients. Methods To validate the antibody reagent concentrations along with the optimization of a specifically designed panel composition flow cytometry was used. The antibody reagents in a panel were compared to the individual antibody by flow cytometry to observe consistency in the fluorescence intensity of the antibody in a panel versus individual reagent. The Application Settings were applied to the flow cytometer so as to obtained comparable measurements when acquiring samples on different days. Kinetic assays were performed to study the effect of IFN-gamma; on PD-1, PD-L1 and PD-L2 in relation to time to achieve optimal upregulation of the antigens on myeloma cells and T cells. The cells from BM samples from MM patients were cultured overnight in complete media in presence or absence of IFN-gamma;. They were then labeled with monoclonal antibody cocktails and acquired by flow cytometry. The geometric mean fluorescence intensity was used to determine the measurements of the expression levels of PD-1, PD-L1 and PD-L2. Results The data obtained from all processed samples revealed that plasma cells from MM patients do express PD-L1 and PD-L2 but not PD-1. Although, IFN-gamma; was found to upregulate the expression of PD-L1 and PD-L2 in agreement with the literature; no modulation in the expression of PD-1 was observed. T cells were observed to express both PD-1 and PD-L1, consistent with the literature, however, IFN-gamma failed to modulate the expression of PD-1 and PD-L1 on T cells from BM samples from MM patients. A comparison between diseased marrow sample group and normal marrow sample group revealed that the diseased group showed a higher expression of PD-1, PD-L1 and PD-L2 on plasma cells and PD-1 and PD-L1 on T cells. The effect of IFN-gamma; on PD-L1 and PD-L2 expression on plasma cells was found to be higher in the diseased marrow group when compared to the normal group. The expression of PD-1 and PD-L1 on T cells was observed not to be modulated upon incubation with IFN-gamma; in either group. Conclusion In conclusion, it was observed that T cells expressed PD-1 and PD-L1 while plasma cells expressed PD-L1 and PD-L2 but not PD-1. IFN-gamma; was observed to have no modulation on the expression of PD-1 and PD-L1 on T cells. On plasma cells the expression of PD-L1 was found to be modulated by stimulation with IFN-gamma; while PD-1 and PD-L2 expression remained unchanged. A comparison between expression of PD-1, PD-L1 and PD-L2 on cells from "diseased marrow group" and "normal marrow group" showed that the diseased marrow group expressed higher levels of PD-1, PD-L1 and PD-L2 than the normal marrow group.