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. 2016 Jun 7;12(6):e1005688.
doi: 10.1371/journal.ppat.1005688. eCollection 2016 Jun.

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

Shaobin Shang  1 , Sarah Siddiqui  1 , Yao Bian  1 , Jie Zhao  1 , Chyung-Ru Wang  1
Affiliations

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

Shaobin Shang et al. PLoS Pathog. .

Abstract

MHC Ib-restricted CD8+ T cells have been implicated in host defense against Mycobacterium tuberculosis (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 (Kb-/-Db-/-), MHC Ia/H2-M3 (Kb-/-Db-/-M3-/-), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in Listeria infection, we found that M3-restricted CD8+ T cells only represented a small proportion of the CD8+ T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8+ T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8+ T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8+ T cells was mostly β2m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8+ T cells in Kb-/-Db-/-M3-/- mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8+ T cells were detected. Our findings indicate that nonclassical CD8+ T cells other than the known M3, CD1, and MR1-restricted CD8+ T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8+ 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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Non-M3, MHC Ib-restricted CD8+ T cells contribute to protective immunity against Mtb.
C57BL/6, Kb-/-Db-/-, Kb-/-Db-/-M3-/- and β2m-/- mice were infected with aerosolic Mtb H37Rv (~200 CFU) and sacrificed at indicated time-points. (A) Representative dot-plots depict CD8+ and CD4+ 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+ population. (B, C) Bar graphs depict the mean ± SEM of the percentage (B) and total number (C) of CD8+ 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), Kb-/-Db-/- (n = 9–14), Kb-/-Db-/-M3-/- (n = 7–15) and β2m-/- (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 Kb-/-Db-/-M3-/- mice received CD8 depleting mAb or control IgG at day 28 post-infection. Data shown are the mean ± SEM from 9 mice per group. *P <0.05, **P <0.01, ***P <0.001, ns, no statistical significance.
Fig 2
Fig 2. Non-M3, MHC Ib-restricted CD8+ T cells preferentially expand in the lung and express elevated levels of KLRG1 during Mtb infection.
WT and Kb-/-Db-/-M3-/- mice were sacrificed at indicated time-points, single cells from the lung and spleen were prepared for phenotypic analysis of CD8+ T cells by flow cytometry. (A) Kinetic changes of total number of CD8+ T cells in the lung and spleen from C57BL/6 (n = 4–9) and Kb-/-Db-/-M3-/- (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 CD44hiCD62LloCD8+ effector T cells (TEFF) in lung and spleen from C57BL/6 (n = 4–6) and Kb-/-Db-/-M3-/- (n = 4–7) mice during infection. (C, D) Representative histograms show the expression of KLRG1 (C) and PD-1 (D) on CD8 TEFF 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+ effector and memory (CD44hiCD62Lhi) 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+ Tcm. (CD127+CD44hiCD62Lhi) cells in the lung of C57BL/6 (n = 4–6) and Kb-/-Db-/-M3-/- (n = 4–7) at indicated time points after infection. *P <0.05, **P <0.01, ***P <0.001.
Fig 3
Fig 3. CD8+ T cells from Mtb-infected Kb-/-Db-/-M3-/- mice mount a more robust Mtb antigen-specific IFN-γ response as compared to CD8+ T cells from B6 mice.
CD8+ T cells from the lungs of Mtb-infected C57BL/6 and Kb-/-Db-/-M3-/- mice were stimulated ex vivo for 18h with Mtb antigen-pulsed C57BL/6 and Kb-/-Db-/-M3-/- BMDCs, respectively, and harvested for intracellular staining of IFN-γ. (A) Representative dot-plots of IFN-γ-producing CD8+ T cells in the lungs of C57BL/6 and Kb-/-Db-/-M3-/- mice at day 30 after infection upon stimulation with Mtb whole cell lysates (WCL)-pulsed BMDCs. (B, C) The changes of percentage (B) and total number (C) of WCL-specific CD8+ T cells detected in the lungs of Kb-/-Db-/-M3-/- (n = 3–9) and C57BL/6 (n = 3–7) mice during the course of infection. (D) Comparison of the magnitude of Mtb-specific MHC Ib-restricted CD8+ T cell responses in C57BL/6 and Kb-/-Db-/-M3-/- mice. CD8+ T cells from the lung of C57BL/6 and Kb-/-Db-/-M3-/- mice were stimulated ex vivo with WCL-pulsed or un-pulsed Kb-/-Db-/-M3-/- BMDCs for 18h, and harvested for intracellular staining of IFN-γ. Bar graphs depict the mean ± SEM of the percentage IFN-γ-producing CD8+ T cells in C57BL/6 (n = 3) and Kb-/-Db-/-M3-/- (n = 5) mice at day 30 post-infection. Data shown are pooled from two independent experiments. **P <0.01, ***P <0.001.
Fig 4
Fig 4. Non-M3, MHC Ib-restricted CD8+ T cells recognize Mtb protein antigens in a β2m-dependent and TAP-independent manner.
CD8+ T cells from the lung of Kb-/-Db-/-M3-/- 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+ T cells detected from Kb-/-Db-/-M3-/- mice upon stimulation with Kb-/-Db-/-M3-/- 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+ T cells detected from Kb-/-Db-/-M3-/- (n = 3) mice in response to different Mtb antigen fractions. (C) Percentage of IFN-γ+TNF-α+CD8+ T cells detected upon stimulation with CFP-pulsed BMDCs from C57BL/6, Kb-/-Db-/-M3-/-, β2m-/-, TAP-/-, MyD88-/- 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. ***P <0.001.
Fig 5
Fig 5. Non-M3, MHC Ib-restricted CD8+ T cells exhibit polyfunctional characteristics.
CD8+ T cells from the lung or spleen of C57BL/6 and Kb-/-Db-/-M3-/- mice were stimulated ex vivo for 18h with Mtb antigen-pulsed C57BL/6 and Kb-/-Db-/-M3-/- BMDCs, respectively, then harvested for intracellular staining of IFN-γ, TNF-α and IL-2. (A) Representative dot-plots of multiple cytokine-producing CD8+ T cells in the lung of C57BL/6 and Kb-/-Db-/-M3-/- mice at day 30 after infection upon stimulation with Mtb culture filtrate proteins (CFP). (B) Pie graphs show relative proportion of TNF-α+, IL-2+ and TNF-α+IL-2+ CD8+ T cells among IFN-γ+CD8+ T cells from the lung of Mtb-infected Kb-/-Db-/-M3-/- and C57BL/6 mice. (C) IL-17A production from enriched CD8+ T cells in Mtb-infected Kb-/-Db-/-M3-/- mice detected by ELISA after stimulation with CFP. ***P <0.001.
Fig 6
Fig 6. Non-M3, MHC Ib-restricted CD8+ T cells recognize various Mtb antigens.
At day 30 post-infection, CD8+ T cells from Mtb-infected Kb-/-Db-/-M3-/- and B6 mice were used in ELISPOT assays using various Mtb antigen-pulsed Kb-/-Db-/-M3-/- or B6 BMDCs as stimulators. (A, B) The frequency of IFN-γ-producing CD8+ T cells detected in the lung (A) and spleen (B) of Kb-/-Db-/-M3-/- mice (n = 3) in response to in vitro stimulation with various Mtb antigens. (C, D) The frequency of IFN-γ-producing CD8+ 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.
Fig 7
Fig 7. Qa-2-restricted T cells comprise a significant proportion of the expanded CD8+ T cells during Mtb infection.
CD8+ T cells from the lungs of Mtb-infected Kb-/-Db-/-M3-/- 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+ 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+CD8+ 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+ T cells from spleens or lungs of Kb-/-Db-/-M3-/- (D) and C57BL/6 (E) mice were stimulated with un-pulsed or CFP-pulsed Kb-/-Db-/-M3-/- BMDCs in the presence of anti-Qa-2 (20-8-4) or control IgG (anti-Kb, 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). **P <0.01, ***P <0.001.
Fig 8
Fig 8. MR1-restricted T cells do not represent a significant population of Mtb-specific T cells in Kb-/-Db-/-M3-/- mice after Mtb infection.
(A) Lymphocytes isolated from the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice were stimulated with Mtb culture supernatant-pulsed BMDCs from C57BL/6, MR1-/- and β2m-/- mice. Percentages of cytokine-producing CD8+ and CD4-CD8- (DN) T cells were determined by intracellular cytokine staining. Data are representative of two independent experiments, and are the mean ± SEM. (n = 3). ***P<0.001. (B) CD8+ T cells isolated from the lung, mediastinal lymph node and spleen of naïve and Mtb-infected Kb-/-Db-/-M3-/- 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+ T cells in naïve and Mtb-infected Kb-/-Db-/-M3-/- mice (n = 5) at day 30 post-infection.
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