CD4+ T cell-dependent IFN-γ production by CD8+ effector T cells in Mycobacterium tuberculosis infection

Abstract

Both CD4+ and CD8+ T cells contribute to immunity to tuberculosis, and both can produce the essential effector cytokine IFN-γ. However, the precise role and relative contribution of each cell type to in vivo IFN-γ production are incompletely understood. To identify and quantitate the cells that produce IFN-γ at the site of Mycobacterium tuberculosis infection in mice, we used direct intracellular cytokine staining ex vivo without restimulation. We found that CD4+ and CD8+ cells were predominantly responsible for production of this cytokine in vivo, and we observed a remarkable linear correlation between the fraction of CD4+ cells and the fraction of CD8+ cells producing IFN-γ in the lungs. In the absence of CD4+ cells, a reduced fraction of CD8+ cells was actively producing IFN-γ in vivo, suggesting that CD4+ effector cells are continually required for optimal IFN-γ production by CD8+ effector cells. Accordingly, when infected mice were treated i.v. with an MHC-II-restricted M. tuberculosis epitope peptide to stimulate CD4+ cells in vivo, we observed rapid activation of both CD4+ and CD8+ cells in the lungs. Indirect activation of CD8+ cells was dependent on the presence of CD4+ cells but independent of IFN-g responsiveness of the CD8+ cells. These data provide evidence that CD4+ cell deficiency impairs IFN-γ production by CD8+ effector cells and that ongoing cross-talk between distinct effector T cell types in the lungs may contribute to a protective immune response against M. tuberculosis. Conversely, defects in these interactions may contribute to susceptibility to tuberculosis and other infections.

Figure 1. IFN-γ producing cells in the lungs of M. tuberculosis infected mice

(A) Concentrations of IFN-γ protein in lung homogenates during the initial nine weeks of infection, as measured by IFN-γ ELISA. Asterisks indicate statistical significance of differences in concentration of IFN-γ observed in n = 4 mice, between adjacent time points. ** p<0.005; *** p<0.001. (B) CD3⁺ T cells account for 90% of the IFN-γ⁺ cells in the lungs on day 28 post-infection as measured by direct intracellular cytokine staining followed by flow cytometry. IFN-γ-producing cells were identified relative to isotype control antibody staining. Flow cytometry plots show lung cells from a representative mouse. (C) Graphic representation of data from B, showing mean data from n=4 mice. (D) Frequency of IFN-γ producing cells within a given cell subset. Lung cells from day 28 post-infection were gated on relevant markers: CD3⁺, CD4⁺; CD3⁺, CD8⁺; or CD3⁻, DX5⁺. (E) Graphic representation of data from D, showing mean data from n=5 mice. All figures represent data from 2 or more experimental replicates. Data are representative of at least 2 independent experiments.

Figure 2. IFN-γ production by CD8⁺ effector T cells is influenced by CD4⁺ T cells

(A) IFN-γ production by CD4⁺ or CD8⁺ T cells from the lungs of individual mice on day 28 post-infection. Flow cytometry plots show CD3⁺, CD4⁺ (top row) or CD8⁺ (bottom row) cells; values indicate the frequency of IFN-γ⁺ cells within each population. (B) Linear correlation between the frequency of IFN-γ producing T cells in CD4⁺ and CD8⁺ subsets: graphic depiction of data in A, n=5 mice, with linear correlation performed with “x” indicating slope (p = 0.018) and “R²” indicating goodness of fit for linear curve. Data shown in Panels (A) and (B) are from independent experiments. (C) Requirement of CD4⁺ T cells for IFN-γ production by CD8⁺ T cells. C57BL/6 or CD4⁺ T cell deficient MHCII^−/− mice were infected with M. tuberculosis and the frequency of IFN-γ⁺ CD8⁺ T cells in the lungs was compared, n=4 mice. * p<0.05. Data are representative of at least 2 independent experiments.

Figure 3. Production of IFN-γ by CD8⁺ T cells requires continual presence of CD4⁺ T cells

C57BL/6 mice infected with M. tuberculosis were treated with one intraperitoneal dose of monoclonal antibody: either anti-CD4⁺ or isotype control. Cells were isolated from lungs 24 hours later. The frequency of CD8⁺ T cells producing IFN-γ in each group was determined by flow cytometry. (A) Flow cytometry plots indicate CD3⁺ CD8⁺ cells from the lungs of a representative mouse from either treatment group. Values indicate the frequency of IFN-γ⁺ cells within each subset 24 hours after treatment. (B) Graphic representation of data from A, indicating the frequency of IFN-γ⁺ CD8⁺ T cells in the lungs of n=4 mice. * p<0.05. Data are representative of 2 independent experiments.

Figure 4. Activation of CD4⁺ effector T cells activates CD8⁺ effector T cells in vivo

(A) C57BL/6 mice infected with M. tuberculosis were injected intravenously with a synthetic peptide (100 µg) containing a known CD4⁺ T cell epitope from M. tuberculosis Ag85B, peptide 25 (aa 240–254). 2 hours after injection, the frequency of IFN-γ⁺ CD4⁺ or CD8⁺ T cells was compared to those in infected mice not receiving peptide. Flow cytometry plots show CD3⁺CD4⁺ (top row) or CD8⁺ (bottom row) cells from the lungs of a representative mouse. Values indicate frequency of IFN-γ⁺ cells in each subset. (B) CD8⁺ T cell activation upon peptide 25 injection is not due to direct recognition of peptide 25 by CD8⁺ T cells. Graph depicts the fraction of IFN-γ⁺ CD8⁺ T cells in the lungs of either C57BL/6 or MHCII^−/− mice, either untreated or 2 hours after injection with 100 µg of either Ag85B peptide 25 (a CD4⁺ T cell epitope) or Mtb32A_309–318 peptide (a CD8⁺ T cell epitope). Asterisk indicates statistical significance of differences in frequency of IFN-γ⁺ cells detected among one group of cells from n=4 mice of one genetic background between different treatment groups. * p<0.05; “n.s.”: not significant. Data are representative of 2 independent experiments.

Figure 5. Indirect activation of CD8⁺ T cells with peptide 25 is independent of IFN-γ signaling

C57BL/6 or IFN-γR^−/− mice infected with M. tuberculosis were injected intravenously with Ag85B peptide 25 to activate CD4⁺ T cells. 2 hours after injection, the frequency of IFN-γ⁺ T cells was compared to those in infected mice not receiving peptide. Graphs depict the fraction of IFN-γ⁺ among CD3⁺, CD4⁺ (left) or CD3⁺, CD8⁺ (right) T cells in the lungs. Asterisks indicate statistical significance of differences in frequency of IFN-γ⁺ cells detected among one cell type between different treatment groups. * p<0.05; “n.s.”: not significant. Data are representative of 2 independent experiments.