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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 human MDSC in myeloma cell conditioned mediumA. Human PBMC were cultured in control medium or HMCL-CM (RPMI8226, OPM2 and LP1) for 6 days and T cell proliferation was determined by FACS staining (n = 4). B. Peripheral blood mononuclear cells derived from healthy donor blood samples were cultured for 24 h in MM-CM (RPMI8226, OPM2, LP1) and viability was analyzed by CellTiter-Glo assay (n = 3). C. After 72 h in HMCL-CM, PBMC were analyzed by flow cytometry for MDSC markers CD11b, CD33, CD14 and HLA-DRlow (n = 4). Gating strategy is shown. D. Human CD11b+ cells, cultured in RPMI8226-CM (3 days) and stained by the May-Grünwald-Giemsa method, are shown. Bright-field pictures were taken with a Nikon Eclipse 90i microscope at 400x original magnification. * indicate p < 0.05 (Mann–Whitney U-test). Error bars represent the SD.
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Figure 5: Generation of human MDSC in myeloma cell conditioned mediumA. Human PBMC were cultured in control medium or HMCL-CM (RPMI8226, OPM2 and LP1) for 6 days and T cell proliferation was determined by FACS staining (n = 4). B. Peripheral blood mononuclear cells derived from healthy donor blood samples were cultured for 24 h in MM-CM (RPMI8226, OPM2, LP1) and viability was analyzed by CellTiter-Glo assay (n = 3). C. After 72 h in HMCL-CM, PBMC were analyzed by flow cytometry for MDSC markers CD11b, CD33, CD14 and HLA-DRlow (n = 4). Gating strategy is shown. D. Human CD11b+ cells, cultured in RPMI8226-CM (3 days) and stained by the May-Grünwald-Giemsa method, are shown. Bright-field pictures were taken with a Nikon Eclipse 90i microscope at 400x original magnification. * indicate p < 0.05 (Mann–Whitney U-test). Error bars represent the SD.

Mentions: We also investigated the induction of human MDSC in MM-CM. Since it is difficult to obtain BM from healthy donors we used peripheral blood mononuclear cells. Total CFSE-labeled PBMC were stimulated with anti-CD3/CD28 dynabeads and cultured in control medium or CM derived from HMCL (RPMI8226, OPM2 and LP1). A significantly decreased CD4+ and CD8+ T cell proliferation was observed in the cultures with MM-CM (Figure 5A) indicating that an immunosuppressive population is generated in MM-CM. Similar to mouse, an increased viability of total PBMC in the presence of HMCL-CM could be observed (Figure 5B). Furthermore, the presence of human MDSC was analyzed based on CD11b, CD33, HLA-DR, CD14 and CD15 expression. We did not detect differences in the total percentage of CD11b+CD33+ cells between the control condition and HMCL-CM. However, within the CD11b+CD33+ population, the percentages of mononuclear MDSC (identified as CD14+HLA-DRlow) were increased compared to control conditions (Figure 5C). Interestingly, human CD11b+ cells with a monocytic and polymorphonuclear morphology could be observed 1–2 days after culture in MM-CM. After 3 days of culture, monocytic cells remained while cells with a polymorphonuclear morphology disappeared (Figure 5D). These data indicate that soluble factor(s) secreted by MM cells have the capacity to induce MDSC with T cell suppressive ability. Additionally, we investigated cytokines responsible for this MDSC induction. As GM-CSF is not expressed by human MM cell lines (data not shown), we investigated the effects of anti-VEGF, anti-IL-10 and anti-M-CSF, cytokines which are associated with MDSC expansion and produced by human MM cells. Anti-IL-10 was able to partially decrease the accumulation of CD14+HLA-DRlow in RPMI8226-CM, however we could not observe this effect in the presence of LP1-CM (Supplementary 3C–3D). Anti-VEGF and anti-M-CSF had no effect on the presence of MDSC.


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 human MDSC in myeloma cell conditioned mediumA. Human PBMC were cultured in control medium or HMCL-CM (RPMI8226, OPM2 and LP1) for 6 days and T cell proliferation was determined by FACS staining (n = 4). B. Peripheral blood mononuclear cells derived from healthy donor blood samples were cultured for 24 h in MM-CM (RPMI8226, OPM2, LP1) and viability was analyzed by CellTiter-Glo assay (n = 3). C. After 72 h in HMCL-CM, PBMC were analyzed by flow cytometry for MDSC markers CD11b, CD33, CD14 and HLA-DRlow (n = 4). Gating strategy is shown. D. Human CD11b+ cells, cultured in RPMI8226-CM (3 days) and stained by the May-Grünwald-Giemsa method, are shown. Bright-field pictures were taken with a Nikon Eclipse 90i microscope at 400x original magnification. * indicate p < 0.05 (Mann–Whitney U-test). Error bars represent the SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Generation of human MDSC in myeloma cell conditioned mediumA. Human PBMC were cultured in control medium or HMCL-CM (RPMI8226, OPM2 and LP1) for 6 days and T cell proliferation was determined by FACS staining (n = 4). B. Peripheral blood mononuclear cells derived from healthy donor blood samples were cultured for 24 h in MM-CM (RPMI8226, OPM2, LP1) and viability was analyzed by CellTiter-Glo assay (n = 3). C. After 72 h in HMCL-CM, PBMC were analyzed by flow cytometry for MDSC markers CD11b, CD33, CD14 and HLA-DRlow (n = 4). Gating strategy is shown. D. Human CD11b+ cells, cultured in RPMI8226-CM (3 days) and stained by the May-Grünwald-Giemsa method, are shown. Bright-field pictures were taken with a Nikon Eclipse 90i microscope at 400x original magnification. * indicate p < 0.05 (Mann–Whitney U-test). Error bars represent the SD.
Mentions: We also investigated the induction of human MDSC in MM-CM. Since it is difficult to obtain BM from healthy donors we used peripheral blood mononuclear cells. Total CFSE-labeled PBMC were stimulated with anti-CD3/CD28 dynabeads and cultured in control medium or CM derived from HMCL (RPMI8226, OPM2 and LP1). A significantly decreased CD4+ and CD8+ T cell proliferation was observed in the cultures with MM-CM (Figure 5A) indicating that an immunosuppressive population is generated in MM-CM. Similar to mouse, an increased viability of total PBMC in the presence of HMCL-CM could be observed (Figure 5B). Furthermore, the presence of human MDSC was analyzed based on CD11b, CD33, HLA-DR, CD14 and CD15 expression. We did not detect differences in the total percentage of CD11b+CD33+ cells between the control condition and HMCL-CM. However, within the CD11b+CD33+ population, the percentages of mononuclear MDSC (identified as CD14+HLA-DRlow) were increased compared to control conditions (Figure 5C). Interestingly, human CD11b+ cells with a monocytic and polymorphonuclear morphology could be observed 1–2 days after culture in MM-CM. After 3 days of culture, monocytic cells remained while cells with a polymorphonuclear morphology disappeared (Figure 5D). These data indicate that soluble factor(s) secreted by MM cells have the capacity to induce MDSC with T cell suppressive ability. Additionally, we investigated cytokines responsible for this MDSC induction. As GM-CSF is not expressed by human MM cell lines (data not shown), we investigated the effects of anti-VEGF, anti-IL-10 and anti-M-CSF, cytokines which are associated with MDSC expansion and produced by human MM cells. Anti-IL-10 was able to partially decrease the accumulation of CD14+HLA-DRlow in RPMI8226-CM, however we could not observe this effect in the presence of LP1-CM (Supplementary 3C–3D). Anti-VEGF and anti-M-CSF had no effect on the presence of MDSC.

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