2.3. MDSC Isolation and Flow Cytometry Analysis

Peripheral blood samples were obtained from 118 patients with pathologically and clinically confirmed HNSCC (Table 3). To assess the proportion of MDSCs among peripheral blood mononuclear cells (PBMCs), multicolor fluorescence-activated cell sorting (FACS) was performed using the FACS Caliber flow cytometer (BD Biosciences, San Jose, CA, USA). Human low-density neutrophils and granulocytic MDSCs are closely related, and presently there is no generally accepted consensus on mutually exclusive definitions for these cell types [22]. In the majority of oncological studies, human granulocytic MDSCs are characterized as CD14⁻CD15⁺CD11b⁺HLA-DR⁻ cells [7]. Accordingly, the human MDSC subset characterized as CD11b⁺CD14⁻HLA-DR⁻ cells was sorted from the peripheral blood. The leukocytes were separated from the peripheral blood using a Ficoll gradient before analysis or sorting. Multicolor cell analysis was performed using the following antibodies: PerCP-Cy5.5-conjugated CD14, polyethylene (PE)-conjugated CD11b, and fluorescein isothiocyanate-conjugated HLA-DR. The percentage of MDSCs was measured using multicolor flow cytometry, and isotype-specific antibodies were used as negative controls.

Characteristics of head and neck squamous cell carcinoma (HNSCC) patients with curative-intent treatment.

MDSC: myeloid-derived suppressor cell; *: Statistically significant covariate.