Mv. Kopp et al., Culture conditions for the detection of allergen-specific T-cell reactivity in cord blood: Influence of cell number, PEDIAT A IM, 11(1), 2000, pp. 4-11
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.