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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: host resistance, passive immunization, peptide-specific CD8 T cells, primary immune response
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 LLO9199-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 LLO9199 (the most immunodominant of the known LM-specific class Ia-restricted epitopes). The f-MIGWII-immune response also developed more rapidly, peaking on day 57 versus day 79 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 OVA257264 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 1. 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 SDSPAGE. Protein concentrations were measured after solubilization in SDS using the BCA method (Pierce, Rockford, IL).
|
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- 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
(Ly-2/53-6.7) and biotin-labeled rat anti-mouse L-selectin (CD62L/MEL-14) followed by streptavidinPerCP. The surface-stained cells were next fixed and permeabilized using formaldehyde and saponin, and finally stained for intracytoplasmic IFN-
using FITC-labeled, rat anti-mouse IFN-
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- 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- 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--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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Both f-MIGWII- and f-MIVIL-induced responses (Fig. 2A and B) 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.
|
|
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 23 months after their last inoculation than animals receiving a single antigen dose (Fig. 3).
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. 4 and Table 2
).
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The CD8 responses we observed clearly require the presence of the hydrophobic lemA733 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 lemA733 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 lemA733-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 proteinadjuvant 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 NP396404 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 lemA733 in our constructs clearly was necessary, it alone could not ensure large responses. Neither r-semA, containing lemA733 linked to the conventional class Ia-restricted peptide OVA257264, nor a mixture of r-lemA and OVA257264 could elicit >0.1% OVA257264-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 (1214). 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 (13), but not all effectors are equally potent. Recent studies using LM engineered to express the same LCMV NP118126 epitope associated with a secreted or a non-secreted product found that NP118126-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 (1214).
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 |
---|
![]() |
Abbreviations |
---|
LM Listeria monocytogenes |
OVA ovalbumin |
R10 RPMI 1640 supplemented with FCS, L-glutamine, penicillin, streptomycin and 2-mercaptoethanol |
![]() |
Notes |
---|
accepted 5 November 2001.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|