Mycobacterium tuberculosis growth control by lung macrophages and CD8 cells from patient contacts

Abstract

Rationale: Healthy household contacts (HHCs) of patients with active pulmonary tuberculosis are exposed aerogenically to Mycobacterium tuberculosis (Mtb), thus permitting the study of protective local immunity.

Objectives: To assess alveolar macrophage (AM) and autologous blood CD4 and CD8 T-cell-mediated Mtb growth control in HHCs and healthy, unexposed community control subjects (CCs).

Methods: AMs were infected with Mtb strains H(37)Ra and H(37)Rv at multiplicities of infection 0.1 and 1. Mtb colony-forming units were evaluated on Days 1, 4, and 7.

Main results: CD8 T cells from HHCs in 1:1 cocultures with AMs significantly (p < 0.05) increased Mtb growth control by AMs. In CCs, no detectable contribution of CD8 T cells to Mtb growth control was observed. CD4 T cells did not increase Mtb growth control in HHCs or in CCs. IFN-gamma, nitric oxide, and tumor necrosis factor were determined as potential mediators of Mtb growth control in AMs and AM/CD8 and AM/CD4 cocultures. IFN-gamma production in AM/CD4 was twofold higher than that in AM/CD8 cocultures in both HHCs and CCs (p < 0.05). Nitric oxide production from AMs of HHCs increased on Days 4 and 7 and was undetectable in AMs from CCs. IFN-gamma and nitric acid concentrations and Mtb growth control were not correlated. Tumor necrosis factor levels were significantly increased in AM/CD8 cocultures from HHCs compared with AM/CD8 cocultures from CCs (p < 0.05).

Conclusion: Aerogenic exposure to Mtb in HHCs leads to expansion of Mtb-specific effector CD8 T cells that limit Mtb growth in autologous AMs.

Figure 1.

Inhibition by peripheral CD8 T cells of intracellular mycobacterial growth in alveolar macrophages (AMs) from healthy household contacts (HHCs) of patients with tuberculosis (TB). AMs alone (closed circles) and AMs in coculture with autologous CD8 T cells in a 1:1 ratio (AM/CD8; open circles) were infected with Mtb H₃₇Ra (n = 11) or H₃₇Rv (n = 13) at 0.1:1 (A, C) or 1:1 (B, D) bacteria/AM ratios for 1 h, 4 and 7 d (Days 0, 4, and 7). AM/CD8 colony-forming units (cfu) were lower than AM cfu in 8 of 11 subjects for H₃₇Ra 0.1:1 (Day 7), and in 9 of 11 subjects for H₃₇Ra 1:1 (Days 4 and 7). Mean cfu ± SEM cfu and significant differences (p < 0.05) are shown.

Figure 2.

IFN-γ production in AMs and AM/CD8 cocultures from HHCs. IFN-γ production by AMs alone (black bars) or AMs in coculture with autologous CD8 T cells in a 1:1 ratio (AM/CD8; white bars) from HHCs was assessed in culture supernatants by ELISA on Days 0, 4, and 7 (Day 0, 4, and 7) (A–D). Experiments were performed as indicated in Figure 1. Bars represent mean values ± SEM. Significant differences between IFN-γ concentrations from AMs and AM/CD8 cocultures (p < 0.05) are shown.

Figure 3.

Effect of CD4 T cells on intracellular mycobacterial growth (cfu; A) and IFN-γ (B) production in AM cultures from HHCs (n = 4). AMs alone (black circles) or AMs in coculture with autologous CD4 T cells in a 1:1 ratio (AM/CD4; white circles) were infected with Mtb H₃₇Ra (A) or H₃₇Rv (B) at 0.1:1 bacteria/AM ratio for 1 h, 4 d, and 7 d (Days 0, 4, and 7), and cfu were determined. IFN-γ was assessed by ELISA in culture supernatants from AMs alone (black bars) and from AM/CD4 cocultures (gray bars). Mean cfu and IFN-γ concentrations ± SEM are shown.

Figure 4.

Effect of autologous peripheral CD8 and CD4 T cells on the mycobacterial growth in AMs from concurrent non–Mtb-exposed community control subjects (CCs). AMs (black circles) were infected with Mtb H₃₇Ra (A; n = 8) or H₃₇Rv (B; n = 9) at a 1:1 bacteria/AM ratio, and incubated alone or with autologous peripheral CD8 (white circles) or CD4 (inverted black triangles) T cells for 1 h (Day 0), 4 d (Day 4), or 7 d (Day 7). Mean cfu ± SEM are shown. No significant differences were found at any time point or condition tested.

Figure 5.

IFN-γ production by AMs alone (black bars) or by AMs in 1:1 coculture with autologous CD8 (AM/CD8; light gray bars) or CD4 (AM/CD4; dark gray bars) T cells in CCs. AMs were infected with Mtb H₃₇Ra (A; n = 8) or H₃₇Rv (B; n = 11) in the indicated conditions. Supernatants were harvested at the indicated time points and IFN-γ assessed by ELISA. Bars represent means ± SEM. Significant differences (p < 0.05) were found between AMs and AM/CD8 or AM/CD4 cocultures and between AM/CD8 and AM/CD4 cocultures.