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Sections of Infectious Diseases and Immunobiology, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ag processing has been shown to play a pivotal role in generating T cell responses. For example, studies have shown that changing the flanking residues of T cell epitopes can alter the antigenicity of a protein (5, 6, 7, 8). Targeting of peptides to the endoplasmic reticulum can also enhance T cell responsiveness (9). The rate of peptide dissociation from MHC molecules has also been implicated in determining T cell response sizes (10, 11, 12). The overall complexity of the T cell response to a pathogen can influence the magnitude of the response to individual epitopes, as demonstrated in the MHC class I-restricted response to SV40 T Ag (2), influenza virus (13), and the MHC class II-restricted response to the staphylococcal nuclease (1). Other studies have implicated the TCR repertoire as a major determinant of the size of responding T cell populations (14, 15, 16, 17). The effect of TCR repertoire differences on immunodominance has been suggested to result from differences in positive selection (17). Several studies have demonstrated the dramatic influence of the CD28 and CTLA-4 costimulatory molecules on the size of in vivo T cell responses following immunization (18, 19).
Another factor that influences T cell responses is the number of epitopes presented on the cell surface. In vitro analyses of MHC class II-restricted CD4 T cells showed that variations in Ag dose swayed responding T cells to secrete either Th1 or Th2 cytokines (20, 21). Similar in vitro analyses with MHC class I-restricted CD8 T cells have shown that different concentrations of peptide epitopes during T cell priming can dramatically influence the kinetics of T cell proliferation and IL-2 expression (22). CTL clones vary in their sensitivity to surface epitope concentrations, with some requiring as few as 10 and others requiring thousands of surface epitopes for activation (23). The extent of in vitro activation of T cell clones can be determined by different epitope concentrations (24, 25). The effects of different epitope concentrations on in vivo T cell priming and expansion, however, are not known.
Listeria monocytogenes is a Gram-positive, pathogenic bacterium that enters the cytosol of infected cells. Mice infected with L. monocytogenes mount MHC class I-restricted CTL responses specific for several peptides presented by H2-Kd and H2-M3 molecules (26). Quantitative studies of the presentation of three H2-Kd-restricted epitopes revealed that they are presented by infected cells in vastly different amounts (8, 27). Thus, LLO 91–99, p60 217–225, and p60 449–457 are present in infected cells at a ratio of approximately 1:4:14, respectively. In contrast, the ratio of T cells specific for these three epitopes at the peak of the response is approximately 20:10:1, respectively (4). Thus, in comparing T cell responses with different epitopes, the efficiency of epitope presentation does not predict the magnitude of T cell responses.
In this study, we determine the influence of Ag-processing efficiency on the magnitude of in vivo CTL responses. Without changing the virulence, infectivity, or p60 secretion rate of L. monocytogenes, we have mutated a flanking residue of the dominant p60 217–225 epitope. This has generated a panel of L. monocytogenes strains that are identical, except that the relative amount of p60 217–225 that is presented upon cellular infection varies from 0 to more than 4000 epitopes per cell. We find that the magnitude of the in vivo T cell response to p60 217–225 does not directly reflect the amount of epitope that is presented. Over a fivefold range of epitope concentrations the size of the T cell response remains unchanged. However, once the efficiency of p60 217–225 drops below a threshold level, even though epitopes are being generated, there is no detectable T cell response. Our investigations indicate that the ultimate size of the T cell response is not determined by the number of epitopes that are presented in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CTL clones L9.6 (specific for p60 217–225) and WP11.12 (specific for p60 449–457) were maintained by weekly restimulation with L. monocytogenes-infected J774 cells, as described previously (8). P815 mastocytoma cell line (H-2d) and J774 macrophage cells (H-2d) were obtained from American Type Culture Collection (Rockville, MD) and maintained in RP10 medium supplemented with 10% FCS, as described (27). BALB/c and CB6 mice (C57BL/6 x BALB/c F1; H-2bxd) were obtained from The Jackson Laboratory (Bar Harbor, ME).
Bacterial strains
L. monocytogenes strain 10403S was obtained from Dr. Daniel Portnoy (University of Pennsylvania, Philadelphia, PA) and cultured in brain-heart infusion broth (BHI3).
Generation of L. monocytogenes mutant strains
L. monocytogenes p60 216 mutant strains were generated by changing wild-type valine 216 codon to alanine, histidine, glycine, and aspartic acid within the p60 gene. A promoterless copy of the p60 gene spanning bp 431 to 1941 was amplified from genomic DNA of L. monocytogenes using the PCR with Vent polymerase (NEB, Beverly, MA). The 5' oligonucleotide sequence was 5'-GAGAGGAGTCATATGAATATGAAAAAAGCAACTG-3', and the 3' oligonucleotide sequence was 5'-CGCTTAAGGAACTGCTTGCTCCACAGGTT-3'. The 1.5-kb PCR product was cloned into ptz19U (Bio-Rad, Hercules, CA) after an intermediate cloning step into the TA vector (Invitrogen, San Diego, CA). ssDNA was generated from ptz19U for mutagenesis (28) and served as a template for generation of the various mutants in codon 216 and codon 218 of p60. The wild-type 216 valine codon was changed to alanine, histidine, glycine, and aspartic acid, and wild-type 218 tyrosine was changed to serine with the following primers:
Alanine: 5'-CATAATGTCTTGTACACTAACACCGTATTTTGGGGATAAAGCCC-3'
Histidine: 5'-CATAATGTCTTGTACACTAACACCGTATTTGTGGGATAAAGCCC-3'
Glycine: 5'-CATAATGTCTTGTACACTAACACCGTATTTTCCGGATAAAGCCC-3'
Aspartic acid: 5'-CATAATGTCTTGTACACTAACACCGTATTTGTCGGATAAAGCCC-3'
Serine: 5'-CATAATGTCTTGTACACTAACACCAGATTTTACGGATAAAGCCC-3'
The above primers also incorporated a unique, silent BsrGI restriction site (that is underlined) in p60 to simplify screening of the chromosomal mutants. All mutations were confirmed by DNA sequencing. Mutated p60 genes were cloned into the thermosensitive plasmid, pKSV7, to enable homologous recombination of these mutations into the chromosome of L. monocytogenes (29, 30). Briefly, the plasmid pKSV7 containing the mutant p60 gene was electroporated into L. monocytogenes 10403S and plated onto BHI plates containing 10 µg/ml chloramphenicol. Cultures of chloramphenicol-resistant colonies were passaged at 41°C to integrate the plasmid into the homologous chromosomal p60 gene of L. monocytogenes. The cultures were then passaged at 30°C in the absence of chloramphenicol, which resulted in excision of the plasmid. After several such passages, colonies were screened for the presence of the chromosomal mutation by replica plating on BHI plates with and without chloramphenicol. Genomic DNA from chloramphenicol-sensitive colonies carrying the unique restriction site was PCR amplified and sequenced to confirm the presence of the desired mutation.
Quantitation of p60 217–225 and p60 449–457 CTL epitope numbers from J774 cell line (H2-Kd)
CTL epitopes were isolated from cell pellets, as described previously (27, 31). Briefly, J774 cell pellets obtained after a 6-h infection with L. monocytogenes were resuspended in 10 ml of 0.1% trifluoroacetic acid (TFA), dounce homogenized, sonicated, and centrifuged at 100,000 x g for 35 min. Supernatants were concentrated by lyophilization, resuspended in 2 ml 0.1% TFA, and passed through a Centricon-10 membrane (Beverly, MA). The filtrate was HPLC fractionated, and fractions were lyophilized and resuspended in 200 µl of PBS. Fractions 28 and 29 were tested for p60 217–225 and p60 449–457, respectively, in a 4-h chromium-release assay using CTL clones L9.6 and WP11.12 (8) with 51Cr-labeled P815 cells. The concentration of epitope in HPLC fractions was determined by titration of HPLC fractions, and comparison with the percent specific lysis obtained with a standard curve of known concentrations of synthetic peptide (27). The number of epitopes per infected cell was calculated by correcting for a 50% extraction efficiency for p60 217–225, as described previously (27).
p60 217–225 epitope and bacterial quantitation from the spleens of Listeria-infected mice
BALB/c mice were infected with 1 x 106 wild-type L. monocytogenes and each of the L. monocytogenes 216 mutant strains. Spleens were removed 48 h after infection and homogenized with a sintered glass homogenizer, followed by dounce homogenization (32). p60 217–225 epitopes were extracted in 0.1% TFA and HPLC fractionated, as described above. HPLC fractions were assayed for p60 217–225 with P815 cells in a 51Cr release assay using the CTL clone L9.6 (specific for p60 217–225) at an E:T ratio of 20:1. The concentrations of epitopes in HPLC fractions of spleen extracts were calculated by comparing the specific lysis obtained with a standard curve generated with known concentrations of synthetic p60 217–225. For bacterial quantitation from infected spleens, a small sample of the infected spleen was washed and then passed through a wire mesh and solubilized in 0.05% Triton X-100, and an appropriately diluted aliquot was plated onto BHI plates.
The ELISPOT assay for quantitating the number of IFN-secreting T cells
The ELISPOT assay, as described previously (4, 33), was used to
determine the number of IFN-
-secreting T cells specific for LLO
91–99, p60 217–225, and p60 449–457 in spleens of immunized mice.
Briefly, 96-well nitrocellulose plates (Millipore, Bedford, Ma) were
coated overnight with rat anti-IFN- Ab. Immune splenocytes
(105) were placed into wells along with
105 P815 cells that were precoated (or left uncoated) for
1 h at 37°C with 10-6 M of the three synthetic
epitopes. Splenocytes were incubated with peptide-coated APCs for
24 h with 30 U/ml IL-2. Wells were then washed, and the production
of IFN- was detected by development of the plates, as described (4).
The number of spots were counted under a dissecting microscope, and the
magnitude of the in vivo CTL response was plotted as number of
IFN-secreting T cells per 105 splenocytes.
Epitope-specific in vitro restimulation of Listeria-immune splenocytes from wild-type, p60 Asp216, and Ser218 strains of L. monocytogenes
CB6 mice were immunized by i.v. injection with 1000 L. monocytogenes wild-type, Asp216, and Ser218 strains. After 1 wk, splenocytes were harvested and restimulated in vitro with naive, irradiated syngeneic splenocytes that had been coated with 10-6 M of LLO 91–99, p60 217–225, and p60 449–457, as previously described (8). After 5 days of incubation at 37°C, responders were tested either undiluted or at serial threefold dilutions for recognition of 51Cr-labeled P815 target cells in the presence and absence of 10-6 M of the respective stimulating peptides. The percent specific lysis was calculated as previously described (8).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies have suggested that the magnitude of the T
cell response to three different H2-Kd-restricted
L. monocytogenes epitopes does not correlate with the
efficiency of their presentation (4). Because of the difficulties
inherent in comparisons of T cell responses different epitopes, we
decided to determine the influence of different Ag-processing
efficiencies on the magnitude of the in vivo CTL response to one
epitope. We therefore generated strains of L. monocytogenes
with mutations in position 216 of p60, the amino acid that flanks the N
terminus of the immunodominant CTL epitope p60 217–225. The codon for
amino acid 216 of p60 was mutated from wild-type serine to alanine,
histidine, glycine, and aspartic acid. The mutant p60 genes were
incorporated into the chromosome of L. monocytogenes by
homologous recombination, thus enabling stable expression of mutant
p60, even upon infection of mice (Fig. 1
). Amino acid 218 of p60 was also
mutated from tyrosine to serine, thereby replacing an essential anchor
residue for binding to H2-Kd. This strain therefore served
as a knockout of the p60 217–225 epitope during immunization studies.
All of the mutant L. monocytogenes strains were evaluated
for growth rate, p60 secretion, and J774 cell infectivity, and were
found to be identical to the wild-type strain (data not shown).
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To determine whether mutations in amino acid 216 altered the
efficiency of p60 217–225 generation, we infected J774 cells with the
mutant L. monocytogenes strains and TFA-extracted and
HPLC-fractionated MHC class I-associated peptides, as previously
described (27). Relevant HPLC fractions were assayed for the presence
of p60 217–225 and p60 449–457 in 51Cr release assay
using CTL clone L9.6 (specific for p60 217–225) and CTL clone WP11.12
(specific for p60 449–457). As shown in Figure 2A, target cells coated with
HPLC fractions from cells infected with the different L.
monocytogenes strains were lysed to different extents by CTL clone
L9.6. Fractions obtained from L. monocytogenes
Ala216-infected cells reproducibly contained the largest
amount of targeting peptide, while, as expected, no p60 217–225
targeting activity could be detected in extracts of L.
monocytogenes Ser218-infected cells. L.
monocytogenes wild-type, His216-,
Gly216-infected cells yielded intermediate amounts of p60
217–225, while L. monocytogenes Asp216-infected
cells yielded a smaller amount of the epitope. These results indicate
that changes in amino acid 216 of p60 affect the efficiency of p60
217–225 generation. The amount of p60 449–457, as determined with CTL
clone WP11.12, is similar in cells infected with the different L.
monocytogenes strains. This indicates that altering residue 216 of
p60 affected only the processing of the p60 217–225 epitope and not
the generation of p60 449–457. Metabolic labeling and pulse/chase
analyses of L. monocytogenes-infected J774 cells revealed
that wild-type and mutant forms of p60 were degraded with similar
kinetics (results not shown).
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In vivo Ag processing following infection with mutant L. monocytogenes strains
Since T cell priming occurs in response to in vivo Ag processing,
we determined whether our in vitro findings on the cellular processing
of wild-type and mutant p60 approximated in vivo Ag processing. Mice
were infected with L. monocytogenes Ala216,
wild-type, His216, Gly216, and
Asp216, and p60 217–225 epitopes were extracted from
infected spleens. TFA extracts of infected spleens were HPLC
fractionated, and p60 217–225-containing fractions were assayed for
specific lysis with CTL clone L9.6. The concentration of p60 217–225
in the HPLC fractions of spleens infected with the different L.
monocytogenes strains was determined by comparison with a standard
curve of known concentrations of synthetic peptide p60 217–225 (27).
The concentration of p60 217–225 in infected spleens reflects a range
of cellular infections, with some highly and some minimally infected
cells. As shown in Figure 3A,
a hierarchy of epitope concentrations was present in HPLC fractions of
infected spleens, similar to that obtained with infected J774 cells
(Fig. 2
, A and C). This indicates that in
vitro analyses of Ag processing in L. monocytogenes-infected
J774 cells closely approximate in vivo Ag processing in infected
spleens. Our assays did not detect p60 217–225 in spleens infected
with L. monocytogenes Asp216. Although it is
possible that no p60 217–225 is generated in this situation, we
believe it is far more likely that the number of epitopes produced
falls below the detection sensitivity of our assay. To determine
whether the mutant L. monocytogenes strains were of similar
virulence upon murine infection, we quantified the number of bacteria
in spleens of mice 48 h after i.v. infection. As shown in Figure 3B, the number of live bacteria obtained per gram of spleen
is similar in mice infected with each of the L.
monocytogenes strains, indicating similar extents of
infection.
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To determine whether the efficiency of p60 217–225 generation
influences the magnitude of the in vivo T cell response, mice were
immunized with wild-type and the mutant L. monocytogenes
strains. One week later, the number of p60 217–225-specific T
cells was quantified by ELISPOT. The magnitude of the T cell response
to LLO 91–99, p60 217–225, and p60 449–457 was similar in mice
infected with wild-type and L. monocytogenes
Ala216, Gly216, and His216 (Fig. 4). As expected, no p60 217–225
epitope-specific T cells are generated in mice infected with L.
monocytogenes Ser218. Interestingly, immunization with
L. monocytogenes Asp216 did not prime a T cell
response to p60 217–225, while the response to LLO 91–99 and p60
449–457 was normal (Fig. 4). These results suggest that the amount of
p60 217–225 generated by cells infected with L.
monocytogenes Asp216 falls below a threshold required
to elicit a T cell response. It is possible that a linear relationship
between CTL response magnitude and Ag-processing efficiency exists in
the narrow window between the Asp216 and Gly216
mutants. Nevertheless, our results demonstrate that once the threshold
is exceeded, incremental increases in the amount of CTL epitope do not
augment the magnitude of the in vivo T cell response.
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A, splenocytes from L. monocytogenes wild-type
infected mice were readily restimulated with LLO 91–99,
p60 217–225 (Fig. 5D),
and p60 449–457 (Fig. 5G). As expected, splenocytes
from L. monocytogenes Ser218-infected mice did
not respond to restimulation with p60 217–225 (Fig. 5F), but did respond to LLO 91–99 (Fig. 5C) and p60 449–457 (Fig. 5I).
Similarly, splenocytes from L. monocytogenes
Asp216-immunized mice also did not respond to p60 217–225
(Fig. 5E), while LLO 91–99 (Fig. 5B) and p60 449–457 (Fig. 5H)
stimulation readily expanded specific CTL. This result confirms that
infection with L. monocytogenes Asp216 does not
prime p60 217–225 epitope-specific T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 4). Roughly 17 p60 molecules with alanine
in position 216 must be degraded to generate one
H2-Kd-bound p60 217–225 epitope. In contrast, roughly 350
p60 molecules with aspartic acid in position 216 are degraded to
generate one p60 217–225 epitope. These experiments confirm the
importance of flanking residues on the efficiency of epitope generation
and presentation (5, 6, 8). Furthermore, these experiments demonstrate
for the first time that quantitative analyses of Ag processing in
infected J774 cells nicely reflect Ag processing occurring in vivo
following L. monocytogenes infection. How the size of a T cell response is determined in vivo is an important, unanswered question. Two factors, the number of responding T cells and their extent of replication, determine the ultimate magnitude of an epitope-specific T cell response. One might expect that higher in vivo epitope concentrations would stimulate a broader range of T cells and prolong the duration of T cell expansion. Our results suggest that neither of these circumstances is realized during in vivo T cell priming and expansion in response to L. monocytogenes infection. Instead, our experiments indicate that a relatively constant T cell response is activated by a range of suprathreshold epitope numbers. Although the mechanism underlying the constant T cell response to different concentrations of surface MHC/epitope complexes is unclear, several conclusions flow from our findings. First, it is likely that a population of CD8 T cells with similar avidities for H2-Kd/p60 217–225 complexes responds to p60 217–225. Thus, changing the concentration of epitope in vivo generates uniform behavior among responding T cells (i.e., either no response at subthreshold or a full response at suprathreshold epitope concentrations). Second, the extent of naive T cell priming and expansion is not related in a linear fashion to the amount of presented epitope. Thus, adding more epitopes during T cell priming does not appear to either recruit more or enhance the replication of T cells. Our findings suggest that the extent of T cell expansion during the response to L. monocytogenes infection is not influenced by the epitope concentration. Thus, synchronous expression of a T cell surface molecule such as CTLA-4 at a given time point following infection may curtail T cell expansion independent of epitope concentrations (37, 38). Alternatively, epitopes may be cleared from the infected focus at a fixed time point following infection, perhaps by CTL-mediated destruction of infected cells, regardless of the efficiency of Ag presentation.
The question of how T cell populations expand, contract, and enter the memory compartment following infection by pathogenic organisms is one of the most important in the field of immunology. Our findings suggest that the relationship between Ag presentation and immunodominance may be more complex than previously suspected, and may have important implications for vaccine design and development.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric G. Pamer, Yale University School of Medicine, Infectious Diseases Section, 803 LCI, P.O. Box 208022, New Haven, CT 06520-8022.
3 Abbreviations used in this paper: BHI, brain-heart infusion; TFA, trifluoroacetic acid.
Received for publication November 14, 1997. Accepted for publication December 22, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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