title: Myeloid-derived suppressor cell measurements in fresh and cryopreserved blood samples.
abstract: Myeloid-derived suppressor cells (MDSC) present in the human peripheral blood, represent a heterogeneous population of cells with monocytic and granulocytic features. To provide guidelines for reliable assessments of the frequency and function of MDSC, we compared fresh vs. cryopreserved peripheral blood mononuclear cell (PBMC) samples obtained from normal controls and patients with cancer. PBMC were obtained from 4 healthy donors and 21 patients with cancer. They were stained with labeled antibodies, and the frequency of DR⁻/LIN⁻/CD11b+, DR⁻/LIN⁻/CD15+, DR⁻/LIN⁻/CD33+ and DR(-/low)/CD14+ cells was determined by flow cytometry before and after cryopreservation. CFSE-based suppressor assays were used to test inhibitory functions of MDSC. Arginase I expression and reactive oxygen species (ROS) upregulation in MDSC subsets were evaluated by flow cytometry. The DR(-/low)/CD14+ and DR⁻/LIN⁻/CD11b+ subsets of MDSC were found to be more resistant to the cryopreservation/thawing procedure compared to the DR⁻/LIN⁻/CD15+ and DR⁻/LIN⁻/CD33+ subsets. The frequency of the latter two MDSC subsets was significantly reduced after cryopreservation. All but DR⁻/LIN⁻/CD15+ cells inhibited proliferation of autologous CSFE-labeled CD4+ cells but lost suppressor activity after cryopreservation. Only DR⁻/LIN-/CD15+ cells were positive for Arginase I, but lost its expression after cryopreservation. Only fresh DR⁻/LIN⁻/CD11b+ and DR⁻/LIN⁻/CD15+ cells produced ROS after in vitro stimulation. Studies of human MDSC should be performed in fresh blood samples. If samples have to be cryopreserved, monitoring of CD11b+ and CD14+ MDSC subsets provides the most reliable results. Arginase I expression or stimulated ROS production assessed by flow cytometry are useful markers for MDSC subsets only in fresh samples.
authors.affiliation: Departments of Pathology, Medical Oncology, Immunology and Otolaryngology, University of Pittsburgh Cancer Institute and University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Kotsakis
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Harasymczuk
authors.forename: Bastian
authors.initials: B
authors.lastname: Schilling
authors.forename: Vasilis
authors.initials: V
authors.lastname: Georgoulias
authors.forename: Athanassios
authors.initials: A
authors.lastname: Argiris
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Whiteside
mesh_terms: D015703:Antigens, CD / Q000097:blood; D015214:Antigens, Differentiation, Myelomonocytic / Q000097:blood; D001119:Arginase / Q000097:blood; D015925:Cryopreservation; D005434:Flow Cytometry / Q000379:methods; D006098:Granulocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D006684:HLA-DR Antigens / Q000097:blood; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D016256:Lewis X Antigen / Q000097:blood; D018950:Lipopolysaccharide Receptors / Q000097:blood; D009000:Monocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D022423:Myeloid Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D009369:Neoplasms / Q000097:blood* / Q000276:immunology / Q000473:pathology; D017382:Reactive Oxygen Species / Q000097:blood; D063268:Sialic Acid Binding Ig-like Lectin 3
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D006684:HLA-DR Antigens; D016256:Lewis X Antigen; D018950:Lipopolysaccharide Receptors; D017382:Reactive Oxygen Species; D063268:Sialic Acid Binding Ig-like Lectin 3; D001119:Arginase