Impaired response to Listeria in H2-M3–deficient mice reveals a nonredundant role of MHC class Ib–specific T cells in host defense

The major histocompatibility complex (MHC) class Ib molecule H2-M3 primes the rapid expansion of CD8+ T cells by presenting N-formylated bacterial peptides. However, the significance of H2-M3–restricted T cells in host defense against bacteria is unclear. We generated H2-M3–deficient mice to investigate the role of H2-M3 in immunity against Listeria monocytogenes (LM), a model intracellular bacterial pathogen. H2-M3–deficient mice are impaired in early bacterial clearance during primary infection, with diminished LM-specific CD8+ T cell responses and compromised innate immune functions. Although H2-M3–restricted CD8+ T cells constitute a significant proportion of the anti-listerial CD8+ T cell repertoire, the kinetics and magnitude of MHC class Ia–restricted T cell responses are not altered in H2-M3–deficient mice. The fact that MHC class Ia–restricted responses cannot compensate for the H2-M3–mediated immunity suggests a nonredundant role of H2-M3 in the protective immunity against LM. Thus, the early H2-M3–restricted response temporally bridges the gap between innate and adaptive immune responses, subsequently affecting the function of both branches of the immune system.

The supply of endogenous N-formylated mitochondrial peptides is limited, likely explaining the relatively low surface expression of H2-M3 as compared with MHC class Ia molecules (11). A signifi cant pool of H2-M3 exists intracellularly and can be rapidly mobilized to the surface when provided with appropriate N-formylated peptides (11). This observation suggests that a rapid and eff ective display of bacterial peptides by H2-M3 may occur during bacteria infection. Despite low levels of surface expression and a limited repertoire of selfligands, H2-M3 plays an active role in thymic selection (12,13). A recent study showed that H2-M3-restricted CD8 + T cells could be The MHC contains a large number of class I genes, most of which belong to the MHC class Ib family and are distinguished from MHC class Ia molecules by their lack of polymorphism. The MHC class Ib molecules are thought to serve a more specialized function than MHC class Ia molecules, possibly through the presentation of structurally distinct, yet conserved, microbial antigens to T cells. H2-M3 is one of the best studied MHC class Ib molecules in the mouse. H2-M3 was originally shown to present a polymorphic N-formylated peptide, derived from the mitochondrially encoded ND1 protein, as a histocompatibility antigen to CD8 + T cells (1)(2)(3). Further studies have demonstrated that H2-M3 specifi cally binds N-formylated hydrophobic peptides, exhibiting 100-1,000-fold effi ciently positively selected by H2-M3-expressing hematopoietic cells, unlike thymic epithelial cell-mediated selection of MHC class Ia-restricted CD8 + T cells (14,15). The disparate thymic selection may, in part, account for the distinct behavior of MHC class Ia-and class Ib-restricted T cells in response to infection.
LM is a Gram-positive facultative intracellular bacterial pathogen that causes disseminated infection in immunecompromised human hosts. Experimental listeriosis of mice has been instrumental in analyzing cellular immunity against intracellular bacteria. Investigations of this model have demonstrated that the immune response to LM proceeds in two stages: an early innate immune response that controls the initial bacterial burden (16); and then a later "sterilizing" adaptive immune response that involves antigenspecifi c T cells, particularly CD8 + T cells, which clear the infection and are responsible for a protective memory response (17). In addition to an MHC class Ia-restricted CD8 + T cell response, mice infected with LM also develop a robust H2-M3-restricted CD8 + T cell response. To date, three listerial peptides presented by H2-M3 have been identifi ed, including Fr38 (fMIVIL; reference 9), LemA (fMIGWII; reference 8), and AttM (fMIVTLF; reference 7). One unique feature of the H2-M3-restricted CD8 + T cell response to LM is that it occurs earlier than the MHC class Iarestricted response during primary infection (peaking at days 5-7 vs. days 7-9 after infection; references 18 and 19). However, upon secondary LM infection H2-M3-restricted T cells fail to undergo signifi cant expansion while MHC class Ia-restricted T cells undergo very vigorous expansion (18)(19)(20). The distinct kinetics of H2-M3-restricted and MHC class Ia-restricted T cells during LM infection suggest that these two CD8 + T cell populations may fulfi ll nonoverlapping functions in host defense against intracellular bacteria. A protective role of H2-M3-restricted T cells in both primary and secondary LM infection has been implicated in studying the function of residual CD8 + T cells in mice lacking MHC class Ia molecules (K b−/− D b−/− ; references 19, 21, and 22). However, these experiments are complicated by the contribution of other MHC class Ibrestricted responses, such as the LM-specifi c Qa-1-restricted response (23)(24)(25).
In this study, we specifi cally investigated the role of H2-M3 in protective immunity against LM and examined the interrelationship between MHC class Ia-and class Ib-restricted T cells during LM infection using H2-M3-defi cient (M3 −/− ) mice. We showed that H2-M3 is important for the protective anti-listerial response, as demonstrated by higher bacterial burdens in the liver and the spleen, as well as increased susceptibility to infection in M3 −/− mice. Although naive M3 −/− mice do not display an apparent defect in the CD8 + T cell compartment, we observed signifi cantly reduced numbers of eff ector CD8 + T cells, concurrent with lower levels of LM-specifi c IFN-γ production and cytotoxicity in M3 −/− mice during primary LM infection. These results suggest that H2-M3-restricted T cells comprise a substantial proportion of the anti-listerial CD8 + T cell population. In addition, lack of early activation of H2-M3-restricted T cells resulted in reduced functions of macrophages and NK cells. In contrast, H2-M3 defi ciency has no signifi cant eff ect on anti-listeria immunity during secondary infection. Interestingly, the kinetics and magnitude of LM-specifi c MHC class Ia-restricted T cell responses were not signifi cantly altered when the H2-M3-restricted responses were absent. Our fi nding that MHC class Ia-restricted T cells were not able to completely recompense the CD8 + T cell compartment in M3 −/− mice during (A) Genomic confi guration of the H2-M3 locus before (top) and after (bottom) homologous recombination with the targeting construct (middle). Exons (black boxes), lengths of diagnostic restriction fragments, and the probes used for Southern analysis are shown. An arrow indicates the transcription orientation of the neo gene. B, BglII; N, NheI; E, EcoRI; S, SacI; A, ApaI; H, HindIII; X, XbaI. (B) Splenocytes from WT and M3 −/− mice were incubated overnight with (fi lled histogram) or without (open histogram) 10 μM LemA peptide. Cells were stained with an anti-H2-M3 mAb 130, followed by FITC-anti-hamster IgG. (C) H2-M3-specifi c · CTL clone 4E3 was incubated with LPS blasts from WT and M3 −/− during a 51 Cr release assay. The effector-target ratio is indicated in the fi gure. (D) Identifi cation of LM-specifi c H2-M3-restricted T cells after i.v. infection with LM. Peripheral blood lymphocytes were isolated from LM-infected M3 −/− mice (N6) or WT controls on day 7 after infection. Cells were stained with FITC-anti-CD8α and PE-H2-M3/LemA-tetramer. CD8 + tetramer + T cells are highlighted by the gate, and the proportion of gated cells is indicated.

ARTICLE
LM infection suggests a unique role of H2-M3-restricted T cells in host defense against bacteria infection.

Generation and characterization of H2-M3-defi cient (M3 −/− ) mice
To assess the function of H2-M3, we generated M3 −/− mice by a gene-targeting approach in which exons 4-6 of the H2-M3 gene were replaced by a neomycin (neo)-resistance gene ( Fig. 1 A). Flow cytometric analysis and CTL assays were performed to demonstrate that mice homozygous for the H2-M3 mutation do not express H2-M3 on the cell surface. As shown in Fig. 1 B, surface expression of H2-M3 cannot be induced in splenocytes from M3 −/− mice by incubating with H2-M3-restricted LemA peptide. Splenocytes from WT mice, but not from M3 −/− mice, can be recognized by a mitochondrial ND1 peptide-specifi c, H2-M3-restricted CTL clone ( Fig.  1 C). To confi rm that H2-M3 defi ciency aff ects its restricted CTL response, we infected WT and M3 −/− mice with LM and measured reactivity with H2-M3/LemA tetramer at day 7 after infection. As shown in Fig. 1 D, a substantial population of tetramer + CD8 + T cells was elicited in WT mice, but was not detectable in M3 −/− mice. Thus, M3 protein and its restricted CD8 + T cells were completely absent in M3 −/− mice.

Effect of H2-M3 on T cell development
To determine the eff ect of H2-M3 defi ciency on T cell development, we performed fl ow cytometric analysis on lymphocytes isolated from the thymus, spleen, liver, bone marrow, and lymph nodes of M3 −/− and control mice. We found no signifi cant diff erences in the number and proportions of CD4 + , CD8 + , CD4 − CD8 − , CD4 + CD8 + , αβ, and γδ T cells between the two groups (not depicted). It has been shown that the residual CD8 + T cells in K b−/− D b−/− mice are enriched for cells expressing activation markers, suggesting that MHC class Ib molecule(s) may be critical in the development of this unique subset of CD8 + T cells (19). We compared CD8 + cells from M3 −/− and WT mice for the expression of various activation markers, including CD44, CD69, CD122, and Ly6C. Although there is a trend toward the frequencies of CD8 + CD44 hi T cells from the spleen and mesenteric lymph nodes being slightly decreased in M3 −/− mice, this diff erence is not statistically signifi cant with the sample size analyzed (see Table S1, available at http:// www. jem. org/ cgi/ content/ full/ jem. 20051866/ DC1). Our data suggests that H2-M3-restricted T cells may represent only a small fraction of the total CD8 + T cell population in naive animals.

H2-M3 defi ciency increases susceptibility to Listeria infection
H2-M3-restricted T cells show a peak immune response that precedes their MHC class Ia-restricted counterparts after LM infection (18,19). To investigate the relative significance of H2-M3-restricted and MHC class Ia-restricted CD8 + T cell responses in protective immunity against LM, we infected M3 −/− , K b−/− D b−/− , and WT mice with a sublethal dose of LM (5 × 10 3 CFU) and measured bacterial burden at various time points after infection. Compared with WT mice, M3 −/− mice showed signifi cantly higher bacterial burdens (‫-001-01ف‬fold) in both the spleen and liver at early time points (days 3 and 5) after infection (Fig. 2 A). In contrast, K b−/− D b−/− and  WT mice clear LM from the spleen and liver with similar kinetics, consistent with the previous report (22). By day 7 after infection, LM was nearly absent from all three strains of mice. This data suggests that H2-M3-restricted responses may play an important and even more dominant role than MHC class Ia-restricted responses in protective immunity against LM during the early phase of infection. In agreement with this notion, M3 −/− mice were more susceptible to a higher dose (5 × 10 4 CFU) of LM infection than WT controls (Fig. 2 B). Thus, H2-M3-restricted responses contribute signifi cantly to host defense against LM infection. and examined for CTL activity to 51 Cr-labeld LM-infected J774-K b target cells. Each data line represents the mean from two mice. Data are representative of results obtain from three independent experiments. Specifi c lysis for uninfected J774-K b targets was below 5% from all effectors tested. (D and E) Decreased LM-specifi c, IFN-γ-producing CD8 + T cells in M3 −/− mice. Splenocytes and hepatic leukocytes from indicated mice were harvested on day 7 after LM infection and stimulated overnight with HKLM. Cells were stained for CD8 expression and intracellular IFN-γ, and analyzed by fl ow cytometry. The percentages of CD8 + IFN-γ + and CD8 − IFN-γ + cells are indicated (D). Results are representative from eight mice per group. Bar graph depicts mean ± SEM for total number of LM-specifi c CD8 + cells in the spleens and livers of M3 −/− and WT mice (E). M3 −/− and WT controls used in these experiments are from N10 backcross.

The frequency of activated CD8 + T cells is reduced in LM-infected M3 −/− mice
To determine whether H2-M3-restricted CD8 + T cells constitute a signifi cant proportion of the anti-listerial CD8 + T cell repertoire, we compared the CD8 + population in M3 −/− to WT mice during the course of LM infection. LM infection leads to expansion of LM-specifi c eff ector CD8 + T cells, which reach a peak number around day 7. On day 5 after LM infection, the frequency (Fig. 3) and total number (not depicted) of CD8 + CD44 + T cells were slightly reduced in M3 −/− mice. On day 7 after infection, the percentage of CD8 + CD44 + T cells in M3 −/− mice was decreased by nearly 50% compared with WT controls. The eff ect of H2-M3 defi ciency is specifi c for CD8 + T cells, as no signifi cant diff erences in the CD4 + population can be detected between LM-infected M3 −/− and control mice (not depicted).

The effector functions of CD8 + T cells are diminished in LM-infected M3 −/− mice
To investigate the mechanisms by which H2-M3-restricted T cells contribute to anti-listerial immunity, we compared the antigen-specifi c cytotoxic response and IFN-γ production between M3 −/− and WT mice. For comparison purposes, we also included K b−/− D b−/− mice in our analysis. First, an in vivo cytotoxicity assay was used to assess the cytotoxic potential of H2-M3-restricted T cells during the early phase (day 3) of LM infection, the time point when the difference in bacterial clearance was already apparent. Because MHC class Ia-restricted listerial peptides are not well defi ned in the H-2 b background, we infected mice with a recombinant LM strain expressing OVA protein (rLM-ova) to compare H2-M3-and K b -restricted CTL responses simultaneously. On day 3 after infection, mice were injected with B6 splenocytes pulsed with either H2-M3-restricted LemA peptide or K b -restricted OVA peptide, and the clearance of these target cells was evaluated 15 h later by fl ow cytometry. H2-M3/ LemA-specifi c and K b /OVA-specifi c cytotoxic responses are readily detectable in WT mice (Fig. 4, A and B). However, the cytotoxic response to LemA-coated targets was significantly higher than that to OVA-bearing targets both in the spleens and livers, suggesting that H2-M3/LemA-specifi c CTLs may be more dominant than K b /OVA-specifi c CTLs in WT mice at this time point (Fig. 4, A and B). In M3 −/− mice, no H2-M3/LemA-specifi c response was detected, confi rming the specifi city of the assay. The cytolytic activity to OVA-coated targets was comparable between M3 −/− mice and WT mice, suggesting that the generation/expansion of K b -restricted CTLs was not aff ected in the absence of H2-M3-restricted responses. On the contrary, the H2-M3/ LemA-specifi c cytotoxic response was signifi cantly enhanced in K b−/− D b−/− mice compared with WT mice (Fig. 4, A and  B). Thus, the overall CTL activity is reduced in M3 −/− mice during the early phase of LM infection due to the lack of a compensatory increase of MHC class Ia-restricted CTL responses while remaining largely unchanged in K b−/− D b−/− mice due to augmented H2-M3-restricted responses.
The use of in vivo cytotoxicity assays allowed us to detect relatively small numbers of LM-specifi c CTLs during early LM infection. However, the analysis was limited to a single immunodominant epitope for K b and H2-M3. To further investigate the contribution of H2-M3-restricted T cells to LM-specifi c cytolytic response, we performed in vitro CTL assays using LM-infected J774-K b transfectants as targets, which allowed us to measure overall LM-specifi c, H2-M3-and K b -restricted CTL responses. Splenocytes from M3 −/− , K b−/− D b−/− , or WT mice infected 5 d previously were isolated and stimulated with Con A for 3 d and then used as eff ectors in a 51 Cr release assay. As shown in Fig. 4 C, eff ectors derived from WT and K b−/− D b−/− mice lysed LM-infected J744-K b targets to a similar extent, whereas eff ectors derived from M3 −/− mice displayed much lower lytic activity.
To determine whether H2-M3 defi ciency aff ects the IFN-γ production by CD8 + T cells, intracellular IFN-γ staining was performed. Splenocytes and hepatic leukocytes harvested from M3 −/− or WT mice 7 d after infection were stimulated in vitro with heat-killed LM (HKLM). We found that the frequency and total numbers of LM-specifi c IFN-γ-producing CD8 + T cells were signifi cantly reduced in the spleens and livers of M3 −/− mice compared with WT mice (Fig. 4, D and E). Collectively, these data demonstrate that cytolytic activity and IFN-γ production by CD8 + T cells are impaired in M3 −/− mice during primary LM infection.

H2-M3-defi cient mice have compromised macrophage and NK cell functions
Innate immunity to LM is thought to limit the bacteria burden until the development of specifi c adaptive immunity.
Although several studies have shown that innate immunity can infl uence the development of antigen-specifi c T cells (26,27), the reciprocal eff ect of antigen-specifi c T cells on the innate response is less defi ned. As H2-M3-restricted T cells are functional before the receding of innate immune responses, we examined whether the lack of H2-M3-dependent responses can alter the intensity of the innate immune response to LM infection. At day 3 after LM infection, splenocytes and hepatic leukocytes from M3 −/− and littermate controls were assayed in vitro for nitric oxide production and cytotoxicity to YAC-1 cells to assess the function of macrophages and NK cells, respectively. As shown in Fig. 5 A, splenocytes and hepatic leukocytes from LM-infected M3 −/− mice produced less nitric oxide (as measured by nitrite formation) compared with that of M3 +/− littermate controls. Similar results were obtained from peritoneal exudate macrophages (not depicted). Although splenocytes and hepatic leukocytes from LM-infected M3 −/− mice were able to lyse NK targets, the percentage of killing was lower compared with WT mice (Fig. 5 B). Thus, H2-M3-restricted responses may potentiate the innate immune responses during the early phase of LM infection.

H2-M3 defi ciency does not infl uence the secondary CD8 + T cell response to LM
Although H2-M3-restricted T cells exhibit limited expansion during secondary LM infection, a substantial number of H2-M3-restricted T cells are detectable 3 d after rechallenging with LM (18). To determine whether an H2-M3-restricted response plays a signifi cant role in secondary LM infection, we immunized WT and M3 −/− mice with a sublethal dose of LM and rechallenged them 1 mo later with a high dose of LM. In contrast to the results seen in primary infection, M3 −/− and WT mice displayed equivalent numbers of bacteria in the liver at days 2 and 3 after secondary infection (Fig. 6 A). As early as day 2 after secondary LM infection, the bacterial burdens in the spleens approached the detection limits in both WT and M3 −/− mice (not depicted). In addition, the number of CD8 + T cells and anti-listerial CTL activity are comparable between M3 −/− and WT controls (Fig. 6, B and C). Thus, H2-M3 defi ciency does not have a signifi cant eff ect on the secondary immune response to LM.

Interrelationship between MHC class Ia-and H2-M3-restricted CD8 + T cells
The increased LM burden during the early phase of LM infection in M3 −/− mice could infl uence the antigenic load to which class Ia-restricted CD8 + T cells are exposed during infection, which consequently might aff ect the kinetics and magnitude of class Ia-restricted LM-specifi c CD8 + T responses. To address this question, we used K b /OVA tetramer staining (not depicted) and intracellular IFN-γ staining to compare the K b /OVA-specifi c response between WT and M3 −/− mice after primary and secondary infection with rLM-ova. We found no signifi cant diff erences in the number of K b /OVA-specifi c IFN-γ-producing CD8 + T cells between M3 −/− and WT mice either after primary or secondary LM infection (Fig. 7, A and B), indicating that H2-M3 defi ciency does not aff ect the primary and secondary expansion of MHC class Ia-restricted T cells. In addition, comparable numbers of K b /OVA-specifi c CD8 + T cells were found in WT control and H2-M3-defi cient mice at day 30 after LM infection (2.13 ± 0.41 × 10 5 cells and 2.37 ± 0.38 × 10 5 cells per spleen in WT and M3 −/− mice, respectively), suggesting that H2-M3 defi ciency does not aff ect the development of the class Ia-restricted memory CD8 + T cell population We also assessed the functional avidity of K b /OVA-specifi c CD8 + T cells by incubation of splenocytes from LM-infected WT or M3 −/− mice with a serial dilution of OVA peptide and measured the amount of IFN-γ production by ELISA. No diff erences in functional avidity of K b /OVA-specifi c T cells from WT or M3 −/− mice were observed (not depicted). A reciprocal analysis was performed to examine the eff ect of the lack of MHC class Ia-restricted responses on H2-M3/LemA-specifi c responses. We found  that the number of H2-M3/LemA-specifi c CD8 + T cells is drastically increased in K b−/− D b−/− mice during primary LM infection compared with WT mice (Fig. 7, A and B). A similar trend was also observed during secondary LM infection. Thus, these data suggest that H2-M3 defi ciency has no quantitative or qualitative eff ect on MHC class Ia-restricted T cell responses. In stark contrast, MHC class Ia-restricted responses limit the expansion of H2-M3-restricted T cells during primary as well as secondary LM infection.

DISCUSSION
Emerging evidence suggests that MHC class Ib molecules can contribute to the host immune response by presenting conserved microbial antigens to T cells. However, the relative contributions and dynamic interactions between the MHC class Ia-and class Ib-restricted responses to infections have yet to be defi ned. In this study, we investigated the role of H2-M3 in T cell development and protective immunity against intracellular bacteria using M3 −/− mice in a Listeria infection model. We found that signifi cantly higher numbers of bacteria were recovered from both the spleens and livers of M3 −/− mice than from WT and K b−/− D b−/− mice at days 3 and 5 after LM infection, suggesting that H2-M3-restricted CD8 + T cells may play a dominant role in protective immu-nity against LM during the early phase of infection. The increased susceptibility of M3 −/− mice to LM infection is associated with a decreased number of LM-specifi c IFN-γproducing CD8 + T cells and reduced cytolytic activity. In addition, innate immunity to Listeria infection is lessened in the absence of H2-M3-restricted responses. Lack of H2-M3restricted T cells, however, does not alter the kinetics and magnitude of MHC class Ia-restricted T cell responses during LM infection. Conversely, lack of MHC class Ia-restricted T cells results in an increased number of LM-specifi c H2-M3-restricted T cells after LM infection. This diff erential ability in compensating for the loss of anti-listerial CD8 + T cell responses could be one of the factors underlying the increased susceptibility of M3 −/− mice, but not K b−/− D b−/− mice, to LM infection. Collectively, our results suggest that H2-M3-restricted responses play a unique role in early host defense against listerial infection by contributing to adaptive immunity as well as by modulating the innate response to Listeria in infected mice.
As the number of CD8 + T cells is reduced by 90% in the periphery of naive K b−/− D b−/− mice (28), one may expect that only a small fraction of the CD8 + T cell repertoire is selected by MHC class Ib molecules. Thus, it is not surprising that we were not able to detect signifi cant diff erences in the proportions of T cell subsets between M3 −/− and WT mice. Although H2-M3-restricted T cells only represent a small proportion of the total pool of CD8 + T cells in naive animals, we found that H2-M3-restricted T cells contribute substantially to the overall anti-listerial CD8 + T cell responses during primary infection. The fact that the number of CD8 + CD44 hi cells and LM-specifi c IFN-γ-producing CD8 + T cells is reduced by 50% in LM-infected M3 −/− mice suggests that the small pool of naive M3-restricted T cells are quite capable of expanding to a magnitude similar to their MHC class Ia-restricted counterparts after LM infection. This could be attributed to the accelerated expansion of H2-M3-restricted T cells in response to LM infection as a result of their "memory-like" phenotype (14,19). Another possible explanation could be that the precursor frequency of LM-specifi c T cells within the H2-M3-restricted T cell repertoire is relatively high. It has been shown that some H2-M3-restricted LMspecifi c CTLs are cross-reactive with antigens derived from a variety of Gram-positive and Gram-negative bacteria (29). In addition, deletion of H2-M3-restricted epitopes from LM does not prevent the priming of H2-M3-restricted T cells with specifi city for the deleted epitope, suggesting that promiscuous antigen recognition may be a feature common to many H2-M3-restricted T cells (30)(31)(32). The promiscuous recognition and cross-reactivity to common environmental bacteria may lead to the priming and expansion of LM-reactive H2-M3-restricted T cells in "naive" animals, resulting in increased precursor frequency as well as a "primed" state.
CD8 + CTLs exert their protective functions against intracellular pathogens through cytolytic activity and secretion of cytokines, such as IFN-γ. Both activities are diminished in H2-M3-defi cient mice. To address whether IFN-γ or cytolysis or both are important for M3-dependent immunity to Listeria, we treated LM-infected M3 −/− and WT mice with neutralizing antibody against IFN-γ. CFU data obtained from both the spleens and livers at day 3 after infection showed that anti-IFN-γ-treated M3 −/− mice are still more susceptible to LM infection when compared with anti-IFNγ-treated WT mice (see Fig. S1, available at http://www. jem.org/cgi/content/full/jem.20051866/DC1). Thus, neutralization of IFN-γ does not signifi cantly aff ect the protection mediated by H2-M3-restricted response, suggesting that cytolytic activity may play an important role in H2-M3-mediated early protection during LM infection.
Our results have shown that the LM-specifi c CD8 + T cell population is signifi cantly reduced in M3 −/− mice, suggesting that MHC class Ia-restricted LM-specifi c CD8 + T cells did not undergo compensatory expansion during LM infection. This is further supported by the observations that CD8 + T cell responses to two K b -restricted epitopes, OVA 257-264 and LLO 296-304 , despite the diff erence in magnitude, were not aff ected by the H2-M3 defi ciency ( Fig. 7 and not depicted). One possible explanation is that the majority of naive LM-specifi c MHC class Ia-restricted CD8 + T cell precursors have been recruited in our experimental settings. Thus, MHC class Ia-restricted LM-specifi c CD8 + T cells were not able to contribute further, even when a sizable amount of H2-M3-restricted T cells were absent. Recent studies have shown that the magnitude of LM-specifi c CD8 + T cell responses is determined within the fi rst 24 h of an infection and is independent of the severity and duration of in vivo bacterial infection (33). This notion may further explain the lack of a concordant increase in LM-specifi c class Ia-restricted T cells in M3 −/− mice despite the presence of higher bacterial titers in the spleens and livers of M3 −/− mice after 3-5 d of LM infection. However, it is conceivable that in an infection model where not all naive antigen-specifi c MHC class Ia-restricted T cells were recruited, either due to the presence of a large pool of antigen-specifi c MHC class Ia-restricted T cell precursors or due to the limited supplies of antigens/infl ammatory cytokines, the presence or absence of an H2-M3-restricted response may aff ect the magnitude of MHC class Ia-restricted T cell responses.
Our fi nding that the number of LM-specifi c H2-M3-restricted T cells is signifi cantly higher in K b−/− D b−/− mice than in that of WT controls after primary infection is in accordance with previous observations using an in vitro cytotoxicity assay (22). It is possible that the precursor frequency of H2-M3-restricted CD8 + T cells is increased in K b−/− D b−/− mice. Another possible explanation, although not mutually exclusive, is that lack of MHC class Ia-restricted T cells may allow for maximal expansion of LM-specifi c H2-M3-restricted T cells during infection by providing a favorable environment with more access to APCs, cytokines, and growth factors. A recent study has shown that the expansion of LM-specifi c H2-M3restricted memory T cells can be prevented by LM-specifi c MHC class Ia-restricted memory T cells, possibly though rapid removal of antigen-presenting DCs (34). Comparing the numbers of H2-M3/LemA-specifi c T cells elicited during primary and secondary LM infection, we found that H2-M3-restricted T cells did not experience a similar degree of secondary expansion as observed for MHC class Ia-restricted T cells even in K b−/− D b−/− mice, consistent with the previous fi nding using IFN-γ ELISPOT assays (19). Thus, these data suggest that lack of memory expansion of H2-M3-restricted response during infection may be, at least in part, due to intrinsic diff erences between MHC class Ia-and H2-M3restricted T cells. Alternatively, the presence of other LM-specifi c non-MHC class Ia-restricted memory T cells might also be able to limit the secondary expansion of H2-M3-restricted T cells. Similar to H2-M3-restricted T cells, lack of memory expansion is also evident for Qa-1-restricted T cells during LM infection (23). Thus, MHC class Ib-restricted responses against infectious organisms may play a unique and important role during the early stages of infection, but participate only minimally in the protective memory immune response.
In addition to H2-M3-restricted T cells, LM infection has been shown to activate/induce T cells restricted by other MHC class Ib molecules, such as CD1d and Qa-1 b (22,24,35). To see whether lack of H2-M3-restricted responses has an eff ect on other MHC class Ib-restricted responses, we ARTICLE have examined the CD1d-and Qa-1 b -restricted T cell responses in LM-infected WT and M3 −/− mice. We found no signifi cant diff erences in the number of CD1d-restricted NKT cells or the magnitude of Qa-1 b -specifi c cytolytic activity between LM-infected M3 −/− and control mice at all time points examined (not depicted). This fi nding further supports the nonredundant role of H2-M3-restricted T cells in anti-listerial immunity.
Previous studies have shown that H2-M3-restricted T cells share some characteristics with CD1d-restricted NKT cells, such as the capacity to elicit rapid eff ector function and ability to be positively selected by hematopoietic cells (36,37). However, unlike CD1d-restricted NKT cells that express a limited TCR repertoire (38)(39)(40)(41), the H2-M3-restricted T cells studied to date have diverse TCR usage (42). Consistent with this notion, analysis of TCR Vα and Vβ chains in M3 −/− mice does not reveal signifi cant alterations in Vα and Vβ usage in CD8 + T cell populations compared with control (not depicted). In addition to the diff erences in TCR Vα and Vβ repertoire diversity, the kinetics of H2-M3-restricted T cell responses are also diff erent from that of CD1d-restricted NKT cells in LM infection. Although we can detect H2-M3-restricted cytotoxic responses as early as day 2 after LM infection using an in vivo CTL assay (not depicted), H2-M3-restricted T cell response peaks at days 5-7 after infection (18,19). In contrast, the majority of CD1d-restricted NKT cells are activated within 24 h after LM infection (35 and not depicted). Thus, the distinct kinetics of H2-M3-restricted T cells positions them to fi ll the temporal gap between "innate" T cells and conventional adaptive T cells.
In summary, our study demonstrates that H2-M3-restricted responses play a signifi cant and nonredundant role in protective immunity against intracellular bacterial infection. Similar to H2-M3, other MHC class Ib molecules may serve a specialized role in antimicrobial immunity by selective presentation of conserved molecular signatures of microbial antigens and/or stress-induced ligands. The recognition of specifi c molecular patterns and rapid acquisition of eff ector functions may provide the unique niche for the MHC class Ib-restricted T cells in host defense against microbial pathogens.

MATERIALS AND METHODS
Generation of H2-M3-defi cient (M3 −/− ) mice. A DNA fragment containing H2-M3 was isolated from a 129/sv genomic DNA phage library (Stratagene). The targeting construct was generated by insertion of a 3.8-kb fragment containing exons 1-3 of the H2-M3 and a 5.5-kb fragment containing the 3′ end of H2-M3 into the EcoRI-BamHI and NotI-XhoI sites of pPNT, respectively (43). The targeting construct was designed to delete a 2.5-kb ApaI-SacI fragment containing exons 4-6 of the H2-M3 and replace these coding regions with the neo-resistance gene (Fig. 1 A). Strain 129/svderived R-1 embryonic stem cells were electroporated with 30 μg of NotIlinearized targeting construct, and G418-resistant colonies were screened for homologous recombination by Southern blot hybridization. Embryonic stem clones were injected into blastocysts of B6 mice, and chimeras were mated to B6 females. Off spring heterozygous for the mutation were intercrossed to produce M3 −/− mice. Most of the M3 −/− mice used in this study were backcrossed on to a B6 background (between 6 to 10 generations, N6-N10). The H2-M3 genotype was determined with PCR using the following three primers:  ). H2-M3/LemA tetramers were prepared as described previously (18). For fl ow cytometric analysis, single cell suspensions from the thymus, spleen, lymph node, and liver were prepared by standard procedures. For detecting H2-M3 surface expression, splenocytes from M3 −/− and WT mice were incubated in RPMI 10 (RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 20 mM Hepes, 50 μM 2-mercaptoethanol, penicillin, and streptomycin) with or without 10 μM LemA peptide overnight at 37°C. Cells were harvested and stained with anti-H2-M3 antibody (mAb130), followed by FITC-anti-hamster IgG (11). To examine T cell subsets and various immune cell types, cells were stained with combinations of fl uorescent-conjugated antibodies for 30 min on ice in HBSS (Life Technologies) containing 2% FBS and 0.1% sodium azide (Sigma-Aldrich), followed by washing with the same buff er. The stained cells were analyzed by fl ow cytometry using a FACSCaliber with CELLQuest (Becton Dickinson) or Flowjo (Tree Star) software.

M3-forward-C A G C G A T G G A A C C A C C C A C A A T G A , M3-reverse-C A G A C T A G C A A C G A T G A C C A T G A T G A C , and neo-G A T-T C G C A G C G C A T C G C C T T C T
Bacteria, listeria infections, and CFU assays. LM strain EGD was provided by R. Kurlander (National Institutes of Health, Bethesda, MD) and was grown in brain-heart infusion broth (Difco Laboratories). The recombinant LM strain rLM-ova, which secretes OVA and contains erythromycin marker, was provided by L. Lefrançois (University of Connecticut Health Center, Farmington, CT; reference 45). rLM-ova was grown in brain-heart infusion broth supplemented with 5 μg/ml erythromycin. For quantitation of bacterial titers from tissues, 12-20-wk-old mice were infected i.v. with 5 × 10 3 CFU LM (EGD) for primary infection. Recall infection with 10 5 CFU LM was performed 1 mo after primary infection. The bacterial dose was verifi ed by plating dilutions of the inoculum on BH agar plates. At the indicated times after infection, mice were killed and specimens of liver and spleen were examined for bacterial titers. In brief, organs were homogenized and lysed in sterile water with 0.2% NP-40, serial dilutions of homogenates were plated on BH agar plates, and colonies were counted after incubation at 37°C for 24 h. For comparing the MHC class Ia-restricted and H2-M3-restricted T cell responses, mice were infected i.v. with 5 × 10 4 CFU rLM-ova for the primary infection and 10 6 CFU rLM-ova for the secondary infection. 51 Cr release CTL assays. ND1 α -specifi c, H2-M3-restricted CTL clone (4E3; provided by K.F. Lindahl, Texas Southwestern Medical Center, Dallas, TX) was used as eff ector in CTL assays to determine the expression of H2-M3 on LPS blast from M3 −/− and WT mice. To measure LM-specifi c CTL activity, splenocytes from LM-infected mice (either 5 d after primary infection or 3 d after secondary infection) were cultured in RPMI 10 with 1 μg/ml Con A. After 3 d, cells were harvested and used as eff ectors in a 51 Cr release CTL assay. LM-infected and noninfected J774 transfectant-expressing H2-K b (J774-K b ; provided by J. Forman) were used as targets. In brief, monolayers of J774-K b targets grown in antibiotic-free medium were infected with LM for 1 h at a multiplicity of infection of 5:1. Cells were washed three times with warm PBS and cultured in DMEM containing 40 μg/ml gentamicin for an additional 3 h. All target cells used in CTL assays were labeled with 51 Cr for 1 h at 37°C. Target cells (10 4 ) were added to round-bottom microtiter wells containing serial dilutions of eff ector cells. After 4 h of incubation at 37°C, 100 μl of supernatant from each well was collected to determine the amount of 51 Cr release. The percent-specifi c lysis was calculated as 100 × (experimental cpm − spontaneous cpm) / (maximal cpm − spontaneous cpm). Data presented are the mean of triplicate wells.
In vivo cytotoxicity assay. The in vivo cytotoxicity assay was performed as described by Barber et al. (46). In brief, splenocytes from B6 mice were labeled with red membrane dye PKH26 and either 5 μM, 500 nM, or 50 nM of cytosolic dye CFSE. The labeled cells were then pulsed with 1 μM LemA or OVA peptide for 4 h and transferred retro-orbitally (1:1:1 ratio, 5-10 million cells for each population) into the indicated groups of recipient mice. After 15 h of in vivo killing, lymphocytes were isolated from the spleens and livers and analyzed by fl ow cytometry for target cell clearance. Target cells were diff erentiated from recipient cells based on PKH26 staining and from each other based on diff erent intensity of CFSE staining. Gated on PKH26 + cells, the percentage of specifi c killing was calculated as the following: 100 − [(% peptide pulsed in LM-infected/% unpulsed in LM-infected) / (% peptide pulsed in uninfected/% unpulsed in uninfected)] × 100.
Intracellular IFN-staining. Splenocytes or hepatic leukocytes from rLM-ova-infected mice were cultured in 96-well plates at a concentration of 10 6 cells/well and stimulated with either 5 μM LemA or 1 μM OVA peptide for 5 h, or HKLM overnight. For the last 2 h of stimulation, 10 μM monensin was added to block the secretion of cytokines. Cells were washed and stained for cell surface markers CD8 and CD4. After fi xation with 4% paraformaldehyde and permeabilization with 0.15% saponin, cells were then stained with PE-anti-IFN-γ antibody. Labeled cells were analyzed on a FACSCalibur.
NK cell cytotoxicity assays. Splenocytes and hepatic leukocytes from uninfected mice and mice infected for 3 d with LM were prepared and used as eff ectors. YAC-1, a mouse lymphoma cell line, was used as a target in this assay. Cells were harvested and labeled with 100 μCi of 51 Cr for 1 h at 37°C. Target cells (10 4 ) were added to round-bottom microtiter wells containing serial dilutions of eff ectors. After 4 h of incubation at 37°C, 100 μl of supernatant from each well was collected and assayed for 51 Cr release. Percent specifi c lysis = (experimental cpm − spontaneous cpm) / (maximal cpm − spontaneous cpm) × 100. Data presented are the means of triplicates. Nitrite assay. 3 d after LM infection, hepatic leukocytes and splenocytes from M3 −/− and WT mice were isolated and stimulated with HKLM for 48 h at 4 × 10 5 -10 6 cells/well in 96-well plates. Equivalent population of macrophages and DCs were assessed using PE-anti-F4/80 and PE-anti-CD11c by FACS analysis. Accumulated nitrite in the culture supernatant was measured by a colorimetric assay based on the Griess reaction with NaNO 2 as the standard (47).

Statistical analysis.
Mean values were compared using the unpaired Student's t test. All statistical analyses were performed with the Prism program (GraphPad Software, Inc.). Statistically signifi cant diff erences P < 0.05, P < 0.01, and P < 0.001 are noted with *, **, and ***, respectively. Table S1 shows the expression of various activation markers on CD8 + T cells from M3 −/− and WT mice. Fig.  S1 shows the eff ect of a neutralizing anti-IFN-γ mAb on M3-mediated early protection to LM infection. Table S1 and Fig. S1 are available at http:// www. jem. org/ cgi/ content/ full/ jem. 20051866/ DC1.