Longitudinal study of intracellular T cell cytokine production in infants compared to adults

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.

Fig. 1

Total helper/inducer T cells and cytotoxic/suppressor T cells in 2, 6, 7 and 12-month infant (n = 325) and adult (n = 26) T cells. Blood samples were stimulated for 4 h as described in Materials and methods, and stained with MoAb against CD3-labelled APC, CD4-labelled PerCP, IFN-γ-labelled FITC and IL4-labelled PE. 30 000 leucocytes were counted, and the gated CD3⁺versus side scatter population was analysed for cytokine production. Data are presented as median values on a two-dimensional plot. Age groups are shown on the x-axis, cell numbers per μl and percentages are plotted on the y-axes. Th and Tc cells are expressed as a percentage of T lymphocytes. ▪, Th (%); ○, Tc (%); □, Th (no.); ✦, Tc (no.).

Fig. 2

Intracellular Th1, Tc1 cytokine production in 2, 6, 7 and 12-month infant (n = 325) and adult (n = 26) T cells. Blood samples were stimulated for 4h as described in Materials and methods, and stained with MoAb against CD3-labelled APC, CD4-labelled PerCP, IFN-γ-labelled FITC and IL4-labelled PE. 30 000 leucocytes were counted, and the gated CD3⁺versus side scatter population was analysed for cytokine production. Data are presented as median values on a two-dimensional plot. Age groups are shown on the x-axis, cell numbers per μl and percentages are plotted on the y-axes. Th1 cells are expressed as a percentage of Th lymphocytes and Tc1 cells are expressed as a percentage of Tc lymphocytes. ▪, Th1 (no.); ○, Tc1 (no.); □, Th1 (%); ✦, Tc1 (%).

Fig. 3

Intracellular Th2 and Tc2 cytokine production in 2, 6, 7 and 12-month infant (n = 325) and adult (n = 26) T cells. Blood samples were stimulated for 4h as described in Materials and methods, and stained with MoAb against CD3-labelled APC, CD4-labelled PerCP, IFN-γ-labelled FITC, IL4-labelled PE. 30 000 leucocytes were counted, and the gated CD3⁺versus side scatter population was analysed for cytokine production. Data are presented as median values on a two-dimensional plot. Age groups are shown on the x-axis, cell numbers per μl and percentages are plotted on the y-axes. Th2 cells are expressed as a percentage of Th lymphocytes and Tc2 cells are expressed as a percentage of Tc lymphocytes. ▪, Th2 (no.); ○, Tc2 (no.); □, Th2 (%); ✦, Tc2 (%).