Inhibition of indoleamine 2,3-dioxygenase enhances the T-cell response to influenza virus infection

Abstract

Influenza infection induces an increase in the level of indoleamine 2,3-dioxygenase (IDO) activity in the lung parenchyma. IDO is the first and rate-limiting step in the kynurenine pathway where tryptophan is reduced to kynurenine and other metabolites. The depletion of tryptophan, and production of associated metabolites, attenuates the immune response to infection. The impact of IDO on the primary immune response to influenza virus infection was determined using the IDO inhibitor 1-methyl-D,L-tryptophan (1MT). C57BL/6 mice treated with 1MT and infected with A/HKx31 influenza virus had increased numbers of activated and functional CD4⁺ T-cells, influenza-specific CD8⁺ T-cells and effector memory cells in the lung. Inhibition of IDO increased the Th1 response in CD4⁺ T-cells as well as enhanced the Th17 response. These studies show that inhibition of IDO engenders a more robust T-cell response to influenza virus, and suggests an approach for enhancing the immune response to influenza vaccination by facilitating increased influenza-specific T-cell response.

Fig. 1.

Influenza infection increases IDO activity in the lungs and sera. Mice were treated with 1MT or control 3 days prior to infection. On day 0, i.e. 3 days after treatment, mice were intranasally (i.n.) infected with 10³ p.f.u. X31 in PBS. (a) Serum and (b) lung homogenates were collected every other day from day 0 (uninfected animals) until day 14 p.i. Each time point represents the mean and sem of 3–5 mice per group and shows two independent experiments. (**P value <0.01 and *P value <0.05).

Fig. 2.

1MT treatment does not affect total frequency of T-cells infiltrating the lungs. Mice were treated with 1MT or control 3 days prior to infection and subsequently infected with 10³ p.f.u. X31 i.n. (a) BAL and (b) MLN cell numbers from day 10 p.i. Number of CD8⁺ and CD4⁺ T-cells in the (c) BALs and (d) MLNs. Representative data from one experiment are shown from three independent experiments. (*P value <0.05).

Fig. 3.

1MT treatment enhances the Th1 response. Mice were treated with 1MT or control, as described, and infected with 10³ p.f.u. X31 i.n. Ten days p.i., BAL cells were stimulated for 6 h with UV-inactivated influenza virus and subsequently stained for intracellular expression of IFN-γ, IL-6 and IL-4. (a) Representative dot plots of CD4⁺ T-cells expressing IFN-γ, IL-6 and IL-4. (b, d, f) Proportion and (c, e, g) frequency of CD4⁺ T-cells expressing (b, c) IFN-γ, (d, e) IL-6 and (f, g) IL-4. Representative data from one experiment are shown from three independent experiments. (**P value <0.01 and *P value <0.05).

Fig. 4.

IDO inhibition enhances the influenza-specific response. Mice were treated with 1MT or control, as described, and infected with 10³ p.f.u. X31 i.n. On day 10 p.i., BAL cells were analysed for virus-specific CD8⁺ T-cell numbers as determined by tetramers specific to NP_366–374, PA_224–233 or PB1_703–711. (a) Proportion and (b) frequency of CD8⁺ T-cells are shown. Representative data from one experiment is shown from three independent experiments. (*P value <0.05).

Fig. 5.

IDO inhibition increases the frequency of functional PA-specific CD8⁺ T-cells. Mice were treated with 1MT or control, as described, and infected with 10³ p.f.u. X31 i.n. Ten days p.i., single cells from the BALs were stimulated for 4 h with influenza immunodominant peptides and subsequently stained for intracellular expression of IFN-γ. (a, b) Representative dot plots of (a) CD8⁺ T-cells and (b) influenza-specific CD8⁺ T-cells expressing IFN-γ. (c) Percentage and (d) frequency of CD8⁺ T-cells expressing IFN-γ. (e) Percentage and (f) frequency of influenza-specific CD8⁺ T-cells expressing IFN-γ. Representative data from one experiment are shown from three independent experiments. (*P value <0.05).

Fig. 6.

Inhibition of IDO activity increases the presence of CD8⁺ effector memory cells. Mice were treated with 1MT or control 3 days prior to infection. On day 0, mice were i.n. infected with 10³ p.f.u. X31. (a, b) CD8⁺ and (c, d) CD4⁺ T-cells collected 10 days p.i. from the BALs were stained for the presence of CD44 and CD62L. Representative data from one experiment is shown from two independent experiments. (*P value <0.05).