Gamma interferon-producing CD4+ T lymphocytes in the lung correlate with resistance to infection with Mycobacterium tuberculosis

Abstract

The human immune system efficiently limits the replication of Mycobacterium tuberculosis in most infected individuals. Only 5 to 10% of infected people develop clinical tuberculosis, a sign of the inability of the immune system to control the infection. We have studied the C3H/HeJ (C3H) and C57BL/6 (B6) inbred mouse strains, which differ in their susceptibility to tuberculosis, in order to ascertain the immunological determinants of a successful immune response against M. tuberculosis and to establish a system to identify genes that influence susceptibility to tuberculosis. We found that the resistant B6 mice were able to control infection in both the lung and spleen, while susceptible C3H mice were incapable of limiting bacteria growth, especially in the lung, and succumbed to infection within 4 weeks. We determined that the susceptibility of C3H mice was independent of the Toll-like receptor 4 (tlr4) genetic locus and allelic major histocompatibility complex differences. Although the splenic immune responses were similar in the two mouse strains, the local immune responses in the lungs of the infected mice differed greatly. The pulmonary immune response in resistant B6 mice was characterized by an early influx of both CD4+ and CD8+ lymphocytes that produced gamma interferon (IFN-gamma). In contrast, the immune response of C3H mice in the lung was characterized by a delayed and decreased influx of lymphocytes, which produced little IFN-gamma. These results suggest an important role for the early appearance of IFN-gamma-producing lymphocytes in the lung in resistance to infection with M. tuberculosis.

FIG. 1

Survival of C57BL/6J and C3H/HeJ mice following M. tuberculosis infection. (A) C57BL/6J (solid line) and C3H/HeJ (broken line) mice were inoculated i.v. with 10⁶ CFU of M. tuberculosis (Erdman). The results were pooled from three independent experiments with a total of 33 or 34 mice in each group. The survival curves were generated using the Kaplan-Meier method, and the increased mortality of the C3H/HeJ mice was statistically significant (P < 0.0001 by the log rank test). C3H/HeJ MST = 28 days; C57BL/6 MST > 190 days. (B) Survival of C57BL/6, C3H/HeJ, C3H.SW-(H-2^b)/SnJ, and B6C3F1 mice following M. tuberculosis infection. The survival of C3H/HeJ (H-2^k) and C3H.SW-(H-2^b)/SnJ mice was similar following infection (P, not significant). In contrast, the prolonged survival of C57BL/6 and the F1 mice was statistically significant compared to the inbred C3H mouse strains (P < 0.0001). (C) Survival of C3H/HeJ and C3H/HeOuJ mice following M. tuberculosis infection. C3H/HeJ (dashed line) and C3H/HeOuJ (solid line) mice were infected as described above. Each group contained 10 mice, and the differences in survival between the two mouse strains following infection were not statistically significant by the log rank test. C3H/HeJ MST = 31 days; C3H/HeOuJ MST = 35 days.

FIG. 2

Mycobacterial burden in the target organs from infected mice. C57BL/6J (solid line) and C3H/HeJ (dashed line) mice were inoculated i.v. with 10⁶ CFU of M. tuberculosis. The number of CFU in the lung and spleen was determined weekly as described in Materials and Methods. Error bars represent the standard errors of the means, and the data are representative of four independent experiments.

FIG. 3

Lung pathology from M. tuberculosis-infected mice. Lung tissue was obtained from C57BL/6J mice (panels 1, 3, 5, and 7) and from C3H/HeJ mice (panels 2, 4, 6, and 8) 21 days after intravenous inoculation with M. tuberculosis. The gross appearances of the lungs from M. tuberculosis-infected B6 mice (panel 1) and C3H mice (panel 2) are compared. Shown are representative sections of formalin-fixed paraffin-embedded lung tissue stained with hematoxylin and eosin (panels 3 and 4; magnification, ×25), with trichrome (which stains collagen fibers blue) (panels 5 and 6; magnification, ×50), or for AFB (panels 7 and 8; magnification, ×4,000). See text for details.

FIG. 4

The numbers of CD4⁺ and CD8⁺ cells in the lungs and spleens of C57BL/6J (solid line) and C3H/HeJ (dashed line) mice infected with M. tuberculosis. The data represent the number of cells per half organ. This figure is representative of four independent experiments, and the numbers have been normalized to cells per half organ. The data for uninfected mice (day 0) represent the mean of three independent determinations carried out using pooled tissue from 5 to 10 mice, and these numbers have also been normalized.

FIG. 5

IFN-γ production by lymphocytes from the spleens and the lungs of uninfected and infected C57BL/6J mice. Splenocytes and lung mononuclear cells were pooled from uninfected or infected mice 3 weeks after i.v. inoculation with M. tuberculosis. Cells were cultured in vitro with brefeldin A for 3.5 h, either in the presence (stimulated condition; upper row) or absence (unstimulated condition; lower row) of PMA and ionomycin. Flow cytometry detected IFN-γ production by CD4⁺ lymphocytes as described in Materials and Methods. The numbers in the upper right quadrant of each dot plot are the percentages of lymphoid cells that were CD4⁺ and (in parentheses) the percentages of CD4⁺ cells that produced IFN-γ. These figures are representative of four independent experiments.

FIG. 6

IFN-γ production by lymphocytes from the lungs of C57BL/6J (upper row) and C3H/HeJ (lower row) mice after infection with M. tuberculosis. At weekly time points after i.v. inoculation, lung mononuclear cells were cultured with brefeldin A, PMA, and ionomycin for 3.5 h. Size-gated lymphoid cells were analyzed by flow cytometry to determine IFN-γ production. The numbers in the upper right quadrant of each dot plot are the percentages of lymphoid cells that were CD4⁺ and (in parentheses) the percentages of CD4⁺ cells that produced IFN-γ. These figures are representative of four independent experiments.

FIG. 7

IFN-γ production by CD4⁺ or CD8⁺ lymphocytes from the spleens and the lungs of C57BL/6 (solid line) and C3H/HeJ (dashed line) mice. At weekly time points after infection, splenocytes or lung mononuclear cells were cultured with brefeldin A for 3.5 h, either in the presence or absence of PMA and ionomycin. Cells were analyzed by flow cytometry as described in Materials and Methods. The data represent the number of cells per half organ. These figures are representative of four independent experiments. The data for uninfected mice (day 0) are the averages of three independent determinations carried out using pooled tissue from 5 to 10 mice and have been normalized to cells per half organ.