Phenotypic Characterization and Isolation of Myeloid-Derived Suppressor Cells

Abstract

Myeloid-derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation and induce immunosuppression evident in an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases. Herein, we describe methods to isolate and characterize MDSCs from various murine tissue, as well as to phenotype blood-derived MDSCs from patients. The protocols describe methods for isolation of total MDSCs and their subpopulations, for characterization, and for evaluation of their distribution within tissue, as well as for assessing their maturation stage by flow cytometry, immunofluorescence analyses, and Giemsa staining. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single-cell suspension generation from different tissue Alternate Protocol 1: Single-cell suspension generation from subcutaneous melanoma tumors Basic Protocol 2: Characterization of MDSC phenotype Basic Protocol 3: Cell separation using magnetic beads: Separating pan-MDSCs or PMN-MDSC and M-MDSC subpopulations Alternate Protocol 2: Staining and preparing MDSCs for sorting Support Protocol: PMN-MDSC and M-MDSC gating strategy in mouse Basic Protocol 4: Immunofluorescence analysis of MDSCs Basic Protocol 5: Handling human blood samples and characterizing human MDSCs Alternate Protocol 3: Flow cytometry staining of thawed human whole blood samples.

Keywords: MDSC; chronic inflammation; flow cytometry; immunofluorescence imaging; immunosuppression; inflammatory mouse model; myeloid-derived suppressor cells; patient MDSC.

Figure 1

Growth kinetics of mice injected with B16‐F10 melanoma tumor cells over the course of 28 days. When injecting a small number of cells, tumors will appear about 10 to 14 days after injection, and at the endpoint there might be variability in tumor volume.

Figure 2

General and mouse myeloid‐derived suppressor cell (MDSC) reciprocal gating strategy. (A) Doublet discrimination: side scatter width (SSC‐W) versus side scatter height (SSC‐H) and SSC‐W low population in the SSC gate. (B) Forward scatter width (FSC‐W) versus forward scatter height (FSC‐H) and FSC‐W low population in the FSC gate. (C) The main gate is plotted on SSC area (SSC‐A) versus FSC‐A and excludes dead cells and debris, visible left of the gate. (D) Gr‐1⁺CD11b⁺ mouse pan‐MDSC gating. (E) Mouse MDSC subset gating: lineage exclusion (T, B, and erythroid cells excluded). (F) Ly6G⁺CD11b⁺ mouse PMN‐MDSC gate and Ly6G⁻ gate. (G) Ly6C^hiCD11b⁺ M‐MDSC gate. All percentages of the gated cells are presented separately each plot.

Figure 3

Representative results of control (A) and inflamed (B) total bone marrow cells stained with Giemsa. Cells were mounted to slides using an SLEE Cellspin I cytocentrifuge, fixed with methanol, and stained with Giemsa. The different precursor cells can be distinguished according to nuclear morphology, cell size, and basophilic‐acidophilic properties of the cells. Black arrows indicate granulocytes in a mature state in A, whereas black arrows indicate immature cells in B. Images were taken using an Olympus BX51 microscope at a 10× magnification.

Figure 4

Immunofluorescence images showing the distribution of myeloid‐derived suppressor cells (MDSCs) stained with the pan‐MDSC marker Gr‐1 (green) in the spleens of control (A) and inflamed (B) mice. Evident is the morphology of T (red) and B (orange) zones within the spleen and MDSCs surrounding these structures. Further MDSC subpopulation division can be made by combining staining of the cells using anti‐Ly6C and anti‐Ly6G antibodies. Images were taken using an Olympus BX51 fluorescence microscope at a 10× magnification.

Figure 5

Human myeloid‐derived suppressor cell (MDSC) subset reciprocal gating strategy. (A) Human leukocyte antigen DR isotype (HLA‐DR)^–/lo gate excludes mature myeloid cells and B cells. (B) CD33⁺CD11b⁺ human pan‐MDSC gating presented as percentage of live cells. (C) Subdivision of human MDSCs to CD14⁻ PMN‐MDSCs and CD14⁺ M‐MDSCs presented as percentage of pan‐MDSCs. (D) Discrimination of PMN‐ and M‐MDSCs inside the pan‐MDSC gate: CD33^loCD11b^lo PMN‐MDSCs (red) and CD33^hiCD11b^hi M‐MDSCs (blue) presented as percentage of live cells in the sample.