Myeloid-derived Suppressor Cells

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Myeloid-derived Suppressor Cells

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that fail to differentiate into granulocytes, macrophages, and dendritic cells. These immature cells have the capacity to suppress immune responses mediated by natural killer cells, CD8+ and CD4+ T cells. MDSCs accumulate in the blood, bone marrow, and secondary lymphoid organs of tumor-bearing mice and

cancer patients, where circulating levels of MDSCs have been shown to correlate with clinical stage, metastatic burden, and chemoresistance. As a result, these cells have been suggested to have a causative role in promoting tumor-associated immunosuppression. In mouse, MDSCs are broadly defined as CD11b+ Gr-1+ cells, but the relative expression levels of Ly-6G and Ly-6C define two specific subsets known as

granulocytic and monocytic MDSCs. Mouse granulocytic MDSCs are CD11b+ Gr-1/Ly-6Ghigh Ly-6Clow, while monocytic MDSCs are CD11b+ Gr-1/

Ly-6G–/low Ly-6Chigh. Human MDSCs also commonly express CD11b along with Siglec-3/CD33 and lack HLA-DR and the lineage markers CD3,

CD14, CD19, and CD56. Similar to mouse, human granulocytic (Lin– CD11b+ CD14– CD15+ CD33+ CD66b+ HLA-DR–) and monocytic (Lin-

CD11b+ CD14+ CD15– CD33+ CD66b– HLA-DR–) subsets have been identified, but they are based on the differential expression of CD14, CD15,

and CD66b/CEACAM-8.

MDSCs are of great interest due to their immunosuppressive properties. While the mechanism by which MDSCs inhibit NK cells is currently not well-understood, multiple pathways are responsible for MDSC-mediated T cell suppression including production of arginase 1/ARG1 and upregulation of nitric oxide synthase 2 (iNOS). ARG1 and iNOS metabolize L-arginine and either together, or separately, cause the loss of the TCR zeta chain, promote nitration or nitrosylation of TCR, CD3, CD8, and CCL2, disrupt IL-2 signaling, and inhibit T cell proliferation. Additionally, MDSCs secrete immunosuppressive cytokines and induce regulatory T cell development. Although it is now evident that MDSCs are a major obstacle for immunotherapy, further characterization is necessary to determine how MDSCs accumulate, how they function, and mechanisms by which they can be inhibited. R&D Systems, Novus Biologicals, and Tocris Biosciences together offer a wide range of products for characterizing MDSCs and studying their functions.

Analysis of Human Granulocytic and Monocytic MDSCs by Flow Cytometry

Identification of Human Granulocytic and Monocytic Myeloid-derived Suppressor Cells by Flow Cytometry. (A) Lin–/HLA-DR– cells were detected in human peripheral blood mononuclear

cells by staining with a lineage cocktail containing Alexa Fluor 700-conjugated Mouse Anti-Human CD3e and CD19 Monoclonal Antibodies (R&D Systems, Catalog # FAB100N and # FAB4867N, respectively) and an Alexa Fluor 750-conjugated Mouse Anti-Human HLA-DR Monoclonal Antibody (R&D Systems, Catalog # FAB4869S). Lin–/HLA-DR– cells were gated. (B)

CD11b+/CD33+ cells were detected in the Lin–/HLA-DR– cell population by staining with an APC-conjugated Mouse Anti-Human Siglec-3/CD33 Monoclonal Antibody (R&D Systems,

Catalog # FAB1137A) and an Alexa Fluor 405-conjugated Mouse Anti-Human Integrin aM/CD11b Monoclonal Antibody (R&D Systems, Catalog # FAB16991V). CD11b+/CD33+ cells were

gated. (C) Human granulocytic myeloid-derived suppressor cells (Lin–/CD11b+/CD14–/CD33+/CEACAM-8+/HLA-DR–) and monocytic myeloid-derived suppressor cells (Lin–/CD11b+/

CD14+/CD33+/CEACAM-8–/HLA-DR–) were detected in the Lin–/HLA-DR–/CD11b+/CD33+ cell population by staining with a PE-conjugated Mouse Anti-Human CEACAM-8/CD66b

Monoclonal Antibody (R&D Systems, Catalog # FAB4246P) and a PerCP-conjugated Mouse Anti-Human CD14 Monoclonal Antibody (R&D Systems, Catalog # FAB3832C).

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Detection of Arginase 1 Expression in Human Granulocytic and Monocytic Myeloid-derived Suppressor Cells by Flow Cytometry. Following identification of myeloid-derived suppressor cells (MDSCs) by flow cytometry, expression of Arginase 1 in the gated (A) granulocytic (Lin–/CD11b+/

CD14–/CD33+/CEACAM-8+/HLA-DR–) and (B) monocytic

(Lin–/CD11b+/CD14+/CD33+/CEACAM-8–/HLA-DR–) MDSC

populations was determined by staining with an Alexa Fluor 488-conjugated Mouse Anti-Human Arginase 1/ARG1 Monoclonal Antibody (R&D Systems, Catalog # IC8026G; filled histograms) or an Alexa Fluor 488-conjugated Mouse IgG2B Isotype Control (R&D Systems, Catalog # IC0041G;

open histograms).

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Detection of IDO in Human Granulocytic and Monocytic Myeloid-derived Suppressor Cells by Flow Cytometry. Following identification of myeloid-derived suppressor cells (MDSCs) by flow cytometry, expression of Indoleamine 2,3-dioxygenase/IDO in the gated (A) granulocytic (Lin–/CD11b+/CD14–/CD33+/CEACAM-8+/HLA-DR–) and

(B) monocytic (Lin–/CD11b+/CD14+/CD33+/CEACAM-8–/

HLA-DR–) MDSC populations was determined by staining

with an Alexa Fluor 488-conjugated Mouse Anti-Human Indoleamine 2,3-dioxygenase/IDO Monoclonal Antibody (R&D Systems, Catalog # IC6030G; filled histograms) or an Alexa Fluor 488-conjugated Mouse IgG1 Isotype Control

(R&D Systems, Catalog # IC002G; open histograms).

Analysis of Mouse Granulocytic and Monocytic MDSCs by Flow Cytometry

Identification of Mouse Monocytic and Granulocytic Myeloid-derived Suppressor Cells by Flow Cytometry. Mouse monocytic (CD11b+/Gr-1low/mid/Ly6C+) and granulocytic (CD11b+/Gr-1high/Ly6C–) myeloid-derived suppressor cells (MDSCs)

from C57BL/6 mouse bone marrow cells were identified by staining with an APC-conjugated Rat Anti-Mouse Gr-1/Ly-6G Monoclonal Antibody (R&D Systems, Catalog # FAB1037A) and a PE-conjugated Rat Anti-Mouse Integrin aM/CD11b Monoclonal Antibody (R&D Systems, Catalog # FAB1124P). CD11b+/Gr-1low/mid and CD11b+/Gr-1high cells were gated to show

the two populations of MDSCs.

Fluorochrome-conjugated & Unlabeled Antibodies from R&D Systems and Novus Biologicals

R&D Systems and Novus Biologicals offer a wide selection of unlabeled and fluorochrome-conjugated antibodies for the identification and characterization of mouse and human MDSCs.

Fluorochrome-conjugated & Unlabeled Antibodies from R&D Systems and Novus Biologicals continued

Recombinant Proteins & ELISA Kits from R&D Systems

R&D Systems portfolio includes recombinant proteins for generating MDSC-like cells from bone marrow-derived cells and ELISA Kits for detecting molecules that affect MDSC proliferation or promote immunosuppression.

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