Ectopic activation of Mycobacterium tuberculosis-specific CD4+ T cells in lungs of CCR7-/- mice

Abstract

Initiation of an adaptive cellular immune response depends on intimate interactions with APCs and naive T lymphocytes. We previously reported that activation of naive Mycobacterium tuberculosis-specific CD4+ T cells depends on dendritic cell (DC) transport of live bacteria from the lungs to the mediastinal lymph node (MDLN). Because the migratory paths of DCs are largely governed by the chemokine receptor CCR7, which is expressed on DCs upon maturation by proinflammatory stimuli, we examined the quantitative contribution of CCR7-dependent DC migration in the context of tuberculosis. We found that early trafficking of DCs from the lungs to the MDLN depended on CCR7-mediated signaling, but alternative mechanism(s) are used later in infection. Impaired migration of DCs in CCR7(-/-) mice resulted in delayed dissemination of bacteria to MDLN and spleen and in delayed kinetics of activation of adoptively transferred Ag85B-specific CD4+ T cells. Furthermore, in contrast to control mice, we found that naive Ag85B-specific CD4+ T cells are activated to proliferate in the lungs of CCR7(-/-) mice and, when infected with higher doses of bacteria, resistance to M. tuberculosis infection in CCR7(-/-) mice is compromised compared with wild-type mice.

FIGURE 1

Impaired trafficking of dendritic cells from lungs to MDLN in CCR7^−/− mice. CD11b⁺CD11c⁺ dendritic cells were quantitated in lungs and mediastinal lymph nodes of CCR7^+/+ and CCR7^−/− mice at days 14, 21 and 28 after aerosol infection with a low dose of M. tuberculosis. Unpaired Student’s t test was performed, * P = 0.035, ***P = 0.0005. In an additional independent experiment, we found similar results, and we found that the number of lung and MDLN DCs did not differ between CCR7^−/− and CCR7^+/+ at day 35 post infection.

FIGURE 2

Trafficking of M. tuberculosis from the lungs to the MDLN and spleen is delayed in CCR7^−/− mice compared with controls. CCR7^+/+ and CCR7^−/− mice were sacrificed at the indicated time points after aerosol infection with a low dose of M. tuberculosis and colony-forming units were determined in homogenates of lungs, mediastinal lymph node, and spleen. Data are represented as mean ± SD of four mice per group and per time point. * P< 0.05, ** P<0.005, *** P<0.001 by unpaired Student’s t test.

FIGURE 3

Proliferation of M. tuberculosis Ag85B–specific CD4⁺ T cells is delayed in MLN of CCR7^−/− compared to wild type mice. (A) CFSE dilution profile of cells in the mediastinal lymph node of CCR7^+/+ and CCR7^−/− mice over the course of infection with M. tuberculosis. Plots are representative of four mice per group at each time point. In uninfected CCR7^−/− (but not CCR7^+/+) mice, a small fraction of adoptively-transferred P25TCR-Tg CD4⁺ T cells exhibited fluoresence half as intense as that of undivided cells; the cause of this is unknown. (B) Total number of P25TCR-Tg CD4⁺ T cells was calculated within the CD4⁺ population in mediastinal lymph node of CCR7^+/+ and CCR7^−/− mice. Data are represented as mean ± SD of four mice per group and per time point. ** P < 0.005 by unpaired Student’s t test. (C) 2×10⁶ CFSE-labeled P25TCR-Tg CD4⁺ T cells were transferred either into naive CCR7^+/+ or CCR7^−/− recipients; 4 hours later, mice were euthanized and CFSE⁺ P25TCR-Tg CD4⁺ T cells were isolated from different organs (mean ± SD; n = 4 mice per group).

FIGURE 4

M. tuberculosis Ag85B–specific CD4⁺ T cells proliferate in lungs of CCR7^−/− mice. (A) CFSE dilution profile of P25TCR-Tg CD4⁺ T cells in the lungs of CCR7^+/+ and CCR7^−/− mice over the course of infection with M. tuberculosis. Plots are representative of four mice per group at each time point. (B) Total number of P25TCR-Tg CD4⁺ T cells was calculated within the CD4⁺ population in lung of CCR7^+/+ and CCR7^−/− mice. Data are represented as mean ± SD of four mice per group and per time point. * P = 0.016 by unpaired Student’s t test.

FIGURE 5

Survival of M. tuberculosis-infected CCR7^−/− mice. (A) CCR7^+/+ and CCR7^−/− mice were aerosol infected with a low dose (~100 cfu) or a high dose (~600 cfu) of M. tuberculosis and survival was monitored. n = 6–8 mice per group. (B) CCR7^+/+ and CCR7^−/− mice were sacrificed 28 days after aerosol infection with a high dose of M. tuberculosis (~600cfu) and colony-forming units were determined in lung homogenates. * P = 0.033 by unpaired Student’s t test.