21 research outputs found

    Nonclassical MHC Ib-restricted CD8<sup>+</sup> T Cells Recognize <i>Mycobacterium tuberculosis</i>-Derived Protein Antigens and Contribute to Protection Against Infection

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    <div><p>MHC Ib-restricted CD8<sup>+</sup> T cells have been implicated in host defense against <i>Mycobacterium tuberculosis</i> (Mtb) infection. However, the relative contribution of various MHC Ib-restricted T cell populations to anti-mycobacterial immunity remains elusive. In this study, we used mice that lack MHC Ia (K<sup>b-/-</sup>D<sup>b-/-</sup>), MHC Ia/H2-M3 (K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup>), or β<sub>2</sub>m (β<sub>2</sub>m<sup>-/-</sup>) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in <i>Listeria</i> infection, we found that M3-restricted CD8<sup>+</sup> T cells only represented a small proportion of the CD8<sup>+</sup> T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8<sup>+</sup> T cells expanded preferentially in the lungs of Mtb-infected K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8<sup>+</sup> T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8<sup>+</sup> T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8<sup>+</sup> T cells was mostly β<sub>2</sub>m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8<sup>+</sup> T cells in K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8<sup>+</sup> T cells were detected. Our findings indicate that nonclassical CD8<sup>+</sup> T cells other than the known M3, CD1, and MR1-restricted CD8<sup>+</sup> T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8<sup>+</sup> T cells would facilitate the design of better Mtb vaccines with broader coverage across MHC haplotypes due to the limited polymorphism of MHC class Ib molecules.</p></div

    Non-M3, MHC Ib-restricted CD8<sup>+</sup> T cells preferentially expand in the lung and express elevated levels of KLRG1 during Mtb infection.

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    <p>WT and K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice were sacrificed at indicated time-points, single cells from the lung and spleen were prepared for phenotypic analysis of CD8<sup>+</sup> T cells by flow cytometry. (A) Kinetic changes of total number of CD8<sup>+</sup> T cells in the lung and spleen from C57BL/6 (n = 4–9) and K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> (n = 7–9) mice after infection. Numbers in bracket indicate fold changes of expansion at indicated time points after infection. (B) Kinetic changes of total number of CD44<sup>hi</sup>CD62L<sup>lo</sup>CD8<sup>+</sup> effector T cells (T<sub>EFF</sub>) in lung and spleen from C57BL/6 (n = 4–6) and K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> (n = 4–7) mice during infection. (C, D) Representative histograms show the expression of KLRG1 (C) and PD-1 (D) on CD8 T<sub>EFF</sub> cells from the lung at day 0 and day 60 post-infection. Grey solid areas indicate isotype control. Data shown are representative of three independent experiments. (E) Representative dot plots depict CD127 expression on CD8<sup>+</sup> effector and memory (CD44<sup>hi</sup>CD62L<sup>hi</sup>) cells in the lungs of indicated mice before infection or at day 60 post-infection. (F, G) Bar graphs depict the mean ± SEM of the percentage (F) and total number (G) of CD8<sup>+</sup> Tcm. (CD127<sup>+</sup>CD44<sup>hi</sup>CD62L<sup>hi</sup>) cells in the lung of C57BL/6 (n = 4–6) and K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> (n = 4–7) at indicated time points after infection. *<i>P</i> <0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001.</p

    Non-M3, MHC Ib-restricted CD8<sup>+</sup> T cells contribute to protective immunity against Mtb.

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    <p>C57BL/6, K<sup>b-/-</sup>D<sup>b-/-</sup>, K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> and β<sub>2</sub>m<sup>-/-</sup> mice were infected with aerosolic Mtb H37Rv (~200 CFU) and sacrificed at indicated time-points. (A) Representative dot-plots depict CD8<sup>+</sup> and CD4<sup>+</sup> T cell populations from indicated mice before infection or at day 30 post-infection. Numbers indicate the percentage of cells in each quadrant in the TCRβ<sup>+</sup> population. (B, C) Bar graphs depict the mean ± SEM of the percentage (B) and total number (C) of CD8<sup>+</sup> T cells in the lung and spleen of indicated mice at day 30 post-infection. Data shown are from one of three independent experiments with 3 mice in each group. (D, E) Comparison of CFU between C57BL/6 (n = 5–8), K<sup>b-/-</sup>D<sup>b-/-</sup> (n = 9–14), K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> (n = 7–15) and β<sub>2</sub>m<sup>-/-</sup> (n = 4–6) mice in the lung (D) and spleen (E) at day 14, 30 and 60 post-infection. Data shown are pooled from three independent experiments. (F) Bacterial burden in the lung of K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice received CD8 depleting mAb or control IgG at day 28 post-infection. Data shown are the mean ± SEM from 9 mice per group. *<i>P</i> <0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001, ns, no statistical significance.</p

    Non-M3, MHC Ib-restricted CD8<sup>+</sup> T cells preferentially expand in the lung and express elevated levels of KLRG1 during Mtb infection.

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    <p>WT and K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice were sacrificed at indicated time-points, single cells from the lung and spleen were prepared for phenotypic analysis of CD8<sup>+</sup> T cells by flow cytometry. (A) Kinetic changes of total number of CD8<sup>+</sup> T cells in the lung and spleen from C57BL/6 (n = 4–9) and K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> (n = 7–9) mice after infection. Numbers in bracket indicate fold changes of expansion at indicated time points after infection. (B) Kinetic changes of total number of CD44<sup>hi</sup>CD62L<sup>lo</sup>CD8<sup>+</sup> effector T cells (T<sub>EFF</sub>) in lung and spleen from C57BL/6 (n = 4–6) and K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> (n = 4–7) mice during infection. (C, D) Representative histograms show the expression of KLRG1 (C) and PD-1 (D) on CD8 T<sub>EFF</sub> cells from the lung at day 0 and day 60 post-infection. Grey solid areas indicate isotype control. Data shown are representative of three independent experiments. (E) Representative dot plots depict CD127 expression on CD8<sup>+</sup> effector and memory (CD44<sup>hi</sup>CD62L<sup>hi</sup>) cells in the lungs of indicated mice before infection or at day 60 post-infection. (F, G) Bar graphs depict the mean ± SEM of the percentage (F) and total number (G) of CD8<sup>+</sup> Tcm. (CD127<sup>+</sup>CD44<sup>hi</sup>CD62L<sup>hi</sup>) cells in the lung of C57BL/6 (n = 4–6) and K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> (n = 4–7) at indicated time points after infection. *<i>P</i> <0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001.</p

    MR1-restricted T cells do not represent a significant population of Mtb-specific T cells in K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice after Mtb infection.

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    <p>(A) Lymphocytes isolated from the lungs of Mtb-infected K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice were stimulated with Mtb culture supernatant-pulsed BMDCs from C57BL/6, MR1<sup>-/-</sup> and β<sub>2</sub>m<sup>-/-</sup> mice. Percentages of cytokine-producing CD8<sup>+</sup> and CD4<sup>-</sup>CD8<sup>-</sup> (DN) T cells were determined by intracellular cytokine staining. Data are representative of two independent experiments, and are the mean ± SEM. (n = 3). ***<i>P</i><0.001. (B) CD8<sup>+</sup> T cells isolated from the lung, mediastinal lymph node and spleen of naïve and Mtb-infected K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice (at day 30 post-infection) were used to examine the expression of Vα19-Jα33 transcripts by quantitative RT-PCR (n = 6 per group). qPCR results are presented as relative units normalized to TCRα constant region mRNA. ns, no statistical significance. (C) TCR Vβ usage of CD8<sup>+</sup> T cells in naïve and Mtb-infected K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice (n = 5) at day 30 post-infection.</p

    Qa-2-restricted T cells comprise a significant proportion of the expanded CD8<sup>+</sup> T cells during Mtb infection.

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    <p>CD8<sup>+</sup> T cells from the lungs of Mtb-infected K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice at day 30 post-infection were stimulated with un-pulsed or CFP-pulsed BMDCs or Mtb-infected BMDCs from indicated mice and intracellularly stained for the cytokine IFN-γ and TNF-α. (A, B) The percentage of cytokine-producing CD8<sup>+</sup> T cells in response to stimulation with CFP-pulsed (A) and Mtb-infected (B) BMDCs derived from B6 and various MHC Ib molecule-deficient mice. (C) The percentage of IFN-γ<sup>+</sup>CD8<sup>+</sup> T cells upon stimulation with CFP-pulsed BMDCs that expressed different level of Qa-2. The percentage shown is the percentage of cytokine-producing cells with Ag stimulation minus the baseline without Ag stimulation. (D, E) CD8<sup>+</sup> T cells from spleens or lungs of K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> (D) and C57BL/6 (E) mice were stimulated with un-pulsed or CFP-pulsed K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> BMDCs in the presence of anti-Qa-2 (20-8-4) or control IgG (anti-K<sup>b</sup>, Y3 or MOPC1) and the IFN-γ-secreting cells were quantified in an ELISPOT assay. Data shown are representative of three independent experiments, and are the mean ± SEM (n = 3 per experiment). **<i>P</i> <0.01, ***<i>P</i> <0.001.</p

    Non-M3, MHC Ib-restricted CD8<sup>+</sup> T cells recognize Mtb protein antigens in a β<sub>2</sub>m-dependent and TAP-independent manner.

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    <p>CD8<sup>+</sup> T cells from the lung of K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice at day 30 after infection were stimulated with BMDCs pulsed with or without Mtb antigens, followed by ICS for IFN-γ. (A) Representative dot-plots of IFN-γ-producing CD8<sup>+</sup> T cells detected from K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice upon stimulation with K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> BMDCs pulsed with Mtb whole cell lysate (WCL), culture filtrate proteins (CFP), purified protein derivatives (PPD), total lipids (Tlip), and proteinase K-treated WCL, respectively. (B) The percentage of IFN-γ-producing MHC Ib-restricted CD8<sup>+</sup> T cells detected from K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> (n = 3) mice in response to different Mtb antigen fractions. (C) Percentage of IFN-γ<sup>+</sup>TNF-α<sup>+</sup>CD8<sup>+</sup> T cells detected upon stimulation with CFP-pulsed BMDCs from C57BL/6, K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup>, β<sub>2</sub>m<sup>-/-</sup>, TAP<sup>-/-</sup>, MyD88<sup>-/-</sup> mice, respectively. Shown are the percentages of cytokine-positive cells in responses to CFP-pulsed DCs minus the percentage of cytokine-positive cells in responses to the corresponding un-pulsed DCs. Data shown are representative of three independent experiments. ***<i>P</i> <0.001.</p

    Non-M3, MHC Ib-restricted CD8<sup>+</sup> T cells recognize various Mtb antigens.

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    <p>At day 30 post-infection, CD8<sup>+</sup> T cells from Mtb-infected K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> and B6 mice were used in ELISPOT assays using various Mtb antigen-pulsed K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> or B6 BMDCs as stimulators. (A, B) The frequency of IFN-γ-producing CD8<sup>+</sup> T cells detected in the lung (A) and spleen (B) of K<sup>b-/-</sup>D<sup>b-/-</sup>M3<sup>-/-</sup> mice (n = 3) in response to <i>in vitro</i> stimulation with various Mtb antigens. (C, D) The frequency of IFN-γ-producing CD8<sup>+</sup> T cells detected in the lung (C) and spleen (D) of B6 mice (n = 3) upon stimulation with various Mtb antigens. Dot lines depict the background cytokine production in the absence of Mtb Ag. Data shown are representative of three independent experiments.</p

    Con A induced hepatitis is alleviated in B7- and CD28- deficient mice.

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    <p>Eight-week old B7KO, CD28KO and C57BL/6 mice received intravenous injection of Con A at a dose of 20 mg/kg. Sixteen hours later, serum and liver tissues from individual mice were harvested. A. Serum ALT and AST levels. B. One representative section from each group showing the pattern of liver injury. C. Average percentages of area with injuries from 3–4 sections per mouse and 4–5 mice per group.</p

    Roles for B7-1/2-CD28 interaction in the accumulation of TCR β<sup>+</sup>NK1.1<sup>+</sup> NKT cells in the thymus (A), spleen (B) and liver (C).

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    <p>B7KO, CD28KO and WT C57BL/6 mice were sacrificed at 8-week of age. Total lymphocytes (left column) from viable cells, CD4<sup>−</sup>CD8<sup>−</sup> DN lymphocytes (middle column) and CD4<sup>+</sup> lymphocytes (right column) were analyzed for the expression of TCRβ and NK1.1. One representative profile from each group is presented. The numbers in the panels are percentages (Mean±SD) of gated NKT cells (n = 9). The numbers shown on the side of figures are <i>p</i> value between two indicated groups.</p
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