Antigen-specific CD8+ T cell responses in intestinal tissues during murine listeriosis

Microbes and Infection 6 (2004) 8–16 www.elsevier.com/locate/micinf

Original article

Antigen-specific CD8+ T cell responses in intestinal tissues during murine listeriosis Mischo Kursar a, Kerstin Bonhagen b, Anne Köhler a, Thomas Kamradt b, Stefan H.E. Kaufmann a, Hans-Willi Mittrücker a,* a

Max-Planck Institute for Infection Biology, Schumannstr. 21/22, 10117 Berlin, Germany b Deutsches Rheumaforschungszentrum, Schumannstr. 21/22, 10117 Berlin, Germany Received 12 June 2003; accepted 1 October 2003

Abstract Infection of mice with Listeria monocytogenes induces a strong CD8+ T cell response, which is critical for the control of bacteria and for protection against re-infection. We analyzed the CD8+ T cell response in different intestinal tissues following oral and intravenous (i.v.) L. monocytogenes infection. After oral infection, bacterial titers in small intestine and large intestine, and the listeria-specific CD8+ T cell response in the mucosa of both parts of the intestine, were highly correlated. Oral infection of CD28-deficient mice revealed that this response was strictly dependent on CD28 costimulation. Significant listeria-specific CD8+ T cell responses also occurred in all intestinal tissues analyzed after i.v. infection or after DNA vaccination, indicating that the accumulation of listeria-specific CD8+ T cells in these tissues only partially depends on local antigen presentation and inflammation. © 2003 Elsevier SAS. All rights reserved. Keywords: T lymphocytes; Bacterial infection; Costimulation; Mucosa

1. Introduction The Gram-positive bacterium Listeria monocytogenes is the cause of human listeriosis, a disease characterized by bacterial dissemination from the intestinal lumen into deeper tissues. The course of L. monocytogenes infection has been intensively analyzed in the experimental mouse model [1,2]. After oral application, listeriae reach the small intestine (SI), transmigrate through the mucosa, and spread via the mesenteric lymph nodes (MLNs) into deeper tissue sites. One day after infection, L. monocytogenes can be isolated from its target organs, spleen and liver [3,4]. L. monocytogenes infects macrophages and hepatocytes. Inside these cells, bacteria escape from the phagosome and enter the cytosol, where they replicate. Large numbers of bacteria can be isolated

Abbreviations: IEL, intraepithelial lymphocyte; LI, large intestine; LLO, listeriolysin-O; MLN, mesenteric lymph node; p.o., per os; PP, Peyer’s patches; SI, small intestine. * Corresponding author. Tel.: +49-30-2846-0532; fax: +49-30-2846-0501. E-mail address: [email protected] (H.-W. Mittrücker). © 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2003.10.004

from spleens and livers of infected animals 3–5 days after infection. Eventually, the acquired immune response controls infection, and by day 10, bacteria have been eradicated from all tissues [1,4]. The cytosolic habitat of L. monocytogenes leads to processing and presentation of listerial antigens via the MHC class I pathway. Consequently, infection of mice with L. monocytogenes induces a strong CD8+ T cell response, which is critical for anti-listerial defense [1,5,6]. Several dominant MHC class I-presented T cell epitopes from L. monocytogenes have been characterized, enabling the analyses of the kinetics and the magnitude of the listeria-specific CD8+ T cell response in mice [7]. In these studies, it was demonstrated that an unexpectedly large fraction of CD8+ T cells is involved in the anti-listerial response after intravenous (i.v.) infection [8]. Oral infection with wild type or recombinant L. monocytogenes strains causes strong listeriaspecific CD8+ T cell responses in the intestinal mucosa of the small intestine (SI) as well as in spleen and liver [9–11]. In all organs, CD8+ T cell responses peak around day 9 and then rapidly decline. Subsequently, high frequencies of memory cells can be identified in spleen, liver and lamina propria of the SI. Surprisingly, a strong listeria-specific CD8+ T cell

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response is also observed in the SI after i.v. infection [9,11], suggesting that the strong mucosal CD8+ T cell response is only partially due to local infection but rather the result of preferential enrichment, expansion or survival of effector and memory CD8+ T cells at mucosal sites [12]. Here, we analyzed the CD8+ T cell responses in different intestinal tissues following oral and i.v. L. monocytogenes infection and after DNA immunization. After oral infection, a strong L. monocytogenes-specific CD8+ T cell response developed in the mucosa of the SI and large intestine (LI), and this response was strictly dependent on CD28 costimulation. However, a significant CD8+ T cell response also occurred in these tissues after i.v. infection and after DNA vaccination. Thus, accumulation of listeria-specific CD8+ T cells in the intestinal mucosa is at least in part regulated by mechanisms independent of the site of infection and location of antigen.

2. Materials and methods 2.1. Antibodies Rat IgG antibodies, anti-CD16/CD32 mAb (clone: 2.4G2), anti-IFN-c mAb (clone: R4-6A2, rat IgG1), antiCD8a mAb (clone: YTS169), and anti-CD62L mAb (clone: Mel-14) were purified from rat serum or hybridoma supernatants with protein G sepharose. Antibodies were Cy5- or FITC-conjugated according to standard protocols. PEconjugated anti-IL-2 mAb (clone: JES6-5H4, rat-IgG2b), FITC-conjugated rat-IgG1 isotype control mAb (clone: R334) and PE-conjugated rat-IgG2b isotype control mAb (clone: A95-1) were purchased from Pharmingen, San Diego, CA. 2.2. Bacteria and bacterial infection of mice BALB/c mice and BALB/c CD28–/– mice [13,14] were bred in our facility at the Federal Institute for Health Protection of Consumers and Veterinary Medicine in Berlin, and experiments were conducted according to the German animal protection law. Mice were infected with L. monocytogenes strain EGD, as previously described [4]. For primary i.v. infection, mice received 2 × 103 listeria, for secondary infection 1 × 105 listeria. Listeria were injected in a volume of 200 µl PBS into the lateral tail veins of mice. For primary and secondary per os (p.o.) infection, mice received 1 × 109 and 5 × 109 listeria, respectively, in a total volume of 200 µl PBS by gastric intubation. For determination of listerial burdens, organs were homogenized in PBS. SI, caecum and LI were homogenized, including the luminal content. Serial dilutions of homogenates were plated on PALCAM-listeria selective agar supplemented with selective antibiotics (Merck, Darmstadt, Germany), and colonies were counted after 48 h incubation at 30 °C.

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2.3. DNA immunization The plasmid pChly was constructed by cloning the hly (listeriolysin) gene into the eukaryotic expression plasmid pCI [15]. The plasmid pCMV-GM-CSF contains the GMCSF gene under the control of a cytomegalovirus (CMV) promoter. DNA of the plasmid pChly (1.0 µg) was coprecipitated with 0.8 µg plasmid pCMV-GM-CSF on 0.5-mg gold particles. Phosphothiate-modified oligodeoxynucleotides (ODN) containing a CpG motif (ODN1760) [16] were dissolved in PBS to a final concentration of 0.5 µg/ml. ODN were synthesized by Interactiva Biotechnology (Ulm, Germany). Gene-gun inoculations were performed using a gene gun (Bio-Rad, CA). For immunizations, two nonoverlapping shots per mouse were performed into freshly shaven abdominal skin using 0.5 mg of 1.0-µm DNA-coated gold particles per shot. Subsequently, 10 µg of the CpGcontaining ODN were injected intradermally at the site of particle bombardment. 2.4. Purification of cells from different tissues Single-cell suspensions of spleen cells were prepared using an iron mesh sieve. Spleen cells were treated with Trisbuffered ammonium chloride to lyse red blood cells and then washed twice with RPMI 1640 medium supplemented with glutamine, Na-pyruvate, b-mercaptoethanol, penicillin, streptomycin and 10% heat-inactivated fetal calf serum (complete RPMI medium). Peyer’s patches (PPs) and MLNs were excised, single cell suspensions were prepared using an iron mesh sieve, and cells were washed twice with complete RPMI medium. Intraepithelial lymphocytes (IELs) from SI and LI were isolated as previously described [4,17], with some modifications. Briefly, LI and SI (after excision of PPs of individual mice were cut open and washed twice in PBS, 1% BSA. Intestines were stirred at 37 °C for 20 min in complete RPMI medium and then washed twice by shaking in complete RPMI medium for 0.5 min. Supernatants were filtered through a 70-µm nylon sieve and centrifuged to pellet the cells. Cells were resuspended and centrifuged through a 40%/70% Percoll gradient (Biochrom, Berlin, Germany) for 30 min at 600 × g. Cells were collected from the interface of the gradient and washed in complete RPMI medium. Lamina propria lymphocytes were isolated from SI and LI as described [17]. After IEL isolation, intestines were cut into 5-mm pieces and digested for 60 min at 37 °C in complete RPMI medium supplemented with Collagenase D (Roche, Mannheim, Germany) and Collagenase Type VIII (Sigma, St. Louis, MO). Resulting cell suspensions were filtered through a 70-µm nylon sieve and centrifuged to pellet the cells. Cells were washed in complete RPMI medium and further purified by a 40%/70% Percoll gradient. Livers were perfused with PBS through the vena portae, removed and homogenized using an iron-mesh sieve. Cell suspensions were washed with PBS, centrifuged for 1 min at 50 × g, and supernatants were collected. This step was repeated three

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times. Cells from pooled supernatants were further purified by a 40%/70% Percoll gradient. 2.5. In vitro restimulation of cells and flow cytometric determination of cytokine expression Cells were restimulated and analyzed as previously described [13]. Briefly, 3 × 106 cells were stimulated for 5 h with 10–6 M of the peptide LL091–99. During the final 4 h of culture, 10 µg/ml Brefeldin A (Sigma) was added. Cells were stained with Cy5-conjugated anti-CD8a mAb, fixed, and stained intracellularly with FITC-conjugated anti-IFN-c mAb and PE-conjugated anti-IL-2 mAb, or FITC- and PEconjugated isotype control mAbs. Cells were analyzed using a FACS-Calibur and the CellQuest software (BectonDickinson, Mountain View, CA). 2.6. Generation of MHC class I tetramers and staining of cells with tetramers

3. Results 3.1. L. monocytogenes titers in different organs after p.o. and i.v. infection BALB/c mice were infected p.o. or i.v. with L. monocytogenes, and the bacterial burden in various organs was determined at different time points post-infection (Fig. 1). After i.v. infection, mice suffered from high bacterial titers in spleen and liver at days 1 and 3, but cleared bacteria by day 10. We detected bacteria sporadically in MLNs and in the intestinal lumen. Oral infection led to high bacterial titers in the SI, caecum and LI. L. monocytogenes was eradicated from the SI by day 8 but persisted in the caecum and LI. At day 13, few listeria remained in the distal parts of the intestine. Bacteria were detected in the MLNs at day 1, and reached high numbers in MLNs, spleen and liver by day 3 post-infection. In spleen and MLNs, bacteria were cleared by day 10, and in the liver by day 13. Mice were also analyzed after secondary i.v. and p.o. infection (Fig. 2). At day 1, we detected L. monocytogenes in spleens, and at day 1 and 3 in livers of i.v. infected mice. No

Fig. 1. Course of primary L. monocytogenes infection. BALB/c mice were infected with 2 × 103 listeria i.v. (filled symbols) or with 1 × 109 listeria p.o. (open symbols). Bacterial titers in spleen, liver, MLN, SI, caecum, and LI were determined at the days indicated. Results are representative of two independent experiments.

5

spleen

4

4

SI

3

3 2

log CFU

MHC class I tetramers were constructed as previously described [8,13]. For flow cytometry analysis, 1–2 × 106 cells were incubated for 15 min at 4 °C with rat serum, antiCD16/CD32 mAb and streptavidin (Molecular Probes) in PBS containing 0.5% BSA and 0.01% sodium azide. After incubation, cells were stained for 60 min at 4 °C with Cy5conjugated anti-CD8a mAb, FITC-conjugated anti-CD62L mAb, and PE-conjugated MHC class I-LLO91–99 tetramers (LLO91–99 = listeriolysin-O epitope amino acids 91–99). Subsequently, cells were washed with PBS, 0.5% BSA, 0.01% sodium azide, and diluted in PBS. Propidium iodide was added prior to four-color flow cytometry analysis.

2

4

liver

3

8

caecum

6 4

2

2

4

MLN 8

LI

6 4 2

3 2 13 3

5

8

1

5

8

day post infection Fig. 2. Course of secondary L. monocytogenes infection. BALB/c mice were infected with 2 × 103 listeria i.v. or with 1 × 109 listeria p.o. After 2 months, mice were re-infected via the same route used for primary infection with 1 × 105 listeria i.v. (filled symbols) or 5 × 109 listeria p.o. (open symbols). Bacterial titers in spleen, liver, MLN, SI, caecum, and LI were determined at the days indicated. Results are representative of two independent experiments.

or only low numbers of listeria were found in the intestine and in the MLNs. At day 5, L. monocytogenes was eradicated from all organs. Secondary p.o. infection resulted in high bacterial titers in caecum and LI at day 1 of infection, but bacteria were rapidly cleared from these organs. In the SI,

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bacteria were only occasionally detected. Secondary p.o. infection was followed by limited dissemination, and very low numbers of bacteria could be recovered from spleen, liver, and MLNs. 3.2. Anti-listeria CD8+ T cell response in intestinal tissues To quantify the listeria-specific T cell response, we used MHC class I tetramers, which recognize CD8+ T cells specific for LLO91–99. Mice were infected either i.v. or p.o. with L. monocytogenes. At different time points after infection, lymphocytes from spleen, liver, and various intestinal tissues were analyzed for the presence of LLO91–99-specific T cells. Results of day 9 after primary infection are shown in Fig. 3. Spleen and liver, the main sites of bacterial replication, harbored high frequencies of LLO91–99-specific CD8+ T cells, and in both organs, frequencies were similar regardless of the route of infection. Additionally, high frequencies of memory T cells were detected in these organs (data not shown). Frequencies of LLO91–99-specific CD8+ T cells differed markedly in intestinal tissues, and depended on the route of infection (Fig. 3). After p.o. infection, LLO91–99-specific CD8+ T cells were abundant in the lymphocyte populations isolated from the lamina propria of the SI and LI (SILPLs and LILPLs, respectively). Lower but still significant levels of LLO91–99-specific CD8+ T cells were detected in the MLNs, the PPs and the IEL populations of SI and LI (SIIELs and LIIELs). Compared with the spleen, the kinetics of the T cell response in the intestine was slightly delayed, and we still observed high T cell frequencies at day 12 of infection (data not shown). Frequencies of memory T cells were particularly high in SILPLs and LILPLs, but memory cells could also be found in all other intestinal tissues, although at far lower levels (data not shown). After i.v. infection, we detected listeria-specific CD8+ T cells in all intestinal tissues. However, compared with p.o. infection, the response in the intestine was significantly reduced in magnitude and in the levels of memory CD8+ T cells detected 6–8 weeks after infection (Fig. 3 and not shown). After 6–8 weeks, mice were re-infected via the same route used for the primary infection, and the LLO91–99-specific CD8+ T cell response was quantified (Fig. 4). In all organs analyzed, we observed an accelerated and increased LLO91– + 99-specific CD8 T cell response, which peaked around day 6 of re-infection. After p.o. re-infection, a strong response was observed in all intestinal tissues. The strongest response occurred in the lamina propria of SI and LI. Intravenous re-infection also caused a secondary T cell response in the intestinal tissues—but similarly to the primary infection—the response was significantly reduced compared with p.o. infection. 3.3. Anti-listeria CD8+ T cell response in intestinal tissues depends on CD28 costimulation During systemic infection, the generation of listeriaspecific CD8+ splenocytes partially depends on CD28 co-

Fig. 3. LLO91–99-specific CD8+ T cell response following primary L. monocytogenes infection. BALB/c mice were infected with 2 × 103 listeria i.v. or with 1 × 109 listeria p.o. At day 9, cells from different tissues were isolated, stained with Cy5-conjugated anti-CD8a mAb, FITC-conjugated antiCD62L mAb, and PE-conjugated LLO91–99 MHC class I tetramers. Cells were analyzed by flow cytometry after the addition of propidium iodide. Figures show live-gated, propidium-iodide-negative CD8a+ cells and are representative results for individually analyzed mice, from three mice per group. Numbers give percentage values of cells in the quadrant for CD8+ T cells only. Results are representative of three independent experiments.

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15.0 12.5 10.0 7.5 5.0

0.0

+/+ -/+/+ liver -/+/+ SiLPL -/+/+ SiIEL -/+/+ LiLPL -/-

2.5

spleen

% LLO91-99 tetramer+ CD62Lof CD8+ T cells

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Fig. 5. LLO91–99-specific CD8+ T cell response to L. monocytogenes infection in CD28–/– mice. CD28+/+ and CD28–/– BALB/c mice were infected p.o. with 1 × 109 listeria. After 8 days, mice were killed, and cells isolated from different organs were analyzed as described for Fig. 3. Results are representative of two independent experiments.

reduction in frequency and total numbers of specific CD8+ T cells was equally profound in spleen, liver and intestinal mucosa. Analysis of the activation marker CD62L on CD8+ T cells isolated from spleen and liver revealed that the impaired T cell response was not restricted to LLO91–99-specific CD8+ T cells, since CD28–/– mice demonstrated an overall reduction in frequencies of activated CD62Llow T cells after L. monocytogenes infection (not shown). Consistent with the reduced listeria-specific CD8+ T cell response, CD28–/– mice were more susceptible to oral L. monocytogenes infection and suffered from impaired clearance of L. monocytogenes (not shown). 3.4. Specific T cell response in intestinal tissues after DNA immunization

Fig. 4. LLO91–99-specific CD8+ T cell response during secondary L. monocytogenes infection. BALB/c mice were infected i.v. with 2 × 103 listeria or p.o. with 1 × 109 listeria. After 56 days, mice were re-infected via the same route used for primary infection with 1 × 105 listeria i.v. or 5 × 109 listeria p.o. At day 6 of secondary infection, cells were analyzed as described for Fig. 3. Figure shows representative results for individually analyzed mice, from three mice per group. Results are representative of three independent experiments.

stimulation [13]. However, it remains unclear whether CD8+ T cell responses in peripheral tissues require CD28 costimulation as well, or whether organ-specific mechanisms bypass the need for CD28. To analyze the role of CD28 in mucosal tissues, we infected CD28-deficient mice back-crossed on the BALB/c background and control mice p.o. with L. monocytogenes and investigated the LLO91–99-specific CD8+ T cell response in these mice (Fig. 5 and Table 1). Although LLO91–99-specific T cells were detected in all organs from CD28–/– mice, the response was markedly reduced, and the

The presence of LLO91–99-specific CD8+ T cells in the intestinal mucosa after both i.v. and p.o. infection suggested that the tissue distribution pattern of these T cells was in part independent of the site of primary infection. To analyze this phenomenon in more detail, we immunized mice with the Table 1 LLO91–99-specific CD8+ T cell response to L. monocytogenes infection in CD28–/– mice Organ Spleen Liver SIIELs SILPLs LILPLs

LLO91–99-specific CD8+ T cells per organ CD28–/– mice CD28+/+ mice 1.58 ± 0.64 × 105 0.24 ± 0.10 × 105 8.51 ± 0.86 × 104 0.43 ± 0.27 × 104 4 3.17 ± 1.42 × 10 0.84 ± 0.28 × 104 1.69 ± 0.73 × 105 0.21 ± 0.10 × 105 3 5.95 ± 2.05 × 10 1.04 ± 0.40 × 103

CD28+/+ and CD28–/– mice were infected p.o. with 1 × 109 listeria. After 8 days, mice were killed and cells isolated from different organs were counted and analyzed as described in Fig. 3. Results represent mean ± standard deviation of three individually analyzed mice per group and are representative of two independent experiments.

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plasmid pChly, which contains the gene for LLO under the control of a eukaryotic promoter. In contrast to bacteria, this DNA vector does not replicate in vivo and does not actively spread throughout the body after vaccination. For gene-gun application, the DNA is coupled to gold particles, which further limits dissemination of the DNA. Therefore, presentation of LLO-derived peptides should be virtually restricted to the site of DNA application. Since a single DNA immunization resulted in only very low frequencies of LLO91–99specific CD8+ T cells [15], mice were primed by oral infection with L. monocytogenes and after 3 months, challenged either by a second oral infection or by DNA boost with the LLO gene (Fig. 6). DNA immunization increased frequencies of LLO91–99-specific CD8+ T cells in all organs analyzed. This response was not restricted to lymphoid organs but also detectable in the intestinal mucosa and the liver. The same phenomenon was observed, although with lower T cell frequencies, when gene-gun immunization was used for primary and secondary immunization (data not shown). After p.o. infection or DNA immunization, cells from intestinal tissues were analyzed for specific IL-2 and IFN-c production (Fig. 7). In vitro re-stimulation with peptide induced marginal amounts of IL-2 in CD8+ T cells. However, LLO91–99-induced IFN-c production was detected in CD8+ T

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Fig. 7. LLO91–99-specific IFN-c and IL-2 production of CD8+ T cells after secondary L. monocytogenes infection or DNA boost immunization. Mice were p.o. infected with 1 × 109 listeria. After 2 months, mice were reinfected p.o. with 5 × 109 listeria, immunized with DNA containing the LLO gene (pChly), or left untreated. Six days later, cells isolated from different tissues were incubated for 5 h with 10–6 M LLO91–99 peptide and analyzed for IFN-c and IL-2 production with intracellular cytokine staining and flow cytometry. Figure shows CD8a+ gated cells and are representative results of individually analyzed mice, from three mice per group. Numbers give percentage values of cells in the quadrant. Isotype controls for the cytokine mAb gave less than 0.05% positive cells in all samples analyzed. Results are representative of two independent experiments.

cells from all organs analyzed, including the LI mucosa. These cells were present both after DNA immunization and p.o. infection. Frequencies of IFN-c-producing CD8+ T cells were generally lower than frequencies observed with the corresponding tetramers. After L. monocytogenes infection, we observed ratios of 1:2 to 1:3 for IFN-c+ versus tetramer+ CD8+ T cells. In contrast, after gene-gun immunization, ratios were higher, frequently close to a 1:1 ratio (not shown). These results indicate that both after L. monocytogenes infection and gene-gun immunization, LLO91–99-specific CD8+ T cells isolated from the intestinal mucosa were, at least in terms of IFN-c production, functional T cells. 4. Discussion

Fig. 6. Response to DNA boost immunization of L. monocytogenes-primed mice. Mice were p.o. infected with 1 × 109 listeria. After 2 months, mice were re-infected with 5 × 109 listeria p.o., immunized with DNA containing the LLO gene (pChly), or left untreated. Six days later, cells from different tissues were analyzed as described for Fig. 3. Figure shows representative results for individually analyzed mice, from three mice per group. Results are representative of two independent experiments.

In our study, we used the model of systemic (i.v.) and oral infection of mice with L. monocytogenes to compare the CD8+ T cell responses in different intestinal tissues, liver and spleen. Cossart and colleagues recently demonstrated that internalin A of L. monocytogenes binds with high affinity to human and guinea pig E-cadherin but only with low affinity to mouse E-cadherin. Furthermore, in transgenic mice expressing human E-cadherin on the intestinal epithelium, L.

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monocytogenes shows increased enterocyte invasion and enhanced transmigration through the intestinal mucosa [18]. Thus, the question arises, whether the mouse is a valid model for analyzing oral L. monocytogenes infections. In our experimental model, we used oral infection doses 10–100-fold lower than those used by Cossart and colleagues to infect E-cadherin transgenic mice and still achieved consistent systemic infection of wild-type animals. Currently, we have no explanation for the discrepancy. Since we do not observe L. monocytogenes in spleen and liver at day 1 post-oral infection, we can largely exclude systemic infection due to injury of mice during oral gavage. L. monocytogenes possesses several internalins, and only for some of them the function is known [19,20]. Therefore, it is possible that in addition to internalin A, other internalins are involved in the transmigration of L. monocytogenes through the intestinal wall. After oral infection, listeria were detected in all parts of the intestine. Particularly the caecum and LI contained high L. monocytogenes titers over an extended time period. L. monocytogenes infects cells of the intestinal epithelium and can cross the intestinal mucosa in the absence of M cells and PPs [21–24]. It is, therefore, possible that caecum and LI are main sources for the spread of L. monocytogenes into deeper tissues. Two recent studies demonstrated that L. monocytogenes indeed disseminates from the LI, and it has been argued that the LI is the main source of systemic spreading [3,22]. Consistent with the high numbers of listeria in the SI and LI, we detected high CD8+ T cell frequencies in the lamina propria of both intestinal compartments. IELs contained only modest frequencies of specific CD8+ T cells. LLO91–99-specific T cells from all organs analyzed, including the intestinal epithelium, expressed a CD8a+ CD8b+ and CD4– phenotype characteristic for conventional CD8+ T cells and distinct from the CD8a+ CD8b– phenotype of the majority of intraepithelial CD8+ T cells [25]. When calculated only for the conventional CD8a+ CD8b+ T cell population, frequencies of LLO91–99-specific CD8+ IEL cells were close to frequencies detected in the lamina propria (M.K. and H.W.M., unpublished observation). After oral infection, we observed rapid bacterial dissemination and high bacterial titers in spleen and liver. Spleen and liver also contained high frequencies of listeria-specific CD8+ T cells consistent with a strong T cell response at sites of bacterial replication. In all organs, the CD8+ T cell response peaked at day 9 of the primary response and then declined. Concomitant with the slow clearance of bacteria from the intestine and liver, we observed a delayed contraction period of the CD8+ T cell response in these tissues. Listeria-specific CD8+ memory T cells were most prominent in spleen, liver and LPLs, the tissues with the highest frequencies during the peak of response, but could also be detected in MLNs, PPs and IELs of SI and LI. Our observations for different compartments of the intestine are consistent with and extend results recently published for the mucosa of the SI [9,11]. An unexpected result, which was also reported in these studies, was the low frequencies of specific CD8+ T cells in the PPs and MLNs.

Although PPs line the SI, which harbored L. monocytogenes, and MLNs contained high numbers of bacteria, responses were marginal compared with those in other tissues analyzed. Currently, we have no explanation for this observation. There could be a failure to generate listeria-specific-CD8+ T cells in these tissues, or specific T cells were generated but either rapidly emigrated out of these tissues or died by mechanisms such as activation-induced cell death. After secondary oral infection, listeria were sporadically recovered from the SI. We detected high listerial titers in LI and caecum, but listeria were rapidly cleared from these parts of the intestine. This observation implies the existence of an acquired immune mechanism that is responsible for the fast removal of listeria from the intestine. Currently, we can only speculate on the nature of this mechanism. Listeria-specific antibodies could prevent adhesion of L. monocytogenes to the mucosa or could agglutinate the bacteria. Both mechanisms would result in an accelerated passage of L. monocytogenes through the intestine. It is also possible that listeria in the intestinal lumen are mainly derived from listeria replicating in the intestinal epithelium. Control of L. monocytogenes replication in epithelial cells by listeria-specific T cells would consequently result in reduced shedding of bacteria into the intestinal lumen. Our current studies aim to elucidate these mechanisms. In all internal organs, we observed only small numbers of L. monocytogenes following secondary oral infection, which was probably due to both rapid listerial clearance from the intestine and accelerated secondary immune response. Despite the low bacterial numbers, a secondary LLO91–99specific CD8+ T cell response was identified in all organs, indicating that listerial antigen was available in sufficient amounts to induce the T cell response. This result is consistent with the recent observation that CD8+ T cells require antigen only during the initial phase and are then driven by antigen-independent mechanisms [26–29]. The tissue distribution of the secondary CD8+ T cell response was similar to that observed after primary infection with the strongest responses in liver, spleen and lamina propria of the SI and LI. After oral L. monocytogenes infection of CD28–/– mice, only low numbers of specific CD8+ T cells were detected in all tissues analyzed. CD28, therefore, differs from other co-stimulatory signals such as those provided by CD40/CD40L interaction, for which tissue-specific requirements were observed during L. monocytogenes infection [11,30]. Furthermore, CD28 co-signaling is mandatory in all tissues for the generation of a CD8+ T cell response during L. monocytogenes infection. The CD28 signal is, therefore, unique and cannot be replaced by signals mediated via other co-stimulatory molecules such as the related ICOS molecule or members of the TNF/TNFR family [31], or via local inflammatory or “danger” signals and inflammatory cytokines such as IL-12 [32]. Compared with oral infection, i.v. infection led to a different pattern of bacterial dissemination. We detected high bacterial titers in spleen and liver, but could only sporadically

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find listeria in MLNs and intestine. These observations are in contrast to those of Nichterlein et al. [33] who could detect L. monocytogenes in the feces of i.v. infected animals, which implies infection of intestinal tissues and shedding of listeria into the intestinal lumen. Spleen and liver contained high frequencies of listeria-specific CD8+ T cells at the peak of response and significant populations of CD8+ memory T cells. However, LLO91–99-specific CD8+ T cells were also detected in all intestinal tissues, although at lower levels than after oral infection. Finally, the lamina propria of both the SI and LI harbored a significant population of LLO91–99specific memory CD8+ T cells after systemic infection. Similar results have recently been reported for the SI in the L. monocytogenes infection model and in a virus model using systemic vesicular stomatitis virus (VSV) infection [9–12]. These results, together with our own data, demonstrate accumulation of effector and memory T cells in non-lymphoid tissues such as liver and intestinal mucosa. Although a single DNA immunization of mice previously infected with L. monocytogenes was not very effective in inducing an LLO91– + 99-specific CD8 T cell recall response, we still detected a significant increase in the frequency of these cells in intestinal tissues. Thus, both infection and DNA immunization studies provide intriguing evidence that this accumulation happens, at least in part, independently of local antigen expression or local inflammation. In contrast to infectious agents, the DNA vaccine does not replicate. In addition, by using gene-gun immunization, mice received only small amounts of DNA coupled to gold beads. Therefore, dissemination of DNA should be marginal and largely restricted to the site of application, the abdominal skin, and it is unlikely, although we cannot formally exclude it, that DNA-loaded antigen-presenting cells migrate from the abdominal skin to the intestine. However, we still observed accumulation of specific T cells in liver and intestinal mucosa, indicating that migration of these cells was independent of the presence of antigen. Our observation raises the question of whether the local CD8+ T cell response in the intestinal mucosa following oral infection was only due to migration of specific T cells into these tissues, or whether local priming of naive CD8+ T cells also occurred. In other words, is a listeria-specific CD8+ T cell response induced in the mucosa, independently of the systemic response? After oral infection, L. monocytogenes disseminates into the PPs and the MLNs within hours, and within 1–2 days, bacteria can be isolated from spleen and liver. Due to limits in the sensitivity of our assays, we can detect specific T cells only by day 4–5. Therefore, we cannot directly address the mechanisms and site of initial T cell priming. Indirect evidence points to a common origin for listeria-specific CD8+ T cells of internal organs and the intestinal mucosa. In all tissues, specific CD8+ T cells display a CD8ab+ CD4– phenotype, and the kinetics of the response in the intestine and internal organs is similar after both i.v. and p.o. infection ([9,11], and own results). Yet, evidence for the independent origin of listeria-specific CD8+ T cells in the

15

intestinal mucosa also exists. LLO91–99-specific CD8+ T cells express different TCR-Vb repertoires, and H-2M3restricted CD8+ T cells are underrepresented in the intestinal mucosa. Furthermore, listeria-specific CD8+ T cells of the mucosa are particularly dependent on help from CD4+ T cells and on CD40 co-stimulation [9,11]. However, we cannot exclude that these features are consequences of adaptation of immigrants to the local environment of the intestinal mucosa. Our current aims are to characterize the initial events and the site of CD8+ T cell priming in more detail. In conclusion, our results suggest that accumulation of antigen-specific CD8+ effector and memory T cells in the intestinal mucosa is caused, at least in part, by an antigenindependent migration pattern intrinsic to these cells. This is consistent with the general observation that activated and memory T cells migrate from lymphoid into non-lymphoid tissues. This migration is probably enhanced by the local infection and inflammation in the intestinal mucosa after oral infection. The presence of listeria antigens and the inflammatory environment will then induce proliferation of specific T cells and further enhance accumulation of effector and memory T cells in the intestinal mucosa.

Acknowledgements The authors thank Dr. Robert Hurwitz and Dr. Joachim Fensterle for assistance in the preparation of tetramers and DNA immunization, respectively, and Manuela Stäber for purification and labeling of antibodies. M. Kursar was supported by the Graduiertenkolleg 276/2. S.H.E. Kaufmann acknowledges support from the Fonds Chemie.

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