Age-Stratified T Cell Responses in Children Infected with Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) in young children differs from adult TB in that the risk of rapid progression to active TB (aTB) is higher in children than in adults. The reasons for this increased risk are not fully understood. Early differentiation remains difficult between children at risk to develop aTB from those who will remain healthy and develop a latent TB infection (LTBI). Biomarkers to differentiate aTB from LTBI in children, especially in very young children, are urgently needed. To identify M. tuberculosis-specific functional T cell subsets related to clinical manifestations in children, we enrolled 87 children exposed to M. tuberculosis. After standard clinical assessment, the children were classified as aTB, LTBI, or uninfected. Their CD4⁺ T cell cytokine profiles (IFN-γ, TNF-α, IL-2, IL-17) were analyzed at the single-cell level by flow cytometry after stimulation with three mycobacterial antigens, purified protein derivative (PPD), early-secreted-antigenic target-6 (ESAT-6), or heparin-binding hemagglutinin (HBHA). This approach identified age-related discriminative markers between aTB and LTBI. Whereas among the 3- to 15-year-old children, an excellent discrimination between aTB and LTBI was provided by comparing the ratio between the proportions of ESAT-6-induced IFN-γ^single+ and ESAT-6-induced TNF-α^single+CD4⁺ T lymphocytes, this was not the case for children younger than 3 years. By contrast, in this group (<3years), the analysis of HBHA-induced IL-17^single+CD4⁺ T lymphocytes allowed us to identify children with LTBI by the high proportion of this cellular lymphocyte subset, whereas this was not the case for children with aTB. The analysis at the single-cell level of T cell immune responses induced by mycobacterial antigens are, thus, different in infected children younger or older than 3 years of age. HBHA-induced IL-17 production by CD4⁺ T lymphocytes was associated with protection only in children under 3 years who are at high risk for rapid progression to aTB. This suggests that the HBHA-induced IL-17 production by CD4⁺ T lymphocytes is a potential new correlate of protection against M. tuberculosis in humans, and that the distinction between children with LTBI and those with aTB is possible based on age-related diagnostic markers.

Keywords: children; early-secreted-antigenic target-6; heparin binding hemagglutinin; interferon-γ; interleukin-17; tuberculosis; tumor necrosis factor-α.

Figure 1

Flowchart of inclusion. Eighty-seven eligible children were prospectively enrolled in the study. Using strict criteria as defined in the Figure, they were retrospectively classified as NI, LTBI, or aTB. Twenty-six children were excluded from the final analysis for reasons explained in the Figure.

Figure 2

Mycobacterium tuberculosis-specific cytokine expression by CD4⁺ T lymphocytes. Peripheral blood mononuclear cells were in vitro stimulated during 5 days with purified protein derivative (PPD), early-secreted-antigenic target-6 (ESAT-6), or heparin-binding hemagglutinin (HBHA), and the percentages of CD4⁺ T lymphocytes expressing one or several of the four studied cytokines (IFN-γ, TNF-α, IL-2, IL-17) were measured by flow cytometry. Children with active TB (aTB) were compared to latently TB infected (LTBI) and non-infected (NI) children. Each dot corresponds to the result from an individual child and horizontal bars represent the medians of the results. ***p < 0.001; **p < 0.01.

Figure 3

Poly-functional profile of Mycobacterium tuberculosis-specific CD4⁺ T lymphocytes after in vitro stimulation with purified protein derivative (PPD), early-secreted-antigenic target-6 (ESAT-6), or heparin-binding hemagglutinin (HBHA). Peripheral blood mononuclear cell were in vitro stimulated during 5 days with PPD (A), ESAT-6 (B), or HBHA (C) and the percentages of all possible combinations of the four studied cytokines (IFN-γ, TNF-α, IL-2, and IL-17) were analyzed among the M. tuberculosis-specific cytokine-containing CD4⁺ T lymphocytes. Children with active TB (aTB) are represented by the red dots, whereas latently TB infected (LTBI) children are represented by the blue dots. All the possible combinations of cytokines are represented on the x-axis of the bar charts graphs, and color coded, whereas the percentages of each subpopulation within M. tuberculosis-specific CD4⁺ T cells are shown on the y-axis. Boxes represent interquartile ranges (P25–75). Results were analyzed with SPICE and degrees of significance are based on the “unequal variance T-test.” *p < 0.05; ***p = 0.001. The pie charts average the data for both groups (aTB and LTBI), each slice corresponding to the proportion of M. tuberculosis-specific CD4⁺ T cells positive for a certain combination of cytokines. The colors in the pie chart are defined by the 15 color boxes on the x-axis of the bar chart.

Figure 3

Poly-functional profile of Mycobacterium tuberculosis-specific CD4⁺ T lymphocytes after in vitro stimulation with purified protein derivative (PPD), early-secreted-antigenic target-6 (ESAT-6), or heparin-binding hemagglutinin (HBHA). Peripheral blood mononuclear cell were in vitro stimulated during 5 days with PPD (A), ESAT-6 (B), or HBHA (C) and the percentages of all possible combinations of the four studied cytokines (IFN-γ, TNF-α, IL-2, and IL-17) were analyzed among the M. tuberculosis-specific cytokine-containing CD4⁺ T lymphocytes. Children with active TB (aTB) are represented by the red dots, whereas latently TB infected (LTBI) children are represented by the blue dots. All the possible combinations of cytokines are represented on the x-axis of the bar charts graphs, and color coded, whereas the percentages of each subpopulation within M. tuberculosis-specific CD4⁺ T cells are shown on the y-axis. Boxes represent interquartile ranges (P25–75). Results were analyzed with SPICE and degrees of significance are based on the “unequal variance T-test.” *p < 0.05; ***p = 0.001. The pie charts average the data for both groups (aTB and LTBI), each slice corresponding to the proportion of M. tuberculosis-specific CD4⁺ T cells positive for a certain combination of cytokines. The colors in the pie chart are defined by the 15 color boxes on the x-axis of the bar chart.

Figure 4

Age-related differences in CD4⁺ T cell cytokine profiles. Peripheral blood mononuclear cells were in vitro stimulated during 5 days with early-secreted-antigenic target-6 (ESAT-6), purified protein derivative (PPD), or heparin-binding hemagglutinin (HBHA), and the frequency of cytokine^single+CD4⁺ T cells was analyzed and compared between infected children who were younger or older than 3 years of age, and between active TB (aTB) (hatched columns) and latently TB infected (LTBI) children (open columns). Among older children, the frequency of ESAT-6-induced TNF-α^single+CD4⁺ T cells (A) was higher and the frequencies of both ESAT-6- and PPD-induced IFN-γ^single+CD4⁺ T cells were lower (B,D) among children with aTB compared to LTBI children. This resulted in lower ratios between the proportions of ESAT-6-induced IFN-γ^single+ and ESAT-6-induced TNF-α^single+CD4⁺ T cells in children older than 3 years (C). By contrast, in children younger than 3 years, only the frequency of IL-17^single+CD4⁺ T cells was higher among LTBI children compared to those with aTB (E). Results are illustrated as medians (horizontal bars), 10th–90th percentiles (boxes) and ranges. ***p < 0.005; **p ≤ 0.01; *p < 0.05.