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

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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 × 103 or 100 × 103 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), β‐lactoglobulin (β‐LG), ovalbumin (OVA), house dust mite allergen Dermatophagoides pteronyssinus (Der p 1), and timothy grass allergen Phleum pratense (Phl p 1) for 7 days. The cell concentration ranged from 62.5 × 103 to 100 × 103 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 × 103, 50 × 103, 25 × 103, 12.5 × 103, and 62.5 × 103 cells/well, respectively. In addition, the highest number of positive proliferative responses to specific allergens were obscured at lower cell concentrations. For β‐LG, the maximal number of positive responses were obtained between 25 × 103 (n = 44) and 12.5 × 103 (n = 46) cells/well, for OVA at 25 × 103 (n = 3) cells/well, for Der p 1 at 50 × 103 (n = 5) cells/well, and for Phl p 1 between 25 × 103 and 12.5 × 103 (n = 5) cells/well. Positive proliferation in at least one of the tested assays was observed in 100% of samples in response to β‐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, 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 × 103–50 × 103 cells/well in proliferation experiments.

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