mdscs  (Miltenyi Biotec)

 
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    Myeloid Derived Suppressor Cell Isolation Kit mouse
    Description:
    The Myeloid Derived Suppressor Cell Isolation Kit has been developed for the isolation of Gr 1highLy 6G and Gr 1dimLy 6G myeloid cells from lymphoid tissue This Kit works ideally for spleen and tumor tissues
    Catalog Number:
    130-094-538
    Price:
    720.00
    Category:
    MACS Cell Separation Cell separation reagents MicroBeads and Isolation Kits Myeloid Derived Suppressor Cell Isolation Kit mouse
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    Structured Review

    Miltenyi Biotec mdscs
    Myeloid Derived Suppressor Cell Isolation Kit mouse
    The Myeloid Derived Suppressor Cell Isolation Kit has been developed for the isolation of Gr 1highLy 6G and Gr 1dimLy 6G myeloid cells from lymphoid tissue This Kit works ideally for spleen and tumor tissues
    https://www.bioz.com/result/mdscs/product/Miltenyi Biotec
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mdscs - by Bioz Stars, 2021-07
    86/100 stars

    Images

    1) Product Images from "IL-7 treatment augments and prolongs sepsis-induced expansion of IL-10-producing B lymphocytes and myeloid-derived suppressor cells"

    Article Title: IL-7 treatment augments and prolongs sepsis-induced expansion of IL-10-producing B lymphocytes and myeloid-derived suppressor cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192304

    Sepsis results in a sustained expansion of MDSCs. Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. Gr1 + CD11b + cells from the spleen and bone marrow were analysed 1 week, 1 month and 3.5 months after sepsis induction. (A, B) Representative flow cytometry images from spleen (A) and bone marrow (B) from analysis after 3.5 months. (C) Frequency of Gr1 + CD11b + cells among total spleen cells (top) and among total bone marrow cells (bottom). (D) Number of Gr1 + CD11b + cells in spleen (top) and bone marrow (bottom). n = 6–9 (1 week), 5–12 (1 month), 8–20 (3.5 months). * P
    Figure Legend Snippet: Sepsis results in a sustained expansion of MDSCs. Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. Gr1 + CD11b + cells from the spleen and bone marrow were analysed 1 week, 1 month and 3.5 months after sepsis induction. (A, B) Representative flow cytometry images from spleen (A) and bone marrow (B) from analysis after 3.5 months. (C) Frequency of Gr1 + CD11b + cells among total spleen cells (top) and among total bone marrow cells (bottom). (D) Number of Gr1 + CD11b + cells in spleen (top) and bone marrow (bottom). n = 6–9 (1 week), 5–12 (1 month), 8–20 (3.5 months). * P

    Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry

    MDSCs from septic mice efficiently suppress T cell proliferation. Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. The proliferation of CD4 + T cells in the presence of Gr1 + cells from spleen was analysed 1 week, 1 month and 3.5 months after sepsis induction. (A) Representative flow cytometry images from analysis after 1 month showing proliferation of T cells (determined by dilution of cell proliferation dye, CPD) from septic mice treated with IL-7 when cultured with MDSCs from sham mice (left), sepsis + PBS mice (middle) and sepsis + IL-7 mice (right). (B) Graph representing frequency of proliferating CD4 + T cells from spleen when cultured with MDSCs from spleen from different groups of mice for 3 days. This graph is representative of the experiment performed 1 month post-sepsis induction. n = 3–4 (for all time points). * P
    Figure Legend Snippet: MDSCs from septic mice efficiently suppress T cell proliferation. Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. The proliferation of CD4 + T cells in the presence of Gr1 + cells from spleen was analysed 1 week, 1 month and 3.5 months after sepsis induction. (A) Representative flow cytometry images from analysis after 1 month showing proliferation of T cells (determined by dilution of cell proliferation dye, CPD) from septic mice treated with IL-7 when cultured with MDSCs from sham mice (left), sepsis + PBS mice (middle) and sepsis + IL-7 mice (right). (B) Graph representing frequency of proliferating CD4 + T cells from spleen when cultured with MDSCs from spleen from different groups of mice for 3 days. This graph is representative of the experiment performed 1 month post-sepsis induction. n = 3–4 (for all time points). * P

    Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, Cell Culture

    2) Product Images from "TRAMETINIB DRIVES T CELL-DEPENDENT CONTROL OF K-RAS-MUTATED TUMORS BY INHIBITING PATHOLOGICAL MYELOPOIESIS"

    Article Title: TRAMETINIB DRIVES T CELL-DEPENDENT CONTROL OF K-RAS-MUTATED TUMORS BY INHIBITING PATHOLOGICAL MYELOPOIESIS

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-16-1308

    Osteopontin chemoattracts MDSCs and is reduced by trametinib treatment of tumor cells ( A ) LC-MS/MS data of cytokines found in supernatants of Brpkp110 cells cultured for 40 hrs in vehicle or 200 nM trametinib. Y axis=MS count (abundance) in vehicle supernatants. X axis=fold change. Positive values=(trametinib/vehicle), negative values = −(vehicle/trametinib). ( B ) Osteopontin concentration measured from supernatants of Brpkp110 cells cultured overnight in the indicated conditions. ( C ) Osteopontin concentration from plasma samples collected from Brpkp110-bearing mice (or naïve tumor-free mice) gavaged daily with trametinib on days 7–9, and harvested on day 10. ( D ) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G − populations. Pre- and post-sort cell populations were analyzed for Ly6G and Ly6C expression by flow cytometry. ( E – F ) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G − populations and assayed for their ability to migrate in a transwell assay towards osteopontin (chemotaxis) or within the presence of osteopontin (chemokinesis). ( G ) Osteopontin concentration measured from intratumoral fluid collected from four separate Brpkp110 tumors. * P
    Figure Legend Snippet: Osteopontin chemoattracts MDSCs and is reduced by trametinib treatment of tumor cells ( A ) LC-MS/MS data of cytokines found in supernatants of Brpkp110 cells cultured for 40 hrs in vehicle or 200 nM trametinib. Y axis=MS count (abundance) in vehicle supernatants. X axis=fold change. Positive values=(trametinib/vehicle), negative values = −(vehicle/trametinib). ( B ) Osteopontin concentration measured from supernatants of Brpkp110 cells cultured overnight in the indicated conditions. ( C ) Osteopontin concentration from plasma samples collected from Brpkp110-bearing mice (or naïve tumor-free mice) gavaged daily with trametinib on days 7–9, and harvested on day 10. ( D ) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G − populations. Pre- and post-sort cell populations were analyzed for Ly6G and Ly6C expression by flow cytometry. ( E – F ) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G − populations and assayed for their ability to migrate in a transwell assay towards osteopontin (chemotaxis) or within the presence of osteopontin (chemokinesis). ( G ) Osteopontin concentration measured from intratumoral fluid collected from four separate Brpkp110 tumors. * P

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Cell Culture, Concentration Assay, Mouse Assay, In Vitro, Derivative Assay, Magnetic Cell Separation, Expressing, Flow Cytometry, Cytometry, Transwell Assay, Chemotaxis Assay

    Trametinib reduces the accumulation of Ly6C hi M-MDSCs in tumors ( A – E ) Mice with Brpkp110 subcutaneous tumors were gavaged daily with trametinib or vehicle on days 7–9, harvested on day 10, and analyzed by flow cytometry. ( A – D ) Percentages of cell populations found in dissociated tumors from two independent experiments. ( E ) Representative gating for Ly6C hi and Ly6G + from CD11b + MHCII − cells in tumors. ( F ) CD11b + MHCII − Ly6C hi or CD11b + MHCII − Ly6G + were FACS sorted from advanced Brpkp110 tumor-bearing or naive mice and analyzed by qPCR. Expression normalized to TATA binding protein is shown. ( G ) CD11b + MHCII − Ly6C hi cells were sorted from advanced Brpkp110 tumor-bearing mice and mixed at the indicated ratios with OVA 257-264 -peptide-loaded OT-I splenocytes. Proliferation was measured by CellTrace dilution of CD8 + cells 3 days later. Representative of two experiments. ( H – I ). Percentages or total numbers of cell populations from spleens of Brpkp110 tumor-bearing mice from 3 independent experiments. * P
    Figure Legend Snippet: Trametinib reduces the accumulation of Ly6C hi M-MDSCs in tumors ( A – E ) Mice with Brpkp110 subcutaneous tumors were gavaged daily with trametinib or vehicle on days 7–9, harvested on day 10, and analyzed by flow cytometry. ( A – D ) Percentages of cell populations found in dissociated tumors from two independent experiments. ( E ) Representative gating for Ly6C hi and Ly6G + from CD11b + MHCII − cells in tumors. ( F ) CD11b + MHCII − Ly6C hi or CD11b + MHCII − Ly6G + were FACS sorted from advanced Brpkp110 tumor-bearing or naive mice and analyzed by qPCR. Expression normalized to TATA binding protein is shown. ( G ) CD11b + MHCII − Ly6C hi cells were sorted from advanced Brpkp110 tumor-bearing mice and mixed at the indicated ratios with OVA 257-264 -peptide-loaded OT-I splenocytes. Proliferation was measured by CellTrace dilution of CD8 + cells 3 days later. Representative of two experiments. ( H – I ). Percentages or total numbers of cell populations from spleens of Brpkp110 tumor-bearing mice from 3 independent experiments. * P

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, FACS, Real-time Polymerase Chain Reaction, Expressing, Binding Assay

    3) Product Images from "Overexpression of Dominant Negative Peroxisome Proliferator-Activated Receptor-? (PPAR?) in Alveolar Type II Epithelial Cells Causes Inflammation and T-Cell Suppression in the Lung"

    Article Title: Overexpression of Dominant Negative Peroxisome Proliferator-Activated Receptor-? (PPAR?) in Alveolar Type II Epithelial Cells Causes Inflammation and T-Cell Suppression in the Lung

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2011.01.046

    The suppressive function of MDSCs on CD4 + T cells. A: CFSE-labeled wild-type splenic CD4 + T cells were stimulated with anti-CD3 and anti-CD28 mAb for 4 days in the presence or absence of CD11b + Gr-1 + MDSCs from the lung of doxycycline-treated or untreated
    Figure Legend Snippet: The suppressive function of MDSCs on CD4 + T cells. A: CFSE-labeled wild-type splenic CD4 + T cells were stimulated with anti-CD3 and anti-CD28 mAb for 4 days in the presence or absence of CD11b + Gr-1 + MDSCs from the lung of doxycycline-treated or untreated

    Techniques Used: Labeling

    4) Product Images from "Targeting ornithine decarboxylase by α-difluoromethylornithine inhibits tumor growth by impairing myeloid-derived suppressor cells"

    Article Title: Targeting ornithine decarboxylase by α-difluoromethylornithine inhibits tumor growth by impairing myeloid-derived suppressor cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1500729

    DFMO targets MDSCs to inhibit tumor growth Mice were injected s.c. with 10 6 B16F10 tumor cells. Depletion of MDSC was achieved by either twice-weekly i.p. injection of ( A ) 5-Fluorouracil (5-FU) or ( B ) anti-Gr1 antibodies starting 2 days after tumor challenge (5 mice per group). ( C ) Splenic Gr1 + CD11b + MDSCs from B16F10-bearing mice treated with DFMO or dH 2 O were injected i.v. into B16-bearing mice at d7 and d14. Mice receiving PBS without MDSCs were controls. Tumor volume was measured and plotted at indicated times (5 mice per group). ( D ) Flow cytometry analysis of expression of ki67 and Annexin V in gp100-specific tetramer + CD8 + TCRVβ + cells, and absolute number of these tetramer + CD8 + TCRVβ + cells per 10 6 cells in tumor infiltrates 3d after the initial MDSC transfer (5 mice per group). *, p
    Figure Legend Snippet: DFMO targets MDSCs to inhibit tumor growth Mice were injected s.c. with 10 6 B16F10 tumor cells. Depletion of MDSC was achieved by either twice-weekly i.p. injection of ( A ) 5-Fluorouracil (5-FU) or ( B ) anti-Gr1 antibodies starting 2 days after tumor challenge (5 mice per group). ( C ) Splenic Gr1 + CD11b + MDSCs from B16F10-bearing mice treated with DFMO or dH 2 O were injected i.v. into B16-bearing mice at d7 and d14. Mice receiving PBS without MDSCs were controls. Tumor volume was measured and plotted at indicated times (5 mice per group). ( D ) Flow cytometry analysis of expression of ki67 and Annexin V in gp100-specific tetramer + CD8 + TCRVβ + cells, and absolute number of these tetramer + CD8 + TCRVβ + cells per 10 6 cells in tumor infiltrates 3d after the initial MDSC transfer (5 mice per group). *, p

    Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, Expressing

    Charaterization of phenotypic tumor-associated MDSCs following DFMO treatment ( A ) Percent splenic Gr1 + CD11b + MDSCs were determined by flow cytometry from B16F10-bearing mice. Percent CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs were indicated within plots (5 mice per group). ( B ) Percent Gr1 + CD11b + MDSCs, CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs in spleen and tumor tissues from B16F10-bearing mice were summarized (5 mice per group). ( C ) Measurement of ODC activity in Gr1 + CD11b + cells from naïve and B16F10 tumor-bearing (TB) mice treated by DFMO or dH 2 O (5 mice per group). ( D ) Expression levels of CD39, CD73, CD115, MHC-II, B7H1, DCFDA (ROS indicator) and arginase-I among both tumor-infiltrating CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs were determined by flow cytometry. Cells were collected from B16F10-bearing DFMO treated or control mice 14 days after tumor inoculation (5 mice per group). Data (mean ± SEM) are representative of 2 independent experiments. *, p
    Figure Legend Snippet: Charaterization of phenotypic tumor-associated MDSCs following DFMO treatment ( A ) Percent splenic Gr1 + CD11b + MDSCs were determined by flow cytometry from B16F10-bearing mice. Percent CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs were indicated within plots (5 mice per group). ( B ) Percent Gr1 + CD11b + MDSCs, CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs in spleen and tumor tissues from B16F10-bearing mice were summarized (5 mice per group). ( C ) Measurement of ODC activity in Gr1 + CD11b + cells from naïve and B16F10 tumor-bearing (TB) mice treated by DFMO or dH 2 O (5 mice per group). ( D ) Expression levels of CD39, CD73, CD115, MHC-II, B7H1, DCFDA (ROS indicator) and arginase-I among both tumor-infiltrating CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs were determined by flow cytometry. Cells were collected from B16F10-bearing DFMO treated or control mice 14 days after tumor inoculation (5 mice per group). Data (mean ± SEM) are representative of 2 independent experiments. *, p

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Activity Assay, Expressing

    5) Product Images from "Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia"

    Article Title: Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02686-14

    Increased PD-L1 expression in MDSCs from PcP mice. MDSCs (MDSCs/PcP) were isolated from PcP mice at 5 weeks post- Pneumocystis infection. Control Gr1BM cells were isolated from uninfected mice immunosuppressed by weekly injection of anti-CD4 (L3T4) antibody. (A) Total RNA was isolated from the cells, and PD-L1 gene expression was determined by real-time RT-PCR. The average PD-L1 expression level in Gr1BM cells was set as 1, and that in MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) The cells were examined by flow cytometry using anti-Gr-1 and anti-PD-L1 antibodies. The result shown is representative of three independent experiments.
    Figure Legend Snippet: Increased PD-L1 expression in MDSCs from PcP mice. MDSCs (MDSCs/PcP) were isolated from PcP mice at 5 weeks post- Pneumocystis infection. Control Gr1BM cells were isolated from uninfected mice immunosuppressed by weekly injection of anti-CD4 (L3T4) antibody. (A) Total RNA was isolated from the cells, and PD-L1 gene expression was determined by real-time RT-PCR. The average PD-L1 expression level in Gr1BM cells was set as 1, and that in MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) The cells were examined by flow cytometry using anti-Gr-1 and anti-PD-L1 antibodies. The result shown is representative of three independent experiments.

    Techniques Used: Expressing, Mouse Assay, Isolation, Infection, Injection, Quantitative RT-PCR, Flow Cytometry, Cytometry

    6) Product Images from "TRAF6 Regulates the Immunosuppressive Effects of Myeloid-Derived Suppressor Cells in Tumor-Bearing Host"

    Article Title: TRAF6 Regulates the Immunosuppressive Effects of Myeloid-Derived Suppressor Cells in Tumor-Bearing Host

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.649020

    TRAF6 knockdown impairs the immunosuppressive effects of MDSCs in vitro . Specific siRNA (siTRAF6) was used to knockdown the expression of TRAF6 in MDSCs, and the efficiency of siTRAF6 knockdown was validated by qRT-PCR (A) and Western blotting (B) . (C) Tumor-derived MDSCs were transfected with siTRAF6 and cocultured with CFSE-labeled CD4 + T cells, and proliferation was measured by flow cytometry after 72 h. (D) Statistical analyses of the percentage of proliferating CD4 + T cells co-cultured with MDSCs transfected with siTRAF6. After TRAF6 knockdown, the activity of Arg1 was measured by a QuantiChrom arginase assay kit (E) , and the concentration of NO was determined via a Griess reagent system kit (F) . *** p
    Figure Legend Snippet: TRAF6 knockdown impairs the immunosuppressive effects of MDSCs in vitro . Specific siRNA (siTRAF6) was used to knockdown the expression of TRAF6 in MDSCs, and the efficiency of siTRAF6 knockdown was validated by qRT-PCR (A) and Western blotting (B) . (C) Tumor-derived MDSCs were transfected with siTRAF6 and cocultured with CFSE-labeled CD4 + T cells, and proliferation was measured by flow cytometry after 72 h. (D) Statistical analyses of the percentage of proliferating CD4 + T cells co-cultured with MDSCs transfected with siTRAF6. After TRAF6 knockdown, the activity of Arg1 was measured by a QuantiChrom arginase assay kit (E) , and the concentration of NO was determined via a Griess reagent system kit (F) . *** p

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Transfection, Labeling, Flow Cytometry, Cell Culture, Activity Assay, Arginase Assay, Concentration Assay

    TRAF6 expression was augmented in MDSCs from lung cancer patients. To examine the modulation of TRAF6 in MDSCs from lung cancer patients, the level of TRAF6 in MDSCs was measured in the lung cancer patient group (LC) and healthy control group (HC). (A) The proportions of MDSCs in the PBMCs of lung cancer patients and healthy persons were analyzed by flow cytometry. Representative dot plots of CD11b + CD33 + HLA-DR − MDSCs in the blood of patients with LC and healthy controls are shown. (B) The mean fluorescence intensity (MFI) of TRAF6 in MDSCs was determined by flow cytometry. (C) The MFI of arginase-1 in MDSCs was determined by flow cytometry. (D) The correlation between TRAF6 and arginase-1 in MDSCs was analyzed. *** p
    Figure Legend Snippet: TRAF6 expression was augmented in MDSCs from lung cancer patients. To examine the modulation of TRAF6 in MDSCs from lung cancer patients, the level of TRAF6 in MDSCs was measured in the lung cancer patient group (LC) and healthy control group (HC). (A) The proportions of MDSCs in the PBMCs of lung cancer patients and healthy persons were analyzed by flow cytometry. Representative dot plots of CD11b + CD33 + HLA-DR − MDSCs in the blood of patients with LC and healthy controls are shown. (B) The mean fluorescence intensity (MFI) of TRAF6 in MDSCs was determined by flow cytometry. (C) The MFI of arginase-1 in MDSCs was determined by flow cytometry. (D) The correlation between TRAF6 and arginase-1 in MDSCs was analyzed. *** p

    Techniques Used: Expressing, Flow Cytometry, Fluorescence

    TRAF6 is highly expressed in MDSCs derived from the tumor tissue of tumor-bearing mice. Approximately 1 × 10 6 LLC cells were s.c. injected in the backs of C57BL/6 mice for 28 d to establish a tumor-bearing (TB) mouse model. MDSCs were isolated by immunomagnetic beads from the spleens of TB mice, the tumor tissue of TB mice or the spleens of wild-type (WT) mice. (A) The purity of the isolated MDSCs was determined using flow cytometry via the detection of the CD11b + Gr1 + phenotype. The expression of TRAF6 in MDSCs derived from different sources was determined by qRT-PCR (B) or Western blotting (C) . The mRNA expression of TRAF6 in PMN-MDSCs and M-MDSCs derived from the spleen (D) or tumor tissue (E) . (F) CFSE-labeled CD4 + T cells were co-cultured with MDSCs derived from the spleen or tumor tissue in the presence of CD3 and CD28 stimulation. After 72 h, the proliferation of CD4 + T cells was tested via flow cytometry. (G) Statistical analyses of the percentage of proliferating CD4 + T cells co-cultured with MDSCs derived from the spleen or tumor tissue of TB mice. The mRNA expression levels of Arg1 (H) and iNOS (I) in MDSCs were measured by qRT-PCR. *** p
    Figure Legend Snippet: TRAF6 is highly expressed in MDSCs derived from the tumor tissue of tumor-bearing mice. Approximately 1 × 10 6 LLC cells were s.c. injected in the backs of C57BL/6 mice for 28 d to establish a tumor-bearing (TB) mouse model. MDSCs were isolated by immunomagnetic beads from the spleens of TB mice, the tumor tissue of TB mice or the spleens of wild-type (WT) mice. (A) The purity of the isolated MDSCs was determined using flow cytometry via the detection of the CD11b + Gr1 + phenotype. The expression of TRAF6 in MDSCs derived from different sources was determined by qRT-PCR (B) or Western blotting (C) . The mRNA expression of TRAF6 in PMN-MDSCs and M-MDSCs derived from the spleen (D) or tumor tissue (E) . (F) CFSE-labeled CD4 + T cells were co-cultured with MDSCs derived from the spleen or tumor tissue in the presence of CD3 and CD28 stimulation. After 72 h, the proliferation of CD4 + T cells was tested via flow cytometry. (G) Statistical analyses of the percentage of proliferating CD4 + T cells co-cultured with MDSCs derived from the spleen or tumor tissue of TB mice. The mRNA expression levels of Arg1 (H) and iNOS (I) in MDSCs were measured by qRT-PCR. *** p

    Techniques Used: Derivative Assay, Mouse Assay, Injection, Isolation, Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot, Labeling, Cell Culture

    TRAF6 knockdown attenuates the ability of MDSCs to accelerate tumor progression in tumor-bearing mice. To investigate the effects of TRAF6 on the suppressive activity of MDSCs in vivo , 2 groups of wild-type C57BL/6 mice were s.c. injected with 1 × 10 6 LLC cells and 1 × 10 6 MDSCs transfected with siTRAF6 (siTRAF6 group) or MDSCs transfected with siNC (control group). (A) Tumor growth was constantly monitored. The width “a” and length “b” were measured, and tumor volume was calculated. (B,C) On the 28th day after the inoculation of LLC cells, the mice were sacrificed, and the tumor image and weights were showed in both groups. (D) The proportion of CD4 + IFN-γ + Th1 cells in the tumor tissue of both groups was analyzed by FCM. (E) The proportion of CD8 + IFN-γ + CTLs in the tumor tissue of both groups was analyzed by FCM. * p
    Figure Legend Snippet: TRAF6 knockdown attenuates the ability of MDSCs to accelerate tumor progression in tumor-bearing mice. To investigate the effects of TRAF6 on the suppressive activity of MDSCs in vivo , 2 groups of wild-type C57BL/6 mice were s.c. injected with 1 × 10 6 LLC cells and 1 × 10 6 MDSCs transfected with siTRAF6 (siTRAF6 group) or MDSCs transfected with siNC (control group). (A) Tumor growth was constantly monitored. The width “a” and length “b” were measured, and tumor volume was calculated. (B,C) On the 28th day after the inoculation of LLC cells, the mice were sacrificed, and the tumor image and weights were showed in both groups. (D) The proportion of CD4 + IFN-γ + Th1 cells in the tumor tissue of both groups was analyzed by FCM. (E) The proportion of CD8 + IFN-γ + CTLs in the tumor tissue of both groups was analyzed by FCM. * p

    Techniques Used: Mouse Assay, Activity Assay, In Vivo, Injection, Transfection

    7) Product Images from "Neonatal granulocytic MDSCs possess phagocytic properties during bacterial infection"

    Article Title: Neonatal granulocytic MDSCs possess phagocytic properties during bacterial infection

    Journal: bioRxiv

    doi: 10.1101/2019.12.30.891077

    MDSCs and monocytes produce inflammatory cytokines during infection. CD66 + MDSCs and CD14 + monocytes isolated from human umbilical cord blood PBMCs were infected with an MOI 10 of E. coli O1:K1:H7 and incubated at 37°C for 6 hours. Supernatants were collected for inflammatory cytokine measurements. Cells were then lysed in TRI Reagent for RNA extraction, cDNA synthesis, and gene expression analysis of inflammatory cytokines. (A) Gene expression analysis of TNFα levels during infections. Infection levels were normalized relative to uninfected MDSC controls. Representative of 4 independent experiments, 10 replicates total per group. (B) Serum cytokine levels of TNFα measured via ELISA for infections of both MDSCs and monocytes. Cytokine levels were normalized based on standard curves for each protein. A representative of 2 independent experiments with 8 replicates total per group is shown. Statistical analysis was performed using an unpaired t-test for all panels. * p ≤ 0.05, **** p ≤ 0.0001.
    Figure Legend Snippet: MDSCs and monocytes produce inflammatory cytokines during infection. CD66 + MDSCs and CD14 + monocytes isolated from human umbilical cord blood PBMCs were infected with an MOI 10 of E. coli O1:K1:H7 and incubated at 37°C for 6 hours. Supernatants were collected for inflammatory cytokine measurements. Cells were then lysed in TRI Reagent for RNA extraction, cDNA synthesis, and gene expression analysis of inflammatory cytokines. (A) Gene expression analysis of TNFα levels during infections. Infection levels were normalized relative to uninfected MDSC controls. Representative of 4 independent experiments, 10 replicates total per group. (B) Serum cytokine levels of TNFα measured via ELISA for infections of both MDSCs and monocytes. Cytokine levels were normalized based on standard curves for each protein. A representative of 2 independent experiments with 8 replicates total per group is shown. Statistical analysis was performed using an unpaired t-test for all panels. * p ≤ 0.05, **** p ≤ 0.0001.

    Techniques Used: Infection, Isolation, Incubation, RNA Extraction, Expressing, Enzyme-linked Immunosorbent Assay

    MDSC-derived extracellular DNA affects bacterial viability in MDSC-only cultures, but does not affect monocyte phagocytosis and killing of bacteria. CD66 + MDSCs and CD14 + monocytes isolated from human umbilical cord blood PBMCs were either single or co-culture infected with an MOI of 10 of E. coli O1:K1:H7 and incubated at 37°C for 6 hours. DNAse I (100 U) was added to appropriate cultures. For extracellular bacterial burdens, 50 μL of supernatant was collected from each infection, diluted ten-fold in PBS, and plated on TSA for standard plate counting. For intracellular burdens, 100 μL of 1% saponin was added to each well for 15 minutes, cell lysates were diluted ten-fold in PBS, and bacteria was plated on TSA for standard enumeration. Colony forming units (CFUs) of combined extracellular and intracellular bacteria in single or co-culture infections of MDSCs and monocytes untreated or treated with DNAse I at 6 hours post-infection. Statistical analysis was performed using a two-way ANOVA. P -values are as follows: interaction variation, p=0.3818, row factor variation (untreated vs. treated), p=0.4288, column factor variation (MDSCs vs. Monocytes vs. Mixed cultures), p=0.1156.
    Figure Legend Snippet: MDSC-derived extracellular DNA affects bacterial viability in MDSC-only cultures, but does not affect monocyte phagocytosis and killing of bacteria. CD66 + MDSCs and CD14 + monocytes isolated from human umbilical cord blood PBMCs were either single or co-culture infected with an MOI of 10 of E. coli O1:K1:H7 and incubated at 37°C for 6 hours. DNAse I (100 U) was added to appropriate cultures. For extracellular bacterial burdens, 50 μL of supernatant was collected from each infection, diluted ten-fold in PBS, and plated on TSA for standard plate counting. For intracellular burdens, 100 μL of 1% saponin was added to each well for 15 minutes, cell lysates were diluted ten-fold in PBS, and bacteria was plated on TSA for standard enumeration. Colony forming units (CFUs) of combined extracellular and intracellular bacteria in single or co-culture infections of MDSCs and monocytes untreated or treated with DNAse I at 6 hours post-infection. Statistical analysis was performed using a two-way ANOVA. P -values are as follows: interaction variation, p=0.3818, row factor variation (untreated vs. treated), p=0.4288, column factor variation (MDSCs vs. Monocytes vs. Mixed cultures), p=0.1156.

    Techniques Used: Derivative Assay, Isolation, Co-Culture Assay, Infection, Incubation

    MDSCs are less efficient at bacterial elimination compared to monocytes. CD66 + MDSCs and CD14 + monocytes isolated from human umbilical cord blood PBMCs were infected with an MOI of 20 of E. coli O1:K1:H7 and incubated at 37°C for 1 hour. Media was replaced at this time point for gentamicin-supplemented media and cells were incubated for 2, 6, 18, and 24 hours post gentamicin exposure. At each time point, cells were permeabilized with 1% saponin, diluted ten-fold, and plated on TSA for standard bacterial enumeration. The graph represents bacterial recovery between MDSCs and monocytes at 6, 18, and 24 hours post exposure. All time points were normalized to the 2 hour time point. The data shown is representative of 5 independent experiments. Statistical analysis was performed using a Mann-Whitney U test. * p ≤ 0.05, ** p ≤ 0.01, n.s. not significant.
    Figure Legend Snippet: MDSCs are less efficient at bacterial elimination compared to monocytes. CD66 + MDSCs and CD14 + monocytes isolated from human umbilical cord blood PBMCs were infected with an MOI of 20 of E. coli O1:K1:H7 and incubated at 37°C for 1 hour. Media was replaced at this time point for gentamicin-supplemented media and cells were incubated for 2, 6, 18, and 24 hours post gentamicin exposure. At each time point, cells were permeabilized with 1% saponin, diluted ten-fold, and plated on TSA for standard bacterial enumeration. The graph represents bacterial recovery between MDSCs and monocytes at 6, 18, and 24 hours post exposure. All time points were normalized to the 2 hour time point. The data shown is representative of 5 independent experiments. Statistical analysis was performed using a Mann-Whitney U test. * p ≤ 0.05, ** p ≤ 0.01, n.s. not significant.

    Techniques Used: Isolation, Infection, Incubation, MANN-WHITNEY

    Neonatal human MDSCs have characteristic cell surface markers and suppress T cell proliferation. CD66 + MDSCs from human umbilical cord blood PBMCs were either labeled with cell surface markers for cell marker profiling or were co-cultured at a 1:1 ratio with CD4 + T cells for 4 days for a T cell proliferation assay. For cell marker profiling, MDSCs were labeled with antibodies for CD66, HLA-DR, CD33, and CD14, fixed in 4% paraformaldehyde, and resuspended in PBS prior to collection on the flow cytometer. For T cell proliferation assays, MDSCs were incubated with T cells stimulated with IL-2 (100 U) for 4 days. Cells were collected each day and fixed in 4% paraformaldehyde for flow cytometry analysis. Cells supplemented with CD3/CD28 DynaBeads were imaged on a Lionheart FX automated microscope to visualize clonal expansion of T cells surrounding beads during proliferation. (A) Shown are representative histogram overlay plots of cell surface markers for MDSCs compared to no stain controls. The top panel shows PE-labeled CD66 and HLA-DR expression on cell surfaces. The bottom panel shows FITC-labeled CD33 and CD14 expression on cell surfaces. Shifts to the right represent increasing fluorescence. Black lines = CD66 or CD33 expression in top and bottom panels, respectively, blue lines = HLA-DR or CD14 expression in top and bottom panels, respectively, grey lines = no stain control in both panels. A representative histogram of 2 independent experiments is shown. (B) Shown are representative histogram plots of T cell proliferation. Stimulated T cells at Day 0, IL-2 stimulated T cells at day 3, and IL-2 stimulated T cells supplemented with MDSCs at day 3 are displayed. The red vertical line on all plots is used to visualize the shift in proliferation in all plots. The data shown is representative of 5 independent experiments. (C) Representative DIC images of T cells supplemented with DynaBeads ± MDSCs are shown. Black coverage is representative of beads associated with proliferating T cells. The data shown is representative of 5 independent experiments. Scale bar = 100 μm.
    Figure Legend Snippet: Neonatal human MDSCs have characteristic cell surface markers and suppress T cell proliferation. CD66 + MDSCs from human umbilical cord blood PBMCs were either labeled with cell surface markers for cell marker profiling or were co-cultured at a 1:1 ratio with CD4 + T cells for 4 days for a T cell proliferation assay. For cell marker profiling, MDSCs were labeled with antibodies for CD66, HLA-DR, CD33, and CD14, fixed in 4% paraformaldehyde, and resuspended in PBS prior to collection on the flow cytometer. For T cell proliferation assays, MDSCs were incubated with T cells stimulated with IL-2 (100 U) for 4 days. Cells were collected each day and fixed in 4% paraformaldehyde for flow cytometry analysis. Cells supplemented with CD3/CD28 DynaBeads were imaged on a Lionheart FX automated microscope to visualize clonal expansion of T cells surrounding beads during proliferation. (A) Shown are representative histogram overlay plots of cell surface markers for MDSCs compared to no stain controls. The top panel shows PE-labeled CD66 and HLA-DR expression on cell surfaces. The bottom panel shows FITC-labeled CD33 and CD14 expression on cell surfaces. Shifts to the right represent increasing fluorescence. Black lines = CD66 or CD33 expression in top and bottom panels, respectively, blue lines = HLA-DR or CD14 expression in top and bottom panels, respectively, grey lines = no stain control in both panels. A representative histogram of 2 independent experiments is shown. (B) Shown are representative histogram plots of T cell proliferation. Stimulated T cells at Day 0, IL-2 stimulated T cells at day 3, and IL-2 stimulated T cells supplemented with MDSCs at day 3 are displayed. The red vertical line on all plots is used to visualize the shift in proliferation in all plots. The data shown is representative of 5 independent experiments. (C) Representative DIC images of T cells supplemented with DynaBeads ± MDSCs are shown. Black coverage is representative of beads associated with proliferating T cells. The data shown is representative of 5 independent experiments. Scale bar = 100 μm.

    Techniques Used: Labeling, Marker, Cell Culture, Proliferation Assay, Flow Cytometry, Incubation, Microscopy, Staining, Expressing, Fluorescence

    MDSCs are less efficient at bacterial uptake compared to monocytes. CD66 + MDSCs and CD14 + monocytes isolated from human umbilical cord blood PBMCs were infected with an MOI of 10 of Syto 9™- or pHrodo Red-labeled E. coli O1:K1:H7 and incubated at 37°C. For flow cytometry, cells were fixed in 4% paraformaldehyde and resuspended in PBS prior to collection. For imaging, cells were imaged and analyzed for pHrodo fluorescence using FIJI. For longitudinal imaging, cells were imaged every 10 minutes over a 6-hour period and quantified for pHrodo fluorescence. (A) Histogram and dot plots are representative of 2 independent experiments with three replicates per experiment. (B and C) Graphs are representative of 3 independent experiments. n = 63 and 70 images analyzed for MDSCs and monocytes, respectively. (D) The graph shown is representative of 2 independent experiments. n = 9 fields of view per cell type averaged at each time point per experiment. (A) Flow cytometry histograms and representative dot plot for MDSCs and monocytes display percent of cells that have not phagocytosed bacteria (none), have phagocytosed a low amount of bacteria (low), or have phagocytosed a high amount of bacteria (high). Grey circle symbols = monocytes, royal blue square symbols = MDSCs. (B) Quantification of the number of fluorescent bacterial particles phagocytosed by MDSCs and monocytes during infection. (C) Quantification of the area of pHrodo fluorescence in pixels phagocytosed by MDSCs and monocytes during infection. (D) Longitudinal phagocytosis of pHrodo bacteria during a 6-hour time course. Images were taken every 10 minutes of both MDSCs (blue line) and monocytes (grey line). Fluorescent bacteria per cell type were quantified at each time point from 9 fields of view. Statistical analyses in (A, B, and C) were performed using a Mann-Whitney U test. ** p ≤ 0.01, **** p ≤ 0.0001, n.s. not significant. Median with interquartile range displayed in all graphs.
    Figure Legend Snippet: MDSCs are less efficient at bacterial uptake compared to monocytes. CD66 + MDSCs and CD14 + monocytes isolated from human umbilical cord blood PBMCs were infected with an MOI of 10 of Syto 9™- or pHrodo Red-labeled E. coli O1:K1:H7 and incubated at 37°C. For flow cytometry, cells were fixed in 4% paraformaldehyde and resuspended in PBS prior to collection. For imaging, cells were imaged and analyzed for pHrodo fluorescence using FIJI. For longitudinal imaging, cells were imaged every 10 minutes over a 6-hour period and quantified for pHrodo fluorescence. (A) Histogram and dot plots are representative of 2 independent experiments with three replicates per experiment. (B and C) Graphs are representative of 3 independent experiments. n = 63 and 70 images analyzed for MDSCs and monocytes, respectively. (D) The graph shown is representative of 2 independent experiments. n = 9 fields of view per cell type averaged at each time point per experiment. (A) Flow cytometry histograms and representative dot plot for MDSCs and monocytes display percent of cells that have not phagocytosed bacteria (none), have phagocytosed a low amount of bacteria (low), or have phagocytosed a high amount of bacteria (high). Grey circle symbols = monocytes, royal blue square symbols = MDSCs. (B) Quantification of the number of fluorescent bacterial particles phagocytosed by MDSCs and monocytes during infection. (C) Quantification of the area of pHrodo fluorescence in pixels phagocytosed by MDSCs and monocytes during infection. (D) Longitudinal phagocytosis of pHrodo bacteria during a 6-hour time course. Images were taken every 10 minutes of both MDSCs (blue line) and monocytes (grey line). Fluorescent bacteria per cell type were quantified at each time point from 9 fields of view. Statistical analyses in (A, B, and C) were performed using a Mann-Whitney U test. ** p ≤ 0.01, **** p ≤ 0.0001, n.s. not significant. Median with interquartile range displayed in all graphs.

    Techniques Used: Isolation, Infection, Labeling, Incubation, Flow Cytometry, Imaging, Fluorescence, MANN-WHITNEY

    8) Product Images from "Repression of MUC1 Promotes Expansion and Suppressive Function of Myeloid-Derived Suppressor Cells in Pancreatic and Breast Cancer Murine Models"

    Article Title: Repression of MUC1 Promotes Expansion and Suppressive Function of Myeloid-Derived Suppressor Cells in Pancreatic and Breast Cancer Murine Models

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22115587

    Phenotypic characterization of MDSCs in the BM of healthy and tumor bearing WT and Muc1KO mice. ( A ) Splenocytes from tumor bearing WT and MUC1KO mice were isolated and labeled with antibodies against Gr1, CD11b, and pSTAT3. pSTAT3 + cells were determined on Gr1 + CD11b + gated cells. ( B ) MFI values of Arginase-1 expression in MDSCs of WT and MUC1KO mice was measured via flow cytometry. Arg-1 + cells were determined on Gr1 + CD11b + BM gated cells. ( C ) Arginase-1 activity on sorted MDSCs was measured using a urea assay. Welch’s t -test was used to compare between WT and MUC1KO groups and p
    Figure Legend Snippet: Phenotypic characterization of MDSCs in the BM of healthy and tumor bearing WT and Muc1KO mice. ( A ) Splenocytes from tumor bearing WT and MUC1KO mice were isolated and labeled with antibodies against Gr1, CD11b, and pSTAT3. pSTAT3 + cells were determined on Gr1 + CD11b + gated cells. ( B ) MFI values of Arginase-1 expression in MDSCs of WT and MUC1KO mice was measured via flow cytometry. Arg-1 + cells were determined on Gr1 + CD11b + BM gated cells. ( C ) Arginase-1 activity on sorted MDSCs was measured using a urea assay. Welch’s t -test was used to compare between WT and MUC1KO groups and p

    Techniques Used: Mouse Assay, Isolation, Labeling, Expressing, Flow Cytometry, Activity Assay

    Cancer bearing MUC1KO mice have higher frequency of MDSCs creating a more immune suppressive environment. ( A ) Splenocytes from healthy and tumor bearing WT and Muc1KO mice were isolated and labeled with anti Gr1-APC and CD11b-PECy7 antibodies. Representative contour plots of Gr1 + CD11b + cells from three separate experiments are shown; the MDSC subsets were obtained by gating on high SSC and high FSC cells using an isotype control antibody and further on high Gr1 + and low Gr1 + CD11b + cells. The red circles represent the percent depicted on the right top quadrant for each of the groups. ( B , C ) Levels of TGF-β was measured in the serum of healthy and cancer bearing WT and MUC1KO mice using ELISA kits. ( D ) Levels of MDSCs and CD4 + FoxP3 + cells were measured in the tumor of WT and MUC1KO mice using flow cytometry. High SSC and FSC was gated for the lymphocyte population, then CD4 + T cells were gated on the lymphocyte population, and the CD25 + FoxP3 + double positive cells were gated on the CD4 + T cell population. Welch’s t -test was used to compare between WT and MUC1KO groups and p
    Figure Legend Snippet: Cancer bearing MUC1KO mice have higher frequency of MDSCs creating a more immune suppressive environment. ( A ) Splenocytes from healthy and tumor bearing WT and Muc1KO mice were isolated and labeled with anti Gr1-APC and CD11b-PECy7 antibodies. Representative contour plots of Gr1 + CD11b + cells from three separate experiments are shown; the MDSC subsets were obtained by gating on high SSC and high FSC cells using an isotype control antibody and further on high Gr1 + and low Gr1 + CD11b + cells. The red circles represent the percent depicted on the right top quadrant for each of the groups. ( B , C ) Levels of TGF-β was measured in the serum of healthy and cancer bearing WT and MUC1KO mice using ELISA kits. ( D ) Levels of MDSCs and CD4 + FoxP3 + cells were measured in the tumor of WT and MUC1KO mice using flow cytometry. High SSC and FSC was gated for the lymphocyte population, then CD4 + T cells were gated on the lymphocyte population, and the CD25 + FoxP3 + double positive cells were gated on the CD4 + T cell population. Welch’s t -test was used to compare between WT and MUC1KO groups and p

    Techniques Used: Mouse Assay, Isolation, Labeling, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    MDSCs from MUC1KO mice produce increased iNOS and c-Myc. ( A ) BM cells from healthy mice were isolated and labeled with antibodies against Gr1 and CD11b. ( B ) Level of iNOS expression by MDSCs from the BM of healthy and tumor bearing mice. Percentages of iNOS + cells on Gr1 + CD11b + BM gated cells. ( C ) MFI values of c-Myc protein expression by MDSCs from the BM of healthy and tumor bearing mice. c-Myc + cells were gated in Gr1 CD11b + cells. Welch’s t -test was used to compare between WT and MUC1KO groups and p
    Figure Legend Snippet: MDSCs from MUC1KO mice produce increased iNOS and c-Myc. ( A ) BM cells from healthy mice were isolated and labeled with antibodies against Gr1 and CD11b. ( B ) Level of iNOS expression by MDSCs from the BM of healthy and tumor bearing mice. Percentages of iNOS + cells on Gr1 + CD11b + BM gated cells. ( C ) MFI values of c-Myc protein expression by MDSCs from the BM of healthy and tumor bearing mice. c-Myc + cells were gated in Gr1 CD11b + cells. Welch’s t -test was used to compare between WT and MUC1KO groups and p

    Techniques Used: Mouse Assay, Isolation, Labeling, Expressing

    9) Product Images from "TRAMETINIB DRIVES T CELL-DEPENDENT CONTROL OF K-RAS-MUTATED TUMORS BY INHIBITING PATHOLOGICAL MYELOPOIESIS"

    Article Title: TRAMETINIB DRIVES T CELL-DEPENDENT CONTROL OF K-RAS-MUTATED TUMORS BY INHIBITING PATHOLOGICAL MYELOPOIESIS

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-16-1308

    Osteopontin chemoattracts MDSCs and is reduced by trametinib treatment of tumor cells ( A ) LC-MS/MS data of cytokines found in supernatants of Brpkp110 cells cultured for 40 hrs in vehicle or 200 nM trametinib. Y axis=MS count (abundance) in vehicle supernatants. X axis=fold change. Positive values=(trametinib/vehicle), negative values = −(vehicle/trametinib). ( B ) Osteopontin concentration measured from supernatants of Brpkp110 cells cultured overnight in the indicated conditions. ( C ) Osteopontin concentration from plasma samples collected from Brpkp110-bearing mice (or naïve tumor-free mice) gavaged daily with trametinib on days 7–9, and harvested on day 10. ( D ) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G − populations. Pre- and post-sort cell populations were analyzed for Ly6G and Ly6C expression by flow cytometry. ( E – F ) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G − populations and assayed for their ability to migrate in a transwell assay towards osteopontin (chemotaxis) or within the presence of osteopontin (chemokinesis). ( G ) Osteopontin concentration measured from intratumoral fluid collected from four separate Brpkp110 tumors. * P
    Figure Legend Snippet: Osteopontin chemoattracts MDSCs and is reduced by trametinib treatment of tumor cells ( A ) LC-MS/MS data of cytokines found in supernatants of Brpkp110 cells cultured for 40 hrs in vehicle or 200 nM trametinib. Y axis=MS count (abundance) in vehicle supernatants. X axis=fold change. Positive values=(trametinib/vehicle), negative values = −(vehicle/trametinib). ( B ) Osteopontin concentration measured from supernatants of Brpkp110 cells cultured overnight in the indicated conditions. ( C ) Osteopontin concentration from plasma samples collected from Brpkp110-bearing mice (or naïve tumor-free mice) gavaged daily with trametinib on days 7–9, and harvested on day 10. ( D ) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G − populations. Pre- and post-sort cell populations were analyzed for Ly6G and Ly6C expression by flow cytometry. ( E – F ) GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G − populations and assayed for their ability to migrate in a transwell assay towards osteopontin (chemotaxis) or within the presence of osteopontin (chemokinesis). ( G ) Osteopontin concentration measured from intratumoral fluid collected from four separate Brpkp110 tumors. * P

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Concentration Assay, Mouse Assay, In Vitro, Derivative Assay, Magnetic Cell Separation, Expressing, Flow Cytometry, Transwell Assay, Chemotaxis Assay

    Trametinib reduces the accumulation of Ly6C hi M-MDSCs in tumors ( A – E ) Mice with Brpkp110 subcutaneous tumors were gavaged daily with trametinib or vehicle on days 7–9, harvested on day 10, and analyzed by flow cytometry. ( A – D ) Percentages of cell populations found in dissociated tumors from two independent experiments. ( E ) Representative gating for Ly6C hi and Ly6G + from CD11b + MHCII − cells in tumors. ( F ) CD11b + MHCII − Ly6C hi or CD11b + MHCII − Ly6G + were FACS sorted from advanced Brpkp110 tumor-bearing or naive mice and analyzed by qPCR. Expression normalized to TATA binding protein is shown. ( G ) CD11b + MHCII − Ly6C hi cells were sorted from advanced Brpkp110 tumor-bearing mice and mixed at the indicated ratios with OVA 257-264 -peptide-loaded OT-I splenocytes. Proliferation was measured by CellTrace dilution of CD8 + cells 3 days later. Representative of two experiments. ( H – I ). Percentages or total numbers of cell populations from spleens of Brpkp110 tumor-bearing mice from 3 independent experiments. * P
    Figure Legend Snippet: Trametinib reduces the accumulation of Ly6C hi M-MDSCs in tumors ( A – E ) Mice with Brpkp110 subcutaneous tumors were gavaged daily with trametinib or vehicle on days 7–9, harvested on day 10, and analyzed by flow cytometry. ( A – D ) Percentages of cell populations found in dissociated tumors from two independent experiments. ( E ) Representative gating for Ly6C hi and Ly6G + from CD11b + MHCII − cells in tumors. ( F ) CD11b + MHCII − Ly6C hi or CD11b + MHCII − Ly6G + were FACS sorted from advanced Brpkp110 tumor-bearing or naive mice and analyzed by qPCR. Expression normalized to TATA binding protein is shown. ( G ) CD11b + MHCII − Ly6C hi cells were sorted from advanced Brpkp110 tumor-bearing mice and mixed at the indicated ratios with OVA 257-264 -peptide-loaded OT-I splenocytes. Proliferation was measured by CellTrace dilution of CD8 + cells 3 days later. Representative of two experiments. ( H – I ). Percentages or total numbers of cell populations from spleens of Brpkp110 tumor-bearing mice from 3 independent experiments. * P

    Techniques Used: Mouse Assay, Flow Cytometry, FACS, Real-time Polymerase Chain Reaction, Expressing, Binding Assay

    10) Product Images from "Myeloid-Derived Suppressor Cells Regulate Natural Killer Cell Response to Adenovirus-Mediated Gene Transfer"

    Article Title: Myeloid-Derived Suppressor Cells Regulate Natural Killer Cell Response to Adenovirus-Mediated Gene Transfer

    Journal: Journal of Virology

    doi: 10.1128/JVI.01595-12

    Production of arginase-1, ROS, and iNOS by naïve and adenovirus-infected G-MDSCs. Mice were infected with Ad-LacZ (Ad) or left uninfected (Naïve). At 24 h after infection, purified G-MDSCs (G-MDSC) or CD8 + T cells (CD8) were used for the
    Figure Legend Snippet: Production of arginase-1, ROS, and iNOS by naïve and adenovirus-infected G-MDSCs. Mice were infected with Ad-LacZ (Ad) or left uninfected (Naïve). At 24 h after infection, purified G-MDSCs (G-MDSC) or CD8 + T cells (CD8) were used for the

    Techniques Used: Infection, Mouse Assay, Purification

    11) Product Images from "Superior GVHD-free, relapse-free survival for G-BM to G-PBSC grafts is associated with higher MDSCs content in allografting for patients with acute leukemia"

    Article Title: Superior GVHD-free, relapse-free survival for G-BM to G-PBSC grafts is associated with higher MDSCs content in allografting for patients with acute leukemia

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-017-0503-2

    Suppressive activity of MDSCs isolated from G-BM and G-PBSC grafts. a The proliferation of purified and CFSE-labled CD3+ T cells in the coculture with isolated MDSCs or not. b The inhibition rate of the proliferation of CD3+ T cells by MDSCs. Isolated MDSCs were added at a 1:1 and 1:5 ratio to purified CD3+ T cells in the presence of anti-CD2/CD3/CD28 biotin beads according to manufacturer instructions (Miltenyi Biotec). Data are representative of three independent experiments (NS P > 0.05, ** P
    Figure Legend Snippet: Suppressive activity of MDSCs isolated from G-BM and G-PBSC grafts. a The proliferation of purified and CFSE-labled CD3+ T cells in the coculture with isolated MDSCs or not. b The inhibition rate of the proliferation of CD3+ T cells by MDSCs. Isolated MDSCs were added at a 1:1 and 1:5 ratio to purified CD3+ T cells in the presence of anti-CD2/CD3/CD28 biotin beads according to manufacturer instructions (Miltenyi Biotec). Data are representative of three independent experiments (NS P > 0.05, ** P

    Techniques Used: Activity Assay, Isolation, Purification, Inhibition

    12) Product Images from "MDSCs drive the process of endometriosis by enhancing angiogenesis and provide a new potential approach for treatment"

    Article Title: MDSCs drive the process of endometriosis by enhancing angiogenesis and provide a new potential approach for treatment

    Journal: European journal of immunology

    doi: 10.1002/eji.201747417

    Characterization of MDSC subsets in mouse model of endometriosis. (A) Gating strategy and exemplary FACS plot and morphology of MDSC subsets in peritoneum. G-MDSCs are CD11b+Ly-6C lo Ly-6G+ and with polymorphonuclear morphology while M-MDSCs are CD11b+Ly-6C hi Ly-6G- with monocytic morphology. (B) Absolute cell number of peritoneal G-MDSC and M-MDSC within 24 hours after transplantation of endometrial fragments (n=10 at each time point). * represent P
    Figure Legend Snippet: Characterization of MDSC subsets in mouse model of endometriosis. (A) Gating strategy and exemplary FACS plot and morphology of MDSC subsets in peritoneum. G-MDSCs are CD11b+Ly-6C lo Ly-6G+ and with polymorphonuclear morphology while M-MDSCs are CD11b+Ly-6C hi Ly-6G- with monocytic morphology. (B) Absolute cell number of peritoneal G-MDSC and M-MDSC within 24 hours after transplantation of endometrial fragments (n=10 at each time point). * represent P

    Techniques Used: FACS, Transplantation Assay

    Analysis of MDSCs and cytokines in endometriosis patients. (A) Gating strategy of human HLA-DR-CD11b+CD33+ MDSC and representative plots of flow cytometric analysis of MDSCs in PBMCs from (B) a normal female control and (C) a patient with endometriosis, respectively. (D) Comparison of the percentage of MDSCs in PBMCs between normal female controls and endometriosis female patients, 10 cases in each group. (E and F) Representative plots of flow cytometric analysis of MDSCs in PBMCs from the same patient with endometriosis before (pre-treatment) and 3 months after (post-treatment) laparoscopic removal of the endometrial lesions, respectively. (G) Comparison of the change of MDSCs in PBMCs from endometriosis patients before and after laparoscopic surgery (n=10). (H) Comparison of the concentrations of cytokines in the plasma between normal controls (n=15) and endometriosis patients (n=30). (H) Comparison of the concentrations of cytokines in the plasma from endometriosis patients between pre-treatment and post-treatment (n=30). All bar graphs show mean+SEM. Mann-Whitney U test was used to determine all the significant difference. * represents P
    Figure Legend Snippet: Analysis of MDSCs and cytokines in endometriosis patients. (A) Gating strategy of human HLA-DR-CD11b+CD33+ MDSC and representative plots of flow cytometric analysis of MDSCs in PBMCs from (B) a normal female control and (C) a patient with endometriosis, respectively. (D) Comparison of the percentage of MDSCs in PBMCs between normal female controls and endometriosis female patients, 10 cases in each group. (E and F) Representative plots of flow cytometric analysis of MDSCs in PBMCs from the same patient with endometriosis before (pre-treatment) and 3 months after (post-treatment) laparoscopic removal of the endometrial lesions, respectively. (G) Comparison of the change of MDSCs in PBMCs from endometriosis patients before and after laparoscopic surgery (n=10). (H) Comparison of the concentrations of cytokines in the plasma between normal controls (n=15) and endometriosis patients (n=30). (H) Comparison of the concentrations of cytokines in the plasma from endometriosis patients between pre-treatment and post-treatment (n=30). All bar graphs show mean+SEM. Mann-Whitney U test was used to determine all the significant difference. * represents P

    Techniques Used: Flow Cytometry, MANN-WHITNEY

    Role of CXCR2 in the development of endometriosis. (A) Exemplary FACS plot and frequency of G-MDSCs and M-MDSCs in CXCR2 KO mice. (B) Development of endometrial lesions (yellow arrows) was investigated in CXCR2 KO mice and CXCR2 KO mice with exogenous G-MDSC transplantation. Endometrial lesions were collected at day7 after transplantation. (C) Comparision of the size and weight of endometrial lesions in wide type (WT) mice, CXCR2 KO mice and CXCR2 KO mice with exogenous G-MDSC transplantation. Data are mean±SEM (n=5). * represent P
    Figure Legend Snippet: Role of CXCR2 in the development of endometriosis. (A) Exemplary FACS plot and frequency of G-MDSCs and M-MDSCs in CXCR2 KO mice. (B) Development of endometrial lesions (yellow arrows) was investigated in CXCR2 KO mice and CXCR2 KO mice with exogenous G-MDSC transplantation. Endometrial lesions were collected at day7 after transplantation. (C) Comparision of the size and weight of endometrial lesions in wide type (WT) mice, CXCR2 KO mice and CXCR2 KO mice with exogenous G-MDSC transplantation. Data are mean±SEM (n=5). * represent P

    Techniques Used: FACS, Mouse Assay, Transplantation Assay

    13) Product Images from "Cholangiocarcinoma presents a distinct myeloid-derived suppressor cell signature compared to other hepatobiliary cancers"

    Article Title: Cholangiocarcinoma presents a distinct myeloid-derived suppressor cell signature compared to other hepatobiliary cancers

    Journal: bioRxiv

    doi: 10.1101/554600

    D2HG blocks mMDSC polarization in vitro. A) Representative flow plots demonstrating the gating strategy for in vitro generated mMDSCs and gMDSCs. B) Proliferation rate of T cells co-cultured with increasing concentrations of MDSCs or nonMDSCs. ** p
    Figure Legend Snippet: D2HG blocks mMDSC polarization in vitro. A) Representative flow plots demonstrating the gating strategy for in vitro generated mMDSCs and gMDSCs. B) Proliferation rate of T cells co-cultured with increasing concentrations of MDSCs or nonMDSCs. ** p

    Techniques Used: In Vitro, Generated, Cell Culture

    14) Product Images from "Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia"

    Article Title: Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02686-14

    Reduced phagocytic activity in AMs incubated with MDSCs. AMs cocultured with control Gr1BM cells (A) or MDSCs (B) overnight were incubated with fluorescein-conjugated zymosan beads for 1 h. The nuclei of AMs were counterstained with DAPI. (C) The number of zymosan beads phagocytosed by AMs incubated with MDSCs or Gr1BM cells were counted under a confocal microscope. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Reduced phagocytic activity in AMs incubated with MDSCs. AMs cocultured with control Gr1BM cells (A) or MDSCs (B) overnight were incubated with fluorescein-conjugated zymosan beads for 1 h. The nuclei of AMs were counterstained with DAPI. (C) The number of zymosan beads phagocytosed by AMs incubated with MDSCs or Gr1BM cells were counted under a confocal microscope. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Activity Assay, Affinity Magnetic Separation, Incubation, Microscopy

    Decreased PU.1 expression in AMs incubated with MDSCs. AMs from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA of AMs was isolated, and PU.1 mRNA levels were determined by real-time PCR. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with either type of cell was compared to it. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Decreased PU.1 expression in AMs incubated with MDSCs. AMs from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA of AMs was isolated, and PU.1 mRNA levels were determined by real-time PCR. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with either type of cell was compared to it. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Expressing, Affinity Magnetic Separation, Incubation, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction

    Increased PD-1 expression in AMs incubated with MDSDs. AMs isolated from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . (A) After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA was isolated from AMs of each group. PD-1 gene expression was determined by real-time RT-PCR. The level of AMs/Gr1BM was set as 1, and that in AMs/MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) AMs incubated with MDSCs or Gr1BM cells were analyzed for surface PD-1 expression by flow cytometry.
    Figure Legend Snippet: Increased PD-1 expression in AMs incubated with MDSDs. AMs isolated from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . (A) After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA was isolated from AMs of each group. PD-1 gene expression was determined by real-time RT-PCR. The level of AMs/Gr1BM was set as 1, and that in AMs/MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) AMs incubated with MDSCs or Gr1BM cells were analyzed for surface PD-1 expression by flow cytometry.

    Techniques Used: Expressing, Affinity Magnetic Separation, Incubation, Isolation, Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry

    Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The C T values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The C T values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Affinity Magnetic Separation, Incubation, Mouse Assay, Sonication, Generated, Chromatin Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction

    Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.

    Techniques Used: DNA Methylation Assay, Affinity Magnetic Separation, Incubation, Mouse Assay, Isolation, CpG Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    Loss of suppressive effect of MDSCs pretreated with anti-PD-L1 antibody on AMs. A total of 1 × 10 5 AMs were cocultured with 5 × 10 5 MDSCs that were treated with anti-PD-L1 antibody or control IgG. (A) After 16 h of incubation, AMs were isolated, and the PU.1 mRNA levels were determined by real-time RT-PCR. Data are presented as means ± SD from three independent experiments. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with MDSCs that were pretreated with or without anti-PD-L1 antibody was compared to it. (B) Phagocytosis was assayed, and the number of zymosan beads phagocytosed by AMs incubated with MDSCs pretreated with anti-PD-L1 antibody or control IgG was counted under a confocal microscope.
    Figure Legend Snippet: Loss of suppressive effect of MDSCs pretreated with anti-PD-L1 antibody on AMs. A total of 1 × 10 5 AMs were cocultured with 5 × 10 5 MDSCs that were treated with anti-PD-L1 antibody or control IgG. (A) After 16 h of incubation, AMs were isolated, and the PU.1 mRNA levels were determined by real-time RT-PCR. Data are presented as means ± SD from three independent experiments. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with MDSCs that were pretreated with or without anti-PD-L1 antibody was compared to it. (B) Phagocytosis was assayed, and the number of zymosan beads phagocytosed by AMs incubated with MDSCs pretreated with anti-PD-L1 antibody or control IgG was counted under a confocal microscope.

    Techniques Used: Affinity Magnetic Separation, Incubation, Isolation, Quantitative RT-PCR, Expressing, Microscopy

    15) Product Images from "IL-7 treatment augments and prolongs sepsis-induced expansion of IL-10-producing B lymphocytes and myeloid-derived suppressor cells"

    Article Title: IL-7 treatment augments and prolongs sepsis-induced expansion of IL-10-producing B lymphocytes and myeloid-derived suppressor cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0192304

    Sepsis results in a sustained expansion of MDSCs. Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. Gr1 + CD11b + cells from the spleen and bone marrow were analysed 1 week, 1 month and 3.5 months after sepsis induction. (A, B) Representative flow cytometry images from spleen (A) and bone marrow (B) from analysis after 3.5 months. (C) Frequency of Gr1 + CD11b + cells among total spleen cells (top) and among total bone marrow cells (bottom). (D) Number of Gr1 + CD11b + cells in spleen (top) and bone marrow (bottom). n = 6–9 (1 week), 5–12 (1 month), 8–20 (3.5 months). * P
    Figure Legend Snippet: Sepsis results in a sustained expansion of MDSCs. Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. Gr1 + CD11b + cells from the spleen and bone marrow were analysed 1 week, 1 month and 3.5 months after sepsis induction. (A, B) Representative flow cytometry images from spleen (A) and bone marrow (B) from analysis after 3.5 months. (C) Frequency of Gr1 + CD11b + cells among total spleen cells (top) and among total bone marrow cells (bottom). (D) Number of Gr1 + CD11b + cells in spleen (top) and bone marrow (bottom). n = 6–9 (1 week), 5–12 (1 month), 8–20 (3.5 months). * P

    Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry

    MDSCs from septic mice efficiently suppress T cell proliferation. Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. The proliferation of CD4 + T cells in the presence of Gr1 + cells from spleen was analysed 1 week, 1 month and 3.5 months after sepsis induction. (A) Representative flow cytometry images from analysis after 1 month showing proliferation of T cells (determined by dilution of cell proliferation dye, CPD) from septic mice treated with IL-7 when cultured with MDSCs from sham mice (left), sepsis + PBS mice (middle) and sepsis + IL-7 mice (right). (B) Graph representing frequency of proliferating CD4 + T cells from spleen when cultured with MDSCs from spleen from different groups of mice for 3 days. This graph is representative of the experiment performed 1 month post-sepsis induction. n = 3–4 (for all time points). * P
    Figure Legend Snippet: MDSCs from septic mice efficiently suppress T cell proliferation. Mice were injected with PBS i.p. (Sham) or subjected to sepsis induction. IL-7 (Sepsis + IL-7) or PBS (Sepsis + PBS) was injected daily for 5 days from day 5–9 post sepsis induction. The proliferation of CD4 + T cells in the presence of Gr1 + cells from spleen was analysed 1 week, 1 month and 3.5 months after sepsis induction. (A) Representative flow cytometry images from analysis after 1 month showing proliferation of T cells (determined by dilution of cell proliferation dye, CPD) from septic mice treated with IL-7 when cultured with MDSCs from sham mice (left), sepsis + PBS mice (middle) and sepsis + IL-7 mice (right). (B) Graph representing frequency of proliferating CD4 + T cells from spleen when cultured with MDSCs from spleen from different groups of mice for 3 days. This graph is representative of the experiment performed 1 month post-sepsis induction. n = 3–4 (for all time points). * P

    Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, Cell Culture

    16) Product Images from "A highly efficient tumor-infiltrating MDSC differentiation system for discovery of anti-neoplastic targets, which circumvents the need for tumor establishment in mice"

    Article Title: A highly efficient tumor-infiltrating MDSC differentiation system for discovery of anti-neoplastic targets, which circumvents the need for tumor establishment in mice

    Journal: Oncotarget

    doi:

    Ex vivo monocytic MDSCs are precursors of granulocytic MDSCs, which represent the terminal differentiation stage (A) Bar graphs representing the relative proportion of monocytic and granulocytic MDSCs (M-MDSCs, G-MDSCs) in 293T-MDSC and B16-MDSC cultures on the indicated days of differentiation. Relevant statistical comparisons are shown. (B) Ly6G-CD11c expression profiles of purified M-MDSCs on day 5 (density flow cytometry plot on the left), and the same cells incubated in CM 293T for 3 additional days. The percentage of G-MDSCs is shown within the graph. (C) Top left, column graph representing the ratio of the number of cells on days 8 versus 5 in 293T-MDSC and B16-MDSC cultures, to calculate cell growth rate. Top right, dead cell staining with fixable viability stain (FVS) of M-MDSC and G-MDSCs in culture. The proportion of viable cells is shown in the legend. Below left, column graph representing the proportion of Ki67-expressing cells within the G-MDSC and M-MDSC subsets from B16-MDSC cultures, as indicated. Below right, the same but representing Ki67 mean fluorescent intensities (MFI). (D) Top histogram, Ly6G expression on day-five 293T-MDSC cultures incubated for three days with either DC medium, or CM 293T , as indicated within the histogram. Percentages and mean fluorescent intensities are indicated in the legend. The same is represented in the histogram below, but plotting CD11c expression. Relevant statistical comparisons are indicated. *, **, ***, represent significant (P
    Figure Legend Snippet: Ex vivo monocytic MDSCs are precursors of granulocytic MDSCs, which represent the terminal differentiation stage (A) Bar graphs representing the relative proportion of monocytic and granulocytic MDSCs (M-MDSCs, G-MDSCs) in 293T-MDSC and B16-MDSC cultures on the indicated days of differentiation. Relevant statistical comparisons are shown. (B) Ly6G-CD11c expression profiles of purified M-MDSCs on day 5 (density flow cytometry plot on the left), and the same cells incubated in CM 293T for 3 additional days. The percentage of G-MDSCs is shown within the graph. (C) Top left, column graph representing the ratio of the number of cells on days 8 versus 5 in 293T-MDSC and B16-MDSC cultures, to calculate cell growth rate. Top right, dead cell staining with fixable viability stain (FVS) of M-MDSC and G-MDSCs in culture. The proportion of viable cells is shown in the legend. Below left, column graph representing the proportion of Ki67-expressing cells within the G-MDSC and M-MDSC subsets from B16-MDSC cultures, as indicated. Below right, the same but representing Ki67 mean fluorescent intensities (MFI). (D) Top histogram, Ly6G expression on day-five 293T-MDSC cultures incubated for three days with either DC medium, or CM 293T , as indicated within the histogram. Percentages and mean fluorescent intensities are indicated in the legend. The same is represented in the histogram below, but plotting CD11c expression. Relevant statistical comparisons are indicated. *, **, ***, represent significant (P

    Techniques Used: Ex Vivo, Expressing, Purification, Flow Cytometry, Cytometry, Incubation, Staining

    Ex vivo myelopoiesis within a simulated tumor environment differentiates bone marrow cells into large numbers of MDSC-like cells (A) Experimental scheme for MDSC production. On top, lentivector construct used to express GM-CSF and puromycin resistance gene. Below, schematic representation of the generation of MDSC cells. Cancer cell lines are transduced with the lentivector (LV-GMCSF-PuroR). As a result, transduced cells generate conditioning medium (CM) that simulates the tumor microenvironment. Bone marrow (BM) cells from a single tumor-free mouse are cultured in CM for a minimum of 5 days. (B) Left, bar graph representing the number of myeloid cells after a 5 day incubation of BM with the indicated percentages of CM, from 293T or B16F0 cells as indicated. Conventional immature DCs were obtained with recombinant GM-CSF (GM-CSF) following standard protocols. Error bars correspond to standard deviations. Right, percentage of surface expression of the indicated markers, as a function of the increasing percentage of CM 293T . (C) CD11c-MHC II expression profiles are shown as flow cytometry density plots. Percentages of CD11c and MHC II expressing myeloid cells are shown within the graph. Control plots of unstained, immature DCs, 293T- and B16-MDSCs are shown on top of the plots. Myeloid cells were collected on day 8 of differentiation. (D) Same as in c but assessing Ly6C-Ly6G (top density plots) and CD62L-Ly6G (bottom density plots) expression profiles in the indicated myeloid cells. LTR, long terminal repeat; SFFV p, spleen focus-forming virus promoter; moGM-CSF, mouse GM-CSF gene; Puromycin R, puromycin resistance gene; UBI p, ubiquitin promoter; SIN, Self-inactivating LTR.
    Figure Legend Snippet: Ex vivo myelopoiesis within a simulated tumor environment differentiates bone marrow cells into large numbers of MDSC-like cells (A) Experimental scheme for MDSC production. On top, lentivector construct used to express GM-CSF and puromycin resistance gene. Below, schematic representation of the generation of MDSC cells. Cancer cell lines are transduced with the lentivector (LV-GMCSF-PuroR). As a result, transduced cells generate conditioning medium (CM) that simulates the tumor microenvironment. Bone marrow (BM) cells from a single tumor-free mouse are cultured in CM for a minimum of 5 days. (B) Left, bar graph representing the number of myeloid cells after a 5 day incubation of BM with the indicated percentages of CM, from 293T or B16F0 cells as indicated. Conventional immature DCs were obtained with recombinant GM-CSF (GM-CSF) following standard protocols. Error bars correspond to standard deviations. Right, percentage of surface expression of the indicated markers, as a function of the increasing percentage of CM 293T . (C) CD11c-MHC II expression profiles are shown as flow cytometry density plots. Percentages of CD11c and MHC II expressing myeloid cells are shown within the graph. Control plots of unstained, immature DCs, 293T- and B16-MDSCs are shown on top of the plots. Myeloid cells were collected on day 8 of differentiation. (D) Same as in c but assessing Ly6C-Ly6G (top density plots) and CD62L-Ly6G (bottom density plots) expression profiles in the indicated myeloid cells. LTR, long terminal repeat; SFFV p, spleen focus-forming virus promoter; moGM-CSF, mouse GM-CSF gene; Puromycin R, puromycin resistance gene; UBI p, ubiquitin promoter; SIN, Self-inactivating LTR.

    Techniques Used: Ex Vivo, Construct, Transduction, Cell Culture, Incubation, Recombinant, Expressing, Flow Cytometry, Cytometry

    17) Product Images from "MIF is necessary for late-stage melanoma patient MDSC immune suppression and differentiation"

    Article Title: MIF is necessary for late-stage melanoma patient MDSC immune suppression and differentiation

    Journal: Cancer immunology research

    doi: 10.1158/2326-6066.CIR-15-0070-T

    CD14 + HLA-DR −/low MDSCs are increased in the peripheral blood of patients with advanced melanoma
    Figure Legend Snippet: CD14 + HLA-DR −/low MDSCs are increased in the peripheral blood of patients with advanced melanoma

    Techniques Used:

    18) Product Images from "STAT3 Silencing and TLR7/8 Pathway Activation Repolarize and Suppress Myeloid-Derived Suppressor Cells From Breast Cancer Patients"

    Article Title: STAT3 Silencing and TLR7/8 Pathway Activation Repolarize and Suppress Myeloid-Derived Suppressor Cells From Breast Cancer Patients

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.613215

    In patients with BC treated with chemotherapy, circulating MDSC levels are decreased, but their MDSCs remain highly T cell-suppressive. (A) In a co-culture experiment, treatment of MDSCs with the TLR7/8 agonist R848 and si-STAT3 reduces MCF-7 BC cells’ proliferation. (B) The frequency of MDSCs in BC patients undergoing chemotherapy was assessed. The flow cytometric results showed that after chemotherapy (right), the circulating MDSC frequencies were decreased. (C) The MDSCs of patients with BC after chemotherapy still had more T cell-suppressive activity than HLA-DR - CD33 + MDSCs isolated from healthy individuals. *P
    Figure Legend Snippet: In patients with BC treated with chemotherapy, circulating MDSC levels are decreased, but their MDSCs remain highly T cell-suppressive. (A) In a co-culture experiment, treatment of MDSCs with the TLR7/8 agonist R848 and si-STAT3 reduces MCF-7 BC cells’ proliferation. (B) The frequency of MDSCs in BC patients undergoing chemotherapy was assessed. The flow cytometric results showed that after chemotherapy (right), the circulating MDSC frequencies were decreased. (C) The MDSCs of patients with BC after chemotherapy still had more T cell-suppressive activity than HLA-DR - CD33 + MDSCs isolated from healthy individuals. *P

    Techniques Used: Co-Culture Assay, Activity Assay, Isolation

    Both total and activated STAT3 levels are increased in MDSCs from BC patients. (A) Expression of STAT3 in MDSCs from healthy individuals and patients with BC. The results were presented as the mean expression level of STAT3 in each group. (B) STAT3 and pSTAT3 protein expression in MDSCs from healthy subjects and patients with BC. Patients with BC exhibit higher expression of the active form of STAT3. Beta-actin was used as a loading control. (C) Histological specimens from patients and healthy subjects were stained and evaluated by IHC for CD33, STAT3, pSTAT3, and Ki67 markers with relevant antibodies; arrows represent cells positive for studied markers. The results showed an increased level of the filtration of CD33 + STAT3 + p-STAT3 + cells into tissue samples of patients with BC; **P = 0.002.
    Figure Legend Snippet: Both total and activated STAT3 levels are increased in MDSCs from BC patients. (A) Expression of STAT3 in MDSCs from healthy individuals and patients with BC. The results were presented as the mean expression level of STAT3 in each group. (B) STAT3 and pSTAT3 protein expression in MDSCs from healthy subjects and patients with BC. Patients with BC exhibit higher expression of the active form of STAT3. Beta-actin was used as a loading control. (C) Histological specimens from patients and healthy subjects were stained and evaluated by IHC for CD33, STAT3, pSTAT3, and Ki67 markers with relevant antibodies; arrows represent cells positive for studied markers. The results showed an increased level of the filtration of CD33 + STAT3 + p-STAT3 + cells into tissue samples of patients with BC; **P = 0.002.

    Techniques Used: Expressing, Staining, Immunohistochemistry, Filtration

    Circulating HLA‐DR ‐ CD33 + myeloid‐derived suppressor cells in BC patients. The frequencies and phenotypes of the studied MDSCs in patients’ blood samples and healthy cases were assessed using multi-color flow cytometry. (A) Shows the characterization and quantification of MDSCs as the HLA‐DR -/low CD33 + population and as a percentage of gated cells via supplying a minimum of 10,000 live events per sample. Histogram plots also indicate the expression rate and percentages of the positive cells for surface markers (CD14 and CD15) on gated HLA‐DR -/low CD33 + cells. HLA‐DR -/low CD33 + cells were negative for the expression of lineage markers CD3, CD19, and CD56. (B) Percentages of MDSC subpopulations, including total MDSCs, monocytic MDSCs, and granulocytic MDSCs in peripheral blood of the study groups ( P
    Figure Legend Snippet: Circulating HLA‐DR ‐ CD33 + myeloid‐derived suppressor cells in BC patients. The frequencies and phenotypes of the studied MDSCs in patients’ blood samples and healthy cases were assessed using multi-color flow cytometry. (A) Shows the characterization and quantification of MDSCs as the HLA‐DR -/low CD33 + population and as a percentage of gated cells via supplying a minimum of 10,000 live events per sample. Histogram plots also indicate the expression rate and percentages of the positive cells for surface markers (CD14 and CD15) on gated HLA‐DR -/low CD33 + cells. HLA‐DR -/low CD33 + cells were negative for the expression of lineage markers CD3, CD19, and CD56. (B) Percentages of MDSC subpopulations, including total MDSCs, monocytic MDSCs, and granulocytic MDSCs in peripheral blood of the study groups ( P

    Techniques Used: Derivative Assay, Flow Cytometry, Expressing

    19) Product Images from "Doxorubicin resistant cancer cells activate myeloid-derived suppressor cells by releasing PGE2"

    Article Title: Doxorubicin resistant cancer cells activate myeloid-derived suppressor cells by releasing PGE2

    Journal: Scientific Reports

    doi: 10.1038/srep23824

    The role of AMPK in the miR-10a expression of MDSCs induced by PGE2. ( A ) Western blotting of p-AMPK and AMPK in BM-derived MDSCs cultured with conditioned medium from 4T1 or Doxorubicin-resistant 4T1 cells (4T1/DOX) for 3 days in the presence of GM-CSF and IL-6. ( B ) BM-derived MDSCs transfected with miR-10a antagomir (miR-10a ASO) or scrambled control were cultured with metformin (10mM) or compound C (Com C, 5 μM) in the presence of or absence of PGE 2 , and the expression of miR-10a was determined by qRT-PCR. ( C ) BM-derived MDSCs transfected with miR-10a mimics or scrambled control were cultured with metformin (10 mM) or Compound C (Com C, 5 μM) in the presence of or absence of PGE 2 , MMP9 and Arg1 mRNA were determined by qRT-PCR. ( D ) MDSCs were cultured as C and activated by LPS (100 ng/ml) and IFN-γ (2 ng/ml), the secretion of IL-10 was examined by ELISA. ( E ) Representative FACS plots showing the percentage of M-MDSCs and G-MDSCs (cells were gated on CD11b + Gr-1 + ) in spleen two weeks after 4T1/DOX cells were intravenously inoculated into BALB/c mice with/without Compound C treatment (25 mg/kg, one time/every three days for four times). ( F ) Quantification of the expression of miR-10 in CD11b + Gr-1 + MDSCs sorted from spleen. Data represent Mean ± SD from 3 individual experiments. ** p
    Figure Legend Snippet: The role of AMPK in the miR-10a expression of MDSCs induced by PGE2. ( A ) Western blotting of p-AMPK and AMPK in BM-derived MDSCs cultured with conditioned medium from 4T1 or Doxorubicin-resistant 4T1 cells (4T1/DOX) for 3 days in the presence of GM-CSF and IL-6. ( B ) BM-derived MDSCs transfected with miR-10a antagomir (miR-10a ASO) or scrambled control were cultured with metformin (10mM) or compound C (Com C, 5 μM) in the presence of or absence of PGE 2 , and the expression of miR-10a was determined by qRT-PCR. ( C ) BM-derived MDSCs transfected with miR-10a mimics or scrambled control were cultured with metformin (10 mM) or Compound C (Com C, 5 μM) in the presence of or absence of PGE 2 , MMP9 and Arg1 mRNA were determined by qRT-PCR. ( D ) MDSCs were cultured as C and activated by LPS (100 ng/ml) and IFN-γ (2 ng/ml), the secretion of IL-10 was examined by ELISA. ( E ) Representative FACS plots showing the percentage of M-MDSCs and G-MDSCs (cells were gated on CD11b + Gr-1 + ) in spleen two weeks after 4T1/DOX cells were intravenously inoculated into BALB/c mice with/without Compound C treatment (25 mg/kg, one time/every three days for four times). ( F ) Quantification of the expression of miR-10 in CD11b + Gr-1 + MDSCs sorted from spleen. Data represent Mean ± SD from 3 individual experiments. ** p

    Techniques Used: Expressing, Western Blot, Derivative Assay, Cell Culture, Transfection, Allele-specific Oligonucleotide, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, FACS, Mouse Assay

    PGE 2 promotes the expansion and M2 polarization of MDSCs via miR-10a. BM cells were treated with PGE 2 in the presence of GM-CSF and IL-6. MiR-10a antagomir (miR-10a ASO) and scrambled oligonucleotides were transfected on the second day. Gr-1 + CD11b + MDSCs were evaluated by flowcytometry after 4 days ( A ). BM cells in ( A ) were cultured with ( B ) or without ( C ) EP4 antagonist (1 μM ONO-AE3-208), rhe relative levels of TNF-α, NOS2, Arg1, MMP9, and TGF-β mRNA in BM-derived MDSCs were detected by qRT-PCR after 3 days of transfection; ( D ) In vitro suppressive ability of MDSCs that transfected with miR-10a antagomir (miR-10a ASO) or scrambled control on naïve CD4 + CD25 − T cells proliferation were analyzed by flowcytometry. CFSE labeled CD4 + CD25 − naïve T cells were incubated with APC (CD4 + T cell depleted splenocytes) and MDSCs, and stimulated with anti-CD3 with/without PGE 2 for 3–5 days. Cell proliferation was measured as a function of CFSE dilution. ( E ) The level of IFN-γ in the supernatant of cocultured cells in D was determined by ELISA. Data represent Mean ± SD from 3–5 individual experiments. *p
    Figure Legend Snippet: PGE 2 promotes the expansion and M2 polarization of MDSCs via miR-10a. BM cells were treated with PGE 2 in the presence of GM-CSF and IL-6. MiR-10a antagomir (miR-10a ASO) and scrambled oligonucleotides were transfected on the second day. Gr-1 + CD11b + MDSCs were evaluated by flowcytometry after 4 days ( A ). BM cells in ( A ) were cultured with ( B ) or without ( C ) EP4 antagonist (1 μM ONO-AE3-208), rhe relative levels of TNF-α, NOS2, Arg1, MMP9, and TGF-β mRNA in BM-derived MDSCs were detected by qRT-PCR after 3 days of transfection; ( D ) In vitro suppressive ability of MDSCs that transfected with miR-10a antagomir (miR-10a ASO) or scrambled control on naïve CD4 + CD25 − T cells proliferation were analyzed by flowcytometry. CFSE labeled CD4 + CD25 − naïve T cells were incubated with APC (CD4 + T cell depleted splenocytes) and MDSCs, and stimulated with anti-CD3 with/without PGE 2 for 3–5 days. Cell proliferation was measured as a function of CFSE dilution. ( E ) The level of IFN-γ in the supernatant of cocultured cells in D was determined by ELISA. Data represent Mean ± SD from 3–5 individual experiments. *p

    Techniques Used: Allele-specific Oligonucleotide, Transfection, Cell Culture, Derivative Assay, Quantitative RT-PCR, In Vitro, Labeling, Incubation, Enzyme-linked Immunosorbent Assay

    20) Product Images from "Superior GVHD-free, relapse-free survival for G-BM to G-PBSC grafts is associated with higher MDSCs content in allografting for patients with acute leukemia"

    Article Title: Superior GVHD-free, relapse-free survival for G-BM to G-PBSC grafts is associated with higher MDSCs content in allografting for patients with acute leukemia

    Journal: Journal of Hematology & Oncology

    doi: 10.1186/s13045-017-0503-2

    Suppressive activity of MDSCs isolated from G-BM and G-PBSC grafts. a The proliferation of purified and CFSE-labled CD3+ T cells in the coculture with isolated MDSCs or not. b The inhibition rate of the proliferation of CD3+ T cells by MDSCs. Isolated MDSCs were added at a 1:1 and 1:5 ratio to purified CD3+ T cells in the presence of anti-CD2/CD3/CD28 biotin beads according to manufacturer instructions (Miltenyi Biotec). Data are representative of three independent experiments (NS P > 0.05, ** P
    Figure Legend Snippet: Suppressive activity of MDSCs isolated from G-BM and G-PBSC grafts. a The proliferation of purified and CFSE-labled CD3+ T cells in the coculture with isolated MDSCs or not. b The inhibition rate of the proliferation of CD3+ T cells by MDSCs. Isolated MDSCs were added at a 1:1 and 1:5 ratio to purified CD3+ T cells in the presence of anti-CD2/CD3/CD28 biotin beads according to manufacturer instructions (Miltenyi Biotec). Data are representative of three independent experiments (NS P > 0.05, ** P

    Techniques Used: Activity Assay, Isolation, Purification, Inhibition

    21) Product Images from "Myeloid-Derived Suppressor Cells Evolve during Sepsis and Can Enhance or Attenuate the Systemic Inflammatory Response"

    Article Title: Myeloid-Derived Suppressor Cells Evolve during Sepsis and Can Enhance or Attenuate the Systemic Inflammatory Response

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00239-12

    Gr1 + CD11b + MDSCs harvested from day 3 (D3) septic mice produce more NO and exhibit lower arginase activity than MDSCs harvested from day 12 (D12) septic mice. Gr1 + CD11b + MDSCs were purified from the bone marrow of a separate group of mice at day 3 and
    Figure Legend Snippet: Gr1 + CD11b + MDSCs harvested from day 3 (D3) septic mice produce more NO and exhibit lower arginase activity than MDSCs harvested from day 12 (D12) septic mice. Gr1 + CD11b + MDSCs were purified from the bone marrow of a separate group of mice at day 3 and

    Techniques Used: Mouse Assay, Activity Assay, Purification

    Levels of circulating cytokines in CLP mice after adaptive transfer of Gr1 + CD11b + MDSCs. Mice were subjected to CLP and received Gr1 + CD11b + MDSCs or saline as described in the legend for . Sera were collected from moribund mice that were sacrificed
    Figure Legend Snippet: Levels of circulating cytokines in CLP mice after adaptive transfer of Gr1 + CD11b + MDSCs. Mice were subjected to CLP and received Gr1 + CD11b + MDSCs or saline as described in the legend for . Sera were collected from moribund mice that were sacrificed

    Techniques Used: Mouse Assay

    Adaptive transfer of Gr1 + CD11b + MDSCs from day 3 septic mice reduces peritoneal bacteria in early sepsis. Peritoneal lavage fluid was collected from moribund mice that were sacrificed at days 2 to 5 after CLP and cultured on 5% sheep blood agar plates.
    Figure Legend Snippet: Adaptive transfer of Gr1 + CD11b + MDSCs from day 3 septic mice reduces peritoneal bacteria in early sepsis. Peritoneal lavage fluid was collected from moribund mice that were sacrificed at days 2 to 5 after CLP and cultured on 5% sheep blood agar plates.

    Techniques Used: Mouse Assay, Cell Culture

    Differentiation of Gr1 + CD11b + MDSCs ex vivo . Total Gr1 + CD11b + MDSCs were harvested from the bone marrows of a separate group of mice at day 3 (D3) and day 12 (D12) after CLP. (A) Flow cytometry of sorted GR1 + CD11b + MDSCs recovered from septic mice
    Figure Legend Snippet: Differentiation of Gr1 + CD11b + MDSCs ex vivo . Total Gr1 + CD11b + MDSCs were harvested from the bone marrows of a separate group of mice at day 3 (D3) and day 12 (D12) after CLP. (A) Flow cytometry of sorted GR1 + CD11b + MDSCs recovered from septic mice

    Techniques Used: Ex Vivo, Mouse Assay, Flow Cytometry, Cytometry

    22) Product Images from "Immunosuppressive myeloid-derived suppressor cells are increased in splenocytes from cancer patients"

    Article Title: Immunosuppressive myeloid-derived suppressor cells are increased in splenocytes from cancer patients

    Journal: Cancer immunology, immunotherapy : CII

    doi: 10.1007/s00262-016-1953-z

    Myeloid-derived suppressor cell subsets identified in human spleen tissue. A. Human splenocytes or PBMC from the same donor were stained with antibodies specific for Lineage markers (CD3, CD19, and CD56), CD11b, and CD33. The frequency of total MDSCs (Lin−, CD11b+, CD33+, HLA-DR−), CD15+ MDSCs (Lin−, CD11b+, CD33+, HLA-DR−, CD15+, CD14−), and CD14+ MDSCs (Lin−, CD11b+, CD33+, HLA-DR−, CD15−, CD14+) was determined by flow cytometry. B. The frequency of MDSCs subsets in total PBMC or splenocytes was compared using ANOVA and compared between PBMC and spleen samples using a paired t test (C).
    Figure Legend Snippet: Myeloid-derived suppressor cell subsets identified in human spleen tissue. A. Human splenocytes or PBMC from the same donor were stained with antibodies specific for Lineage markers (CD3, CD19, and CD56), CD11b, and CD33. The frequency of total MDSCs (Lin−, CD11b+, CD33+, HLA-DR−), CD15+ MDSCs (Lin−, CD11b+, CD33+, HLA-DR−, CD15+, CD14−), and CD14+ MDSCs (Lin−, CD11b+, CD33+, HLA-DR−, CD15−, CD14+) was determined by flow cytometry. B. The frequency of MDSCs subsets in total PBMC or splenocytes was compared using ANOVA and compared between PBMC and spleen samples using a paired t test (C).

    Techniques Used: Derivative Assay, Staining, Flow Cytometry, Cytometry

    23) Product Images from "Activated invariant NKT cells control central nervous system autoimmunity in a mechanism that involves myeloid-derived suppressor cells"

    Article Title: Activated invariant NKT cells control central nervous system autoimmunity in a mechanism that involves myeloid-derived suppressor cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1201718

    α-GalCer-mediated expansion of MDSCs in EAE-induced mice. B6 mice were induced with EAE and treated with vehicle or α-GalCer. ( A ) Splenic and lymph node cellularity in vehicle- or α-GalCer-treated mice induced for development of EAE. B6 mice were induced with EAE and treated with vehicle or α-GalCer. At the indicated times the cellularity of spleen and lymph nodes was determined. The data presented are the mean ± SEM of 6 mice per group and representative of at least 3 individual experiments. *p
    Figure Legend Snippet: α-GalCer-mediated expansion of MDSCs in EAE-induced mice. B6 mice were induced with EAE and treated with vehicle or α-GalCer. ( A ) Splenic and lymph node cellularity in vehicle- or α-GalCer-treated mice induced for development of EAE. B6 mice were induced with EAE and treated with vehicle or α-GalCer. At the indicated times the cellularity of spleen and lymph nodes was determined. The data presented are the mean ± SEM of 6 mice per group and representative of at least 3 individual experiments. *p

    Techniques Used: Mouse Assay

    Inhibition of MOGp-specific Th17 cells by MDSCs. CD4 + T cells were isolated at day 11 after EAE induction and were stimulated with DCs and MOGp in the presence of varying numbers of total MDSCs from wild-type mice ( A ), total MDSCs derived from wild-type or CD1 d ̄ mice ( B ), or G-MDSCs or M-MDSCs ( C ) derived from MOGp-challenged mice treated with vehicle or α-GalCer. IL-17A production in the culture supernatants was measured by ELISA. ( D ) Naïve B6 mice were injected i.p. with 2 μg α-GalCer at days 0, 4, and 7. At day 11 the percentage (top) and absolute numbers (bottom left) of MDSCs, and the ability of these cells to inhibit MOGp-specific Th17 cells (bottom right) was determined. The results are the mean ± SEM of 3 mice and are a representative of at least 3 experiments. *p
    Figure Legend Snippet: Inhibition of MOGp-specific Th17 cells by MDSCs. CD4 + T cells were isolated at day 11 after EAE induction and were stimulated with DCs and MOGp in the presence of varying numbers of total MDSCs from wild-type mice ( A ), total MDSCs derived from wild-type or CD1 d ̄ mice ( B ), or G-MDSCs or M-MDSCs ( C ) derived from MOGp-challenged mice treated with vehicle or α-GalCer. IL-17A production in the culture supernatants was measured by ELISA. ( D ) Naïve B6 mice were injected i.p. with 2 μg α-GalCer at days 0, 4, and 7. At day 11 the percentage (top) and absolute numbers (bottom left) of MDSCs, and the ability of these cells to inhibit MOGp-specific Th17 cells (bottom right) was determined. The results are the mean ± SEM of 3 mice and are a representative of at least 3 experiments. *p

    Techniques Used: Inhibition, Isolation, Mouse Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Injection

    Effects of MDSC depletion or adoptive transfer on EAE. ( A,B ) EAE was induced in B6 mice and these animals were treated with vehicle or α-GalCer. Mice were then treated at days 6 and 9 after EAE induction with PBS or gemcitabine at 20 mg/kg body weight by i.p. injection. ( A ) At day 11 after EAE induction, spleen cells were analyzed for the prevalence of MDSCs. Note that > 80% depletion was observed. ( B ) EAE clinical scores were determined as described in Methods. ( C ) Mice induced with aEAE were treated with vehicle or α-GalCer and sacrificed 11 days after EAE induction. MDSCs were enriched from the spleen using magnetic sorting, pulsed with MOGp at 100 μg/ml for 1 hr and 5×10 6 cells were adoptively transferred into B6 mice on days 1, 4 and 9 following induction of pEAE with MOGp-specific T cells. EAE clinical scores were determined as described for active EAE. Combined data for two experiments with 4 mice in each group are shown.
    Figure Legend Snippet: Effects of MDSC depletion or adoptive transfer on EAE. ( A,B ) EAE was induced in B6 mice and these animals were treated with vehicle or α-GalCer. Mice were then treated at days 6 and 9 after EAE induction with PBS or gemcitabine at 20 mg/kg body weight by i.p. injection. ( A ) At day 11 after EAE induction, spleen cells were analyzed for the prevalence of MDSCs. Note that > 80% depletion was observed. ( B ) EAE clinical scores were determined as described in Methods. ( C ) Mice induced with aEAE were treated with vehicle or α-GalCer and sacrificed 11 days after EAE induction. MDSCs were enriched from the spleen using magnetic sorting, pulsed with MOGp at 100 μg/ml for 1 hr and 5×10 6 cells were adoptively transferred into B6 mice on days 1, 4 and 9 following induction of pEAE with MOGp-specific T cells. EAE clinical scores were determined as described for active EAE. Combined data for two experiments with 4 mice in each group are shown.

    Techniques Used: Adoptive Transfer Assay, Mouse Assay, Injection

    Role of iNOS, Arg1 and cytokines in the protective effects of α-GalCer against EAE. ( A ) B6 and iNOS −/− mice were induced with EAE and treated with vehicle or α-GalCer. The course of EAE disease was monitored and clinical scores were determined. ( B ) CD4 + T cells were isolated at day 11 after EAE induction in B6 mice and were stimulated with DCs and MOGp in the presence of MDSCs derived from mice treated with vehicle or α-GalCer at a MDSC:T cells ratio of 1:4 in HL-1 medium alone or medium containing BEC (50 μM). IL-17A production was measured in the culture supernatant by ELISA as a readout of Th17 cell activation. ( C ) CD4 + T cells were isolated at day 11 after EAE induction and were stimulated with DCs and MOGp in the presence of M-MDSCs derived from mice treated with vehicle or α-GalCer at a M-MDSC:T cell ratio of 1:4 in complete medium containing isotype control or anti-IL-10 antibody (10 μg/ml) (top panel). M-MDSCs derived from wild-type or IL-10 −/− mice induced for EAE and treated with vehicle or α-GalCer were cultured as above at a M-MDSC:T cell ratio of 1:4 (bottom panel). IL-17A production was measured in the culture supernatant by ELISA as a readout of Th17 cell activation. *p
    Figure Legend Snippet: Role of iNOS, Arg1 and cytokines in the protective effects of α-GalCer against EAE. ( A ) B6 and iNOS −/− mice were induced with EAE and treated with vehicle or α-GalCer. The course of EAE disease was monitored and clinical scores were determined. ( B ) CD4 + T cells were isolated at day 11 after EAE induction in B6 mice and were stimulated with DCs and MOGp in the presence of MDSCs derived from mice treated with vehicle or α-GalCer at a MDSC:T cells ratio of 1:4 in HL-1 medium alone or medium containing BEC (50 μM). IL-17A production was measured in the culture supernatant by ELISA as a readout of Th17 cell activation. ( C ) CD4 + T cells were isolated at day 11 after EAE induction and were stimulated with DCs and MOGp in the presence of M-MDSCs derived from mice treated with vehicle or α-GalCer at a M-MDSC:T cell ratio of 1:4 in complete medium containing isotype control or anti-IL-10 antibody (10 μg/ml) (top panel). M-MDSCs derived from wild-type or IL-10 −/− mice induced for EAE and treated with vehicle or α-GalCer were cultured as above at a M-MDSC:T cell ratio of 1:4 (bottom panel). IL-17A production was measured in the culture supernatant by ELISA as a readout of Th17 cell activation. *p

    Techniques Used: Mouse Assay, Isolation, Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Cell Culture

    24) Product Images from "Repression of MUC1 promotes expansion and suppressive function of myeloid-derived suppressor cells in pancreatic ductal adenocarcinoma and breast cancer murine models"

    Article Title: Repression of MUC1 promotes expansion and suppressive function of myeloid-derived suppressor cells in pancreatic ductal adenocarcinoma and breast cancer murine models

    Journal: bioRxiv

    doi: 10.1101/2020.12.07.415299

    Phenotypic characterization of MDSCs in the BM of healthy and tumor bearing WT and Muc1KO mice. A) Splenocytes from tumor bearing WT and MUC1KO mice were isolated and labeled with antibodies against Gr1, CD11b, and pSTAT3. B) Levels of Arginase-1 expression in MDSCs of WT and MUC1KO mice was measured via flow cytometry. Arginase-1 activity in sorted MDSCs was measured using a urea assay.
    Figure Legend Snippet: Phenotypic characterization of MDSCs in the BM of healthy and tumor bearing WT and Muc1KO mice. A) Splenocytes from tumor bearing WT and MUC1KO mice were isolated and labeled with antibodies against Gr1, CD11b, and pSTAT3. B) Levels of Arginase-1 expression in MDSCs of WT and MUC1KO mice was measured via flow cytometry. Arginase-1 activity in sorted MDSCs was measured using a urea assay.

    Techniques Used: Mouse Assay, Isolation, Labeling, Expressing, Flow Cytometry, Activity Assay

    MDSCs from MUC1KO mice have higher frequency and create a more immune suppressive environment. A) Splenocytes from healthy and tumor bearing WT and Muc1KO mice were isolated and labeled with anti Gr1 and CD11b antibodies. Representative plots from three separate experiments are shown. B) Levels of TGF-β was measured in the serum of healthy and cancer bearing WT and MUC1KO mice using ELISA kits. C) Levels of MDSCs and CD4+FoxP3+ cells were measured in the tumor of WT and Muc1KO mice using flow cytometry.
    Figure Legend Snippet: MDSCs from MUC1KO mice have higher frequency and create a more immune suppressive environment. A) Splenocytes from healthy and tumor bearing WT and Muc1KO mice were isolated and labeled with anti Gr1 and CD11b antibodies. Representative plots from three separate experiments are shown. B) Levels of TGF-β was measured in the serum of healthy and cancer bearing WT and MUC1KO mice using ELISA kits. C) Levels of MDSCs and CD4+FoxP3+ cells were measured in the tumor of WT and Muc1KO mice using flow cytometry.

    Techniques Used: Mouse Assay, Isolation, Labeling, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    MDSCs from MUC1KO mice produce increased iNOS and c-Myc A) BM cells from healthy mice were isolated and labeled with antibodies against Gr1 and CD11b. B) Level of iNOS expression by MDSCs from the BM of healthy and tumor bearing mice. C) Level of c-Myc protein expression by MDSCs from the BM of healthy and tumor bearing mice.
    Figure Legend Snippet: MDSCs from MUC1KO mice produce increased iNOS and c-Myc A) BM cells from healthy mice were isolated and labeled with antibodies against Gr1 and CD11b. B) Level of iNOS expression by MDSCs from the BM of healthy and tumor bearing mice. C) Level of c-Myc protein expression by MDSCs from the BM of healthy and tumor bearing mice.

    Techniques Used: Mouse Assay, Isolation, Labeling, Expressing

    25) Product Images from "Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia"

    Article Title: Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02686-14

    Reduced phagocytic activity in AMs incubated with MDSCs. AMs cocultured with control Gr1BM cells (A) or MDSCs (B) overnight were incubated with fluorescein-conjugated zymosan beads for 1 h. The nuclei of AMs were counterstained with DAPI. (C) The number of zymosan beads phagocytosed by AMs incubated with MDSCs or Gr1BM cells were counted under a confocal microscope. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Reduced phagocytic activity in AMs incubated with MDSCs. AMs cocultured with control Gr1BM cells (A) or MDSCs (B) overnight were incubated with fluorescein-conjugated zymosan beads for 1 h. The nuclei of AMs were counterstained with DAPI. (C) The number of zymosan beads phagocytosed by AMs incubated with MDSCs or Gr1BM cells were counted under a confocal microscope. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Activity Assay, Affinity Magnetic Separation, Incubation, Microscopy

    Decreased PU.1 expression in AMs incubated with MDSCs. AMs from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA of AMs was isolated, and PU.1 mRNA levels were determined by real-time PCR. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with either type of cell was compared to it. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Decreased PU.1 expression in AMs incubated with MDSCs. AMs from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA of AMs was isolated, and PU.1 mRNA levels were determined by real-time PCR. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with either type of cell was compared to it. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Expressing, Affinity Magnetic Separation, Incubation, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction

    Increased PD-1 expression in AMs incubated with MDSDs. AMs isolated from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . (A) After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA was isolated from AMs of each group. PD-1 gene expression was determined by real-time RT-PCR. The level of AMs/Gr1BM was set as 1, and that in AMs/MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) AMs incubated with MDSCs or Gr1BM cells were analyzed for surface PD-1 expression by flow cytometry.
    Figure Legend Snippet: Increased PD-1 expression in AMs incubated with MDSDs. AMs isolated from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . (A) After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA was isolated from AMs of each group. PD-1 gene expression was determined by real-time RT-PCR. The level of AMs/Gr1BM was set as 1, and that in AMs/MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) AMs incubated with MDSCs or Gr1BM cells were analyzed for surface PD-1 expression by flow cytometry.

    Techniques Used: Expressing, Affinity Magnetic Separation, Incubation, Isolation, Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry

    Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The C T values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The C T values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Affinity Magnetic Separation, Incubation, Mouse Assay, Sonication, Generated, Chromatin Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction

    Increased PD-L1 expression in MDSCs from PcP mice. MDSCs (MDSCs/PcP) were isolated from PcP mice at 5 weeks post- Pneumocystis infection. Control Gr1BM cells were isolated from uninfected mice immunosuppressed by weekly injection of anti-CD4 (L3T4) antibody. (A) Total RNA was isolated from the cells, and PD-L1 gene expression was determined by real-time RT-PCR. The average PD-L1 expression level in Gr1BM cells was set as 1, and that in MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) The cells were examined by flow cytometry using anti-Gr-1 and anti-PD-L1 antibodies. The result shown is representative of three independent experiments.
    Figure Legend Snippet: Increased PD-L1 expression in MDSCs from PcP mice. MDSCs (MDSCs/PcP) were isolated from PcP mice at 5 weeks post- Pneumocystis infection. Control Gr1BM cells were isolated from uninfected mice immunosuppressed by weekly injection of anti-CD4 (L3T4) antibody. (A) Total RNA was isolated from the cells, and PD-L1 gene expression was determined by real-time RT-PCR. The average PD-L1 expression level in Gr1BM cells was set as 1, and that in MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) The cells were examined by flow cytometry using anti-Gr-1 and anti-PD-L1 antibodies. The result shown is representative of three independent experiments.

    Techniques Used: Expressing, Mouse Assay, Isolation, Infection, Injection, Quantitative RT-PCR, Flow Cytometry, Cytometry

    Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.

    Techniques Used: DNA Methylation Assay, Affinity Magnetic Separation, Incubation, Mouse Assay, Isolation, CpG Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    Loss of suppressive effect of MDSCs pretreated with anti-PD-L1 antibody on AMs. A total of 1 × 10 5 AMs were cocultured with 5 × 10 5 MDSCs that were treated with anti-PD-L1 antibody or control IgG. (A) After 16 h of incubation, AMs were isolated, and the PU.1 mRNA levels were determined by real-time RT-PCR. Data are presented as means ± SD from three independent experiments. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with MDSCs that were pretreated with or without anti-PD-L1 antibody was compared to it. (B) Phagocytosis was assayed, and the number of zymosan beads phagocytosed by AMs incubated with MDSCs pretreated with anti-PD-L1 antibody or control IgG was counted under a confocal microscope.
    Figure Legend Snippet: Loss of suppressive effect of MDSCs pretreated with anti-PD-L1 antibody on AMs. A total of 1 × 10 5 AMs were cocultured with 5 × 10 5 MDSCs that were treated with anti-PD-L1 antibody or control IgG. (A) After 16 h of incubation, AMs were isolated, and the PU.1 mRNA levels were determined by real-time RT-PCR. Data are presented as means ± SD from three independent experiments. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with MDSCs that were pretreated with or without anti-PD-L1 antibody was compared to it. (B) Phagocytosis was assayed, and the number of zymosan beads phagocytosed by AMs incubated with MDSCs pretreated with anti-PD-L1 antibody or control IgG was counted under a confocal microscope.

    Techniques Used: Affinity Magnetic Separation, Incubation, Isolation, Quantitative RT-PCR, Expressing, Microscopy

    26) Product Images from "Blocking Monocytic Myeloid-Derived Suppressor Cell Function via Anti-DC-HIL/GPNMB Antibody Restores the In Vitro Integrity of T Cells from Cancer Patients"

    Article Title: Blocking Monocytic Myeloid-Derived Suppressor Cell Function via Anti-DC-HIL/GPNMB Antibody Restores the In Vitro Integrity of T Cells from Cancer Patients

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-18-0330

    Comparison of anti-DC-HIL and anti-PDL1 mAb in expression and the ability to reverse MDSC function. A, MDSCs from indicated cancer patients were assayed for percentage of DC-HIL positivity versus percentage of PDL1 positivity, and their differential expression levels are plotted in a graph, with the dashed line displaying the same percentage between the two receptors. B, Similarly MDSC-T-cell suppression assays were performed with or without anti-DC-HIL (αDCH), anti-PDL1 (αPDL), combined (Comb), or anti-KLH mAb (αKLH), and mAb effects are shown by IFNγ amounts in the cocultures. C, Data are summarized in a scattered graph, with median percentage, the cutoff value (dashed line), and statistical significance P value between 2 groups. On days 0 and 3 post-coculturing MDSCs ( D ) and T cells ( E ), cells were harvested and examined for MDSC (or CD8 T cells) expression of DC-HIL (or SD4) and PDL1 (or PD1). MDSCs were gated for CD14 + HLA-DR no/lo on day 0 and for CD45 + CD3 neg on day 3. Dot plots are shown with percentage of positive cells within the population. αDCH, anti-DC-HIL mAb; αKLH, anti-KLH mAb; αPDL, anti-PDL1 mAb; CO, colorectal; Comb, combined; PA, pancreatic; PR, prostate. F, MDSCs isolated from varying cancer patients were treated with the continuous presence of mAb or pretreated with mAb before coculturing. *, P
    Figure Legend Snippet: Comparison of anti-DC-HIL and anti-PDL1 mAb in expression and the ability to reverse MDSC function. A, MDSCs from indicated cancer patients were assayed for percentage of DC-HIL positivity versus percentage of PDL1 positivity, and their differential expression levels are plotted in a graph, with the dashed line displaying the same percentage between the two receptors. B, Similarly MDSC-T-cell suppression assays were performed with or without anti-DC-HIL (αDCH), anti-PDL1 (αPDL), combined (Comb), or anti-KLH mAb (αKLH), and mAb effects are shown by IFNγ amounts in the cocultures. C, Data are summarized in a scattered graph, with median percentage, the cutoff value (dashed line), and statistical significance P value between 2 groups. On days 0 and 3 post-coculturing MDSCs ( D ) and T cells ( E ), cells were harvested and examined for MDSC (or CD8 T cells) expression of DC-HIL (or SD4) and PDL1 (or PD1). MDSCs were gated for CD14 + HLA-DR no/lo on day 0 and for CD45 + CD3 neg on day 3. Dot plots are shown with percentage of positive cells within the population. αDCH, anti-DC-HIL mAb; αKLH, anti-KLH mAb; αPDL, anti-PDL1 mAb; CO, colorectal; Comb, combined; PA, pancreatic; PR, prostate. F, MDSCs isolated from varying cancer patients were treated with the continuous presence of mAb or pretreated with mAb before coculturing. *, P

    Techniques Used: Expressing, Isolation

    Anti-DC-HIL mAb restored the suppressed T-cell IFNγ response caused by MDSCs. A, Individual patients with colorectal (CO), pancreatic (PA), or prostate (PR) cancer was determined for percentage of DC-HIL + cells among MDSCs and their ability to suppress T-cell IFNγ response, expressed as percentage of T-cell suppression at cocultures of a 1:1 cell ratio. Values (percentages) were plotted and analyzed for correlation coefficient R 2 . B, Representative data of T-cell restoration assays by 3D5 anti-DC-HIL mAb: MDSCs were isolated from the blood of indicated patients and cocultured with autologous T cells at different cell ratios with costimulations. 3D5 (αDCH) or control anti-KLH mAb (αKLH) was added to 1:1 cocultures. Five days after culturing, IFNγ amounts were determined and shown in median ± SD, n = 3. *, P
    Figure Legend Snippet: Anti-DC-HIL mAb restored the suppressed T-cell IFNγ response caused by MDSCs. A, Individual patients with colorectal (CO), pancreatic (PA), or prostate (PR) cancer was determined for percentage of DC-HIL + cells among MDSCs and their ability to suppress T-cell IFNγ response, expressed as percentage of T-cell suppression at cocultures of a 1:1 cell ratio. Values (percentages) were plotted and analyzed for correlation coefficient R 2 . B, Representative data of T-cell restoration assays by 3D5 anti-DC-HIL mAb: MDSCs were isolated from the blood of indicated patients and cocultured with autologous T cells at different cell ratios with costimulations. 3D5 (αDCH) or control anti-KLH mAb (αKLH) was added to 1:1 cocultures. Five days after culturing, IFNγ amounts were determined and shown in median ± SD, n = 3. *, P

    Techniques Used: Isolation

    27) Product Images from "Differential Regulation of Myeloid-Derived Suppressor Cells by Candida Species"

    Article Title: Differential Regulation of Myeloid-Derived Suppressor Cells by Candida Species

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01624

    Candida -induced MDSCs suppress T cell responses. MDSCs generated by Candida species are able to suppress T-cell proliferation and function in a dose dependent manner. The suppressive effects of CD33 + -MACS-isolated MDSCs on CD4 + (green) and CD8 + (lilac) were assessed by T-cell proliferation (CFSE polyclonal proliferation) assay. MDSCs were generated by incubating PBMCs (5 × 10 5 /ml) from healthy donors with heat killed yeast cells of various Candida species (1 × 10 5 /ml) or C. albicans yeasts for 6 days. (A) Representative CFSE stainings, showing the effect of in vitro C. albicans, C. krusei , and C. glabrata induced MDSCs on CD4 + and CD8 + T-cell proliferation. Different MDSC to T cell ratios were assessed by using a wide range of MDSC:Target ratio (1:2, 1:4, 1:6, 1:8, and 1:16). (B) The bar graphs represent the proliferation index compared to control conditions. Even at a higher MDSC:target ratio of 1:16, MDSCs induced by C. krusei , and C. glabrata show higher suppressive activity in comparison to C. albicans . Data is shown as mean ± SEM ( n = 4) ∗∗ P
    Figure Legend Snippet: Candida -induced MDSCs suppress T cell responses. MDSCs generated by Candida species are able to suppress T-cell proliferation and function in a dose dependent manner. The suppressive effects of CD33 + -MACS-isolated MDSCs on CD4 + (green) and CD8 + (lilac) were assessed by T-cell proliferation (CFSE polyclonal proliferation) assay. MDSCs were generated by incubating PBMCs (5 × 10 5 /ml) from healthy donors with heat killed yeast cells of various Candida species (1 × 10 5 /ml) or C. albicans yeasts for 6 days. (A) Representative CFSE stainings, showing the effect of in vitro C. albicans, C. krusei , and C. glabrata induced MDSCs on CD4 + and CD8 + T-cell proliferation. Different MDSC to T cell ratios were assessed by using a wide range of MDSC:Target ratio (1:2, 1:4, 1:6, 1:8, and 1:16). (B) The bar graphs represent the proliferation index compared to control conditions. Even at a higher MDSC:target ratio of 1:16, MDSCs induced by C. krusei , and C. glabrata show higher suppressive activity in comparison to C. albicans . Data is shown as mean ± SEM ( n = 4) ∗∗ P

    Techniques Used: Generated, Magnetic Cell Separation, Isolation, Proliferation Assay, In Vitro, Activity Assay

    In vitro MDSC generation by different Candida non- albicans species and C. albicans morphotypes. MDSCs were generated by incubating freshly isolated PBMCs (5 × 10 5 /ml) from healthy donors with medium only (negative control) or indicated stimulants. (A) PBMCs were cultured with heat killed yeast cells of C. albicans, C. glabrata, C. krusei, C. parapsilosis , and C. dubliniensis (1 × 10 5 /ml) for 6 days ( n = 11–20) or (B) with heat killed C. albicans yeast cells (1 × 10 5 /ml), filter sterilized C. albicans yeast supernatant (5% SNT), or C. albicans hyphae (1 × 10 5 /ml) for 6 days ( n = 8–13). Granulocytic MDSCs (CD11b + CD33 + CD14 - ) were quantified by using Flow Cytometry. The number of MDSCs in % of all cells in medium only cultures was set to 1-fold for every single experiment. The MDSC induction due to specific stimuli is presented as x-fold compared to medium control (mean ± SEM) and differences compared to controls were analyzed by a one-sample t -test. Significant differences between control and G-MDSCs induction by stimulants are indicated by an asterisk ( ∗ P
    Figure Legend Snippet: In vitro MDSC generation by different Candida non- albicans species and C. albicans morphotypes. MDSCs were generated by incubating freshly isolated PBMCs (5 × 10 5 /ml) from healthy donors with medium only (negative control) or indicated stimulants. (A) PBMCs were cultured with heat killed yeast cells of C. albicans, C. glabrata, C. krusei, C. parapsilosis , and C. dubliniensis (1 × 10 5 /ml) for 6 days ( n = 11–20) or (B) with heat killed C. albicans yeast cells (1 × 10 5 /ml), filter sterilized C. albicans yeast supernatant (5% SNT), or C. albicans hyphae (1 × 10 5 /ml) for 6 days ( n = 8–13). Granulocytic MDSCs (CD11b + CD33 + CD14 - ) were quantified by using Flow Cytometry. The number of MDSCs in % of all cells in medium only cultures was set to 1-fold for every single experiment. The MDSC induction due to specific stimuli is presented as x-fold compared to medium control (mean ± SEM) and differences compared to controls were analyzed by a one-sample t -test. Significant differences between control and G-MDSCs induction by stimulants are indicated by an asterisk ( ∗ P

    Techniques Used: In Vitro, Generated, Isolation, Negative Control, Cell Culture, Flow Cytometry, Cytometry

    28) Product Images from "MUC1 Vaccine for Individuals with Advanced Adenoma of the Colon: A Cancer Immunoprevention Feasibility Study"

    Article Title: MUC1 Vaccine for Individuals with Advanced Adenoma of the Colon: A Cancer Immunoprevention Feasibility Study

    Journal: Cancer prevention research (Philadelphia, Pa.)

    doi: 10.1158/1940-6207.CAPR-12-0275

    PBMC of non-responders contain increased levels of myeloid derived suppressor cells (MDSC). A . Representative PBMC flow cytometry profile of a responder (left) and a non-responder (right) showing a difference in the CD33 +/low, CD11b+ and HLA-DR- cell
    Figure Legend Snippet: PBMC of non-responders contain increased levels of myeloid derived suppressor cells (MDSC). A . Representative PBMC flow cytometry profile of a responder (left) and a non-responder (right) showing a difference in the CD33 +/low, CD11b+ and HLA-DR- cell

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry

    29) Product Images from "Vaccine-induced myeloid cell population dampens protective immunity to SIV"

    Article Title: Vaccine-induced myeloid cell population dampens protective immunity to SIV

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI73518

    MDSCs from PBMCs of the SIV vaccine–immunized (prior to infection) or vaccinated animals after SIVmac251 infection suppressed CD8 + T cell responses.
    Figure Legend Snippet: MDSCs from PBMCs of the SIV vaccine–immunized (prior to infection) or vaccinated animals after SIVmac251 infection suppressed CD8 + T cell responses.

    Techniques Used: Infection

    30) Product Images from "Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells"

    Article Title: Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1186/2051-1426-2-5

    Tolerogenic MDSCs incubated with VSSP are conditioned to cross-prime antigen-specific CD8 + T cells. CD11b + Gr1 + cells isolated from EL4 and EG.7 TB mice, inoculated or not with VSSP, were treated in vitro with 10 μg/mL of both OVA and VSSP (A and C) or left untreated (A and B) . To assess the capacity of these cells to accomplish a detectable cross-priming, antigen-specific CD8 + T cells were isolated from OTI transgenic mice and cocultured at 1:1 ratio with MDSCs for 96 h. BM-DCs previously incubated with OVA, OVA and VSSP or pulsed with SIINFEKL peptide were used as controls (D and E) . Graphs show the percentage of CD69 + cells as a measure of the activation of antigen-specific CD8 + T cells. ANOVA and Tukey’s tests were used for statistical comparison of the groups’ mean. Two experiments with similar results were performed.
    Figure Legend Snippet: Tolerogenic MDSCs incubated with VSSP are conditioned to cross-prime antigen-specific CD8 + T cells. CD11b + Gr1 + cells isolated from EL4 and EG.7 TB mice, inoculated or not with VSSP, were treated in vitro with 10 μg/mL of both OVA and VSSP (A and C) or left untreated (A and B) . To assess the capacity of these cells to accomplish a detectable cross-priming, antigen-specific CD8 + T cells were isolated from OTI transgenic mice and cocultured at 1:1 ratio with MDSCs for 96 h. BM-DCs previously incubated with OVA, OVA and VSSP or pulsed with SIINFEKL peptide were used as controls (D and E) . Graphs show the percentage of CD69 + cells as a measure of the activation of antigen-specific CD8 + T cells. ANOVA and Tukey’s tests were used for statistical comparison of the groups’ mean. Two experiments with similar results were performed.

    Techniques Used: Incubation, Isolation, Mouse Assay, In Vitro, Transgenic Assay, Activation Assay

    Suppressive mechanisms of MDSCs are dampened by VSSP. Mice were treated as described in Figure 2 . FACS analyses of the down-regulation of CD3ζ chain on CD8 + (A) and CD4 + (B) T cells, as well as CD62L on T cells (C) , were performed in splenocytes from five individual mice per group. Data were normalized by the percentage of MDSCs in each mouse. The reduction in Tregs frequency caused by VSSP was further corroborated and resulted more obvious after the normalization procedure (D) . Statistical comparisons between groups were done with Student’s t test for CD62L expression and Tregs percentages, whereas Mann-Whitney’s U test was used to analyze down-regulation of CD3ζ chain. (E-F) MDSCs immunomagnetically enriched from the spleens of VSSP-injected or untreated MCA203 TB mice were cultured at 20% with splenocytes from OTI transgenic mice, in the presence of relevant peptide. Histograms show the expression of CD3ζ chain on CD8 + T cells specifically stimulated with SIINFEKL peptide in the presence or absence of MDSCs. Further characterization of MDSCs isolated from each experimental group was done by RT-PCR and the reduction of Arg1 (G) and Nos 2 (H) gene expression is represented in bar graphs. Three replicates of CD11b + Gr1 + cells isolated from pools of three mice per group were included in the RT-PCR analysis. (I) The capacity of MDSCs to behave as APCs during Con A-stimulated IFN-γ production by CD8 + T cells was measured through ELISPOT assay. CD8 + T cells and CD11b + Gr1 + cells were isolated from TB mice, either treated or not with the adjuvant, and cocultured for 72 h in the presence of Con A mitogen. Graph indicates the mean ± SD of the number of IFN-γ spots per 10 5 CD8 + T cells from one experiment representative of two (T: tumor and V: VSSP). (G-I) The multiple comparisons of mean values were executed with ANOVA and Tukey’s tests.
    Figure Legend Snippet: Suppressive mechanisms of MDSCs are dampened by VSSP. Mice were treated as described in Figure 2 . FACS analyses of the down-regulation of CD3ζ chain on CD8 + (A) and CD4 + (B) T cells, as well as CD62L on T cells (C) , were performed in splenocytes from five individual mice per group. Data were normalized by the percentage of MDSCs in each mouse. The reduction in Tregs frequency caused by VSSP was further corroborated and resulted more obvious after the normalization procedure (D) . Statistical comparisons between groups were done with Student’s t test for CD62L expression and Tregs percentages, whereas Mann-Whitney’s U test was used to analyze down-regulation of CD3ζ chain. (E-F) MDSCs immunomagnetically enriched from the spleens of VSSP-injected or untreated MCA203 TB mice were cultured at 20% with splenocytes from OTI transgenic mice, in the presence of relevant peptide. Histograms show the expression of CD3ζ chain on CD8 + T cells specifically stimulated with SIINFEKL peptide in the presence or absence of MDSCs. Further characterization of MDSCs isolated from each experimental group was done by RT-PCR and the reduction of Arg1 (G) and Nos 2 (H) gene expression is represented in bar graphs. Three replicates of CD11b + Gr1 + cells isolated from pools of three mice per group were included in the RT-PCR analysis. (I) The capacity of MDSCs to behave as APCs during Con A-stimulated IFN-γ production by CD8 + T cells was measured through ELISPOT assay. CD8 + T cells and CD11b + Gr1 + cells were isolated from TB mice, either treated or not with the adjuvant, and cocultured for 72 h in the presence of Con A mitogen. Graph indicates the mean ± SD of the number of IFN-γ spots per 10 5 CD8 + T cells from one experiment representative of two (T: tumor and V: VSSP). (G-I) The multiple comparisons of mean values were executed with ANOVA and Tukey’s tests.

    Techniques Used: Mouse Assay, FACS, Expressing, MANN-WHITNEY, Injection, Cell Culture, Transgenic Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunospot

    31) Product Images from "Targeting ornithine decarboxylase by α-difluoromethylornithine inhibits tumor growth by impairing myeloid-derived suppressor cells"

    Article Title: Targeting ornithine decarboxylase by α-difluoromethylornithine inhibits tumor growth by impairing myeloid-derived suppressor cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1500729

    DFMO targets MDSCs to inhibit tumor growth Mice were injected s.c. with 10 6 B16F10 tumor cells. Depletion of MDSC was achieved by either twice-weekly i.p. injection of ( A ) 5-Fluorouracil (5-FU) or ( B ) anti-Gr1 antibodies starting 2 days after tumor challenge (5 mice per group). ( C ) Splenic Gr1 + CD11b + MDSCs from B16F10-bearing mice treated with DFMO or dH 2 O were injected i.v. into B16-bearing mice at d7 and d14. Mice receiving PBS without MDSCs were controls. Tumor volume was measured and plotted at indicated times (5 mice per group). ( D ) Flow cytometry analysis of expression of ki67 and Annexin V in gp100-specific tetramer + CD8 + TCRVβ + cells, and absolute number of these tetramer + CD8 + TCRVβ + cells per 10 6 cells in tumor infiltrates 3d after the initial MDSC transfer (5 mice per group). *, p
    Figure Legend Snippet: DFMO targets MDSCs to inhibit tumor growth Mice were injected s.c. with 10 6 B16F10 tumor cells. Depletion of MDSC was achieved by either twice-weekly i.p. injection of ( A ) 5-Fluorouracil (5-FU) or ( B ) anti-Gr1 antibodies starting 2 days after tumor challenge (5 mice per group). ( C ) Splenic Gr1 + CD11b + MDSCs from B16F10-bearing mice treated with DFMO or dH 2 O were injected i.v. into B16-bearing mice at d7 and d14. Mice receiving PBS without MDSCs were controls. Tumor volume was measured and plotted at indicated times (5 mice per group). ( D ) Flow cytometry analysis of expression of ki67 and Annexin V in gp100-specific tetramer + CD8 + TCRVβ + cells, and absolute number of these tetramer + CD8 + TCRVβ + cells per 10 6 cells in tumor infiltrates 3d after the initial MDSC transfer (5 mice per group). *, p

    Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, Expressing

    Charaterization of phenotypic tumor-associated MDSCs following DFMO treatment ( A ) Percent splenic Gr1 + CD11b + MDSCs were determined by flow cytometry from B16F10-bearing mice. Percent CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs were indicated within plots (5 mice per group). ( B ) Percent Gr1 + CD11b + MDSCs, CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs in spleen and tumor tissues from B16F10-bearing mice were summarized (5 mice per group). ( C ) Measurement of ODC activity in Gr1 + CD11b + cells from naïve and B16F10 tumor-bearing (TB) mice treated by DFMO or dH 2 O (5 mice per group). ( D ) Expression levels of CD39, CD73, CD115, MHC-II, B7H1, DCFDA (ROS indicator) and arginase-I among both tumor-infiltrating CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs were determined by flow cytometry. Cells were collected from B16F10-bearing DFMO treated or control mice 14 days after tumor inoculation (5 mice per group). Data (mean ± SEM) are representative of 2 independent experiments. *, p
    Figure Legend Snippet: Charaterization of phenotypic tumor-associated MDSCs following DFMO treatment ( A ) Percent splenic Gr1 + CD11b + MDSCs were determined by flow cytometry from B16F10-bearing mice. Percent CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs were indicated within plots (5 mice per group). ( B ) Percent Gr1 + CD11b + MDSCs, CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs in spleen and tumor tissues from B16F10-bearing mice were summarized (5 mice per group). ( C ) Measurement of ODC activity in Gr1 + CD11b + cells from naïve and B16F10 tumor-bearing (TB) mice treated by DFMO or dH 2 O (5 mice per group). ( D ) Expression levels of CD39, CD73, CD115, MHC-II, B7H1, DCFDA (ROS indicator) and arginase-I among both tumor-infiltrating CD11b + Ly6G + Ly6C low (granulocytic) and CD11b + Ly6G − Ly6C high (monocytic) MDSCs were determined by flow cytometry. Cells were collected from B16F10-bearing DFMO treated or control mice 14 days after tumor inoculation (5 mice per group). Data (mean ± SEM) are representative of 2 independent experiments. *, p

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Activity Assay, Expressing

    32) Product Images from "Induction of DNMT3B by PGE2 and IL6 at distant metastatic sites promotes epigenetic modification and breast cancer colonization"

    Article Title: Induction of DNMT3B by PGE2 and IL6 at distant metastatic sites promotes epigenetic modification and breast cancer colonization

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-19-3339

    Inflammatory Mediators PGE2 and IL-6 Induce DNMT3B. a ELISA and Bioplex assays of protein extraction from normal lungs and lungs from 4T1 tumor-bearing mice (n=3–6 lungs). b Western blots for DNMT3B, pSTAT3, pNFκB and pSmad2 of 4T1 cells cultured with lung tissue culture supernatants, and with neutralization of TGF-β, IL-6 or/and PGE2 receptor antagonist (AH-6809), as well as knockdown of NFκB or Smad2 or treatment with STAT3 inhibitor (Stattic). c Western blots for DNMT3B, pSTAT3, pNFκB in an in vitro coculture of 4T1 cells with MDSCs, with or without PGE2 receptor antagonist (AH-6809) and IL-6 neutralizing antibody treatment. d PGE2 ELISA of protein extraction from normal lungs and lungs from tumor-bearing mice with or without Celecoxib treatment. e DNMT3B Western of lung nodules of 4T1 (left panel) and EMT6 (right panel, each lane from an individual mouse) tumor-bearing mice treated with Celecoxib, normal lung or primary tumors are used as controls. Days after MFP tumor injection is indicated. f Locus-specific DNA methylation analysis and RT-qPCR of DNMT3B target genes Cldn9 and Lfng. g Number of lung metastatic nodules from mice that were treated with PGE2 or IL-6 or in combination. The DNMT3B KD or wt control 4T1 tumor cells were injected through tail vein, representative images shown on the right. h Schematic hypothesis: MDCSs, PGE2, and IL-6 are increased in the inflammatory metastatic microenvironment, which induce DNMT3B in the metastatic cancer cells through PGE2/NFκB and IL-6/STAT3 signaling pathways. DNMT3B alters gene methylation and transcriptome thus activates signaling pathways including STAT3, NFκB, PI3K/Akt, β-catenin, as well as Notch signaling pathways that are important in metastatic colonization. Data are presented as mean ± SD. *p
    Figure Legend Snippet: Inflammatory Mediators PGE2 and IL-6 Induce DNMT3B. a ELISA and Bioplex assays of protein extraction from normal lungs and lungs from 4T1 tumor-bearing mice (n=3–6 lungs). b Western blots for DNMT3B, pSTAT3, pNFκB and pSmad2 of 4T1 cells cultured with lung tissue culture supernatants, and with neutralization of TGF-β, IL-6 or/and PGE2 receptor antagonist (AH-6809), as well as knockdown of NFκB or Smad2 or treatment with STAT3 inhibitor (Stattic). c Western blots for DNMT3B, pSTAT3, pNFκB in an in vitro coculture of 4T1 cells with MDSCs, with or without PGE2 receptor antagonist (AH-6809) and IL-6 neutralizing antibody treatment. d PGE2 ELISA of protein extraction from normal lungs and lungs from tumor-bearing mice with or without Celecoxib treatment. e DNMT3B Western of lung nodules of 4T1 (left panel) and EMT6 (right panel, each lane from an individual mouse) tumor-bearing mice treated with Celecoxib, normal lung or primary tumors are used as controls. Days after MFP tumor injection is indicated. f Locus-specific DNA methylation analysis and RT-qPCR of DNMT3B target genes Cldn9 and Lfng. g Number of lung metastatic nodules from mice that were treated with PGE2 or IL-6 or in combination. The DNMT3B KD or wt control 4T1 tumor cells were injected through tail vein, representative images shown on the right. h Schematic hypothesis: MDCSs, PGE2, and IL-6 are increased in the inflammatory metastatic microenvironment, which induce DNMT3B in the metastatic cancer cells through PGE2/NFκB and IL-6/STAT3 signaling pathways. DNMT3B alters gene methylation and transcriptome thus activates signaling pathways including STAT3, NFκB, PI3K/Akt, β-catenin, as well as Notch signaling pathways that are important in metastatic colonization. Data are presented as mean ± SD. *p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Protein Extraction, Mouse Assay, Western Blot, Cell Culture, Neutralization, In Vitro, Injection, DNA Methylation Assay, Quantitative RT-PCR, Methylation

    33) Product Images from "Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells"

    Article Title: Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1186/2051-1426-2-5

    Tolerogenic MDSCs incubated with VSSP are conditioned to cross-prime antigen-specific CD8 + T cells. CD11b + Gr1 + cells isolated from EL4 and EG.7 TB mice, inoculated or not with VSSP, were treated in vitro with 10 μg/mL of both OVA and VSSP (A and C) or left untreated (A and B) . To assess the capacity of these cells to accomplish a detectable cross-priming, antigen-specific CD8 + T cells were isolated from OTI transgenic mice and cocultured at 1:1 ratio with MDSCs for 96 h. BM-DCs previously incubated with OVA, OVA and VSSP or pulsed with SIINFEKL peptide were used as controls (D and E) . Graphs show the percentage of CD69 + cells as a measure of the activation of antigen-specific CD8 + T cells. ANOVA and Tukey’s tests were used for statistical comparison of the groups’ mean. Two experiments with similar results were performed.
    Figure Legend Snippet: Tolerogenic MDSCs incubated with VSSP are conditioned to cross-prime antigen-specific CD8 + T cells. CD11b + Gr1 + cells isolated from EL4 and EG.7 TB mice, inoculated or not with VSSP, were treated in vitro with 10 μg/mL of both OVA and VSSP (A and C) or left untreated (A and B) . To assess the capacity of these cells to accomplish a detectable cross-priming, antigen-specific CD8 + T cells were isolated from OTI transgenic mice and cocultured at 1:1 ratio with MDSCs for 96 h. BM-DCs previously incubated with OVA, OVA and VSSP or pulsed with SIINFEKL peptide were used as controls (D and E) . Graphs show the percentage of CD69 + cells as a measure of the activation of antigen-specific CD8 + T cells. ANOVA and Tukey’s tests were used for statistical comparison of the groups’ mean. Two experiments with similar results were performed.

    Techniques Used: Incubation, Isolation, Mouse Assay, In Vitro, Transgenic Assay, Activation Assay

    Suppressive mechanisms of MDSCs are dampened by VSSP. Mice were treated as described in Figure 2 . FACS analyses of the down-regulation of CD3ζ chain on CD8 + (A) and CD4 + (B) T cells, as well as CD62L on T cells (C) , were performed in splenocytes from five individual mice per group. Data were normalized by the percentage of MDSCs in each mouse. The reduction in Tregs frequency caused by VSSP was further corroborated and resulted more obvious after the normalization procedure (D) . Statistical comparisons between groups were done with Student’s t test for CD62L expression and Tregs percentages, whereas Mann-Whitney’s U test was used to analyze down-regulation of CD3ζ chain. (E-F) MDSCs immunomagnetically enriched from the spleens of VSSP-injected or untreated MCA203 TB mice were cultured at 20% with splenocytes from OTI transgenic mice, in the presence of relevant peptide. Histograms show the expression of CD3ζ chain on CD8 + T cells specifically stimulated with SIINFEKL peptide in the presence or absence of MDSCs. Further characterization of MDSCs isolated from each experimental group was done by RT-PCR and the reduction of Arg1 (G) and Nos 2 (H) gene expression is represented in bar graphs. Three replicates of CD11b + Gr1 + cells isolated from pools of three mice per group were included in the RT-PCR analysis. (I) The capacity of MDSCs to behave as APCs during Con A-stimulated IFN-γ production by CD8 + T cells was measured through ELISPOT assay. CD8 + T cells and CD11b + Gr1 + cells were isolated from TB mice, either treated or not with the adjuvant, and cocultured for 72 h in the presence of Con A mitogen. Graph indicates the mean ± SD of the number of IFN-γ spots per 10 5 CD8 + T cells from one experiment representative of two (T: tumor and V: VSSP). (G-I) The multiple comparisons of mean values were executed with ANOVA and Tukey’s tests.
    Figure Legend Snippet: Suppressive mechanisms of MDSCs are dampened by VSSP. Mice were treated as described in Figure 2 . FACS analyses of the down-regulation of CD3ζ chain on CD8 + (A) and CD4 + (B) T cells, as well as CD62L on T cells (C) , were performed in splenocytes from five individual mice per group. Data were normalized by the percentage of MDSCs in each mouse. The reduction in Tregs frequency caused by VSSP was further corroborated and resulted more obvious after the normalization procedure (D) . Statistical comparisons between groups were done with Student’s t test for CD62L expression and Tregs percentages, whereas Mann-Whitney’s U test was used to analyze down-regulation of CD3ζ chain. (E-F) MDSCs immunomagnetically enriched from the spleens of VSSP-injected or untreated MCA203 TB mice were cultured at 20% with splenocytes from OTI transgenic mice, in the presence of relevant peptide. Histograms show the expression of CD3ζ chain on CD8 + T cells specifically stimulated with SIINFEKL peptide in the presence or absence of MDSCs. Further characterization of MDSCs isolated from each experimental group was done by RT-PCR and the reduction of Arg1 (G) and Nos 2 (H) gene expression is represented in bar graphs. Three replicates of CD11b + Gr1 + cells isolated from pools of three mice per group were included in the RT-PCR analysis. (I) The capacity of MDSCs to behave as APCs during Con A-stimulated IFN-γ production by CD8 + T cells was measured through ELISPOT assay. CD8 + T cells and CD11b + Gr1 + cells were isolated from TB mice, either treated or not with the adjuvant, and cocultured for 72 h in the presence of Con A mitogen. Graph indicates the mean ± SD of the number of IFN-γ spots per 10 5 CD8 + T cells from one experiment representative of two (T: tumor and V: VSSP). (G-I) The multiple comparisons of mean values were executed with ANOVA and Tukey’s tests.

    Techniques Used: Mouse Assay, FACS, Expressing, MANN-WHITNEY, Injection, Cell Culture, Transgenic Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunospot

    34) Product Images from "TRAF6 Regulates the Immunosuppressive Effects of Myeloid-Derived Suppressor Cells in Tumor-Bearing Host"

    Article Title: TRAF6 Regulates the Immunosuppressive Effects of Myeloid-Derived Suppressor Cells in Tumor-Bearing Host

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.649020

    TRAF6 knockdown impairs the immunosuppressive effects of MDSCs in vitro . Specific siRNA (siTRAF6) was used to knockdown the expression of TRAF6 in MDSCs, and the efficiency of siTRAF6 knockdown was validated by qRT-PCR (A) and Western blotting (B) . (C) Tumor-derived MDSCs were transfected with siTRAF6 and cocultured with CFSE-labeled CD4 + T cells, and proliferation was measured by flow cytometry after 72 h. (D) Statistical analyses of the percentage of proliferating CD4 + T cells co-cultured with MDSCs transfected with siTRAF6. After TRAF6 knockdown, the activity of Arg1 was measured by a QuantiChrom arginase assay kit (E) , and the concentration of NO was determined via a Griess reagent system kit (F) . *** p
    Figure Legend Snippet: TRAF6 knockdown impairs the immunosuppressive effects of MDSCs in vitro . Specific siRNA (siTRAF6) was used to knockdown the expression of TRAF6 in MDSCs, and the efficiency of siTRAF6 knockdown was validated by qRT-PCR (A) and Western blotting (B) . (C) Tumor-derived MDSCs were transfected with siTRAF6 and cocultured with CFSE-labeled CD4 + T cells, and proliferation was measured by flow cytometry after 72 h. (D) Statistical analyses of the percentage of proliferating CD4 + T cells co-cultured with MDSCs transfected with siTRAF6. After TRAF6 knockdown, the activity of Arg1 was measured by a QuantiChrom arginase assay kit (E) , and the concentration of NO was determined via a Griess reagent system kit (F) . *** p

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Transfection, Labeling, Flow Cytometry, Cell Culture, Activity Assay, Arginase Assay, Concentration Assay

    TRAF6 expression was augmented in MDSCs from lung cancer patients. To examine the modulation of TRAF6 in MDSCs from lung cancer patients, the level of TRAF6 in MDSCs was measured in the lung cancer patient group (LC) and healthy control group (HC). (A) The proportions of MDSCs in the PBMCs of lung cancer patients and healthy persons were analyzed by flow cytometry. Representative dot plots of CD11b + CD33 + HLA-DR − MDSCs in the blood of patients with LC and healthy controls are shown. (B) The mean fluorescence intensity (MFI) of TRAF6 in MDSCs was determined by flow cytometry. (C) The MFI of arginase-1 in MDSCs was determined by flow cytometry. (D) The correlation between TRAF6 and arginase-1 in MDSCs was analyzed. *** p
    Figure Legend Snippet: TRAF6 expression was augmented in MDSCs from lung cancer patients. To examine the modulation of TRAF6 in MDSCs from lung cancer patients, the level of TRAF6 in MDSCs was measured in the lung cancer patient group (LC) and healthy control group (HC). (A) The proportions of MDSCs in the PBMCs of lung cancer patients and healthy persons were analyzed by flow cytometry. Representative dot plots of CD11b + CD33 + HLA-DR − MDSCs in the blood of patients with LC and healthy controls are shown. (B) The mean fluorescence intensity (MFI) of TRAF6 in MDSCs was determined by flow cytometry. (C) The MFI of arginase-1 in MDSCs was determined by flow cytometry. (D) The correlation between TRAF6 and arginase-1 in MDSCs was analyzed. *** p

    Techniques Used: Expressing, Flow Cytometry, Fluorescence

    TRAF6 is highly expressed in MDSCs derived from the tumor tissue of tumor-bearing mice. Approximately 1 × 10 6 LLC cells were s.c. injected in the backs of C57BL/6 mice for 28 d to establish a tumor-bearing (TB) mouse model. MDSCs were isolated by immunomagnetic beads from the spleens of TB mice, the tumor tissue of TB mice or the spleens of wild-type (WT) mice. (A) The purity of the isolated MDSCs was determined using flow cytometry via the detection of the CD11b + Gr1 + phenotype. The expression of TRAF6 in MDSCs derived from different sources was determined by qRT-PCR (B) or Western blotting (C) . The mRNA expression of TRAF6 in PMN-MDSCs and M-MDSCs derived from the spleen (D) or tumor tissue (E) . (F) CFSE-labeled CD4 + T cells were co-cultured with MDSCs derived from the spleen or tumor tissue in the presence of CD3 and CD28 stimulation. After 72 h, the proliferation of CD4 + T cells was tested via flow cytometry. (G) Statistical analyses of the percentage of proliferating CD4 + T cells co-cultured with MDSCs derived from the spleen or tumor tissue of TB mice. The mRNA expression levels of Arg1 (H) and iNOS (I) in MDSCs were measured by qRT-PCR. *** p
    Figure Legend Snippet: TRAF6 is highly expressed in MDSCs derived from the tumor tissue of tumor-bearing mice. Approximately 1 × 10 6 LLC cells were s.c. injected in the backs of C57BL/6 mice for 28 d to establish a tumor-bearing (TB) mouse model. MDSCs were isolated by immunomagnetic beads from the spleens of TB mice, the tumor tissue of TB mice or the spleens of wild-type (WT) mice. (A) The purity of the isolated MDSCs was determined using flow cytometry via the detection of the CD11b + Gr1 + phenotype. The expression of TRAF6 in MDSCs derived from different sources was determined by qRT-PCR (B) or Western blotting (C) . The mRNA expression of TRAF6 in PMN-MDSCs and M-MDSCs derived from the spleen (D) or tumor tissue (E) . (F) CFSE-labeled CD4 + T cells were co-cultured with MDSCs derived from the spleen or tumor tissue in the presence of CD3 and CD28 stimulation. After 72 h, the proliferation of CD4 + T cells was tested via flow cytometry. (G) Statistical analyses of the percentage of proliferating CD4 + T cells co-cultured with MDSCs derived from the spleen or tumor tissue of TB mice. The mRNA expression levels of Arg1 (H) and iNOS (I) in MDSCs were measured by qRT-PCR. *** p

    Techniques Used: Derivative Assay, Mouse Assay, Injection, Isolation, Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot, Labeling, Cell Culture

    TRAF6 knockdown attenuates the ability of MDSCs to accelerate tumor progression in tumor-bearing mice. To investigate the effects of TRAF6 on the suppressive activity of MDSCs in vivo , 2 groups of wild-type C57BL/6 mice were s.c. injected with 1 × 10 6 LLC cells and 1 × 10 6 MDSCs transfected with siTRAF6 (siTRAF6 group) or MDSCs transfected with siNC (control group). (A) Tumor growth was constantly monitored. The width “a” and length “b” were measured, and tumor volume was calculated. (B,C) On the 28th day after the inoculation of LLC cells, the mice were sacrificed, and the tumor image and weights were showed in both groups. (D) The proportion of CD4 + IFN-γ + Th1 cells in the tumor tissue of both groups was analyzed by FCM. (E) The proportion of CD8 + IFN-γ + CTLs in the tumor tissue of both groups was analyzed by FCM. * p
    Figure Legend Snippet: TRAF6 knockdown attenuates the ability of MDSCs to accelerate tumor progression in tumor-bearing mice. To investigate the effects of TRAF6 on the suppressive activity of MDSCs in vivo , 2 groups of wild-type C57BL/6 mice were s.c. injected with 1 × 10 6 LLC cells and 1 × 10 6 MDSCs transfected with siTRAF6 (siTRAF6 group) or MDSCs transfected with siNC (control group). (A) Tumor growth was constantly monitored. The width “a” and length “b” were measured, and tumor volume was calculated. (B,C) On the 28th day after the inoculation of LLC cells, the mice were sacrificed, and the tumor image and weights were showed in both groups. (D) The proportion of CD4 + IFN-γ + Th1 cells in the tumor tissue of both groups was analyzed by FCM. (E) The proportion of CD8 + IFN-γ + CTLs in the tumor tissue of both groups was analyzed by FCM. * p

    Techniques Used: Mouse Assay, Activity Assay, In Vivo, Injection, Transfection

    35) Product Images from "Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia"

    Article Title: Myeloid-Derived Suppressor Cells Impair Alveolar Macrophages through PD-1 Receptor Ligation during Pneumocystis Pneumonia

    Journal: Infection and Immunity

    doi: 10.1128/IAI.02686-14

    Reduced phagocytic activity in AMs incubated with MDSCs. AMs cocultured with control Gr1BM cells (A) or MDSCs (B) overnight were incubated with fluorescein-conjugated zymosan beads for 1 h. The nuclei of AMs were counterstained with DAPI. (C) The number of zymosan beads phagocytosed by AMs incubated with MDSCs or Gr1BM cells were counted under a confocal microscope. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Reduced phagocytic activity in AMs incubated with MDSCs. AMs cocultured with control Gr1BM cells (A) or MDSCs (B) overnight were incubated with fluorescein-conjugated zymosan beads for 1 h. The nuclei of AMs were counterstained with DAPI. (C) The number of zymosan beads phagocytosed by AMs incubated with MDSCs or Gr1BM cells were counted under a confocal microscope. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Activity Assay, Affinity Magnetic Separation, Incubation, Microscopy

    Decreased PU.1 expression in AMs incubated with MDSCs. AMs from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA of AMs was isolated, and PU.1 mRNA levels were determined by real-time PCR. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with either type of cell was compared to it. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Decreased PU.1 expression in AMs incubated with MDSCs. AMs from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA of AMs was isolated, and PU.1 mRNA levels were determined by real-time PCR. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with either type of cell was compared to it. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Expressing, Affinity Magnetic Separation, Incubation, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction

    Increased PD-1 expression in AMs incubated with MDSDs. AMs isolated from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . (A) After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA was isolated from AMs of each group. PD-1 gene expression was determined by real-time RT-PCR. The level of AMs/Gr1BM was set as 1, and that in AMs/MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) AMs incubated with MDSCs or Gr1BM cells were analyzed for surface PD-1 expression by flow cytometry.
    Figure Legend Snippet: Increased PD-1 expression in AMs incubated with MDSDs. AMs isolated from uninfected mice were cocultured with MDSCs or Gr1BM cells at a ratio of 1:5 for 16 h in a 37°C incubator with 5% CO 2 . (A) After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, total RNA was isolated from AMs of each group. PD-1 gene expression was determined by real-time RT-PCR. The level of AMs/Gr1BM was set as 1, and that in AMs/MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) AMs incubated with MDSCs or Gr1BM cells were analyzed for surface PD-1 expression by flow cytometry.

    Techniques Used: Expressing, Affinity Magnetic Separation, Incubation, Isolation, Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Cytometry

    Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The C T values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Increased histone deacetylation of PU.1 gene in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM cells with anti-Gr-1 antibody-conjugated magnetic microbeads, the AMs were treated with 0.1% formaldehyde to cross-link histone proteins to DNA, lysed, and sonicated to generated chromatin fragments. Chromatin immunoprecipitation was performed using anti-H3K4me3, anti-H3ac, and anti-H3K27me3 antibodies in separate reactions. DNA in the precipitated chromatin was isolated and used as the template for real-time PCR to amplify the 3′URE, 5′URE, and promoter regions of the PU.1 gene. The C T values obtained were used to determine the ratios of percent input of H3K4me3 to H3K27me3 and H3Ac to H3K27me3. Data are presented as means ± SD from three independent experiments.

    Techniques Used: Affinity Magnetic Separation, Incubation, Mouse Assay, Sonication, Generated, Chromatin Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction

    Increased PD-L1 expression in MDSCs from PcP mice. MDSCs (MDSCs/PcP) were isolated from PcP mice at 5 weeks post- Pneumocystis infection. Control Gr1BM cells were isolated from uninfected mice immunosuppressed by weekly injection of anti-CD4 (L3T4) antibody. (A) Total RNA was isolated from the cells, and PD-L1 gene expression was determined by real-time RT-PCR. The average PD-L1 expression level in Gr1BM cells was set as 1, and that in MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) The cells were examined by flow cytometry using anti-Gr-1 and anti-PD-L1 antibodies. The result shown is representative of three independent experiments.
    Figure Legend Snippet: Increased PD-L1 expression in MDSCs from PcP mice. MDSCs (MDSCs/PcP) were isolated from PcP mice at 5 weeks post- Pneumocystis infection. Control Gr1BM cells were isolated from uninfected mice immunosuppressed by weekly injection of anti-CD4 (L3T4) antibody. (A) Total RNA was isolated from the cells, and PD-L1 gene expression was determined by real-time RT-PCR. The average PD-L1 expression level in Gr1BM cells was set as 1, and that in MDSCs was compared to it. Data are presented as means ± SD from three independent experiments. (B) The cells were examined by flow cytometry using anti-Gr-1 and anti-PD-L1 antibodies. The result shown is representative of three independent experiments.

    Techniques Used: Expressing, Mouse Assay, Isolation, Infection, Injection, Quantitative RT-PCR, Flow Cytometry, Cytometry

    Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.
    Figure Legend Snippet: Increased DNA methylation of PU.1 promoter in AMs incubated with MDSCs. AMs from uninfected mice were incubated with MDSCs or Gr1BM cells overnight. After removing MDSCs and Gr1BM, AM genomic DNA was isolated and assessed for CpG methylation by digestion with methylation-dependent and methylation-sensitive restriction enzymes using the EpiTect methyl II enzyme kit (Qiagen). Real-time PCR then was performed to amplify a 100-bp region of the PU.1 promoter. The resulting C T values were entered into the data analysis spreadsheet of the kit to calculate the relative amount of methylated DNA in each sample. Data are presented as means ± SD from three independent experiments.

    Techniques Used: DNA Methylation Assay, Affinity Magnetic Separation, Incubation, Mouse Assay, Isolation, CpG Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    Loss of suppressive effect of MDSCs pretreated with anti-PD-L1 antibody on AMs. A total of 1 × 10 5 AMs were cocultured with 5 × 10 5 MDSCs that were treated with anti-PD-L1 antibody or control IgG. (A) After 16 h of incubation, AMs were isolated, and the PU.1 mRNA levels were determined by real-time RT-PCR. Data are presented as means ± SD from three independent experiments. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with MDSCs that were pretreated with or without anti-PD-L1 antibody was compared to it. (B) Phagocytosis was assayed, and the number of zymosan beads phagocytosed by AMs incubated with MDSCs pretreated with anti-PD-L1 antibody or control IgG was counted under a confocal microscope.
    Figure Legend Snippet: Loss of suppressive effect of MDSCs pretreated with anti-PD-L1 antibody on AMs. A total of 1 × 10 5 AMs were cocultured with 5 × 10 5 MDSCs that were treated with anti-PD-L1 antibody or control IgG. (A) After 16 h of incubation, AMs were isolated, and the PU.1 mRNA levels were determined by real-time RT-PCR. Data are presented as means ± SD from three independent experiments. The level of PU.1 expression in AMs that were not incubated with MDSCs or Gr1BM cells was set as 1, and that in AMs incubated with MDSCs that were pretreated with or without anti-PD-L1 antibody was compared to it. (B) Phagocytosis was assayed, and the number of zymosan beads phagocytosed by AMs incubated with MDSCs pretreated with anti-PD-L1 antibody or control IgG was counted under a confocal microscope.

    Techniques Used: Affinity Magnetic Separation, Incubation, Isolation, Quantitative RT-PCR, Expressing, Microscopy

    36) Product Images from "A highly efficient tumor-infiltrating MDSC differentiation system for discovery of anti-neoplastic targets, which circumvents the need for tumor establishment in mice"

    Article Title: A highly efficient tumor-infiltrating MDSC differentiation system for discovery of anti-neoplastic targets, which circumvents the need for tumor establishment in mice

    Journal: Oncotarget

    doi:

    Ex vivo monocytic MDSCs are precursors of granulocytic MDSCs, which represent the terminal differentiation stage (A) Bar graphs representing the relative proportion of monocytic and granulocytic MDSCs (M-MDSCs, G-MDSCs) in 293T-MDSC and B16-MDSC cultures on the indicated days of differentiation. Relevant statistical comparisons are shown. (B) Ly6G-CD11c expression profiles of purified M-MDSCs on day 5 (density flow cytometry plot on the left), and the same cells incubated in CM 293T for 3 additional days. The percentage of G-MDSCs is shown within the graph. (C) Top left, column graph representing the ratio of the number of cells on days 8 versus 5 in 293T-MDSC and B16-MDSC cultures, to calculate cell growth rate. Top right, dead cell staining with fixable viability stain (FVS) of M-MDSC and G-MDSCs in culture. The proportion of viable cells is shown in the legend. Below left, column graph representing the proportion of Ki67-expressing cells within the G-MDSC and M-MDSC subsets from B16-MDSC cultures, as indicated. Below right, the same but representing Ki67 mean fluorescent intensities (MFI). (D) Top histogram, Ly6G expression on day-five 293T-MDSC cultures incubated for three days with either DC medium, or CM 293T , as indicated within the histogram. Percentages and mean fluorescent intensities are indicated in the legend. The same is represented in the histogram below, but plotting CD11c expression. Relevant statistical comparisons are indicated. *, **, ***, represent significant (P
    Figure Legend Snippet: Ex vivo monocytic MDSCs are precursors of granulocytic MDSCs, which represent the terminal differentiation stage (A) Bar graphs representing the relative proportion of monocytic and granulocytic MDSCs (M-MDSCs, G-MDSCs) in 293T-MDSC and B16-MDSC cultures on the indicated days of differentiation. Relevant statistical comparisons are shown. (B) Ly6G-CD11c expression profiles of purified M-MDSCs on day 5 (density flow cytometry plot on the left), and the same cells incubated in CM 293T for 3 additional days. The percentage of G-MDSCs is shown within the graph. (C) Top left, column graph representing the ratio of the number of cells on days 8 versus 5 in 293T-MDSC and B16-MDSC cultures, to calculate cell growth rate. Top right, dead cell staining with fixable viability stain (FVS) of M-MDSC and G-MDSCs in culture. The proportion of viable cells is shown in the legend. Below left, column graph representing the proportion of Ki67-expressing cells within the G-MDSC and M-MDSC subsets from B16-MDSC cultures, as indicated. Below right, the same but representing Ki67 mean fluorescent intensities (MFI). (D) Top histogram, Ly6G expression on day-five 293T-MDSC cultures incubated for three days with either DC medium, or CM 293T , as indicated within the histogram. Percentages and mean fluorescent intensities are indicated in the legend. The same is represented in the histogram below, but plotting CD11c expression. Relevant statistical comparisons are indicated. *, **, ***, represent significant (P

    Techniques Used: Ex Vivo, Expressing, Purification, Flow Cytometry, Cytometry, Incubation, Staining

    Ex vivo myelopoiesis within a simulated tumor environment differentiates bone marrow cells into large numbers of MDSC-like cells (A) Experimental scheme for MDSC production. On top, lentivector construct used to express GM-CSF and puromycin resistance gene. Below, schematic representation of the generation of MDSC cells. Cancer cell lines are transduced with the lentivector (LV-GMCSF-PuroR). As a result, transduced cells generate conditioning medium (CM) that simulates the tumor microenvironment. Bone marrow (BM) cells from a single tumor-free mouse are cultured in CM for a minimum of 5 days. (B) Left, bar graph representing the number of myeloid cells after a 5 day incubation of BM with the indicated percentages of CM, from 293T or B16F0 cells as indicated. Conventional immature DCs were obtained with recombinant GM-CSF (GM-CSF) following standard protocols. Error bars correspond to standard deviations. Right, percentage of surface expression of the indicated markers, as a function of the increasing percentage of CM 293T . (C) CD11c-MHC II expression profiles are shown as flow cytometry density plots. Percentages of CD11c and MHC II expressing myeloid cells are shown within the graph. Control plots of unstained, immature DCs, 293T- and B16-MDSCs are shown on top of the plots. Myeloid cells were collected on day 8 of differentiation. (D) Same as in c but assessing Ly6C-Ly6G (top density plots) and CD62L-Ly6G (bottom density plots) expression profiles in the indicated myeloid cells. LTR, long terminal repeat; SFFV p, spleen focus-forming virus promoter; moGM-CSF, mouse GM-CSF gene; Puromycin R, puromycin resistance gene; UBI p, ubiquitin promoter; SIN, Self-inactivating LTR.
    Figure Legend Snippet: Ex vivo myelopoiesis within a simulated tumor environment differentiates bone marrow cells into large numbers of MDSC-like cells (A) Experimental scheme for MDSC production. On top, lentivector construct used to express GM-CSF and puromycin resistance gene. Below, schematic representation of the generation of MDSC cells. Cancer cell lines are transduced with the lentivector (LV-GMCSF-PuroR). As a result, transduced cells generate conditioning medium (CM) that simulates the tumor microenvironment. Bone marrow (BM) cells from a single tumor-free mouse are cultured in CM for a minimum of 5 days. (B) Left, bar graph representing the number of myeloid cells after a 5 day incubation of BM with the indicated percentages of CM, from 293T or B16F0 cells as indicated. Conventional immature DCs were obtained with recombinant GM-CSF (GM-CSF) following standard protocols. Error bars correspond to standard deviations. Right, percentage of surface expression of the indicated markers, as a function of the increasing percentage of CM 293T . (C) CD11c-MHC II expression profiles are shown as flow cytometry density plots. Percentages of CD11c and MHC II expressing myeloid cells are shown within the graph. Control plots of unstained, immature DCs, 293T- and B16-MDSCs are shown on top of the plots. Myeloid cells were collected on day 8 of differentiation. (D) Same as in c but assessing Ly6C-Ly6G (top density plots) and CD62L-Ly6G (bottom density plots) expression profiles in the indicated myeloid cells. LTR, long terminal repeat; SFFV p, spleen focus-forming virus promoter; moGM-CSF, mouse GM-CSF gene; Puromycin R, puromycin resistance gene; UBI p, ubiquitin promoter; SIN, Self-inactivating LTR.

    Techniques Used: Ex Vivo, Construct, Transduction, Cell Culture, Incubation, Recombinant, Expressing, Flow Cytometry, Cytometry

    37) Product Images from "Synergistic COX2 induction by IFNγ and TNFα self-limits type-1 immunity in the human tumor microenvironment"

    Article Title: Synergistic COX2 induction by IFNγ and TNFα self-limits type-1 immunity in the human tumor microenvironment

    Journal: Cancer immunology research

    doi: 10.1158/2326-6066.CIR-15-0157

    IFNγ and TNFα are critical mediators of MDSC hyper-activation induced by type-1 immune effector cells. (A) Expression of IDO1, NOS2, IL10, and COX2 in OvCa ascites-isolated MDSCs cultured for 24 h with or without IL18/IFNα-activated
    Figure Legend Snippet: IFNγ and TNFα are critical mediators of MDSC hyper-activation induced by type-1 immune effector cells. (A) Expression of IDO1, NOS2, IL10, and COX2 in OvCa ascites-isolated MDSCs cultured for 24 h with or without IL18/IFNα-activated

    Techniques Used: Activation Assay, Expressing, Isolation, Cell Culture

    Type-1 effector cell-driven hyperactivation of MDSCs requires the intact COX2-PGE 2 axis. (A) Expression of IDO1, NOS2, IL10, COX2, IFNγ, and TNFα in OvCa ascites-isolated MDSCs cultured for 24 h with or without IL18/IFNα-activated
    Figure Legend Snippet: Type-1 effector cell-driven hyperactivation of MDSCs requires the intact COX2-PGE 2 axis. (A) Expression of IDO1, NOS2, IL10, COX2, IFNγ, and TNFα in OvCa ascites-isolated MDSCs cultured for 24 h with or without IL18/IFNα-activated

    Techniques Used: Expressing, Isolation, Cell Culture

    38) Product Images from "The inflammasome component Nlrp3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells"

    Article Title: The inflammasome component Nlrp3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-10-1921

    Superior response to DC vaccines by Nlrp3 −/− mice is due to impaired migration of MDSCs (A) Percentages of MDSCs from the spleen and total number of MDSCs in the TDLN of WT (striped) and Nlrp3 −/− (solid) mice as determined by flow cytometry 14 days after tumor injection. (B) The total number of tumor-associated Gr-1 + , CD11b + cells is increased in WT mice as determined by microscopy. (C) Increased percentage of monocytic MDSCs (left) and granulocytic MDSCs (right) from the tumor of WT (striped) compared to Nlrp3 −/− (solid) mice as determined by flow cytometry. (D) Survival curves of WT and Nlrp3 −/− mice treated with anti-Gr-1 antibody (dotted arrows) and DC vaccine (solid arrows). (E) Contour plots on the left show phenotype of MDSCs isolated from the spleens of WT (upper) and Nlrp3 −/− (lower) mice prior to injection into Nlrp3 −/− mice. Dot plots on the right show cells recovered from B16 tumors in vaccinated Nlrp3 −/− mice. Upper panels are WT EGFP + MDSCs and lower panels are Nlrp3 −/− EGFP + MDSCs. Values represent the average number of cells recovered from each tumor.
    Figure Legend Snippet: Superior response to DC vaccines by Nlrp3 −/− mice is due to impaired migration of MDSCs (A) Percentages of MDSCs from the spleen and total number of MDSCs in the TDLN of WT (striped) and Nlrp3 −/− (solid) mice as determined by flow cytometry 14 days after tumor injection. (B) The total number of tumor-associated Gr-1 + , CD11b + cells is increased in WT mice as determined by microscopy. (C) Increased percentage of monocytic MDSCs (left) and granulocytic MDSCs (right) from the tumor of WT (striped) compared to Nlrp3 −/− (solid) mice as determined by flow cytometry. (D) Survival curves of WT and Nlrp3 −/− mice treated with anti-Gr-1 antibody (dotted arrows) and DC vaccine (solid arrows). (E) Contour plots on the left show phenotype of MDSCs isolated from the spleens of WT (upper) and Nlrp3 −/− (lower) mice prior to injection into Nlrp3 −/− mice. Dot plots on the right show cells recovered from B16 tumors in vaccinated Nlrp3 −/− mice. Upper panels are WT EGFP + MDSCs and lower panels are Nlrp3 −/− EGFP + MDSCs. Values represent the average number of cells recovered from each tumor.

    Techniques Used: Mouse Assay, Migration, Flow Cytometry, Cytometry, Injection, Microscopy, Isolation

    39) Product Images from "Ceramide activates lysosomal cathepsin B and cathepsin D to attenuate autophagy and induces ER stress to suppress myeloid-derived suppressor cells"

    Article Title: Ceramide activates lysosomal cathepsin B and cathepsin D to attenuate autophagy and induces ER stress to suppress myeloid-derived suppressor cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13438

    LCL521 suppresses MDSC accumulation in tumor-bearing mice in vivo A. CMS4-met tumor cells were injected into BALB/c mice sc. Spleen, blood and bone marrow (BM) were collected from tumor-free and tumor-bearing mice thirty days after tumor cell injection. Cells were stained with CD11b- and Gr1-specific mAbs and analyzed by flow cytometry. Shown are representative plots of MDSCs from one mouse of three mice in each group. B. The CD11b + Gr1 + MDSCs were quantified as percentage of total cells. Column: mean, Bar: SD. C. CD3 + T cells were purified from spleens of tumor-free BALB/c mice, and MDSCs were purified from spleens of tumor-bearing mice as described in the materials and methods. The purified cells were analyzed by flow cytometry for purity. The purities of the purified cells are indicated in the plots. D. The purified CD3 + T cells (1.5 x 10 5 cells/well) were labelled with CFSE, and then cultured in anti-CD3 and anti-CD28-coated plates in the absence or presence of purified MDSCs (1x10 6 cells/well) for 3 days. Cell culture mixtures were stained with anti-CD11b and anti-Gr1 mAbs. CD11b - Gr1 - cells were gated (left panel) and analyzed for CFSE intensity (right panel. The unstimulated (resting), and stimulated T cells in the absence (stimulated) or presence (stimulated + MDSCs) of MDSCs were overlayed for CFSE intensity. Shown is a representative images of CSFE intensity of the three groups of cells (right panel). Cells were quantified based on CFSE intensity and presented in the bottom panel. E. Mouse tumor cell line CMS4-met was cultured in the presence of LCL521 at the indicated doses for approximately 24 hours. Cells were stained with PI and analyzed for cell death. % cell death is calculated as % PI + cells. F. CMS4-met tumor cells were injected into BALB/c mice. Thirty days later, tumor-bearing mice were treated with LCL521 at a dose of 75 mg/kg body weight every two days twice. Shown are tumor volumes in control and LCL521-treated mice. G. Spleens and tumors were collected from control and LCL521-treated tumor-bearing mice. Spleen cells were stained with CD11b- and Gr1-specific mAbs and analyzed by flow cytometry. Tumors were digested with collagenase solutions to make single cell suspension. The tumor tissue single cell suspension was then stained with CD11b- and Gr1-specific mAbs and analyzed by flow cytometry. Shown are representative results of one of three pairs of untreated and LCL521-treated tumor-bearing mice. The % CD11b + Gr1 + MDSCs were quantified and presented at the right. Column: Mean, Bar: SD.
    Figure Legend Snippet: LCL521 suppresses MDSC accumulation in tumor-bearing mice in vivo A. CMS4-met tumor cells were injected into BALB/c mice sc. Spleen, blood and bone marrow (BM) were collected from tumor-free and tumor-bearing mice thirty days after tumor cell injection. Cells were stained with CD11b- and Gr1-specific mAbs and analyzed by flow cytometry. Shown are representative plots of MDSCs from one mouse of three mice in each group. B. The CD11b + Gr1 + MDSCs were quantified as percentage of total cells. Column: mean, Bar: SD. C. CD3 + T cells were purified from spleens of tumor-free BALB/c mice, and MDSCs were purified from spleens of tumor-bearing mice as described in the materials and methods. The purified cells were analyzed by flow cytometry for purity. The purities of the purified cells are indicated in the plots. D. The purified CD3 + T cells (1.5 x 10 5 cells/well) were labelled with CFSE, and then cultured in anti-CD3 and anti-CD28-coated plates in the absence or presence of purified MDSCs (1x10 6 cells/well) for 3 days. Cell culture mixtures were stained with anti-CD11b and anti-Gr1 mAbs. CD11b - Gr1 - cells were gated (left panel) and analyzed for CFSE intensity (right panel. The unstimulated (resting), and stimulated T cells in the absence (stimulated) or presence (stimulated + MDSCs) of MDSCs were overlayed for CFSE intensity. Shown is a representative images of CSFE intensity of the three groups of cells (right panel). Cells were quantified based on CFSE intensity and presented in the bottom panel. E. Mouse tumor cell line CMS4-met was cultured in the presence of LCL521 at the indicated doses for approximately 24 hours. Cells were stained with PI and analyzed for cell death. % cell death is calculated as % PI + cells. F. CMS4-met tumor cells were injected into BALB/c mice. Thirty days later, tumor-bearing mice were treated with LCL521 at a dose of 75 mg/kg body weight every two days twice. Shown are tumor volumes in control and LCL521-treated mice. G. Spleens and tumors were collected from control and LCL521-treated tumor-bearing mice. Spleen cells were stained with CD11b- and Gr1-specific mAbs and analyzed by flow cytometry. Tumors were digested with collagenase solutions to make single cell suspension. The tumor tissue single cell suspension was then stained with CD11b- and Gr1-specific mAbs and analyzed by flow cytometry. Shown are representative results of one of three pairs of untreated and LCL521-treated tumor-bearing mice. The % CD11b + Gr1 + MDSCs were quantified and presented at the right. Column: Mean, Bar: SD.

    Techniques Used: Mouse Assay, In Vivo, Injection, Staining, Flow Cytometry, Cytometry, Purification, Cell Culture

    40) Product Images from "SOCS3 Suppression Promoted the Recruitment of CD11b+Gr-1−F4/80−MHCII− Early-Stage Myeloid-Derived Suppressor Cells and Accelerated Interleukin-6-Related Tumor Invasion via Affecting Myeloid Differentiation in Breast Cancer"

    Article Title: SOCS3 Suppression Promoted the Recruitment of CD11b+Gr-1−F4/80−MHCII− Early-Stage Myeloid-Derived Suppressor Cells and Accelerated Interleukin-6-Related Tumor Invasion via Affecting Myeloid Differentiation in Breast Cancer

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01699

    Reversible myeloid differentiation blockage of early-stage MDSCs (e-MDSCs) is interleukin-6 (IL-6) dependent. Bone marrow (BM) cells isolated from tumor-free BALB/c mice were cocultured with rIL-6, tumor cell supernatant, and tumor cells to induce e-MDSCs in vitro . And BM cells treated with GM-CSF (10 ng/mL) were regarded as normal control. (A) The percentage of CD11b + Gr-1 − was detected by flow cytometry. (B) The percentage of F4/80 + and MHCII + cells in CD11b + Gr-1 − myeloid-derived suppressor cells (MDSCs) was detected using flow cytometry. BM cells were isolated from tumor-free mice and cultured with GM-CSF for 3 days with or without 4T1 cells. IL-6 receptor Ab was utilized to block IL-6 downstream signaling. (C) The percentage of CD11b + Gr-1 − , CD11b + Gr-1 + , F4/80 + , and MHCII + cells was detected using flow cytometry. (D) Primary CD11b + Gr-1 − F4/80 − MHCII − cells were isolated using the BD FACSAria™II cell sorter and induced with GM-CSF. The percentage of CD11b + Gr-1 − , CD11b + Gr-1 + , Ly6G (1A8) + cells, F4/80 + cells, and MHCII + cells was detected by flow cytometry (* P
    Figure Legend Snippet: Reversible myeloid differentiation blockage of early-stage MDSCs (e-MDSCs) is interleukin-6 (IL-6) dependent. Bone marrow (BM) cells isolated from tumor-free BALB/c mice were cocultured with rIL-6, tumor cell supernatant, and tumor cells to induce e-MDSCs in vitro . And BM cells treated with GM-CSF (10 ng/mL) were regarded as normal control. (A) The percentage of CD11b + Gr-1 − was detected by flow cytometry. (B) The percentage of F4/80 + and MHCII + cells in CD11b + Gr-1 − myeloid-derived suppressor cells (MDSCs) was detected using flow cytometry. BM cells were isolated from tumor-free mice and cultured with GM-CSF for 3 days with or without 4T1 cells. IL-6 receptor Ab was utilized to block IL-6 downstream signaling. (C) The percentage of CD11b + Gr-1 − , CD11b + Gr-1 + , F4/80 + , and MHCII + cells was detected using flow cytometry. (D) Primary CD11b + Gr-1 − F4/80 − MHCII − cells were isolated using the BD FACSAria™II cell sorter and induced with GM-CSF. The percentage of CD11b + Gr-1 − , CD11b + Gr-1 + , Ly6G (1A8) + cells, F4/80 + cells, and MHCII + cells was detected by flow cytometry (* P

    Techniques Used: Isolation, Mouse Assay, In Vitro, Flow Cytometry, Cytometry, Derivative Assay, Cell Culture, Blocking Assay

    Blocking STAT3 activation or IL-6 receptor (IL-6R) inhibits myeloid-derived suppressor cell (MDSC) development and tumor metastasis in the 4T1 mouse mammary tumor model. The mammary fat pads of BALB/c mice were injected with 4T1 mouse mammary carcinoma cells. After 5 days of tumor implantation, mice were treated intraperitoneally with JSI-124 or IL-6R mAb at for 13 days. (A) Tumor sizes in mice were monitored during JSI-124 or IL-6R mAb treatment. On day 18, mice were sacrificed and the tumors and spleens were separated. (B) Pulmonary metastatic nodules were counted based on H E-stained slides. (C) Tumor-infiltrating CD45 + cells were gated, and MDSCs infiltration in tumors treated with JSI-124 or IL-6R mAb was detected by flow cytometry. (D) SOCS1 and SOCS3 in MDSCs treated with JSI-124 or IL-6R Ab were determined by RT-PCR. (E) Western blotting was performed to detect the expression of p-STAT3, p-STAT1, SOCS1, and SOCS3 in JSI-124 or IL-6R mAb-treated early-stage MDSCs (e-MDSCs). (F) The expression of p-STAT3, p-STAT1, SOCS1, and SOCS3 in JSI-124 or IL-6R mAb-treated e-MDSCs was confirmed by intracellular flow cytometry. And the expression levels were presented by mean fluorescence intensity. JSI-124 and IL-6R mAb-treated CD11b + Gr-1 − e-MDSCs were isolated from tumors and cocultured with T cells. T cell proliferation (G) and apoptosis (H) were determined by flow cytometry. (I) Secreted cytokines by T cells were determined by enzyme-linked immunosorbent assay (* P
    Figure Legend Snippet: Blocking STAT3 activation or IL-6 receptor (IL-6R) inhibits myeloid-derived suppressor cell (MDSC) development and tumor metastasis in the 4T1 mouse mammary tumor model. The mammary fat pads of BALB/c mice were injected with 4T1 mouse mammary carcinoma cells. After 5 days of tumor implantation, mice were treated intraperitoneally with JSI-124 or IL-6R mAb at for 13 days. (A) Tumor sizes in mice were monitored during JSI-124 or IL-6R mAb treatment. On day 18, mice were sacrificed and the tumors and spleens were separated. (B) Pulmonary metastatic nodules were counted based on H E-stained slides. (C) Tumor-infiltrating CD45 + cells were gated, and MDSCs infiltration in tumors treated with JSI-124 or IL-6R mAb was detected by flow cytometry. (D) SOCS1 and SOCS3 in MDSCs treated with JSI-124 or IL-6R Ab were determined by RT-PCR. (E) Western blotting was performed to detect the expression of p-STAT3, p-STAT1, SOCS1, and SOCS3 in JSI-124 or IL-6R mAb-treated early-stage MDSCs (e-MDSCs). (F) The expression of p-STAT3, p-STAT1, SOCS1, and SOCS3 in JSI-124 or IL-6R mAb-treated e-MDSCs was confirmed by intracellular flow cytometry. And the expression levels were presented by mean fluorescence intensity. JSI-124 and IL-6R mAb-treated CD11b + Gr-1 − e-MDSCs were isolated from tumors and cocultured with T cells. T cell proliferation (G) and apoptosis (H) were determined by flow cytometry. (I) Secreted cytokines by T cells were determined by enzyme-linked immunosorbent assay (* P

    Techniques Used: Blocking Assay, Activation Assay, Derivative Assay, Mouse Assay, Injection, Tumor Implantation, Staining, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Fluorescence, Isolation, Enzyme-linked Immunosorbent Assay

    Tumor-derived interleukin-6 (IL-6) promotes the accumulation of CD11b + Gr-1 − F4/80 − MHCII − early-stage MDSCs (e-MDSCs) in tumors. Infiltrated CD45 + cells were gated. (A) The percentage of myeloid-derived suppressor cells (MDSCs) in normal spleen, tumors, and spleens resulting from 4T1 mammary cancer cells detected by flow cytometry. (B) The percentage of the indicated cell subsets in tumors and spleens from mice bearing different IL-6-expressing tumors was determined by flow cytometry. CD11b + Gr-1 − e-MDSCs from spleens and tumors were isolated, and the percentage of CD11b + Gr-1 − F4/80 + MHCII − cells and CD11b + Gr-1 − F4/80 − MHCII + cells in spleens (C) and tumors (D) was determined using flow cytometry. (E) The correlation analysis between tumor volumes and the percentage of tumor-infiltrating CD11b + Gr-1 − F4/80 − MHCII − e-MDSCs or CD11b + Gr-1 + MDSCs ( R 2 = 0.4491 vs. R 2 = 0.1482, P
    Figure Legend Snippet: Tumor-derived interleukin-6 (IL-6) promotes the accumulation of CD11b + Gr-1 − F4/80 − MHCII − early-stage MDSCs (e-MDSCs) in tumors. Infiltrated CD45 + cells were gated. (A) The percentage of myeloid-derived suppressor cells (MDSCs) in normal spleen, tumors, and spleens resulting from 4T1 mammary cancer cells detected by flow cytometry. (B) The percentage of the indicated cell subsets in tumors and spleens from mice bearing different IL-6-expressing tumors was determined by flow cytometry. CD11b + Gr-1 − e-MDSCs from spleens and tumors were isolated, and the percentage of CD11b + Gr-1 − F4/80 + MHCII − cells and CD11b + Gr-1 − F4/80 − MHCII + cells in spleens (C) and tumors (D) was determined using flow cytometry. (E) The correlation analysis between tumor volumes and the percentage of tumor-infiltrating CD11b + Gr-1 − F4/80 − MHCII − e-MDSCs or CD11b + Gr-1 + MDSCs ( R 2 = 0.4491 vs. R 2 = 0.1482, P

    Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Mouse Assay, Expressing, Isolation

    CD11b + Gr-1 − F4/80 − MHCII − early-stage MDSCs (e-MDSCs) could switch to CD11b + Gr-1 + myeloid-derived suppressor cells (MDSCs) and exerted stronger T cell immunosuppression. (A) BrdU (50 mg/kg) was injected in 4T1 WT -bearing mice by tail vein. Seventy-two hours later, CD11b + Gr-1 − MDSCs and CD11b + Gr-1 + MDSCs labeled with BrdU in tumors were detected by flow cytometry. (B) CD11b + Gr-1 − cells were isolated from 4T1 WT tumors and labeled with CSFE (0.5 µM) for 20 min in vitro . Then, the labeled cells were transferred back to normal BALB/c mice. After 96 h, CD11b + Gr-1 + cells in spleens were gated, and cells labeled with CSFE were detected. (C) CD11b + Gr-1 − MDSCs were separated from 4T1 WT tumors and transferred into mice bearing 4T1 IL-6low tumors (2 × 10 6 /twice/week), and the size of tumors was monitored. CD11b + Gr-1 + MDSC or CD11b + Gr-1 − e-MDSC was isolated from tumors and incubated with T cells in vitro . (D) The proliferation of T cells was detected by flow cytometry for Brdu + cells. (E) Apoptotic T cells (Annexin V + PI − ) were determined by flow cytometry. tMDSCs WT , tMDSCs NC , or tMDSCs IL-6low were isolated from tumors and cocultured with T cells, respectively. (F) The proliferation of T cells was detected by flow cytometry for Brdu + cells. (G) Apoptotic T cells (Annexin V + PI − ) were determined by flow cytometry. (H) IFN-γ, IL-10, and TGF-β secreted by T cells were detected using enzyme-linked immunosorbent assay (ELISA). sMDSCs WT , sMDSCs NC , or sMDSCs IL-6low were isolated from spleens and cocultured with T cells, respectively. (I) The proliferation of T cells was detected by flow cytometry for Brdu + cells. (J) Apoptotic T cells (Annexin V + PI − ) were determined by flow cytometry. (K) IFN-γ, IL-10, and TGF-β secreted by the T cells were detected using ELISA (** P
    Figure Legend Snippet: CD11b + Gr-1 − F4/80 − MHCII − early-stage MDSCs (e-MDSCs) could switch to CD11b + Gr-1 + myeloid-derived suppressor cells (MDSCs) and exerted stronger T cell immunosuppression. (A) BrdU (50 mg/kg) was injected in 4T1 WT -bearing mice by tail vein. Seventy-two hours later, CD11b + Gr-1 − MDSCs and CD11b + Gr-1 + MDSCs labeled with BrdU in tumors were detected by flow cytometry. (B) CD11b + Gr-1 − cells were isolated from 4T1 WT tumors and labeled with CSFE (0.5 µM) for 20 min in vitro . Then, the labeled cells were transferred back to normal BALB/c mice. After 96 h, CD11b + Gr-1 + cells in spleens were gated, and cells labeled with CSFE were detected. (C) CD11b + Gr-1 − MDSCs were separated from 4T1 WT tumors and transferred into mice bearing 4T1 IL-6low tumors (2 × 10 6 /twice/week), and the size of tumors was monitored. CD11b + Gr-1 + MDSC or CD11b + Gr-1 − e-MDSC was isolated from tumors and incubated with T cells in vitro . (D) The proliferation of T cells was detected by flow cytometry for Brdu + cells. (E) Apoptotic T cells (Annexin V + PI − ) were determined by flow cytometry. tMDSCs WT , tMDSCs NC , or tMDSCs IL-6low were isolated from tumors and cocultured with T cells, respectively. (F) The proliferation of T cells was detected by flow cytometry for Brdu + cells. (G) Apoptotic T cells (Annexin V + PI − ) were determined by flow cytometry. (H) IFN-γ, IL-10, and TGF-β secreted by T cells were detected using enzyme-linked immunosorbent assay (ELISA). sMDSCs WT , sMDSCs NC , or sMDSCs IL-6low were isolated from spleens and cocultured with T cells, respectively. (I) The proliferation of T cells was detected by flow cytometry for Brdu + cells. (J) Apoptotic T cells (Annexin V + PI − ) were determined by flow cytometry. (K) IFN-γ, IL-10, and TGF-β secreted by the T cells were detected using ELISA (** P

    Techniques Used: Derivative Assay, Injection, Mouse Assay, Labeling, Flow Cytometry, Cytometry, Isolation, In Vitro, Incubation, Enzyme-linked Immunosorbent Assay

    Interleukin-6 (IL-6)-mediated suppressed expression of SOCS3 resulting in the hyperactivation of the JAK/STAT pathway in CD11b + Gr-1 − early-stage MDSCs (e-MDSCs). Western blotting was performed to detect the JAK/STAT (A) and MAPK (B) on the whole-cell extracts of primary CD11b + Gr-1 − e-MDSCs isolated from different IL-6-expressing tumors. (C) The protein expression of SOCS1–3 in primary CD11b + Gr-1 − e-MDSCs. (D) The mRNA of SOCS1–3 in primary CD11b + Gr-1 − e-MDSCs was examined by RT-PCR. (E) Bone marrow (BM) cells were treated with 4T1 NC and 4T1 IL-6low separately to induce iMDSCs NC and iMDSCs IL-6low . The activation status of the JAK/STAT pathway downstream of IL-6 signaling in induce MDSCs (iMDSCs) was detected at different time point. BM cells exposed to IL-6 (40 ng/mL) for 15 min were as control. The levels of phosphorylated JAK1 (p-JAK1), p-JAK2, p-TYK2, p-STAT1, p-STAT3, p-P38, p-ERK, and p-JNK were compared using the density ratio of phosphorylated protein to total protein. The levels of the suppressor of cytokine signaling (SOCS) protein were compared using the density ratio of the indicated protein to β-actin (* P
    Figure Legend Snippet: Interleukin-6 (IL-6)-mediated suppressed expression of SOCS3 resulting in the hyperactivation of the JAK/STAT pathway in CD11b + Gr-1 − early-stage MDSCs (e-MDSCs). Western blotting was performed to detect the JAK/STAT (A) and MAPK (B) on the whole-cell extracts of primary CD11b + Gr-1 − e-MDSCs isolated from different IL-6-expressing tumors. (C) The protein expression of SOCS1–3 in primary CD11b + Gr-1 − e-MDSCs. (D) The mRNA of SOCS1–3 in primary CD11b + Gr-1 − e-MDSCs was examined by RT-PCR. (E) Bone marrow (BM) cells were treated with 4T1 NC and 4T1 IL-6low separately to induce iMDSCs NC and iMDSCs IL-6low . The activation status of the JAK/STAT pathway downstream of IL-6 signaling in induce MDSCs (iMDSCs) was detected at different time point. BM cells exposed to IL-6 (40 ng/mL) for 15 min were as control. The levels of phosphorylated JAK1 (p-JAK1), p-JAK2, p-TYK2, p-STAT1, p-STAT3, p-P38, p-ERK, and p-JNK were compared using the density ratio of phosphorylated protein to total protein. The levels of the suppressor of cytokine signaling (SOCS) protein were compared using the density ratio of the indicated protein to β-actin (* P

    Techniques Used: Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Activation Assay

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    Magnetic Cell Separation:

    Article Title: Blocking Migration of Polymorphonuclear Myeloid-Derived Suppressor Cells Inhibits Mouse Melanoma Progression
    Article Snippet: Briefly, 2.5 × 106 C57BL/6 BM cells were cultured in RPMI-1640 with GlutaMAX supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, 1 mM MEM non-essential amino acids (all Thermo Fisher Scientific, Waltham, MA, USA), 40 ng/mL GM-CSF, and 40 ng/mL IL-6 (PeproTech, Inc., Rocky Hill, NJ, USA) for 4 days. .. Then PMN-MDSC and M-MDSC were isolated from the culture using magnetic-activated cell sorting (MACS® , Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany, Cat. 130-094-538, cell purity was more than 90%) according to the manufacturer’s instructions. .. NK cells were isolated from the spleens of normal C57BL/6 mice using MACS® (MACS®, Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany, Cat. 130-115-818, the purity of isolated cell was more than 90%) according to the manufacturer’s instructions and stimulated with IL-2 (0.1 ng/mL), IL-12 (1 ng/mL), IL-18 p80 (20 ng/mL) (all from PeproTech, Inc., Rocky Hill, NJ, USA) for 3 h. Afterwards, NK cells were washed and cultured alone or with PMN-MDSC or M-MDSC for 4 h followed by the staining of NK cells for intracellular IL-16 and IFN-γ and measurement by flow cytometry.

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    Miltenyi Biotec mdsc isolation kit
    MIF-deficiency or small molecule inhibition reduces splenic <t>MDSC</t> immune suppression in tumor bearing mice GR-1 hi <t>Ly-6G</t> + granulocytic and GR-1 dim Ly-6G − monocytic MDSCs were isolated from spleens of MIF +/+ and MIF −/− C57BL/6 mice bearing a s.c. melanoma tumor (n = 10). (A, B) Cells were either untreated or (C, D) pre-treated with 4-IPP (50 μm) or DMSO (vehicle control). Cells were cultured in triplicates at the indicated cell number ratios for 16 hours. Splenocytes from OT-1 mice were then added to wells containing MDSCs in presence of the ovalbumin (200 μg/ml) and cultured for additional 72 hours. Eighteen hours before harvesting, co-cultures were pulsed with [ 3 H]-thymidine. Data represents the average ± SD of triplicate samples representative of two independent experiments. P values = *, p≤0.05; **, p≤0.005.
    Mdsc Isolation Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mdsc isolation kit/product/Miltenyi Biotec
    Average 96 stars, based on 1 article reviews
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    97
    Miltenyi Biotec gr 1 cd11b mdsc
    <t>Gr-1</t> + <t>CD11b</t> + cells coinjected with Pan02 tumor cells facilitate more rapid tumor engraftment
    Gr 1 Cd11b Mdsc, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gr 1 cd11b mdsc/product/Miltenyi Biotec
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gr 1 cd11b mdsc - by Bioz Stars, 2021-07
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    MIF-deficiency or small molecule inhibition reduces splenic MDSC immune suppression in tumor bearing mice GR-1 hi Ly-6G + granulocytic and GR-1 dim Ly-6G − monocytic MDSCs were isolated from spleens of MIF +/+ and MIF −/− C57BL/6 mice bearing a s.c. melanoma tumor (n = 10). (A, B) Cells were either untreated or (C, D) pre-treated with 4-IPP (50 μm) or DMSO (vehicle control). Cells were cultured in triplicates at the indicated cell number ratios for 16 hours. Splenocytes from OT-1 mice were then added to wells containing MDSCs in presence of the ovalbumin (200 μg/ml) and cultured for additional 72 hours. Eighteen hours before harvesting, co-cultures were pulsed with [ 3 H]-thymidine. Data represents the average ± SD of triplicate samples representative of two independent experiments. P values = *, p≤0.05; **, p≤0.005.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Control of tumor-associated macrophage alternative activation by MIF

    doi: 10.4049/jimmunol.1201650

    Figure Lengend Snippet: MIF-deficiency or small molecule inhibition reduces splenic MDSC immune suppression in tumor bearing mice GR-1 hi Ly-6G + granulocytic and GR-1 dim Ly-6G − monocytic MDSCs were isolated from spleens of MIF +/+ and MIF −/− C57BL/6 mice bearing a s.c. melanoma tumor (n = 10). (A, B) Cells were either untreated or (C, D) pre-treated with 4-IPP (50 μm) or DMSO (vehicle control). Cells were cultured in triplicates at the indicated cell number ratios for 16 hours. Splenocytes from OT-1 mice were then added to wells containing MDSCs in presence of the ovalbumin (200 μg/ml) and cultured for additional 72 hours. Eighteen hours before harvesting, co-cultures were pulsed with [ 3 H]-thymidine. Data represents the average ± SD of triplicate samples representative of two independent experiments. P values = *, p≤0.05; **, p≤0.005.

    Article Snippet: GR-1hi Ly-6G+ granulocytic and GR-1dim Ly-6G− monocytic MDSCs were isolated from the spleens using the MDSC isolation kit (Miltenyi Biotec).

    Techniques: Inhibition, Mouse Assay, Isolation, Cell Culture

    gp130/STAT3 signaling and MDSCs in colitis. WHAT IS CURRENT KNOWLEDGE Myeloid derived suppressor cells (MDSCs) are a heterogeneous group of immature cells that includes precursors of macrophages, granulocytes, dendritic cells and myeloid cells at earlier stages of differentiation. They are defined by their co-expression of Gr-1 and CD11b. MDSCs regulate immune responses and tissue repair in healthy individuals and MDSCs rapidly expands during inflammation, infection and cancer. It is predominantly (70-90%) the granulocytic subset of MDSCs (G-MDSC) that expands, which has a CD11b + Ly6G + Ly6C low phenotype. G-MDSCs have increased activity of signal transducer and activator of transcription 3 (STAT3), which is activated by binding of cytokines to the glycoprotein (gp)130 receptor and regulates the expansion and survival of G-MDSC subsets. Activation of G-MDSCs leads to the upregulation of arginase 1 (ARG1), which causes the suppression of T cell responses, and to the increased production of other suppressive cytokines, such as interleukin (IL-)10 and transforming growth factor-β (TGF-β). WHAT IS NEW HERE gp130 757F/F mice with a phenylalanine (F) for tyrosine (Y) substitution at the 757 residue of the gp130 receptor show sustained STAT3 signalling due to the inhibition of negative feedback loops, which normally activate alternative signalling pathways downstream of gp130. Experimental colitis, induced with 3% dextran sulphate sodium (DSS) in drinking water in gp130 757F/F mice and compared to water (H 2 O)-treated gp130 757F/F and wildtype (gp130 757Y/Y ) mice, resulted in reduced disease severity and amelioration of intestinal inflammation with expansion of G-MDSCs in the colon, increased arginase 1 expression and activity and increased expression of the protective cytokines IL-19 and IL-33 in the colon. LysMcre/Stat3 flox mice with myeloid-specific STAT3 deficiency, were not protected from DSS-induced colitis and colons showed reduced numbers of G-MDSCs, increased gene expression of pro-inflammatory interferon γ (IFNγ) and inducible nitric oxide synthase (iNos), and decreased expression of IL-19 and IL-33. Additionally, G-MDSCs of LysMcre/Stat3 flox mice produced significantly less anti-inflammatory cytokines, such as IL-4, IL-10 and IL-13, compared to gp130 757Y/Y mice. We suggest that the resistance to DSS-induced colitis in gp130 757F/F mice is via myeloid-cell specific STAT3 activation, MDSC expansion in the colon and increased production of suppressive and protective cytokines.

    Journal: Scientific Reports

    Article Title: Altered gp130 signalling ameliorates experimental colitis via myeloid cell-specific STAT3 activation and myeloid-derived suppressor cells

    doi: 10.1038/srep20584

    Figure Lengend Snippet: gp130/STAT3 signaling and MDSCs in colitis. WHAT IS CURRENT KNOWLEDGE Myeloid derived suppressor cells (MDSCs) are a heterogeneous group of immature cells that includes precursors of macrophages, granulocytes, dendritic cells and myeloid cells at earlier stages of differentiation. They are defined by their co-expression of Gr-1 and CD11b. MDSCs regulate immune responses and tissue repair in healthy individuals and MDSCs rapidly expands during inflammation, infection and cancer. It is predominantly (70-90%) the granulocytic subset of MDSCs (G-MDSC) that expands, which has a CD11b + Ly6G + Ly6C low phenotype. G-MDSCs have increased activity of signal transducer and activator of transcription 3 (STAT3), which is activated by binding of cytokines to the glycoprotein (gp)130 receptor and regulates the expansion and survival of G-MDSC subsets. Activation of G-MDSCs leads to the upregulation of arginase 1 (ARG1), which causes the suppression of T cell responses, and to the increased production of other suppressive cytokines, such as interleukin (IL-)10 and transforming growth factor-β (TGF-β). WHAT IS NEW HERE gp130 757F/F mice with a phenylalanine (F) for tyrosine (Y) substitution at the 757 residue of the gp130 receptor show sustained STAT3 signalling due to the inhibition of negative feedback loops, which normally activate alternative signalling pathways downstream of gp130. Experimental colitis, induced with 3% dextran sulphate sodium (DSS) in drinking water in gp130 757F/F mice and compared to water (H 2 O)-treated gp130 757F/F and wildtype (gp130 757Y/Y ) mice, resulted in reduced disease severity and amelioration of intestinal inflammation with expansion of G-MDSCs in the colon, increased arginase 1 expression and activity and increased expression of the protective cytokines IL-19 and IL-33 in the colon. LysMcre/Stat3 flox mice with myeloid-specific STAT3 deficiency, were not protected from DSS-induced colitis and colons showed reduced numbers of G-MDSCs, increased gene expression of pro-inflammatory interferon γ (IFNγ) and inducible nitric oxide synthase (iNos), and decreased expression of IL-19 and IL-33. Additionally, G-MDSCs of LysMcre/Stat3 flox mice produced significantly less anti-inflammatory cytokines, such as IL-4, IL-10 and IL-13, compared to gp130 757Y/Y mice. We suggest that the resistance to DSS-induced colitis in gp130 757F/F mice is via myeloid-cell specific STAT3 activation, MDSC expansion in the colon and increased production of suppressive and protective cytokines.

    Article Snippet: Granulocytic MDSCs were isolated by indirect magnetic labelling of Gr1high Ly6G+ cells with Anti-Ly6G-Biotin and Anti-Biotin MicroBeads using a mouse myeloid-derived suppressor cell kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and subsequent magnetic separation using LS columns and a MidiMACS and QuadroMACS separator (all Miltenyi Biotec, Bergisch Gladbach, Germany).

    Techniques: Derivative Assay, Expressing, Infection, Activity Assay, Binding Assay, Activation Assay, Mouse Assay, Inhibition, Produced

    Inflammasome-dependent IL-1 family cytokines and alarmins induce MDSCs in vitro. a IL-1 family members IL-1β, Il-1α, and IL-18 are able to induce MDSCs. PBMCs were isolated from heparinized fresh blood from healthy donors using Ficoll density gradient sedimentation. MDSCs were induced by incubating PBMCs (5 × 10 5 /ml) with complete medium only (medium control), 10 ng/ml GM-CSF (positive control), or different concentrations (1 ng/ml to 1 µg/ml) of the respective IL-1 family cytokines. MDSCs were determined as SSC high CD33 + CD14 - cells. The number of MDSCs as a percentage of all cells in medium-only cultures (mean 1.69%; median 1.3%) were set to 1-fold for every single experiment. The MDSC induction due to specific stimuli is presented as x-fold compared to medium control (mean ± SEM), and differences compared to controls were analyzed by a one-sample t test. b S100 proteins are able to slightly induce MDSCs. General culture conditions were as described under a . S100 proteins S100A7, S100A8, S100A9, or S100A12 were added in different concentrations ranging from 1 ng/ml to 1 µg/ml. The x-fold induction of MDSCs compared to medium control conditions is depicted as means ± SEM, and differences were analyzed by a one-sample t test. c Calprotectin is able to induce MDSCs. General culture conditions were as described under a . Calprotectin was added in different concentrations ranging from 1 ng/ml to 2 µg/ml. The x-fold induction of MDSCs compared to medium control conditions is depicted as means ± SEM, and differences were analyzed by a one-sample t test. d SAA is able to induce MDSCs. General culture conditions were as described in a . Recombinant hybrid SAA was added in different concentrations ranging from 0.1 ng/ml to 2 µg/ml. The x-fold induction of MDSCs compared to medium control conditions is depicted as means ± SEM, and differences were analyzed by a one-sample t test. * p

    Journal: Journal of Innate Immunity

    Article Title: Induction of Myeloid-Derived Suppressor Cells in Cryopyrin-Associated Periodic Syndromes

    doi: 10.1159/000446615

    Figure Lengend Snippet: Inflammasome-dependent IL-1 family cytokines and alarmins induce MDSCs in vitro. a IL-1 family members IL-1β, Il-1α, and IL-18 are able to induce MDSCs. PBMCs were isolated from heparinized fresh blood from healthy donors using Ficoll density gradient sedimentation. MDSCs were induced by incubating PBMCs (5 × 10 5 /ml) with complete medium only (medium control), 10 ng/ml GM-CSF (positive control), or different concentrations (1 ng/ml to 1 µg/ml) of the respective IL-1 family cytokines. MDSCs were determined as SSC high CD33 + CD14 - cells. The number of MDSCs as a percentage of all cells in medium-only cultures (mean 1.69%; median 1.3%) were set to 1-fold for every single experiment. The MDSC induction due to specific stimuli is presented as x-fold compared to medium control (mean ± SEM), and differences compared to controls were analyzed by a one-sample t test. b S100 proteins are able to slightly induce MDSCs. General culture conditions were as described under a . S100 proteins S100A7, S100A8, S100A9, or S100A12 were added in different concentrations ranging from 1 ng/ml to 1 µg/ml. The x-fold induction of MDSCs compared to medium control conditions is depicted as means ± SEM, and differences were analyzed by a one-sample t test. c Calprotectin is able to induce MDSCs. General culture conditions were as described under a . Calprotectin was added in different concentrations ranging from 1 ng/ml to 2 µg/ml. The x-fold induction of MDSCs compared to medium control conditions is depicted as means ± SEM, and differences were analyzed by a one-sample t test. d SAA is able to induce MDSCs. General culture conditions were as described in a . Recombinant hybrid SAA was added in different concentrations ranging from 0.1 ng/ml to 2 µg/ml. The x-fold induction of MDSCs compared to medium control conditions is depicted as means ± SEM, and differences were analyzed by a one-sample t test. * p

    Article Snippet: For MDSC isolation, cells were obtained from the PBMC fraction and labeled with anti-CD66b-FITC followed by two sequential anti-FITC magnetic bead separation steps (Miltenyi Biotec), according to the manufacturer's protocol.

    Techniques: In Vitro, Isolation, Sedimentation, Positive Control, Recombinant

    Gr-1 + CD11b + cells coinjected with Pan02 tumor cells facilitate more rapid tumor engraftment

    Journal: Cancer immunology, immunotherapy : CII

    Article Title: Pancreatic adenocarcinoma induces bone marrow mobilization of myeloid derived suppressor cells which promote primary tumor growth

    doi: 10.1007/s00262-011-1178-0

    Figure Lengend Snippet: Gr-1 + CD11b + cells coinjected with Pan02 tumor cells facilitate more rapid tumor engraftment

    Article Snippet: 100,000 CFSE-labeled splenocytes obtained from OT-1 mice were co-cultured in 96-well plates with 1μm SIINFEKL peptide and varying concentrations of Gr-1+ CD11b+ MDSC isolated from mature tumors, spleen, or bone marrow after successful CD11b+ magnetic bead separation (Miltenyi Biotech), according to the manufacturer’s instructions.

    Techniques: