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The Journal of Immunology, 2003, 171: 6032-6038.
Copyright © 2003 by The American Association of Immunologists

Activation of Antigen-Specific CD8 T Cells Results in Minimal Killing of Bystander Bacteria 1

Jiu Jiang2, Lauren A. Zenewicz, Lani R. San Mateo3, Lisa L. Lau4 and Hao Shen5

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Memory CD8 T cells play a critical role in protective immunity against intracellular pathogens. In addition to their ability to specifically recognize and lyse infected targets, activated CD8 T cells secrete cytokines that induce phagocytic cells to engulf and kill bacterial pathogens. In this study, we asked whether activation of Ag-specific CD8 T cells results in nonspecific killing of bystander bacteria during a mixed infection. Mice with epitope-specific memory CD8 T cells were coinfected with two isogenic strains of recombinant Listeria monocytogenes that differ in the cognate epitope. Recall responses by epitope-specific CD8 T cells rapidly inhibited the growth of epitope-bearing bacteria, impeding the course of infection within 6 h after challenge. This rapid inhibition was highly specific and did not affect the growth of coinfecting bacteria without the epitope. CTL recall did not enhance activation of innate immune cells, as evidenced by the absence of inducible NO synthase production in infectious foci. Our observations demonstrate the remarkable specificity of the bactericidal mechanisms of CTL and reveal the possibility for escape mutants to prevail in the hostile environment of a specific immune response. This implication has a bearing on subunit vaccine design strategies and understanding failure of immunization against bacterial infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigen-specific CD8 T cells employ a number of effector mechanisms that participate in protective immunity against intracellular microbial pathogens (1, 2). Studies involving knockout mice with specific effector deficiencies have revealed the contributions of both lytic and nonlytic mechanisms to protective immunity against the intracellular bacterium Listeria monocytogenes (LM).6 LM is a Gram-positive bacterium that, upon i.v. inoculation, is taken up by phagocytes in the spleen and liver (3). Most bacteria are killed within phagolysosomes, although a few escape from phagocytic vacuoles, replicate within the host cell cytoplasm, and spread to adjacent cells via host-actin polymerization (4). Clearance of LM infection involves the action of IFN-{gamma} and the development of LM-specific T cells, particularly cytotoxic CD8 T lymphocytes or CTL (5, 6, 7). In the immune host, memory CTL are key to the control of LM infection (8). Recognition of infected target cells by epitope-specific CD8 T cells through MHC-peptide:TCR interactions results in lysis of target cells primarily through a pathway involving release of the pore-forming protein, perforin, and apoptosis-inducing granzymes from secretory granules of CD8 T cells (2, 9). Perforin-deficient LM-specific CD8 T cells are greatly impaired in their ability to restrict growth of LM in the spleens of infected mice (10); however, evidence for TNF-dependent protective immunity conferred by perforin-deficient CD8 T cells supports a role for cytokines in clearance of secondary LM infections (11).

Despite the considerable information gained from these experiments, the exact mechanism for CD8 T cell-mediated LM killing remains largely undefined. Until recently, it was believed that lysis of infected cells by CTL does not kill LM directly but only releases intracellular bacteria to the extracellular environment. Activated CD8 T cells, upon engaging target cells, are known to secrete cytokines including IFN-{gamma} and TNF that recruit phagocytes (3). LM killing has been attributed to oxidative microbicidal mechanisms of these freshly recruited phagocytic cells (12, 13). Thus, the prevailing model postulates that CD8 T cell-mediated immunity ultimately involves participation of the nonspecific, innate components of the immune system. CD8 T cell responses induced by specific Ags could therefore result in nonspecific killing of bacteria in the vicinity, that is, bystander killing. However, recent studies have shown that CD8 T cells can directly kill intracellular bacteria by releasing an antimicrobial protein, granulysin, during lysis of infected target cells (14). These new findings suggest that CD8 T cell responses induced by specific Ags may not result in a significant level of nonspecific killing of bystander bacteria.

In this study, we examined the issue of bystander killing of bacteria during a CTL recall response using an experimental model of murine listeriosis. By using rLM strains that differ in a heterologous CTL epitope and carry different selection markers, we asked whether activation of epitope-specific CD8 T cells leads to nonspecific killing of bystander bacteria. Our results show that epitope-specific CD8 T cells begin to hinder the course of infection with epitope-bearing bacteria within the first 6 h after challenge. This rapid inhibition is highly specific, is accompanied by little or no activation of innate immunity, and does not affect the growth of bystander bacteria. The high degree of specificity of CTL-mediated bacterial inhibition reveals the possibility for CD8 epitope escape mutants to prevail in an immune individual. This implication has a bearing on subunit vaccine design strategies and understanding failure of immunization against bacterial infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Specific pathogen-free female BALB/c mice (National Cancer Institute, Frederick, MD), aged 8–10 wk, were housed in insulator cages and cared for in accordance with institutional animal care and use committee-approved protocols at the University of Pennsylvania School of Medicine animal facility.

Bacterial strains and virus

rLM strain XFL303 has been previously described (15). Integrated into the chromosome of XFL303 are a secreted fusion protein containing the H-2Ld-restricted nuclear protein (NP)118–126 lymphocytic choriomeningitis virus (LCMV)-derived CD8 T cell epitope and a gene (aph-A3) whose protein product confers kanamycin resistance. A new rLM strain, JJL101, was constructed in a similar manner and expressed an identical fusion protein except for the omission of NP118–126. In addition, JJL101 was constructed with the antibiotic resistance gene cat, conferring resistance to chloramphenicol, to allow for differentiation from XFL303 on antibiotic-containing agar plates. Strains were maintained as -80°C stocks in brain heart infusion (BHI)/50% glycerol. Before each experiment, each strain was inoculated onto BHI agar. A single colony was inoculated into BHI broth, and the culture was incubated overnight at 37°C with aeration. For clarity, XFL303 and JJL101 will be referred to as rLM-NP118+ and rLM-NP118-, respectively, throughout this paper. The LD50 of both strains is ~1 x 104 CFU in BALB/c mice by i.v. infection. LCMV Armstrong and recombinant vaccinia virus expressing NP118–126 (rVV) were propagated as previously described (16, 17).

Western blot

Western blots were performed as previously described (15). Briefly, secreted proteins were TCA precipitated from rLM culture supernatants, separated on SDS-PAGE gels, and transferred to Immobilon-P membrane (Millipore, Bedford, MA). Membranes were blocked with 5% milk in 1% Tween 20/PBS, incubated with two primary mAbs, anti-HA (clone 12CA5; Roche, Indianapolis, IN) and anti-VSV-G (clone P5D4; Sigma-Aldrich, St. Louis, MO), washed, and then incubated with peroxidase-conjugated anti-mouse polyclonal Ab. mAb binding was detected using the ECL detection system (Amersham Pharmacia, Piscataway, NJ).

LCMV and rVV immunization and LM challenge

To generate LCMV- or rVV-immune mice, mice were injected i.p. with 2 x 105 PFU LCMV Armstrong or 1 x 107 PFU rVV. rLM challenge was performed at least 3 wk postimmunization. Overnight cultures of rLM were serially diluted in PBS to the desired dose and injected into the lateral tail veins of mice. Inocula were plated to verify dose. At different time points postinfection, mice were sacrificed, and spleens and livers were aseptically removed, homogenized, and serially diluted in 0.1% Triton X-100/PBS. Dilutions were plated on BHI plates containing 5 µg/ml chloramphenicol or 5 µg/ml kanamycin. Bacterial colonies were counted after incubation at 37°C for 24–48 h. The limits of detection were 200 CFU/spleen and 300 CFU/g of liver.

Intracellular cytokine staining

Splenocytes were resuspended in RPMI 1640 containing 5% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 4 mM L-glutamine. To analyze IFN-{gamma} production in response to NP118–126, splenocytes were incubated with 50 U/ml recombinant human IL-2 and GolgiStop (BD PharMingen, San Diego, CA) and with or without 1 µM synthetic NP118–126 peptide (RPQASGVYM) at 37°C with 5% CO2 for 5 h. Cells were surface stained with anti-CD8 mAb (clone 53-6.7; BD PharMingen) in 1% BSA/PBS, permeabilized with Cytofix/Cytoperm solution (BD PharMingen), and then stained with anti-IFN-{gamma} mAb (clone XMG1.2; BD PharMingen) and fixed with paraformaldehyde. Splenocytes were analyzed with a FACSCalibur (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo, version 3.6.1 (Tree Star, San Carlos, CA).

Preparation of spleens for immunofluorescence microscopy

Frozen 5-µm-thick spleen sections were fixed with cold acetone and blocked for nonspecific binding with 3% nonimmune goat serum for 20 min. Adjacent sections were incubated with Listeria O rabbit polyclonal Ab (Difco, Detroit, MI) and/or anti-inducible NO synthase (iNOS) rabbit polyclonal Ab (Transduction Laboratories, Lexington, KY) for 1 h at room temperature, washed, and then incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR) to detect both primary Abs. Prepared sections were viewed using a Leica (Wetzlar, Germany) DM R fluorescence microscope and photographed with a digital charge-coupled device camera (Hamamatsu, Hamamatsu City, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isogenic strains of rLM differing in expression of a model CD8 T cell epitope and selection markers

To examine nonspecific bacterial killing by CD8 T cells in vivo, we created two isogenic strains of rLM containing chromosomal Ag cassettes constructed as described previously (15). One strain, rLM-NP118+, secretes a fusion protein with an embedded H-2Ld-restricted LCMV CD8 T cell epitope, NP118–126. A second strain, rLM-NP118-, secretes the same fusion protein but without the NP118–126 nonamer epitope. The two rLM strains carry different antibiotic selection markers to allow us to follow the growth of each individual strain in mixed infections (Fig. 1A). The two rLM strains secrete similar amounts of the fusion protein as detected by Western blot analysis (Fig. 1B), and the expression of the fusion has no measurable effect on the ability of these bacteria to invade and grow in J774 cells, and to form plaques in vitro (data not shown). The two rLM strains grew similarly after sublethal infections of naive mice (Fig. 1C), demonstrating that the two strains are equally virulent. In LCMV-immune BALB/c mice, which have memory CD8 T cells specific to the NP118–126 epitope, the rLM-NP118+ strain was rapidly cleared by a vigorous recall response to the NP118–126 epitope. In contrast, the rLM-NP118- strain lacking the NP118–126 epitope grew to similar levels in LCMV-immune and naive control mice. Thus, LCMV-immune mice mount a vigorous NP118–126-specific CTL recall response that controls infection with rLM-NP118+ but not with rLM-NP118-.



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  		FIGURE 1. Two isogenic strains of rLM expressing a secreted fusion protein with and without the H-2Ld-restricted CD8 T cell epitope NP118–126 from LCMV. A, Diagram of Ag cassettes inserted into a specific site between the lecithinase and lactate dehydrogenase (LDH) operons in the chromosome of LM. Strains rLM-NP118- and rLM-NP118+ are identical except for the following two features: 1) rLM-NP118+ contains the NP118–126 epitope, whereas rLM-NP118- does not, and 2) rLM-NP118+ carries an aminoglycoside phosphotransferase gene (aph A3) allowing selection on kanamycin, whereas rLM-NP118- carries the chloramphenicol acetyl transferase (cat) gene conferring resistance to chloramphenicol. Other features of the dihydrofolate reductase (DHFR) fusion proteins include a secretion signal sequence (ss) and mAb epitope tags, ha and vg, derived from influenza virus hemagglutinin and vesicular stomatitis virus glycoprotein, respectively. B, In vitro expression of rLM-secreted fusion proteins. Secreted proteins from culture supernatants were separated by SDS-PAGE, transferred to membrane, and probed with mAbs against ha and vg. Wild-type LM was the parent strain for rLM-NP118- and rLM-NP118+. C, Growth of the rLM strains in naive and LCMV-immunized BALB/c mice. Naive (N) and LCMV-immune (I) BALB/c mice were infected i.v. with 5 x 104 CFU rLM-NP118- or rLM-NP118+, and bacterial loads were determined 2 days postinfection. Bars depict mean + SD from three mice per group; the dotted line depicts the limit of detection (200 CFU/spleen).

 
Absence of nonspecific bacterial killing by NP118–126-specific CD8 T cells in the context of mixed infection

LCMV-immune and naive control mice were infected with a mixture of 5 x 104 CFU each of the two rLM strains, rLM-NP118+ and rLM-NP118-. At different time points after i.v. infection, bacterial numbers in the spleen and liver of each mouse were determined by plating organ homogenates on agar containing either kanamycin (to select for rLM-NP118+) or chloramphenicol (to select for rLM-NP118-). Both rLM strains replicated to comparably high levels in naive control mice, leading to severe debilitation by day 3 postinfection (Fig. 2). In LCMV-immune mice, rLM-NP118+ was rapidly eliminated in the spleens and livers by a vigorous NP118–126-specific recall CTL response. In contrast, the rLM-NP118- strain exhibited similar infection kinetics in LCMV-immune and naive mice despite the vigorous NP118–126-specific CTL recall response in the LCMV-immune mice induced by coinfection with rLM-NP118+ (Fig. 2). rLM-NP118- was eliminated in the spleens and livers of both naive and LCMV-immunized mice by day 7 postinfection, and clearance of this strain was also not altered by the presence of rLM-NP118+ during a mixed infection (data not shown). Thus, activation of epitope-specific CD8 T cells had no effect on the growth of bystander bacteria not expressing the epitope.



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  		FIGURE 2. Absence of bystander killing of rLM-NP118- in LCMV-immunized mice challenged with a mixture of rLM-NP118+ and rLM-NP118-. Naive or LCMV-immune mice were coinfected with 5 x 104 CFU each of rLM-NP118- and rLM-NP118+. At different time points postinfection, mouse spleens (A) and livers (B) were homogenized and plated on BHI agar with chloramphenicol or kanamycin to quantify viable rLM-NP118- and rLM-NP118+, respectively. Symbols represent individual mice, and the dotted line represents the limit of detection. C and D, Side-by-side comparison of the same rLM strain in naive vs immune mice. Bars represent the mean bacterial loads (+SD) from data shown in A and B, while numbers represent p values between the naive and immunized groups as determined by Student’s t test. Dotted lines indicate the limit of detection.

 
Next, we examined whether bystander killing by NP118–126-specific recall CTL would occur in coinfections with a large dose of rLM-NP118+ mixed with a small dose of rLM-NP118-. We infected LCMV-immune and naive control mice with rLM-NP118+ and rLM-NP118- at a ratio of 1000:1 (5 x 105 CFU rLM-NP118+ + 5 x 102 CFU rLM-NP118-) or in a ratio of 1:1 as before (5 x 104 CFU of each strain). Inoculation of LCMV-immune mice with such a large dose of NP118–126-secreting bacteria resulted in a NP118–126-specific CTL recall response of much greater magnitude than that observed after infection with 5 x 104 CFU (Fig. 3, A and B). This robust recall response controlled the high-dose infection with rLM-NP118+, whereas the growth of rLM-NP118- remained unaffected (Fig. 3, C and D). These experiments demonstrate that a minor population of bacteria without the CTL epitope can survive and supplant the majority population of epitope-bearing bacteria in the context of a vigorous epitope-specific recall CD8 T cell response.



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  		FIGURE 3. A minor rLM-NP118- population survived and supplanted the majority rLM-NP118+ population in LCMV-immunized mice challenged with a 1:1000 mixture of rLM-NP118- and rLM-NP118+. Naive or LCMV-immune mice were challenged with either a 1:1 ratio of rLM-NP118- to rLM-NP118+ (5 x 104 CFU each) (A and C) or a 1:1000 ratio of rLM-NP118- to rLM-NP118+ (5 x 102 CFU and 5 x 105 CFU, respectively) (B and D). NP118–126-specific splenic CD8 T cell responses were determined by ex vivo intracellular IFN-{gamma} staining at day 2 postchallenge (A and B). Numbers represent the percentages of specific CD8 T cells of total live splenocytes (means of three mice). Bacterial loads in the spleens were determined 2 days postchallenge (C and D). Bars show mean + SD of three to five mice per group; dotted line represents the limit of detection. The experiment was performed twice with similar results.

 
Rapid clearance of bacteria without apparent phagocyte activation

The previous series of experiments showed that activation of NP118–126-specific CD8 T cells had no impact on the growth of bacteria that do not express NP118–126. This result is inconsistent with the current model whereby CD8 T cell recall responses, through cytokine production, enhance activation of phagocytic cells, which in turn phagocytose and kill bacteria in a nonspecific fashion. We thus asked whether recall CD8 T cells enhance early recruitment of phagocytic cells to sites of infection and activate them to participate in bacterial clearance. We infected LCMV-immune mice with rLM-NP118+ or rLM-NP118- and, at different time points after infection, examined accumulation and activation of phagocytes at the site of infection by immunofluorescence microscopy using Abs against LM and iNOS. On day 2 after infection, rLM-NP118- bacteria were visible in sections of the spleen, with activated iNOS+ cells surrounding bacterial foci of infection (Fig. 4A). Because the anti-LM and anti-iNOS primary Abs used were both generated in rabbits, it was not possible to differentiate the two signals by using different fluorochrome-conjugated secondary Abs. However, bacteria were visually distinguishable from iNOS+ cells on the basis of their size and rod shape. Infection of LCMV-immune mice with rLM-NP118- resulted in increasing bacterial burden over time (Fig. 4, B and C). In agreement with data derived from plating spleen homogenates, greater numbers of bacteria were seen in spleen sections taken at later time points. At each time point observed, foci of rLM-NP118- infection were surrounded by iNOS+ cells. Increasing numbers of activated iNOS+ cells were seen over time, coinciding with the increase in bacterial burden. In contrast, few bacteria were visible in spleen sections of LCMV-immune mice infected with rLM-NP118+, and there was no evidence for enhanced macrophage recruitment and activation (Fig. 4B). Our evidence suggests that rapid recall of specific CTL eliminated rLM-NP118+ before bacterial proliferation could occur. In fact, examination of splenic bacterial burdens at early time points after infection showed significant control of bacteria within 6 h of infection (Fig. 4C). These experiments demonstrate that clearance of bacteria by epitope-specific CTL is rapid, and does not involve enhanced activation of innate microbicidal mechanisms.



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  		FIGURE 4. Memory CD8 T cells rapidly controlled rLM-NP118+ without substantial activation of phagocytic cells. LCMV-immune mice were challenged with 5 x 104 CFU rLM-NP118- or rLM-NP118+. A, Two days postinfection, adjacent frozen spleen sections from rLM-NP118--challenged mice were stained with anti-LM Ab to localize bacteria, anti-iNOS Ab to detect activation of phagocytes, and both Ab simultaneously. Ab were visualized using secondary Ab conjugated to Alexa Fluor 488. Bacteria appear as small rods with intense green fluorescence, iNOS+ cells show more diffuse green staining (x200). B and C, Production of iNOS (x1000) (B) and bacterial numbers (C) were assessed in spleens at different times postinfection with rLM-NP118- or rLM-NP118+. Bars represent mean + SD from three mice per group; dotted line represents the limit of detection. The experiment was repeated twice with similar results.

 
Minimal killing of bystander bacteria in mice with lower levels of CD8 T cell memory

The level of memory NP118-specific CD8 T cells is very high in LCMV-immune mice. The presence of such a high level of Ag-specific CTLs may allow for rapid clearance of rLM-NP118+ without the sustained immune activation that could otherwise lead to bystander killing of bacteria. Therefore, we examined whether bystander killing of bacteria would occur in mice with a lower frequency of Ag-specific memory CTL. Mice were immunized with a rVV that expresses the NP118 epitope (17). This generated considerably less NP118-specific memory cells as compared with LCMV immunization, 0.83 vs 19.4% of CD8 T cells (Fig. 5A). rVV-immune and naive mice were challenged with a mixture of rLM-NP118+ and rLM-NP118- (2 x 104 CFU of each), and 2 days postinfection, rLM was recovered from spleens as described previously. Both rLM strains replicated to similar levels in naive mice, as observed before (Fig. 5B). In rVV-immunized mice, levels of rLM-NP118+ were significantly less than in naive mice due to the NP118-specific recall response (Fig. 5B). In contrast, there was no statistically significant difference in rLM-NP118- levels between naive and rVV-immunized mice (Fig. 5B). These data demonstrate that activation of a lower frequency of NP118-specific CD8 T cells also results in minimal killing of bystander bacteria during a recall response that reduces levels of Ag-specific bacteria.



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  		FIGURE 5. Minimal killing of bystander bacteria in rVV-immunized mice. A, NP118-specific CD8 T cells in rVV- or LCMV-immunized mice. Splenocytes from rVV- or LCMV-immune mice were stimulated ex vivo with or without NP118 peptide; cells were surface stained with anti-CD8 and anti-CD11a, a T cell activation marker, and then intracellularly stained for IFN-{gamma}. Numbers above the gates show percentage of IFN-{gamma}-producing cells of total CD8 T cells. Each FACS plot shows one representative mouse of three. B, Naive or rVV-immune mice were coinfected with a mixture of rLM-NP118+ and rLM-NP118- (2 x 104 CFU of each). Two days postinfection, bacteria were recovered from spleens as described in Fig. 2. Bar graphs represent mean + SD, and numbers are p values, as determined by Student’s t test. The experiment was performed twice with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The clearance of intracellular bacteria by CD8 T cells has long been assumed to rely on two distinct effector functions: the lysis of infected cells leading to release of intracellular bacteria; and the production of cytokines or chemokines for the recruitment and activation of phagocytic cells (9). This model for CTL-dependent immunity to bacteria posits the involvement of innate immune mechanisms in the manifestation of adaptive immunity. One prediction based on this model is that a robust recall of CTL memory would induce a strong innate immune response, resulting in increased nonspecific bacterial killing by phagocytic cells. We tested this prediction by measuring the extent of bystander bacterial killing during an active Ag-specific recall CD8 T cell response. The results from our mixed infection experiments indicate that Ag-specific CTL responses are remarkably specific, resulting in efficient control of Ag-expressing bacteria and minimal inhibition of nonspecific bystander bacteria, contrary to predictions of the above model.

One possible explanation for the lack of bystander effect may lie in the intracellular life cycle of LM, which allows this bacterium to replicate inside host cells and spread directly from cell to cell. This suggests that intracellular bacteria may not be exposed to bystander killing by the phagocytic cells that are activated by Ag-specific CD8 T cells. However, several lines of evidence indicate that bacteria are not found exclusively within host cells during LM infection. LM replicates rapidly within host cells to high levels, resulting in lysis of host cells and release of intracellular bacteria in vitro (18). In addition, histological examination reveals that many bacteria are found extracellularly in lesions in the spleen of LM-infected mice (19). These data indicate that, although LM has an intracellular life cycle, its rapid intracellular growth can result in lysis of infected cells and release of intracellular bacteria, which can reinfect other cells but also become exposed to killing by phagocytic cells. Indeed, the actA mutant deficient in cell-to-cell spread, although attenuated, is capable of establishing an infection and replicates to relatively high numbers (20). More importantly, clearance of LM during a primary infection is critically dependent on the presence of macrophages and neutrophils (21, 22). Thus, it is surprising that we find minimal inhibition of nonspecific bystander bacteria by the activation of Ag-specific memory CD8 T cells.

Our microscopic observations show that the recall response by memory CD8 T cells did not enhance iNOS production at infectious foci, suggesting minimal activation of phagocytic microbicidal mechanisms by specific CD8 T cells. This result is consistent with previous reports of significant CTL-mediated protective immunity against LM in mice depleted of granulocytes (23). However, our results do not exclude the participation of innate immune mechanisms for killing of Ag-bearing bacteria released upon host cell lysis. Such specific bacterial killing by nonspecific mechanisms would explain the reduction of protective immunity seen in granulocyte-depleted mice. Our data suggest a scenario wherein a bacterium is killed only upon CTL-mediated lysis of its host cell. CTL lysis could result in bacterial killing directly, through the release of bactericidal proteins such as granulysin and defensins, along with other granule components such as perforin and granzymes (14, 24), or indirectly, through the release of live intracellular bacteria for killing by neutrophils and macrophages (22, 25). Our experiments showing significantly lower bacterial burdens in LCMV-immune compared with naive control mice within 6 h of rLM-NP118+ challenge suggest that memory CTL provide protection by impeding the normal course of infection, eliminating the colonizing bacteria before significant bacterial replication has occurred. Previous data from our laboratory show that memory CTL protect against rLM challenge primarily by blocking direct cell-cell spread and preventing bacterial dissemination and growth (20). Thus, in the context of specific CTL memory, it is unlikely that granulocytes are required to kill large numbers of bacteria released upon CTL lysis.

Our results demonstrating the absence of nonspecific killing of bystander bacteria by CTL reflect a strict specificity of the CD8 T cell response that might allow for the evolution and selection of bacterial mutants capable of CTL escape. Our experiments showed survival and ascendancy of the few rLM-NP118- bacteria after mixed infection with 1000-fold more rLM-NP118+ during an active NP118–126-specific recall CTL response. This result suggests that a single mutant with an altered CTL epitope might survive and take over an infection in the context of effective immunity. Mutations in T cell epitopes have allowed viruses including LCMV (26), influenza (27), and SIV/HIV (28, 29, 30) to evade adaptive immune responses. Viral escape of immune surveillance also occurs through antigenic variation, or the production of viral variants through reassortment (31). Antigenic variation has also been described in bacteria, in which variation is thought to allow Ab evasion and functional heterogeneity (32). For example, Neisseria gonorrhoeae produces a changing array of outer membrane Opa proteins by variable expression from a family of 11 genes, in addition to amino acid variation in pili proteins and production of multiple lipo-oligosaccharides (33, 34). Such variation is thought to contribute to the inability of human hosts to develop protective immunity against gonorhea. A similar mechanism for variation of proteins with CTL epitopes could allow for bacterial escape mutants to arise in populations subject to selection pressure exerted by CD8 T cell-mediated immunity. Although no such CTL escape mutants have yet been isolated in bacteria, several lines of evidence suggest that they can evolve. Recent studies have revealed microbial genomes to have more plasticity than previously thought (35, 36). Comparative genome analysis of different Escherichia coli strains has demonstrated that DNA sequence variation exists throughout the chromosome, and that the genome contains a high number of putative open reading frames and mobile genetic elements (37). A number of studies have also demonstrated the ability of bacteria to increase mutation rates in vivo, giving rise to increased genetic variability and increased chances of persistent infection in unstable environments. For example, chronic Pseudomonas aeruginosa infection in cystic fibrosis patients is characterized by colonization with a hypermutator strain that allows continued infection in the context of antibiotic selection (38). Together, these considerations suggest that bacterial mutants capable of CTL escape can evolve in vivo, and the results of this study suggest that once such mutants evolve, these mutants may be able to survive and prevail in the context of CTL surveillance. The results of this study may have minimal relevance to polyclonal immune responses in the context of natural infections. However, the possibility for bacterial escape of CTL surveillance has important implications for the design of the new generation of vaccines specific to single bacterial components. The emergence of CTL escape mutants in a population of immunized individuals would result in potentially catastrophic vaccine failure.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI-45025 and AI-46184 (to H.S.). L.A.Z. was supported by National Research Scientist Award Training Grant 5-T32-GM07229-26. L.R.S.M. was supported by a research supplement for underrepresented minorities from the National Institute of Allergy and Infectious Diseases. Back

2 Current address: Department of Microbiology and Immunology, Drexel University, College of Medicine, 2900 Queen Lane, G44H, Philadelphia, PA 19129. Back

3 Current address: Centocor, Inc., 200 Great Valley Parkway, Malvern, PA 19355. Back

4 Current address: Office of Technology Transfer, Thomas Jefferson University, 1020 Locust Street, M60, Philadelphia, PA 19107. Back

5 Address correspondence and reprint requests to Dr. Hao Shen, Department of Microbiology, University of Pennsylvania School of Medicine, 225 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6076. E-mail address: hshen{at}mail.med.upenn.edu Back

6 Abbreviations used in this paper: LM, Listeria monocytogenes; NP, nuclear protein; LCMV, lymphocytic choriomeningitis virus; BHI, brain heart infusion; rVV, recombinant vaccinia virus expressing NP118; iNOS, inducible NO synthase. Back

Received for publication November 6, 2002. Accepted for publication September 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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