Re-appraising the role of T-cell derived interferon gamma in restriction of Mycobacterium tuberculosis in the murine lung: T-cell derived IFNγ is required to restrict pulmonary Mtb

Abstract

T cells producing interferon gamma (IFNγ) 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 which achieved protection by adoptively transferred Mtb-specific IFNγ^-/- T cells. Using IFNγ^-/- T cell chimeric mice and adoptive transfer of IFNγ^-/- T cells into TCRβ^-/-δ^-/- mice, we demonstrate that control of lung Mtb burden is in fact dependent on T cell-derived IFNγ, and furthermore, mice selectively deficient in T cell-derived IFNγ develop exacerbated disease compared to T cell-deficient controls despite equivalent lung bacterial burdens. Deficiency in T cell-derived IFNγ skews infected and bystander monocyte-derived macrophages (MDMs) to an alternative M2 phenotype, and promotes neutrophil and eosinophil influx. Our studies support an important role for T cell-derived IFNγ in pulmonary immunity against TB.

Figure 1:. IFNγ^−/− T cells do not reduce Mtb bacterial burden, and exacerbate disease in T cell chimeric mice.

(A) Schematic of the preparation of TCRβ^−/−δ^−/−, WT, and IFNγ^−/−, T cell chimeric mice, followed by infection with aerosolized Mtb. (B–C) Bacterial burden in lungs and spleens of Mtb-infected T cell chimeric mice at 25dpi 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 IFNγ^−/− T cell chimeric mice. Statistical significance was determined by Tukey’s range test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. (D) Weight trends of Mtb-infected T cell chimeric mice through 29dpi.

Figure 2:. Adoptively transferred IFNγ^−/− CD4+ T cells do not reduce Mtb burden, and exacerbate disease in TCRβ^−/−δ^−/− mice.

(A) Schematic of adoptive transfer of zero (none) or 3*10⁶ WT, 50/50% mixed, or IFNγ^−/− CD4+ T cells to T cell-deficient host mice after infection with aerosolized Mtb. (B) Bacterial burden in lungs and spleens of Mtb-infected adoptive transfer mice at 36dpi. Results are representative of two independent experiments. Statistical significance was determined by Tukey’s range test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. (C) Weight trends of Mtb-infected adoptive transfer mice through 36dpi.

Figure 3:. IFNγ^−/− 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 25dpi. Results are representative of six independent experiments. Statistical significance was determined by Tukey’s range test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. AM: Alveolar macrophage, MDM: monocyte-derived macrophage, Neut: neutrophil, Eo: eosinophil (B) Representative confocal microscopy demonstrating AMs (SiglecF+, CD68+), MDMs (SiglecF-, CD68+), neutrophils (CD177+), and eosinophils (SiglecF+, CD11c-) in TB lesions in T cell chimeric mice. (C) Representative images of TB lesions in H&E-stained sections of T cell chimeric mice at 25dpi. 40x: arrows represent TB lesions. 200x: asterisks represent perivascular and peribronchiolar lymphocyte aggregates. 400x: arrows represent neutrophilic infiltrates. (D) Principal component analysis of fifteen histopathologic features assessed in representative sections of fixed and hematoxylin-eosin (H&E) stained lung of T cell chimeric mice at 25dpi. PVLA: perivascular lymphoid aggregates. PBLA: peribronchial lymphoid aggregates. MNGC: multinucleated giant cells.

Figure 4:. IFNγ^−/− T cells drive a Th2 cytokine milieu and alternative activation of monocyte-derived macrophages 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 25dpi. (B) Concentration of Type 2 cytokines in lung lysates from Mtb-infected T cell chimeric mice at 25dpi, as measured by cytokine bead array. Results are representative of two independent experiments. (C) Concentration of IFNγ in lung lysates from Mtb-infected T cell chimeric mice at 25dpi, as measured by Luminex assay. Results are representative of two independent experiments. (D) Representative confocal microscopy demonstrating expression of iNOS and ARG1 in lungs of Mtb-infected T cell chimeric mice at 25dpi.