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Conventional T Cells Are Sufficient for Innate and Adaptive Immunity Against Enteric Listeria monocytogenes¹

Søren Bregenholt^2,*, Patrick Berche, Frank Brombacher and James P. Di Santo^3,*

^* Unité des Cytokines et Développement Lymphoïde, Institut Pasteur, Paris, France; Laboratoire de Microbiologie, Institut National de la Santé et de la Recherche Médicale, Unité 411, Faculté de Médecine Necker-Enfants Malades, Paris, France; and University of Cape Town, Cape Town, South Africa

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have begun to dissect the cellular requirements for generation of immunity against enteric infection by Listeria monocytogenes using a novel T^- B^- NK^- mouse strain (mice double deficient for the common cytokine receptor -chain (_c) and the recombinase-activating gene-2 (RAG2/_c mice). Initial experiments showed that C57BL/6 mice and alymphoid RAG2/_c mice had similar kinetics of bacterial accumulation in the spleen, liver, and brain early after intragastric L. monocytogenes infection (up to day 3), calling into question the physiologic role of gut-associated lymphoid cells during the passage of this enterobacterium into the host. However, in contrast to C57BL/6 mice, RAG2/_c mice rapidly succumbed to disseminated infection by day 7. Polyclonal lymph node CD4⁺ and CD8⁺ T cells were able to confer RAG2/_c mice with long-lasting protection against enteric L. monocytogenes infection in the absence of T, NK, and NK-T cells. Moreover, these T-reconstituted RAG2/_c mice produced IFN- at levels comparable to C57BL/6 mice in response to L. monocytogenes both in vitro and in vivo. Protection was IFN- dependent, as RAG2/_c mice reconstituted with IFN--deficient T cells were unable to control enteric L. monocytogenes infection. Furthermore, T cell-reconstituted RAG2/_c mice were able to mount memory responses when challenged with lethal doses of L. monocytogenes. These data suggest that NK, NK-T, T, and B cells are functionally redundant in the immunity against oral L. monocytogenes infection, and that in their absence T cells are able to mediate the early IFN- production required for both innate and adaptive immunity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Gram-positive bacteria Listeria monocytogenes is a human pathogen causing severe disease, including fever and bacterial encephalitis in immunocompromised individuals and abortions in pregnant women (1). The immune response against L. monocytogenes mainly depends on the ability of the host to mount a Th1-like immune response. Hence, L. monocytogenes-infected macrophages have been demonstrated to produce IL-12, thereby inducing IFN- production in NK cells and CD4⁺ T cells (2, 3). In turn, IFN- induces IL-12 production in macrophages (thus establishing a positive feedback loop) and enhances macrophage bactericidal mechanisms that help to limit L. monocytogenes replication within these cells (4, 5). A pivotal role for the IFN--IL-12 loop in protection against L. monocytogenes has been demonstrated by the increased susceptibility of mice deficient for IFN-, for the IFN- receptor, for components of IFN- signaling, and for IL-12 and in mice treated with neutralizing anti-IL-12 Abs to this pathogen (6, 7, 8, 9, 10, 11). However, control of the primary L. monocytogenes infection and generation of memory responses have been reported in both IFN-- and IL-12-deficient mice either infected with attenuated L. monocytogenes or following low dose inoculation (6, 10), suggesting that other pathways are involved. TNF- is also important in the control of L. monocytogenes, as demonstrated by the heightened susceptibility of TNF- and p55 TNF- receptor-deficient mice to this pathogen (12, 13, 14). Moreover, TNF- treatment partially restores the ability of IFN- receptor-deficient mice to control L. monocytogenes infection (15).

Previous studies have suggested a crucial role for NK cells in the early phase of L. monocytogenes infection (16). NK cells can rapidly produce IFN- and thus participate in the IFN--IL-12 loop (17). SCID (4) mice lacking mature T and B cells, but harboring NK cells, are able to control primary L. monocytogenes infection; however, T cells are pivotal for generating sterilizing immunity, since SCID mice eventually succumb to chronic listeriosis with bacterial accumulation in liver and spleen (18). Studies with MHC class I- and class II-deficient mice have revealed important roles for both CD8⁺ and CD4⁺ T cells in primary and secondary immune responses to L. monocytogenes (19), although the generation of Ag-specific CD8⁺ T cells appeared essential for the complete clearance of L. monocytogenes and for subsequent memory responses (18). These studies using gene-ablated mice have provided important information on the essential cellular subsets and cytokines required for the protection against L. monocytogenes. However, transfer studies aimed at identifying the cells and factors sufficient for providing immunity against L. monocytogenes are lacking.

Another important shortcoming of previous studies involving L. monocytogenes has been the use of i.v. infection protocols. However, L. monocytogenes is an enterobacterium that normally enters the body via invasion through the epithelial barrier of the small intestine (1, 20). Thus, infection via the i.v. route might bypass important regulatory mechanisms existing in the gut, such as unspecific barrier functions, epithelial cell-derived anti-bacterial cationic peptides, and cellular components residing in the small intestinal mucosa, including granulocytes, macrophages, and various lymphoid subsets. In addition, the role of these gut-resident lymphoid cells and other morphologically defined structures, such as Peyer’s patches (PP)⁴ and cryptopatches, in the defense against enteric L. monocytogenes remains to be carefully defined.

Here, we use a novel alymphoid mouse model, RAG2/_c mice, to address the cellular requirements for immunity against L. monocytogenes. This strain comprises mutations in the recombinase-activating gene-2 (RAG2) and the common cytokine receptor -chain (_c). As such, RAG2/_c mice are devoid of NK cells due to the _c mutation that blocks IL-15 responsiveness and are devoid of T and B cells, as the RAG2 mutation blocks Ag receptor development (reviewed in Ref. 21). Using alymphoid RAG2/_c mice as hosts for T cell transfer experiments, we demonstrate that NK, NK-T, and T cells are not required for the control of enteric L. monocytogenes infection and for the generation of protective memory responses. Furthermore, we show that the full complement of the gut-associated lymphoid system appears redundant for immunity against enteric L. monocytogenes.

	Materials and Methods

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Mice

RAG2/_c mice have been previously described (22) and were the 10th backcross to the C57BL/6 background. Mice were housed at the animal facilities at Institut National de la Santé et de la Recherche Médical, Unité 429, Hôpital Necker, and at Institut Pasteur (Paris, France). C57BL/6 mice were purchased from IFFA-CREDO (L’Arbresle, France). IFN--deficient C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

Preparation of bacterial strains

Bacteria, L. monocytogenes reference strain LO28 (23), were grown to the exponential phase in brain-heart infusion medium (Difco, Detroit, MI; Becton Dickinson, Mountain View, CA), harvested, washed, and stored at -80°C in aliquots of 10⁹ bacteria/ml in PBS. Bacteria were thawed immediately before their use for infection.

Isolation of lymphoid cells

For isolation of lymphoid cells from peripheral lymphoid organs, mice were killed, and the mesenteric lymph nodes (LN), spleen, liver, and intestines were removed. Single-cell suspensions were generated from mesenteric LN 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 an 80/40% Percoll gradient and subsequent erythrocyte lysis. Lamina propria lymphocytes were isolated as described previously (24).

Transfer of T cells or NK cells into RAG2/_c mice

The mesenteric, axial, and inguinal LN were aseptically recovered from C57BL/6 mice, and single-cell suspensions were prepared. In initial experiments, RAG2/_c mice were transplanted i.p. with 5 x 10⁶ total LN cells. The transferred population consisted of approximately 60% T cells, <1% T cells, 35% B cells, and 1% NK cells as analyzed by flow cytometry (data not shown). In separate experiments B cells were depleted by passing the LN cells preparation over T cell enrichment columns according to the manufacturer’s instructions (Cedarlane, Cambridge, MA). These populations were 96% T cells, 1% T cells, <1% B cells, and 1% NK cells. In separate experiments RAG2/_c mice were transplanted with LN cells isolated from IFN--deficient C57BL/6 mice. The composition of the transplant in these experiments was similar to that described above. NK cells were purified from the spleens of RAG2 mice using mini-MACS columns and anti-NK cell-specific beads (DX5, Miltenyi Biotec, Auburn, CA).

L. monocytogenes infection and CFU determination

For oral infection with 5 x 10 ⁸ L. monocytogenes strain LO28, groups of mice were gavaged intragastrically with an 18-gauge dumb-end feeding needle. For rechallenge experiments mice were injected i.v. via the tail vein with 10⁶ bacteria.

At the indicated time points after infection, mice were sacrificed, the liver and spleen were aseptically removed, and homogenates were prepared by grinding organs in sterile PBS with a motorized Teflon pestle. Bacteria CFUs were enumerated by plating organ homogenates in serial, 10-fold dilutions on tryptic soy agar (Acumedia, Baltimore, MD).

Flow cytometry

The following Abs were used as FITC, PE, Tricolor, or biotin conjugates: TCR, TCR, DX5, NK1.1, and CD19 (all from PharMingen, Mountain View, CA), and CD4 and CD8 (from Caltag, San Francisco, CA). FITC- and PE-conjugated streptavidin were obtained from Caltag.

For 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 mAb, washed three times, incubated for 30 min with the relevant streptavidin conjugate, and then washed three times before analysis. Samples were analyzed using a FACScan flow cytometer (Becton Dickinson); data were analyzed using CellQuest software (Becton Dickinson).

In vitro culture and ELISA

Spleens were aseptically removed, single-cell suspensions were generated, and erythrocytes were lysed in NH₄Cl. Splenocytes (10⁶ cells/ml) were cultured in RPMI 1640 supplemented with 10% FCS, glutamine, and antibiotics in the presence or the absence of 10⁷ bacterial equivalents of heat-killed L. monocytogenes (HKLM). Following 48 h of incubation, the supernatants were harvested for ELISA analysis.

The concentrations of IFN- in serum and culture supernatants were determined by a commercial cytokine-specific sandwich ELISA kit according to the manufacturer’s instructions (Geneset, Cambridge, MA).

Statistics

Statistical significance was evaluated using the Mann-Whitney t test. p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alymphoid RAG2/_c mice are susceptible to enteric L. monocytogenes infection

We have recently developed a novel alymphoid mouse model, RAG2/_c mice, which comprises mutations in RAG2 and _c. RAG2/_c mice are thereby genetically deficient in B and T cells (due to the RAG2 mutation, which blocks Ag receptor development) and in NK cells (due to the _c mutation, which blocks IL-15R signaling (reviewed in Ref. 21). Moreover, RAG2/_c mice lack macroscopically and microscopically discernable Peyer’s patch structures (25) (data not shown) and cryptopatches (26) and are therefore completely deficient in gut-associated lymphoid structures and cells. A number of enteric pathogens, including Listeria, Shigella, and Salmonella, cross the intestinal barrier to infect the host. The mechanisms by which these microorganisms gain entry may include passage through specialized epithelial M cells overlying the PP of the small intestine or via other nonspecific pathways (20, 27). We therefore investigated whether RAG2/_c mice were susceptible to enteric L. monocytogenes infection to assess the role of gut-associated lymphoid cells in this process. Wild-type (wt) C57BL/6 and RAG2/_c mice were infected with 5 x 10⁸ L. monocytogenes strain LO28 via intragastric gavage, and bacterial CFUs in liver and spleen were determined at various time points after infection. Fig. 1 shows that the kinetics of bacterial accumulation in the liver of wt and RAG2/_c mice in the first 3 days after oral L. monocytogenes infection were remarkably similar. The kinetics of bacterial accumulation in the spleen and brain of these mice were also comparable (data not shown). However, while wt mice had cleared the bacteria by day 7 after infection, RAG2/_c mice did not control the infection and started to succumb to disseminated listeriosis at this time point (Fig. 1). These data highlight the dispensable role for gut-associated lymphoid structures (PP, cryptopatches) and lymphoid cells (intraepithelial lymphocytes) in the early response to L. monocytogenes, in contrast to the essential role of peripheral lymphocytes in providing protection against this pathogen.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Kinetics of enteric L. monocytogenes infection in C57BL/6 and RAG2/c mice. C57BL/6 (•) and RAG2/c () mice were gavaged with 5 x 108 L. monocytogenes strain LO28, and the CFU in liver were determined at the indicated time points after infection. Data represent the mean ± SD from groups of three mice. Similar results were seen in one additional experiment.

Selective lymphoid reconstitution of RAG2/_c mice

To dissect the cellular requirements for generation of immunity to enteric L. monocytogenes, we employed an experimental system in which RAG2/_c mice were reconstituted with 5 x 10⁶ LN cells from immunocompetent congenic C57BL/6 mice. Three weeks after injection, CD8⁺ and CD4⁺ TCR⁺ T cells and B cells were identified in the liver and spleen of the hosts (Fig. 2A and Table I). In contrast, TCR⁺ T cells, NK cells, or NK-T cells could not be found in these organs of transplanted RAG2/_c mice (Fig. 2B and Table I), despite the small contamination with T cells and NK cells in the initial transplanted cell preparations. The mesenteric LN, the lamina propria, and the intraepithelial compartment of the gut were also selectively repopulated with CD8⁺ and CD4⁺ TCR⁺ T cells in these mice (Table I and data not shown), which we will refer to as T reconstituted RAG2/_c mice.

View larger version (37K): [in this window] [in a new window]   FIGURE 2. T reconstitution (recon.) of peripheral lymphoid organs in RAG2/c mice after LN cells transplantation. TCR, NK1.1, CD4, and CD8 expression in the spleen (A) and liver (B) of wt and T-reconstituted RAG2/c mice 4 wk after transplantation. Representative staining from one of six experiments is shown.

View this table: [in this window] [in a new window]   Table I. Lymphoid reconstitution of RAG2/c mice by peripheral T cells1

T reconstituted RAG2/_c mice control enteric L. monocytogenes infection

Next, C57BL/6 mice, RAG2/_c mice, and T-reconstituted RAG2/_c mice were gavaged with 5 x 10⁸ L. monocytogenes, and liver and spleen CFUs were measured 7 days after infection. At this time point, wt mice were able to control L. monocytogenes infection, while RAG2/_c mice did not and showed bacterial accumulation in the liver and spleen (Fig. 3). In contrast, T-reconstituted RAG2/_c mice were able to control bacterial infection similar to wt mice (Fig. 3). These data argue that T cells might be sufficient to control enteric L. monocytogenes infection in the absence of T cells, NK-T cells, and NK cells. An equivalent response was seen using RAG2/_c mice reconstituted with whole LN cells or B cell-depleted LN cells (data not shown), underscoring the redundant role for B cells in the innate immune response to L. monocytogenes (18).

View larger version (18K): [in this window] [in a new window]   FIGURE 3. T-reconstituted RAG2/c mice control enteric L. monocytogenes infection. C57BL/6 (), RAG2/c (), T-reconstituted RAG2/c (), and IFN--deficient T-reconstituted RAG2/c mice () were gavaged with 5 x 108 L. monocytogenes strain LO28, and the CFU in liver and spleen were determined 7 days after infection. Data represent the mean ± SD of groups (comprising 8–16 mice) from three different experiments.

Role for IFN- in responses of T-reconstituted RAG2/_c mice to L. monocytogenes

Since IFN- production appears crucial for immunity to L. monocytogenes, we analyzed whether T-reconstituted RAG2/_c mice generated a functional systemic IFN- response upon infection. These studies showed that wt and T-reconstituted RAG2/_c mice had comparable levels of circulating IFN- levels on day 3 after infection, whereas unmanipulated RAG2/_c mice were unable to mount an IFN- response (Table II). In addition, the kinetics of the IFN- response were similar in wt and T-reconstituted RAG2/_c mice, reaching a peak on day 3 postinfection (data not show). Importantly, systemic IFN- was not detected in T-reconstituted RAG2/_c mice before infection (Table II). To determine the source of IFN-, we cultured splenocytes from the three groups of mice in the presence or the absence of HKLM. Table II shows that splenocytes from both wt and T-reconstituted RAG2/_c mice produced high amounts of IFN- in response to HKLM. In contrast, IFN- could not be detected in the supernatants from RAG2/_c splenocytes, in agreement with the absence of systemic IFN- production in these mice (Table II).

View this table: [in this window] [in a new window]   Table II. IFN- production in L. monocytogenes-infected mice

Generation of memory responses in T-reconstituted RAG2/_c mice

In subsequent experiments we analyzed adaptive immunity and the generation of specific memory responses to L. monocytogenes in T-reconstituted RAG2/_c mice. To assess whether mice were able to provide long term, sterilizing immunity after L. monocytogenes infection, survival was followed for a 4-wk period after oral L. monocytogenes infection. Fig. 4 shows that nonmanipulated RAG2/_c mice (6 of 13) became moribund starting on day 6 after infection, and all had died (13 of 13) by day 13, whereas wt mice (10 of 10) survived for the entire observation period. In contrast to RAG2/_c mice, approximately 90% (15 of 17) of the T-reconstituted RAG2/_c mice were alive 4 wk after infection, strongly suggesting that these mice had cleared L. monocytogenes through adaptive immunity. Since bacterial clearance is dependent on the generation of specific T cell immunity (18), we tested whether the primed T-reconstituted RAG2/_c mice were able to control a lethal innoculum of L. monocytogenes. To this end, four groups of mice were infected with a lethal i.v. L. monocytogenes innoculum (10⁶ bacteria); these included wt or T-reconstituted RAG2/_c mice that had never received L. monocytogenes (naive) and wt or T-reconstituted RAG2/_c mice that had been infected with L. monocytogenes 4 wk earlier (immune). The bacterial burden in the liver and spleen was assessed 2 days after infection. Naive wt and T-reconstituted RAG2/_c mice were unable to control a lethal infection and displayed bacterial dissemination in the liver and spleen (Fig. 5). In contrast, immune wt and T-reconstituted RAG2/_c mice demonstrated a 3.5–4 log reduction in the bacterial burden in the liver and spleen at this time point (Fig. 5), demonstrating efficient generation of memory responses.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. T-reconstituted RAG2/c mice do not succumb to chronic listeriosis. C57BL/6 (), RAG2/c (), and repopulated RAG2/c () mice were infected orally with 5 x 108 L. monocytogenes strain LO28, and their survival was monitored for 30 days after infection. Pooled data from two identical experiments with a total of 8–16 mice/group are shown.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. T-reconstituted RAG2/c mice develop functional memory responses to L. monocytogenes after enteric infection. C57BL/6 () and T-reconstituted RAG2/c () were gavaged with 5 x 108 L. monocytogenes strain LO28. Four weeks later, these immune mice as well as naive C57BL/6 and T-reconstituted RAG2/c mice were challenged with 106 L. monocytogenes strain LO28 i.v. Three days after rechallenge, CFU in the liver and spleen were determined. The relative protection was calculated as (CFU of naive mice - CFU of immune mice). Pooled data from two identical experiments, representing the mean ± SD of groups of eight mice, are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A number of enteric pathogens, such as Salmonella, Yersinia, and Shigella, may enter the body through the specialized M cells located in the follicle-associated epithelium overlaying the PP of the small intestine (20). While L. monocytogenes may use this particular route of entry, transepithelial migration of L. monocytogenes was recently demonstrated in the area of the small bowel devoid of macroscopically discernable PP (27). Cryptopatches are small, discrete, microscopic lymphoid accumulations (numbering 200) that have been identified throughout the length of the small bowel (26). Ingested carbon particles localize to structures reminiscent of cryptopatches (28), which argues that these structures might also collect intraluminal microorganisms, and thereby serve functions similar to PP. Thus, L. monocytogenes could enter through these non-PP structures or nonspecifically through breaks in the epithelium. Our observations in RAG2/_c mice identify a nonspecific route of entry for L. monocytogenes, as these mice lack both cryptopatches and PP (25, 29) yet are susceptible to enteric infection. The mechanisms responsible for uptake of L. monocytogenes into epithelial cells are unknown, but could involve invasion of these cells via the internalin/E-cadherin receptor pair (30, 31).

The earliest innate resistance to L. monocytogenes is believed mainly to be mediated by unspecific professional phagocytes, such as polymorphonuclear granulocytes and monocytic macrophages, without the contribution of lymphoid cells (18). Insights into the role played by gut lymphocytes in the earliest defense against L. monocytogenes are provided by infection studies in alymphoid RAG2/_c and normal mice. Hence, identical bacterial accumulation in target organs was found during the first 3 days after enteric infection, arguing that the full complement of gut-associated lymphoid cells (intraepithelial T cells and lamina propria T, B, and NK cells) is redundant in the early immune response against this enterobacterium.

Using the alymphoid RAG2/_c mouse strain, we have begun to decipher the minimal cellular components required for systemic immunity against L. monocytogenes. We find that mice selectively repopulated with T cells can provide both innate and adaptive immunity following oral infection with L. monocytogenes. While one might argue that contaminating NK cells in the transplant are responsible for the anti-L. monocytogenes immunity reported here, this seems unlikely, since NK cells were not found in T-reconstituted RAG2/_c mice at the time of oral infection. In addition, RAG2/_c mice transplanted with purified NK cells showed no early protection after L. monocytogenes infection and had levels of bacterial accumulation similar to that seen in unmanipulated RAG2/_c mice after infection. We conclude that anti-L. monocytogenes immunity can be generated in the absence of T cells, NK-T cells, and NK cells.

The observation that RAG2/_c mice selectively repopulated with T cells can control primary L. monocytogenes infection as efficiently as wild-type mice is contradictory to the view that NK cells play a unique role in the innate defense by providing a rapid source of IFN- (16, 32). A recent publication has suggested that dendritic cells may also produce IFN- (33). Concerning our results, it should be noted that RAG2/_c mice can develop dendritic cells (34), although these mice are not competent to produce IFN- following infection, thereby arguing against any role for dendritic cell-derived IFN- during early listeriosis. Our observations that wt and T-reconstituted RAG2/_c mice had similar levels and kinetics of IFN- production in response to enteric L. monocytogenes argues that T cells are able to substitute for NK cells as an early source of IFN- to up-regulate macrophage bactericidal mechanisms. This view is supported by the inability of RAG2/_c mice reconstituted with IFN--deficient T cells to control enteric L. monocytogenes infection and is consistent with previous reports showing an essential role of IFN- in the control of early L. monocytogenes infection (5, 6, 7, 35). Thus, in the absence of NK cells, T cells might acquire innate functions by providing rapid IFN- production in the early phases of L. monocytogenes infection.

Despite their ability to control primary infection in SCID and RAG mice, NK cells cannot mediate sterilizing immunity. Memory responses to L. monocytogenes rely on CD8⁺ T cells, although the contribution of CD4⁺ T cells and T cells in memory responses to L. monocytogenes has also been demonstrated (19, 36). Here we show that T cells alone are sufficient to mount memory responses similar (although somewhat less efficiently) to those seen in wt mice (Fig. 5). It might be speculated that this small difference reflects the absence of T cells in the repopulated RAG2/_c mice. Although memory responses can be generated in the absence of IFN- or IL-12 by immunization with attenuated bacteria or low doses of wt bacteria (6, 10), IFN- has been shown to be crucial for the immunization with higher doses of wt bacteria (5, 6, 7). Our data point toward T cells as a potential physiological source of IFN- in the absence of NK cells, thus providing the basis for the generation of T cell-mediated memory responses to L. monocytogenes.

We recently reported that _c single-deficient (_c^-) mice lacking NK cells were resistant to early infection by L. monocytogenes and that the protection was mediated by the few circulating _c^- T cells found in these mice (37). However, due to aberrant thymic and peripheral selection, the T cells found in _c^- mice have an activated phenotype and are refractory to restimulation in vitro (38). This default T cell activation pattern resulted in elevated levels of systemic IFN- even in noninfected _c^- mice, which probably provided an explanation for their resistance to L. monocytogenes infection. In the present study a fraction of the CD4⁺ T cells in T-reconstituted RAG2/_c mice appeared partially activated (as evidenced by low levels of CD69 expression; data not shown), but, importantly, systemic IFN- could not be detected in these mice before infection. Thus, constitutive T cell-mediated IFN- production could not be responsible for protection in T-reconstituted RAG2/_c mice. Taken together, our observations suggest that T cells are sufficient to provide both innate and adaptive immunity against enteric L. monocytogenes infection in the absence of T cells, NK-T cells, and NK cells. The ability of T cells to provide innate functions may be an important physiological mediator in the early defense against enterobacterial infections.

	Acknowledgments

 
We thank Drs. D. Guy-Grand, O. Gaillot, and F. Colluci for fruitful discussions.

	Footnotes

 
¹ This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Necker-Enfants Malades (Paris V), Association pour la Recherche sur le Cancer, Ligue Nationale Contre le Cancer, Le Cooperation France/Sud Afrique, and a postdoctoral fellowship from the Danish Research Agency (to S.B.).

² Current address: Islet Discovery Research, Novo Nordisk A/S, Novo Alle, DK-2880 Bagsværd, Denmark.

³ Address correspondence and reprint requests to Dr. James P. Di Santo, Unité des Cytokines et Développement Lymphoïde, Institut Pasteur, 25 rue du Docteur Roux, Cedex 15 Paris, France.

⁴ Abbreviations used in this paper: PP, Peyer’s patch; HKLM, heat-killed Listeria monocytogenes; LN, lymph node; RAG2, recombinase-activating gene-2; wt, wild-type; _c, common cytokine receptor -chain.

Received for publication May 4, 2000. Accepted for publication November 1, 2000.

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