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

Antibodies

Rat IgG Abs, anti-CD16/CD32 mAb (clone: 2.4G2), anti-IFN-γ 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-α 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 × 10³ bacteria i.v., or 5 × 10⁹ bacteria p.o. After 5–8 wk, mice were secondary infected with 10⁵ bacteria i.v. or 5 × 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 × 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 × 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 × 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.

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.

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FIGURE 1.

Course of primary L. monocytogenes infection. Mice were infected i.v. with 5 × 10³ Listeria (⋄), or p.o. with 5 × 10⁹ Listeria (♦), and bacterial titers in spleen, liver, MLN, and small intestine were determined at the time points indicated.

Mice were also analyzed after secondary i.v. and p.o. infection (Fig. 2⇓). Only at day 1, low numbers of Listeria were detected in spleens and livers of secondary i.v. infected animals. No or only very low numbers of Listeria were detected in MLN and small intestine. After secondary p.o. infection, Listeria were found in small intestine and MLN at days 3 and 5 postinfection. Compared with the primary infection, titers were lower and Listeria were cleared from most mice by day 5 postinfection. Only sporadically were Listeria isolated from spleens and livers at day 1 after secondary oral infection.

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FIGURE 2.

Course of secondary L. monocytogenes infection. Mice were infected i.v. with 5 × 10³ Listeria, or p.o. with 5 × 10⁹ Listeria. After 2 mo, mice were reinfected via the same route used for primary infection with i.v. 1 × 10⁵ Listeria (⋄), or p.o. with 5 × 10⁹ Listeria (♦). Bacterial titers in spleen, liver, MLN, and small intestine were determined at the time points indicated.

Ag-specific production of cytokines by CD4⁺ T cells from mice infected with L. monocytogenes

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-γ-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-γ⁺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-γ⁺CD4⁺ T cells were observed in cells isolated from spleen, liver, lamina propria, and intestinal epithelium. In contrast, frequencies of IFN-γ⁺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-γ⁺IL-2⁻ and IFN-γ⁺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-α 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-α resulted in CD4⁺ T cell frequencies similar to those for IFN-γ (Fig. 4⇓ and data not shown).

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FIGURE 3.

IL-2 and IFN-γ production of CD4⁺ T cells after peptide restimulation in vitro. Mice were infected p.o. with 5 × 10⁹ L. monocytogenes. After 8 days, cells from different tissues were restimulated for 5 h with the peptide LLO_189–201 (10⁻⁶ M). Cells were stained extracellularly with Cy5-conjugated anti-CD4 mAb, intracellularly with FITC-conjugated anti-IFN-γ 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).

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FIGURE 4.

Kinetics of LLO_189–201-specific CD4⁺ T cells in different organs after oral L. monocytogenes infection. Mice were infected p.o. with 5 × 10⁹ L. monocytogenes. At the indicated days, cells from different tissues were restimulated with the peptide LLO_189–201 for 5 h. Cells were stained with Cy5-conjugated anti-CD4 mAb and FITC-conjugated IFN-γ mAb or TNF-α mAb, and analyzed by flow cytometry. Graphs indicate percent-values of IFN-γ⁺ and TNF-α⁺ cells of CD4⁺ cells from different tissues and represent mean ± SD of three mice per group and time point.

Kinetics of Ag-specific IFN-γ and TNF-α 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-α⁺ and IFN-γ⁺CD4⁺ T cells were comparable in all tissues and at all time points analyzed. LLO_189–201-specific IFN-γ and TNF-α 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-γ⁺ or TNF-α⁺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-γ⁺ and TNF-α⁺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.

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FIGURE 5.

Primary and secondary LLO_189–201-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 × 10⁹) or i.v. (5 × 10³). After 37 or 45 days, mice were reinfected p.o. and i.v. with 5 × 10⁹ and 1 × 10⁵ L. monocytogenes, respectively. At the days indicated after primary and secondary infection, cells from different organs were restimulated with the peptide LLO_189–201. Cells were stained with Cy5-conjugated anti-CD4 mAb and FITC-conjugated IFN-γ 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.

Ag-specific CD4⁺ T cell responses to p.o. and i.v. infection with L. monocytogenes

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 × 10⁴, and 1.5 × 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-α⁺CD4⁺ T cells (data not shown).

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FIGURE 6.

Total numbers of recovered LLO_189–201-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 × 10⁹) or i.v. (5 × 10³). After 37 or 45 days, mice were reinfected p.o. and i.v. with 5 × 10⁹ and 1 × 10⁵ L. monocytogenes, respectively. At the days indicated after primary and secondary infection, cells from different organs were restimulated with the peptide LLO_189–201. Cells were stained with Cy5-conjugated anti-CD4 mAb and intracellularly with FITC-conjugated IFN-γ 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-γ⁺ cells. Bars represent mean ± SD of tissues from three mice. Results are representative for at least two individual experiments per time point.

Ag-specific CD4⁺ T cell responses to secondary p.o. and i.v. infection with L. monocytogenes

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-γ 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-γ⁺ and IL-2⁻IFN-γ⁺-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.

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FIGURE 7.

IL-2 and IFN-γ production of LLO_189–201-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 × 10⁹ L. monocytogenes. Eight days after primary and 7 days after secondary infection, cells from different organs were restimulated with the peptide LLO_189–201. Cells were stained with Cy5-conjugated anti-CD4 mAb and intracellularly with FITC-conjugated IFN-γ 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-γ⁺ of CD4⁺ cells; black bars, percentage of IL-2⁻IFN- γ⁺ of CD4⁺ cells.