Differential IFN-γ production by adult and neonatal blood CD56+ natural killer (NK) and NK-like-T cells in response to Trypanosoma cruzi and IL-15

Abstract

Early interferon-gamma (IFN-γ) release by innate cells is critical to direct type 1 immune response able to control intracellular pathogens like Trypanosoma cruzi. Although CD56(bright) natural killer (NK) cells are reported to be potent early IFN-γ producers, other CD56(+) cells like CD56(dim) NK cells and NK-like T cells have recently been shown to also release IFN-γ. We have here studied the contribution of each CD56(+) lymphocyte populations in early IFN-γ production in both adults and neonates. On this purpose, we analysed the kinetics of IFN-γ production by RT-PCR, ELISA and flow cytometry from 2 h onwards after T. cruzi and IL-15 stimulation and sought for the responding CD56(+) cells. CD56(bright) and CD56(dim) CD16(-) NK cells were the more potent IFN-γ early producers in response to IL-15 and parasites in adults and neonates. In both age groups, the majority of IFN-γ producing cells were NK cells. However, on the contrary to neonates, CD3(+) CD56(+) NK-like T cells and CD3(+) CD56(-) 'classical' T cells also contributed to early IFN-γ production in adults. Altogether, our results support that whereas NK cells responded almost similarly in neonates and adults, cord blood innate CD56(+) and CD56(-) T cells displayed major quantitative and qualitative defects that could contribute to the well-known neonatal immune immaturity.

Keywords: Interferon-gamma; T cell; Trypanosoma cruzi; human neonate and adult; innate immunity; natural killer cell.

Figure 1

Kinetics of IFN-γ mRNA accumulation and protein secretion in/by cord and adult cells. Kinetics of IFN-γ mRNA accumulation and protein secretion following stimulation of PBMC (a,c,e) or CBMC (b,d,f) with Trypanosoma cruzi trypomastigotes at a ratio of 1 parasite per cell (a,b), IL-15 (20 ng/mL) (c,d) or both (e,f). mRNA amounts were calculated by the method of 2^∧ΔΔCT in relation to unstimulated cells cultured for the same time and GAPDH. Results are shown as median of 5–6 independent experiments. †P < 0·05 as compared with the effect of IL-15 alone (Wilcoxon paired test), $P < 0·05 as compared with cord blood cells (Mann–Whitney U-test). CBMC, cord blood mononuclear cells; PBMC, peripheral blood mononuclear cells.

Figure 2

Kinetics of intracellular IFN-γ production in different CD56⁺ adult cells. Kinetics of IFN-γ production by CD56⁺ cells following stimulation of PBMC with Trypanosoma cruzi trypomastigotes at a ratio of 1 parasite per cell (a–d), IL-15 (20 ng/mL) (e–h) or both (i–l). Results are shown as box-and-whisker plots of proportions of blood CD56^bright (a, e, i), CD56^dim CD16⁺ (b, f, j), CD56^dim CD16⁻ (c, g, k) natural killer (NK) cells and CD3⁺CD56⁺ (d, h, l) NK-like T cells (n = 5). *P < 0·05 as compared with unstimulated cells, †P < 0·05 as compared with the effect of IL-15 alone (Wilcoxon paired test), $P < 0·05 as compared with cord blood cells (Mann–Whitney U-test). PBMC, peripheral blood mononuclear cells.

Figure 3

Kinetics of intracellular IFN-γ production in different CD56⁺ cord cells. Kinetics of IFN-γ production by CD56⁺ cells following stimulation of CBMC with Trypanosoma cruzi trypomastigotes at a ratio of 1 parasite per cell (a–d), IL-15 (20 ng/mL) (e–h) or both (i–l). Results are shown as box-and-whisker plots of proportions of blood CD56^bright (a, e, i), CD56^dim CD16⁺ (b, f, j), CD56^dim CD16⁻ (c, g, k) natural killer (NK) cells and CD3⁺CD56⁺ (d, h, l) NK-like T cells (n = 6). *P < 0·05 as compared with unstimulated cells, †P < 0·05 as compared with the effect of IL-15 alone (Wilcoxon paired test). CBMC, cord blood mononuclear cells.

Figure 4

IL-15 dose-dependent IFN-γ response by different cord blood natural killer (NK) cell subsets. IFN-γ production by NK cells following 24 h stimulation of CBMC with different concentrations of rhIL-15 (0–20 ng/mL) in the presence or absence of Trypanosoma cruzi trypomastigotes at a ratio of 1 parasite per cell. Results are shown as box-and-whisker plots of proportions of IFN-γ positive cells amongst blood CD56^bright (a), CD56^dim CD16⁺ (b) and CD56^dim CD16⁻ (C) NK cells (n = 6). †P < 0·05 as compared with the condition without parasite at the same concentration of IL-15 (Wilcoxon paired test). CBMC, cord blood mononuclear cells.

Figure 5

Relative proportions of different natural killer (NK) subsets, NK-like T cells and ‘classical’ T cells in total or IFN-γ⁺ mononuclear cells in adult and cord cells. Relative proportions of NK and T populations in total (a,b) or IFN-γ⁺ (c,d) adult (a,c) or neonatal (b,d) CD56⁺ and/or CD3⁺ mononuclear cells after 24 h stimulation with IL-15 and Trypanosoma cruzi. We determined the proportion of ‘classical’ T cells (CD3⁺CD56⁻), natural killer (NK)-like T cells (CD56⁺CD3⁺) and the three subpopulations of NK cells (CD56⁺CD3⁻): CD56^bright CD16^−/low cells, CD56^dim CD16⁺ cells and CD56^dim CD16⁻ cells. Data are shown as medians of six donors. $P < 0·05 to neonates (Mann–Whitney U-test).