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Organ-Specific CD4⁺ T Cell Response During Listeria monocytogenes Infection¹

Mischo Kursar^2,*, Kerstin Bonhagen^2,, Anne Köhler^*, Thomas Kamradt, Stefan H. E. Kaufmann^* and Hans-Willi Mittrücker^3,*

^* Max-Planck-Institute for Infection Biology and Deutsches Rheumaforschungszentrum, Berlin, Germany

	Abstract

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The immune response against the intracellular bacterium Listeria monocytogenes involves both CD4⁺ and CD8⁺ T cells. We used the MHC class II-presented peptide listeriolysin_189–201 to characterize the organ-specific CD4⁺ T cell response during infection. Systemic listeriosis resulted in a strong peptide-specific CD4⁺ T cell response with frequencies of 1/100 and 1/30 CD4⁺ splenocytes at the peak of primary and secondary response, respectively. This response was not restricted to lymphoid organs, because we detected specific CD4⁺ T cells in all tissues analyzed. However, the tissue distribution of the T cell response was dependent on the route of infection. After i.v. infection, the strongest CD4⁺ T cell response and the highest levels of memory cells were observed in spleen and liver, the major sites of L. monocytogenes replication. After oral infection, we detected a strong response in the liver, the lamina propria, and the intestinal epithelium. These tissues also harbored the highest frequencies of listeriolysin_189–201-specific CD4⁺ memory T cells 5–8 wk post oral infection. Our results show that kinetics and magnitude of the CD4⁺ T cell response and the accumulation of CD4⁺ memory T cells depend on the route of infection and are regulated in a tissue-specific way.

	Introduction

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The course of Listeria monocytogenes infection has been extensively characterized in the mouse (1). After oral uptake, bacteria reach the small intestine, from where they translocate across the intestinal mucosa into the Peyer’s patches (PP).⁴ From there, bacteria spread via the mesenteric lymph nodes (MLN) into spleen and liver. In these organs, L. monocytogenes infects and replicates in macrophages and hepatocytes, and for several days high numbers of bacteria can be isolated from infected organs. Eventually, the acquired immune response controls infection, and bacteria are eradicated from the organism by day 10 of infection (1).

The cytosolic habitat of L. monocytogenes promotes processing and presentation of listerial Ags through the MHC class I pathway. As a consequence, a potent CD8⁺ T cell response is induced which is critical for antilisterial defense (1, 2). Different MHC class I presented T cell epitopes expressed by wild type, and recombinant L. monocytogenes strains have been used to analyze the Listeria-specific CD8⁺ T cell response (3, 4, 5, 6, 7). These studies show strong CD8⁺ T cell responses in spleen, liver, lamina propria, and intestinal epithelium. However, there were differences in the magnitude and kinetics of responses between different organs, indicating an organ-specific regulation of CD8⁺ T cells (4, 5, 6, 7).

L. monocytogenes infection also induces a CD4⁺ T cell response, and there is evidence that CD4⁺ T cells participate in protection (1, 8, 9). Transfer of CD4⁺ T cells from infected into naive mice confers partial protection in recipients and MHC class II-deficient mice, or mice in which CD4⁺ T cells were depleted by Ab treatment suffer from reduced protection against listeriosis (2, 9, 10). During L. monocytogenes infection, CD4⁺ T cells differentiate into Th1 cells and production of Th1 cell-derived cytokines, such as IFN-, is regarded central to CD4⁺ T cell-mediated protection (1, 11). There is very limited information on the kinetics, magnitude, and the tissue distribution of the CD4⁺ T cell response against L. monocytogenes (8), and only recently immunodominant CD4⁺ T cell epitopes have been identified which allow tracking of Listeria-specific CD4⁺ T cells during infection (12, 13, 14, 15).

In this study, we use an immunodominant CD4⁺ T cell epitope derived from listeriolysin (LLO) to characterize and quantify a Listeria-specific CD4⁺ T cell response in different lymphoid and nonlymphoid organs after both oral and systemic infection.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibodies

Rat IgG Abs, anti-CD16/CD32 mAb (clone: 2.4G2), anti-IFN- mAb (clone: R4-6A2, rat IgG1), and anti-CD4 mAb (clone: YTS191.1) were purified from rat serum or hybridoma supernatants with protein G-Sepharose. Abs were Cy5- or FITC-conjugated according to standard protocols. FITC-conjugated anti-TNF- mAb (clone: MP6-XT22, rat IgG1), PE-conjugated anti-IL-10 mAb (clone: JES5-16E3, rat IgG2b), PE-conjugated anti-IL-2 mAb (clone: JES6-5h4, rat IgG2b), FITC- and PE-conjugated rat IgG1 isotype control mAb (clone: R3-34), and rat IgG2b isotype control mAb (clone: A95-1) were purchased from BD PharMingen (San Diego, CA).

Infection of mice

C57BL/6 mice were bred in our facility, and experiments were conducted according to the German animal protection laws. Mice were infected with L. monocytogenes strain EGD. Bacteria were grown overnight in tryptic soy broth (TSB), washed twice in PBS, aliquoted in PBS/10% glycerol, and stored at -80°C. Aliquots were thawed and bacterial titers were determined by plating serial dilutions on TSB agar plates. For i.v. infection, bacteria were diluted and injected in a volume of 200 µl PBS into the lateral tail veins. For per os (p.o.) infection, L. monocytogenes was grown overnight in TSB and washed twice in PBS. Bacterial density was determined by absorption at 600 nm, and bacteria were appropriately diluted in PBS (an OD₆₀₀ value of 1 is equivalent to 10⁹ bacteria/ml). Bacteria were applied in 200 µl PBS by gastric intubation. The bacterial dose was controlled by plating dilutions of the inoculum on TSB agar plates. Mice were primary infected with 5 x 10³ bacteria i.v., or 5 x 10⁹ bacteria p.o. After 5–8 wk, mice were secondary infected with 10⁵ bacteria i.v. or 5 x 10⁹ bacteria p.o.

For determination of bacterial burdens in organs, mice were killed and organs were homogenized in PBS. The small intestine was 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 a 48-h incubation at 30°C.

Purification of cells from different tissues

Spleens were removed and single-cell suspensions were prepared using an iron mesh sieve. RBCs were lysed and spleen cells were washed twice with RPMI 1640 medium supplemented with glutamine, Na-pyruvate, 2-ME, penicillin, streptomycin, and 10% heat-inactivated FCS (complete RPMI medium). PP and MLN were excised, single-cell suspensions were prepared using an iron mesh sieve, and cells were washed twice with complete RPMI medium. Intraepithelial lymphocytes (IEL) were isolated as previously described (16). Briefly, after the excision of PP, small intestines of individual mice were cut open and washed twice in PBS/1% BSA. Small 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 x g. Cells were collected from the interface of the gradient and washed in complete RPMI medium. Lamina propria lymphocytes were isolated by a modified version of the protocol described (16). After IEL isolation, the small intestine was 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 Typ VIII (Sigma-Aldrich, 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 x g, and the supernatants were collected. This step was repeated four times. Cells from pooled supernatants were further purified by a 40%/70% Percoll gradient.

In vitro restimulation of cells and flow cytometric determination of cytokine expression

Cells (4 x 10⁶) were cultured in a volume of 2 ml complete RPMI medium. Spleen cells were stimulated for 5 h with 10^-6 M of the peptide LLO_189–201 (WNEKYAQAYPNVS; Jerini Bio Tools, Berlin, Germany). During the final 4 h of culture, 10 µg/ml Brefeldin A (Sigma-Aldrich) were added. Cultured cells were washed and incubated for 10 min with rat IgG Abs and anti-CD16/CD32 mAb to block nonspecific Ab binding. Subsequently, cells were stained with Cy5-conjugated anti-CD4 mAb, and after 30 min on ice, cells were washed with PBS and fixed for 20 min at room temperature with PBS/4% paraformaldehyde. Cells were washed with PBS/0.1% BSA, permeabilized with PBS/0.1% BSA/0.5% saponin (Sigma-Aldrich), and incubated in this buffer with rat IgG Abs and anti-CD16/CD32 mAb. After 5 min, FITC- or PE-conjugated anti-cytokine or isotype control mAb were added. After a further 20 min at room temperature, cells were washed with PBS and fixed with PBS/1% paraformaldehyde. Cells were analyzed using a FACSCalibur and the CellQuest software (BD Biosciences, Mountain View, CA). With the exception of PP samples, we routinely acquired 50,000–100,000 lymphocyte-gated CD4⁺ T cells from each sample.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
L. monocytogenes titers in different organs after i.v. and p.o. infection

Mice were infected i.v. or p.o. with L. monocytogenes, and bacterial burdens in various organs were determined at different time points postinfection (Fig. 1). After i.v. infection, mice showed high bacterial titers in spleen and liver at days 3 and 5, followed by rapid bacterial clearance in most of the mice analyzed. Only sporadically did we detect bacteria in MLN and small intestine. In contrast, oral infection resulted in high bacterial titers in the small intestine. Clearance of Listeria was slow, and at day 12 postinfection, low numbers of Listeria were still recovered from the small intestine. Bacteria were detected in the MLN at day 1, and reached high numbers at days 3 and 5 postinfection. In spleens, bacteria were isolated at days 3, 5, and 8 post oral infection, but titers did not reach the levels observed after i.v. infection. Oral infection resulted in high bacterial titers in the liver, and bacterial clearance from this organ was delayed compared with that after i.v. infection.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Course of primary L. monocytogenes infection. Mice were infected i.v. with 5 x 103 Listeria (), or p.o. with 5 x 109 Listeria (), and bacterial titers in spleen, liver, MLN, and small intestine were determined at the time points indicated.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Course of secondary L. monocytogenes infection. Mice were infected i.v. with 5 x 103 Listeria, or p.o. with 5 x 109 Listeria. After 2 mo, mice were reinfected via the same route used for primary infection with i.v. 1 x 105 Listeria (), or p.o. with 5 x 109 Listeria (). Bacterial titers in spleen, liver, MLN, and small intestine were determined at the time points indicated.

A series of immunodominant CD4⁺ T cell epitopes from LLO and p60 has been characterized for BALB/c and C57BL/6 mice (12). Among these epitopes, LLO_190–201 was particularly strong, and induced a high number of IFN--positive spots in the ELISPOT assay from spleens of C57BL/6 mice infected with L. monocytogenes (12). To determine whether we could detect a specific response by intracellular cytokine staining, C57BL/6 mice were infected p.o. with L. monocytogenes, and at day 8 postinfection, spleen cells were restimulated with LLO_189–201 and analyzed for cytokine expression by flow cytometry. LLO_190–201, the peptide described by Geginat et al. (12), and the peptide LLO_189–201, which was used throughout this study, resulted in identical frequencies for cytokine positive cells (data not shown). Fig. 3 shows that IFN-⁺CD4⁺ T cells were detected in spleens from infected but not from naive mice. LLO_189–201-specific CD4⁺ T cells were not restricted to lymphoid tissues but were also detected in the liver and intestinal tissues. High frequencies of IFN-⁺CD4⁺ T cells were observed in cells isolated from spleen, liver, lamina propria, and intestinal epithelium. In contrast, frequencies of IFN-⁺CD4⁺ T cells in the MLN and in PP were only slightly above background levels defined by isotype control staining and staining of nonstimulated cells. In all tissues, two distinct populations of IFN-⁺IL-2^- and IFN-⁺IL-2⁺CD4⁺ T cells were identified. Staining for IL-2 already gave relatively high frequencies in cells that were not restimulated with peptide, and there was no significant increase after peptide restimulation. Therefore, our results do not allow estimates of LLO_189–201-specific IL-2⁺CD4⁺ T cells, particularly at early time points of infection with low frequencies of specific T cells. Cells were also analyzed for the expression of TNF- and IL-10. Restimulation with peptide did not increase frequencies of IL-10⁺CD4⁺ T cells compared with nonstimulated controls at any time point of infection and in all tissues analyzed. Intracellular staining for TNF- resulted in CD4⁺ T cell frequencies similar to those for IFN- (Fig. 4 and data not shown).

View larger version (35K): [in this window] [in a new window]   FIGURE 3. IL-2 and IFN- production of CD4+ T cells after peptide restimulation in vitro. Mice were infected p.o. with 5 x 109 L. monocytogenes. After 8 days, cells from different tissues were restimulated for 5 h with the peptide LLO189–201 (10-6 M). Cells were stained extracellularly with Cy5-conjugated anti-CD4 mAb, intracellularly with FITC-conjugated anti-IFN- mAb and PE-conjugated anti-IL-2 mAb or the corresponding isotype control mAb, and were analyzed by flow cytometry. Dot blots show CD4-gated lymphoid cells from individual mice and are representative for three mice per group and three independent experiments. Figures give percent-values of cytokine-positive cells of CD4-gated lymphoid cells. Staining with FITC-conjugated rat-IgG1 usually resulted in <0.05%, and with PE-conjugated rat-IgG2b in <0.10% positive events for CD4-gated cells (data not shown). LPL, lamina propria lymphocytes (small intestine).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Kinetics of LLO189–201-specific CD4+ T cells in different organs after oral L. monocytogenes infection. Mice were infected p.o. with 5 x 109 L. monocytogenes. At the indicated days, cells from different tissues were restimulated with the peptide LLO189–201 for 5 h. Cells were stained with Cy5-conjugated anti-CD4 mAb and FITC-conjugated IFN- mAb or TNF- mAb, and analyzed by flow cytometry. Graphs indicate percent-values of IFN-+ and TNF-+ cells of CD4+ cells from different tissues and represent mean ± SD of three mice per group and time point.

Kinetics of Ag-specific IFN- and TNF- production by CD4⁺ T cells in response to oral L. monocytogenes infection

Mice were infected p.o. with L. monocytogenes, and the kinetic of the LLO_189–201-specific CD4⁺ T cell response was investigated in secondary lymphoid and in nonlymphoid tissues (Fig. 4). Overall, frequencies of TNF-⁺ and IFN-⁺CD4⁺ T cells were comparable in all tissues and at all time points analyzed. LLO_189–201-specific IFN- and TNF- production was visible in all tissues by day 6 and peaked at day 8 postinfection. In the experiment shown, we identified up to 1% of cytokine-secreting CD4⁺ T cells in spleen and liver, and >4% in the lamina propria. After p.o. infection, we observed some variation in the frequencies of LLO_189–201-specific IFN-⁺ or TNF-⁺CD4⁺ T cells in the lamina propria between different experiments (mean values varied between 1 and 4% of CD4⁺ T cells at the peak of response; data not shown and Fig. 5). In spleens, frequencies of cytokine-secreting CD4⁺ T cells rapidly declined, and by days 12–18, a level of 0.2–0.4% of cytokine-secreting cells was reached, which remained stable throughout the observation period of 6 wk. The decline of LLO_189–201-specific CD4⁺ T cell frequencies was delayed in liver and lamina propria, and memory frequencies of >0.5% were observed in the lamina propria 6 wk after infection. In MLN and PP, frequencies of LLO_189–201-specific IFN-⁺ and TNF-⁺CD4⁺ T cells showed only a moderate increase after p.o. infection; and particularly in the PP, there was a high degree of variation, which can be explained by the very low frequencies of cytokine-secreting CD4⁺ T cells in these tissues, that was only slightly above our detection limit.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Primary and secondary LLO189–201-specific CD4+ T cells responses in different tissues after p.o. and i.v. L. monocytogenes infection. Mice were infected with L. monocytogenes p.o. (5 x 109) or i.v. (5 x 103). After 37 or 45 days, mice were reinfected p.o. and i.v. with 5 x 109 and 1 x 105 L. monocytogenes, respectively. At the days indicated after primary and secondary infection, cells from different organs were restimulated with the peptide LLO189–201. Cells were stained with Cy5-conjugated anti-CD4 mAb and FITC-conjugated IFN- mAb, and analyzed by flow cytometry. Bars represent mean ± SD of tissues from three mice per time point. Results are representative for at least two individual experiments per time point.

To analyze the localization of CD4⁺ T cell responses following different routes of infection, mice were infected p.o. and i.v., and frequencies and total numbers of LLO_189–201-specific CD4⁺ T cells were determined in different tissues (Figs. 5 and 6). After oral infection, we observed strong responses in spleen, liver, and lamina propria, and weaker responses in intestinal epithelium, MLN, and PP. At day 8 postinfection, 10⁵, 6 x 10⁴, and 1.5 x 10⁴ specific CD4⁺ T cells were recovered from spleen, liver, and lamina propria, respectively (Fig. 6). Six weeks postinfection, significant numbers of specific CD4⁺ T cells were still detectable in spleen, liver, MLN, lamina propria, and intestinal epithelium. Compared with the peak of the response, numbers were particularly high in the lamina propria and the intestinal epithelium. Primary i.v. infection resulted in a different tissue distribution of the response. Compared with the p.o. infection, higher numbers of LLO_189–201-specific CD4⁺ T cells were recovered from the spleen and reduced numbers from the liver and intestinal tissues. After 5 wk of infection, levels of memory cells were high in spleen and liver but low in the intestinal mucosa. Similar results were determined for TNF-⁺CD4⁺ T cells (data not shown).

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Total numbers of recovered LLO189–201-specific CD4+ T cells from different organs after p.o. and i.v. L. monocytogenes infection. Mice were infected with L. monocytogenes p.o. (5 x 109) or i.v. (5 x 103). After 37 or 45 days, mice were reinfected p.o. and i.v. with 5 x 109 and 1 x 105 L. monocytogenes, respectively. At the days indicated after primary and secondary infection, cells from different organs were restimulated with the peptide LLO189–201. Cells were stained with Cy5-conjugated anti-CD4 mAb and intracellularly with FITC-conjugated IFN- mAb and analyzed by flow cytometry. Total cell numbers were calculated from the numbers of recovered cells per tissue and the percent-values of CD4+ and IFN-+ cells. Bars represent mean ± SD of tissues from three mice. Results are representative for at least two individual experiments per time point.

Mice were secondary infected with L. monocytogenes via the same route used for primary infection. Both secondary p.o. and i.v. infection accelerated the response, peaking at days 5–7 postinfection (Figs. 5 and 6, and data not shown). Compared with primary i.v. infection, secondary i.v. infection induced a 2- and 4-fold increase in frequencies and numbers of LLO_189–201-specific CD4⁺ T cells in spleens and livers, respectively. In addition, numbers of specific CD4⁺ T cells recovered from lamina propria and intestinal epithelium during secondary i.v. infection were significantly increased. In contrast, we observed only a modest response after secondary p.o. infection with L. monocytogenes. Although frequencies and numbers of LLO_189–201-specific CD4⁺ T cells were increased compared with the level observed 5–8 wk after primary infection, numbers did only marginally exceed or were even lower than those observed at the peak of the primary response.

We also compared the expression profile of IFN- and IL-2 in lymphoid and nonlymphoid organs during primary and secondary L. monocytogenes infection (Fig. 7). During both primary and secondary responses, we observed two populations of IL-2⁺IFN-⁺ and IL-2^-IFN-⁺-specific CD4⁺ T cells. Although there were some variations between individual mice (data not shown), both populations had roughly equal sizes and this pattern was maintained in lymphoid and nonlymphoid organs, and during both primary and memory responses.

View larger version (26K): [in this window] [in a new window]   FIGURE 7. IL-2 and IFN- production of LLO189–201-specific CD4+ T cells during primary and secondary L. monocytogenes infection. Mice were p.o. infected and after 8 wk reinfected p.o. with 5 x 109 L. monocytogenes. Eight days after primary and 7 days after secondary infection, cells from different organs were restimulated with the peptide LLO189–201. Cells were stained with Cy5-conjugated anti-CD4 mAb and intracellularly with FITC-conjugated IFN- mAb and PE-conjugated IL-2 mAb, and analyzed by flow cytometry. Dot blots show CD4-gated lymphoid cells. Figures give percent-values of cytokine-positive cells of CD4-gated lymphoid cells. The bar diagrams show the results from experiments with three mice per group. Left panel, Primary d8; right panel, Secondary d7, gray bars, percentage of IL-2+IFN-+ of CD4+ cells; black bars, percentage of IL-2-IFN- + of CD4+ cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our detection system is based on short-term in vitro restimulation and quantification of cytokine-producing T cells. Ag-specific T cells could be refractory or could produce other cytokines not analyzed in our assays. Because L. monocytogenes induces a strong Th1 response (11), we consider this possibility unlikely and are confident that our assays for TNF- and IFN- detect the vast majority of LLO_189–201-specific CD4⁺ T cells. Due to the relatively high background levels, our IL-2 assay does not allow an interpretation of results for specific CD4⁺ T cells producing only IL-2 during infection. However, it does allow to discriminate between IL-2^-IFN-⁺ and IL-2⁺IFN-⁺ LLO_189–201-specific CD4⁺ T cells. During the first 5 days of infection, frequencies of LLO_189–201-specific CD4⁺ T cells were too low to allow determination of their cytokine profile with our assay. Therefore, we cannot state on changes in the profile during the initial phase of CD4⁺ T cell activation and differentiation. However, in the later stages of the response, the cytokine profile of specific CD4⁺ T cells was relatively stable. Although the ratios of the IL-2^-IFN-⁺ and IL-2⁺IFN-⁺CD4⁺ T cell populations varied between individual mice and different organs, there was no clear distinction between lymphoid and nonlymphoid tissues, and both CD4⁺ T cell subpopulations were detected in all organs over the whole observation period of primary and secondary responses. Although we focused on a limited set of cytokines, our results suggest that during the T cell response against L. monocytogenes, specific CD4⁺ T cells acquire a particular cytokine profile which is then maintained throughout infection and independent from the tissue harboring these cells.

After primary i.v. infection, we observed high Listeria titers in spleens and slightly lower titers in livers of infected animals. No Listeria were isolated from MLN or the small intestine. Oral infection resulted in a different profile for Listeria burden. We detected lower titers in spleen, and high titers in liver, MLN, and small intestine. Furthermore, clearance of Listeria from liver and small intestine was delayed. The CD4⁺ T cell response correlated for most tissues with the dissemination pattern of Listeria. After i.v. infection, strong T cell responses and high numbers of memory cells were observed in spleen and liver, the major sites of L. monocytogenes replication. After oral infection, we detected lower frequencies of specific CD4⁺ T cells in the spleen, but strong responses in the liver and intestinal mucosa, particularly in the lamina propria. Consistent with the delayed bacterial clearance from liver and small intestine, there was also a delay in the contraction phase of the specific CD4⁺ T cell response. LLO_189–201-specific CD4⁺ memory T cells persisted in these tissues 5–8 wk postinfection. Frequencies of Listeria-specific CD4⁺ T cells were low in the PP and MLN. Because both tissues are involved in listerial invasion from the intestinal lumen into central organs, the weak T cell response is surprising. However, this observation is not unique to CD4⁺ T cells. A similar phenomenon has been observed for Listeria-specific CD8⁺ T cells (Refs. 4 , 5 , and 7 and our unpublished results). Currently, it is unclear whether the low T cell frequencies observed in PP and MLN are caused by impaired T cell responses, or whether they are due to local apoptosis in, or rapid emigration of Listeria-specific T cells from these tissues following activation.

After secondary i.v. infection, we detected Listeria only at day 1 in spleen and liver, and only in low numbers. However, the transient presence of Listeria apparently sufficed for a secondary CD4⁺ T cell response. Compared with the primary response, this response was accelerated and higher numbers of specific CD4⁺ T cells were recovered from spleen, liver, and intestinal mucosa. In contrast, the response to secondary p.o. infection was weak and not increased compared with the primary response. A similar phenomenon has been observed for CD8⁺ T cells (5). It has been suggested that high frequencies of memory T cells rapidly restrict bacterial replication. Consequently, the availability of listerial Ag should be restricted, resulting in limited T cell activation (5). Our results support this notion, because after secondary oral infection, only a few mice showed bacterial dissemination into deeper tissues. Even in MLN and small intestine, only low numbers of Listeria were detected, and most mice had cleared Listeria from these tissues by day 5. At present, the acquired immune mechanisms responsible for rapid clearance of bacteria from the intestine remain unclear, and current studies in our laboratory are aimed at identifying these mechanisms.

Several features of the CD4⁺ T cell response described in this study are reminiscent of the CD8⁺ T cell response against L. monocytogenes (1, 2, 3, 4, 5, 6, 7). In the spleen, both the CD4⁺ and the CD8⁺ T cell response peak around day 9 post i.v. infection. This peak is followed by a rapid contraction phase for both T cell populations. However, the contraction is less pronounced for CD4⁺ T cells, and in contrast to CD8⁺ T cells, significant numbers of specific CD4⁺ T cells can be recovered from the spleen 5–8 wk postinfection. After p.o. infection, frequencies of Listeria-specific CD4⁺ and CD8⁺ T cells in the liver and the lamina propria exceed frequencies observed in the spleen, and the response in these nonlymphoid organs is extended and shows a prolonged contraction phase. Furthermore, after p.o. infection, both Ag-specific CD4⁺ and CD8⁺ T cell populations generate high frequencies of memory T cells in the intestinal mucosa.

A surprising feature of the CD8⁺ T cell responses against L. monocytogenes was that independent of the route of infection, large populations of effector and memory T cells accumulated in the intestinal mucosa (4, 5, 6, 7). This accumulation was not caused by a spread of L. monocytogenes into mucosal tissues, because the mucosa was devoid of bacteria after systemic infection at all time points analyzed (4). Rather, CD8⁺ T cell migration from lymphoid tissues into nonlymphoid tissues during infection appears to be a general phenomenon (7). In contrast, our results reveal that the tissue accumulation of Listeria-specific CD4⁺ T cells mainly depends on the site of bacterial replication (i.v. infection: spleen and liver; p.o. infection: liver and intestinal mucosa). Furthermore, there was a direct correlation between the initial strength of response in the tissue and the frequency of memory T cells recovered from this tissue. However, during systemic L. monocytogenes infection Listeria-specific CD4⁺ T cells were also identified in the lamina propria and the intestinal epithelium, indicating that the Ag-independent migration pattern also applies for Listeria-specific CD4⁺ T cells. Therefore, homeostasis of CD4⁺ effector and memory T cells appears to be regulated by the local infection as well as general migration patterns of these cells. Further investigations are aimed at elucidating the underlying mechanisms.

	Acknowledgments

 
We thank Manuela Stäber for her excellent technical assistance.

	Footnotes

 
¹ M.K. was supported by the Graduiertenkolleg 276/2, and this work will be part of his PhD thesis.

² M.K. and K.B. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Hans-Willi Mittrücker, Max-Planck-Institute for Infection Biology, Schumannstr. 21/22, 10117 Berlin, Germany. E-mail address: mittruecker{at}mpiib-berlin.mpg.de

⁴ Abbreviations used in this paper: PP, Peyer’s patch; IEL, intraepithelial lymphocyte; LLO, listeriolysin; MLN, mesenteric lymph node; p.o., per os; TSB, tryptic soy broth.

Received for publication November 6, 2001. Accepted for publication April 11, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kaufmann, S. H. E.. 1993. Immunity to intracellular bacteria. Annu. Rev. Immunol. 11:129.[Medline]
2. Ladel, C. H., I. E. Flesch, J. Arnoldi, S. H. E. Kaufmann. 1994. Studies with MHC-deficient knockout mice reveal impact of both MHC I- and MHC II-dependent T cell responses on Listeria monocytogenes infection. J. Immunol. 153:3116.[Abstract]
3. Busch, D. H., I. M. Pilip, S. Vijh, E. G. Pamer. 1998. Coordinate regulation of complex T cell populations responding to bacterial infections. Immunity 8:353.[Medline]
4. Huleatt, J. W., I. Philip, K. Kerksiek, E. G. Pamer. 2001. Intestinal and splenic T cell responses to enteric Listeria monocytogenes infection: distinct repertoires of responding CD8⁺ T lymphocytes. J. Immunol. 166:4065.[Abstract/Free Full Text]
5. Pope, C., S.-K. Kim, A. Marzo, K. Williams, J. Jiang, H. Shen, L. Lefrancois. 2001. Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection. J. Immunol. 166:3402.[Abstract/Free Full Text]
6. Masopust, D., J. Jiang, H. Shen, L. Lefrancois. 2001. Direct analysis of the dynamics of the intestinal mucosa CD8 T cell response to systemic virus infection. J. Immunol. 166:2348.[Abstract/Free Full Text]
7. Masopust, D., V. Vezys, A. L. Marzo, L. Lefrancois. 2001. Preferential localization of effector memory cells in nonlymphoid tissues. Science 291:2413.[Abstract/Free Full Text]
8. Mittrücker, H.-W., A. Köhler, S. H. E. Kaufmann. 2000. Substantial in vivo proliferation of CD4⁺ and CD8⁺ T lymphocytes during secondary Listeria monocytogenes infection. Eur. J. Immunol. 30:1053.[Medline]
9. Kaufmann, S. H. E., E. Hug, U. Vath, I. Müller. 1985. Effective protection against Listeria monocytogenes and delayed-type hypersensitivity to listerial antigens depend on cooperation between specific L3T4⁺ and Lyt2⁺ T cells. Infect. Immun. 48:263.[Abstract/Free Full Text]
10. Baldridge, J. R., R. A. Barry, D. J. Hinrichs. 1990. Expression of systemic protection and delayed-type hypersensitivity to Listeria monocytogenes is mediated by different T-cell subsets. Infect. Immun. 58:654.[Abstract/Free Full Text]
11. Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O’Garra, K. M. Murphy. 1993. Development of TH1 CD4⁺ T cells through IL-12 produced by Listeria-induced macrophages. Science 260:547.[Abstract/Free Full Text]
12. Geginat, G., S. Schenk, M. Skoberne, W. Göbel, H. Hof. 2001. A novel approach of direct ex vivo epitope mapping identifies dominant and subdominant CD4 and CD8 T cell epitopes from Listeria monocytogenes. J. Immunol. 166:1877.[Abstract/Free Full Text]
13. Campbell, D. J., N. Shastri. 1998. Bacterial surface proteins recognized by CD4⁺ T cells during murine infection with Listeria monocytogenes. J. Immunol. 161:2339.[Abstract/Free Full Text]
14. Safley, S. A., P. E. Jensen, P. A. Reay, H. K. Ziegler. 1995. Mechanisms of T cell epitope immunodominance analyzed in murine listeriosis. J. Immunol. 155:4355.[Abstract]
15. Safley, S. A., C. W. Cluff, N. E. Marshall, H. K. Ziegler. 1991. Role of listeriolysin-O (LLO) in the T lymphocyte response to infection with Listeria monocytogenes: identification of T cell epitopes of LLO. J. Immunol. 146:3604.[Abstract]
16. Bonhagen, K., S. Thoma, P. Bland, S. Bregenholt, A. Rudolphi, M. H. Claesson, J. Reimann. 1996. Cytotoxic reactivity of gut lamina propria CD4⁺ T cells in SCID mice with colitis. Eur. J. Immunol. 26:3074.[Medline]

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