Reappraising the Role of T Cell-Derived IFN-γ in Restriction of Mycobacterium tuberculosis in the Murine Lung

Abstract

T cells producing IFN-γ have long been considered a stalwart for immune protection against Mycobacterium tuberculosis (Mtb), but their relative importance to pulmonary immunity has been challenged by murine studies that achieved protection by adoptively transferred Mtb-specific IFN-γ-/- T cells. Using IFN-γ-/- T cell chimeric mice and adoptive transfer of IFN-γ-/- T cells into TCRβ-/-δ-/- mice, we demonstrate that control of lung Mtb burden is in fact dependent on T cell-derived IFN-γ, and, furthermore, mice selectively deficient in T cell-derived IFN-γ develop exacerbated disease compared with T cell-deficient control animals, despite equivalent lung bacterial burdens. Deficiency in T cell-derived IFN-γ skews infected and bystander monocyte-derived macrophages to an alternative M2 phenotype and promotes neutrophil and eosinophil influx. Our studies support an important role for T cell-derived IFN-γ in pulmonary immunity against tuberculosis.

Graphical abstract

FIGURE 1.

IFN-γ^−/− T cells do not reduce Mtb bacterial burden, and they exacerbate disease in T cell chimeric mice. (A) Schematic of the preparation of TCRβ^−/−δ^−/−, WT, and IFN-γ^−/− T cell chimeric mice, followed by infection with aerosolized Mtb. (B and C) Bacterial burden in lungs and spleens of Mtb-infected T cell chimeric mice at 25 dpi in one representative experiment (B), as well as group means from six independent experiments (C). Total n = 35 TCRβ^−/−δ^−/−, n = 34 WT, and n = 33 IFN-γ^−/− T cell chimeric mice. (D) Weight trends of Mtb-infected T cell chimeric mice through 29 dpi for n = 45 TCRβ^−/−δ^−/−, n = 3 WT, and n = 6 IFN-γ^−/− T cell chimeric mice in one representative experiment. Statistical significance is shown for weights at 29 dpi. Statistical significance was determined by Tukey’s range test (B and D) and paired t test (C). *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001. BM, bone marrow.

FIGURE 2.

Adoptively transferred IFN-γ^−/− CD4⁺ T cells do not reduce Mtb burden, and they exacerbate disease in TCRβ^−/−δ^−/− mice. (A) Schematic of adoptive transfer of zero (none) or 3 × 10⁶ WT, 50%/50% mixed WT⁺IFN-γ^−/−, or IFN-γ^−/− CD4⁺ T cells to T cell–deficient host mice postinfection with aerosolized Mtb. (B) Bacterial burden in lungs and spleens of Mtb-infected adoptive transfer mice at 36 dpi. Results are representative of two independent experiments. (C) Weight trends of Mtb-infected adoptive transfer mice through 36 dpi. Statistical significance is shown for weights at 36 dpi. n = 5 none, n = 4 WT, n = 5 WT⁺IFN-γ^−/−, and n = 5 IFN-γ^−/− adoptive transfer mice. Results are representative of two independent experiments. Statistical significance was determined by Dunnett’s test using “none” as the control group. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001.

FIGURE 3.

IFN-γ^−/− T cells promote neutrophil and eosinophil recruitment to pulmonary lesions in T cell chimeric mice infected with Mtb. (A) Absolute number of each indicated cell type among live, parenchymal (IV-) cells in the right lung of T cell chimeric mice at 25 dpi. Results are representative of six independent experiments. Statistical significance was determined by Tukey’s range test. *p ≤ 0.05, ****p ≤ 0.0001. AM, alveolar macrophage; Eo, eosinophil; Neut, neutrophil. (B) Representative confocal microscopy demonstrating AMs (Siglec F⁺, CD68⁺), MDMs (Siglec F⁻, CD68⁺), neutrophils (CD177⁺), and eosinophils (Siglec F⁺, CD68⁻) in TB lesions in T cell chimeric mice. (C) Principal component analysis of 15 histopathologic features assessed in representative sections of fixed and H&E-stained lung of T cell chimeric mice at 25 dpi. MNGC, multinucleated giant cells; PBLA, peribronchial lymphoid aggregates; PVLA, perivascular lymphoid aggregates.

FIGURE 4.

IFN-γ^−/− T cells drive a type 2 cytokine milieu and alternative activation of MDMs in T cell chimeric mice infected with Mtb. (A) Relative expression of classical (M1) and alternative (M2) genes in FACS-sorted bystander and Mtb-infected MDMs in lungs of T cell chimeric mice at 25 dpi. Results are representative of two independent experiments. (B) Concentration of type 2 cytokines in lung lysates from Mtb-infected T cell chimeric mice at 25 dpi, as measured by cytokine bead array. n = 4 TCRβ^−/−δ^−/−, n = 5 WT, and n = 5 IFN-γ^−/− T cell chimeric mice. Results are representative of three independent experiments. (C) Concentration of IFN-γ in lung lysates from Mtb-infected T cell chimeric mice at 25 dpi, as measured by Luminex assay. n = 5 TCRβ^−/−δ^−/−, n = 5 WT, and n = 6 IFN-γ^−/− T cell chimeric mice. Results are representative of three independent experiments. (D) Representative confocal microscopy demonstrating expression of iNOS and ARG1 in lungs of Mtb-infected T cell chimeric mice at 25 dpi.