Measurement of T-helper cytokines secreted by Cord Blood Mononuclear Cells (CBMC) in response to allergens

In utero factors may influence the development of childhood atopy. To test this hypothesis it is necessary to measure T-helper cell (Th) cytokines secreted by CBMC stimulated by allergens, but this has proved technically difficult for the key Th2 cytokine interleukin-4 (IL-4). We have adapted a sensitive novel cell based ELISA (celELISA) method and have applied it to 100 randomly selected cord blood samples. CBMC were cultured for 8 days, stimulated by timothy grass pollen extract (TG), house dust mite extract (HDM), the mitogen, conA, and the control antigens, purified protein derivative (PPD) and keyhole limpet haemocyanin (KLH).. Proliferative responses on days 4-8 were assessed by incorporation of tritiated thymidine. On day 5, aliquots of cells were layered onto capture antibody and the cytokines secreted over a 24 hour period quantified by a standard ELISA detection protocol. Sensitivity for IL-4 was 1pg/ml and for IFN-γ, 20pg/ml. Significant proliferative and cytokine responses were, respectively, 3 times and twice that of unstimulated controls. The celELISA was up to 20 times more sensitive than conventional ELISA methods. The proportion of samples with significant proliferative and cytokine responses are outlined below. Stimulant % proliferation +ve % IL-4 +ve % IFN-γ +ve TG 69% 17% 44% HDM 35% 20% 21% Con A 95% 22% 90% PPD 89% 27% 71% KLH 85% 27% 68% The results enabled us to identify individuals whose CBMC cytokine profiles demonstrated Th1, Th2 or Th0 bias. Analysis of T-cell phenotypes indicated that a small (≈25%) proportion of CBMC TG responses were secondary, suggesting in utero sensitisation. Conclusion: The celELISA method reliably detects allergen specific CBMC IL-4 secretion, it is cheap and simple, and identifies individuals in a general population whose CBMC exhibit different cytokine biases in response to allergens.