CD4 T Cell-Derived IFN-γ Plays a Minimal Role in Control of Pulmonary Mycobacterium tuberculosis Infection and Must Be Actively Repressed by PD-1 to Prevent Lethal Disease

Abstract

IFN-γ-producing CD4 T cells are required for protection against Mycobacterium tuberculosis (Mtb) infection, but the extent to which IFN-γ contributes to overall CD4 T cell-mediated protection remains unclear. Furthermore, it is not known if increasing IFN-γ production by CD4 T cells is desirable in Mtb infection. Here we show that IFN-γ accounts for only ~30% of CD4 T cell-dependent cumulative bacterial control in the lungs over the first six weeks of infection, but >80% of control in the spleen. Moreover, increasing the IFN-γ-producing capacity of CD4 T cells by ~2 fold exacerbates lung infection and leads to the early death of the host, despite enhancing control in the spleen. In addition, we show that the inhibitory receptor PD-1 facilitates host resistance to Mtb by preventing the detrimental over-production of IFN-γ by CD4 T cells. Specifically, PD-1 suppressed the parenchymal accumulation of and pathogenic IFN-γ production by the CXCR3+KLRG1-CX3CR1- subset of lung-homing CD4 T cells that otherwise mediates control of Mtb infection. Therefore, the primary role for T cell-derived IFN-γ in Mtb infection is at extra-pulmonary sites, and the host-protective subset of CD4 T cells requires negative regulation of IFN-γ production by PD-1 to prevent lethal immune-mediated pathology.

Fig 1. CD4 T cell-derived IFN-γ contributes little to control of Mtb infection in the lung but is essential in the spleen.

(A-C) Naïve CD4 T cells isolated from either WT or IFN-γ KO mice were adoptively transferred into day-7 infected RAG1 KO recipients (A), and survival of mice (B) and bacterial load in the tissues (C) were monitored. Data are representative of two independent experiments. (n = 4-5/group/experiment). **, P<0.005. (D) The areas between the log transformed bacterial growth curves in untreated control mice and WT CD4 T cell recipients or control mice and IFN-γ KO CD4 T cell recipients shown in (C) were calculated for the lungs and spleens. The ratio of IFN-γ KO:WT CD4 T cell areas was then calculated to estimate the proportion of cumulative bacterial reductions that were due to CD4 T cell-derived IFN-γ. Each dot represents the result obtained from two independent experiments. Data are mean ± 95% confidence interval. ****, P<0.0001 (bootstrap test).

Fig 2. Increased IFN-γ–production by CD4 T cells exacerbates pulmonary Mtb infection and leads to the early host mortality, despite enhancing bacterial control in the spleen.

RAG1 KO mice were infected with Mtb 7 days earlier and reconstituted with CD4 T cells from uninfected donors at increasing ratios of either WT or ARE-Del CD4 T cells mixed with IFN-γ KO CD4 T cells (A). All mice received the same total number of donor CD4 T cells, as only the fractions of IFN-γ–producing CD4 T cells varied (either WT or IFN-γ–overproducing). On day 42 IFN-γ concentrations in the lung homogenates (B) and bacterial load in the tissues were measured (C). Data are representative of two independent experiments (n = 5/group/experiment). (D) A mixture of WT and ARE-Del CD4 T cells (at 1:1 ratio) were co-transferred into day-7 infected TCRα KO mice, and on day 60 IFN-γ production by donor CD4 T cells was measured by DrxICS. Data are pooled from two independent experiments (n = 6/experiment) and each connecting line represents an individual animal. ****, P<0.0001 (E) Correlation between IFN-γ levels and bacterial numbers in the lungs of RAG1 KO mice. Data shown are replotted from the values shown in (B and C) to illustrate the correlation. (F) TCRα KO mice were infected with Mtb 7 days before and received with WT, ARE-Del or a 1:1 mixture of WT and ARE-Del naïve CD4 T cells and mouse survival was monitored. Data are representative of three independent experiments. (n = 4-5/group/experiment). **, P<0.002; compared to control group received WT CD4 T cells alone.

Fig 3. CD4 T cell-derived TNF has a minor contribution to inhibition of Mtb growth in both lung and spleen.

CD4 T cells isolated from naïve WT or TNF KO mice were adoptively transferred into day-7 infected RAG1 KO recipients at increasing ratios of WT cells to TNF KO cells. All mice received the same total number of donor CD4 T cells, as only the fractions of TNF–producing CD4 T cells varied (A). TNF levels in the tissue homogenates (B) and bacterial load in the tissues (C) were measured on day 42 p.i. Data are representative of two independent experiments (n = 5/group/experiment).

Fig 4. Mtb-specific CD4 T cells in the lungs of PD-1 KO mice are less differentiated, more parenchymally localized and produce increased amounts of IFN-γ.

(A) Survival of WT and PD-1 KO mice after Mtb infection. Data are representative of at least three independent experiments. (n = 5-6/experiment). (B) IFN-γ levels in the lung homogenates of WT and PD-1 KO mice on day 30 p.i. Data are pooled from three independent experiments (n = 3-4/experiment). (C) PD-1 expression on the parenchymal and intravascular I-A^bESAT-6_4–17 or I-A^bEsxG_46–61–specific CD4 T cells in WT lung on day 30 p.i. (D) Iv-staining of I-A^bESAT-6_4–17–specific CD4 T cells in the lung of WT and PD-1 KO mice on day 30 p.i. Data are pooled from three independent experiments (n = 3-4/experiment). (E) Phenotypic analysis of I-A^bESAT-6_4–17–specific CD4 T cells in WT and PD-1 KO lungs on day 30 p.i. (F) Intracellular IFN-γ staining of lung CD4 T cells in WT and PD-1 KO mice after in vitro stimulation with ESAT-6_1–20 peptide on day 30 p.i. Data are pooled from three independent experiments (n = 3-4/experiment). (G) Direct ex vivo IFN-γ staining for I-A^bESAT-6_4–17–specific CD4 T cells in the lungs of WT and PD-1 KO mice on day 30 p.i. Data are pooled from three independent experiments (n = 3-4/experiment). Cells in (A) and (E) were pooled from n = 3/experiment for FACS analysis. ***, P<0.0005; ****, P<0.0001.

Fig 5. PD-1 expression on CD4 T cells inhibits accumulation of and IFN-γ production by lung parenchymal CD4 T cells during Mtb infection.

(A) Correlation between bacterial load and frequency of parenchymal CD4 T cells in the lungs of Mtb infected mice. Data are pooled from experiments performed at different times p.i. ranging from day 0 to day 180. (B-F) FACS purified naïve CD4 T cells from WT (CD45.1) and PD-1 KO (Thy1.1) mice were mixed at a 1:1 ratio and co-transferred into day-7 infected WT mice (B). On day 28 an iv-stain was performed in the recipient mice and donor CD4 T cells were identified by their congenic markers (C). The frequency of donor total CD44^hi effector CD4 T cells in the recipient lungs (D) and in the lung parenchyma (E). (F) The frequency of KLRG1^- cells in the donor CD44^hi effector CD4 T cells. (G) The frequency of donor naïve CD4 T cells accumulating in the lung parenchyma. (H) IFN-γ production of donor effector CD4 T cells was determined by DrxICS. Data are representative of two independent experiments (n = 5/experiment) and each connecting line represents an individual mouse (n = 5/experiment). *, P<0.02; **, P<0.006.

Fig 6. PD-1 KO CD4 T cells require IFN-γ production to drive early mortality after Mtb infection.

(A and B) Parenchymal iv^- and intravascular iv⁺ effector CD44^hi CD4 T cells were FACS purified from the lungs of day-30 infected WT mice and adoptively transferred into TCRα KO mice that had been infected with Mtb 7 days previously (A) and mouse survival was monitored (B). **, P<0.001. (C and D) Parenchymal iv^- effector CD44^hi CD4 T cells were FACS purified from the lungs of WT and PD-1 KO mice on day 30 p.i., and adoptively transferred into day-7 infected TCRα KO mice (C) and mouse survival was monitored (D). Data are representative of two independent experiments (n = 5/group/experiment). **, P<0.002; compared to mice received iv^- WT CD4 T cells alone. (E and F) TCRα KO mice infected with Mtb 7 days earlier were reconstituted with WT, PD-1 KO or a mixture of WT and PD-1 KO or PD-1/IFN-γ double KO CD4 T cells (E) and survival was monitored (F). **, P<0.002 for WT + PD-1 KO (dotted red line) versus WT + PD-1/IFN-γ double KO (blue line). Data are representative of three independent experiments (n = 4-5/group/experiment).