The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huleatt, J. W.
Right arrow Articles by Pamer, E. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huleatt, J. W.
Right arrow Articles by Pamer, E. G.
The Journal of Immunology, 2001, 166: 4065-4073.
Copyright © 2001 by The American Association of Immunologists

Intestinal and Splenic T Cell Responses to Enteric Listeria monocytogenes Infection: Distinct Repertoires of Responding CD8 T Lymphocytes1

James W. Huleatt2, Ingrid Pilip3, Kristen Kerksiek and Eric G. Pamer3

Sections of Infectious Diseases and Immunobiology, Yale School of Medicine, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Listeria monocytogenes is an intracellular bacterium that causes systemic infections after traversing the intestinal mucosa. Clearance of infection and long term protective immunity are mediated by L. monocytogenes-specific CD8 T lymphocytes. In this report, we characterize the murine CD8 T cell response in the lamina propria and intestinal epithelium after enteric L. monocytogenes infection. We find that the frequency of MHC class Ia-restricted, L. monocytogenes-specific T cells is ~4- to 5-fold greater in the lamina propria than in the spleen of mice after oral or i.v. infection. Although the kinetics of T cell expansion and contraction are similar in spleen, lamina propria, and intestinal epithelium, high frequencies of Ag-specific T cells are detected only in the lamina propria 1 mo after infection. In contrast to MHC class Ia-restricted T cells, the frequency of H2-M3-restricted, L. monocytogenes-specific T cells is decreased in the intestinal mucosa relative to that found in the spleen. In addition to this disparity, we find that MHC class Ia-restricted CD8 T cells specific for a dominant L. monocytogenes epitope have different TCR V{beta} repertoires in the spleen and intestinal mucosa of individual mice. These findings indicate that the intestinal mucosa is a depot where L. monocytogenes-specific effector CD8 T cells accumulate during and after infection irrespective of immunization route. Furthermore, our results demonstrate that CD8 T cell populations in these two sites, although overlapping in Ag specificity, are distinct in terms of their repertoire.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Listeria monocytogenes is a Gram-positive bacterium that causes severe infection of immunocompromised patients and pregnant women (1). The most common clinical syndromes caused by L. monocytogenes infection are sepsis, meningitis and, during pregnancy, chorioamnionitis (1). Natural infection with L. monocytogenes results from ingestion of contaminated food, and listeriosis outbreaks have been attributed to contaminated salads, soft cheeses, pate, chocolate milk, and corn (2, 3, 4, 5). After the ingestion of a large bacterial inoculum, L. monocytogenes can cause enteric infection, manifested by gastroenteritis with diarrhea and abdominal pain (4, 5).

L. monocytogenes is virulent because it can survive within macrophages by escaping the phagosomal vacuole, a process that is strictly dependent on bacterial secretion of listeriolysin O (LLO),4 the major virulence factor (6, 7, 8). On entry into the host cell cytosol, L. monocytogenes further disseminates by polymerizing actin at one pole, a process that enables bacterial infection of neighboring cells (9, 10, 11). L. monocytogenes can also directly invade nonphagocytic epithelial cells by expressing two surface proteins, internalin A and internalin B, which bind to E-cadherin expressed on the surface of mammalian cells (12). It is believed that internalin-mediated invasion is the major pathway for invasion of intestinal epithelial cells. Although early studies of enteric infection with L. monocytogenes demonstrated infection of intestinal epithelial cells (13), it is unclear whether murine or human infection involves epithelial cell invasion or uptake by intestinal M cells.

The murine immune response to systemic L. monocytogenes infection involves early recruitment of neutrophils (14), NK cells (15), and {gamma}{delta} T cells (16) to the liver and spleen and is followed by the activation and expansion of Ag-specific CD4 and CD8 T lymphocytes. Clearance of L. monocytogenes after primary infection and long term protective immunity are mediated by T lymphocytes, with CD8 T cells providing the bulk of the defense (17). CD8 T cells responding to L. monocytogenes infection can be divided into CTL that recognize peptides derived from bacterially secreted proteins in the context of the H2-Kd MHC class Ia molecule, and CTL that recognize bacterial formylmethionine peptides in the context of the H2-M3 MHC class Ib molecule (18). Although phenotypically similar, these two CD8 T cell populations have distinct kinetics of expansion and memory formation in the spleen after primary infection with L. monocytogenes (19). Although H2-M3-restricted T cells undergo more rapid in vivo expansion after primary L. monocytogenes infection than H2-Kd-restricted T cells, memory responses to H2-M3-restricted peptides are dwarfed by the massive H2-Kd-restricted memory response (19). Most studies of T cell responses to L. monocytogenes infection have been performed in animals infected by the i.v. or i.p. route and have focused on the immune responses in the spleen. The mucosal T cell response to enteric infection with L. monocytogenes is largely undefined (20).

Recent studies have demonstrated that systemic infection with vesicular stomatitis virus (VSV) elicits strong Ag-specific CD8 T cell responses in the lamina propria (LP) and intraepithelial lymphocyte (IEL) compartments of the small intestine (21, 22). Interestingly, the frequency of Ag-specific CD8 T cell responses is greater in the intestine than in the spleen and persists longer (23). In this report, we investigate the MHC class Ia and MHC class Ib-restricted T cell response to enteric infection of mice with L. monocytogenes. Although the immunodominance hierarchies of MHC class Ia-restricted T cells in the spleen and intestine are similar, we find that the TCR V{beta} repertoire of Ag-specific CD8{alpha}{beta}, TCR{alpha}{beta} T cells differs substantially among LP, IEL, and splenic T cells. Additionally, we find that H2-M3-restricted T cells are underrepresented in the LP and IEL compartments relative to splenic T cell populations derived from the same animal. The differing effector T cell repertoires responding to L. monocytogenes infection in the spleen demonstrate that Ag-specific T cells populating these two compartments are distinct.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, bacteria, and infection

Female 8- to 10-wk-old C57BL/6 x BALB/c F1 (CB6F1J) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were infected i.v. with 104 (via lateral tail vein injection) or p.o. with 109 (via gavage) CFU of the streptomycin-resistant L. monocytogenes strain 10403s (originally obtained from Daniel Portnoy, University of California, Berkeley, CA). Viable bacterial counts within spleen, liver, and mesenteric lymph nodes were determined by homogenizing the tissue in PBS containing 0.05% Triton X-100 and plating on brain-heart infusion (BHI) agar plates (Life Technologies, Gaithersburg, MD). The number of intestinal L. monocytogenes organisms was determined by sequential dissociation of intestinal tissues and plating on BHI plates containing streptomycin (100 µg/ml) and nalidixic acid (50 µg/ml) to inhibit the growth of endogenous bacterial flora. L. monocytogenes colonies were identified by their characteristic morphology and by Gram staining.

Isolation of T cell populations

Splenic and mesenteric lymph nodes were removed, and single-cell suspensions were prepared with a glass tissue homogenizer. Spleen cells were then treated with Tris-buffered ammonium chloride to lyse RBC. The resulting preparations were then filtered through Nytex, and the filtrate was centrifuged to pellet the cells.

IEL were isolated as previously described (24). Briefly, the small intestines of individual mice were cut into 5-mm pieces and washed twice in HEPES-buffered HBSS medium after the excision of Peyer’s patches. Intestinal pieces were stirred at 37°C for 20 min in HBSS containing 1 mM dithioerythritol. This step was repeated, the resulting supernatants were rapidly filtered through nylon wool, and the filtrate was centrifuged through a 44%/67.5% Percoll gradient. The cells at the interface of the Percoll gradient were collected and prepared for flow cytometry.

LP lymphocytes were isolated by a modified version of the protocols published by Poussier et al. (25) and Kramer et al. (26). After IEL isolation, residual epithelial cells were removed by constant shaking in 1.3 mM EDTA in Ca2+, Mg2+-free HBSS at 37°C for 30 min. This was repeated, and both supernatants were discarded. The intestinal tissue was then stirred in RPMI 1640 (Life Technologies) containing 5% FCS at 22°C for 20 min until clear, and any released cells were discarded. LP lymphocytes were then isolated after double digestion in RPMI supplemented with 100 U/ml collagenase (Life Technologies), 1 mM CaCl2, 1 mM MgCl2, and 5% FCS at 37°C for 30 min. Released cells were then washed in PBS containing 5% FCS and subjected to Percoll fractionation as described above for isolation of IEL.

Generation of MHC class I tetramer-peptide complexes

Tetrameric H2-Kd/LLO91–99 and p60217–225 and H2-M3/fMIGWII complexes were generated as previously described (19, 27). In brief, refolded and biotinylated MHC-peptide complexes were multimerized with the addition of PE-conjugated streptavidin (Molecular Probes, Eugene, OR). Tetrameric complexes were purified by gel filtration over a Superdex 200 HR column to eliminate the substantial proportion of lower multimeric forms after incubation with PE-conjugated streptavidin. Purified tetramers were stored at 2–5 mg/ml at 4°C in PBS (pH 8.0) containing 0.02% sodium azide, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 0.5 mM EDTA. The reagents were frequently tested on Ag-specific T cell lines to document staining intensity.

Flow cytometric analysis

Lymphocytes were resuspended in staining buffer (PBS, 0.2% BSA, 0.1% NaN3) at a concentration of 1 x 106–1 x 107 cells/ml and incubated at 4°C with Fc-Block (PharMingen, San Diego, CA) for 30 min. Samples were then stained with the addition of properly diluted mAb and/or tetramer MHC/peptide complexes for 45–60 min on ice. Subsequently, cells were washed three times in PBS, 0.2% BSA, 0.1% NaN3 and then fixed in 1% paraformaldehyde-PBS (pH 7.45). Flow cytometry was performed using a FACScalibur, and data were further analyzed with CellQuest software (Becton Dickinson, Mountain View, CA). The following mAbs were used (all obtained from PharMingen): PerCP- and FITC-conjugated anti-CD8{alpha} (clone 53-6.7), FITC-conjugated anti-CD8{beta} (clone 53-5.8), FITC-conjugated anti-TCR{alpha}{beta} (clone H57-597), FITC-conjugated anti-TCR{gamma}{delta} (clone GL3), and FITC-conjugated anti-TCR V{beta} segments (TCR V{beta}2, 4, 6, 7, 8.1/.2, 8.1-3, and 9). Statistical significance was evaluated by a two-tailed Student t test.

CTL assays

Standard chromium release assays using 51Cr-labeled P815 (H2-Kd) target cells were performed as previously described (28). After the preparation of single-cell suspension, CD8+ T cells from the spleen, LP and intestinal epithelial fractions were enriched by panning on plastic culture plates coated with the anti-CD8{alpha} mAb 3.168. This resulted in a 2- to 3-fold enrichment of CD8+ T cells. T cell populations were then incubated at 37°C for 6 h in the presence of 2.5 x 103 51Cr-labeled P815 target cells in complete RPMI containing 10-6 M targeting peptide. The percentage of specific lysis was calculated as: 100 x [(cpm released with effectors) - (cpm released spontaneously)]/[(cpm released by detergent) - (cpm released spontaneously)]. Spontaneous release in all experiments was <5%. To compare cytolytic activity in distinct T cell populations, E:T is based on the number of TCR{alpha}{beta} CD8+ T cells added per well.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bacterial growth and clearance after i.v. and p.o. infection with L. monocytogenes

The kinetics and magnitude of in vivo immune responses to numerous viral infections have been studied extensively (29, 30, 31). Although most of these studies focused on T cell responses in secondary lymphoid organs after systemic infection, a smaller number of studies characterized virus-specific T cells in nonlymphoid organs. Studies of lung after respiratory infection with influenza virus or of intestine after systemic VSV infection demonstrated strong Ag-specific T cell responses that differ in magnitude and kinetics from responses in lymph nodes and spleen (21, 23, 30). Because Ag-specific T cell responses after intestinal bacterial infection have not been previously characterized, we set forth to examine intestine immune responses to infection with L. monocytogenes.

Previous studies examining p.o. infection with L. monocytogenes demonstrated that 1–5 x 109 CFU were necessary to elicit immunity (32). As a first step toward investigating Ag-specific T cell responses to intestinal infection with L. monocytogenes, we quantified the number of viable bacteria in the intestinal lumen, intestinal mucosa, spleen and liver after i.v. or p.o. infection with L. monocytogenes. Within the spleen and liver, p.o. infection with 109 bacteria produced an infection that was similar to i.v. infection with 104 bacteria (Fig. 1Go, A and B). Mice infected i.v. and p.o. had increasing numbers of bacteria in the spleen, liver, and mesenteric lymph node, with peak bacterial infection occurring 3 days after inoculation. Viable bacteria could not be detected in any of these sites beyond the 9th day after infection. Mesenteric lymph nodes of animals infected p.o. had higher bacterial counts (10- to 1000-fold) than those infected i.v. on days 2 through 5.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 1. Quantification of viable bacteria in spleen, liver, and mesenteric lymph nodes after i.v. and p.o. infection with L. monocytogenes. The bacterial numbers within the spleen (top), liver (middle) and mesenteric lymph nodes (bottom) from were determined on sequential days after i.v. and p.o. infection with 104 and 109 CFU of L. monocytogenes (strain 10403S), respectively. Listeria colonies were then enumerated after serial dilution of samples in 0.1% Triton X-100 and plating on BHI agar plates. L. monocytogenes colonies were verified by Gram staining. Data shown are the mean and SD of three animals per interval and are representative of results from three independent experiments.

 
The ability to detect and monitor specific pathogens within the compartments of the intestine is compromised by the presence of resident microbial flora. To perform quantitative measurements of L. monocytogenes infection within the small intestine, we exploited the streptomycin and nalidixic acid resistance of L. monocytogenes strain 10403S. The number of L. monocytogenes in the lumen of the small intestine, as determined by growth on BHI agar plates containing streptomycin and nalidixic acid, was maximal 3–4 days after p.o. infection and then decreased until bacteria were cleared from the lumen on the 9th day (Fig. 2Go). The peak number of bacteria in intestine-associated tissues were detected 3 days after p.o. infection, with bacterial clearance by the 7th day (Fig. 2Go). Of note, i.v. infection with L. monocytogenes did not result in detectable infection of the intestine or intestinal lumen (Fig. 2Go). To determine whether bacteria were present in the epithelial layer of the small intestine or the LP, we cultured these two compartments separately and found that viable L. monocytogenes were undetectable in the epithelial layer at any time after p.o. infection. In contrast to the epithelial layer, the LP contained viable bacteria for 5 days after inoculation (results not shown). These findings demonstrate that p.o. infection with L. monocytogenes results in intestinal infection that is most severe during the first 3 days after inoculation. Intestinal infection with L. monocytogenes is followed by systemic spread to the liver and spleen, with peak bacterial infection in these sites also occurring on the 3rd day after inoculation (Fig. 1Go). Immune clearance of systemic infection is similar after p.o. or i.v. infection.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 2. Quantification of L. monocytogenes colonization within the intestinal mucosa after i.v. and p.o. infection. The number of L. monocytogenes was examined in the lumen and intestinal epithelium + LP on sequential days after i.v. or p.o. infection with L. monocytogenes, as described in Fig. 1Go. Lumen contents and intestinal tissues were plated on BHI agar plates containing streptomycin and nalidixic acid after serial dilution, as described in Materials and Methods. Data shown are the mean and SD of three animals per interval and are representative of results from three independent experiments.

 
Characterization of CD8 T cell responses after i.v. and p.o. immunization with L. monocytogenes

CD8 T cells play a major role in the defense against L. monocytogenes infection and are also well represented in the subcompartments of the murine small intestine. To determine the effect of L. monocytogenes on CD8 T cell dynamics in the intestine, we infected mice p.o. and i.v. and isolated lymphocytes from spleen, intestinal epithelium, and LP. Analysis of splenocytes 9 days after p.o. or i.v. infection did not reveal any detectable alterations in the frequency of TCR{alpha}{beta}- or CD8{alpha}{beta}-expressing T cells when compared with uninfected control animals (Fig. 3Go, top). In contrast, the proportion of TCR{alpha}{beta}-expressing T cells among IEL increased after both p.o. (37.2 ± 3.1%) (p = 0.05) and i.v. (34.2 ± 3.9%) (p = 0.03) infection when compared with uninfected control IEL (28.2 ± 4.3%). The increase in TCR{alpha}{beta} IELs after p.o. infection was almost entirely attributable to an increase in CD8{alpha}{beta} T cells, whereas i.v. infection with L. monocytogenes resulted in the expansion of both CD8{alpha}{alpha} and CD8{alpha}{beta} T cells (Fig. 3Go, middle). The expansion of TCR{alpha}{beta} T cells induced by L. monocytogenes infection was even more striking in the LP. Whereas only 28.4 ± 5.2% of LPL express TCR{alpha}{beta} in uninfected mice, p.o. and i.v. infection results in the expansion of TCR{alpha}{beta} T cells to 62.5 ± 4.4% (p = 0.005) and 78.7 ± 5.2% (p < 0.005), respectively. The proportion of TCR{alpha}{beta} T cells that express CD8{alpha}{beta} in the LP also increases with infection, from 24.2 ± 4.2% in uninfected mice to 36.9 ± 5.4% (p < 0.02) and 33.1 ± 3.1% (p < 0.01) after either an i.v. and p.o. infection with L. monocytogenes, respectively (Fig. 3Go, bottom). These findings demonstrate that p.o. and i.v. infections with L. monocytogenes result in an increase of TCR{alpha}{beta} CD8{alpha}{beta} T cells within the intestinal mucosa.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 3. Characterization of T lymphocyte populations responding to i.v. or p.o. infection with L. monocytogenes. The response of splenic, intestinal epithelial, and LP T cells was examined in uninfected and i.v. or p.o. L. monocytogenes-infected animals. Lymphocytes were isolated on day 9 postinfection, as described in Materials and Methods, stained with Abs specific for TCR{alpha}{beta}, CD8{alpha}, and CD8{beta} and evaluated by flow cytometry. Histograms depict the expression of TCR{alpha}{beta}, and the dot plots depict expression of CD8{alpha} (y-axis) vs CD8{beta} (x-axis), gated on viable TCR{alpha}{beta}+ T cells. The percentage of TCR{alpha}{beta} T cells expressing CD8{alpha}{alpha} and CD8{alpha}{beta} are indicated in the upper left and right quadrants, respectively, of the dot plots. Statistical significance between groups were evaluated by Student’s t test and are included within the text. Data are representative of results obtained from six to eight mice per group. Results depict the staining of T cell populations isolated from the spleen, IEL, and LPL of individual animals.

 
Ag specificity of intestinal CD8+ T cells after p.o. or i.v. L. monocytogenes infection

Although numerous studies have identified and characterized Ag-specific CD8 T lymphocytes within lymph nodes and spleen after infection, the specificity of intestinal T cells after bacterial infection remains largely undefined. Tetrameric MHC class I-peptide complexes have been useful reagents for the direct identification and quantitation of L. monocytogenes-specific CD8 T lymphocytes (19, 27). For the following studies, we used H2-Kd and H2-M3 tetramers complexed with a panel of L. monocytogenes peptides to identify Ag-specific T cells in the intestinal mucosa after i.v. and p.o. infection. In mice expressing the H2-Kd MHC class I molecule, the nonamer peptide LLO91–99 elicits an immunodominant CD8 T cell response. We therefore evaluated the response to LLO91–99 in the spleen, intestinal epithelium, and the LP of C56BL/6 mice after infection (Fig. 4Go). Consistent with our previous findings, LLO91–99-specific T cells were readily identified among splenocytes, with frequencies of 3.5 ± 0.7 and 2.1 ± 0.9% (p < 0.03) of CD8 T cells on the 9th day after i.v. or p.o. immunization, respectively. Within the IEL compartment, LLO91–99-specific T cells account for ~1.2 ± 0.6% and 2.1 ± 0.4% (p = 0.02) of the total CD8{alpha}+ T cell population after i.v. and p.o. infection, respectively. Furthermore, specific analysis of CD8{alpha}+ TCR{alpha}{beta}+ IEL revealed LLO-specific T cell responses were greater after p.o. infection (3.7 ± 1.2%) than after i.v. infection (1.3 ± 0.8%) (p < 0.005), with tetramer reactivity limited to T cells expressing CD8{beta} (Fig. 4GoB). Surprisingly, LLO91–99-specific T cells were most prevalent in the LP, constituting 9.7 ± 2.7 and 15.7 ± 4.3% (p < 0.01) of TCR{alpha}{beta} CD8{alpha} T cells among LPL after i.v. and p.o. immunization, respectively.



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 4. Identification of LLO91–99-specific T cell responses after i.v. and p.o. infection with L. monocytogenes. Splenic, IEL, and LPL populations were isolated from uninfected and i.v.- or p.o.-infected animals on day 9 postinoculation. Lymphocytes were stained with mAbs to CD8{alpha}, and TCR{gamma}{delta} and H2-Kd tetramers complexed with the immunodominant LLO91–99 epitope (A). Dot plots are gated on viable CD8{alpha} T cells and measure TCR{gamma}{delta} expression on the horizontal axis and H2-Kd/LLO91–99 tetramer staining on the vertical axis. The percentages of tetramer staining CD8{alpha}+ TCR{alpha}{beta}+ within each plot are indicated. B, Staining with MHC class I/peptide tetramer complexes is detected on CD8{alpha}{beta}+, but not CD8{alpha}{alpha}+ TCR{alpha}{beta}+ T cells from the intestinal epithelium. IEL were stained with H-2Kd tetramers complexed with the immunodominant LLO91–99 epitope and mAb toward TCR{alpha}{beta}+, CD8{alpha}, and CD8{beta}. Plots are gated on CD8{alpha} TCR{alpha}{beta}+ T cells and depict the expression of CD8{beta} expression on the horizontal axis and H2-Kd/LLO91–99 tetramer staining on the ordinate. Blots depict the staining of T cell populations isolated from the spleen, IEL, and LPL of individual animals, representative of results obtained from five animals per group. The percentages of tetramer-staining TCR{alpha}{beta}+ CD8{alpha}+ are indicated. Statistical analyses of results obtained is evaluated by Student’s t test and are included within the text.

 
We also quantified CD8 T cell responses to another dominant H2-Kd-restricted L. monocytogenes-derived epitope, p60217–225 and to the H2-M3-restricted formylmethionine epitope, fMIGWII. Similar to our findings with the LLO91–99-specific T cell response, the frequency of CD8 T cells specific for p60217–225 was ~3 times greater in the LP than in the spleen (Fig. 5Go, A and B). In contrast, however, the frequency of H2-M3-restricted T cells specific for f-MIGWII was lower in the LP than in the spleen (Fig. 5GoC). Within the small intestinal epithelium, although H2-Kd-restricted T cells were readily detectable, we could not detect any H2-M3-restricted T cells (Fig. 5Go). Not surprisingly, because L. monocytogenes does not cause substantial infection of lymph nodes after either p.o. or i.v. infection, we did not detect significant frequencies of Ag-specific, CD8 T cells in peripheral or mesenteric lymph nodes. Nevertheless, whereas MHC class Ia-restricted T cells constitute a significant portion of the mucosal T cell response to L. monocytogenes infection, T cells restricted by H2-M3 appear to play a lesser role.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 5. H2-Kd and H2-M3-restricted T cell responses after p.o. infection with L. monocytogenes. Spleen, mesenteric lymph node, intestinal epithelium, and LP lymphocytes were isolated and stained with Abs specific for CD8{alpha}, TCR{gamma}{delta}, and with H2-Kd tetramers complexed with LLO91–99 (A) or p60217–225 (B) or with H2-M3 tetramers complexed with the L. monocytogenes peptide f-MIGWII (C). H2-Kd-restricted responses were measured on the 9th day after infection, whereas the H2-M3-restricted T cell responses were measured on the 5th day after infection. H2-M3-restricted T cell responses did not increase in any tissues at later time points. The bar graphs depict the percentage (mean + SD) of tetramer reactive of CD8{alpha}+ TCR{alpha}{beta}+ T cells isolated from the spleen, MLN, IEL, and LPL compartments of three individual mice. Data are representative of results from three independent experiments.

 
Kinetics of L. monocytogenes-specific CD8 T cell responses

In previous studies, we found that i.v. L. monocytogenes infection elicits MHC class Ia-restricted T cell responses that increase in size for 8 to 9 days after inoculation and then decrease in frequency until stable memory populations remain (19). To determine whether the kinetics of T cell expansion and contraction were similar in secondary lymphoid tissues and the intestinal mucosa, we infected mice p.o. or i.v. and isolated lymphocytes from spleen, LP, and intestinal epithelium 6, 9, 14, and 30 days after inoculation. The frequency of TCR{alpha}{beta}CD8{alpha}+ T cells specific for LLO91–99 was maximal in all three sites on the 9th day after p.o. or i.v. infection and then decreased over the ensuing 20 days (Fig. 6Go). Remarkably, the frequency of LLO91–99-specific T cells remained at nearly 3% in the LP, suggesting that this site is an important reservoir for L. monocytogenes-specific memory T cells (Fig. 6Go, middle). In contrast, the frequency of LLO91–99-specific T cells in the spleen and intestinal epithelium decreased to nearly undetectable frequencies.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 6. Expansion and contraction of Ag-specific T cell responses after i.v. and p.o. infection with L. monocytogenes. Antigenic specific T cell responses to LLO91–99 were examined in mice infected p.o. with 109 bacteria or i.v. with 104 bacteria. On days 6, 9, 14, and 30 after infection, splenic, IEL, and LPL T cells were isolated and stained with H2-Kd LLO91–99 tetramers and mAbs specific for TCR V{beta} and CD8{alpha}. The bar graphs depict the percentage (mean + SD) of CD8{alpha}-, TCR{alpha}{beta}-expressing T cells from three individual animals that stained with LLO91–99 tetramers after i.v. or p.o. infection. Data are representative of results obtain from five experiments.

 
Cytolytic activity of mucosal MHC class Ia-restricted CD8 T cells

We next examined whether L. monocytogenes-specific CD8 T cells identified in the LP and intestinal epithelium were functional. CB6F1 mice were infected either p.o. or i.v. with L. monocytogenes, and 9 days later CD8 T cells were enriched from spleen, intestinal epithelium, and LP and tested for cytolytic activity against target cells incubated with LLO91–99. CD8-enriched splenocytes lysed LLO91–99-coated target cells after either i.v. or p.o. infection, albeit to a greater extent after i.v. inoculation (Fig. 7GoA). Similarly, CD8 T cells enriched from the LP also lysed LLO91–99 target cells, to a greater extent than CD8-enriched splenocytes (Fig. 7GoC). Interestingly, p.o. infection resulted in greater cytolytic activity in the LP than i.v. infection. In marked contrast to the cytolytic activity detected in spleen and LP, IELs did not exhibit detectable levels of Ag-specific lysis to LL091–99 (Fig. 7GoB) or p60217–225 (data not shown)-coated targets. Although the percentage of LLO91–99-specific T cells is lower among IELs than LPLs or splenocytes, the absence of peptide-specific cytolytic activity suggests that this function may have been lost from L. monocytogenes-specific IELs.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 7. Ag-specific cytolytic activity of CD8 T cells in the spleen, intestinal epithelium, and LP after infection with L. monocytogenes. CD8+ T cell populations from the spleen (left), intestinal epithelium (middle), and LP (right) from i.v.- and p.o.-infected animals were examined for CTL activity to 51Cr-labeled P815 (H2-Kd) target cells alone or which had be coated with LLO91–99 peptide (as described in Materials and Methods) on day 9 postinfection. E:T is based on the number of H2-Kd LLO91–99 tetramer+ TCR{alpha}{beta}+CD8{alpha}{beta}+ effectors per well.

 
TCR V{beta} repertoire of splenic and mucosal LLO91–99-specific T cells

Direct ex vivo staining of LLO91–99-specific T cell with a panel of TCR V{beta}-specific mAbs demonstrated that primary infection with L. monocytogenes elicits a repertoire of T cells that is maintained for months after the resolution of infection (33). While the relative representation of different TCR V{beta}-chains within an Ag-specific T cell population does not change over time within an individual mouse, repertoire comparisons of Ag-specific T cell populations between different mice occasionally uncover substantial disparities in the utilization of TCR V{beta}-chains (33, 34). Since T cells of the intestinal mucosa are characterized by a rather distinctive TCR repertoire, we decided to evaluate the TCR repertoire of Ag-specific T cells in the intestinal mucosa after L. monocytogenes infection. CB6F1 mice were infected with L. monocytogenes and 9 days later splenocytes, IELs, and LPLs were stained with a panel of TCR V{beta}-specific mAbs (V{beta}2, -4, -6, -7, 8.1/8.2, -8.1-3, and -9) and with H2-Kd tetramers complexed with LLO91–99. Staining of LLO91–99-specific T cells for TCR V{beta}6, -8, and -9 is demonstrated in Fig. 8GoA for a p.o. immunized mouse. Although only 3.5% of LLO91–99-specific T cells in the spleen expressed TCR V{beta}6, >24 and 11% of LLO91–99-specific CD8 T cells among IELs and LPLs, respectively, expressed TCR V{beta}6. Differences in the expression of TCR V{beta}8 on LLO91–99-specific CD8 T cells isolated from these compartments were also detected. Comparisons of the TCR V{beta} repertoires among splenocytes, IELs and LPLs from 10 mice demonstrate substantial differences in TCR usage between these different anatomic sites (Fig. 8Go, b and c). The disparities in TCR V{beta} usage are similar after p.o. or i.v. infection (compare Fig. 8Gob), suggesting that the route of bacterial infection does not determine these repertoire differences.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 8. Ag-specific T cells in the intestinal mucosa express a distinct TCR V{beta} repertoire. The TCR V{beta} repertoire of Ag-specific T cells after i.v. and p.o. infection was examined on day 9 after inoculation. After isolation, T cells from splenic, IEL, and LPL populations were stained with LLO91–99 tetramer, anti-CD8{alpha}, and individual mAbs specific for TCR V{beta} segments 2, 4, 6, 7, 8(.1/.2), 8(.3), and 9, as described in Materials and Methods. The percentage of CD8{alpha} expressing, LLO91–99 tetramer-staining T cells that express individual TCR V{beta} segments was then determined by flow cytometry. A, Representative analysis from one mouse, staining splenocytes, IEL, and LPL with LLO91–99 tetramers on the ordinate and with TCR V{beta}6-, -8-, and -9-specific Abs on the horizontal axis. The percentage of tetramer-positive cells that stain with TCR V{beta} Abs is indicated in each plot. B, Percentage of tetramer-positive cells that stain with each of the TCR V{beta} Abs in five p.o. (a, c, e, g, and i)- and five i.v.-infected mice (b, d, f, h, and j). a–f, Results obtained from BALB/c infected mice; g–j, Results from CB6F1-infected mice. Data are representative of three independent experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The intestinal mucosa provides an extensive and intimate interface with the microbial world that is perpetually confronted with potentially invasive pathogens (35). Although it is assumed that intestinal T lymphocytes, in particular IEL, protect the host from microbial invasion (36), the specificity of intestinal T cells after bacterial infection has not been previously determined. In this report, we demonstrate that oral and i.v. infection of mice with L. monocytogenes induces a robust and readily detectable, Ag-specific T cell response in the small intestinal epithelium and the LP. The Ag specificity of CD8 T cells in these compartments overlaps with that previously described for CD8 T cell responses in the spleen. Although the T cell response to LLO is large in both the spleen and intestine after L. monocytogenes infection, we find that the TCR repertoires of CD8 CTLs in the two compartments differ and that the ratio of MHC class Ib to class Ia-restricted, L. monocytogenes-specific T cells is greater in the spleen than in the intestinal mucosa. Our findings have uncovered disparities between CD8 T cell responses in the intestine and secondary lymphoid organs that indicate that the distinction between these two T cell populations extends beyond simply differences in tissue localization.

We were surprised to find that the LLO91–99-specific T cell response in the LP of the small intestine was, in terms of frequency, larger and more prolonged than the CD8 T cell response to the same epitope in the spleen. These findings are consistent with studies of the H2-Kb-restricted CTL response to OVA-derived SIINFEKL in LP after systemic infection with OVA encoding VSV (37). Kinetic analysis of the response after L. monocytogenes infection in the LP vs the spleen suggests that the expansion of Ag-specific T cells is concurrent in both compartments. The greater proportion of LLO-specific cells in LP up to 30 days after resolution of L. monocytogenes infection may reflect their enhanced survival in this site. Alternatively, it is possible that circulating, Ag-specific T cells travel to the LP and are retained there. Another alternative is that the LP supports the proliferation of Ag-specific memory T cells to a greater extent than the spleen. It is tempting to postulate that production of IL-15, a cytokine that has recently been implicated in promoting the proliferation of memory CD8 T cells (38, 39), may be enhanced in intestinal LP. Further studies will be required to test these hypotheses.

The magnitude and kinetics of the intestinal CTL response to L. monocytogenes were similar after p.o. or systemic infection. Systemic infection induced by p.o. inoculation of 109 L. monocytogenes or i.v. inoculation of 104 bacteria similarly peaked on day 3 and were cleared by day 9. However, only p.o. infection resulted in the isolation of viable L. monocytogenes from the intestinal lumen and LP (Fig. 2Go). It is surprising, therefore, that the intestinal immune response is similar after either p.o. or i.v. infection, because the amount of Ag in the intestine is presumably greater after p.o. infection. It is possible that bacterial Ags or APCs migrate to the LP after systemic L. monocytogenes infection and drive T lymphocyte expansion in the mucosa. Alternatively, it is possible that L. monocytogenes-specific T lymphocytes are activated in the spleen and other secondary lymphoid tissues and then migrate to the intestinal mucosa (40, 41). Although neither of these mechanisms can be excluded by our studies, our finding that the TCR repertoire of splenic and intestinal CD8 T cells differs suggests that mucosal T cell populations do not simply represent an "open door" influx from secondary lymphoid organs.

Similarly, it is interesting that i.v. infection produced higher Ag-specific responses in the spleen to LLO91–99 (Figs. 4Go and 6Go) and p60217–225 (data not shown) than p.o. infection, given that p.o. immunization resulted in similar if not higher levels of bacterial numbers in the spleen (Fig. 1Go). One possible explanation is that an infection initiated in the intestinal mucosa influences the migration and recruitment of Ag-specific T cells in secondary lymphoid tissues, including the spleen. Additionally, we have recently reported that the magnitude of Ag-specific CD8 T cell responses to L. monocytogenes is determined within the first 24 h of an infection and is independent of the severity and duration of in vivo bacterial infection (42). Consequently, although p.o. immunization resulted in higher (100-fold) bacterial titers in the spleen (day 3) than i.v. infection, the absence of a concordant increase in Ag-specific CD8 T cells is not surprising.

Murine resistance to intestinal infection with L. monocytogenes has been previously appreciated and can be attributed, at least in part, to sequence differences between murine and human E-cadherin. Recent studies have demonstrated that L. monocytogenes internalin exhibits decreased association with murine E-cadherin compared with human E-cadherin (43), thereby substantially reducing infection of intestinal epithelial cells. It is possible, therefore, that enteric infection of mice with L. monocytogenes disseminates primarily through M cells lining the small intestine, a route that is believed to be followed by many different enteric bacterial pathogens (44). This may also explain our finding that viable L. monocytogenes are not detectable in the intestinal epithelium after p.o. infection (Fig. 2Go).

Previous studies from our laboratory established that the MHC class Ia-restricted CTL response to L. monocytogenes was characterized by a very predictable immunodominance hierarchy (45). The dominant epitope, LLO91–99 , elicits the largest response, p60217–225 elicits an intermediate response, while the responses to p60449–457 and mpl84–92 are small. The current studies of the intestinal CD8 T cell response to L. monocytogenes infection demonstrate that this hierarchic response is also present in the intestine. The rate and duration of in vivo T cell expansion to the four epitopes have been shown previously to be highly synchronized in vivo, suggesting that the magnitudes of the individual CD8 T cell responses reflect differences in T cell repertoire rather than differences in the duration of Ag presentation (27). Intestinal and splenic T cells expressing CD8{alpha}{beta} are selected in the thymus, and our results indicate that the pool of T cells that are available for stimulation in the intestine or in the spleen are similar with respect to their ability to respond to the different L. monocytogenes epitopes.

TCR repertoire analysis of Ag-specific T cells within individual mice demonstrated that primary L. monocytogenes infection elicits a complex population of LLO91–99-specific T cells that express a highly reproducible pattern of TCR V{beta}-chains (33). On the other hand, the pattern of TCR V{beta} expression between genetically identical mice often demonstrated distinctions that were maintained over time within individual mice. One interpretation of this result is that activation of naive, LLO-specific precursor T cells is incomplete after primary infection. In this setting, more prevalent clones will always be represented in all mice, but less prevalent clones, by random chance, may be activated only in occasional mice. The responses that are common to all mice have been referred to as public, while sporadic responses have been called private (46). In our repertoire analyses, for example, the majority of LLO-specific CD8 T cell populations express TCR V{beta}8, whereas the frequencies of the less prevalent TCR V{beta}6, -7, and -9 chains are more variable. Our findings indicate that the variability detected between splenic T cell responses in different animals can also be found within the same animal if different compartments are investigated.

Although differences in the activation of naive T cells in the intestine or spleen may account for the repertoire differences we have identified, there are other possible explanations. Studies using TCR-transgenic T cells specific for OVA have shown that Ag-specific T cells move into the LP and IEL compartments only on Ag challenge (21). It is possible that different T cell clones that share Ag specificity but express different TCRs differ in their ability to migrate into the LP or epithelium of the small intestine. Alternatively, it is possible that distinct subpopulations of Ag-specific T cells differ in their ability to survive in the intestine vs the spleen. Determining the basis for our findings will require a deeper understanding of the T cell trafficking between secondary lymphoid tissues and the intestine.

Why is the H2-M3-restricted T cell response to enteric L. monocytogenes infection small? We have previously hypothesized that H2-M3-restricted T cells might play a prominent role in the intestine (19). The findings in this report do not support this hypothesis. It is possible that the prevalence of formyl peptides in the intestine, derived from the polymicrobial intestinal flora, results in the depletion of formyl peptide-specific T cells due to activation-induced cell death. Alternatively, trafficking of H2-M3-restricted T cells may differ from that of MHC class Ia-restricted T cells, resulting in the dearth of M3-restricted T cells in the LP and IEL compartments. It is interesting to speculate that the M3-restricted T cell response is particularly suited for the detection of formylmethionine peptides in the systemic immune compartments, a location where the presence of bacterial products would be far more alarming than in the intestinal mucosa. The recent finding that intestinal epithelial cells do not express significant amounts of H2-M3 is consistent with this hypothesis (47).

Our studies indicate that L. monocytogenes infection induces a substantial mucosal T cell response with specificity for peptides derived from bacterially secreted proteins irrespective of immunization route. Although it remains unclear whether L. monocytogenes-specific CD8 T cells in the LP or within the intestinal epithelial layer provide protective immunity, their abundance and active effector status in the LP suggests that they are capable of providing surveillance against reinfection. Although direct cytolytic destruction of infected epithelial cells may be a protective mechanism, abundant evidence indicates that perforin and Fas ligand-deficient T cells can confer protective immunity in the setting of systemic L. monocytogenes infection (48). Similarly, production of IFN-{gamma} is dispensable for the protection mediated by CTL after systemic infection (49). The murine model of intestinal L. monocytogenes infection provides a wonderful system to determine the mechanism of T cell-mediated mucosal immunity.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI39031 and AI42135. J.W.H. was supported by a fellowship from the Arthritis Foundation. K.K. was supported by National Institutes of Health Training Grant 5T32-AI07019. Back

2 Address correspondence and reprint requests to Dr. James W. Huleatt, Section of Immunobiology, Yale University, School of Medicine, New Haven, CT 06520. Back

3 Current address: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Back

4 Abbreviations used in this paper: LLO, listeriolysin O; p.o., oral(ly); VSV, vesicular stomatitis virus; LP, lamina propria; IEL, intraepithelial lymphocyte; BHI, brain-heart infusion. Back

Received for publication October 13, 2000. Accepted for publication January 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gellin, B. G., C. V. Broome. 1989. Listeriosis. JAMA 261:1313.[Medline]
  2. Gellin, B. G., C. V. Broome, W. F. Bibb, R. E. Weaver, S. Gaventa, L. Mascola. 1991. The epidemiology of listeriosis in the United States: 1986. Am. J. Epidemiol. 133:392.[Abstract/Free Full Text]
  3. Linnan, M. J., L. Mascola, X. D. Lou, V. Goulet, S. May, C. Salminen, D. W. Hird, M. L. Yonekura, P. Hayes, R. Weaver, et al 1988. Epidemic listeriosis associated with Mexican-style cheese. N. Engl. J. Med. 319:823.[Abstract]
  4. Aureli, P., G. C. Fiorucci, D. Caroli, G. Marchiaro, O. Novara, L. Leone, S. Salmaso. 2000. An outbreak of febrile gastroenteritis associated with corn contaminated by Listeria monocytogenes. N. Engl. J. Med. 342:1236.[Abstract/Free Full Text]
  5. Dalton, C. B., C. C. Austin, J. Sobel, P. S. Hayes, W. F. Bibb, L. M. Graves, B. Swaminathan, M. E. Proctor, P. M. Griffin. 1997. An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk. N. Engl. J. Med. 336:100.[Abstract/Free Full Text]
  6. Gaillard, J. L., P. Berche, P. Sansonetti. 1986. Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect. Immun. 52:50.[Abstract/Free Full Text]
  7. Kathariou, S., P. Metz, H. Hof, W. Goebel. 1987. Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes. J. Bacteriol. 169:1291.[Abstract/Free Full Text]
  8. Portnoy, D. A., P. S. Jacks, D. J. Hinrichs. 1988. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167:1459.[Abstract/Free Full Text]
  9. Pistor, S., T. Chakraborty, K. Niebuhr, E. Domann, J. Wehland. 1994. The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton. EMBO J. 13:758.[Medline]
  10. Kocks, C., E. Gouin, M. Tabouret, P. Berche, H. Ohayon, P. Cossart. 1992. L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell 68:521.[Medline]
  11. Theriot, J. A., T. J. Mitchison, L. G. Tilney, D. A. Portnoy. 1992. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature 357:257.[Medline]
  12. Mengaud, J., H. Ohayon, P. Gounon, R. M. Mege, P. Cossart. 1996. E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell 84:923.[Medline]
  13. Racz, P., K. Tenner, E. Mero. 1972. Experimental Listeria enteritis. I. An electron microscopic study of the epithelial phase in experimental Listeria infection. Lab Invest. 26:694.[Medline]
  14. Czuprynski, C. J., P. M. Henson, P. A. Campbell. 1985. Enhanced accumulation of inflammatory neutrophils and macrophages mediated by transfer of T cells from mice immunized with Listeria monocytogenes. J. Immunol. 134:3449.[Abstract]
  15. Teixeira, H. C., S. H. E. Kaufmann. 1994. Role of NK1.1+ cells in experimental listeriosis. J. Immunol. 152:1873.[Abstract]
  16. Hiromatsu, K., Y. Yoshikai, G. Matsuzaki, S. Ohga, K. Muramori, K. Matsumoto, J. A. Bluestone, K. Nomoto. 1992. A protective role of {gamma}/{delta} T cells in primary infection with Listeria monocytogenes in mice. J. Exp. Med. 175:49.[Abstract/Free Full Text]
  17. Ladel, C. H., I. E. F. Flesch, J. Arnoldi, S. H. E. Kaufmann. 1994. Studies with MHC-deficient knock-out mice reveal impact of both MHC I- and MHC II-dependent T cell responses on Listeria monocytogenes infection. J. Immunol. 153:3116.[Abstract]
  18. Busch, D. H., K. Kerksiek, E. G. Pamer. 1999. Processing of Listeria monocytogenes antigens and the in vivo T cell response to bacterial infection. Immunol. Rev. 172:163.[Medline]
  19. Kerksiek, K. M., D. H. Busch, I. M. Pilip, S. E. Allen, E. G. Pamer. 1999. H2–M3 restricted T cells in bacterial infection: rapid primary but diminished memory responses. J. Exp. Med. 190:195.[Abstract/Free Full Text]
  20. Havell, E. A., G. R. Beretich, P. B. Carter. 1999. The mucosal phase of Listeria infection. Immunobiology 201:164.[Medline]
  21. Kim, S. K., D. S. Reed, S. Olson, M. S. Schnell, J. K. Rose, P. A. Morton, L. Lefrancois. 1998. Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals. Proc. Natl. Acad. Sci. USA 95:10814.[Abstract/Free Full Text]
  22. Lefrancois, L., C. M. Parker, S. Olson, W. Muller, N. Wagner, L. Puddington. 1999. The role of {beta}7 integrins in CD8 T cell trafficking during an antiviral immune response. J. Exp. Med. 189:1631.[Abstract/Free Full Text]
  23. Kim, S. K., K. S. Schluns, L. Lefrancois. 1999. Induction and visualization of mucosal memory CD8 T cells following systemic virus infection. J. Immunol. 163:4125.[Abstract/Free Full Text]
  24. Goodman, T., L. Lefrancois. 1988. Expression of the {gamma}{delta} T-cell receptor on intestinal CD8 intraepithelial lymphocytes. Nature 333:855.[Medline]
  25. Poussier, P., P. Edouard, C. Lee, M. Binnie, M. Julius. 1992. Thymus-independent development and negative selection of T cells expressing T cell receptor {alpha}/{beta} in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes. J. Exp. Med. 176:187.[Abstract/Free Full Text]
  26. Kramer, D. R., J. J. Cebra. 1995. Early appearance of "natural" mucosal IgA responses and germinal centers in suckling mice developing in the absence of maternal antibodies. J. Immunol. 154:2051.[Abstract]
  27. Busch, D. H., I. M. Pilip, S. Vijh, E. G. Pamer. 1998. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8:353.[Medline]
  28. Pamer, E. G.. 1994. Direct sequence identification and kinetic analysis of an MHC class I-restricted Listeria monocytogenes CTL epitope. J. Immunol. 152:686.[Abstract]
  29. Murali-Krishna, M., J. D. Altman, M. Suresh, D. J. D. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177.[Medline]
  30. Flynn, K. J., G. T. Belz, J. D. Altman, R. Ahmed, D. L. Woodland, P. C. Doherty. 1998. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 8:683.[Medline]
  31. Butz, E. A., M. J. Bevan. 1998. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 8:167.[Medline]
  32. Ohtsuka, K., K. Sato, H. Watanabe, M. Kimura, H. Asakura, T. Abo. 1995. Unique order of the lymphocyte subset induction in the liver and intestine of mice during Listeria monocytogenes infection. Cell. Immunol. 161:112.[Medline]
  33. Busch, D. H., I. Pilip, E. G. Pamer. 1998. Evolution of a complex T cell receptor repertoire during primary and recall bacterial infection. J. Exp. Med. 188:61.[Abstract/Free Full Text]
  34. Bousso, P., A. Casrouge, J. D. altman, M. Haury, J. Kanellopoulos, J.-P. Abastado, P. Kourilsky. 1998. Individual variations in the murine T cell response to a specific peptide reflect variability in naive repertoires. Immunity 9:169.[Medline]
  35. Neutra, M. R., E. Pringault, J. P. Kraehenbuhl. 1996. Antigen sampling across epithelial barriers and induction of mucosal immune responses. Annu. Rev. Immunol. 14:275.[Medline]
  36. Lefrancois, L., B. Fuller, J. W. Huleatt, S. Olson, L. Puddington. 1997. On the front lines: intraepithelial lymphocytes as primary effectors of intestinal immunity. Springer Semin. Immunopathol. 18:463.[Medline]
  37. Lefrancois, L., S. Olson, D. Masopust. 1999. A critical role for CD40-CD40 ligand interactions in amplification of the mucosal CD8 T cell response. J. Exp. Med. 190:1275.[Abstract/Free Full Text]
  38. Zhang, X., S. Sun, I. Hwang, D. F. Tough, J. Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8 T cells in vivo by IL-15. Immunity 8:591.[Medline]
  39. Ku, C. C., M. Murakami, A. Sakamoto, J. Kappler, P. Marrack. 2000. Control of homeostasis of CD8 memory T cells by opposing cytokines. Science 288:675.[Abstract/Free Full Text]
  40. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
  41. Kim, S. K., D. S. Reed, W. R. Heath, F. Carbone, L. Lefrancois. 1987. Activation and migration of CD8 T cells in the intestinal mucosa. J. Immunol. 159:4295.[Abstract]
  42. Mercadao, R., S. Vijh, S. E. Allen, K. Kersiek, I. M. Pilip, E. G. Pamer. 2000. Early response of T cell populations to bacterial infection. J. Immunol. 165:6833.[Abstract/Free Full Text]
  43. Lecuit, M., S. Dramsi, C. Gottardi, M. Fedor-Chaiken, B. Gumbiner, P. Cossart. 1999. A single amino acid in E-cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes. EMBO J. 18:3956.[Medline]
  44. Jones, B., L. Pascopella, S. Falkow. 1995. Entry of microbes into the host: using M cells to break mucosal the barrier. Curr. Opin. Immunol. 7:474.[Medline]
  45. Vijh, S., E. G. Pamer. 1997. Immunodominant and subdominant CTL responses to Listeria monocytogenes infection. J. Immunol. 158:3366.[Abstract]
  46. Cibotti, R., J. P. Cabaniols, C. Pannetier, C. Delarbre, I. Vergnon, J. M. Kanellopoulos, P. Kourilsky. 1994. Public and private V{beta} T cell receptor repertoires against hen egg lysozyme in nontransgenic versus HEL transgenic mice. J. Exp. Med. 180:861.[Abstract/Free Full Text]
  47. Chiu, N. M., T. Chun, M. Fay, M. Mandal, C. R. Wang. 1999. The majority of H2–M3 is retained intracellularly in a peptide-receptive state and traffics to the cell surface in the presence of N-formylated peptides. J. Exp. Med. 190:423.[Abstract/Free Full Text]
  48. Harty, J. T., M. J. Bevan. 1999. Responses of CD8 T cells to intracellular bacteria. Curr. Opin. Immunol. 11:89.[Medline]
  49. Harty, J. T., M. J. Bevan. 1995. Specific immunity to Listeria monocytogenes in the absence of INF {gamma}. Immunity 3:109.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
K. Heiss, N. Janner, B. Mahnss, V. Schumacher, F. Koch-Nolte, F. Haag, and H.-W. Mittrucker
High Sensitivity of Intestinal CD8+ T Cells to Nucleotides Indicates P2X7 as a Regulator for Intestinal T Cell Responses
J. Immunol., September 15, 2008; 181(6): 3861 - 3869.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
K. Brandl, G. Plitas, B. Schnabl, R. P. DeMatteo, and E. G. Pamer
MyD88-mediated signals induce the bactericidal lectin RegIII{gamma} and protect mice against intestinal Listeria monocytogenes infection
J. Exp. Med., August 6, 2007; 204(8): 1891 - 1900.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
S. Lee, S. A. Miller, D. W. Wright, M. T. Rock, and J. E. Crowe Jr.
Tissue-Specific Regulation of CD8+ T-Lymphocyte Immunodominance in Respiratory Syncytial Virus Infection
J. Virol., March 1, 2007; 81(5): 2349 - 2358.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. Masopust, V. Vezys, E. J. Wherry, D. L. Barber, and R. Ahmed
Cutting Edge: Gut Microenvironment Promotes Differentiation of a Unique Memory CD8 T Cell Population
J. Immunol., February 15, 2006; 176(4): 2079 - 2083.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. M. Freeman and H. K. Ziegler
Simultaneous Th1-Type Cytokine Expression Is a Signature of Peritoneal CD4+ Lymphocytes Responding to Infection with Listeria monocytogenes
J. Immunol., July 1, 2005; 175(1): 394 - 403.
[Abstract] [Full Text] [PDF]

Home page