MAIT cells are critical for optimal mucosal immune responses during in vivo pulmonary bacterial infection

Abstract

Mucosa-associated invariant T (MAIT) cells are "innate" T cells that express an invariant T-cell receptor α-chain restricted by the nonclassical MHC class I molecule MHC-related protein 1 (MR1). A recent discovery that MR1 presents vitamin B metabolites, presumably from pathogenic and/or commensal bacteria, distinguishes MAIT cells from peptide- or lipid-recognizing αβ T cells in the immune system. MAIT cells are activated by a wide variety of bacterial strains in vitro, but their role in defense against infectious assaults in vivo remains largely unknown. To investigate how MAIT cells contribute to mucosal immunity in vivo, we used a murine model of pulmonary infection by using the live vaccine strain (LVS) of Francisella tularensis. In the early acute phase of infection, MAIT cells expanded robustly in the lungs, where they preferentially accumulated after reaching their peak expansion in the late phase of infection. Throughout the course of infection, MAIT cells produced the critical cytokines IFN-γ, TNF-α, and IL-17A. Mechanistic studies showed that MAIT cells required both MR1 and IL-12 40 kDa subunit (IL-12p40) signals from infected antigen presenting cells to control F. tularensis LVS intracellular growth. Importantly, pulmonary F. tularensis LVS infection of MR1-deficient (MR1(-/-)) mice, which lack MAIT cells, revealed defects in early mucosal cytokine production, timely recruitment of IFN-γ-producing CD4(+) and CD8(+) T cells to the infected lungs, and control of pulmonary F. tularensis LVS growth. This study provides in vivo evidence demonstrating that MAIT cells are an important T-cell subset with activities that influence the innate and adaptive phases of mucosal immunity.

Keywords: respiratory infection; tularemia.

Fig. 1.

MAIT cells expand in the lungs in a murine model of pulmonary F. tularensis LVS infection. WT C57BL/6 mice were infected i.n. with a sublethal dose of 2 × 10² cfu LVS, and (A) total lung cells were harvested at the indicated time points after infection to evaluate the presence of TCRβ⁺ DN T cells (circled). (Exclusion channel: anti-CD4, CD8, B220, γδTCR Abs.) Data are representative of four independent experiments. (B) TCRβ⁺ DN T cells were purified from the lungs of LVS-infected mice at the indicated time points by FACS and evaluated for expression of MAIT cell invariant TCR Vα19-Jα33 transcripts by qPCR (relative units normalized to TCRα constant region mRNA). Lung single cell suspensions were pooled from 20 mice for sorting; data are representative of three independent experiments. (C) Quantification of CD3 and Vβ6+8 expression by DN T cells in the lungs of mice on day 14 after LVS i.n. infection; in the flow cytometry dot plot shown, cells were first gated on TCRβ⁺ DN T cells as indicated in A. Data are representative of three independent experiments.

Fig. 2.

Kinetics of MAIT cell accumulation in the lungs during pulmonary F. tularensis LVS infection. (A) Flow cytometry analyses of TCRβ⁺ DN T cell accumulation in the lungs of WT and MR1^−/− mice infected with a sublethal dose of 2 × 10² cfu LVS i.n. (exclusion channel, anti-CD4, CD8, B220, γδTCR Abs); in the representative dot plots, total live lung cells from individual mice are shown. (B) Enumeration of the total number of TCRβ⁺ DN T cells in the lungs of WT and MR1^−/− mice during sublethal LVS i.n. infection (n = 5 per group). Data are representative of three independent experiments, and are the mean ± SEM (*P < 0.01 vs. LVS-infected MR1^−/− mice at the same time point, analyzed by Student t test.) (C) Growth of LVS in the lungs of WT mice following sublethal LVS i.n. infection. Data are representative of three independent experiments, and values shown are the mean cfu count per lung ± SEM of viable bacteria for five mice per group at each time point. (D) qPCR analyses of expression of MAIT cell invariant TCR Vα19-Jα33 transcripts in total cells harvested from the lungs of WT mice at the indicated time points after a sublethal LVS i.n. infection (n = 5 per group). qPCR results are presented as relative units normalized to TCRα constant region mRNA (*P < 0.01 vs. naive mice by Student t test).

Fig. 3.

MAIT cells preferentially accumulate in the lungs during pulmonary F. tularensis LVS infection. (A) Flow cytometry analyses of total TCRβ⁺ cells in the lungs, livers, spleens, mediastinal lymph nodes (Med LN), and mesenteric lymph nodes (Mes LN) of WT and MR1^−/− mice on day 14 after a sublethal (2 × 10² cfu) LVS i.n. infection; in the representative dot plots shown, cells from different tissues were first gated on total live CD45⁺ cells, and then gated on TCRβ⁺ cells. The red box denotes the TCRβ⁺ DN T cell population in LVS-infected WT or MR1^−/− mice. (B) qPCR analyses of expression of MAIT cell invariant TCR Vα19-Jα33 transcripts in total cells harvested from the lungs, livers, spleens, mediastinal lymph nodes, and mesenteric lymph nodes of WT mice on day 14 after a sublethal LVS i.n. infection (n = 5 pooled mice per group). qPCR results are presented as units normalized to TCRα constant region mRNA. Data are expressed as the mean relative units ± SEM, and are representative of three independent experiments.

Fig. 4.

MR1^−/− mice display impaired antibacterial responses during pulmonary F. tularensis LVS infection. WT and MR1^−/− mice were infected i.n. with LVS at a sublethal dose of 2 × 10² cfu. Bacterial burdens of LVS (in cfu) in the lungs were enumerated at the indicated time points (A). Values shown are the mean cfu count per lung ± SEM of viable bacteria for five mice per group at each time point. (*P < 0.01 vs. cfu count in LVS-infected WT mice, as analyzed by Student t test.) qPCR analyses of expression of IFN-γ (B), TNF (C), IL-17A (D), and iNOS (E) in the lungs of LVS-infected mice. Values shown are the mean expression ± SEM of the indicated transcript relative to GAPDH for five mice per group. (*P < 0.05 vs. LVS-infected WT mice at the same time point, as analyzed by Student t test.) Data are representative of three independent experiments.

Fig. 5.

MAIT cells produce robust levels of critical cytokines in the lungs of WT mice at different stages of pulmonary infection of F. tularensis LVS. (A) TCRβ⁺ DN, CD4⁺, and CD8⁺ T cells were purified from the lungs of WT mice on day 8 after a sublethal (2 × 10² cfu) i.n. infection of LVS by FACS, and evaluated for expression of IFN-γ, TNF, and IL-17A by qPCR (relative units normalized to GAPDH mRNA). Lung cells were pooled from 10 mice; data include values ± SEM from three replicates, and are representative of two independent experiments. (B) Representative flow cytometry dot plots of IFN-γ, TNF, and IL-17A production by TCRβ⁺ cells in the lungs of WT and MR1^−/− mice on day 10 after sublethal LVS i.n. infection. In the representative dot plots shown, cells were first gated on total live lung TCRβ⁺ cells. Data are representative of three independent experiments. The red box denotes the cytokine-positive TCRβ⁺ DN T cell population in WT and MR1^−/− mice. (C) TCRβ⁺ DN, CD4⁺, and CD8⁺ T cells were purified from mouse lungs on day 14 after sublethal LVS i.n. infection by FACS, and evaluated for expression of IFN-γ, TNF, and IL-17A by qPCR (relative units normalized to GAPDH mRNA). Lung cells were pooled from 10 mice; data include values ± SEM from three replicates, and are representative of two independent experiments.

Fig. 6.

Naive transgenic MAIT cells release cytokines and control F. tularensis LVS intracellular growth in macrophages. BMMØs from WT mice were infected with LVS and cultured alone or with Thy1.2⁺ cells enriched from naive Vα19iTg-MR1^+/+ mice. In some cases, as indicated, neutralizing Abs or NMMA were added to the cultures at the time of addition of Vα19iTg-MR1^+/+ T cells. (A) Growth of LVS in the BMMØ monolayer was determined after 72 h coculture. Values shown are the mean cfu counts per milliliter ± SEM of triplicate cultures. (*P < 0.01 vs. “Infected BMMØ alone” by Student t test.) Supernatants were collected after 72 h of coculture and the amount of IFN-γ (B), TNF (C), IL-17A (D), and nitrite (E) was evaluated. Values shown are the mean pg/mL (cytokines) or µM (nitrite) ± SEM of triplicate cultures. (*P < 0.01 vs. Vα19iTg-MR1^+/+ T-cell cultures containing isotype control Ab.) Data are representative of three independent experiments of similar design.

Fig. 7.

Recruitment of activated CD4⁺ and CD8⁺ T cells to the lungs is delayed in MR1^−/− mice during F. tularensis LVS pulmonary infection. WT and MR1^−/− mice were infected i.n. with a sublethal dose of 2 × 10² cfu LVS, and (A) total numbers of TCRβ⁺ CD4⁺ and CD8⁺ T cells were enumerated in the lungs at the indicated time points by flow cytometry. Data are presented as mean ± SEM (n = 5 per group; *P < 0.05 vs. LVS-infected WT mice, as analyzed by Student t test). (B) Flow cytometry staining for activation marker expression and IFN-γ production by TCRβ⁺ CD4⁺ and CD8⁺ T cells in the lungs of LVS-infected WT mice (gray line, gray shaded histograms) and LVS-infected MR1^−/− mice (black line, unshaded histograms) on day 8 after infection (in the IFN-γ plot, isotype control Ab staining is indicated by a dotted line). (C) Comparison of the total numbers of activated (positively stained for both CD69 and CD44) TCRβ⁺ CD4⁺ or CD8⁺ T cells in the lungs of LVS-infected WT and MR1^−/− mice at the indicated time points. Data are presented as mean ± SEM [n = 5 per group; *P < 0.05 vs. LVS-infected WT mice; **P < 0.01 vs. naive mice (day 0) of the same genotype; ns, not significant, i.e., P > 0.05 vs. naive mice (day 0) of the same genotype, as analyzed by Student t test]. (D) Numbers of IFN-γ–producing cells in TCRβ⁺ CD4⁺ and CD8⁺ T cells in the lungs of LVS-infected WT and MR1^−/− mice at the indicated time points. Data are presented as mean ± SEM (n = 5 per group; *P < 0.05 vs. WT mice by Student t test). All data are representative of three independent experiments of similar design.

Fig. 8.

MAIT cells maintain a long-term chronic F. tularensis LVS infection in the absence of CD4⁺ and CD8⁺ T cells. WT and MR1^−/− mice were depleted of CD4⁺ and CD8⁺ T cells before and throughout a sublethal (2 × 10² cfu) LVS i.n. infection by administration of anti-CD4 and anti-CD8 Abs (“depleted”). (A) Flow cytometry analyses of TCRβ⁺ DN T-cell accumulation (boxed) in the lungs of mice on day 14 after LVS i.n. infection (exclusion channel, anti-CD4, CD8, B220, γδTCR Abs); representative dot plots are shown of total lung cells from individual mice. (B) LVS cfu counts in the lungs of LVS-infected WT and MR1^−/− mice depleted of CD4 and CD8 cells at the indicated time points after sublethal LVS i.n. infection. Data are presented as mean ± SEM (n = 5 per group; *P < 0.01 vs. “WT depleted” mice by Student t test). (C) Survival of LVS-infected WT and MR1^−/− mice depleted of CD4 and CD8 cells, as well as of LVS-infected TCRα^−/− mice, after sublethal LVS i.n. infection (n = 5 per group). Data are representative of three independent experiments.