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Depletion of CD4⁺ T Cells during Immunization with Nonviable Listeria monocytogenes Causes Enhanced CD8⁺ T Cell-Mediated Protection against Listeriosis¹

Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany

	Abstract

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Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8⁺ T cell response and consequently only limited protection against subsequent L. monocytogenes infection. We have recently demonstrated that depletion of regulatory CD4⁺ T cells during immunization significantly enhances CD8⁺ T cell responses. In the present study, we determined the impact of CD4⁺ T cell depletion on the CD8⁺ T cell response against heat-killed Listeria. Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8⁺ T cells and improved protection against subsequent infection with L. monocytogenes. During challenge infection, numbers of Listeria-specific CD8⁺ T cells were enhanced, and these cells expressed effector functions in terms of IFN- production. In summary, we demonstrate that combining nonviable L. monocytogenes vaccination and CD4⁺ T cell depletion improves generation of long-lasting and functional Listeria-specific CD8⁺ memory T cells.

	Introduction

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Experimental infection of mice with L. monocytogenes is a well-established model for the analysis of acquired immunity against intracellular bacteria and for the evaluation of vaccination strategies against this type of pathogen (1). L. monocytogenes provokes a profound T cell response including both CD4⁺ and CD8⁺ T cells (2, 3, 4). Due to the intracytoplasmic habitat of L. monocytogenes, CD8⁺ T cells are particularly important for the control of infection and are major mediators of protection against reinfection (1, 5). In BALB/c mice, a large fraction of CD8⁺ T cells is directed against a few dominant Listerial proteins (2, 6). The most dominant CD8⁺ T cell epitope is listeriolysin O (LLO)_91–99,³ a peptide derived from the secreted pore-forming toxin LLO. At the peak of a primary anti-Listerial response, 3–4% of the CD8⁺ splenocytes are specific for LLO_91–99, and during secondary infection, LLO_91–99-specific T cells reach levels as high as 15% of all splenic CD8⁺ T cells (2).

Vaccination approaches using nonviable Listeria have generally proven ineffective in inducing long-lasting protection against subsequent challenges with viable L. monocytogenes (7, 8, 9). Only repeated injections of heat-killed Listeria (HKL) in short intervals or the combination of HKL with IL-12 or anti-CD40 mAb elicited protection (10, 11, 12, 13). Closer analysis revealed that vaccination-induced protection was mediated by both CD4⁺ and CD8⁺ T cells (10, 11). Since nonviable Listeria fail to egress from phagosomes into the cytoplasm, it was assumed that the failure of HKL to induce protection was mainly due to insufficient induction of Listeria-specific CD8⁺ T cells. A recent study by Lauvau et al. (14) challenged this assumption by demonstrating that immunization with HKL generates Listeria-specific CD8⁺ T cells. However, in this study, specific CD8⁺ T cells were functionally impaired in terms of IFN- production and cytotoxicity, and therefore, failed to confer protection (14).

In a recent study, using a DNA vaccine coding for LLO or immunization with the LLO_91–99 peptide, we observed that depletion of CD4⁺ T cells during boost immunization significantly enhances memory CD8⁺ T cell responses against LLO_91–99 (15). A more detailed analysis revealed that the enhanced response was most likely due to the removal of CD25⁺CD4⁺ T cells, a T cell subpopulation, which has been described to contain a major pool of suppressor or regulatory T cells (16, 17). Overall, these results suggest that memory CD8⁺ T cell responses are controlled by regulatory T cells, and that removal of these T cells enhances CD8⁺ T cell responses.

In the present study, we analyzed the impact of CD4⁺ T cell depletion on the generation of a protective CD8⁺ T cell response against listeriosis. We demonstrate that depletion of CD4⁺ T cells during boost immunization with HKL enhanced the generation of long-lasting Listeria-specific CD8⁺ memory T cells and improved protection against subsequent challenge with viable L. monocytogenes.

	Materials and Methods

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Bacterial infection of mice

BALB/c mice were bred in our facility at the Federal Institute for Health Protection of Consumers and Veterinary Medicine (Berlin, Germany), and experiments were conducted according to the German animal protection law. Mice were infected with L. monocytogenes strain EGD. Bacteria were injected in a volume of 200 µl of PBS into the lateral tail vein of mice. The bacterial dose was controlled by plating dilutions of the inoculum on tryptic soy broth (TSB) agar plates. For determination of bacterial burdens in organs, mice were killed, livers and spleens were homogenized in PBS, serial dilutions of homogenates were plated on TSB agar plates, and colonies were counted after incubation at 37°C overnight (18).

Immunization with HKL

For the production of HKL, an overnight culture of L. monocytogenes was washed twice and incubated at 80°C for 2 h. Bacterial numbers were determined by absorption at 600 nm (OD of 1 is equivalent to 1 x 10⁹ bacteria). Effective killing was validated by plating HKL onto TSB agar plates. Mice were injected into the lateral tail vein with 3 x 10⁹ HKL in a volume of 200 µl of PBS.

Antibodies

Rat Ig, anti-CD16/CD32 mAb (2.4G2), anti-CD8 mAb (YTS169), anti-CD4 mAbs (YTS191.1 and GK1.5), anti-CD62L mAb (Mel-14), and anti-IFN- mAb (clone: R4-6A2, IgG1) were purified from rat serum or hybridoma supernatants with protein G-Sepharose. Abs were Cy5- or FITC-conjugated according to standard protocols. FITC-conjugated rat-IgG1 isotype control mAb (R3-34) was purchased from BD PharMingen (San Diego, CA).

In vivo mAb application and adoptive transfer experiments

CD4⁺ T cells were depleted by i.p. injection of 300 µg of anti-CD4 mAb YTS191.1 at intervals of 5 days starting 3 days before immunization. Efficacy of depletion was controlled with the anti-CD4 mAb GK1.5 and was always >95% (15).

For adoptive transfer experiments, donor mice were left untreated or were prime-boost immunized or infected as indicated. Seven days after the boost immunization, mice were killed. Single-cell suspensions of pooled spleen cells were prepared using an iron mesh sieve. Spleen cells were treated with Tris-buffered ammonium chloride to lyse RBC and then washed twice with PBS 10% and passed through a 100-µm filter. Cell numbers equivalent to one donor spleen were i.v. injected into recipient mice. One day later, recipient mice were infected i.v. with 1 x 10⁴ L. monocytogenes.

Flow cytometric determination of cytokine expression and MHC class I tetramer staining

Intracellular cytokine staining after short-term in vitro restimulation was performed as described (18). Briefly, spleen cells were stimulated for 5 h with 10^-6 M of the peptide LLO_91–99. During the final 4 h of culture, 10 µg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) were added. Cultured cells were extracellularly stained with Cy5-conjugated anti-CD8 mAb, and intracellularly stained with FITC-conjugated anti-IFN- mAb or FITC-conjugated isotype control mAb. Cells were analyzed using a FACSCalibur and CellQuest software (BD Biosciences, Mountain View, CA). Generation of LLO_91–99/H-2K^d-tetramers and analysis of cells with tetramers has been described previously (2, 18).

Statistical analysis

Bacterial titers were analyzed with the Mann-Whitney U test, and frequencies and numbers of tetramer-positive or cytokine-expressing cells with the unpaired Student’s t test. _*, p < 0.05; NS, p > 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Depletion of CD4⁺ T cells during boost immunization with HKL enhances frequencies of Listeria-specific CD8⁺ T cells

Immunization of mice with nonviable L. monocytogenes is insufficient in inducing protection against L. monocytogenes infection. Limited protection is probably in large part due to inefficient induction of Listeria-specific CD8⁺ T cells (10, 14). Recently, we demonstrated that depletion of regulatory CD4⁺ T cells during boost immunization with a DNA vaccine coding for LLO or with the LLO_91–99 peptide significantly increased LLO_91–99-specific CD8⁺ T cells (15). Moreover, the majority of the LLO_91–99-specific CD8⁺ T cells generated in this way appeared to be functional CD8⁺ effector T cells in terms of IFN- and TNF- production and, to some degree, cytotoxicity (15).

To determine whether depletion of CD4⁺ T cells could enhance the Listeria-specific CD8⁺ T cell response upon administration of HKL, mice were immunized twice with 3 x 10⁹ HKL i.v. During the boost immunization, one group of mice was treated with anti-CD4 mAb (YTS191.1) to deplete CD4⁺ cells. Depletion efficacy was controlled with a second anti-CD4 mAb (GK1.5), which recognizes an independent epitope on the CD4 molecule. Depletion efficacy was always >95% (data not shown). At different days after boost immunization, frequencies and numbers of LLO_91–99-specific CD8⁺ T cells were determined with MHC class I tetramers (LLO_91–99 in the context of H-2K^d) and CD62L staining (Fig. 1). CD62L is a surface molecule of CD8⁺ T cells that is down regulated following T cell activation. Therefore, costaining with tetramers and CD62L allows the precise determination of LLO_91–99-specific CD8⁺ T effector cells. Before secondary HKL immunization, we detected only low frequencies and numbers of LLO_91–99-specific CD8⁺ T cells and immunization with HKL without any further treatment did not result in a visible enlargement of this cell population. In contrast, depletion of CD4⁺ T cells induced asignificant increase in frequencies and numbers of specific CD8⁺ T cells, which reached a maximum at day 7 after boost immunization and then slowly declined.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Frequencies and numbers of LLO91–99-specific CD8+ T cells after secondary immunization with HKL. BALB/c mice were prime-boost immunized with 3 x 109 HKL i.v. in an interval of 35 days. During the boost immunization, mice were left untreated () or received 300 µg of anti-CD4 mAb i.p. at days -3 and +2 of immunization (). At the days indicated, spleen cells were counted and stained with FITC-conjugated of anti-CD62L mAb, Cy5-conjugated anti-CD8 mAb, and PE-labeled LLO91–99-MHC class I tetramers. Cells were analyzed by flow cytometry after the addition of propidium iodide. Bars represent mean values ± SD for spleen cells of three individually analyzed mice. Results are representative for two independent experiments. *, Difference between anti-CD4 mAb-treated and untreated groups: p < 0.05.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Frequencies of IFN--producing LLO91–99-specific CD8+ T cells after secondary immunization with HKL. BALB/c mice were prime-boost immunized with 3 x 109 HKL i.v. in an interval of 35 days. During the boost immunization, mice were left untreated or received 300 µg of anti-CD4 mAb i.p. at days -3 and +2 of immunization. At the days indicated, mice were killed and spleen cells were incubated for 5 h with () or without () 10-6 M peptide LLO91–99. For the final 4 h, brefeldin A was added to the cultures. Cells were stained extracellularly with Cy5-conjugated anti-CD8 mAb and intracellularly with FITC-conjugated anti-IFN- mAb or FITC-conjugated isotype control mAb. Values for isotype control staining were <0.05% (not shown). Background frequencies and numbers of IFN--producing cells determined in cultures without peptides were subtracted from frequencies and numbers derived from cultures with peptides. Bars depict mean ± SD for LLO91–99-specific IFN-+CD8+T cells of three individually analyzed mice per experimental group. The experiment shown is representative for two similar experiments. *, Difference between anti-CD4 mAb-treated and untreated groups: p < 0.05.

View larger version (13K): [in this window] [in a new window]   FIGURE 3. LLO91–99-specific CD8+ T cell responses in mice treated with anti-CD4 mAb before secondary immunization with HKL. BALB/c mice were prime-boost immunized with 3 x 109 HKL i.v. in an interval of 35 days. During the boost immunization, mice were left untreated (HKL only) or received 300 µg of anti-CD4 mAb i.p. either at day -7 (anti-CD4 before HKL), or at days -3 and +2 of immunization (anti-CD4 + HKL). At day 7 after HKL immunization, spleen cells were counted and stained with FITC-conjugated anti-CD62L mAb, Cy5-conjugated anti-CD8 mAb, and PE-labeled LLO91–99-MHC class I tetramers. Cells were analyzed by flow cytometry after the addition of propidium iodide. Bars represent mean values ± SD for spleen cells of three individually analyzed mice. *, Difference between anti-CD4 mAb-treated and untreated groups: p < 0.05.

Since depletion of CD4⁺ T cells during boost immunization induced an enhanced Listeria-specific CD8⁺ T cell response, we tested whether this treatment caused enhanced cell-mediated protection against L. monocytogenes infection. Mice were prime-boost immunized with 3 x 10⁹ HKL i.v. One group of mice received in addition anti-CD4 mAb during boost immunization. Seven days later, spleen cells from immunized mice were adoptively transferred into naive BALB/c recipients. In parallel, groups of mice received spleen cells from naive mice and from mice infected with L. monocytogenes 5 wk before the transfer. All recipient mice were infected with 1 x 10⁴ Listeria (1 x LD50), and 4 days postinfection, Listeria titers in the spleen were determined (Fig. 4). Transfer of spleen cells from Listeria-primed mice caused protection in recipient mice with highly reduced Listeria titers. Transfer of cells from HKL and HKL + anti-CD4-treated mice lowered the Listeria titers in spleens of recipient mice. Notably, reduction in titers in spleens of mice transferred with HKL + anti-CD4 mAb treated mice was stronger than that observed in mice receiving spleen cells from HKL-treated mice.

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Transfer of cells from mice prime-boost immunized with HKL into naive mice and subsequent challenge of recipients with L. monocytogenes. BALB/c mice were prime-boost immunized with 3 x 109 HKL i.v. in an interval of 35 days. During the boost immunization, mice were left untreated or received 300 µg of anti-CD4 mAb i.p. at days -3 and +2 of immunization. In addition, one group of mice was infected with 2 x 103 L. monocytogenes i.v. in parallel to the primary HKL immunization. Seven days after boost immunization, spleen cells from naive, immunized, and L. monocytogenes-infected mice were transferred into naive mice. After 24 h, recipients were infected with 1 x 104 L. monocytogenes i.v., and after a further 4 days, L. monocytogenes titers in the spleens of recipients were determined. *, Difference between mice receiving spleen cells from naive mice and mice receiving spleen cells from pretreated mice: p < 0.05.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Bacterial burdens in mice immunized with HKL and challenged with L. monocytogenes. BALB/c mice were prime-boost immunized with 3 x 109 HKL i.v. in an interval of 35 days. During the boost immunization, mice were left untreated (HKL) or received 300 µg of antiCD4 mAb i.p. at days -3 and +2 of immunization (HKL + CD4). After a further 3 mo, mice were challenged with 2 x 104 L. monocytogenes i.v. In parallel, naive mice and mice that were infected 3 mo earlier with 2 x 103 L. monocytogenes i.v. were infected with the same dose. At day 5 after the challenge infection, mice were killed, and bacterial titers in spleen and liver were determined. Results are representative for two independent experiments with at least four mice in each experimental group. Statistical difference in spleens: naive vs HKL, p = 0.06; naive vs HKL + anti-CD4 mAb, p = 0.03; HKL vs HKL + anti-CD4 mAb, p = 0.03. Statistical difference in liver: naive vs HKL, p = 0.11; naive vs HKL + anti-CD4 mAb, p = 0.03; HKL vs HKL + anti-CD4 mAb, p = 0.34.

To decide whether the enhanced protection observed in mice immunized with HKL and treated with anti-CD4 mAb correlated with improved Listeria-specific CD8⁺ T cell responses, spleen cells were isolated 5 days after the challenge infection and analyzed with LLO_91–99-tetramers (Fig. 6). In naive mice, only marginal frequencies and numbers of LLO_91–99-specific CD8⁺ T cells were detected at this time point of infection. This result is in accordance with the observation that during a primary L. monocytogenes-infection, significant populations of LLO_91–99-specific CD8⁺ T cells are usually not detectable before days 6–7 of infection (2, 18). In contrast, mice vaccinated with a sublethal dose of L. monocytogenes displayed a strong and rapid response typically observed following secondary infection (2, 18). Mice immunized with HKL also showed this rapid and enhanced response, indicating that HKL treatment led to priming of LLO_91–99-specific CD8⁺ T cells. In correlation with protection, treatment of mice with anti-CD4 mAb during the HKL boost immunization significantly enhanced LLO_91–99-specific CD8⁺ T cell responses. Compared with mice immunized with HKL, frequencies as well as numbers of LLO_91–99-specific CD8⁺ T cells were enlarged severalfold.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. Listeria-specific CD8+ T cell response in HKL-immunized mice challenged with L. monocytogenes. BALB/c mice were prime-boost immunized with 3 x 109 HKL i.v. in an interval of 35 days. During the boost immunization, mice were left untreated (HKL) or received 300 µg of anti-CD4 mAb i.p. at days -3 and +2 of immunization (HKL + anti-CD4 mAb). After a further 3 mo, mice were challenged with 2 x 104 L. monocytogenes i.v. In parallel, naive mice and mice that were infected 3 mo earlier with 2 x 103 L. monocytogenes i.v. were infected with the same dose. At day 5, mice were killed and spleen cells were analyzed as described in Fig. 1. Dot plots depict representative CD62L and LLO91–99-tetramer staining of viable CD8-gated T cells. The percentage of LLO91–99-tetramer+CD62Llow T cells of CD8+ T cells is indicated above the upper left quadrant (mean ± SD of three individually analyzed mice). Figures on the right give the total number of LLO91–99-tetramer+CD62LlowCD8+ splenocytes (mean ± SD of three individually analyzed mice). The experiment shown is representative for two similar experiments.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. Listeria-specific IFN- production of CD8+ T cells in HKL-immunized mice challenged with L. monocytogenes. BALB/c mice were immunized and challenged as described in Fig. 6. Five days after the challenge, spleen cells were counted and cultured for 5 h with (LLO91–99) or without (control) 10-6 M peptide LLO91–99. For the final 4 h, brefeldin A was added to cultures. Cells were stained extracellularly with Cy5-conjugated anti-CD8 mAb and intracellularly with FITC-conjugated anti-IFN- mAb or FITC-conjugated isotype control mAb. Dot plots depict representative stainings and show CD8-gated cells. Numbers above the regions give mean percentages ± SD of IFN-+ cells of CD8+ T cells for three individually analyzed mice per group. Values for isotype control staining were <0.05% (data not shown). Figures on the right give the total number of LLO91–99-specific IFN-+CD8+ T cells per spleen (mean ± SD of three individually analyzed mice per experimental group). The experiment shown is representative for two similar experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In a recent study, Lauvau et al. (14) found that HKL immunization generates a Listeria-specific CD8⁺ T cell response, however, these CD8⁺ T cells were functionally impaired in terms of IFN- production and cytotoxicity, and consequently did not confer protection against challenge infection with viable L. monocytogenes (14). In our experiments, the CD8⁺ T cell response detected in mice challenged with L. monocytogenes after immunization with HKL alone already had the hallmarks of a memory response suggesting that HKL treatment induces Listeria-specific CD8⁺ T cells. The main effect of anti-CD4 mAb treatment was the enhancement of this response, thus allowing detection of specific CD8⁺ T cells already after HKL boost immunization. However, both immunization with HKL alone or with HKL + anti-CD4 mAb induced similar frequencies of LLO_91–99-tetramer⁺ and LLO_91–99-specific IFN--producing CD8⁺ T cells following challenge infection with L. monocytogenes. Thus the majority of LLO_91–99-specific CD8⁺ T cells produced IFN-. Consistent with this result are the high frequencies of LLO_91–99-specific IFN- producers shortly after boost immunization in anti-CD4 mAb-treated mice. Currently, we have no satisfactory explanation for the different results between our study and that of Lauvau et al. (14). The discrepancy could be due to differences in the vaccination protocols, however, this issue needs further investigations.

Our results reveal the paradox situation that depletion of a T cell population, namely the CD4⁺ T cells, does not impair but rather enhances a protective T cell response against a bacterial pathogen. In a previous study, we demonstrated that this effect was caused by the elimination of regulatory CD4⁺ T cells with suppressive functions (15). Even though the detailed mechanisms underlying the enhanced CD8⁺ T cell response following HKL immunization and anti-CD4 mAb treatment were not further investigated in the current study, we propose that similar mechanisms are involved. In conclusion, our results suggest that by interfering with a negative regulatory CD4⁺ T cell-mediated mechanism, pathogen-specific CD8⁺ T cell responses and the generation of pathogen-specific CD8⁺ memory T cells can be improved.

	Acknowledgments

 
We thank Dr. Robert Hurwitz for assistance in the preparation of tetramers and Manuela Stäber for purification and labeling of Abs.

	Footnotes

 
¹ M.K. was supported by the Graduiertenkolleg 276/2, and S.H.E.K. gratefully acknowledges support by the Fond Chemie.

² Address correspondence and reprint requests to Dr. Hans-Willi Mittrücker, Max Planck Institute for Infection Biology, Schumannstrasse 21/22, 10117 Berlin, Germany. E-mail address: mittruecker{at}mpiib-berlin.mpg.de

³ Abbreviations used in this paper: LLO, listeriolysin O; HKL, heat-killed Listeria; TSB, tryptic soy broth.

Received for publication December 30, 2003. Accepted for publication January 5, 2004.

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