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

<div><p>MHC Ib-restricted CD8⁺ 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^b-/-D^b-/-), MHC Ia/H2-M3 (K^b-/-D^b-/-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 <i>Listeria</i> 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 K^b-/-D^b-/-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 K^b-/-D^b-/-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.</p></div

Non-M3, MHC Ib-restricted CD8⁺ T cells recognize Mtb protein antigens in a β₂m-dependent and TAP-independent manner.

<p>CD8⁺ T cells from the lung of K^b-/-D^b-/-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 K^b-/-D^b-/-M3^-/- mice upon stimulation with K^b-/-D^b-/-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 K^b-/-D^b-/-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, K^b-/-D^b-/-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. ***<i>P</i> <0.001.</p

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

<p>WT and K^b-/-D^b-/-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 K^b-/-D^b-/-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 CD44^hiCD62L^loCD8⁺ effector T cells (T_EFF) in lung and spleen from C57BL/6 (n = 4–6) and K^b-/-D^b-/-M3^-/- (n = 4–7) mice during infection. (C, D) Representative histograms show the expression of KLRG1 (C) and PD-1 (D) on CD8 T_EFF 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 (CD44^hiCD62L^hi) 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⁺CD44^hiCD62L^hi) cells in the lung of C57BL/6 (n = 4–6) and K^b-/-D^b-/-M3^-/- (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

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

<p>CD8⁺ T cells from the lungs of Mtb-infected K^b-/-D^b-/-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 K^b-/-D^b-/-M3^-/- (D) and C57BL/6 (E) mice were stimulated with un-pulsed or CFP-pulsed K^b-/-D^b-/-M3^-/- BMDCs in the presence of anti-Qa-2 (20-8-4) or control IgG (anti-K^b, 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⁺ T cells contribute to protective immunity against Mtb.

<p>C57BL/6, K^b-/-D^b-/-, K^b-/-D^b-/-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), K^b-/-D^b-/- (n = 9–14), K^b-/-D^b-/-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 K^b-/-D^b-/-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. *<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⁺ T cells preferentially expand in the lung and express elevated levels of KLRG1 during Mtb infection.

<p>WT and K^b-/-D^b-/-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 K^b-/-D^b-/-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 CD44^hiCD62L^loCD8⁺ effector T cells (T_EFF) in lung and spleen from C57BL/6 (n = 4–6) and K^b-/-D^b-/-M3^-/- (n = 4–7) mice during infection. (C, D) Representative histograms show the expression of KLRG1 (C) and PD-1 (D) on CD8 T_EFF 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 (CD44^hiCD62L^hi) 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⁺CD44^hiCD62L^hi) cells in the lung of C57BL/6 (n = 4–6) and K^b-/-D^b-/-M3^-/- (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^b-/-D^b-/-M3^-/- mice after Mtb infection.

<p>(A) Lymphocytes isolated from the lungs of Mtb-infected K^b-/-D^b-/-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). ***<i>P</i><0.001. (B) CD8⁺ T cells isolated from the lung, mediastinal lymph node and spleen of naïve and Mtb-infected K^b-/-D^b-/-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 K^b-/-D^b-/-M3^-/- mice (n = 5) at day 30 post-infection.</p

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

<p>At day 30 post-infection, CD8⁺ T cells from Mtb-infected K^b-/-D^b-/-M3^-/- and B6 mice were used in ELISPOT assays using various Mtb antigen-pulsed K^b-/-D^b-/-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 K^b-/-D^b-/-M3^-/- mice (n = 3) in response to <i>in vitro</i> 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.</p

Qa-1 expression is upregulated during aerosol Mtb infection.

<p>(A) Expression of Qa-1 on leukocytes from mediastinal lymph node (MLN) of low-dose Mtb-infected C57BL/6 mice at 2, 4, and 6 weeks post-infection, as compared to uninfected B6 mice or isotype control. (B) MFI of Qa-1 expression on B cells (B220⁺ CD11c^-), dendritic cells (DC) (CD11c⁺), T cells (TCRβ⁺), and macrophages (Mϕ) (CD11b⁺ F4/80⁺) from MLN of high-dose-infected B6 mice at 4 weeks post-infection, as compared to uninfected mice. Representative of 3 independent experiments, n ≥ 2 per group. * p < 0.05, ** p < 0.01, *** p < 0.001.</p

T cells from infected Qa-1^-/- mice have increased expression of inhibitory NK receptors.

<p>Lung lymphocytes from Qa-1^<b>+/+</b> and Qa-1^<b>-/-</b> mice were harvested at 4 weeks p.i. and surface expression of indicated markers analyzed by flow cytometry. (A) Representative dot plots of CD94 and NKG2A expression by cell type. (B-C) Frequency and total number of CD94- and NKG2A-expressing CD8⁺ T cells, CD4⁺ T cells, and NK cells. Data representative of at least 3 independent experiments, n≥4 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001.</p