Recombinant lemA without adjuvant induces extensive expansion of H2-M3-restricted CD8 effectors, which can suppress primary listeriosis in mice

Abdul Tawab, Janet Fields, Elizabeth Chao and Roger J. Kurlander

Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda,MD 20892-1508, USA

Correspondence to: R. Kurlander; E-mail: rkurlander{at}mail.cc.nih.gov


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice infected with Listeria monocytogenes (LM) produce large numbers of H2-M3-restricted CD8 T cells directed against the formylated peptides, f-MIGWII and f-MIVIL. To examine responsiveness to these epitopes in the absence of infection, we inoculated mice with recombinant lemA (r-lemA) containing f-MIGWII or r-vemA (a variant of r-lemA containing f-MIVIL in place of f-MIGWII) without adjuvant. To monitor responses, we measured peptide-specific cytoplasmic IFN-{gamma} production ex vivo by freshly harvested splenocytes at varying times post-inoculation. B6 mice inoculated with r-lemA produced substantial numbers of epitope-specific CD8 cells with peak levels on day 7 when there were 1.1 x 106 f-MIGWII-specific CD8 cells in the spleen (8.2% of total CD8 splenocytes). The r-vemA-treated animals accumulated 0.25 x 106 cells (1.8% of total CD8 cells) at this time point. Comparable responses were observed after rechallenge of immunized animals. Other elements in the lemA moiety distinct from the immunogenic peptide were required since mice did not respond to equimolar amounts of synthetic f-MIGWII or f-MIVIL alone. In comparative studies, B6 and C3H/HeJ mice responded to r-lemA much more vigorously than BALB/c animals. When r-lemA- or r-vemA-treated B6 animals were challenged i.v. with LM 7 days later, they suppressed splenic accumulation of bacteria much more effectively than controls. On the other hand, antigen-treated animals were not protected against infection 1 month later. Thus, responsive strains of mice respond vigorously to H2-M3-restricted epitopes, even in the absence of bacterial infection or adjuvant. The resulting effectors acutely enhance antimicrobial resistance but do not confer long-term memory protection.

Keywords: host resistance, passive immunization, peptide-specific CD8 T cells, primary immune response


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice infected with the Gram-positive bacterial pathogen Listeria monocytogenes (LM) develop an extensive CD8 T cell response within 5–7 days, which is essential for normal resolution of infection (1–3). Previous studies have identified subsets of effectors directed against a variety of bacterial epitopes. At least four listerial epitopes, LLO91–99, P60217–225, P60449–457 and mpl84–92 are presented by the classical class Ia MHC product H-2Kd (4), and LLO297–304 is presented by H-2Kb (5). Three others directed against the N-terminal sequences f-MIGWII from lemA (6), f-MIVTLF from AttM polypeptide (7) and f-MIVIL from an as yet unidentified bacterial product (8) are presented by the class Ib MHC product H2-M3 (9,10). Although other interactions are as yet less well characterized, infected mice also generate LM-specific CD8 cells restricted by other class Ib MHC products (11).

Because they share a common, conserved H2-M3 allotype (9,10), BALB/c, C3H/HeJ and C57Bl/6 (B6) mice all generate f-MIGWII- and f-MIVIL-specific CD8 T cells in response to LM infection. Nonetheless, there are substantial, as yet unexplained, strain-specific differences in the magnitude of these H2-M3-restricted responses. BALB/c mice consistently generate 3- to 4-fold fewer f-MIGWII- or f-MIVIL-specific effectors than B6 or C3H/HeJ animals (12,13).

Using C57Bl/6xBALB/c F1 mice to simultaneously study the time course of H-2Kd- and H2-M3-restricted CD8 responses to LM infection in vivo, Kerksiek et al. recently demonstrated significant differences between the patterns of expansion of LLO91–99-specific and f-MIGWII-specific effectors (12,13). Both expanded extensively during the first 9 days of primary LM infection and then diminished during the following 2 weeks, but the H2-M3-restricted response to f-MIGWII was ~3-fold larger than the response against LLO91–99 (the most immunodominant of the known LM-specific class Ia-restricted epitopes). The f-MIGWII-immune response also developed more rapidly, peaking on day 5–7 versus day 7–9 for H-2Kd-restricted cells. When mice were rechallenged with LM, the pattern of dominance was reversed. Recall f-MIGWII- and f-MIVIL-specific responses were substantially smaller than primary responses. By contrast, the secondary response against H-2Kd-restricted peptides was ~20-fold greater than the initial response (4). Consequently class Ia-restricted effectors numerically dominated the secondary response (12,13). Others using B6 mice and ELISpot-based methods have also observed similar differences between classical class I MHC product, and H2-M3-restricted primary and secondary responses (14).

The strain-related variations in H2-M3-restricted responses and the unusual features of CD8 responses against H2-M3-restricted antigens noted above potentially could be pathogen-specific, linked to one or more distinctive aspects of the interaction between invasive LM and the murine host. Alternatively they could reflect more general, intrinsic differences in responsiveness. To examine H2-M3-associated CD8 responses against LM-derived antigens in isolation, we inoculated mice with a recombinant truncated variant of lemA (r-lemA) expressing the immunogenic peptide f-MIGWII (15) and r-vemA, a chimeric molecule based on r-lemA, expressing f-MIVIL. Both antigens stimulated extensive, strain-specific, CD8 effector responses (comparable to those observed after LM infection), indicating that responsiveness to H2-M3-restricted antigens is pre-existent in some strains of mice and is not critically dependent upon exposure to other LM components, invasive infection or acute inflammation. Interestingly, the resulting CD8 effectors demonstrate significant protective potential against acute LM infection in vivo, but do not provide long-term protection against subsequent LM exposure.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Male C57BL/6, BALB/c and C3H/HeJ mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in a barrier facility within the National Institutes of Health. Animals used in these studies were 7–16 weeks old.

Peptides
The synthetic oligopeptides SIINFEKL, f-MIGWII and f-MIVIL were purchased from Genetics Research (Atlanta, GA). f-MFFINILTLLVP (f-ND1) and f-MFINRWLFS (f-CO1) were kindly provided by C.-R. Wang (University of Chicago, IL). Formylated and unformylated variants of MNIFTTSIL (f-ND5) were provided by R. Rich (Emory University School of Medicine, Atlanta, GA).

Preparation of r-lemA, r-vemA and r-semA
The methods used to prepare r-lemA, a recombinant construct containing the first 33 amino acids of the bacterial product lemA (6) followed by a brief linker sequence and a poly-histidine tail, have been described previously (15).

A variant of r-lemA, designated r-vemA, containing an initial f-MIVIL sequence (in place of f-MIGWII) was prepared. To this end, a new PCR product was generated using r-lemA plasmid as a template, the previously described polynucleotide 5'GTCGACACGGTTACGGTATTTTACAAGGCTG-3 as a reverse primer (15) and a new 46 nucleotide sequence 5'-ATTACATATGATCGTCATACTTGCTATCGCTGTTGTTGTCA-TTTTA-3' as a forward primer. The resulting product was cloned into the plasmid pCR2.1 (Invitrogen, Carlsbad, CA), excised using the restriction enzymes NdeI and SalI, and then transferred into the expression vector pET24a (Novagen, Milwaukee, WI).

The construct r-semA, containing the ovalbumin (OVA) sequence OVA257–264 preceded by a methionine, i.e. MSIINFEKL, in place of f-MIGWII was prepared in an analogous manner using a forward primer 5'-CATATGAGTATAATCAACTTTGAAAAACTTGCTATCGCTGTTGTTGTCATTTTAG-3.

After sequencing to confirm constructs were correct, r-lemA, r-vemA and r-semA were each expressed in BL21(DE3) bacteria induced with 1 mM IPTG. The sequence of each protein product is shown in Table 1Go. The resulting products were solubilized in 1% n-octyl-glucoside, purified on Talon metal affinity resin (Clontech, Palo Alto, CA) and then equilibrated in PBS (15). The final products were all insoluble proteins which migrated with an apparent mol. wt of ~5.5 kDa by SDS–PAGE. Protein concentrations were measured after solubilization in SDS using the BCA method (Pierce, Rockford, IL).


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Table 1. Amino acid sequence of recombinant antigens
 
Antigen inoculation of mice
Five nanomoles of the recombinant products r-lemA, r-vemA and r-semA (25 µg) or of the synthetic oligopeptides f-MIGWII (5 µg), f-MIVIL (4 µg) and SIINFEKL (5 µg) in PBS were inoculated s.c. without adjuvant (in a volume of 100–200 µl) into the base of the tail of mice. Secondary responses were evaluated after repeat inoculation with the same dose of antigen 30 days after initial exposure.

Ex vivo detection of peptide-specific CD8 cells
Spleens were obtained from inoculated mice at varying times after primary or recall antigen injection. Splenocyte suspensions prepared by dissociation between a pair of sterile glass slides were treated with ACK (Biowhittaker, Walkersville, MD) to lyse erythrocytes, washed, and resuspended in RPMI 1640 containing 10% FCS, 1 mM L-glutamine, 200 U/ml penicillin, 200 µg/ml streptomycin and 50 µM mercaptoethanol (R10).

To stimulate peptide-specific IFN-{gamma} production in vitro, splenocytes (1x106/well in flat-bottom microtiter plates) were suspended in 200 µl of R10 supplemented with 20 units/ml of IL-2 and monensin, with or without 100 ng/ml representing 130 nM of f-MIGWII or 160 nM of f-MIVIL (16). After incubation at 37°C for 5 h, cells were incubated with phycoerythrin-conjugated rat anti-mouse CD8{alpha} (Ly-2/53-6.7) and biotin-labeled rat anti-mouse L-selectin (CD62L/MEL-14) followed by streptavidin–PerCP. The surface-stained cells were next fixed and permeabilized using formaldehyde and saponin, and finally stained for intracytoplasmic IFN-{gamma} using FITC-labeled, rat anti-mouse IFN-{gamma} antibody (XMG1.2). All mAb, the permeabilizing solution (Cytofix/Cytoperm) and monensin (Golgi-Stop) were purchased from PharMingen (San Diego, CA), and used as suggested by the manufacturer.

The number of CD8+, CD62Llow cells containing intracytoplasmic IFN-{gamma} was quantitated by flow cytometry using a FACSCalibur (Becton Dickinson, Mountain View, CA). The cytometer was calibrated with QC3 microbeads (Flow Cytometry Standards, Fishers, IN) using CellQuest software (Becton Dickinson). In general, 0.5x106 viable splenocytes were sampled for each data point using CellQuest software (Becton Dickinson). The results were analyzed and displayed using FlowJo software (Three Star, San Carlos, CA).

To assess whether other peptides can interfere with epitope-specific IFN-{gamma} production ex vivo, splenocytes from r-lemA- or r-vemA-treated animals were preincubated for 10 min with potential competitors (50 µM) before addition of f-MIGWII or f-MIVIL at ~100 nM concentration as described above.

Quantitation of LM proliferation after primary LM infection in vivo
LM strain 10403S (17) was grown from frozen stock in brain heart infusion broth. To study the impact of effector immune CD8 cells on host resistance to LM, mice immunized 7 days earlier with recombinant proteins or PBS were infected i.v. with 15,000 c.f.u. of log-phase LM suspended in 300 µl of PBS. Mice were sacrificed 3 days later. Spleens were harvested, homogenized using 0.05% Triton X-100 in PBS and plated in serial dilutions on brain heart infusion agar. The number of colonies was determined after incubation at 37°C for 24 h and the results expressed as the log bacteria per spleen. The same procedure was used to study protection by long-term memory CD8 cells. In this case animals were infected with live LM 4 weeks after recombinant protein inoculation.

Statistics
The significance of differences in the number or frequency of IFN-{gamma}-producing cells between two groups of animals was evaluated using the Student's t-test. The significance of multi-group differences in splenic accumulation of bacteria was assessed using a logarithmic transformation to normalize the distribution of results and Dunnett's test (18).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Murine CD8 T cell responses to r-lemA and r-vemA in vivo
B6 mice were injected s.c. with 5 nmol of r-lemA, f-MIGWII, r-vemA and f-MIVIL. CD8 splenocytes were then harvested and assayed ex vivo 7 days later for evidence of peptide-specific intracytoplasmic IFN-{gamma} production. r-lemA and r-vemA both stimulated extensive intracellular IFN-{gamma} production by CD62Llow CD8 T cells (>5 and 1% of splenic CD8 cells respectively), but analogous responses were not noted in mice sensitized using synthetic f-MIGWII or f-MIVIL (Fig. 1Go). Though lemA7–33 is necessary for the f-MIGWII and f-MIVIL-immune responses we observed, this element is not sufficient to stimulate extensive class Ia MHC product-restricted responses. Spleens from mice inoculated with comparable amounts of r-semA (containing the H-2Kb-restricted peptide OVA257–264 covalently linked to lemA7–33) or an equimolar mixture of r-lemA and synthetic OVA257–264 contained <0.1% OVA257–264-specific CD8 cells 7 days later.



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Fig. 1. r-lemA and r-vemA stimulate an extensive epitope-specific CD8 response in vivo. Splenocytes from animals receiving each 5 nmol of either recombinant protein and from control animals treated with the same amount of the corresponding immunogenic peptide alone (see the left margin) were harvested 7 days later. The cells were stimulated for 5 h ex vivo with f-MIGWII, f-MIVIL or PBS alone (indicated on the top) in the presence of IL-2 and Brefeldin, and then stained for surface expression of CD8 and CD62L, and for intracytoplasmic IFN-{gamma}. The percentage of total CD8 cells expressing low CD62L and increased intracellular IFN-{gamma} is indicated in the upper left quadrant of each panel.

 
The H2-M3-restricted responses we observed were predominantly peptide-specific, but small numbers of r-lemA-induced and of r-vemA-induced cells cross-reacted after ex vivo exposure to f-MIVIL and f-MIGWII respectively (Fig. 1Go). On the other hand, neither set of effectors responded to f-ND-1 or f-CO-1 (data not shown), formylated mitochondrial peptides capable of binding avidly to H2-M3 (19).

Both f-MIGWII- and f-MIVIL-induced responses (Fig. 2A and BGo) could be competitively inhibited ex vivo by an excess of avid H2-M3-binding ligands like f-ND1 and f-CO1, but not by f-ND5 or unformylated ND5, peptides which bind very poorly to H2-M3 (19). This pattern of selective inhibition (20) supports peptide binding to H2-M3 as a critical step in recognition by our CD8 cells of f-MIGWII or f-MIVIL epitopes.



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Fig. 2. Impact of competing peptides on the responsiveness of epitope-specific CD8 cells ex vivo. r-lemA-immune (A) and r-vemA-immune (B) CD8 cells were harvested 7 days after ex vivo sensitization, were stimulated with f-MIGWII (A) or f-MIVIL (B) in the presence or absence of a 500-fold excess of unrelated peptides. Formylated peptides capable of binding avidly to H2-M3 (fND1 and fCO1), inhibited epitope-specific responses, but peptides binding poorly to H2-M3 (fND5 and ND5) did not.

 
Time course of primary and secondary responses
A measurable f-MIGWII-specific CD8 response in r-lemA-treated B6 mice becomes detectable by day 5 and peaks on day 7. The mean response at this time was 1.1 ± 0.1x106 cells per spleen or 8.2% of total CD8 cells. f-MIGWII-specific CD8 T cells decreased in frequency after day 9, but residual memory cells were still detected even 2 months after challenge (Fig. 3AGo). When mice were rechallenged with the same dose of r-lemA 30 days after primary exposure, the peak secondary response also was observed on day 7 (Fig. 3BGo). The mean response at this time was larger (2.4 ± 1.0x106 f-MIGWII-immune cells per spleen), but more variable than the primary response. Consequently, with the sample size employed for these studies, this difference was not statistically significant.



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Fig. 3. Time course for primary and recall responses to recombinant antigens. To study primary responses splenocytes were harvested from B6 mice at varying times after inoculation with 5 nmol of purified r-lemA (A) or r-vemA (C). Recall responses were monitored in a similar manner in animals rechallenged 30 days after an initial exposure to r-lemA (B) or r-vemA (D). Harvested cells were treated with the indicated peptide ex vivo, stained and analyzed as described above. The mean number of epitope-specific CD8 cells per spleen is plotted ± SE at the indicated times after primary or recall inoculation. Numbers in parentheses represent the percent of IFN-{gamma}+ CD8 cells on the indicated days post-inoculation. Each point represents the mean for two to seven mice.

 
The time course for accumulation of f-MIVIL-immune CD8 cells after primary and secondary r-vemA inoculation was qualitatively similar to the r-lemA response (Fig. 3C and DGo), but the magnitude of response was smaller. There were 2.5 ± 0.7x105 f-MIVIL-responsive CD8 cells/spleen or 1.8 ± 1% of total CD8 cells on day 7 of primary response, and 2.0 ± 0.4x105 specific cells/spleen representing 1.6 ± 0.4% of total CD8 cells after rechallenge. As with r-lemA, differences between peak primary and secondary responses were not statistically significant.

In our limited experience (two animals per group), animals rechallenged with r-lemA or r-vemA retained larger numbers of residual f-MIGWII- and f-MIVIL-specific memory cells 2–3 months after their last inoculation than animals receiving a single antigen dose (Fig. 3Go).

Strain variations in the response to r-lemA
Although B6, C3H/HeJ and BALB/c mice express an identical H2-M3 molecule, Kerksiek et al. have demonstrated substantial strain variations in the magnitude of the f-MIGWII-specific response to primary LM infection (12,13). We found an identical pattern of response after passive immunization with r-lemA. B6 mice were most responsive, C3H/HeJ demonstrate comparable responsiveness and BALB/c were least responsive (Fig. 4Go and Table 2Go).



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Fig. 4. Representative example of the strain variations in the magnitude of the epitope-specific response to r-lemA. The number of IFN-{gamma}-producing cells generated in B6, C3H/HeJ or BALB/c mice was measured 7 days after inoculation with 5 nmol of purified r-lemA. The percentage of CD8 cells producing IFN-{gamma} in response to f-MIGWII is shown in the upper left quadrant. Two to seven animals of each strain were studied.

 

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Table 2. Strain variations in the magnitude of the epitope-specific response to r-lemAa
 
f-MIGWII- and f-MIVIL-immune CD8 cells enhance resistance to primary LM infection
To assess the impact of mono-specific H2-M3-restricted CD8 cells on bacterial accumulation during LM infection, we infected B6 mice i.v. with 15,000 c.f.u. of live LM, 7 days after inoculation with PBS alone, r-semA, r-lemA or r-vemA. At the time of sacrifice 3 days after infection, animals immunized with r-vemA had 70-fold lower bacterial counts and mice immunized with r-lemA had almost 1400-fold fewer bacteria than PBS-treated control animals. These differences were highly significant (P < 0.005). They were not a non-specific consequence of the infusion of recombinant products since bacterial counts in r-semA- and PBS-treated animals were not significantly different (Fig. 5Go).



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Fig. 5. r-lemA and r-vemA protect mice against LM accumulation in vivo. Groups of four mice were pretreated with 5 nmol of r-lemA, r-vemA or r-semA, or an equivalent volume of PBS. Seven days later, all mice were infected i.v. with 15,000 c.f.u. of LM. Mice were sacrificed 3 days later and the bacterial load in each spleen was quantitated. Each point represents the bacteria count in one animal and the horizontal bar reflects the log mean value for each group. Data are mean log10 c.f.u. ± SD, n = 4 mice per group. The statistical significance of the differences observed is indicated using Dunnett's test. Similar results were obtained in a second independent study.

 
Memory cells generated after exposure to r-lemA or r-vemA do not significantly protect mice against later exposure to LM
To examine the impact of f-MIGWII and f-MIVIL memory cells on the early host response to infection, we infected mice with 45,000 c.f.u. of live LM, 4 weeks after immunization with PBS, r-semA, r-vemA or r-lemA. Though the splenic burden of LM at the time of sacrifice 3 days later was slightly reduced in r-lemA- and r-vemA-treated compared to control animals, these differences were not statistically significant (Fig. 6Go).



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Fig. 6. r-lemA- and r-vemA-induced memory CD8 T cells do not significantly protect mice against early LM accumulation in vivo. Groups of four or five mice were pretreated with 5 nmol of r-lemA, r-vemA or r-semA, or an equivalent volume of PBS. Four weeks later, all mice were infected i.v. with 45,000 c.f.u. of LM. Mice were sacrificed 3 days later and the bacterial load in each spleen was quantitated. Results were analyzed as described in Fig. 5Go.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we have used recombinant constructs based on the LM protein lemA to examine the antigen responsiveness of H2-M3-restricted CD8 cells in vivo. r-lemA (in the absence of pore-forming listeriolysin O or adjuvant) evokes an f-MIGWII-specific CD8 response roughly comparable in magnitude and kinetics to the primary response observed in LM-infected animals (12,13). r-vemA stimulates a smaller f-MIVIL-specific response, which is also quite similar to that induced by LM infection (13). The marked similarities in the numbers of f-MIGWII- and f-MIVIL-specific cells generated after primary infection and passive antigen challenge imply that the initial expansion of these H2-M3-restricted effectors is not critically dependent upon bacterial infection of antigen-presenting cells or co-stimulatory factors released in response to infection and inflammation.

The CD8 responses we observed clearly require the presence of the hydrophobic lemA7–33 element, since inoculation of equivalent molar amounts of synthetic f-MIGWII or f-MIVIL did not stimulate any measurable antigen-specific responses. Indeed, f-MIGWII is a poor immunogen even after infusion with incomplete Freund's adjuvant (21). We do not address the mechanism underlying this enhanced response in vivo directly, but prior studies have demonstrated that lemA7–33 facilitates endosomal processing and presentation of the f-MIGWII sequence in r-lemA and also stabilizes its immunogenic portion from extracellular proteases in vitro (15,22). The particulate character of the hydrophobic lemA7–33-containing constructs may also facilitate their processing as an exogenous antigen (23).

The r-lemA- and r-vemA-dependent responses described in this manuscript are noteworthy for their magnitude. In prior studies using protein–adjuvant mixtures (16), peptide-pulsed dendritic cells (24) or even a sophisticated lipopeptide construct containing a CD4 helper sequence and the extremely antigenic lymphocytic choriomeningitis virus NP396–404 epitope (25) to stimulate class Ia MHC product-restricted responses, peak epitope-specific CD8 cell levels in lymphoid tissue or spleen barely exceeded 1% of total CD8 cells.

While the inclusion of lemA7–33 in our constructs clearly was necessary, it alone could not ensure large responses. Neither r-semA, containing lemA7–33 linked to the conventional class Ia-restricted peptide OVA257–264, nor a mixture of r-lemA and OVA257–264 could elicit >0.1% OVA257–264-specific CD8 cells. Thus, the epitope specificity of our H2-M3-restricted constructs must also be an important determinant of responsiveness.

Our findings could reflect a qualitative difference in antigen presentation or an intrinsic advantage of H2-M3-restricted over class Ia MHC product-restricted CD8 cells in recognizing antigens or proliferating in response to antigen challenge. To date, however, there is no evidence to support these hypotheses. More likely, `naive' B6 mice respond so well to these H2-M3-restricted antigens because they have an unusually large pool of pre-existent peptide-specific precursors. Prior studies have noted that a subset of f-MIGWII-immune CD8 cells can recognize other, irrelevant formylated peptide antigens and heat-killed preparations of unrelated bacteria (26,27). Although the r-lemA- and r-vemA-immune effectors examined in these studies and in recent studies reported by Kerksiek et al. (13) were predominantly `specific' using our limited antigenic peptide panel, they nonetheless could represent descendents of cells previously primed and expanded peripherally by cross-reactive bacterial antigens common in the environment (14,27). Indeed, the failure to observe any statistically significant increase in f-MIGWII- or f-MIVIL-specific responses after rechallenge with recombinant antigen or bacteria suggests preformed memory cells may have already expanded to a maximally sustainable level even before the animals' initial contact with LM-associated epitopes.

In prior studies using LM-infected animals, secondary responses to f-MIGWII and f-MIVIL were substantially smaller than primary responses (12–14). Since we failed to observe comparable declines in animals rechallenged with mono-specific recombinant antigens, this finding may reflect competition (28) between H2-M3-restricted cells and rapidly expanding class Ia-restricted effectors stimulated by repeat exposure to other LM epitopes for access to antigen-presenting cells or other critical resources.

Although all common strains of mice express the same H2-M3 molecule, previous studies using LM infection to stimulate effectors have noted significant differences in the magnitude of H2-M3-restricted responses in BALB/c, B6 and C3H/HeJ mice (12,13). The interpretation of these prior findings, however, is complicated by known strain-specific differences in susceptibility to LM (29), which could markedly influence the ultimate antigenic load to which CD8 cells are exposed during infection. Furthermore, MHC-dependent variations in the number and specificity of CD4, CD8 and B cells generated in response to other LM antigens could also indirectly affect H2-M3-restricted proliferation, e.g. by shaping the cytokine milieu. Since we observed an identical pattern of strain responsiveness using a constant antigen dose, in the absence of other LM-derived epitopes or frank inflammation, the current studies allow us to exclude these factors as probable explanations for the observed diversity. A more systematic immunogenetic approach will be needed to explain strain-specific differences in presentation by an invariant MHC product.

CD8 cells clearly play an important role in the host response against LM infection (1–3), but not all effectors are equally potent. Recent studies using LM engineered to express the same LCMV NP118–126 epitope associated with a secreted or a non-secreted product found that NP118–126-specific CD8 memory cells could protect mice infected with the former (which can be rapidly processed intracytoplasmically in infected cells via the endogenous pathway), but not LM expressing the latter, which first must be released from nonviable bacteria and before processing within the endosomal compartment (30,31).

Since lemA lacks an initial signal peptide sequence (6), these studies raise the possibility that CD8 cells directed against this antigen may be unable to contain LM infection in vivo. Yet, we and others have demonstrated that cloned f-MIGWII-specific effectors can adoptively transfer LM resistance to naive mice (21,22). In addressing this discrepancy directly in immunized animals, we demonstrate significant functional differences between antigen-specific effectors and memory cells. The former, generated in vivo by r-lemA sensitization 1 week earlier, have substantial protective activity against LM infection. Yet, memory cells generated concurrently from these effectors do not. This contrasts with the marked protection, which can be produced by class Ia-restricted CD8 memory cells under comparable conditions (30,32).

This disparity in protective function may be linked to differences in the prevalence of antigen-specific cells 1 week and 1 month after initial exposure to r-lemA or LM. At the earlier time point, the spleen contains large numbers of lemA-specific CD8 effectors. Even if LM-infected cells present lemA less efficiently than some other antigens, this number of effectors appears to be sufficient to provide substantial protection to the infected host. On the other hand, by 1 month after antigen immunization, only a small number of antigen-specific cells are still present and these are in a `resting' memory state. In this setting, where rapid effector expansion is essential, CD8-mediated containment of infection may be much more seriously compromised by inefficient antigen presentation or by possible limitations in the vigor of H2-M3-restricted recall responses noted by others (12–14).

Since high responder strains such as B6 or C3H/HeJ accumulate substantial numbers of f-MIGWII- and f-MIVIL-immune effectors during the critical phases when LM are typically destroyed during primary infection (33), these cells probably make a significant contribution in the CD8-mediated containment of primary infection. On the other hand, H2-M3-restricted effectors accumulate in much smaller numbers in BALB/c mice. Consequently, they probably play a much less important role in control of primary infection in this strain. Indeed, the lower level of responsiveness to H2-M3-restricted antigens could be one of the factors underlying the increased susceptibility of this strain to primary infection (29).

Despite the marked antigenicity of r-lemA and r-vemA, and the capacity of the resulting effectors to enhance host resistance against ongoing LM infection, our studies to date suggest these products cannot induce long-term CD8- mediated protection. It may, however, be premature to conclude that H2-M3 restricted memory cells are intrinsically unsuited for such a role. Mice lacking class Ia MHC products H-2Kb and H-2Db nonetheless generate an effective CD8 memory response after primary LM infection, and much of this response is presumed to be H2-M3 restricted (14). Protection in this setting could reflect the presence of cells directed against other H2-M3-restricted epitopes, which may be presented more efficiently by bacterially infected cells than f-MIGWII and f-MIVIL. Alternatively, f-MIGWII- and f-MIVIL-immune memory cells generated in response to virulent infection may respond to rechallenge better than our cells, which arose in the absence of co-stimulation. It remains to be seen whether the protective activity of CD8 memory T cells generated by r-lemA or r-vemA can be meaningfully enhanced using adjuvants, more intensive inoculation schedules or other immunologic manipulations. Such studies may provide practical insight into the feasibility of enhancing the effectiveness of CD8 responses by manipulating the conditions used during sensitization.


    Acknowledgments
 
We wish to thank Chyung-Ru Wang for generously donating peptide reagents for these studies, Bob Wesley and Dee Koziol for statistical assistance and Jay Berzofsky for critically reviewing the manuscript.


    Abbreviations
 
LM Listeria monocytogenes
OVA ovalbumin
R10 RPMI 1640 supplemented with FCS, L-glutamine, penicillin, streptomycin and 2-mercaptoethanol

    Notes
 
Transmitting editor:M. J. Bevan

accepted 5 November 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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