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Comparative Study
. 2011 Jan 1;186(1):489-98.
doi: 10.4049/jimmunol.1002639. Epub 2010 Nov 22.

Nonconventional CD8+ T cell responses to Listeria infection in mice lacking MHC class Ia and H2-M3

Hoonsik Cho  1 , Hak-Jong Choi, Honglin Xu, Kyrie Felio, Chyung-Ru Wang
Affiliations
Comparative Study

Nonconventional CD8+ T cell responses to Listeria infection in mice lacking MHC class Ia and H2-M3

Hoonsik Cho et al. J Immunol. .

Abstract

CD8(+) T cells restricted to MHC class Ib molecules other than H2-M3 have been shown to recognize bacterial Ags. However, the contribution of these T cells to immune responses against bacterial infection is not well defined. To investigate the immune potential of MHC class Ib-restricted CD8(+) T cells, we have generated mice that lack both MHC class Ia and H2-M3 molecules (K(b-/-)D (b-/-)M3(-/-)). The CD8(+) T cells present in K(b-/-)D (b-/-)M3(-/-) mice display an activated surface phenotype and are able to secrete IFN-γ rapidly upon anti-CD3 and anti-CD28 stimulation. Although the CD8(+) T cell population is reduced in K(b-/-)D (b-/-)M3(-/-) mice compared with that in K(b-/-)D (b-/-) mice, this population retains the capacity to expand significantly in response to primary infection with the bacteria Listeria monocytogenes. However, K(b-/-)D (b-/-)M3(-/-) CD8(+) T cells do not expand upon secondary infection, similar to what has been observed for H2-M3-restricted T cells. CD8(+) T cells isolated from Listeria-infected K(b-/-)D (b-/-)M3(-/-) mice exhibit cytotoxicity and secrete proinflammatory cytokines in response to Listeria-infected APCs. These T cells are protective against primary Listeria infection, as Listeria-infected K(b-/-)D (b-/-)M3(-/-) mice exhibit reduced bacterial burden compared with that of infected β(2)-microglobulin-deficient mice that lack MHC class Ib-restricted CD8(+) T cells altogether. In addition, adoptive transfer of Listeria-experienced K(b-/-)D (b-/-)M3(-/-) splenocytes protects recipient mice against subsequent Listeria infection in a CD8(+) T cell-dependent manner. These data demonstrate that other MHC class Ib-restricted CD8(+) T cells, in addition to H2-M3-restricted T cells, contribute to antilisterial immunity and may contribute to immune responses against other intracellular bacteria.

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Figures

Figure 1
Figure 1. Generation of Kb−/−Db−/−M3−/− mice
(A) Schematic detailing the meiotic intra-H2 recombination required to generate Kb−/−Db−/−M3+/− mice. (B) Flow cytometric analysis of H2-Kb, H2-Db and H2-M3 cell surface expression on B220+ splenocytes isolated from WT (thick line) and Kb−/−Db−/−M3−/− (dotted line) mice. Isotype controls are shown for comparison as shaded histograms. To detect H2-M3 expression, splenocytes from indicated mice were incubated overnight with 10 μM LemA peptide.
Figure 2
Figure 2. Characterization of CD8+ T cells in naïve Kb−/−Db−/−M3−/− mice
(A–C) Flow cytometric analysis of CD4+ and CD8β+ T cell populations in WT, Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice. Data shown are representative of three independent experiments. (A) Lymphocytes were isolated from the spleen, liver and lymph nodes. Numbers indicate the percentage of cells in each quadrant in the lymphocyte gate. (B) Bar graphs indicate the percentage of CD8+ T cells. Data are presented as the mean ± SEM using six mice per genotype. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C) Cell surface expression of activation markers on TCRβ+CD8+ splenocytes in naïve WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice. (D) Ex vivo anti-CD3 and anti-CD28 antibody stimulation of CD8+ T cells enriched from the spleen of WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice. Intracellular staining for IFN-γ was performed at 12 h post-stimulation. Data shown are representative of three experiments.
Figure 3
Figure 3. Expansion of CD8+ T cells in Kb−/−Db−/−M3−/− mice during LM infection
(A) WT, Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice were infected with 2 × 103 CFU of LM. 7 days following infection, splenocytes and hepatic leukocytes were harvested and stained with antibodies against CD8β and TCRβ. Bar graphs depict the mean ± SEM for the percentage of CD8+ T cells in the lymphocyte gate for uninfected and LM-infected WT, Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001. (B) Splenocytes and hepatic leukocytes were harvested from LM-infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice at the indicated time points and stained with antibodies against CD8α, TCRβ and CD44. Bar graphs depict the mean ± SEM for the absolute number of CD44highCD8+ T cells for each indicated genotype. Results from 3–9 mice per genotype are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 4
Figure 4. Protective role of residual CD8+ T cells in Kb−/−Db−/−M3−/− mice
(A) Kb−/−Db−/−, Kb−/−Db−/−M3−/− and β2m−/− mice were infected with 2 × 103 CFU of LM. Bacterial burden in the spleen and liver was determined at indicated time points post-infection. Data are presented as the mean ± SEM from 3–6 mice per genotype for each time point. *, p < 0.05. (B) WT recipient mice were adoptively transferred with 2 × 107 splenocytes isolated from naïve WT or Kb−/−Db−/−M3−/− mice or from WT or Kb−/−Db−/−M3−/− mice that had been infected with 2 × 103 LM 7 days prior. In some cases, splenocytes from infected donor mice were depleted of CD8+ T cells. 30–60 min following cell transfer, recipient mice were infected with 5 × 104 LM. Three days post-infection, spleen and liver were harvested and the bacterial burden was determined. Data shown are the mean ± SEM from six mice for each transfer group. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 5
Figure 5. Effector function of LM-specific CD8+ T cells in Kb−/−Db−/−M3−/− mice
(A–C) Splenocytes and hepatic leukocytes were harvested from Kb−/−Db−/−M3−/− mice 7 days post LM infection. (A) Cells were stimulated with HKLM for 7 h and stained with antibodies against CD8β and TCRβ. Cells were then intracellularly stained for IFN-γ and analyzed by flow cytometry. The percentages of CD8+IFN-γ+ and CD8−IFN-γ+ cells within the TCRβ+ gate are indicated. Results are representative of three experiments. (B) Splenocytes were harvested from Kb−/−Db−/−M3−/− mice 7 days post LM infection and enriched for CD8+ T cells. These T cells were then cultured with LM-infected BMDC for 48 h. Culture supernatants were then harvested to determine the presence of cytokines using a Cytometric Bead Array kit (for TNF, IFN-γ, and IL-6) or by ELISA (for IL-17A). Bar graphs depict means of duplicate wells ± SEM from two representative experiments pooling two mice per genotype. (C) Splenocytes were isolated from Kb−/−Db−/−M3−/− mice 7 days post LM infection, enriched for CD8+ T cells, and activated with ConA. After 3 days of ConA stimulation, splenocytes were used as effectors in a 51Cr release CTL assay at the indicated effector:target cell ratios. Uninfected or LM-infected J774 cells were labeled with 51Cr and used as targets. Graph depicts the mean ± SEM for the percentage of LM-specific killing pooling two mice per genotype. Data are representative of three independent experiments.
Figure 6
Figure 6. LM-specific Kb−/−Db−/−M3−/− CD8+ T cells are not restricted to Qa-1b, Qa2 or MR1
(A) Splenocytes were harvested from LM-infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice at 7 days post-infection and enriched for CD8+ T cells. These T cells were then used as effectors in an IFN-γ ELISPOT assay. Uninfected or LM-infected BMDC were used as stimulators and were incubated with CD8+ T cells in medium alone, in the presence of isotype control Ab, or with mAb against either H2-M3, Qa-1b, Qa-2 or MR1. Results are presented as the mean ± SEM of the number of IFN-γ spot-forming units from two pooled animals per genotype and are representative of three independent experiments. **, p < 0.01. (B, C) T2 CTL, a CD8+ T cell line derived from LM-infected Kb−/−Db−/−M3−/− mice, were used as effector cells in a 51Cr release CTL assay. Results are representative of three independent experiments. (B) BMDC derived from Kb−/−Db−/−M3−/− or β2m−/− mice were labeled with 51Cr and used as targets at the indicated effector: target cell ratios. Some target cells were incubated overnight with HKLM prior to assay setup. (C) BMDC derived from Kb−/−Db−/−M3−/− mice were labeled with 51Cr and used as targets at the indicated effector: target cell ratios. Some target cells were incubated overnight with HKLM prior to assay setup. In addition, some target cells were incubated with blocking mAb against Qa-1b, Qa-2, or MR1 30 min prior to labeling.
Figure 7
Figure 7. Non-M3 MHC class Ib-restricted CD8+ T cell responses to secondary LM infection
(A) Splenocytes were harvested from naïve Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice, from mice that had been infected with 2×103 CFU of LM 1 mo previously, and from mice that had been infected 1 mo previously and subsequently rechallenged with 5×104 CFU of LM. Cells were stained with antibodies against CD8β and TCRβ at 3 and 5 days post-rechallenge and flow cytometry was performed to determine the proportion of CD8+ T cells in the TCRβ+ gate. Results shown are presented as the mean ± SEM from 3–5 mice per experimental group and are representative of two experiments. *, p < 0.05; **, p < 0.01. (B) Splenocytes isolated from Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice at 3 days after secondary LM infection were stimulated ex vivo with HKLM. The proportion of IFN-γ-producing CD8+ T cells in the TCRβ+ gate was determined by intracellular staining. Results are representative of three individual experiments. (C) Bar graphs depict the mean ± SEM for the number of LM-specific IFN-γ-producing CD8+ T cells in spleens of Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice 7 days after primary LM infection, 1 mo after primary infection or 3 days after secondary infection. Results shown are from 3–5 mice per experimental group are representative of two individual experiments.
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