Innate Invariant NKT Cells Recognize Mycobacterium tuberculosis–Infected Macrophages, Produce Interferon-γ, and Kill Intracellular Bacteria

Cellular immunity to Mycobacterium tuberculosis (Mtb) requires a coordinated response between the innate and adaptive arms of the immune system, resulting in a type 1 cytokine response, which is associated with control of infection. The contribution of innate lymphocytes to immunity against Mtb remains controversial. We established an in vitro system to study this question. Interferon-γ is produced when splenocytes from uninfected mice are cultured with Mtb-infected macrophages, and, under these conditions, bacterial replication is suppressed. This innate control of bacterial replication is dependent on CD1d-restricted invariant NKT (iNKT) cells, and their activation requires CD1d expression by infected macrophages as well as IL-12 and IL-18. We show that iNKT cells, even in limiting quantities, are sufficient to restrict Mtb replication. To determine whether iNKT cells contribute to host defense against tuberculosis in vivo, we adoptively transferred iNKT cells into mice. Primary splenic iNKT cells obtained from uninfected mice significantly reduce the bacterial burden in the lungs of mice infected with virulent Mtb by the aerosol route. Thus, iNKT cells have a direct bactericidal effect, even in the absence of synthetic ligands such as α-galactosylceramide. Our finding that iNKT cells protect mice against aerosol Mtb infection is the first evidence that CD1d-restricted NKT cells mediate protection against Mtb in vivo

Naïve splenocytes cultured with <i>Mtb</i>-infected Mφ produce IFN-γ and suppress bacterial replication.

<p>(A) <i>Mtb</i>-infected Mφ secrete IL-12. Uninfected (open bars) and <i>Mtb</i>-infected (solid bars) were cultured in the presence or absence of splenocytes (Spln). IL-12p40 was measured after 24 hrs. Bars represent the mean+/−SD of triplicate cultures. (B) <i>Mtb</i>-infected Mφ stimulate IFN-γ secretion by splenocytes. Uninfected (open bars) and <i>Mtb</i>-infected (solid bars) were cultured in the presence or absence of splenocytes. IFN-γ was measured 24 hrs after coculture. The bars represent the mean+/−SD of triplicate cultures. (C) Coculture of splenocytes with infected Mφ leads to reduced <i>Mtb</i> replication. <i>Mtb</i>-infected Mφ were cultured in the presence or absence of splenocytes from uninfected mice for 3 days and then CFU were determined. Bar represents mean+/−SD from replicate cultures (n = 6). (D) Naïve splenocytes reproducibly suppress intracellular <i>Mtb</i> replication. To determine the average CFU reduction, the data from 18 independent experiments were normalized by setting the CFU recovered from the infected Mφ cultured alone to 100%. Bars, mean±SEM.</p

IFN-γ production by innate lymphocytes upregulates NO synthesis and is dependent on IL-12 and IL-18.

<p><i>Mtb</i>-infected Mφ were cultured alone or with splenocytes. In addition, some cultures included monoclonal antibodies to IL-12, IL-18, or both, or an irrelevant isotype control antibody. After 24 hrs, IFN-γ (A) and NO₂ (B) were measured in the culture supernatant, and after 72 hrs the total CFU were determined (C).</p

iNKT cells are sufficient to inhibit intracellular bacterial replication.

<p>(A) <i>Mtb</i>-infected Mφ were cultured alone or with WT splenocytes (spln) or with iNKT cells. Bacterial growth in the <i>Mtb-</i>infected Mφ was assessed by determining CFU on day 1 and day 4 post-infection. On day 1, WT splenocytes or the NKT cell line were added to the <i>Mtb</i>-infected Mφ, and CFU were determined 72 hrs later on day 4. *, p<0.05, as determined by a one-way ANOVA with Tukey's Multiple Comparison Test. (B) <i>Mtb-</i>infected Mφ were cultured alone or with WT splenocytes or with different numbers of NKT cells as indicated. After 24 hours of coculture, IFN-γ was measured in the culture supernatants by ELISA. (C) After 72 hrs, the cultures from (B) were analyzed for the total CFU and compared to <i>Mtb</i>-infected Mφ alone on day 1 and day 4. All conditions containing splenocytes or iNKT cells had significantly (p<0.05) fewer CFU compared to <i>Mtb</i>-infected Mφ on day 4 post-infection.</p

CD69 is upregulated on T cells following culture of splenocytes with <i>Mtb</i>-infected Mφ.

<p>Splenocytes were cultured with uninfected (top row) or infected (bottom row) Mφ for 24 hrs. Nonadherent cells were recovered and analyzed by flow cytometry. Cells were gated by size and positive staining with CD1d tetramer (left), CD4 (middle), or anti-CD8 (right). CD69 expression was measured as an indication of cell activation.</p

iNKT cells protect mice against <i>Mtb</i>.

<p>C57BL/6 mice were sublethally irradiated and used as recipients in an adoptive transfer experiment (n = 5/group). As a control, one group did not receive any cells (no transfer). Mice received an iNKT cell line, or splenic CD8⁻CD62L⁻ cells from Jα281<i>^−/−</i> mice or WT C57BL/6 mice. All mice were infected within 24 hrs with <i>Mtb</i> by the aerosol route. Three weeks after infection, the bacterial burden in the lung and spleen were determined. Each point represents data from an individual mouse, and the bar represents the mean. The comparison between untransferred mice and mice receiving the NKT cell line was performed twice with similar results. *, p<0.05 by a one-way ANOVA.</p

Suppression of <i>Mtb</i> replication by splenocytes is T cell–dependent.

<p>Statistical testing was done using a one-way ANOVA with Dunnett's post-test comparing each experimental group to infected Mφ cultured alone. Bars, mean±SEM of replicate cultures (n = 6–8). **, ***, p<0.05; ns, not significant. (A) Splenocytes (spln) from RAG<i>^−/−</i> mice are unable to limit bacterial replication. Infected Mφ were cultured alone or with splenocytes from uninfected WT or RAG<i>^−/−</i> mice. Coculture with WT splenocytes but not RAG<i>^−/−</i> splenocytes led to a significant reduction in bacterial CFU after 3 days. (B) T cell enriched cellular fractions have increased anti-mycobacterial activity. Infected Mφ were cultured alone or with cell fractions from WT splenocytes (spleen). CD90 and CD19 immunomagnetic beads were used to enrich or deplete T cells or B cells from splenocytes. (C) Both CD4⁺ and CD4⁻ cells suppress <i>Mtb</i> replication. Infected Mφ were cultured alone or with CD4⁺ or CD4⁻ cell fractions obtained from WT splenocytes.</p

Suppression of bacterial replication requires cognate recognition of <i>Mtb</i>-infected Mφ by iNKT cells.

<p>Statistical testing was done using a one-way ANOVA with Dunnett's post-test comparing each experimental group to infected Mφ cultured alone. Bars, mean±SEM of replicate cultures (n = 6–8). *, ***, p<0.05; ns, not significant. (A) The presence of iNKT cells is required for the <i>Mtb</i> suppressive activity of splenocytes. Infected Mφ were cultured alone or with splenocytes (spln) from WT, CD1d<i>^−/−</i>, or Ja281<i>^−/−</i> mice. Only addition of WT splenocytes led to a significant reduction in CFU. (B) CD1d expression by <i>Mtb</i>-infected Mφ is required for the control of bacterial replication. Mφ obtained from WT (left) or CD1d<i>^−/−</i> (right) mice were infected with <i>Mtb</i>. Infected Mφ were cocultured with WT splenocytes (spln). Addition of splenocytes to WT Mφ but not CD1d<i>^−/−</i> Mφ led to a significant CFU reduction.</p