title: Whole blood cultures to assess the immunostimulatory activities of CpG oligodeoxynucleotides.
abstract: Specially designed oligodeoxynucleotide (ODN) sequences known as 'CpG' ODNs elicit innate and acquired immune responses. In general, screening of new CpG ODNs has been conducted by conventional lymphoproliferative assays or expression of activation markers in peripheral blood mononuclear cell (PBMC) cultures. Here, we compared conventional in vitro human PBMC assays with whole blood assays for screening the immunostimulatory properties of CpG ODNs. Commercially available DNA preparations and mycobacterial-based adjuvants were used as comparators. Activation was assessed by flow cytometry and cytokine production. CpG ODNs, identified by four-letter codes, consisted of 2006 (strong human cell stimulant), 1826 (strong murine cell stimulant), 1840 (weak immunostimulant), and 2041, a non-CpG ODN. In both test systems, and in accordance with previous reports, 2006 was an effective up-regulator of CD40 on human dendritic cells (DC1, DC2), monocytes, and B cells, and of CD69 on NK cells. In contrast to murine cells exposed to CpG ODNs, IL-12 (p40) and IFN-gamma production in human immune cells was negligible, but greatly enhanced by adding GM-CSF. Like 2006, two comparator mycobacterial adjuvant formulations activated DC1, DC2, monocytes and natural killer (NK) cells, but only 2006 had a strong effect on B cells. The usefulness of the whole blood assay was further demonstrated by studies in small volumes of umbilical cord mononuclear cells, that like adult blood cells, showed up-regulation of CD40 expression on B cells, DC, and monocytes, and CD69 on NK cells. The whole blood assay, in conjunction with flow cytometry, is useful for assessing the immunological properties of CpG ODN sequences.
authors.affiliation: Department of Immunology and Medicine, AFRIMS, 315/6 Rajuithi Road, Bangkok, Thailand. sathitp@thai.amedd.army.mil
authors.forename: S
authors.initials: S
authors.lastname: Pichyangkul
authors.forename: K
authors.initials: K
authors.lastname: Yongvanitchit
authors.forename: U
authors.initials: U
authors.lastname: Kum-arb
authors.forename: A M
authors.initials: AM
authors.lastname: Krieg
authors.forename: D G
authors.initials: DG
authors.lastname: Heppner
authors.forename: D S
authors.initials: DS
authors.lastname: Walsh
mesh_terms: D000328:Adult; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D019013:CD40 Antigens / Q000096:biosynthesis; D002417:Cattle; D002478:Cells, Cultured; D018899:CpG Islands / Q000276:immunology*; D004269:DNA, Bacterial / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology; D006801:Humans; D007231:Infant, Newborn; D007700:Kinetics; D037181:Lectins, C-Type; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D009838:Oligodeoxyribonucleotides / Q000276:immunology*; D013950:Thymus Gland; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D019013:CD40 Antigens; D004269:DNA, Bacterial; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D037181:Lectins, C-Type; D009838:Oligodeoxyribonucleotides; D000099192:CD69 Antigens