doi: 10.1128/IAI.00279-12.
Epub 2012 Jul 9.
Polyclonal mucosa-associated invariant T cells have unique innate functions in bacterial infection
Affiliations
- PMID: 22778103
- PMCID: PMC3418730
- DOI: 10.1128/IAI.00279-12
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Polyclonal mucosa-associated invariant T cells have unique innate functions in bacterial infection
Wei-Jen Chua et al.
Infect Immun.
2012 Sep.
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Abstract
Mucosa-associated invariant T (MAIT) cells are a unique population of αβ T cells in mammals that reside preferentially in mucosal tissues and express an invariant Vα paired with limited Vβ T-cell receptor (TCR) chains. Furthermore, MAIT cell development is dependent upon the expression of the evolutionarily conserved major histocompatibility complex (MHC) class Ib molecule MR1. Using in vitro assays, recent studies have shown that mouse and human MAIT cells are activated by antigen-presenting cells (APCs) infected with diverse microbes, including numerous bacterial strains and yeasts, but not viral pathogens. However, whether MAIT cells play an important, and perhaps unique, role in controlling microbial infection has remained unclear. To probe MAIT cell function, we show here that purified polyclonal MAIT cells potently inhibit intracellular bacterial growth of Mycobacterium bovis BCG in macrophages (MΦ) in coculture assays, and this inhibitory activity was dependent upon MAIT cell selection by MR1, secretion of gamma interferon (IFN-γ), and an innate interleukin 12 (IL-12) signal from infected MΦ. Surprisingly, however, the cognate recognition of MR1 by MAIT cells on the infected MΦ was found to play only a minor role in MAIT cell effector function. We also report that MAIT cell-deficient mice had higher bacterial loads at early times after infection compared to wild-type (WT) mice, demonstrating that MAIT cells play a unique role among innate lymphocytes in protective immunity against bacterial infection.
Figures
Innate MR1-restricted MAIT cells inhibit mycobacterial growth in MΦ. (a and b) M. bovis BCG-infected MΦ derived from WT mice were cultured alone or cocultured with naïve T cells from MR1−/−, WT, Vα19iTgMR1−/−, or Vα19iTgMR1+/+ mice for 72 h. The intracellular growth of mycobacteria in MΦ was quantitated by liquid scintillation counting of tritiated uridine uptake (disintegrations per minute) (a). (b) The percentage of mycobacterial growth inhibition was assessed from radioactivity in panel a to demonstrate the extent of suppression of mycobacterial growth by naïve T cells from different mice. See Materials and Methods for the formula used in the calculation of the percentage. (c) Nitric oxide (NO) production by uninfected MΦ, infected MΦ, or infected MΦ cocultured with naïve T cells (Vα19iTgMR1−/− or Vα19iTgMR1+/+). Supernatants were collected after 72 h of culture. The amount of nitrite (NO2−) in supernatants was measured. Results are means ± standard errors of the means (SEM) of the triplicate of different culture conditions. Statistical analysis was performed using a one-way analysis of variance (ANOVA) with Tukey's multiple comparison posttest comparing every culture condition (***, P ≤ 0.001; ns, not significant). The dotted line in panel c indicates that the uninfected MΦ were not included in statistical comparison. Data are results from one of three independent experiments.
MAIT cells are a potent source of innate IFN-γ enhancing the microbicidal activity of infected MΦ. (a) IFN-γ production by naïve T cells from MR1−/−, WT, Vα19iTgMR1−/−, and Vα19iTgMR1+/+ mice when cocultured with uninfected or M. bovis BCG-infected MΦ. Supernatants were collected after 72 h of coculture. The concentration of IFN-γ in supernatants was measured by ELISA. (b) Effect of IFN-γ blockage on the inhibition of mycobacterial growth by Vα19iTgMR1+/+ T cells. M. bovis-infected MΦ were cultured alone or cocultured with Vα19iTgMR1+/+ T cells in the absence or presence of an anti-IFN-γ MAb for 72 h. The intracellular growth of mycobacteria in MΦ was quantitated by liquid scintillation counting of tritiated uridine uptake (disintegrations per minute). (c) Effect of IFN-γ blockage on the enhancement in NO production of infected MΦ by Vα19iTgMR1+/+ T cells. Supernatants from the cultures used for panel b were collected before cell lysis for the radiolabeling of mycobacteria. The amount of NO2− in supernatants was measured. Results are the means ± SEM from triplicate assays of each set of culture conditions. Statistical analysis was performed using a one-way ANOVA with Tukey's multiple comparison posttest comparing all culture conditions (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant). Data are results from one of three independent experiments.
Innate MAIT cells produce IL-17A in response to infection. (a) IL-17A production of Vα19iTgMR1+/+ T cells when cocultured with uninfected or M. bovis BCG-infected MΦ. Supernatants were collected after 72 h of coculture. The concentration of IL-17A in supernatants was measured by ELISA. (b) Effect of IL-17A blockage on the inhibition of mycobacterial growth by Vα19iTgMR1+/+ T cells. M. bovis BCG-infected MΦ were cultured alone or cocultured with Vα19iTgMR1+/+ T cells in the absence or presence of an anti-IL-17A MAb for 72 h. The intracellular growth of mycobacteria in MΦ was quantitated by liquid scintillation counting of tritiated uridine uptake (disintegrations per minute). Results are means ± SEM from triplicate assays of each set of culture conditions. Statistical analysis was performed using a one-way ANOVA with Tukey's multiple comparison posttest comparing all cultures (***, P ≤ 0.001; ns, not significant). Data are results from one of three independent experiments.
Activation of MAIT cells is dependent on the innate signal IL-12 from infected MΦ. (a, c, and e) MAIT cell activation in the presence of anti-MR1 or anti-IL-12/23p40 MAbs. Vα19iTgMR1+/+ T cells were cocultured with M. bovis BCG-infected MΦ, and supernatants were collected after 72 h of coculture. The concentrations of IFN-γ (a), IL-17A (c), and NO2− (e) in supernatants were measured. Results are means ± SEM from triplicate assays of each set of culture conditions. Statistical analysis was performed using a one-way ANOVA with Tukey's multiple comparison posttest comparing all cultures. ***, P ≤ 0.001; ns, not significant (for comparisons to results obtained with MΦ cocultured with Vα19iTgMR1+/+ T cells in the absence of blocking MAbs). The dotted line in panel e indicates that the NO production by MΦ cultured alone was not included in the statistical comparison. Data are the results from one of three independent experiments. (b, d, and f) Activation of MAIT cells cocultured with WT, MR1−/−, or p40−/− MΦ infected with M. bovis BCG. Vα19iTgMR1+/+ T cells were cocultured with WT, MR1−/−, or p40−/− MΦ infected with M. bovis BCG and supernatants, were collected after 72 h of coculture. The concentration of IFN-γ (b), IL-17A (d), and NO2− (f) in supernatants were measured. Results are means ± SEM from triplicate assays of each set of culture conditions. Statistical analysis was performed using a one-way ANOVA with Tukey's multiple comparison posttest comparing all cultures. ***, P ≤ 0.001; ns, not significant (for comparisons to results obtained with WT MΦ cocultured with Vα19iTgMR1+/+ T cells). In panel f, the dotted line indicates that statistical analyses were performed separately for MΦ alone and MΦ cocultured with Vα19iTgMR1+/+ T cells. There was no statistical difference in NO production among infected MΦ of different genotypes cultured in the absence of Vα19iTgMR1+/+ T cells. Data are results from one of two independent experiments.
Control of mycobacterial growth by MAIT cells depends on IL-12. (a and c) The inhibition of mycobacterial growth by MAIT cells in the presence of anti-MR1 or anti-IL-12/23p40 MAbs. M. bovis-infected MΦ were cultured alone, or cocultured with Vα19iTgMR1+/+ T cells in the absence or presence of anti-MR1 MAbs or an anti-IL-12/23p40 MAb for 72 h. The intracellular growth of mycobacteria in MΦ was quantitated by liquid scintillation counting of tritiated uridine uptake (disintegrations per minute) (a). The percentage of mycobacterial growth inhibition (c) was assessed from radioactivity in panel a to demonstrate the extent of suppression of mycobacterial growth by Vα19iTgMR1+/+ T cells in the absence or presence of blocking MAbs. See Materials and Methods for the formula used to calculate inhibition. Results are means ± SEM from triplicate assays of each set of culture conditions. Statistical analysis was performed using a one-way ANOVA with Tukey's multiple comparison posttest comparing all cultures. **, P ≤ 0.01; ns, not significant (for comparison to results obtained with MΦ cocultured with Vα19iTgMR1+/+ T cells in the absence of blocking MAbs). The dotted line in panel a indicates that MΦ cultured alone were not included in statistical comparison. Data are results from one of three independent experiments. (b and d) Inhibition of mycobacterial growth in WT, MR1−/−, or p40−/− MΦ by MAIT cells. WT, MR1−/−, or p40−/− MΦ infected with M. bovis BCG were cultured alone or cocultured with Vα19iTgMR1+/+ T cells for 72 h. The intracellular growth of mycobacteria in MΦ was quantitated by liquid scintillation counting of tritiated uridine uptake (disintegrations per minute) (b). The percentage of mycobacterial growth inhibition (d) was assessed based on genotype. Results are means ± SEM of the triplicate of different culture conditions. Statistical analysis was performed using a one-way ANOVA with Tukey's multiple comparison posttest comparing all cultures. ***, P ≤ 0.001; ns, not significant (for comparison to results obtained with the coculture of infected WT MΦ with Vα19iTgMR1+/+ T cells). In panel b, the dotted line indicates that statistical analyses were performed separately for MΦ alone and MΦ cocultured with Vα19iTgMR1+/+ T cells. There was no statistical difference in mycobacterial growth among infected MΦ of different genotypes cultured in the absence of Vα19iTgMR1+/+ T cells. Data are results from one of two independent experiments.
The role of MR1 in MAIT cell activation. (a and b) MAIT cell activation by p40−/− MΦ in the presence of anti-MR1 MAbs. Vα19iTgMR1+/+ T cells were cocultured with p40−/− MΦ infected with M. bovis BCG in the absence or presence of anti-MR1 MAbs. Supernatants were collected after 24 or 72 h of coculture. (c) The inhibition of mycobacterial growth in p40−/− MΦ by Vα19iTgMR1+/+ T cells in the absence or presence of anti-MR1 MAbs. Results are means ± SEM from triplicate assays of each set of culture conditions. Statistical analysis was performed using Student's t test. Although there was a trend for cytokine secretion of Vα19iTgMR1+/+ T cells to be lower in the presence of MR1 blockage, the difference was not statistically significant. Data are results from two time points in two independent experiments.
MR1-restricted MAIT cells produce IFN-γ in response to recombinant IL-12 in the absence of infection. Vα19iTgMR1−/− or Vα19iTgMR1+/+ T cells were incubated with uninfected WT MΦ in the presence of recombinant IL-12 at various concentrations. Supernatants were collected after 24 h of incubation. The concentration of IFN-γ in supernatants was measured by ELISA. Results are means ± SEM from triplicate assays of each set of culture conditions. Statistical analysis was performed using a two-way ANOVA (***, P ≤ 0.001). Data are results from one of two independent experiments.
MR1-restricted MAIT cells play a unique role in providing protective immunity against mycobacterial infection in vivo. (a) The bacterial burden in WT and MR1−/− mice in the low-dose aerosol infection of M. bovis BCG. WT and MR1−/− mice were infected with M. bovis BCG via the aerosol route. Each mouse received 100 to 300 infectious bacilli in lung. Ten and 30 days after infection, lung tissues were harvested to determine the bacterial burden. Results are given as the mean ± SEM of five mice per group. Each dot represents the CFU count for one mouse. The difference in bacterial burden between WT and MR1−/− mice at day 10 postinfection is statistically significant as analyzed by Student's t test (P = 0.0008). Data are results from one of two independent experiments. (b) IFN-γ responses of WT and MR1−/− mice to intranasal infection with M. bovis BCG. BAL cells were obtained from WT and MR1−/− mice on day 10 and day 30 after intranasal infection of M. bovis BCG (107 CFU per mouse). The frequency of IFN-γ-producing cells in the BAL cells was measured by ELISPOT assay. BAL cells from the same genotype were pooled and stimulated with M. bovis BCG (MOI = 3) and various M. tuberculosis-derived antigens: culture filtrate (CF, 10 μg/ml), cell wall (CW, 50 μg/ml), and an Ag85b peptide pool (Ag85bpp, 2 μg/ml). The numbers of IFN-γ SFUs per one million BAL cells were assessed. Results are means ± SEM from triplicate assays of each set of culture conditions. Data are results from one of two independent experiments. (c) IL-17A production in the supernatant of BAL cells stimulated by the CW fraction of M. tuberculosis
ex vivo. Results are means ± SEM from triplicate assays. Data are results from one of two independent experiments.
References
-
- Andersen P, et al. 1992. Identification of immunodominant antigens during infection with Mycobacterium tuberculosis. Scand. J. Immunol. 36:823–831 - PubMed
-
- Annibali V, et al. 2011. CD161(high)CD8+T cells bear pathogenetic potential in multiple sclerosis. Brain 134:542–554 - PubMed
-
- Berzins SP, Smyth MJ, Baxter AG. 2011. Presumed guilty: natural killer T cell defects and human disease. Nat. Rev. Immunol. 11:131–142 - PubMed
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