Invariant V14+ NKT Cells Participate in the Early Response to Enteric Listeria monocytogenes Infection1
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免疫学杂志 2005年第14期
Abstract
Invariant V14+ NKT cells are a specialized CD1-reactive T cell subset implicated in innate and adaptive immunity. We assessed whether V14+ NKT cells participated in the immune response against enteric Listeria monocytogenes infection in vivo. Using CD1d tetramers loaded with the synthetic lipid -galactosylceramide (CD1d/GC), we found that splenic and hepatic V14+ NKT cells in C57BL/6 mice were early producers of IFN- (but not IL-4) after L. monocytogenes infection. Adoptive transfer of V14+ NKT cells derived from TCR° V14-J18 transgenic (TCR°V14Tg) mice into alymphoid Rag°c° mice demonstrated that V14+ NKT cells were capable of providing early protection against enteric L. monocytogenes infection with systemic production of IFN- and reduction of the bacterial burden in the liver and spleen. Rechallenge experiments demonstrated that previously immunized wild-type and J18° mice, but not TCR° or TCR°V14Tg mice, were able to mount adaptive responses to L. monocytogenes. These data demonstrate that V14+ NKT cells are able to participate in the early response against enteric L. monocytogenes through amplification of IFN- production, but are not essential for, nor capable of, mediating memory responses required to sterilize the host.
Introduction
The primary control of infection by the intracellular pathogen Listeria monocytogenes relies on the ability of the host to mount an efficient Th1-like immune response (reviewed in Ref. 1). Production of IFN- in the early phases of infection is essential to enhance IL-12 production and activate bactericidal mechanisms in macrophages (2, 3). NK cells have been identified as a source of early IFN- production (4). Thus, SCID mice (T–, B–, NK+) are able to control primary L. monocytogenes infection in an IFN--dependent manner. Eventually SCID mice succumb to chronic listeriosis, demonstrating that NK cells alone are unable to fully protect the host against L. monocytogenes (5, 6). Instead, sterilizing immunity relies on the generation of cytotoxic CD8+ T cells which clear infected macrophages and hepatocytes and thereby eliminate the bacteria (reviewed in Refs. 1 and 7). The participation of other cell types has been described in the protection against L. monocytogenes. Several studies have defined a role for CD4+ T cells in both primary and secondary L. monocytogenes infection (8, 9). In addition, T cells play a role in the defense against L. monocytogenes, since they are able to control primary infections in the absence of TCR cells. However, TCR cells are not able to mediate sterilizing immunity after infection (10).
NKT cells constitute a heterogeneous subset of T cells expressing both NK and T cell surface markers. One well-characterized NKT subset includes a thymus-derived population expressing a canonical V14-J18 TCR -chain associated with a limited set of TCR subfamilies (reviewed in Ref. 11). These invariant V14+ NKT cells, which are either CD4+ or CD4–CD8– double negative, are selected on the nonclassical MHC class I molecule CD1. V14+ NKT cells recognize an endogenous lysosomal glycosphingolipid, isoglobotrihexosylceramide (12), and when activated through their TCR or by soluble factors (such as IL-12) can produce both IFN- and IL-4 (13, 14). Moreover, V14+ NKT cells have been shown to transactivate B, T, and NK cells in vivo (15, 16). Along these lines, V14+ NKT cells may act as sentinels to integrate initial signals following immune stimulation and thereby serve to orient subsequent immune responses.
V14+ NKT have been implicated in a number of immune-mediated pathologies including graft-vs-host disease, autoimmune hepatitis, and in fetal loss (17, 18, 19). In addition, a disease-controlling role for NKT cells has been shown in V14-J18 transgenic (Tg)5 nonobese diabetic mice (20). V14+ NKT cells may participate in antitumor responses by counteracting invasion and metastasis (reviewed in Ref. 21). Finally, a role for V14+ NKT cells has been proposed for protection against parasites (Toxoplasma gondii, Plasmodium yoelii, and Plasmodium berghei) and intracellular pathogens (mycobacteria and L. monocytogenes) (reviewed in Ref. 22). V14+ NKT cells could provide a protective role via IFN- in sustaining Th1 responses (23). Alternatively, IL-4 production from V14+ NKT cells could either have a deleterious role by deviating Th1 responses toward Th2 or act as an amplifier of Th2 responses in the context of extracellular parasites (24, 25). The precise role of V14+ NKT cells in infection immunity is clearly not defined and could vary depending on the pathogen.
Concerning L. monocytogenes, previous studies have demonstrated that NKT-deficient mice can resist infection by L. monocytogenes similar to wild-type mice (8, 26), excluding an essential role for these cells in antilisterial immunity. In contrast, Kaufmann and coworkers found that NKT cells are selectively depleted from the liver of L. monocytogenes-infected mice and that treatment of infected mice with CD1-specific Abs ameliorated the antilisterial response via increased IFN-, TNF-, and IL-12 production (27, 28). This group proposed that NKT cells could play a negative role in the immunity against intracellular bacteria, possibly through production of TGF- (28). Considering these contradictory findings, we decided to re-examine the role for V14+ NKT cells in the antilisterial response. Using several approaches in wild-type, Ja18°, and V14 transgenic mice, we demonstrate that invariant V14+ NKT cells clearly contribute to the pro-Th1 response following infection with L. monocytogenes but are not essential for or capable of mediating memory responses to this pathogen.
Materials and Methods
Mice
Rag° and Rag°c° mice (29) were from the 10th backcross to the C57BL/6 background. TCR° mice and V14-J18Tg on the TCR-deficient C57BL/6 background (TCR°V14Tg) mice (20) as well as J18° mice (30) have been previously described. C57BL/6 mice were purchased at IFFA-CREDO. Mice were housed at the Institut Pasteur (Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 668) and at Necker Hospital (INSERM Unité 411). All animal studies were evaluated and approved by a local institutional review board.
Abs and reagents
Abs were obtained from BD Pharmingen and were used as FITC, PE, biotin, and allophycocyanin conjugates. Biotinylated Abs were revealed with FITC-, PE (Caltag Laboratories) or PerCP-conjugated streptavidin (BD Pharmingen). Anti-CD19 microbeads and LS+ magnetic separation columns were obtained from Miltenyi Biotec. RPMI 1640, FCS, and antibiotics were purchased from Invitrogen Life Technologies. Percoll was purchased from Pharmacia. Brain-heart infusion medium (BHI) was obtained from Acumedia
Preparation of bacterial strains
Listeria monocytogenes (strain LO28) (31), was grown to exponential phase in BHI medium and harvested in the exponential growth phase, washed, and stored at –80°C in aliquots of 109 bacteria/ml in PBS.
Isolation of lymphoid cells
For isolation of lymphoid cells from peripheral lymphoid organs, mice were sacrificed and the mesenteric lymph node (mLN), spleen, and liver were removed. Single-cell suspensions were generated from mLN and spleen by teasing the organs through a metal mesh followed by erythrocyte lysis. Single-cell suspensions were generated from liver by teasing the organs through a metal mesh followed by centrifugation on a Percoll gradient (40/80%) and erythrocyte lysis.
Cell sorting and adoptive transfer into Rag°c° mice
For electronic cell sorting, single-cell suspensions were generated from the mLN of TCR°V14Tg mice. Following erythrocyte lysis, lymph nodes cells were depleted of B cells using MACS anti-CD19 microbeads and LS columns according to the manufacturer’s instructions. Subsequently, the cells were incubated with biotinylated anti-CD5 mAb, PE, anti-CD8 mAb and allophycocyanin anti-NK1.1 mAb as described below. Biotinylated Ab was revealed by incubation with FITC-streptavidin. NKT cells were sorted as NK1.1+/CD8–/CD5+ cells using a MoFlo cell sorter (DakoCytomation). Post-sort analysis confirmed that these cells were >98% NKT cells and contained <0.4% contaminating NK cells. Nonirradiated Rag°c° mice (3–6 wk of age) were transplanted i.v. with 5 x 105 purified NK1.1+ T cells 4 days before infection.
Infection and determination of CFU
For intragastric (i.g.) infection with 5 x 108 L. monocytogenes strain LO28, groups of mice were gavaged i.g. using an 18-gauge dumb-end feeding needle. For rechallenge experiments, mice were injected i.v. in the lateral tail vein with 2 x 106 bacteria.
At the indicated time points after infection, mice were sacrificed and the livers and spleens were aseptically removed. Homogenates of liver and spleen were prepared by grinding organs in sterile PBS with a motorized Teflon pestle. Bacterial CFU were enumerated by plating organ homogenates in 10-fold, serial dilutions on BHI agar plates. After incubation at 35°C for 36–48 h, the bacterial colonies were counted.
Flow cytometry
For surface Ab staining, cells were washed twice in PBS supplemented with 1% BSA (PBS-BSA), incubated on ice for 30 min with Abs, and subsequently washed twice in PBS-BSA before analysis. When appropriate, cells were incubated with biotin-conjugated Abs, washed three times, and then incubated for 30 min with the relevant streptavidin conjugate and then washed three times before analysis. Samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and the data were analyzed using CellQuest software (BD Biosciences).
For intracellular cytokine detection, total cell suspensions were incubated for 1 h in RPMI 1640/5% FCS containing brefeldin A (10 μg/ml) to block cytokine secretion. Surface-stained cells (TCR+, tetramer+) were fixed for 1 h in PBS containing 2% paraformaldehyde, and intracellular cytokines were detected using a PE-conjugated IFN- (XMG1.2) or control rat IgG1 (R3-34) mAbs in PBS containing 0.5% saponin.
Tetramer staining
Single-cell suspensions were stained for 20 min on ice with -galactosylceramide (-GC)-loaded allophycocyanin-conjugated CD1d tetramers (derived from mCD1d/2-microglobulin expression vector as described in Ref. 32). Cells were then washed twice with ice-cold PBS-BSA and subsequent Ab surface staining with FITC anti-TCR mAb and PE anti-NK1.1 mAb was performed as described above. Nonspecific binding was controlled by staining using CD1d tetramers without -GC (data not shown).
ELISA
Serum was obtained (days 0 and 3 postinfection) and the concentrations of IFN- were determined using a specific sandwich ELISA kit (Genzyme) according to the manufacturer’s instructions.
Statistics
Statistical significance was evaluated using the Mann-Whitney U test. Values of p < 0.05 were considered to be significant.
Results
V14+ NKT cells participate directly in the antilisterial response in vivo
We used several independent and complementary approaches to assess the role of V14+ NKT cells in antilisterial immunity. We first used CD1d tetramers loaded with the synthetic lipid -GC to follow V14+ NKT cell activation and cytokine production after i.g. L. monocytogenes infection of wild-type mice. Uninfected C57BL/6 mice harbored a population of CD1d/-GC-reactive T cells which, on a percentage basis, were more abundant in the liver (6 ± 1.2%) than in the spleen (0.5 ± 0.1%; Fig. 1A and data not shown). These cells were mainly NK1.1+ and did not constitutively synthesize IFN- (Fig. 1, B and C). As early as 24 h after i.g. infection with L. monocytogenes, invariant V14+ NKT cells became activated and began to produce IFN- (Fig. 1B), but not IL-4 (data not shown). It should be emphasized that the protocol used for ex vivo analysis of cytokine production by V14+ NKT cells did not involve a TCR restimulation in vitro. By day 2 after L. monocytogenes infection, about one-half of the CD1d tetramer-reactive T cells in the liver and spleen were active in IFN- production, and this fraction persisted at day 3 after infection (Fig. 1B and data not shown). Interestingly, the percentage of CD1d tetramer-reactive T cells decreased by days 2 and 3 after infection, which was correlated with a decreased density of NK1.1 expression (Fig. 1, A and C), although CD1d tetramer staining was still clearly observed. This "loss" of V14+ NKT cells likely corresponds to a partial down-modulation of TCR and NK1.1 expression rather than an actual disappearance of the cells. These results clearly demonstrate the participation of NKT cells in response to L. monocytogenes via IFN- production, a cytokine required for the control of this pathogen.
Discussion
Using a combination of approaches, including analysis with CD1d tetramers, V14+ NKT cell transgenic and knockout mice and selective reconstitution of alymphoid mice with highly purified V14+ NKT cells, we have reassessed the role of V14+ NKT cells in the immunity against enteric infection with the intracellular bacterium L. monocytogenes. Although previous reports suggested a negative impact of NKT cells on antilisterial immunity (27, 28), we found that V14+ NKT cells were stimulated to produce IFN- in vivo following enteric L. monocytogenes infection and were able to provide early protection of highly susceptible alymphoid mice against L. monocytogenes. In contrast, we demonstrated that V14+ NKT cells do not provide adaptive immunity to this pathogen under conditions of recall stimulation.
The capacity of -GC-loaded CD1d tetramers to unambiguously identify invariant V14+ T cells provided an essential tool for our studies. Previous reports have demonstrated the specificity of this reagent in wild-type mice and in transgenic mice bearing a functionally rearranged V14-J18 TCR chain that develops increased numbers of V14+ NKT cells (32, 34). These TCR°V14Tg mice provided us with the means to directly assess the functional capacity of V14+ NKT cells to provide early protection after L. monocytogenes infection. One caveat of our experiments is whether the NKT cells derived from TCR°V14Tg mice faithfully represent their counterparts from wild-type mice. Previous studies have shown that CD1d-reactive NK1.1+ T cells from TCR°V14Tg mice have a TCR repertoire and cell surface phenotype that closely matches NK1.1+ T cells from C57BL/6 mice (34). Moreover, NKT cells from TCR°V14Tg mice, like their normal counterparts, have the capacity to rapidly produce cytokines (IL-4, IFN-) following in vitro stimulation (20, 34). Thus, by several distinct criteria, the V14+ NKT cells from TCR°V14Tg mice appear to faithfully represent their normal C57BL/6 counterparts.
CD1d-reactive V14+ T cells from both C57BL/6 and TCR°V14Tg mice harbor a subset of NK1.1– cells. Previous studies from Benlagha et al. (14) have demonstrated that these cells in C57BL/6 mice likely represent precursors of the NK1.1+ cells. Using CD1d tetramers, these authors found that the NK1.1– subset of V14+ T cells bore an immature phenotype and selectively produced IL-4, but not IFN-, after stimulation. The presence of NK1.1–V14+ T cells in the spleen suggested that these precursors could exit the thymus and further differentiate into NK1.1+ IFN- secreting mature V14+ NKT cells in the periphery. Additional experiments showed that purified NK1.1– CD1d-reactive T cells could give rise after adoptive transfer to NK1.1+ progeny. The presence of two phenotypically and functionally distinct V14+ T cell subsets in the periphery of mice could allow for flexibility in the ways that immune responses could be oriented.
The ability of TCR° mice to control primary L. monocytogenes infection is consistent with the previously recognized capacity of TCR and NK cells to participate in innate immunity against this pathogen (3, 6, 10). No difference in the bacterial burden or early survival was observed among wild-type, TCR°, and TCR°V14Tg mice following enteric L. monocytogenes infection. This observation argues against any predominant regulatory role for V14+ NKT cells in the immunity against enteric L. monocytogenes, in contrast with previous studies (27, 28) that reported an amelioration of listeriosis in mice treated with anti-CD1 mAbs. These authors deduced that the blockade of CD1 interfered with the activation of NKT cells, resulting in decreased TGF- levels and increased IFN-, TNF-, and IL-12 production. Since TCR°V14Tg mice were as resistant as TCR° mice to primary infection, our results are incompatible with a dominant negative activity of V14+ NKT cells during L. monocytogenes infection. Still, NKT cells could impact on L. monocytogenes infections under conditions when NK and/or T cells are limiting.
We used adoptive transfer of V14+ NKT cells from TCR°V14Tg mice to assess the capacity of these cells to confer protection against L. monocytogenes when transplanted into alymphoid Rag°c° mice. We observed a beneficial effect of V14+ NKT cells in this setting, which correlated with IFN- (but not IL-4) production. It is interesting to consider our results in light of the observations that V14+ NKT cells can produce both IFN- and IL-4 following TCR stimulation in vitro. In contrast, V14+ NKT cells can preferentially produce either IL-4 or IFN- following stimulation with cytokines (37). The restricted biological activity of NKT cells after L. monocytogenes infection could indicate that these cells do not receive TCR stimulation via CD1d complexes in vivo in the setting. Recent studies by Brenner and colleagues (38) reported that Salmonella infection activated V14+ NKT cells in a TCR- and IL-12-dependent fashion. We also have preliminary evidence that MHC-deficient Rag°c° mice (which lack expression of CD1 molecules) reconstituted with NKT are able to resist early L. monocytogenes infection (T, Ranson and J. P. Di Santo, unpublished observations). These results would suggest that V14+ NKT cells are recruited to respond to certain types of intracellular infections dependent on the cytokine milieu; a pro-Th1 (IL-12)-rich environment would then favor V14+ NKT production of IFN-. Following L. monocytogenes infection, TCR°V14Tg mice displayed systemic IFN- levels comparable to those of wild-type mice and 3- to 4-fold higher levels than found in TCR° mice. Early IFN- production by V14+ NKT cells therefore represents a likely antilisterial mechanism in our experiments, although direct NKT cell-mediated killing of L. monocytogenes-infected macrophages cannot be ruled out (39).
In our transfer experiments, we found that NKT cells were able to substantially reduce the bacterial burden in the liver and spleen of the Rag°c° hosts (by almost 2 logs) after enteric L. monocytogenes infection. The level of protection afforded by the injected NKT cells is even more impressive considering the limited number of NKT cells transferred and the fact that homeostatic expansion of these cells only results in the generation of 105 NKT cells in the liver and spleen of the recipient hosts (36). In addition, the transplanted V14+ NKT cells might have undergone apoptosis following stimulation in vivo (40). Thus, despite being unable to completely eradicate the bacterial inoculum, NKT cells demonstrated potent antilisterial activity which resulted in protection of the reconstituted mice for at least 3 wk.
V14+ NKT cells have been shown to "cross-talk" with other lymphocytes, including NK, B, and T cells (15, 16). In particular, it has been shown that NKT-NK cell interactions may play an important role in tumor surveillance in vivo (reviewed in Ref. 21). Our results using adoptive transfer showed that NKT cells alone provide early protection after L. monocytogenes infection. Still, functional synergy between NKT and NK cells may allow for an even better protection after infectious challenge. The use of CD1d/-GC tetramers allowed us to directly demonstrate that V14+ NKT cells in C57BL/6 mice respond after L. monocytogenes infection by production of IFN-. Comparisons of C57BL/6 and J18° mice revealed a major difference in NK cell IFN- production after L. monocytogenes infection, consistent with V14+ NKT cell transactivation of NK cells in vivo.
The fact that TCR° and TCR°V14Tg mice did not mount functional memory responses to L. monocytogenes is consistent with previous reports demonstrating a pivotal role for cytotoxic CD8+ T cells in the generation of antilisterial memory responses (reviewed in Refs. 1 and 7). Our observations indicate that NKT cells do not play an essential role in recall responses to L. monocytogenes. Nevertheless, NKT cells could amplify memory responses via transactivation of previously established CD8 memory T cells. The capacity for NKT cells to rapidly produce IFN- and to potentiate its production by other lymphocytes (NK cells, T cells, CD8 memory T cells) after L. monocytogenes infection provides an important physiological example of the important role of NKT cells as a bridge between innate and adaptive immunity.
Acknowledgments
We thank Dr. D. Guy-Grand for helpful discussions. We are indebted to Pharmaceutical Research Laboratory, Kirin Brewery Company, for providing -GC and to P. Van Endert and M. Kronenberg for help in generating CD1d tetramers.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Institut Pasteur, INSERM, Association pour la Recherche sur le Cancer, and Ligue National Contre le Cancer. S.B. was the recipient of a postdoctoral fellowship from the Danish Research Agency.
2 T.R. and S.B. contributed equally to this work.
3 Current address: Symphogen, DK-2800 Lyngby, Denmark.
4 Address correspondence and reprint requests to Dr. James P. Di Santo, Unité des Cytokines et Développement Lympho?de, INSERM Unité 668, Institut Pasteur, 25 Rue du Docteur Roux, Cedex 15, Paris, France. E-mail address: disanto@pasteur.fr
5 Abbreviations used in this paper: Tg, transgenic; BHI, brain-heart infusion; c, common -chain; Tg, transgene; -GC, -galactosylceramide; mLN, mesenteric lymph node; i.g., intragastric.
Received for publication January 26, 2005. Accepted for publication April 21, 2005.
References
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Eberl, G., H. R. MacDonald. 1998. Rapid death and regeneration of NKT cells in anti-CD3- or IL-12-treated mice: a major role for bone marrow in NKT cell homeostasis. Immunity 9: 345-355.(Thomas Ranson2,*, S?ren B)
Invariant V14+ NKT cells are a specialized CD1-reactive T cell subset implicated in innate and adaptive immunity. We assessed whether V14+ NKT cells participated in the immune response against enteric Listeria monocytogenes infection in vivo. Using CD1d tetramers loaded with the synthetic lipid -galactosylceramide (CD1d/GC), we found that splenic and hepatic V14+ NKT cells in C57BL/6 mice were early producers of IFN- (but not IL-4) after L. monocytogenes infection. Adoptive transfer of V14+ NKT cells derived from TCR° V14-J18 transgenic (TCR°V14Tg) mice into alymphoid Rag°c° mice demonstrated that V14+ NKT cells were capable of providing early protection against enteric L. monocytogenes infection with systemic production of IFN- and reduction of the bacterial burden in the liver and spleen. Rechallenge experiments demonstrated that previously immunized wild-type and J18° mice, but not TCR° or TCR°V14Tg mice, were able to mount adaptive responses to L. monocytogenes. These data demonstrate that V14+ NKT cells are able to participate in the early response against enteric L. monocytogenes through amplification of IFN- production, but are not essential for, nor capable of, mediating memory responses required to sterilize the host.
Introduction
The primary control of infection by the intracellular pathogen Listeria monocytogenes relies on the ability of the host to mount an efficient Th1-like immune response (reviewed in Ref. 1). Production of IFN- in the early phases of infection is essential to enhance IL-12 production and activate bactericidal mechanisms in macrophages (2, 3). NK cells have been identified as a source of early IFN- production (4). Thus, SCID mice (T–, B–, NK+) are able to control primary L. monocytogenes infection in an IFN--dependent manner. Eventually SCID mice succumb to chronic listeriosis, demonstrating that NK cells alone are unable to fully protect the host against L. monocytogenes (5, 6). Instead, sterilizing immunity relies on the generation of cytotoxic CD8+ T cells which clear infected macrophages and hepatocytes and thereby eliminate the bacteria (reviewed in Refs. 1 and 7). The participation of other cell types has been described in the protection against L. monocytogenes. Several studies have defined a role for CD4+ T cells in both primary and secondary L. monocytogenes infection (8, 9). In addition, T cells play a role in the defense against L. monocytogenes, since they are able to control primary infections in the absence of TCR cells. However, TCR cells are not able to mediate sterilizing immunity after infection (10).
NKT cells constitute a heterogeneous subset of T cells expressing both NK and T cell surface markers. One well-characterized NKT subset includes a thymus-derived population expressing a canonical V14-J18 TCR -chain associated with a limited set of TCR subfamilies (reviewed in Ref. 11). These invariant V14+ NKT cells, which are either CD4+ or CD4–CD8– double negative, are selected on the nonclassical MHC class I molecule CD1. V14+ NKT cells recognize an endogenous lysosomal glycosphingolipid, isoglobotrihexosylceramide (12), and when activated through their TCR or by soluble factors (such as IL-12) can produce both IFN- and IL-4 (13, 14). Moreover, V14+ NKT cells have been shown to transactivate B, T, and NK cells in vivo (15, 16). Along these lines, V14+ NKT cells may act as sentinels to integrate initial signals following immune stimulation and thereby serve to orient subsequent immune responses.
V14+ NKT have been implicated in a number of immune-mediated pathologies including graft-vs-host disease, autoimmune hepatitis, and in fetal loss (17, 18, 19). In addition, a disease-controlling role for NKT cells has been shown in V14-J18 transgenic (Tg)5 nonobese diabetic mice (20). V14+ NKT cells may participate in antitumor responses by counteracting invasion and metastasis (reviewed in Ref. 21). Finally, a role for V14+ NKT cells has been proposed for protection against parasites (Toxoplasma gondii, Plasmodium yoelii, and Plasmodium berghei) and intracellular pathogens (mycobacteria and L. monocytogenes) (reviewed in Ref. 22). V14+ NKT cells could provide a protective role via IFN- in sustaining Th1 responses (23). Alternatively, IL-4 production from V14+ NKT cells could either have a deleterious role by deviating Th1 responses toward Th2 or act as an amplifier of Th2 responses in the context of extracellular parasites (24, 25). The precise role of V14+ NKT cells in infection immunity is clearly not defined and could vary depending on the pathogen.
Concerning L. monocytogenes, previous studies have demonstrated that NKT-deficient mice can resist infection by L. monocytogenes similar to wild-type mice (8, 26), excluding an essential role for these cells in antilisterial immunity. In contrast, Kaufmann and coworkers found that NKT cells are selectively depleted from the liver of L. monocytogenes-infected mice and that treatment of infected mice with CD1-specific Abs ameliorated the antilisterial response via increased IFN-, TNF-, and IL-12 production (27, 28). This group proposed that NKT cells could play a negative role in the immunity against intracellular bacteria, possibly through production of TGF- (28). Considering these contradictory findings, we decided to re-examine the role for V14+ NKT cells in the antilisterial response. Using several approaches in wild-type, Ja18°, and V14 transgenic mice, we demonstrate that invariant V14+ NKT cells clearly contribute to the pro-Th1 response following infection with L. monocytogenes but are not essential for or capable of mediating memory responses to this pathogen.
Materials and Methods
Mice
Rag° and Rag°c° mice (29) were from the 10th backcross to the C57BL/6 background. TCR° mice and V14-J18Tg on the TCR-deficient C57BL/6 background (TCR°V14Tg) mice (20) as well as J18° mice (30) have been previously described. C57BL/6 mice were purchased at IFFA-CREDO. Mice were housed at the Institut Pasteur (Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 668) and at Necker Hospital (INSERM Unité 411). All animal studies were evaluated and approved by a local institutional review board.
Abs and reagents
Abs were obtained from BD Pharmingen and were used as FITC, PE, biotin, and allophycocyanin conjugates. Biotinylated Abs were revealed with FITC-, PE (Caltag Laboratories) or PerCP-conjugated streptavidin (BD Pharmingen). Anti-CD19 microbeads and LS+ magnetic separation columns were obtained from Miltenyi Biotec. RPMI 1640, FCS, and antibiotics were purchased from Invitrogen Life Technologies. Percoll was purchased from Pharmacia. Brain-heart infusion medium (BHI) was obtained from Acumedia
Preparation of bacterial strains
Listeria monocytogenes (strain LO28) (31), was grown to exponential phase in BHI medium and harvested in the exponential growth phase, washed, and stored at –80°C in aliquots of 109 bacteria/ml in PBS.
Isolation of lymphoid cells
For isolation of lymphoid cells from peripheral lymphoid organs, mice were sacrificed and the mesenteric lymph node (mLN), spleen, and liver were removed. Single-cell suspensions were generated from mLN and spleen by teasing the organs through a metal mesh followed by erythrocyte lysis. Single-cell suspensions were generated from liver by teasing the organs through a metal mesh followed by centrifugation on a Percoll gradient (40/80%) and erythrocyte lysis.
Cell sorting and adoptive transfer into Rag°c° mice
For electronic cell sorting, single-cell suspensions were generated from the mLN of TCR°V14Tg mice. Following erythrocyte lysis, lymph nodes cells were depleted of B cells using MACS anti-CD19 microbeads and LS columns according to the manufacturer’s instructions. Subsequently, the cells were incubated with biotinylated anti-CD5 mAb, PE, anti-CD8 mAb and allophycocyanin anti-NK1.1 mAb as described below. Biotinylated Ab was revealed by incubation with FITC-streptavidin. NKT cells were sorted as NK1.1+/CD8–/CD5+ cells using a MoFlo cell sorter (DakoCytomation). Post-sort analysis confirmed that these cells were >98% NKT cells and contained <0.4% contaminating NK cells. Nonirradiated Rag°c° mice (3–6 wk of age) were transplanted i.v. with 5 x 105 purified NK1.1+ T cells 4 days before infection.
Infection and determination of CFU
For intragastric (i.g.) infection with 5 x 108 L. monocytogenes strain LO28, groups of mice were gavaged i.g. using an 18-gauge dumb-end feeding needle. For rechallenge experiments, mice were injected i.v. in the lateral tail vein with 2 x 106 bacteria.
At the indicated time points after infection, mice were sacrificed and the livers and spleens were aseptically removed. Homogenates of liver and spleen were prepared by grinding organs in sterile PBS with a motorized Teflon pestle. Bacterial CFU were enumerated by plating organ homogenates in 10-fold, serial dilutions on BHI agar plates. After incubation at 35°C for 36–48 h, the bacterial colonies were counted.
Flow cytometry
For surface Ab staining, cells were washed twice in PBS supplemented with 1% BSA (PBS-BSA), incubated on ice for 30 min with Abs, and subsequently washed twice in PBS-BSA before analysis. When appropriate, cells were incubated with biotin-conjugated Abs, washed three times, and then incubated for 30 min with the relevant streptavidin conjugate and then washed three times before analysis. Samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences) and the data were analyzed using CellQuest software (BD Biosciences).
For intracellular cytokine detection, total cell suspensions were incubated for 1 h in RPMI 1640/5% FCS containing brefeldin A (10 μg/ml) to block cytokine secretion. Surface-stained cells (TCR+, tetramer+) were fixed for 1 h in PBS containing 2% paraformaldehyde, and intracellular cytokines were detected using a PE-conjugated IFN- (XMG1.2) or control rat IgG1 (R3-34) mAbs in PBS containing 0.5% saponin.
Tetramer staining
Single-cell suspensions were stained for 20 min on ice with -galactosylceramide (-GC)-loaded allophycocyanin-conjugated CD1d tetramers (derived from mCD1d/2-microglobulin expression vector as described in Ref. 32). Cells were then washed twice with ice-cold PBS-BSA and subsequent Ab surface staining with FITC anti-TCR mAb and PE anti-NK1.1 mAb was performed as described above. Nonspecific binding was controlled by staining using CD1d tetramers without -GC (data not shown).
ELISA
Serum was obtained (days 0 and 3 postinfection) and the concentrations of IFN- were determined using a specific sandwich ELISA kit (Genzyme) according to the manufacturer’s instructions.
Statistics
Statistical significance was evaluated using the Mann-Whitney U test. Values of p < 0.05 were considered to be significant.
Results
V14+ NKT cells participate directly in the antilisterial response in vivo
We used several independent and complementary approaches to assess the role of V14+ NKT cells in antilisterial immunity. We first used CD1d tetramers loaded with the synthetic lipid -GC to follow V14+ NKT cell activation and cytokine production after i.g. L. monocytogenes infection of wild-type mice. Uninfected C57BL/6 mice harbored a population of CD1d/-GC-reactive T cells which, on a percentage basis, were more abundant in the liver (6 ± 1.2%) than in the spleen (0.5 ± 0.1%; Fig. 1A and data not shown). These cells were mainly NK1.1+ and did not constitutively synthesize IFN- (Fig. 1, B and C). As early as 24 h after i.g. infection with L. monocytogenes, invariant V14+ NKT cells became activated and began to produce IFN- (Fig. 1B), but not IL-4 (data not shown). It should be emphasized that the protocol used for ex vivo analysis of cytokine production by V14+ NKT cells did not involve a TCR restimulation in vitro. By day 2 after L. monocytogenes infection, about one-half of the CD1d tetramer-reactive T cells in the liver and spleen were active in IFN- production, and this fraction persisted at day 3 after infection (Fig. 1B and data not shown). Interestingly, the percentage of CD1d tetramer-reactive T cells decreased by days 2 and 3 after infection, which was correlated with a decreased density of NK1.1 expression (Fig. 1, A and C), although CD1d tetramer staining was still clearly observed. This "loss" of V14+ NKT cells likely corresponds to a partial down-modulation of TCR and NK1.1 expression rather than an actual disappearance of the cells. These results clearly demonstrate the participation of NKT cells in response to L. monocytogenes via IFN- production, a cytokine required for the control of this pathogen.
Discussion
Using a combination of approaches, including analysis with CD1d tetramers, V14+ NKT cell transgenic and knockout mice and selective reconstitution of alymphoid mice with highly purified V14+ NKT cells, we have reassessed the role of V14+ NKT cells in the immunity against enteric infection with the intracellular bacterium L. monocytogenes. Although previous reports suggested a negative impact of NKT cells on antilisterial immunity (27, 28), we found that V14+ NKT cells were stimulated to produce IFN- in vivo following enteric L. monocytogenes infection and were able to provide early protection of highly susceptible alymphoid mice against L. monocytogenes. In contrast, we demonstrated that V14+ NKT cells do not provide adaptive immunity to this pathogen under conditions of recall stimulation.
The capacity of -GC-loaded CD1d tetramers to unambiguously identify invariant V14+ T cells provided an essential tool for our studies. Previous reports have demonstrated the specificity of this reagent in wild-type mice and in transgenic mice bearing a functionally rearranged V14-J18 TCR chain that develops increased numbers of V14+ NKT cells (32, 34). These TCR°V14Tg mice provided us with the means to directly assess the functional capacity of V14+ NKT cells to provide early protection after L. monocytogenes infection. One caveat of our experiments is whether the NKT cells derived from TCR°V14Tg mice faithfully represent their counterparts from wild-type mice. Previous studies have shown that CD1d-reactive NK1.1+ T cells from TCR°V14Tg mice have a TCR repertoire and cell surface phenotype that closely matches NK1.1+ T cells from C57BL/6 mice (34). Moreover, NKT cells from TCR°V14Tg mice, like their normal counterparts, have the capacity to rapidly produce cytokines (IL-4, IFN-) following in vitro stimulation (20, 34). Thus, by several distinct criteria, the V14+ NKT cells from TCR°V14Tg mice appear to faithfully represent their normal C57BL/6 counterparts.
CD1d-reactive V14+ T cells from both C57BL/6 and TCR°V14Tg mice harbor a subset of NK1.1– cells. Previous studies from Benlagha et al. (14) have demonstrated that these cells in C57BL/6 mice likely represent precursors of the NK1.1+ cells. Using CD1d tetramers, these authors found that the NK1.1– subset of V14+ T cells bore an immature phenotype and selectively produced IL-4, but not IFN-, after stimulation. The presence of NK1.1–V14+ T cells in the spleen suggested that these precursors could exit the thymus and further differentiate into NK1.1+ IFN- secreting mature V14+ NKT cells in the periphery. Additional experiments showed that purified NK1.1– CD1d-reactive T cells could give rise after adoptive transfer to NK1.1+ progeny. The presence of two phenotypically and functionally distinct V14+ T cell subsets in the periphery of mice could allow for flexibility in the ways that immune responses could be oriented.
The ability of TCR° mice to control primary L. monocytogenes infection is consistent with the previously recognized capacity of TCR and NK cells to participate in innate immunity against this pathogen (3, 6, 10). No difference in the bacterial burden or early survival was observed among wild-type, TCR°, and TCR°V14Tg mice following enteric L. monocytogenes infection. This observation argues against any predominant regulatory role for V14+ NKT cells in the immunity against enteric L. monocytogenes, in contrast with previous studies (27, 28) that reported an amelioration of listeriosis in mice treated with anti-CD1 mAbs. These authors deduced that the blockade of CD1 interfered with the activation of NKT cells, resulting in decreased TGF- levels and increased IFN-, TNF-, and IL-12 production. Since TCR°V14Tg mice were as resistant as TCR° mice to primary infection, our results are incompatible with a dominant negative activity of V14+ NKT cells during L. monocytogenes infection. Still, NKT cells could impact on L. monocytogenes infections under conditions when NK and/or T cells are limiting.
We used adoptive transfer of V14+ NKT cells from TCR°V14Tg mice to assess the capacity of these cells to confer protection against L. monocytogenes when transplanted into alymphoid Rag°c° mice. We observed a beneficial effect of V14+ NKT cells in this setting, which correlated with IFN- (but not IL-4) production. It is interesting to consider our results in light of the observations that V14+ NKT cells can produce both IFN- and IL-4 following TCR stimulation in vitro. In contrast, V14+ NKT cells can preferentially produce either IL-4 or IFN- following stimulation with cytokines (37). The restricted biological activity of NKT cells after L. monocytogenes infection could indicate that these cells do not receive TCR stimulation via CD1d complexes in vivo in the setting. Recent studies by Brenner and colleagues (38) reported that Salmonella infection activated V14+ NKT cells in a TCR- and IL-12-dependent fashion. We also have preliminary evidence that MHC-deficient Rag°c° mice (which lack expression of CD1 molecules) reconstituted with NKT are able to resist early L. monocytogenes infection (T, Ranson and J. P. Di Santo, unpublished observations). These results would suggest that V14+ NKT cells are recruited to respond to certain types of intracellular infections dependent on the cytokine milieu; a pro-Th1 (IL-12)-rich environment would then favor V14+ NKT production of IFN-. Following L. monocytogenes infection, TCR°V14Tg mice displayed systemic IFN- levels comparable to those of wild-type mice and 3- to 4-fold higher levels than found in TCR° mice. Early IFN- production by V14+ NKT cells therefore represents a likely antilisterial mechanism in our experiments, although direct NKT cell-mediated killing of L. monocytogenes-infected macrophages cannot be ruled out (39).
In our transfer experiments, we found that NKT cells were able to substantially reduce the bacterial burden in the liver and spleen of the Rag°c° hosts (by almost 2 logs) after enteric L. monocytogenes infection. The level of protection afforded by the injected NKT cells is even more impressive considering the limited number of NKT cells transferred and the fact that homeostatic expansion of these cells only results in the generation of 105 NKT cells in the liver and spleen of the recipient hosts (36). In addition, the transplanted V14+ NKT cells might have undergone apoptosis following stimulation in vivo (40). Thus, despite being unable to completely eradicate the bacterial inoculum, NKT cells demonstrated potent antilisterial activity which resulted in protection of the reconstituted mice for at least 3 wk.
V14+ NKT cells have been shown to "cross-talk" with other lymphocytes, including NK, B, and T cells (15, 16). In particular, it has been shown that NKT-NK cell interactions may play an important role in tumor surveillance in vivo (reviewed in Ref. 21). Our results using adoptive transfer showed that NKT cells alone provide early protection after L. monocytogenes infection. Still, functional synergy between NKT and NK cells may allow for an even better protection after infectious challenge. The use of CD1d/-GC tetramers allowed us to directly demonstrate that V14+ NKT cells in C57BL/6 mice respond after L. monocytogenes infection by production of IFN-. Comparisons of C57BL/6 and J18° mice revealed a major difference in NK cell IFN- production after L. monocytogenes infection, consistent with V14+ NKT cell transactivation of NK cells in vivo.
The fact that TCR° and TCR°V14Tg mice did not mount functional memory responses to L. monocytogenes is consistent with previous reports demonstrating a pivotal role for cytotoxic CD8+ T cells in the generation of antilisterial memory responses (reviewed in Refs. 1 and 7). Our observations indicate that NKT cells do not play an essential role in recall responses to L. monocytogenes. Nevertheless, NKT cells could amplify memory responses via transactivation of previously established CD8 memory T cells. The capacity for NKT cells to rapidly produce IFN- and to potentiate its production by other lymphocytes (NK cells, T cells, CD8 memory T cells) after L. monocytogenes infection provides an important physiological example of the important role of NKT cells as a bridge between innate and adaptive immunity.
Acknowledgments
We thank Dr. D. Guy-Grand for helpful discussions. We are indebted to Pharmaceutical Research Laboratory, Kirin Brewery Company, for providing -GC and to P. Van Endert and M. Kronenberg for help in generating CD1d tetramers.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Institut Pasteur, INSERM, Association pour la Recherche sur le Cancer, and Ligue National Contre le Cancer. S.B. was the recipient of a postdoctoral fellowship from the Danish Research Agency.
2 T.R. and S.B. contributed equally to this work.
3 Current address: Symphogen, DK-2800 Lyngby, Denmark.
4 Address correspondence and reprint requests to Dr. James P. Di Santo, Unité des Cytokines et Développement Lympho?de, INSERM Unité 668, Institut Pasteur, 25 Rue du Docteur Roux, Cedex 15, Paris, France. E-mail address: disanto@pasteur.fr
5 Abbreviations used in this paper: Tg, transgenic; BHI, brain-heart infusion; c, common -chain; Tg, transgene; -GC, -galactosylceramide; mLN, mesenteric lymph node; i.g., intragastric.
Received for publication January 26, 2005. Accepted for publication April 21, 2005.
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