title: Culture conditions for the detection of allergen-specific T-cell reactivity in cord blood: influence of cell number.
abstract: Raised T-cell proliferation of cord blood mononuclear cells (CBMC) in response to various ingestant and inhalant allergens has been reported in newborns, suggesting a prenatal allergen contact. In general, for in vitro proliferation assays a concentration of 50 x 10(3) or 100 x 10(3) cells/well are used. The aim of this study was to analyze whether cell concentration influences T-cell reactivity in cord blood cells and to study differences of T-cell reactivity triggered by inhalant and ingestant allergens. CBMC from 51 neonates (34 females: 22 with and 29 without a family history of allergy, i.e. FH+ or FH-) were incubated with interleukin-2 (IL-2), beta-lactoglobulin (beta-LG), ovalbumin (OVA), house dust mite allergen Dermatophagoides pteronyssinus (Der p 1), and timothy grass allergen Phleum pratense (Ph1 p 1) for 7 days. The cell concentration ranged from 6.25 x 10(3) to 100 x 10(3) cells/well. Proliferation was assessed by incorporation of [3H]-thymidine and was expressed as counts per minute (c.p.m.). In unstimulated cells, a decreasing cell concentration paralleled a steep drop of background activity. In response to IL-2, a decreasing cell concentration led to a slow decrease of c.p.m. The corresponding mean stimulation indices (SI) were 9, 32, 77, 47, and 21 for 100 x 10(3), 50 x 10(3), 25 x 10(3), 12.5 x 10(3), and 6.25 x 10(3) cells/well, respectively. In addition, the highest number of positive proliferative responses to specific allergens were obscured at lower cell concentrations. For beta-LG, the maximal number of positive responses were obtained between 25 x 10(3) (n = 44) and 12.5 x 10(3) (n = 46) cells/well, for OVA at 25 x 10(3) (n = 3) cells/well, for Der p 1 at 50 x 10(3) (n = 5) cells/well, and for Ph1 p 1 between 25 x 10(3) and 12.5 x 10(3) (n = 5) cells/well. Positive proliferation in at least one of the tested assays was observed in 100% of samples in response to beta-LG, in 22% in response to Ph1 p 1, and in 14% in response to OVA and Der p 1. T-cell reactivity did not differ between samples of newborns with or without a family history of atopy. Therefore, sensitivity of T-cell proliferation measurement is highly influenced by background proliferation of unstimulated cells. Hence, proliferation assays with lower cell numbers unmask T-cell reactivity in response to ingestant and inhalant allergens. We suggest the use of concentrations of 12.5 x 10(3)-50 x 10(3) cells/well in proliferation experiments.
authors.affiliation: University Children's Hospital, Vienna, Austria. kopp@kkl200.ukl.uni-freiburg.de
authors.forename: M V
authors.initials: MV
authors.lastname: Kopp
authors.forename: J
authors.initials: J
authors.lastname: Pichler
authors.forename: G
authors.initials: G
authors.lastname: Halmerbauer
authors.forename: J
authors.initials: J
authors.lastname: Kuehr
authors.forename: T
authors.initials: T
authors.lastname: Frischer
authors.forename: R
authors.initials: R
authors.lastname: Urbanek
authors.forename: Z
authors.initials: Z
authors.lastname: Szépfalusi
mesh_terms: D000485:Allergens / Q000276:immunology* / Q000302:isolation & purification; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D004306:Dose-Response Relationship, Immunologic; D018984:Epitopes, T-Lymphocyte / Q000276:immunology* / Q000302:isolation & purification; D005260:Female; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology*; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000235:genetics / Q000276:immunology; D007231:Infant, Newborn; D007782:Lactoglobulins / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D008297:Male; D016176:T-Lymphocyte Subsets / Q000166:cytology* / Q000276:immunology
chemical_list: D000485:Allergens; D018984:Epitopes, T-Lymphocyte; D007782:Lactoglobulins