Production of Proinflammatory Cytokines by CD4+ and CD8+ T Cells in Response to Mycobacterial Antigens among Children and Adults with Tuberculosis

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of pediatric morbidity and mortality. Young children are at high risk of TB following Mtb exposure, and this vulnerability is secondary to insufficient host immunity during early life. Our primary objective was to compare CD4+ and CD8+ T-cell production of proinflammatory cytokines IFN-gamma, IL-2, and TNF-alpha in response to six mycobacterial antigens and superantigen staphylococcal enterotoxin B (SEB) between Ugandan adults with confirmed TB (n = 41) and young Ugandan children with confirmed (n = 12) and unconfirmed TB (n = 41), as well as non-TB lower respiratory tract infection (n = 39). Flow cytometry was utilized to identify and quantify CD4+ and CD8+ T-cell cytokine production in response to each mycobacterial antigen and SEB. We found that the frequency of CD4+ and CD8+ T-cell production of cytokines in response to SEB was reduced in all pediatric cohorts when compared to adults. However, T-cell responses to Mtb-specific antigens ESAT6 and CFP10 were equivalent between children and adults with confirmed TB. In contrast, cytokine production in response to ESAT6 and CFP10 was limited in children with unconfirmed TB and absent in children with non-TB lower respiratory tract infection. Of the five additional mycobacterial antigens tested, PE3 and PPE15 were broadly recognized regardless of TB disease classification and age. Children with confirmed TB exhibited robust proinflammatory CD4+ and CD8+ T-cell responses to Mtb-specific antigens prior to the initiation of TB treatment. Our findings suggest that adaptive proinflammatory immune responses to Mtb, characterized by T-cell production of IFN-gamma, IL-2, and TNF-alpha, are not impaired during early life.

Keywords: Mycobacterium tuberculosis; T cells; adaptive immunity; cytokines; pediatric.

Figure 1

Proportion of children and adults with detectable CD4+ and CD8+ T-cell cytokine response to six mycobacterial antigens. PBMC isolated from children with confirmed (n = 12), unconfirmed TB (n = 41), non-TB lower respiratory tract infection (n = 39) and adults with confirmed TB (n = 41), were thawed in batches, rested overnight, and CD4+ and CD8+ T-cell production of IFN-γ, IL-2, and TNF-α in response to 6 peptide pools representing mycobacterial antigens quantified by intracellular flow cytometry following 18 h of stimulation (ICS). A positive response to each peptide pool was defined as ≥0.05% of CD4+ (A) or CD8+ (B) T cells producing a cytokine following background correction. * Indicates adjusted p-value ≤ 0.05. * Illustrates significant differences between adults and children with confirmed TB.

Figure 2

Distribution of CD4+ and CD8+ T cells that produce cytokines in response to ESAT6/CFP10. Median percentages (frequencies) of CD4+ (A) and CD8+ (B) T cells that produce IFN-γ, IL-2, or TNF-α in response to EC peptide pools were quantified by ICS (n = 12 PedTB; n = 41 AdultTB; n = 41 PedUncTB; n = 39 PedLRTI). Background correction (subtraction of the unstimulated/resting responses) was performed, and responses < 0.05% set to zero. Comparisons of medians among cohorts were performed using Kruskal–Wallis test; post hoc comparisons were performed using Dunn’s test of multiple comparisons. Shown are medians with IQR. Pie charts visualize the fraction of donors within each cohort with unresponsive CD4+ (C) and CD8+ T cells (D) (grey slices). The colored slices visualize the fraction of exclusively single, double, or triple positive CD4+ (C) or CD8+ (D) T cells among those participants with a detectable cytokine response within each cohort. * Indicates adjusted p-value < 0.05.

Figure 3

Distribution of CD4+ and CD8+ T cells that produce cytokines in response to PE3. Median percentages (frequencies) of CD4+ (A) and CD8+ (B) T cells that produce IFN-γ, IL-2, or TNF-α in response to PE3 peptide pool were quantified by ICS (n = 12 PedTB; n = 41 AdultTB; n = 38 PedUncTB; n = 39 PedLRTI). Background correction (subtraction of the unstimulated/resting responses) was performed, and responses <0.05% set to zero. Comparisons of medians among cohorts were performed using Kruskal–Wallis test; post hoc comparisons were performed using Dunn’s test of multiple comparisons. Shown are medians with IQR. Pie charts visualize the fraction of donors within each cohort with unresponsive CD4+ (C) and CD8+ T cells (D) (grey slices). The colored slices visualize the fraction of exclusively single, double, or triple positive CD4+ (C) or CD8+ (D) T cells among those participants with a detectable cytokine response within each cohort. * Indicates adjusted p-value < 0.05.

Figure 4

Distribution of CD4+ and CD8+ T cells that produce cytokines in response to PPE15. Median percentages (frequencies) of CD4+ (A) and CD8+ (B) T cells that produce IFN-γ, IL-2, or TNF-α in response to PPE15 peptide pool were quantified by ICS (n = 12 PedTB; n = 41 AdultTB; n = 38 PedUncTB; n = 39 PedLRTI). Background correction (subtraction of the unstimulated/resting responses) was performed, and responses <0.05% set to zero. Comparisons of medians among cohorts were performed using Kruskal–Wallis test; post hoc comparisons were performed using Dunn’s test of multiple comparisons. Shown are medians with IQR. Pie charts visualize the fraction of donors within each cohort with unresponsive CD4+ (C) and CD8+ T cells (D) (grey slices). The colored slices visualize the fraction of exclusively single, double, or triple positive CD4+ (C) or CD8+ (D) T cells among those participants with a detectable cytokine response within each cohort. * Indicates adjusted p-value < 0.05.

Figure 5

Distribution of CD4+ and CD8+ T cells that produce cytokines in response to SEB. Median percentages (frequencies) of CD4+ (A) and CD8+ (B) T cells that produce IFN-γ, IL-2, or TNF-α in response to SEB were quantified by ICS (n = 12 PedTB; n = 41 AdultTB; n = 41 PedUncTB; n = 39 PedLRTI). Background correction (subtraction of the unstimulated/resting responses) was performed, and responses <0.05% set to zero. Comparisons of medians among cohorts were performed using Kruskal–Wallis test; post hoc comparisons were performed using Dunn’s test of multiple comparisons. Shown are medians with IQR. Pie charts visualize the fraction of donors within each cohort with unresponsive CD4+ (C) and CD8+ T cells (D) (grey slices). The colored slices visualize the fraction of exclusively single, double, or triple positive CD4+ (C) or CD8+ (D) T cells among those participants with a detectable cytokine response within each cohort. * Indicates adjusted p-value < 0.05.