Antigen-specific CD8+ T cells and the development of central memory during Mycobacterium tuberculosis infection

Abstract

Whether true memory T cells develop in the face of chronic infection such as tuberculosis remains controversial. To address this question, we studied CD8+ T cells specific for the Mycobacterium tuberculosis ESAT6-related Ags TB10.3 and TB10.4. The shared epitope TB10.3/10.4(20-28) is presented by H-2 K(d), and 20-30% of the CD8+ T cells in the lungs of chronically infected mice are specific for this Ag following respiratory infection with M. tuberculosis. These TB10.3/10.4(20-28)-specific CD8+ T cells produce IFN-gamma and TNF and express CD107 on their cell surface, which indicates their likely role as CTL in vivo. Nearly all of the Ag-specific CD8+ T cells in the lungs of chronically infected mice had a T effector cell phenotype based on their low expression of CD62L and CD45RB. In contrast, a population of TB10.3/10.4(20-28)-specific CD8+ T cells was identified in the lymphoid organs that express high levels of CD62L and CD45RB. Antibiotic treatment to resolve the infection led to a contraction of the Ag-specific CD8+ T cell population and was accompanied by an increase in the proportion of CD8+ T cells with a central memory phenotype. Finally, challenge of memory-immune mice with M. tuberculosis was accompanied by significant expansion of TB10.3/10.4(20-28)-specific CD8+ T cells, which suggests that these cells are in fact functional memory T cells.

FIGURE 1

Distribution of TB10.4_20–28-specific CD8⁺ T cells during respiratory M. tuberculosis infection. A, The frequency of TB10.3/10.4_20–28-specific CD8⁺ T cells in different tissues from M. tuberculosis-infected mice. Lymphocytes were gated by size and by CD8⁺ staining. The percentage of CD8⁺ cells that stain with the TB10.3/10.4_20–28-loaded H-2 K^d tetramer (TB10.4 K^d tetramer) is indicated in each dot plot. This value is referred to as the percentage of CD8⁺tet⁺ throughout the following figures. Representative FACS plots are shown for a cohort of mice that were analyzed 11 wk after aerosol infection. Ax, axillary. B, The percentage of CD8⁺ cells in the lung, spleen, and PLN that stain with the TB10.4 K^d tetramer at various time points after respiratory M. tuberculosis infection. Each data point represents the mean ± SE of between three and eight determinations for each time point (except for weeks 11 and 32, for which there was only a single determination), representing a total of 82 determinations. The data were generated from the analysis of individual mice and from pooled tissue. C, The absolute number of TB10.3/10.4_20–28-specific CD8⁺ T cells in the lungs of infected mice from a representative experiment. Each data point represents the mean of three mice ± SE. D, The relationship between Ag-specific CD8⁺ T cells detected in blood and the lung. The frequency of TB10.4_20–28-specific CD8⁺ T cells in the blood and the lung of M. tuberculosis-infected mice was determined by flow cytometry using tetramer analysis. Individual mice were analyzed 4–11 wk after infection. The lines indicated paired samples from the same individual.

FIGURE 2

Cytokine production by TB10.4_20–28-specific CD8⁺ T cells. A, Intracellular cytokine production after stimulation with the TB10.4_20–28 peptide. Three months after M. tuberculosis infection, lung MNC were cultured in vitro in the absence (top row) or presence (bottom row) of 10 µM TB10.4_20–28 for 12 h. The cells were gated by size, and CD8⁺tet⁺ cells were analyzed for their production of IFN-γ or TNF. The percentage of CD8⁺tet⁺ cells producing IFN-γ or TNF is indicated in each contour plot. This experiment is representative of three independent experiments. B, Cytokine production by TB10.4_20–28-specific CD8⁺ cells from infected lung (left) and spleen (right) after in vitro culture in the absence (none) or presence (peptide) of the TB10.4_20–28 peptide. Intracellular cytokine staining was performed, as described. Nonspecific staining determined using an isotype control was subtracted from the specific staining obtained with or without the peptide. The data are an average of three independent experiments, and the bars represent the mean ± SE. The percentage of CD8⁺tet⁺ T cells secreting cytokine in the absence or presence of peptide was significantly different, as determined by a two-way ANOVA with Bonferroni’s posttest (*, p < 0.05; **, p < 0.01). The percentage of CD8⁺tet⁺ T cells producing IFN-γ vs TNF was not significantly different. □, IFN-γ; ■, TNF.

FIGURE 3

Expression of granzyme B and CD107A/B by CD8⁺ T cells. A, Expression of granzyme B and CD107A/B by pulmonary CD8⁺ T cells. Lung MNC were obtained from mice 4 wk after infection, and CD8⁺tet⁻ (left and middle panels) and CD8⁺tet⁺ (right panel) T cells were analyzed for their ex vivo expression of intracellular granzyme B and cell surface CD107A/B. The staining with the isotype control (left) is compared with the expression of granzyme B and CD107A/B by CD8⁺tet⁻ cells (middle panel) and CD8⁺tet⁺ cells (right panel). B, Expression of CD107A/B by pulmonary and splenic CD8⁺ T cells. Lung or spleen MNC were obtained from infected mice, and the expression of CD107A/B by CD8⁺tet⁺ cells was determined by flow cytometry. The results from three independent experiments are shown. Each symbol represents individual or pooled tissue samples obtained from mice 4 to 17 wk after infection. □, Isotype control; ■, CD107A/B. Line, mean.

FIGURE 4

Appearance of TB10.4_20–28-specific CD8⁺ T cells with a central memory phenotype in the lymphoid organs of M. tuberculosis-infected mice. A, The expression of CD62L by CD8⁺tet⁻ (top row) and CD8⁺tet⁺ (bottom row) T cells in the lung, PLN, and spleen of M. tuberculosis-infected BALB/c mice. The numbers indicate the percentage of gated CD8⁺ cells that express high levels of CD62L. B, The expression of CD62L and CD45RB by CD8⁺tet⁻ (●) and CD8⁺tet⁺ (○) T cells from the lung, PLN, and spleen of M. tuberculosis-infected BALB/c mice. The percentage of CD8⁺tet⁻ and CD8⁺tet⁺ T cells that express high levels of CD62L (CD62L^high) or CD45RB (CD45RB^high) is represented in the graphs. Data represent 10–17 determinations from seven experiments, done 4–13 wk after infection (some of the symbols are superimposed). Bar, mean. A t test was done to determine whether the expression of these markers was statistically different between CD8⁺tet⁻ and CD8⁺tet⁺ T cells.

FIGURE 5

Reduction in the frequency of TB10.4_20–28-specific CD8⁺ T cells and shift toward T_CM phenotype during resolution of M. tuberculosis infection. A, The frequency of pulmonary TB10.4_20–28-specific CD8⁺ T cells in infected mice and antibiotic-treated memory-immune mice. The percentage of CD8⁺ T cells that stained with the TB10.4 K^d tetramer was determined. Data represent nine determinations from three experiments. No Rx, untreated infected mice; Abx, antibiotic-treated mice (e.g., memory-immune mice); bar, mean. The difference between the groups was tested for statistical significance using a t test (p < 0.0001). B, The absolute number of TB10.4_20–28-specific CD8⁺ T cells in the lungs of infected mice and antibiotic-treated memory-immune mice. Data represent six determinations from two experiments. No Rx, untreated infected mice; Abx, antibiotic-treated mice; bar, mean. The difference between the groups was tested for statistical significance using the Mann-Whitney U test (p = 0.0022). C, Representative flow cytometric analysis of CD8⁺ T cells from the spleen of an infected mouse (No Rx; top row) and an antibiotic-treated mouse (Abx; bottom row). Mice were infected by the aerosol route and, after 3 wk, half of the mice were started on INH and rifabutin. The data represented in this figure were obtained 12 wk later. CD8⁺tet⁻- and CD8⁺tet⁺-staining cell populations were gated (left panels), and the CD62L and CD45RB expression by these CD8⁺tet⁻ (middle panels), and CD8⁺tet⁺ (right panels) T cells was determined. The percentages in each quadrant are an average of four individual subjects that were analyzed. D, The expression of CD62L and CD45RB by CD8⁺tet⁺ T cells from the lung, PLN, and spleen of M. tuberculosis-infected mice. Mice were infected for 3 wk with M. tuberculosis and then were treated with antibiotics (Abx) for 4 (top row) or 12 wk (bottom row), or as a control were left untreated (No Rx). Differences in the phenotype of CD8⁺tet⁺ T cells in Abx-treated and untreated mice were tested for their statistical significance with a t test.

FIGURE 6

Expansion of TB10.4_20–28-specific CD8⁺ T cells following challenge of memory-immune mice with M. tuberculosis. As described in Materials and Methods, mice were infected by the aerosol route and, after 3 wk, half of the mice were treated with INH and rifabutin. After 3 wk, half of the antibiotic-treated mice (memory) were rechallenged (secondary) with aerosolized M. tuberculosis together with age-matched previously uninfected mice (primary). Four mice were analyzed per group, and these results are representative of three experiments. These results were analyzed by a one-way ANOVA with Bonferroni’s post test: *, p < 0.05; **, p < 0.01; ***, p < 0.001. A, The splenic CFU were simultaneously determined in the three experimental groups. Each symbol represents an individual mouse, and the line represents the mean. B, The frequency of TB10.4_20–28-specific CD8⁺ T cells in the lungs of mice following a primary or secondary response, and compared with memory-immune mice. Ag-specific CD8⁺ T cells were enumerated by an IFN-γ ELISPOT assay using the TB10.4_20–28 synthetic peptide (left) and by staining lung cells with TB10.4_20–28 K^d tetramers. The bars represent the mean ± SE. C, The absolute number of total (right panel) and TB10.4_20–28-specific (left panel) CD8⁺ T cells isolated using anti-CD8 immunomagnetic beads from lung MNC obtained from mice following a primary or secondary response, and compared with memory-immune mice. Ag-specific CD8⁺ T cells were enumerated using TB10.4_20–28 K^d tetramers. Bars, Mean ± SE. D, PLN pooled from four mice were analyzed by flow cytometry. CD8⁺tet⁻- and CD8⁺tet⁺-staining cell populations were gated, and the CD62L and CD45RB expression by these CD8⁺tet⁻ (left panels) and CD8⁺tet⁺ (right panels) T cells was determined.