Nonconventional CD8+ T Cell Responses to Listeria Infection in Mice Lacking MHC Class Ia and H2-M3

Hoonsik Cho, Hak-Jong Choi, Honglin Xu, Kyrie Felio and Chyung-Ru Wang
J Immunol January 1, 2011, 186 (1) 489-498; DOI: https://doi.org/10.4049/jimmunol.1002639

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 (Kb−/−D b−/−M3−/−). The CD8+ T cells present in Kb−/−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 Kb−/−D b−/−M3−/− mice compared with that in Kb−/−D b−/− mice, this population retains the capacity to expand significantly in response to primary infection with the bacteria Listeria monocytogenes. However, Kb−/−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 Kb−/−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 Kb−/−D b−/−M3−/− mice exhibit reduced bacterial burden compared with that of infected β2-microglobulin–deficient mice that lack MHC class Ib-restricted CD8+ T cells altogether. In addition, adoptive transfer of Listeria-experienced Kb−/−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.

Effector CD8+ T cells restricted to the classical MHC class I (MHC class Ia) Ag-presenting molecules have been shown to play critical roles in the clearance of bacterial and viral infection. MHC class Ib molecules are structurally related to MHC class Ia and are likewise composed of three Ig-like domains that noncovalently associate with β2-microglobulin (β2m) (1). Although the mammalian genome encodes many more MHC class Ib molecules than MHC class Ia molecules, comparatively little is known regarding their immunological function. However, the conservation of these molecules in mammals indicates that they play important roles that are nonredundant to those of MHC class Ia (1–4). Genes encoding MHC class Ib molecules can be found linked to the MHC (e.g., H2-M3, Qa-1/HLA-E, Qa-2) on chromosome 6 in humans and on chromosome 17 in mice, as well as elsewhere in the genome (e.g., CD1, MR1) (1). In general, MHC class Ib molecules are significantly less polymorphic, are more restricted in their tissue distribution, and have lower cell surface expression than MHC class Ia (1, 5), although in some cases these expression levels can be increased in the presence of Ag (6, 7). Importantly, over the past decade, emerging studies have found that MHC class Ib molecules can contribute to host immune responses through the presentation of microbial Ags to T cells (1, 8, 9).

Some MHC class Ib molecules, such as CD1 and H2-M3, have Ag-binding regions specialized to accommodate Ags that are unique in structure, perhaps positioning them to recognize hallmarks of microbial infection. The hydrophobic binding cleft of CD1 allows it to accommodate and present bacterial lipid Ags to T cells (10–17), whereas H2-M3 preferentially binds peptides that have N-terminal formylation, a signature of bacterial peptide synthesis, with up to a thousand-fold stronger affinity than that of nonformylated peptides (18, 19). H2-M3–restricted T cells have been shown to recognize peptides derived from many bacteria, including Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella typhimurium, and Chlamydia pneumoniae (20–25). We have previously demonstrated that H2-M3–restricted CD8+ T cells play a nonredundant role in host responses against L. monocytogenes and that mice lacking H2-M3 (M3−/−) have an increased susceptibility to L. monocytogenes infection (26). In addition to H2-M3, there is some evidence that Qa-1 can present listerial Ags (27–30). Qa-1 and its human homologue, HLA-E, have been shown to present peptides derived from Salmonella to CD8+ T cells (8, 31, 32). HLA-E–restricted T cells can also respond to Ags derived from M. tuberculosis (33) and have been isolated from M. tuberculosis-infected patients (34). Recent studies have demonstrated that mucosal-associated invariant T (MAIT) cells can be activated by MR1-expressing APCs that have been cultured with various bacteria, indicating that they recognize bacterial Ags presented by MR1 (35, 36). In addition to bacterial peptides, both HLA-E and Qa-2 have been shown to present peptides of viral origin to CD8+ T cells, suggesting that MHC class Ib molecules are also involved in antiviral immune responses (9, 37).

Like MHC class Ia-restricted CD8+ T cells, most MHC class Ib-restricted T cells are cytotoxic and can secrete inflammatory cytokines such as IFN-γ upon stimulation with Ag (31, 38–40). However, other characteristics of MHC class Ib-restricted T cells distinguish them from conventional T cells. Whereas the majority of T cells restricted by the MHC-linked MHC class Ib molecules studied to date express the CD8 coreceptor (2, 9, 19, 20, 22, 24, 25, 33), the T cells restricted by MHC-unlinked MHC class Ib molecules are predominately CD8− (38, 41). Many MHC class Ib-restricted T cells, including H2-M3–restricted CD8+ T cells, CD1d-restricted NKT cells, and MAIT cells, display an activated cell surface phenotype in the absence of infection (41–45). This preactivated status may contribute to the unique kinetics of MHC class Ib-restricted T cell responses, as H2-M3–restricted T cells, NKT cells, and MAIT cells all respond more rapidly to antigenic stimulation than do conventional T cells (40, 42, 43, 45–47). Notably, although their responses to primary stimuli are rapid, H2-M3–restricted T cell and NKT cell responses to secondary stimulation lack the accelerated responses and significant expansion that characterize secondary conventional T cell responses (42, 43, 46–50). It is not clear whether this limited responsiveness is a general feature of MHC class Ib-restricted T cells upon secondary stimulation.

L. monocytogenes infection has long been used to study conventional CD8+ T cell responses to intracellular bacteria (51–54) but has also proved to be a useful model for studying MHC class Ib-restricted responses. Studies performed using MHC class Ia-deficient (Kb−/−Db−/−) mice have demonstrated that MHC class Ib-restricted CD8+ T cells are protective against listerial infection and that antilisterial responses are not limited to H2-M3–restricted T cells (30, 42, 55). As listerial Ags that bind H2-M3 have been identified, it has been easiest to examine H2-M3–restricted CD8+ T cell responses to L. monocytogenes infection using L. monocytogenes Ag-loaded H2-M3 tetramers (46). However, due to the magnitude of H2-M3–restricted immune responses to L. monocytogenes infection and the limited availability of reagents that can detect other MHC class Ib-restricted CD8+ T cells, it has not been possible to investigate the relative contribution of non–H2-M3 MHC class Ib-restricted CD8+ T cell responses even in Kb−/−Db−/− mice.

To address this issue, we have generated mice that lack MHC class Ia as well as H2-M3 (Kb−/−Db−/−M3−/−). Using this novel animal model, we found that the non–H2-M3 MHC class Ib-restricted CD8+ T cell population exhibits an activated phenotype and responds to both primary and secondary L. monocytogenes infection with kinetics that resemble H2-M3–restricted T cell responses. In addition, we demonstrate that although non–H2-M3 MHC class Ib-restricted CD8+ T cells are few in number, they are cytotoxic, secrete proinflammatory cytokines, and can protect against L. monocytogenes infection. Given that MHC class Ib-restricted T cells display significantly less polymorphism than MHC class Ia, these new findings position MHC class Ib molecules and their bound bacterial Ags as attractive vaccine targets that could be widely recognized across the general population to protect against bacterial infection.

Materials and Methods

Mice

C57BL/6 and β2m−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). M3−/− and Kb−/−Db−/− mice (back-crossed with C57BL/6 [B6] mice for at least 10 generations) were generated or maintained in house as previously described (26). Kb−/−Db−/−M3−/− were generated by crossing Kb−/−Db−/− mice with M3−/− mice. F1 offspring were intercrossed, and all resulting F2 progeny that lacked surface expression of H2-Kb and -Db on PBLs were screened for an intra-H2 recombinant using PCR analysis using the following primer set: M3 forward (5′-CAGCGATGGAACCCACCCACAATGA-3′), M3 reverse (5′-AGACTAGCAACGATGACCATGATGAC-3′), and Neo (5′-GATTCGCAGCGCATCGCCTTCTA-3′) (26). Of 165 F2 offspring tested, one male mouse was found to carry the desired intra-H2 recombination (Kb−/−Db−/−M3+/−). We bred this male with Kb−/−Db−/− females to produce Kb−/−Db−/−M3+/− offspring, which were then intercrossed to generate Kb−/−Db−/−M3−/− mice. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MD). The protocol was approved by the Animal Care and Use Committee of Northwestern University (Chicago, IL).

Primary cell preparation and dendritic cell generation

Single-cell suspensions were prepared from whole tissues by mechanical disruption in HBSS/2% FBS or as described (56). T cells were purified from splenic lymphocytes using a Pan T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA). To enrich for CD8+ T cells, splenocytes were labeled with biotin-conjugated anti-B220, anti-CD4, anti-CD11b, anti-CD11c, anti-CD49b, and anti-NK1.1 Abs (eBioscience, San Diego, CA) followed by anti-biotin–conjugated magnetic beads. CD8+ T cells were then isolated to a purity of ∼95% by negative selection according to the manufacturer’s instruction (Miltenyi Biotec). Bone marrow-derived dendritic cells (BMDCs) were derived from mouse bone marrow progenitors using GM-CSF and IL-4 (PeproTech, Rocky Hill, NJ) as previously described (57).

Abs and flow cytometry

FITC-conjugated mAbs specific for CD8β, CD44, CD62L, hamster IgG, H2-Kb, Vβ2, Vβ 5.1/5.2, Vβ 6, Vβ8.1/8.2, Vβ8.3, Vβ12, and Vβ13, FITC-conjugated streptavidin, PE-conjugated Abs specific for B220, CD8α, CD8β, Ly6C, Vβ3, and Vβ11, PerCP-conjugated mAb specific for CD4 and TCRβ, and biotinylated mAb specific for H2-Db were purchased from BD Pharmingen (San Diego, CA). Cells were incubated with 2.4G2 Fcγ RII/RIII blocking mAb (hybridoma supernatant) for 15 min, then stained in HBSS containing 2% FBS for 30 min at 4°C. For detection of H2-M3 surface expression, splenocytes from B6 and Kb−/−Db−/−M3−/− mice were cultured overnight at 37°C in RPMI 10 containing 10 μM LemA peptide (f-MIGWII). Cells were stained first with the anti–H2-M3 Ab 130 (7) followed by staining with anti-hamster IgG. Flow cytometric analysis was performed using a FACSCantoII (BD Biosciences, San Jose, CA) and FlowJo software (Tree Star, Ashland, OR).

Cytokine assay

For polyclonal TCR stimulation, enriched CD8+ T cells (5 × 105 cells/well) from naive wild-type (WT), Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice were stimulated with anti-CD3 (3 μg/ml) and anti-CD28 (3 μg/ml) mAb and cultured in RPMI 10. After 12 h in culture, intracellular staining for IFN-γ was performed as described later. For L. monocytogenes-specific cytokine responses, BMDCs were pulsed with heat-killed Listeria monocytogenes (HKLM) or infected with L. monocytogenes as described later. Responders (5 × 106 CD8+ T cells/well) were stimulated with various BMDCs (1 × 105) in 200 μl RPMI 10 for 12 h (intracellular staining) or 48 h (ELISA and cytometric bead array). IFN-γ production was measured by intracellular staining and flow cytometry. IL-17A levels were quantitated by sandwich ELISA using anti–IL-17A mAb pairs (eBioscience), whereas other cytokines were measured using an Inflammatory and Th1/Th2 Cytometric Bead Array Kit (BD Biosciences) according to the manufacturer’s instructions. To generate HKLM, 1 × 1010 CFU/ml L. monocytogenes culture in PBS was incubated at 70°C for 3 h, then stored at −20°C.

Intracellular cytokine staining

Splenocytes or hepatic leukocytes from naive or L. monocytogenes-infected mice were cultured in 96-well plates and either stimulated or left unstimulated for 7 h at 37°C. For the last 2 h of stimulation, 10 μM monensin (Sigma, St. Louis, MO) was added to block cytokine secretion. Cells were washed and stained for the cell surface markers CD8β, CD4, and TCRβ. After fixation with 4% paraformaldehyde and permeabilization with 0.15% saponin (Sigma), cells were stained with an allophycocyanin-conjugated anti–IFN-γ Ab (eBioscience) for 30 min in PBS containing 1% BSA and 0.1% saponin. Flow cytometry was performed as described earlier.

Listeria infection and CFU assay

The L. monocytogenes EGD strain was provided by R. Kurlander (National Institutes of Health). Bacteria were grown in brain–heart infusion broth (Difco Laboratories, Detroit, MI), and virulent stocks were maintained by repeated passage through B6 mice. For primary infection, mice were i.v. infected with 2 × 103 CFU (one tenth LD50) L. monocytogenes. For rechallenge with L. monocytogenes, mice were rested for 1 mo and then infected with 5 × 104 CFU L. monocytogenes. Bacterial CFUs in the spleen and liver were determined at indicated time points postinfection. Briefly, organs were homogenized in sterile water with 0.2% Nonidet P-40, and serial dilutions were plated onto brain–heart infusion agar plates. CFUs were counted after incubation at 37°C for 24 h.

Adoptive transfer study

Donor WT and Kb−/−Db−/−M3−/− mice were immunized with 2 × 103 CFU L. monocytogenes. Seven days later, splenocytes from L. monocytogenes immune mice were isolated and were divided into two groups. One donor group was incubated with anti-CD8α mAb (3.155) at 4°C for 30 min. Cells were then washed and incubated with 10% rabbit complement (Cedarlane Labs, Burlington, NC) at 37°C for 30 min to deplete CD8+ T cells. All donor cells from naive spleen, CD8+ T cell-depleted immune spleen, and nondepleted immune spleen were washed twice with PBS. Naive WT mice received an i.v. injection of 2 × 107 donor splenocytes and were then challenged with 5 × 104 CFU L. monocytogenes 30–60 min after cell transfer. Three days postinfection, spleen and liver were removed from the recipients, and the bacterial CFUs per organ was determined as described earlier.

ELISPOT assay

Multiscreen-IP plates (Millipore, Bedford, MA) were coated with anti–IFN-γ mAb (eBioscience) at 5 μg/ml in PBS. BMDCs were infected with L. monocytogenes 4 h before each assay as described earlier. For blocking experiments, L. monocytogenes-infected BMDCs were preincubated with mouse IgG or mAb against CD1d (3H3; in-house) (58), H2-M3 (130; in-house) (7), MR1 (26.5) (4), Qa-1b (6A8.6F10.1A6; American Type Culture Collection, Manassas, VA) (31, 59), and Qa-2 (M46) (60) for 30 min at 37°C prior to assay set up (42). Enriched CD8+ T cells (104–105) were mixed with BMDC stimulator cells (5 × 104/well) in RPMI 10 medium and plated in triplicate wells. After 18 h incubation at 37°C, plates were washed free of cells using PBS–Tween (PBS and 0.05% Tween 20) and incubated overnight at 4°C with biotinylated anti–IFN-γ mAb (eBioscience) at 1 μg/ml. Plates were washed and incubated with streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA). After 1 h incubation at room temperature, plates were developed with a BCIP/NBT substrate kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Spots were counted using an ImmunoSpot reader (Cellular Technology, Shaker Heights, OH).

CTL assay

To examine L. monocytogenes-specific CTL ex vivo, splenocytes from L. monocytogenes-infected mice (at day 7 postinfection) were enriched for CD8+ T cells and cultured in RPMI 10 with 1 μg/ml Con A. After 3 d in culture, cells were used as effectors in a 51Cr release CTL assay. Monolayers of the macrophage cell line J774 were grown in antibiotic-free medium and infected with L. monocytogenes for 1 h at a multiplicity of infection 5:1. Cells were then washed with warm PBS and cultured in DMEM containing 40 μg/ml gentamicin for an additional 3 h. Both uninfected and L. monocytogenes-infected J774 target cells were labeled with 51Cr for 1 h at 37°C. To examine cytotoxicity in the T2 CTL line, BMDCs were labeled with 51Cr and used as target cells. BMDC targets were derived from β2m−/− or Kb−/−Db−/−M3−/− mice and either left untreated or treated overnight with HKLM. Some target cells were additionally pretreated with blocking mAb against MR1, Qa-1b, or Qa-2 before labeling as described earlier. Target cells (104) were added to a round-bottom 96-well plate containing varying concentrations of effector cells. Four hours after incubation, 100 μl supernatant was collected from each well, and the amount of 51Cr release was determined using a TopCount scintillation counter. The percentage of specific lysis was calculated as 100 × (experimental cpm − spontaneous cpm) / (maximal cpm − spontaneous cpm).

Generation of Kb−/−Db−/−M3−/− CTL lines

To generate the T2 CTL line, Kb−/−Db−/−M3−/− mice were immunized with 1 × 106 HKLM-pulsed BMDCs. Seven days postimmunization, splenocytes were harvested and placed in culture for 1 wk in RPMI 10. Cells were subsequently cultured in supplemented Mischell Dutton medium with 20 U/ml IL-2 (partially purified from EL4.IL2 cell supernatant) and 20 ng/ml IL-7 (PeproTech). Cells were restimulated weekly with HKLM-pulsed irradiated Kb−/−Db−/−M3−/− BMDCs.

Statistical analysis

Mean values were compared using unpaired Student t tests. All statistical analyses were performed with the Prism program (GraphPad, La Jolla, CA).

Results

Generation and characterization of Kb−/−Db−/−M3−/− mice

To investigate the relative contribution of H2-M3 and other MHC class Ib molecules to CD8+ T cell development and associated responses to bacterial infection, we generated Kb−/−Db−/−M3−/− mice by crossing Kb−/−Db−/− and M3−/− mice (26). F1 offspring were intercrossed, and the resulting F2 offspring were then screened for intra-H2 recombination using FACS and PCR analysis (Fig. 1A). The genetic distance between H2-D and H2-M3 is ∼0.7 cM (61), necessitating extensive screening. Of 165 F2 offspring tested, one mouse was found to carry each targeted locus on the same chromosome (Kb−/−Db−/−M3+/−) and was selected for further breeding to establish Kb−/−Db−/−M3−/− mice. Flow cytometry confirmed that Kb−/−Db−/−M3−/− mice do not express H2-Kb, H2-Db, or H2-M3 molecules on the cell surface (Fig. 1B). To determine the respective role of H2-M3 and other MHC class Ib molecules in the development of CD8+ T cells, we compared the CD8+ T cell populations in the spleen, liver, and lymph nodes of WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice. Total lymphocyte numbers were comparable between these three genotypes (data not shown). However, compared with WT mice, the percentage of CD8+ T cells was profoundly reduced in the spleens and lymph nodes of Kb−/−Db−/− mice and was further reduced, albeit modestly, in Kb−/−Db−/−M3−/− mice (Fig. 2A, 2B). Notably, the reduction in CD8+ T cell percentage was less profound in the livers of Kb−/−Db−/− mice compared with that of WT mice, whereas Kb−/−Db−/−M3−/− mice exhibited a 2- to 3-fold reduction in the percentage of hepatic CD8+ T cells compared with that of Kb−/−Db−/− mice (Fig. 2A, 2B). Judging from the enumeration of CD8+ T cell populations in Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice, H2-M3–restricted CD8+ T cells constitute ∼20–30% of the MHC class Ib-restricted CD8+ T cells found in the peripheral lymphoid tissues of naive animals but contribute 50–75% of the MHC class Ib-restricted CD8+ T cell population in the liver. These data indicate that CD8+ T cells restricted by different MHC class Ib molecules may have distinct tissue distributions, with H2-M3–restricted CD8+ T cells being particularly enriched in the liver.

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 naive 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 naive WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice. D, Ex vivo anti-CD3 and anti-CD28 Ab stimulation of CD8+ T cells enriched from the spleens of WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice. Intracellular staining for IFN-γ was performed at 12 h poststimulation. Data shown are representative of three experiments.

Phenotypic and functional analysis of residual CD8+ T cells in naive Kb−/−Db−/−M3−/− mice

Two unconventional subsets of MHC class Ib-restricted T cells, namely CD1d-restricted invariant NKT cells and MR1-restricted MAIT cells, exhibit restricted TCR usage (45, 62). To examine the diversity of the TCR Vβ region expressed by H2-M3–restricted and non–H2-M3 MHC class Ib-restricted CD8+ T cells, splenocytes from WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice were stained with Abs against various TCR Vβ-chains. Although the CD8+ T cell population found in Kb−/−Db−/− M3−/− mice exhibited a decreased representation of Vβ2, Vβ5, Vβ6, Vβ8.3, Vβ11, and Vβ13 compared with that of the CD8+ T cell population found in WT mice, residual Kb−/−Db−/− M3−/− CD8+ T cells still exhibit a large diversity in TCRβ usage (Supplemental Fig. 1). This observation could indicate that the CD8+ T cell population found in Kb−/−Db−/− M3−/− mice does not exhibit restricted TCR Vβ-chain usage or could merely be reflective of a polyclonal population of CD8+ T cells recognizing diverse restriction elements. However, the residual CD8+ T cells found in Kb−/−Db−/− mice exhibit an increase in the proportion of Vβ8.1/8.2 and Vβ13 usage that is not observed in Kb−/−Db−/−M3−/− mice, suggesting that H2-M3–restricted CD8+ T cells may display a preferential usage for these Vβ-chains.

It has been shown that a large proportion of CD8+ T cells in naive Kb−/−Db−/− mice display an activated/memory-like phenotype (42–44). However, it is not clear whether H2-M3–restricted and/or other MHC class Ib-restricted CD8+ T cells contribute to this phenotype. To address this question, we compared the expression of various activation markers on CD8+ T cells isolated from WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice. Similar to Kb−/−Db−/− mice, the majority of CD8+ T cells in Kb−/−Db−/−M3−/− mice exhibit an activated T cell phenotype that is CD44high and Ly6Chigh (Fig. 2C). In addition, the percentages of CD62Llow cells are also increased in both Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice compared with that of WT mice. These data indicate that in the absence of MHC class Ia-restricted CD8+ T cells, most H2-M3–restricted as well as other MHC class Ib-restricted CD8+ T cells are activated in naive mice.

To examine the functional properties of non–H2-M3 MHC class Ib-restricted CD8+ T cells, CD8+ T cells were enriched from WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice and stimulated with anti-CD3 and anti-CD28 Abs. After in vitro stimulation, intracellular staining revealed that the majority of MHC class Ib-restricted Kb−/−Db−/− and Kb−/−Db−/−M3−/− CD8+ T cells robustly secreted IFN-γ, whereas a significantly smaller proportion of WT CD8+ T cells was able to do so (Fig. 2D). These data indicate that, consistent with their activated phenotype, MHC class Ib-restricted CD8+ T cells more readily produce proinflammatory cytokines compared with MHC class Ia-restricted CD8+ T cells.

Non–H2-M3 MHC class Ib-restricted CD8+ T cells expand upon primary infection with Listeria

To assess whether residual CD8+ T cells from Kb−/−Db−/−M3−/− mice expand in response to bacterial infection, WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice were infected with a sublethal dose of L. monocytogenes. Seven days after infection, splenocytes and hepatic leukocytes were harvested, and the CD4+ and CD8+ T cell populations were analyzed by flow cytometry. Compared with naive mice, a significant increase in overall cellularity (∼2-fold in spleen and ∼3- to 5-fold in liver) was detected in all three L. monocytogenes-infected mouse strains (data not shown). In addition, the percentages of CD8+ T cells increased by 2- to 4-fold and 5- to 7-fold in L. monocytogenes-infected Kb−/−Db−/−M3−/− and Kb−/−Db−/− mice, respectively (Fig. 3A). These data indicate that, similar to H2-M3–restricted CD8+ T cells, CD8+ T cells restricted to other MHC class Ib molecules are able to undergo extensive proliferation after primary infection with L. monocytogenes.

FIGURE 3.
FIGURE 3.

Expansion of CD8+ T cells in Kb−/−Db−/−M3−/− mice during L. monocytogenes infection. A, WT, Kb−/−Db−/−, and Kb−/−Db−/−M3−/− mice were infected with 2 × 103 CFU L. monocytogenes. Seven days after infection, splenocytes and hepatic leukocytes were harvested and stained with Abs against CD8β and TCRβ. Bar graphs depict the mean ± SEM for the percentage of CD8+ T cells in the lymphocyte gate for uninfected and L. monocytogenes (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 L. monocytogenes-infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice at the indicated time points and stained with Abs 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 three to nine mice per genotype are shown. *p < 0.05; **p < 0.01; ***p < 0.001.

To compare the kinetics of H2-M3–restricted and non–H2-M3 MHC class Ib-restricted CD8+ effector T cells during primary L. monocytogenes infection, we infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice with L. monocytogenes and examined the CD44highCD8+ T cell populations in these mice at various time points after infection. Both L. monocytogenes-infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice exhibited a steady increase in the total number of CD44highCD8+ T cells during the first week of infection even though the number of CD44highCD8+ T cells was significantly greater in Kb−/−Db−/− mice than in Kb−/−Db−/−M3−/− mice (Fig. 3B). The number of CD44highCD8+ T cells in L. monocytogenes-infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice was significantly increased in the spleen (∼5-fold) and liver (∼17-fold) at day 5 postinfection compared with that at day 3 postinfection. At day 7 postinfection, the number of CD44highCD8+ T cells was further increased in both the spleen (∼1.5-fold) and liver (∼4.5-fold) of Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice. These data suggest that the kinetics of non–H2-M3 MHC class Ib-restricted CD8+ T cells are similar to those of H2-M3–restricted CD8+ T cells in response to primary L. monocytogenes infection. Notably, as observed in L. monocytogenes-infected Kb−/−Db−/− mice, hepatic CD8+ effector T cells in L. monocytogenes-infected Kb−/−Db−/−M3−/− mice underwent a more vigorous expansion than did splenic CD8+ effector T cells. These data indicate that, similar to H2-M3–restricted T cells, non–H2-M3 class Ib-restricted effector CD8+ T cells may preferentially expand in the liver or may be preferentially recruited to the liver during L. monocytogenes infection.

CD8+ T cells in Kb−/−Db−/−M3−/− mice protect against Listeria infection

To examine the protective capacity of non–H2-M3 MHC class Ib-restricted CD8+ T cells during L. monocytogenes infection, we compared the bacterial burden in both the spleens and livers of infected Kb−/−Db−/−and Kb−/−Db−/−M3−/− mice with that of infected β2m−/− mice that lack most MHC class Ib-restricted CD8+ T cells. At 5 and 7 d postinfection, the bacterial burden was significantly lower in both the spleens and livers of Kb−/−Db−/−M3−/− mice compared with that of β2m−/− mice (Fig. 4A), suggesting that non–H2-M3 MHC class Ib-restricted CD8+ T cells contribute to bacterial clearance. At day 7 postinfection, bacterial burdens were further reduced in Kb−/−Db−/− mice compared with those in Kb−/−Db−/−M3−/− mice (Fig. 4A), confirming a protective role for H2-M3–restricted T cells against L. monocytogenes infection (26).

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 L. monocytogenes. Bacterial burden in the spleen and liver was determined at indicated time points postinfection. Data are presented as the mean ± SEM from three to six mice per genotype for each time point. *p < 0.05. B, WT recipient mice were adoptively transferred with 2 × 107 splenocytes isolated from naive WT or Kb−/−Db−/−M3−/− mice or from WT or Kb−/−Db−/−M3−/− mice that had been infected with 2 × 103 L. monocytogenes (LM) 7 d prior. In some cases, splenocytes from infected donor mice were depleted of CD8+ T cells. Thirty to sixty minutes after cell transfer, recipient mice were infected with 5 × 104 L. monocytogenes. Three days postinfection, 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 < 0.01; ***p < 0.001.

To demonstrate that the protective effect observed in L. monocytogenes-infected Kb−/−Db−/−M3−/− mice is mediated by CD8+ T cells, we adoptively transferred either splenocytes or CD8+ T cell-depleted splenocytes isolated from naive or L. monocytogenes-infected WT or Kb−/−Db−/−M3−/− mice into naive WT recipient mice. The recipient mice were then challenged with a lethal dose of L. monocytogenes, and protective immunity was evaluated on day 3 postinfection by determining the bacterial burden in the spleen and liver. Transfer of splenocytes from L. monocytogenes-vaccinated WT or Kb−/−Db−/−M3−/− mice provided significant protection to recipient mice against subsequent L. monocytogenes challenge compared with that provided by transfer of naive splenocytes (Fig. 4B). However, this protective effect was abolished when CD8+ T cells were depleted from WT or Kb−/−Db−/−M3−/− splenocytes mice prior to transfer. Collectively, these data indicate that residual CD8+ T cells in Kb−/−Db−/−M3−/− mice can contribute to protective immunity against L. monocytogenes infection.

Non–H2-M3 MHC class Ib-restricted CD8+ T cells are cytotoxic and secrete proinflammatory cytokines in response to Listeria infection

To determine the effector function of non–H2-M3 MHC class Ib-restricted CD8+ T cells during L. monocytogenes infection, we harvested splenocytes and hepatic leukocytes from L. monocytogenes-infected Kb−/−Db−/−M3−/− mice and stimulated them ex vivo with HKLM. Consistent with the kinetics of total CD8+ effector T cells upon L. monocytogenes infection, the number of L. monocytogenes-specific non–H2-M3 MHC class Ib-restricted CD8+ T cells was significantly increased at day 5 after infection and was further increased by day 7 (Supplemental Fig. 2). A significant proportion of Kb−/−Db−/−M3−/− CD8+ T cells from L. monocytogenes-infected mice was able to produce IFN-γ when stimulated with HKLM, whereas unstimulated CD8+ T cells did not (Fig. 5A and Supplemental Fig. 3).

FIGURE 5.
FIGURE 5.

Effector function of L. monocytogenes-specific CD8+ T cells in Kb−/−Db−/−M3−/− mice. A–C, Splenocytes and hepatic leukocytes were harvested from Kb−/−Db−/−M3−/− mice 7 d after L. monocytogenes infection. A, Cells were stimulated with HKLM for 7 h and stained with Abs 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 d after L. monocytogenes infection and enriched for CD8+ T cells. These T cells were then cultured with L. monocytogenes (LM)-infected BMDCs 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 d after L. monocytogenes infection, enriched for CD8+ T cells, and activated with Con A. After 3 d of ConA stimulation, splenocytes were used as effectors in a 51Cr release CTL assay at the indicated effector:target cell ratios. Uninfected or L. monocytogenes (LM)-infected J774 cells were labeled with 51Cr and used as targets. Graph depicts the mean ± SEM for the percentage of L. monocytogenes-specific killing pooling two mice per genotype. Data are representative of three independent experiments.

To characterize further the cytokines produced by the CD8+ T cells found in L. monocytogenes-infected Kb−/−Db−/−M3−/− mice, CD8+ T cells were enriched from the spleens of Kb−/−Db−/−M3−/− mice at 7 d post-L. monocytogenes infection and stimulated ex vivo with L. monocytogenes-infected BMDCs. We found that CD8+ T cells isolated from L. monocytogenes-infected Kb−/−Db−/−M3−/− mice were able to produce significant amounts of TNF-α, IFN-γ, IL-6, and IL-17A in response to stimulation with L. monocytogenes-infected BMDCs (Fig. 5B).

To investigate the cytolytic capacity of non–H2-M3 MHC class Ib-restricted CD8+ T cells during L. monocytogenes infection, we performed an in vitro CTL assay using Con A-activated CD8+ effector T cells enriched from L. monocytogenes-infected Kb−/−Db−/−M3−/− mice. Effector T cells enriched from L. monocytogenes-infected Kb−/−Db−/−M3−/− mice preferentially lysed L. monocytogenes-infected J774 target cells but not uninfected targets, indicating that the residual CD8+ T cell population found in Kb−/−Db−/−M3−/− mice contains CTL specific to L. monocytogenes (Fig. 5C). The combined abilities of non–H2-M3 MHC class Ib-restricted L. monocytogenes-specific CD8+ cells to lyse L. monocytogenes-infected cells and to produce proinflammatory cytokines in response to listerial Ags likely contribute to their ability to protect against L. monocytogenes infection.

Antilisterial CD8+ T cells from Kb−/−Db−/−M3−/− mice do not recognize previously characterized MHC class Ib molecules

Although more than 40 MHC class Ib molecules are encoded in the murine genome, only H2-M3 and Qa-1 have been shown to function as restriction elements for L. monocytogenes-specific CTLs (20–22, 27–30). However, H2-M3– and Qa-1–restricted T cell responses can account for only a fraction of the MHC class Ib-restricted responses observed during L. monocytogenes infection. To investigate which MHC class Ib molecules might serve as restriction elements for the CD8+ T cells found in L. monocytogenes-infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice, we enriched splenic CD8+ T cells from these mice and cultured them with L. monocytogenes-infected BMDCs that had been pretreated with blocking Abs against CD1d, H2-M3, Qa-1b, Qa-2, or MR1 in an ELISPOT assay. L. monocytogenes-specific IFN-γ secretion by CD8+ T cells isolated from Kb−/−Db−/− mice was significantly reduced in the presence of anti–H2-M3 Ab, confirming that a substantial fraction of L. monocytogenes-specific CD8+ T cells in Kb−/−Db−/− mice are restricted to H2-M3 (Fig. 6A and Supplemental Fig. 4). However, the presence of blocking Abs against H2-M3, Qa-1b, Qa-2, and MR1 had no effect on L. monocytogenes-specific responses produced by CD8+ T cells isolated from Kb−/−Db−/−M3−/− mice. These data suggest that neither Qa-1b, Qa-2, nor MR1 serve as major restriction elements for the MHC class Ib-restricted antilisterial CTLs found in Kb−/−Db−/−M3−/− mice.

FIGURE 6.
FIGURE 6.

L. monocytogenes-specific Kb−/−Db−/−M3−/− CD8+ T cells are not restricted to Qa-1b, Qa-2, or MR1. A, Splenocytes were harvested from L. monocytogenes-infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice at 7 d postinfection and enriched for CD8+ T cells. These T cells were then used as effectors in an IFN-γ ELISPOT assay. Uninfected or L. monocytogenes-infected BMDCs 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 and C, T2 CTLs, a CD8+ T cell line derived from L. monocytogenes-infected Kb−/−Db−/−M3−/− mice, were used as effector cells in a 51Cr release CTL assay. Results are representative of three independent experiments. B, BMDCs 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, BMDCs 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.

To attempt to identify novel MHC class Ib molecules that are capable of presenting bacterial Ags to CD8+ T cells, we established T cell lines from HKLM-immunized Kb−/−Db−/−M3−/− mice. One of these CD8+ T cell lines, T2 CTL, preferentially lysed HKLM-pulsed BMDCs derived from Kb−/−Db−/−M3−/− mice but not BMDCs derived from β2m−/− mice, suggesting that T2 CTL recognizes a β2m-associated MHC class Ib molecule (Fig. 6B). In addition, blocking Abs against Qa-1b, Qa-2, and MR1 did not inhibit the reactivity of T2 CTL, suggesting that this T cell line recognizes a novel MHC class Ib molecule (Fig. 6C).

Non–H2-M3 MHC class Ib-restricted memory T cells do not exhibit enhanced recall responses to L. monocytogenes

Previous studies have shown that H2-M3–restricted CD8+ T cells do not undergo significant expansion after secondary infection with L. monocytogenes (42, 43, 46, 47). To determine whether non–H2-M3 MHC class Ib-restricted CD8+ T cells can persist as long-lasting memory T cells and expand in response to secondary L. monocytogenes infection, we compared the percentage of CD8+ T cells in the spleens of naive Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice with those in mice that had been infected 1 mo previously with L. monocytogenes and either had been allowed to recover or had received a secondary lethal dose of L. monocytogenes. The proportion of CD8+ T cells in Kb−/−Db−/− mice was increased 2-fold at 1 mo post-primary L. monocytogenes infection, compared with that in naive mice, but did not increase further upon secondary challenge (Fig. 7A). Compared with naive Kb−/−Db−/−M3−/− mice, Kb−/−Db−/−M3−/− mice that had been infected 1 mo previously also exhibited a 2-fold increase in the proportion of CD8+ T cells (Fig. 7A). However, neither the percentage nor the total number of Kb−/−Db−/−M3−/− CD8+ T cells changed significantly upon rechallenge with L. monocytogenes (Fig. 7A; data not shown), suggesting that similar to H2-M3–restricted CD8+ T cells, non–H2-M3 MHC class Ib-restricted CD8+ T cells do not proliferate extensively during recall responses to L. monocytogenes infection. Compared with the analogous CD8+ T cell population in rechallenged Kb−/−Db−/− mice, the percentage of CD8+ T cells in rechallenged Kb−/−Db−/−M3−/− mice is substantially lower, suggesting that H2-M3–restricted CD8+ T cells remain the dominant MHC class Ib-restricted T cell population during secondary L. monocytogenes infection (Fig. 7A). However, no differences in bacterial burden were observed between Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice upon rechallenge with L. monocytogenes (Supplemental Fig. 5).

FIGURE 7.
FIGURE 7.

Non-M3 MHC class Ib-restricted CD8+ T cell responses to secondary L. monocytogenes infection. A, Splenocytes were harvested from naive Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice, from mice that had been infected with 2 × 103 CFU L. monocytogenes 1 mo previously, and from mice that had been infected 1 mo previously and subsequently rechallenged with 5 × 104 CFU L. monocytogenes. Cells were stained with Abs against CD8β and TCRβ at 3 and 5 d 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 three to five 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 d after secondary L. monocytogenes 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 L. monocytogenes-specific IFN-γ–producing CD8+ T cells in spleens of Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice 7 d after primary L. monocytogenes infection, 1 mo after primary infection, or 3 d after secondary infection. Results shown are from three to five mice per experimental group and are representative of two individual experiments.

Despite their lack of expansion, CD8+ T cells isolated from both Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice at 3 d post-secondary L. monocytogenes infection are capable of secreting IFN-γ upon ex vivo stimulation with listerial Ags (Fig. 7B), suggesting that both H2-M3–restricted and non–H2-M3 MHC class Ib-restricted CD8+ T cells can survive for extended periods of time postinfection and maintain effector function. Although secondary L. monocytogenes infection led to an increase in total numbers of L. monocytogenes-specific IFN-γ–producing CD8+ T cells in both Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice, the magnitude of L. monocytogenes-specific CD8+ T cell responses to secondary infection was significantly lower compared with CD8+ T cell responses in Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice after primary infection (Fig. 7C). These data indicate that the lack of ability to undergo memory cell expansion may be a common feature of MHC class Ib-restricted CD8+ T cell responses to L. monocytogenes infection.

Discussion

Emerging evidence suggests that MHC class Ib molecules can contribute to host immune responses through the presentation of unusual bacterial Ags to T cells. H2-M3 exemplifies this strategy by presenting N-formylated bacterial peptides to CD8+ cytotoxic T cells (20–22). Recent studies by our laboratory have shown that H2-M3–restricted CD8+ T cells play a significant and nonredundant role during L. monocytogenes infection that bridges innate and adaptive immunity (26, 42). However, the relative contribution of other MHC class Ib-restricted CD8+ T cells to bacterial infection has yet to be defined. In this report, we have demonstrated an important role for non–H2-M3 MHC class Ib-restricted CD8+ T cells in the protection of mice from L. monocytogenes infection. We have shown that the CD8+ T cells found in Kb−/−Db−/−M3−/− mice can expand rapidly, secrete inflammatory cytokines, and exert Ag-specific cytolytic activities in response to L. monocytogenes infection. By comparing the CD8+ T cell responses observed in L. monocytogenes-infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice, we found that H2-M3–restricted and non–H2-M3 MHC class Ib-restricted CD8+ T cells respond with similar kinetics during L. monocytogenes infection, characterized by rapid and extensive proliferation during primary infection and limited expansion during secondary infection.

H2-M3–restricted T cells make up a large proportion of MHC class Ib-restricted CD8+ T cells during both primary and secondary L. monocytogenes infection, which is likely reflective of a significant H2-M3–restricted T cell pool in naive mice. Indeed, a comparison of the residual CD8+ T cell population in naive Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice revealed that H2-M3–restricted CD8+ T cells constitute ∼20–30% and 50–75% of the MHC class Ib-restricted CD8+ T cell population in the spleen and liver, respectively. The enrichment of H2-M3–restricted CD8+ T cells in the liver is reminiscent of CD1d-restricted NKT cells and might be due at least in part to their unique developmental requirement that positive selection be mediated by hematopoietic-derived cells in the thymus (63). The fact that we observed significantly fewer CD8+ T cells in the periphery of Kb−/−Db−/−M3−/− mice than in the periphery of Kb−/−Db−/− mice demonstrates that T cells restricted by non–H2-M3–restricted MHC class Ib molecules are not able to fill the CD8+ T cell compartment, even in the absence of MHC class Ia-restricted and H2-M3–restricted CD8+ T cells. These data suggest that non–H2-M3 MHC class Ib-restricted CD8+ T cells may be limited in their ability to undergo homeostatic expansion.

The majority of L. monocytogenes-specific MHC class Ib-restricted CD8+ T cells isolated to date are restricted to H2-M3, which appears to play a dominant role in antilisterial immunity (26, 42). Qa-1 is the only other MHC class Ib molecule known to present L. monocytogenes Ags (27–30). However, the L. monocytogenes-specific CD8+ T cell responses observed in infected Kb−/−Db−/−M3−/− mice were not noticeably inhibited by Abs specific to several known MHC class Ib molecules, including Qa-1b, Qa-2, MR1, and CD1d (Fig. 6A and Supplemental Fig. 4), suggesting that these L. monocytogenes-specific CD8+ T cells are restricted by a previously uncharacterized MHC class Ib molecule(s). Indeed, CD8+ T cells isolated from infected Kb−/−Db−/−M3−/− mice failed to lyse Qa-1b-transfectants (Supplemental Fig. 6). Of the more than 40 MHC-linked MHC class Ib genes present in the mouse, only three definitively present microbial Ags to CD8+ T cells while little is known regarding the expression status and function of the remaining MHC class Ib genes. Thus, T cell lines derived from L. monocytogenes-infected Kb−/−Db−/−M3−/− mice would be useful tools for identifying novel Ag-presenting MHC class Ib molecules.

Analysis of the surface phenotype and functional properties of the residual CD8+ T cell populations found in the peripheral lymphoid organs of Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice indicated that most H2-M3–restricted and non–H2-M3 MHC class Ib-restricted CD8+ T cells display an activated/memory phenotype. However, most of the CD8+ T cells in the thymus of Kb−/−Db−/−M3−/− mice are CD44low (data not shown), suggesting that the acquisition of a memory phenotype by non–H2-M3 MHC class Ib-restricted CD8+ T cells is mainly a postthymic event. It is possible that these non–H2-M3 MHC class Ib-restricted CD8+ T cells recognize commensal Ags and thereby become primed once they enter the periphery. The residual CD8+ T cells in Kb−/−Db−/−M3−/− mice can rapidly produce IFN-γ after TCR stimulation. In addition, culture of Kb−/−Db−/−M3−/− CD8+ T cells with IL-12 and IL-18, cytokines induced by L. monocytogenes infection as well as by infection with other pathogens, can induce IFN-γ secretion (data not shown). These data suggest that non–H2-M3 MHC class Ib-restricted CD8+ T cells may respond to L. monocytogenes infection in an Ag- and cytokine-dependent manner to contribute to the early phase of acquired immune responses against infection. Indeed, Kb−/−Db−/−M3−/− mice are more resistant to primary L. monocytogenes infection compared with β2m−/− mice that lack all MHC class Ib-restricted CD8+ T cells. We cannot eliminate the possibility that the observed differences in susceptibility to L. monocytogenes infection between β2m−/− and Kb−/−Db−/−M3−/− mice may in part be due to differential NK cell activity, as it has been shown that ligand expression during NK cell development can affect their functional activity (64, 65). However, adoptive transfer of splenocytes isolated from L. monocytogenes-immunized Kb−/−Db−/−M3−/− mice confers significant protection upon recipient mice against lethal L. monocytogenes infection in a CD8+ T cell-dependent manner. These data provide direct evidence that CD8+ T cells restricted by MHC class Ib molecules other than H2-M3 can contribute to protective immunity against bacterial pathogens. Although H2-M3 appears to be the dominant restriction element for MHC class Ib-restricted responses during L. monocytogenes infection, our preliminary studies suggest that other MHC class Ib-restricted CD8+ T cells may play a more prominent role during M. tuberculosis infection, as we do not detect significant differences in CD8+ T cell expansion when comparing M. tuberculosis-infected Kb−/−Db−/− and Kb−/−Db−/−M3−/− mice (data not shown). Thus, CD8+ T cells restricted by distinct MHC class Ib molecules may play differential roles in host defense against different bacterial infections.

A recent study has shown that HLA-E can present a panel of M. tuberculosis-derived peptides to CD8+ T cells that have both cytotoxic and immunoregulatory activity (34). In addition, M. tuberculosis-reactive MR1-restricted CD8+ T cells were found to be enriched in the lungs of patients with active tuberculosis and responded to M. tuberculosis-infected MR1-expressing lung epithelial cells (35). These studies suggest that MHC class Ib-restricted CD8+ T cells may participate in immune responses against bacterial infection in humans. Further studies of the in vivo function of MHC class Ib-restricted CD8+ T cells during microbial infection using mice deficient in MHC class Ia and various MHC class Ib molecules would provide insight into the relative contribution of different MHC class Ib-restricted T cell populations to antimicrobial immunity. In addition, they may lead to the identification of novel Ag-presenting MHC class Ib molecules, which could be further explored as targets for vaccines against intracellular bacteria. Whereas the highly polymorphic nature of MHC Ia molecules complicates vaccine design, vaccines that induce MHC class Ib-restricted T cell responses by targeting the relatively non-polymorphic MHC class Ib molecules would likely be effective in most human individuals.

Acknowledgments

We thank Dr. James Forman for providing the HeLa cell line and HeLa–Qa-1b transfectant, Dr. Ted Hansen for providing anti-MR1 Ab, and Dr. Iwana Stroynowski for providing anti–Qa-2 Ab. We also acknowledge Jessica Rojas, Ashley Rohr, Sharmila Shanmuganad, Stephen Wood, and Chunting Yang for technical assistance.

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Grants AI40310 and AI57460 (to C.-R.W.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this paper:

    B6
    C57BL/6
    BMDC
    bone marrow-derived dendritic cell
    HKLM
    heat-killed Listeria monocytogenes
    β2m
    β2-microglobulin
    MAIT
    mucosal-associated invariant T
    WT
    wild-type.

  • Received August 3, 2010.
  • Accepted October 23, 2010.

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