Culture conditions for the detection of allergen-specific T-cell reactivity in cord blood: Influence of cell number

Raised T-cell proliferation of cord blood mononuclear cells (CBMC) in respo nse to various ingestant and inhalant allergens has been reported in newbor ns, suggesting a prenatal allergen contact. In general for in vitro prolife ration assays a concentration of 50 x 10(3) or 100 x 10(3) cells/well are u sed. The aim of this study was to analyze whether cell concentration influe nces T-cell reactivity in cord blood cells and to study differences of T-ce ll reactivity triggered by inhalant and ingestant allergens. CBMC from 51 n eonates (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 pter onyssinus (Der p 1), and timothy grass allergen Phleum pratense (Phl p 1) f or 7 days. The cell concentration ranged from 6.25 x 10(3) to 100 x 10(3) c ells/well. Proliferation was assessed by incorporation of [H-3]-thymidine a nd was expressed as counts per minute (c.p.m.). In unstimulated cells, a de creasing cell concentration paralleled a steep drop of background activity. In response to IL-2, a decreasing cell concentration led to a slow decreas e 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 posit ive proliferative responses to specific allergens were obscured at lower ce ll concentrations. For beta-LG, the maximal number of positive responses we re obtained between 25 x 10(3) (n = 44) and 17.5 x 10(3) (n = 46) cells/wel l, 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 Phl 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 wa s observed in 100% of samples in response to beta-LG, in 22% in response to Phl 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, proli feration assays with lower cell numbers unmask T-cell reactivity in respons e 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.