Abstract
Intracellular cytokine production in lymphocytes obtained longitudinally from 325 healthy infants aged 2-12 months was compared with adult lymphocytes using four-colour flow cytometry. Peripheral blood samples (180 microlitres) were stimulated with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A to induce production and intracellular accumulation of cytokines. The method was validated by assessing reproducibility, repeatibility, ruggedness (i.e. fresh versus day-old blood samples), precision, linearity and sensitivity. Among infants, the number and percentage of T lymphocytes (helper/inducer T cell subsets and cytotoxic/suppressor T cell subsets) producing IFN-gamma (type 1) and IL4 (type 2) increased over the first year of life but remained significantly lower than levels found in adults. In both infants and adults more CD4- T cells than CD4+ T cells were induced to make IFN-gamma. Infant Th1/Th2 ratios revealed modest Th1-skewed (predominant) profiles compared to adults, which were 5-10 times higher. Infant Tc1/Tc2 ratios revealed Tc1-skewed responses which were equal to adult ratios by age 12 months. At 12 months infant Th2 responses were closer to adult levels than were Th1 cells. Intracellular cytokine detection by flow cytometry is a rapid, sensitive, rugged and precise method to characterize immune status changes over time.