Phenotypic definition of effector and memory T-lymphocyte subsets in mice chronically infected with Mycobacterium tuberculosis

Abstract

The bacterium Mycobacterium tuberculosis remains one of the world's most successful pathogens, a situation that is aggravated by the fact that the existing vaccine, Mycobacterium bovis BCG, is not effective in adults. As with any vaccine, the purpose of giving BCG vaccination is to establish a long-lived state of memory immunity, but whether this is successfully completely established is still unclear. It is generally accepted that memory T cells can be divided into central and effector memory populations by function and by phenotype; however, the majority of data supporting this division have been generated using transgenic mouse models or mice that have recovered from acute viral infections. Tuberculosis, on the other hand, represents a persistent, chronic state of immunity in which the presence of memory T cells is far less well defined. We show here that mice vaccinated with BCG or chronically infected with M. tuberculosis establish antigen-specific populations of cells within the lungs that predominantly express a cellular phenotype consistent with their being effector or effector memory cells. In contrast, cells with a central memory phenotype exist in much lower numbers in the lungs but can be found in significantly larger numbers in the spleen, where they may represent a potential reservoir. These data suggest that the effector-to-central-memory T-cell transition may well be minimal in these persisting mycobacterial infections, and they support a novel hypothesis that this may explain the fundamental basis of the failure of the BCG vaccine in humans.

FIG. 1.

Changes in CD4 and CD8 T-cell subsets expressing markers for T_EM and T_CM in target organs over the course of the study. (A) Representative gating strategy to identify and quantitate CD62L^lo CCR7^lo CD44^hi T_EM and CD62L^hi CCR7^hi CD44^hi T_CM populations. (B) Numbers of CD4 T_EM, CD8 T_EM, CD4 T_CM, and CD8 T_CM in the lungs versus time of the infection. Test groups were mice chronically infected with M. tuberculosis (▪), mice that had been subcutaneously vaccinated with BCG (▴), or age-matched uninfected controls (▾). In each case, the data point represents the log₁₀ mean value for cells recovered from the lungs or other organs of four or five mice; standard errors of the means (SEM) are omitted for clarity and did not exceed 10%. In all cases, numbers of cells were increased in the infected animals compared to uninfected age-matched controls (for M. tuberculosis infection at all time points, P < 0.01 or lower for T_EM values and P < 0.02 for T_CM values; for BCG, P < 0.04 [day 40 CD4 and CD8 T_EM] and P < 0.03 or lower [for all other data points]).

FIG. 2.

Numbers of CD4 T_EM and CD8 T_EM (top panels) and CD4 T_CM and CD8 T_CM (bottom panels) in the spleen and draining mediastinal lymph nodes versus time. Test groups were mice chronically infected with M. tuberculosis (▪), mice that had been subcutaneously vaccinated with BCG (▴), or age-matched uninfected controls (▾). In each case, the data point represents the log₁₀ mean value for cells recovered from the lungs or other organs of four or five mice; SEM are omitted for clarity and did not exceed 10%. All data points for the spleen were significantly higher than for uninfected controls (P < 0.02 or lower). In the lymph nodes, T_EM data points were significant (P < 0.04 or lower), but BCG data points did not reach significance (P < 0.05) until the day 90 time point. Similarly, values for T_CM did not reach significance until day 90 and were borderline (P < 0.05).

FIG. 3.

Percentages of tetramer-positive CD4 and CD8 cells (means ± SEM; n = 4 or 5) in target organs in the two infection groups, analyzed on day 100 of each infection. BM, bone marrow aspirates. CD4 cells against a major epitope of ESAT-6 and CD8 cells against a major epitope of Mtb32 were detected.

FIG. 4.

Changes in numbers in a CD8 subset expressing the CD62L^lo CCR7^lo CD44^hi CD45RB^hi phenotype. (A) Gating strategy. (B) Numbers of cells recovered from organs of mice chronically infected with M. tuberculosis (▪), mice that had been subcutaneously vaccinated with BCG (▴), or age-matched uninfected controls (▾). In each case, the data point represents the log₁₀ mean value for cells recovered from the organs of four or five mice; SEM are omitted for clarity and did not exceed 10%. All data points for the lungs and spleen were raised over uninfected control values (P < 0.03 or lower). Similar ranges of P values were seen in the lymph nodes and bone after day 90 of the study.

FIG. 5.

Changes in numbers of CD4 and CD8 T_EM cells that stained positive for IFN-γ. (A) Gating strategy. (B) Numbers of CD4 cells and CD8 cells recovered from the indicated organs from mice chronically infected with M. tuberculosis (▪), mice that had been subcutaneously vaccinated with BCG (▴), or age-matched uninfected controls (▾). In each case, the data point represents the log₁₀ mean value for cells recovered from the organs of four or five mice; SEM are omitted for clarity and did not exceed 10%. All data points were higher than uninfected control values (P < 0.02 or lower), except for the lungs of BCG-vaccinated mice at the first two time points.

FIG. 6.

Changes in numbers of CD4 and CD8 T_CM cells that stained positive for IFN-γ. Data shown are from mice chronically infected with M. tuberculosis (▪), mice that had been subcutaneously vaccinated with BCG (▴), or age-matched uninfected controls (▾). In each case, the data point represents the log₁₀ mean value for cells recovered from the organs of four or five mice; SEM are omitted for clarity and did not exceed 10%. In the case of CD4 cells, only values for the M. tuberculosis-infected group were raised above control values (P < 0.03 or lower), whereas for CD8 cells, these were raised in both test groups after day 60 (P < 0.04). As noted, however, these values were at the limit of assay detection and should be viewed with caution.

FIG. 7.

Analysis of CD127 and CD45RB subsets. (A) Gating strategies. (B) Changes in numbers of CD4 and CD8 T-cell subsets expressing the CD62L^lo CCR7^hi CD44^hi CD127^hi phenotype. Data shown are from mice chronically infected with M. tuberculosis (▪), mice that had been subcutaneously vaccinated with BCG (▴), or age-matched uninfected controls (▾). In each case, the data point represents the log₁₀ mean value for cells recovered from the organs of four or five mice; SEM are omitted for clarity and did not exceed 10%. Numbers of CD4 cells in the lungs were not significant; all other data points were raised above control values (P < 0.04 or lower). (C) Changes in numbers of CD4 and CD8 T-cell subsets expressing the CD62L^lo CCR7^hi CD44^hi CD45RB^lo phenotype. Data shown are from mice chronically infected with M. tuberculosis (▪), mice that had been subcutaneously vaccinated with BCG (▴), or age-matched uninfected controls (▾). In each case the data point represents the log₁₀ mean value for cells recovered from the organs of four or five mice; SEM are omitted for clarity and did not exceed 10%. Values for CD4 cells were raised above uninfected control values (P < 0.04 or lower), whereas only those in the M. tuberculosis-infected test group were raised (P < 0.01) in the case of CD8 cells.