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Multiple myeloma induces Mcl-1 expression and survival of myeloid-derived suppressor cells.

De Veirman K, Van Ginderachter JA, Lub S, De Beule N, Thielemans K, Bautmans I, Oyajobi BO, De Bruyne E, Menu E, Lemaire M, Van Riet I, Vanderkerken K, Van Valckenborgh E - Oncotarget (2015)

Bottom Line: Interestingly, in vivo MDSC targeting by anti-GR1 antibodies and 5-Fluorouracil resulted in a significant reduced tumor load in 5TMM-diseased mice.Finally, increased Mcl-1 expression was identified as underlying mechanism for MDSC survival.In conclusion, our data demonstrate that soluble factors from MM cells are able to generate MDSC through Mcl-1 upregulation and this cell population can be considered as a possible target in MM disease.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

ABSTRACT
Myeloid-derived suppressor cells (MDSC) are contributing to an immunosuppressive environment by their ability to inhibit T cell activity and thereby promoting cancer progression. An important feature of the incurable plasma cell malignancy Multiple Myeloma (MM) is immune dysfunction. MDSC were previously identified to be present and active in MM patients, however little is known about the MDSC-inducing and -activating capacity of MM cells. In this study we investigated the effects of the tumor microenvironment on MDSC survival. During MM progression in the 5TMM mouse model, accumulation of MDSC in the bone marrow was observed in early stages of disease development, while circulating myeloid cells were increased at later stages of disease. Interestingly, in vivo MDSC targeting by anti-GR1 antibodies and 5-Fluorouracil resulted in a significant reduced tumor load in 5TMM-diseased mice. In vitro generation of MDSC was demonstrated by increased T cell immunosuppressive capacity and MDSC survival was observed in the presence of MM-conditioned medium. Finally, increased Mcl-1 expression was identified as underlying mechanism for MDSC survival. In conclusion, our data demonstrate that soluble factors from MM cells are able to generate MDSC through Mcl-1 upregulation and this cell population can be considered as a possible target in MM disease.

No MeSH data available.


Related in: MedlinePlus

Generation of murine MDSC in myeloma cell conditioned mediumA. Mouse CD11b+ cells were cultured in 5T33MMvt-CM for 3 days at different MDSC/spleen cell ratios. CFSE labeled cells were activated with anti-CD3/CD28 Dynabeads and T cell proliferation was determined by FACS staining (n = 3). B. CD11b+ cells were isolated from the BM of naive C57BL/KaLwRij mice and cultured in 5T33MMvt-CM for 48 h. Viability was measured by CellTiter-Glo assay (n = 5). C and D. Apoptosis was determined by AnnexinV FITC (n = 4) and active Caspase 3 FITC (n = 3) with flow cytometry. In some conditions, 10 μg/ml anti GM-CSF or control rat IgG1 was added. E. Cytospins of murine CD11b+ cells, cultured in 5T33MMvt-CM (2 days) and stained by the May-Grünwald-Giemsa method, are shown. * indicate p < 0.05, ** indicate p < 0.01 (Mann–Whitney U-test). Error bars represent the SD.
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Figure 4: Generation of murine MDSC in myeloma cell conditioned mediumA. Mouse CD11b+ cells were cultured in 5T33MMvt-CM for 3 days at different MDSC/spleen cell ratios. CFSE labeled cells were activated with anti-CD3/CD28 Dynabeads and T cell proliferation was determined by FACS staining (n = 3). B. CD11b+ cells were isolated from the BM of naive C57BL/KaLwRij mice and cultured in 5T33MMvt-CM for 48 h. Viability was measured by CellTiter-Glo assay (n = 5). C and D. Apoptosis was determined by AnnexinV FITC (n = 4) and active Caspase 3 FITC (n = 3) with flow cytometry. In some conditions, 10 μg/ml anti GM-CSF or control rat IgG1 was added. E. Cytospins of murine CD11b+ cells, cultured in 5T33MMvt-CM (2 days) and stained by the May-Grünwald-Giemsa method, are shown. * indicate p < 0.05, ** indicate p < 0.01 (Mann–Whitney U-test). Error bars represent the SD.

Mentions: As we could observe an induction of MDSC in early stages of MM development, we further wanted to evaluate the underlying mechanisms of MDSC induction in vitro stimulated by conditioned medium (CM) derived from 5T33MMvt cells. In first instance, the immunosuppressive capacity was measured as this is the major characteristic of MDSC. Murine CD11b+ cells from naïve bone marrow were cultured in presence or absence of 5T33MMvt-CM with total CFSE-labeled spleen cells at different MDSC/spleen cell ratios. T cells were stimulated with anti-CD3/CD28 dynabeads and analyzed for proliferation by flow cytometry. CD11b+ cells cultured in the presence of MM-CM were able to significantly decrease T cell proliferation compared to control (Figure 4A). We further investigated the effect of MM-CM on the survival of CD11b+ cells. Interestingly, CD11b+ cells cultured in CM derived from 5T33MMvt cells, have an increased viability and reduced apoptosis (reduced % Annexin V+ and cleaved caspase-3+ cells) as compared to CD11b+ cells cultured in control medium (Figure 4B–4D). Cytospin stainings showed the presence of both monocytic and polymorphonuclear cells 2 days after incubation with MM-CM for murine CD11b+ cells (Figure 4E). This is in accordance with the observation that both sorted Ly6Glow and Ly6Ghigh CD11b+ cells incubated with 5T33MMvt-CM have a survival benefit (data not shown). Next, we aimed to identify the soluble factor involved in this phenomenon. As GM-CSF, a major survival factor for MDSC was identified in the 5T33MMvt-CM by cytokine array (Supplementary Figure 2), we investigated the effect of a GM-CSF blocking antibody and found that the pro-survival effect of the MM-CM could be abrogated (Figure 4B–4C). Other MDSC survival factors like VEGF and IL-10 are also produced by 5T33MMvt cells, but no effects of anti-VEGF and anti-IL-10 antibodies could be observed (Supplementary Figure 3A–3B).


Multiple myeloma induces Mcl-1 expression and survival of myeloid-derived suppressor cells.

De Veirman K, Van Ginderachter JA, Lub S, De Beule N, Thielemans K, Bautmans I, Oyajobi BO, De Bruyne E, Menu E, Lemaire M, Van Riet I, Vanderkerken K, Van Valckenborgh E - Oncotarget (2015)

Generation of murine MDSC in myeloma cell conditioned mediumA. Mouse CD11b+ cells were cultured in 5T33MMvt-CM for 3 days at different MDSC/spleen cell ratios. CFSE labeled cells were activated with anti-CD3/CD28 Dynabeads and T cell proliferation was determined by FACS staining (n = 3). B. CD11b+ cells were isolated from the BM of naive C57BL/KaLwRij mice and cultured in 5T33MMvt-CM for 48 h. Viability was measured by CellTiter-Glo assay (n = 5). C and D. Apoptosis was determined by AnnexinV FITC (n = 4) and active Caspase 3 FITC (n = 3) with flow cytometry. In some conditions, 10 μg/ml anti GM-CSF or control rat IgG1 was added. E. Cytospins of murine CD11b+ cells, cultured in 5T33MMvt-CM (2 days) and stained by the May-Grünwald-Giemsa method, are shown. * indicate p < 0.05, ** indicate p < 0.01 (Mann–Whitney U-test). Error bars represent the SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496373&req=5

Figure 4: Generation of murine MDSC in myeloma cell conditioned mediumA. Mouse CD11b+ cells were cultured in 5T33MMvt-CM for 3 days at different MDSC/spleen cell ratios. CFSE labeled cells were activated with anti-CD3/CD28 Dynabeads and T cell proliferation was determined by FACS staining (n = 3). B. CD11b+ cells were isolated from the BM of naive C57BL/KaLwRij mice and cultured in 5T33MMvt-CM for 48 h. Viability was measured by CellTiter-Glo assay (n = 5). C and D. Apoptosis was determined by AnnexinV FITC (n = 4) and active Caspase 3 FITC (n = 3) with flow cytometry. In some conditions, 10 μg/ml anti GM-CSF or control rat IgG1 was added. E. Cytospins of murine CD11b+ cells, cultured in 5T33MMvt-CM (2 days) and stained by the May-Grünwald-Giemsa method, are shown. * indicate p < 0.05, ** indicate p < 0.01 (Mann–Whitney U-test). Error bars represent the SD.
Mentions: As we could observe an induction of MDSC in early stages of MM development, we further wanted to evaluate the underlying mechanisms of MDSC induction in vitro stimulated by conditioned medium (CM) derived from 5T33MMvt cells. In first instance, the immunosuppressive capacity was measured as this is the major characteristic of MDSC. Murine CD11b+ cells from naïve bone marrow were cultured in presence or absence of 5T33MMvt-CM with total CFSE-labeled spleen cells at different MDSC/spleen cell ratios. T cells were stimulated with anti-CD3/CD28 dynabeads and analyzed for proliferation by flow cytometry. CD11b+ cells cultured in the presence of MM-CM were able to significantly decrease T cell proliferation compared to control (Figure 4A). We further investigated the effect of MM-CM on the survival of CD11b+ cells. Interestingly, CD11b+ cells cultured in CM derived from 5T33MMvt cells, have an increased viability and reduced apoptosis (reduced % Annexin V+ and cleaved caspase-3+ cells) as compared to CD11b+ cells cultured in control medium (Figure 4B–4D). Cytospin stainings showed the presence of both monocytic and polymorphonuclear cells 2 days after incubation with MM-CM for murine CD11b+ cells (Figure 4E). This is in accordance with the observation that both sorted Ly6Glow and Ly6Ghigh CD11b+ cells incubated with 5T33MMvt-CM have a survival benefit (data not shown). Next, we aimed to identify the soluble factor involved in this phenomenon. As GM-CSF, a major survival factor for MDSC was identified in the 5T33MMvt-CM by cytokine array (Supplementary Figure 2), we investigated the effect of a GM-CSF blocking antibody and found that the pro-survival effect of the MM-CM could be abrogated (Figure 4B–4C). Other MDSC survival factors like VEGF and IL-10 are also produced by 5T33MMvt cells, but no effects of anti-VEGF and anti-IL-10 antibodies could be observed (Supplementary Figure 3A–3B).

Bottom Line: Interestingly, in vivo MDSC targeting by anti-GR1 antibodies and 5-Fluorouracil resulted in a significant reduced tumor load in 5TMM-diseased mice.Finally, increased Mcl-1 expression was identified as underlying mechanism for MDSC survival.In conclusion, our data demonstrate that soluble factors from MM cells are able to generate MDSC through Mcl-1 upregulation and this cell population can be considered as a possible target in MM disease.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

ABSTRACT
Myeloid-derived suppressor cells (MDSC) are contributing to an immunosuppressive environment by their ability to inhibit T cell activity and thereby promoting cancer progression. An important feature of the incurable plasma cell malignancy Multiple Myeloma (MM) is immune dysfunction. MDSC were previously identified to be present and active in MM patients, however little is known about the MDSC-inducing and -activating capacity of MM cells. In this study we investigated the effects of the tumor microenvironment on MDSC survival. During MM progression in the 5TMM mouse model, accumulation of MDSC in the bone marrow was observed in early stages of disease development, while circulating myeloid cells were increased at later stages of disease. Interestingly, in vivo MDSC targeting by anti-GR1 antibodies and 5-Fluorouracil resulted in a significant reduced tumor load in 5TMM-diseased mice. In vitro generation of MDSC was demonstrated by increased T cell immunosuppressive capacity and MDSC survival was observed in the presence of MM-conditioned medium. Finally, increased Mcl-1 expression was identified as underlying mechanism for MDSC survival. In conclusion, our data demonstrate that soluble factors from MM cells are able to generate MDSC through Mcl-1 upregulation and this cell population can be considered as a possible target in MM disease.

No MeSH data available.


Related in: MedlinePlus