CD4 and CD8 T-cell responses to mycobacterial antigens in African children

Abstract

Rationale: The current tuberculosis (TB) vaccine, bacille Calmette-Guérin (BCG), does not provide adequate protection against TB disease in children. Furthermore, more efficacious TB vaccines are needed for children with immunodeficiencies such as HIV infection, who are at highest risk of disease.

Objectives: To characterize mycobacteria-specific T cells in children who might benefit from vaccination against TB, focusing on responses to antigens contained in novel TB vaccines.

Methods: Whole blood was collected from three groups of BCG-vaccinated children: HIV-seronegative children receiving TB treatment (n = 30), HIV-infected children (n = 30), and HIV-unexposed healthy children (n = 30). Blood was stimulated with Ag85B and TB10.4, or purified protein derivative, and T-cell cytokine production by CD4 and CD8 was determined by flow cytometry. The memory phenotype of antigen-specific CD4 and CD8 T cells was also determined.

Measurements and main results: Mycobacteria-specific CD4 and CD8 T-cell responses were detectable in all three groups of children. Children receiving TB treatment had significantly higher frequencies of antigen-specific CD4 T cells compared with HIV-infected children (P = 0.0176). No significant differences in magnitude, function, or phenotype of specific T cells were observed in HIV-infected children compared with healthy control subjects. CD4 T cells expressing IFN-gamma, IL-2, or both expressed a CD45RA(-)CCR7(-)CD27(+/-) effector memory phenotype. Mycobacteria-specific CD8 T cells expressed mostly IFN-gamma in all groups of children; these cells expressed CD45RA(-)CCR7(-)CD27(+/-) or CD45RA(+)CCR7(-)CD27(+/-) effector memory phenotypes.

Conclusions: Mycobacteria-specific T-cell responses could be demonstrated in all groups of children, suggesting that the responses could be boosted by new TB vaccines currently in clinical trials.

Figure 1.

Functional characterization of Ag-specific T-cell cytokine expression by intracellular cytokine staining and flow cytometry. (A) Representative density plots from a single healthy child showing the gating strategy used to identify CD4 and CD8 T cells from whole blood. From left to right, upper plots, cell doublets were excluded using forward scatter/side scatter (FSC/SSC)-area and FSC-height parameters. Small lymphocytes were then selected. Lower plots, selection of T cells by gating on CD3⁺ cells, followed by selection of CD4 and CD8 T cells. (B) Representative plots showing IL-2 and IFN-γ expression by unstimulated (Nil) or Ag85B/TB10.4-stimulated CD4 or CD8 T cells. FITC = fluorescein isothiocyanate.

Figure 2.

Mycobacteria-specific T-cell responses measured by intracellular cytokine staining and flow cytometry in whole blood from the three groups of children. Each symbol represents an individual, and for each plot the median is represented by the horizontal line. (A) Frequencies of Ag85B/TB10.4-specific, total cytokine⁺ (IFN-γ⁺ and/or IL-2⁺) CD4 T cells, total IFN-γ⁺, or total IL-2⁺ CD4 T cells. (B) Frequencies of purified protein derivative (PPD)-specific CD4 T cells. (C) Frequencies of CD8 T cells expressing IFN-γ⁺ after stimulation with Ag85B/TB10.4 (left) or PPD (right) or total IL-2⁺ CD4 T cells. Differences between any two groups were calculated using the Mann Whitney U test. HC = healthy control subjects; HIV = children with HIV-1 infection; TB = children with tuberculosis.

Figure 3.

Analysis of the quality of mycobacteria-specific T cells measured by intracellular cytokine staining and flow cytometry in whole blood from the three groups of children. Each symbol represents an individual and for each plot, the median is represented by the horizontal line. (A) Frequencies of bifunctional or monofunctional Ag85B/TB10.4-specific CD4 T cells. (B) Pie charts representing the median proportions of cells coexpressing IFN-γ and/or IL-2, among the total CD4 T-cell response, after stimulation with Ag85B/TB10.4 or purified protein derivative (PPD). (C) Frequencies of bifunctional or monofunctional Ag85B/TB10.4-specific CD8 T cells. (D) Pie charts representing the median proportions of cells coexpressing IFN-γ and/or IL-2, among the total CD8 T-cell response, after stimulation with Ag85B/TB10.4. Differences between any two groups were calculated using the Mann Whitney U test. HC = healthy control subjects; HIV = children with HIV-1 infection; TB = children with tuberculosis.

Figure 4.

Memory phenotype of mycobacteria-specific T cells in healthy children. (A) Flow cytometric analysis of CCR7, CD45RA, and CD27 expression on total CD4 T cells (red background) and Ag85B/TB10.4-specific CD4 T cells, expressing IFN-γ (black dots, upper plots) or IL-2 (black dots, lower plots). (B) Relative proportion of memory phenotype marker coexpression by the indicated cytokine-producing CD4 T-cell populations, or the total CD4 T-cell population, after stimulation of whole blood with Ag85B/TB10.4. (C) Relative proportion of memory phenotype marker coexpression by IFN-γ⁺ CD8 T cells, or the total CD8 T cell population, after stimulation of whole blood with Ag85B/TB10.4.

Figure 5.

Comparison of Ag85B/TB10.4-specific T-cell memory phenotype between the three groups of children. Relative proportions of memory phenotype marker coexpression by (A) IFN-γ⁺ CD4 T cells, or (B) IL-2⁺ CD4 T cells for the different groups. (C) Relative proportions of memory phenotype marker coexpression by IFN-γ⁺ CD8 T cells. No significant differences were observed. HC = healthy control subjects; HIV = children with HIV-1 infection; TB = children with tuberculosis.