Samenvatting
Multiple Myeloma (MM) is an incurable malignancy of terminally differentiated plasma cells, which are predominantly localized in the bone marrow. The presence of an immunosuppressive bone marrow microenvironment and dysfunction of immune cells are both described in MM patients; however the mechanisms controlling this immunosuppression are not well defined. Recently, a heterogeneous population of immature myeloid cells, the so called myeloid-derived suppressor cells (MDSC), were identified to be present and active in MM. MDSC are thought to promote cancer progression by their T-cell suppressive capacity, however little is known about the generation and activation of MDSC by MM cells. Also, it is not known whether MDSC are involved in the development of drug resistance.
In this study we investigated the effects of myeloma cells on MDSC induction using the 5T33MM mouse model and healthy donor blood samples. In a first instance, myeloid cells (CD11b+) were isolated from the bone marrow of naïve C57BL/KaLwRij mice and cultured in conditioned medium (CM) derived from 5T33MMvt cells to measure survival with CellTiterGlo Assay and AnnexinV staining. Increased viability and reduced apoptosis of CD11b+ cells cultured in MM-CM compared to control medium were observed. In literature GM-CSF is described as a major survival factor for MDSC. The pro-survival effect induced by MM-CM could be abrogated by the use of a GM-CSF blocking antibody. In addition, we analyzed the effect of CM of human myeloma cell lines (HMCL) RPMI8226, LP-1 and OPM2 on the viability of peripheral blood mononuclear cells (PBMC) of healthy individuals. Human myeloma CM increased the viability of PBMC compared to control medium. When we analyzed the phenotype of PBMC using MDSC markers, we did not detect differences in the total CD11b+CD33+ percentage between control condition and HMCL-CM. However, in the CD11b+CD33+ population, the percentages of mononuclear MDSC (identified as CD14+HLA-DRlow) and polymorphonuclear MDSC (identified as CD11b+CD33+CD15+) were increased compared to control conditions. The immunosuppressive capacity of the generated population was analyzed. For mouse as well as for human experiments, we could observe a decreased proliferation of T-cells in CM compared to control medium. To unravel the mechanism of MM-induced MDSC survival, the expression of apoptosis regulating proteins from the Bcl-2 family (bcl-2,bcl-xl,mcl-1) and survival proteins (pSTAT3,STAT3) were investigated by western blot. Mouse CD11b+ cells incubated with 5T33MMvt CM had a clear increase in pSTAT3 compared to control. Moreover the anti-apoptotic proteins mcl-1 and bcl-xl were increased in 5T33MMvt-CM compared to control, while bcl-2 was not affected. We were able to confirm the results of pSTAT3, bcl-2 and mcl-1 using human CD11b+ cells incubated in control or RPMI8226-CM. In addition, we investigated the sensitivity of MM cells to drugs currently used in the clinic (melphalan and bortezomib) in the presence of MDSC. 5T33MMvv cells incubated with MDSC could survive better than 5T33MMvv cells cultured without MDSC. This phenomenon was also observed when drugs were added, indicating a pro-survival effect of MDSC on MM cells.
From our experiments we can conclude that soluble factor(s) from myeloma cells are able to reduce T-cell proliferation. Since we could observe a better survival of CD11b+ cells and the appearance of MDSC phenotypes in myeloma CM, this immunosuppression is likely caused by the generation and increased survival of MDSC in myeloma CM with an increased STAT3 phosphorylation, mcl-1 and bcl-xl expression as underlying mechanisms.
In this study we investigated the effects of myeloma cells on MDSC induction using the 5T33MM mouse model and healthy donor blood samples. In a first instance, myeloid cells (CD11b+) were isolated from the bone marrow of naïve C57BL/KaLwRij mice and cultured in conditioned medium (CM) derived from 5T33MMvt cells to measure survival with CellTiterGlo Assay and AnnexinV staining. Increased viability and reduced apoptosis of CD11b+ cells cultured in MM-CM compared to control medium were observed. In literature GM-CSF is described as a major survival factor for MDSC. The pro-survival effect induced by MM-CM could be abrogated by the use of a GM-CSF blocking antibody. In addition, we analyzed the effect of CM of human myeloma cell lines (HMCL) RPMI8226, LP-1 and OPM2 on the viability of peripheral blood mononuclear cells (PBMC) of healthy individuals. Human myeloma CM increased the viability of PBMC compared to control medium. When we analyzed the phenotype of PBMC using MDSC markers, we did not detect differences in the total CD11b+CD33+ percentage between control condition and HMCL-CM. However, in the CD11b+CD33+ population, the percentages of mononuclear MDSC (identified as CD14+HLA-DRlow) and polymorphonuclear MDSC (identified as CD11b+CD33+CD15+) were increased compared to control conditions. The immunosuppressive capacity of the generated population was analyzed. For mouse as well as for human experiments, we could observe a decreased proliferation of T-cells in CM compared to control medium. To unravel the mechanism of MM-induced MDSC survival, the expression of apoptosis regulating proteins from the Bcl-2 family (bcl-2,bcl-xl,mcl-1) and survival proteins (pSTAT3,STAT3) were investigated by western blot. Mouse CD11b+ cells incubated with 5T33MMvt CM had a clear increase in pSTAT3 compared to control. Moreover the anti-apoptotic proteins mcl-1 and bcl-xl were increased in 5T33MMvt-CM compared to control, while bcl-2 was not affected. We were able to confirm the results of pSTAT3, bcl-2 and mcl-1 using human CD11b+ cells incubated in control or RPMI8226-CM. In addition, we investigated the sensitivity of MM cells to drugs currently used in the clinic (melphalan and bortezomib) in the presence of MDSC. 5T33MMvv cells incubated with MDSC could survive better than 5T33MMvv cells cultured without MDSC. This phenomenon was also observed when drugs were added, indicating a pro-survival effect of MDSC on MM cells.
From our experiments we can conclude that soluble factor(s) from myeloma cells are able to reduce T-cell proliferation. Since we could observe a better survival of CD11b+ cells and the appearance of MDSC phenotypes in myeloma CM, this immunosuppression is likely caused by the generation and increased survival of MDSC in myeloma CM with an increased STAT3 phosphorylation, mcl-1 and bcl-xl expression as underlying mechanisms.