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Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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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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Previously, we reported that a dichotomy exists in the CD8 T cell response between L. monocytogenes expressing a secreted or nonsecreted fusion protein containing a H-2d-restricted epitope, nucleoprotein (NP)118–126,3 derived from lymphocytic choriomeningitis virus (LCMV) (11). Both forms of Ag are capable of priming NP118–126-specific CD8 T cells and establishing functional memory. However, infection of LCMV-immunized mice with L. monocytogenes expressing either the secreted or nonsecreted LCMV-derived Ag produces unexpected results. L. monocytogenes expressing the secreted Ag is rapidly cleared from the mice, whereas L. monocytogenes expressing the nonsecreted Ag is cleared at the same rate as in naive mice. These results indicate that both secreted and nonsecreted Ag can induce CD8 T cell responses, while only secreted Ag can serve as protective Ag for CD8 T cell-mediated immunity. This dichotomy as a result of Ag compartmentalization reveals an intriguing aspect of CD8 T cell surveillance during L. monocytogenes infection. Several critical questions remain. Does this phenomenon hold true for other Ags and therefore does it apply to bacterial Ags in general? What is the underlying mechanism for the dichotomy between priming and protective immunity?
Two possible mechanisms have been postulated to explain why nonsecreted bacterial Ag induces CD8 T cell responses, yet cannot serve as a protective target (11). One mechanism relates to the presentation of nonsecreted Ag by only a subset of infected cells. L. monocytogenes can infect both phagocytic and nonphagocytic cells. Secreted bacterial Ag in the host cell cytosol is presented by the endogenous MHC class I pathway, which is operative in most cell types. On the other hand, presentation of nonsecreted Ag may be limited to cross-presentation, which, in turn, is restricted to professional APCs, such as dendritic cells. Thus, Ag-specific memory CD8 T cells specific for a nonsecreted epitope may be unable to control infection, since they cannot recognize most infected cells, including hepatocytes, which are a major site of L. monocytogenes replication (2). The second possibility is suggested by the long-standing hypothesis that nonsecreted Ag does not induce a strong CD8 T cell response during the pivotal early stages of infection. While our previous results have shown that nonsecreted Ag induces CD8 T cell memory to a level similar (only 3- to 5-fold lower) to that induced by its secreted counterpart, it is not known whether similar levels of CD8 T cell responses are induced by secreted and nonsecreted Ags at early time points during infection. Furthermore, protective immunity against L. monocytogenes is mediated by the recall response of memory CD8 T cells, which was not examined in our previous study. Thus, it is possible that the lack of protection against recombinant L. monocytogenes expressing nonsecreted Ag is due to the absence of a strong recall response to nonsecreted Ag.
In this study we extend our analysis of Ag compartmentalization to include another epitope that is recognized in a different haplotype, the H-2b-restricted gp33–41 epitope of LCMV. The results of this study confirmed our previous observations, thus demonstrating that the dichotomy between CD8 T cell priming and protective immunity as a result of Ag compartmentalization is not limited to a particular epitope or MHC haplotype. In addition, we further examined whether secreted and nonsecreted Ags induce similar levels of primary and secondary CD8 T cell responses during the early stages of infection. Our results showed that secreted Ag induced a much more robust response than nonsecreted Ag at the onset of a primary CD8 T cell response, supporting the hypothesis that secreted Ags induce a more effective early immune response than nonsecreted Ags. However, memory CD8 T cells mounted rapid recall responses similar in magnitude and kinetics to both secreted and nonsecreted Ags. Therefore, the lack of protection against L. monocytogenes expressing nonsecreted Ags cannot be attributed to the absence of a strong recall response by memory CD8 T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c and C57BL/6 female mice (National Cancer Institute, Frederick, MD), aged 6–10 wk, were housed in insulator cages and cared for in accordance with institutional animal care and use committee-approved protocols at University of Pennsylvania School of Medicine animal facility. Mice within experiments were age-matched. P14 TCR transgenic mice (specific for H-2Db-gp33–41) (12) and C57BL/6 Thy1.1 mice (The Jackson Laboratory, Bar Harbor, ME) were bred to produce an F1 generation (P14 Thy1.1/Thy1.2), which was used as donors in adoptive transfer experiments.
Bacteria and virus
Recombinant L. monocytogenes expressing secreted (XFL703) or nonsecreted (XFL704) fusion proteins containing CD8 T cell epitopes were constructed as previously described (11). Briefly, DNA sequences encoding dihydrofolate reductase (DHFR), the hly promoter, and the hly signal sequence and five additional amino acids (only in XFL703) were PCR amplified from pEH18 (13) and L. monocytogenes chromosomal DNA, respectively. Two complementary oligonucleotides were synthesized for sequences encoding the gp33–41 and NP118–126 epitopes and the mAb HA epitope, derived from influenza virus hemagglutinin (14) or the vesicular stomatitis virus epitope, derived from vesicular stomatitis virus glycoprotein (15). Ag cassettes were cloned into delivery vector pHS-LV and introduced into the genome of wild-type L. monocytogenes 10403S as previously described (16). 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 in BHI, and the culture was grown overnight at 30°C with aeration. For clarity, XFL703 and XFL704 will be referred to as rLM-sAg and rLM-nsAg, respectively, throughout this paper. LCMV Armstrong was propagated as previously described (17).
Western blot
Overnight cultures of 10403S, rLM-sAg, and rLM-nsAg were subcultured 1/200 in BHI with 100 mM MOPS in duplicate. Cultures were incubated at 37°C with aeration until an OD600 of 1.0 was reached. To isolate secreted proteins, TCA was added to one set of cultures to a final concentration of 10%. After incubation on ice, the samples were centrifuged, washed with acetone, and then resuspended in 4% SDS/0.5 M Tris-Cl, pH 8.0, which solubilizes secreted and cell surface-associated proteins, but does not lyse bacteria. To isolate proteins from the bacteria, another set of cultures was centrifuged, and pellets were resuspended in PBS with 2.5 mg/ml lysozyme and 25% sucrose and incubated at 37°C for 2 h. To lyse the bacteria, lysis buffer with 0.75 mg/ml pronase was added, and the samples were incubated at 37°C for 30 min. Proteins were then precipitated with 10% TCA, washed with acetone, and resuspended in PBS. 2x SDS sample buffer with DTT was added to all protein samples, and the samples were then boiled. Samples were separated on a 12% polyacrylamide gel, transferred to Immobilon-P membrane (Millipore, Bedford, MA), and blocked overnight at 4°C in 5% nonfat milk/1% Tween 20/PBS. After washing with 1% Tween 20/PBS, the blot was incubated with primary mAb specific for the hemagglutinin tag diluted 1/3000 (clone 12CA5; Roche, Indianapolis, IN), washed, and incubated with secondary Ab, peroxidase-labeled anti-mouse polyclonal Ab diluted 1/1000. mAb binding was detected using the ECL detection system (Amersham Pharmacia, Piscataway, NJ).
L. monocytogenes invasion of and intracellular growth in J774 cells
J774 cells (ATCC TIB-67; American Type Culture Collection,
Manassas, VA) were seeded into six-well plates. Confluent monolayers
were infected with stationary phase rLM-sAg or rLM-nsAg in RPMI 1640
with a multiplicity of infection of 
10. After 1 h of incubation
at 37°C with 5% CO2, monolayers were washed,
and fresh RPMI 1640 containing 50 µg/ml gentamicin was added. After
further incubation, monolayers were lysed at different time
points after gentamicin addition with 1% Triton X-100/PBS, serially
diluted, and plated onto BHI agar, and CFU per well were
calculated.
L. monocytogenes infection and LCMV immunization of mice
Overnight cultures were washed once with PBS, serially diluted in PBS to the desired dose, and injected into the lateral tail veins of mice. Inocula were plated to verify the dose. C57BL/6 mice were infected with 1 x 104 CFU (0.1 of the LD50), and BALB/c mice were infected with 5 x 103 CFU (0.1 of the LD50). Bacterial load was determined by plating 10-fold serial dilutions of spleen and liver homogenates in sterile 1% Triton X-100/PBS. To generate LCMV-immunized mice, mice were injected i.p. with 2 x 105 PFU LCMV Armstrong strain. Challenges with L. monocytogenes were performed at least 21 days after LCMV immunization.
Splenocyte preparation
Spleens were aseptically removed from sacrificed mice, placed into cold RPMI 1640, and passed through a wire-mesh screen. RBC were lysed with 0.83% ammonium chloride. Splenocytes were resuspended in complete RPMI 1640 medium containing 5% FCS, 4 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Aliquots were diluted in 0.1% trypan blue/PBS to calculate the number of viable cells per spleen.
Surface and intracellular staining
For surface staining, 1% BSA/PBS was added to splenocytes to
block nonspecific mAb binding. All mAb and reagents were purchased from
BD PharMingen (San Diego, CA) unless otherwise specified. Cells were
stained with mAb 
-CD8-cychrome c or PerCP (clone 53-6.7)
or -CD4-PerCP (clone RM4-5), and with mAbs to one of several
activation markers, including -CD44 (clone IM7) and -CD43 (clone
1B11) in 1% BSA/PBS. After several washes in 1% BSA/PBS, splenocytes
were fixed with 2% paraformaldehyde. For intracellular staining to
analyze IFN-
production, splenocytes were incubated with 50 U/ml
recombinant human IL-2 and Golgistop and with or without 1 µM
synthetic peptide in complete RPMI 1640 medium. Peptides included
gp33–41 (KAVYNFATM), NP118–126 (RPQASGVYM),
listeriolysin O (LLO)91–99 (GYKDGNEYI),
LLO190–201 (NEKYAQAYPNVS), and
p60217–225 (KYGVSVQDI). After 5-h incubation at
37°C with 5% CO2, the cells were
surface-stained as described above. According to the manufacturer’s
protocol, splenocytes were then permeabilized with Cytofix/Cytoperm
solution, then stained with -IFN--allophycocyanin mAb (clone
XMG1.2) and fixed with paraformaldehyde. Splenocytes were analyzed with
a FACSCalibur (BD Biosciences, San Jose, CA), and data were analyzed
using Flow-Jo, version 3.4 (TreeStar, San Carlos, CA).
ELISPOT
ELISPOT assays were performed as previously described with minor
alterations (18). Briefly, MultiScreen-HA membrane plates
(Millipore) were coated with 4 µg/ml of purified anti-mouse IFN-
mAb (clone R4-6A2) and then blocked with 10% FCS/RPMI 1640. Serial
dilutions (1 x 105, 3 x
104, 1 x 104, 3
x 103) of splenocytes from experimental mice
(effector cells) were stimulated with 1 µM peptide in the presence of
syngenic 5 x 105 splenocytes from
naive mice (feeder cells) and 50 U/ml of IL-2 in complete RPMI 1640
medium for 24 h at 37°C with 5% CO2.
Plates were then washed, incubated with biotinylated anti-mouse IFN-
mAb (clone XMG1.2), and developed with HRP-streptavidin conjugate
(Vector, Burlingame, CA) and aminoethylcarbazole dye solution
(Sigma-Aldrich, St. Louis, MO).
Adoptive transfer
Spleens from female P14 Thy1.1/Thy1.2 transgenic mice (at least
8 wk old) were aseptically removed and prepared as described above.
Splenocytes were resuspended in PBS, labeled with 5 µM CFSE
(Molecular Probes, Eugene, OR), and quenched with FCS. After washing,
cells were resuspended in PBS, and 1–2 x
107 splenocytes/mouse were injected i.v. into
C57BL/6 (Thy1.2) female mice. Mice were injected i.v. 18 h later
with either 1 x 104 CFU rLM-sAg or
rLM-nsAg. Mice were sacrificed at days 2, 3, and 4 postinfection
(p.i.). Cells were stained for with mAb -CD8 and -Thy1.1-PE
(clone OX-7) to identify transferred Thy1.1+ T
cells and then were stained for intracellular IFN- as described
above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To extend our previous studies on the effect of Ag
compartmentalization to the CD8 T cell response, we constructed new
isogenic strains of L. monocytogenes, rLM-sAg and rLM-nsAg,
that express two CD8 T cell epitopes as secreted or nonsecreted fusion
proteins, respectively (Fig. 1
A). Both rLM-sAg and rLM-nsAg
expressed gp33–41 and NP118–126 epitopes from
LCMV within a DHFR fusion protein under the control of a virulence gene
promoter (Phly). The fusion proteins in rLM-sAg
and rLM-nsAg were identical, with the exception of an N-terminal signal
sequence in rLM-sAg that is cleaved upon secretion from the bacterium.
The cassettes encoding the fusion proteins were integrated into the
chromosome of wild-type L. monocytogenes 10403S between the
lecithinase and lactate dehydrogenase operons as previously described
(11). gp33–41 and NP118–126 are recognized
in C57BL/6 and BALB/c mice, respectively. Inclusion of both epitopes
within the same recombinant L. monocytogenes strain makes it
possible to examine the CD8 T cell response in two mouse strains,
thus providing cross-validation of our results. Addition of the
gp33–41 epitope also allows the use of P14 TCR transgenic cells in an
adoptive transfer model to visualize in vivo proliferation of
Ag-specific CD8 T cells.
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B). The fusion protein of rLM-sAg was present only in the
supernatant, demonstrating efficient secretion of the fusion protein
from the bacterium. On the other hand, the nonsecreted fusion protein
of strain rLM-nsAg was present only in the bacterial pellet, as
expected. Fusion proteins expressed by rLM-nsAg and secreted by rLM-sAg
had an apparent size of 28 kDa on the Western blot, consistent with
their predicted Mr. The fusion protein expressed
by rLM-sAg appeared slightly larger than the protein expressed by
rLM-nsAg due to the addition of five amino acid residues after the
secretion signal cleavage site that were included to ensure cleavage
efficiency.
The expression of foreign proteins can affect bacterial in vitro and in
vivo growth, particularly when they are fused to a signal sequence but
cannot be efficiently translocated (19). It is
hypothesized that these proteins congest bacterial secretory pathways,
thus blocking the secretion of virulence factors. While DHFR is
efficiently secreted in many organisms, including L.
monocytogenes (11), we nevertheless assayed the in
vitro and in vivo virulence of the recombinant strains. In J774 cells,
a murine macrophage-like cell line, rLM-sAg and rLM-nsAg invaded with
equal efficiency and replicated to similar numbers (Fig. 1C). After infection of mice with a sublethal dose of
rLM-sAg or rLM-nsAg, similar levels of bacteria were recovered from
spleens in both C57BL/6 (Fig. 1D) and BALB/c (data not
shown) mice on days 2 and 3 p.i. Thus, rLM-sAg and rLM-nsAg were
equally virulent, and therefore any differences observed in the immune
responses to these strains were not due to differences in levels of
infection.
To determine whether secreted or nonsecreted gp33–41 can prime naive
CD8 T cells, C57BL/6 mice were infected with wild-type L.
monocytogenes, rLM-sAg or rLM-nsAg. At least 20 days p.i., the
number of gp33–41-specific CD8 T cells in the spleen was quantitated
by intracellular IFN- staining after in vitro peptide
stimulation. Both rLM-sAg and rLM-nsAg generated
gp33–41-specific CD8 T cell populations (Fig. 2A). The rLM-sAg generated a
5- to 10-fold greater number of Ag-specific cells than rLM-nsAg (Fig. 2B). The parental strain, wild-type L.
monocytogenes, did not induce gp33–41-specific CD8 T cells above
background levels (Fig. 2A).
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C). In LCMV-immunized mice, rLM-sAg was cleared by day
3 p.i.; however, LCMV-immunized mice challenged with rLM-nsAg had
bacterial loads equal to those seen in naive challenged mice (Fig. 2C). Thus, nonsecreted gp33–41 was not protective, although
it was capable of priming CD8 T cells. These results extend our
previous findings to include another Ag in a different mouse strain,
thus confirming our observation of a dichotomy between CD8 T cell
priming and protective immunity as a result of Ag compartmentalization
(11). Secreted bacterial Ags induce more robust primary CD8 T cell responses at early time points than nonsecreted Ags
As a first step toward understanding why nonsecreted bacterial Ags
are not protective, we examined the kinetics of the primary immune
response to secreted and nonsecreted Ag. C57BL/6 mice were infected
with rLM-sAg or rLM-nsAg, and at different days p.i., the numbers of
gp33–41-specific CD8 T cells per spleen were quantitated by
intracellular IFN- staining assay (Fig. 3A). Ag-specific CD8 T cells
were first detected on day 5 p.i. in the spleens of mice infected
with either rLM-sAg or rLM-nsAg. In mice infected with rLM-sAg, the
number of Ag-specific cells peaked on day 8 p.i. with
106 cells/spleen and then contracted to
105 cells by day 14 p.i. Interestingly,
the response to nonsecreted gp33–41 expanded and contracted almost
synchronously with the response to secreted gp33–41 (Fig. 3A), although the magnitude of the response to nonsecreted
Ag was 5- to 10-fold less throughout the expansion and contraction
phases. This difference was not due to variation in the levels of
L. monocytogenes infection, since we observed that the CD4 T
cell response to the endogenous epitope
LLO190–201 (7) was similar in mice
infected with rLM-sAg and those infected with rLM-nsAg (data not
shown).
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B). In mice infected with rLM-sAg,
NP118–126-specific CD8 T cells were first
detected on day 5 p.i., expanded to a peak of 3 x
105 cells/spleen on day 7 p.i., and then
contracted to a smaller population of 5 x
104 cells/spleen by day 14 p.i. The kinetics
of the response to nonsecreted NP118–126 were
similar, although the magnitude was 5- to 10-fold less at each of the
time points examined. These results are consistent with the
gp33–41-specific response observed in C57BL/6 mice (Fig. 3A) and with our previous findings that secreted
NP118–126 induces a 3- to 5-fold higher level of
memory (11). As internal controls, we examined the
kinetics of the CD8 T cell response to endogenous secreted L.
monocytogenes proteins. LLO91–99- and
p60217–225-specific responses in mice infected
with rLM-sAg or rLM-nsAg were similar in magnitude and synchronous in
kinetics (Fig. 3B). Thus, these two bacterial strains
induced similar overall immune responses, and any differences observed
in the response to recombinant secreted or nonsecreted Ag were not due
to differences in the virulence of the two bacterial strains.
Furthermore, responses to secreted and nonsecreted
NP118–126 were nearly synchronous with the
responses to endogenous LLO91–99 and
p60217–225 epitopes.
The earliest time point at which Ag-specific CD8 T cells could be
detected by IFN- intracellular staining or ELISPOT was day 5
following L. monocytogenes infection of normal mice. To
further examine earlier time points, we employed an adoptive transfer
model using CFSE-labeled TCR transgenic P14 cells that are specific to
the H-2Db-restricted gp33–41 epitope.
CFSE-labeled splenocytes from P14 mice (Thy1.1/Thy1.2) were adoptively
transferred into congenic C57BL/6 (Thy1.2) mice, which were then
infected with rLM-sAg or rLM-nsAg (Fig. 4A). On day 2 p.i.,
transferred P14 cells had not divided significantly in mice infected
with either rLM-sAg or rLM-nsAg. By day 3, transferred P14 cells began
to respond to both secreted and nonsecreted gp33–41, although there
was substantially more proliferation of P14 cells in rLM-sAg infected
mice, with 85% of P14 cells having divided compared with 30% in mice
infected with rLM-nsAg. By day 4 p.i., the population of P14 cells
in rLM-sAg infected mice was comprised of mostly (97%) daughter cells,
while only 57% of P14 cells had divided in mice infected with
rLM-nsAg. P14 cells that had divided in response to infection with
either rLM-sAg or rLM-nsAg produced IFN- when stimulated with
gp33–41 peptide in vitro (Fig. 4B). Thus, both secreted and
nonsecreted Ags prime functional CD8 T cells, but secreted Ag induces a
stronger early response than nonsecreted Ag. In summary, our results
show that CD8 T cells begin to respond to both secreted and nonsecreted
bacterial Ags at about the same time (between days 2 and 3 p.i.).
These responding populations of Ag-specific cells then expand and
contract almost synchronously, although the magnitude of the response
to secreted Ags is 5- to 10-fold greater.
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Infection of mice with rLM-nsAg induced less proliferation of
Ag-specific CD8 T cells during the primary immune response, suggesting
that the lack of protective immunity may be due to nonsecreted Ag
inducing a less robust response early during infection. However,
protective immunity is mediated by memory CD8 T cells, which are
present at a greater frequency and respond faster than naive CD8 T
cells (20). The recall responses to secreted and
nonsecreted Ags by memory CD8 T cells have not been examined, and it is
possible that the lack of protection against rLM-nsAg is due to the
absence or delay of a recall response to nonsecreted Ag. To determine
whether memory CD8 T cells mount a response against nonsecreted Ag, we
examined the secondary immune response on days 1 and 2 after challenge.
Effective recall responses by memory CD8 T cells at these early time
points are critical for conferring protective immunity. LCMV-immunized
and age-matched naive C57BL/6 mice were infected with rLM-sAg or
rLM-nsAg. Similar numbers of rLM-sAg or rLM-nsAg were detected in the
spleens of naive mice, while LCMV-immunized mice quickly cleared
infection by rLM-sAg, but not by rLM-nsAg (Fig. 5C). Total numbers of
gp33–41-specific CD8 T cells were comparable in LCMV-immunized mice
challenged with rLM-sAg or rLM-nsAg (Fig. 5B). Therefore,
LCMV-immunized mice were protected from rLM-sAg, but not rLM-nsAg,
infection. This disparity in protective immunity was not due to
differences in the number of Ag-specific CD8 T cells in the spleen that
were capable of deploying effector functions, such as IFN-
secretion. To extend our analysis, we distinguished between memory and
effector Ag-specific CD8 T cells by using the mAb 1B11 that recognizes
two core O-glycans on CD43. Glycosylation of mucin-type
glycoproteins is up-regulated on primary and secondary effector cells,
but down-regulated on memory cells, and mAb 1B11 epitope expression has
been demonstrated to directly correlate with effector function
(21). In LCMV-immunized mice, most of the
gp33–41-specific CD8 T cells had low levels of O-glycan
CD43 expression, consistent with their memory phenotype. Upon challenge
with either rLM-sAg or rLM-nsAg, a substantial number of
gp33–41-specific CD8 T cells up-regulated O-glycan CD43
expression and thus had become effectors (Fig. 5A).
Interestingly, similar levels of gp33–41-specific effector cells were
generated in mice challenged with either rLM-sAg or rLM-nsAg.
Thus, secreted and nonsecreted gp33–41 induced similar levels of
differentiation of gp33–41-specific memory CD8 T cells to effector
cells. Taken together, these data shown that the lack of protective
immunity was not due to the inability of Ag-specific memory CD8 T cells
to respond to nonsecreted Ag.
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). Infection with both rLM-sAg and
rLM-nsAg increased the total number of Ag-specific CD8 T cells in
LCMV-immunized mice compared with unchallenged immune mice, from 2
x 105 to 1 x 106
cells/spleen. Thus, although rLM-sAg was rapidly cleared, and rLM-nsAg
persisted in the mice, there were no detectable differences in the
recall responses induced by these bacteria.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20% of activated
CD8 T cells during L. monocytogenes infection
(23). In contrast, in viral infections such as LCMV,
influenza, and EBV, known epitopes account for >70% of the activated
CD8 T cells (24, 25, 26). These data suggest that many CD8 T
cell epitopes in L. monocytogenes have yet to be identified,
some of which may be derived from nonsecreted proteins. While T cells
that proliferate in response to nonsecreted Ags have been shown to
exist by in vitro culturing with bacterial lysate (27),
CD8 epitopes from nonsecreted proteins have not yet been identified due
to an experimental bias toward secreted proteins. Early studies using
in vitro stimulation assays to identify L. monocytogenes
epitopes relied on isolation of CD8 T cell clones specific to L.
monocytogenes (4, 5). In these studies splenocytes
from L. monocytogenes-immunized mice were generated and
maintained by stimulation with L. monocytogenes-infected
J774 cells. Since J774 cells are nonbactericidal, nonsecreted proteins
are unlikely to be presented in this in vitro culture to stimulate and
maintain CD8 T cells. A recent study employed overlapping synthetic
dodecamer peptides to screen for epitopes using the ELISPOT assay with
splenocytes from L. monocytogenes-immunized mice
(7). While this approach should allow identification of
epitopes from nonsecreted L. monocytogenes proteins, it is
costly and was only applied to secreted proteins, which are
thought to be more likely vaccine targets because of their localization
in the host cell cytosol. However, recent advances in our understanding
of T cell activation in vivo have unveiled the importance of dendritic
cells in priming a CD8 T cell response through cross-presentation
(28). While nonsecreted bacterial Ags are not accessible
to the classical endogenous MHC class I pathway, our study using two
model Ags has clearly demonstrated that nonsecreted proteins can induce
a CD8 T cell response. Our results therefore support the role of
cross-presentation in initiating a CD8 T cell response to
infection. A long-standing hypothesis postulates that secreted proteins induce a more robust response than nonsecreted proteins at the onset of infection and therefore are more relevant vaccine targets. While this hypothesis has been used as a guideline in choosing vaccine Ags, the validity of this hypothesis has not been rigorously tested. A direct test of this hypothesis has been difficult because available assays for measuring Ag-specific responses are not sensitive enough to measure the extremely low levels of Ag-specific CD8 T cells at the onset of the immune response. To overcome this limitation, we employed an adoptive transfer model using CFSE-labeled TCR transgenic cells. Our results show that Ag-specific CD8 T cells did not respond to secreted or nonsecreted Ags by day 2 p.i. However, on days 3 and 4 there was substantially more proliferation of Ag-specific CD8 T cells responding to secreted than nonsecreted Ag. While it was difficult to precisely quantitate the number of P14 cells recruited and the number of times they divided due to limited resolution of CFSE peaks in our in vivo experiments, the results suggest that 1) the timing of recognition and the extent of proliferation of recruited cells were similar during infection with rLM-sAg and rLM-nsAg; and 2) the difference in the numbers of Ag-specific CD8 T cells was mostly due to a difference in the number of precursors recruited into division. These results from early time points in adoptive transfer experiments are consistent with data from later time points taken from normal mice and show that secreted Ag induced a relatively greater CD8 T cell response during primary infection compared with nonsecreted Ag. Furthermore, CD8 T cell populations responding to secreted and nonsecreted recombinant Ags expanded and contracted synchronously with those specific to epitopes from endogenous secreted bacterial proteins. These results provide a direct comparison of the responses induced by two MHC class I pathways and demonstrate that the kinetics are similar regardless of whether Ag is accessible to the endogenous MHC class I pathway or is restricted to cross-presentation. The difference in the magnitudes of the responses is probably due to different numbers of APC presenting the Ag and the amount of Ag presented on APC. We compared the amounts of secreted and nonsecreted recombinant Ag presented during the early stages of L. monocytogenes infection using a modified direct ex vivo Ag detection assay (29, 30). Antigenic peptides were readily measurable in splenocytes of mice infected with rLM-sAg, but were barely detectable in splenocytes of rLM-nsAg-infected mice (data not shown). These differences in Ag availability and in the magnitude of the responses to secreted and nonsecreted Ag probably reflect distinct features of cross-presentation in inducing the activation and expansion of CD8 T cells, which are currently under investigation.
Protective immunity against L. monocytogenes is mediated by
the recall response of memory CD8 T cells. Bacteria will gain a
foothold and multiply rapidly within their intracellular niche if an
effective immune response is not initiated soon after the onset of
infection. Thus, the decisive battle between pathogen and host is
fought at the onset of infection. Ineffective induction of a recall
response has been hypothesized to contribute to the lack of protection
against L. monocytogenes expressing nonsecreted Ag
(11). However, our results demonstrate that both L.
monocytogenes expressing secreted or nonsecreted Ag induced
differentiation of memory CD8 T cells by day 1 p.i., and
Ag-specific CD8 T cells had similar expansion kinetics during a recall
response. Thus, less effective induction of an immune response by
nonsecreted Ag is unlikely to be a critical factor contributing to the
lack of protective immunity. Why do memory CD8 T cells not provide
protection despite the fact that they are activated to produce
cytokines such as IFN-? We are currently investigating the
possibility that the lack of protective immunity relates to the
presentation of nonsecreted Ag by only a subset of infected cells
and/or dendritic cells that have acquired exogenous Ags. L.
monocytogenes infects many different cell types that are incapable
of presenting Ag through cross-presentation. These infected cells will
not display peptides from nonsecreted bacterial proteins on their
surface and therefore will not be recognized. These intracellular
bacteria will continue to multiply and disseminate by direct
cell-to-cell spread despite an ongoing CD8 T cell response to
nonsecreted Ags presented by APCs. This is true even when CD8 T cells
are activated to produce IFN-, since activated CD8 T cells exert
little bystander control of intracellular bacteria. Memory CD8 T cells
provided protection only against rLM-sAg, but not wild-type L.
monocytogenes, when LCMV-immunized mice were challenged with a mix
of rLM-sAg and wild-type bacteria (J. Jiang and H. Shen, unpublished
observations).
In summary, we have extended our previous findings to another Ag restricted by a different MHC haplotype, thus validating the conclusion that protein secretion in bacteria is a major factor in determining whether an Ag can serve as a protective target. While both secreted and nonsecreted bacterial Ags can prime CD8 T cell responses, only recognition of secreted Ags by memory CD8 T cells results in protective immunity against L. monocytogenes. We show that the kinetics of the primary responses to secreted and nonsecreted bacterial Ags are similar, although the magnitude of the response to secreted Ags is relatively greater. We further demonstrate that nonsecreted bacterial proteins induce recall CD8 T cell responses, but do not serve as protective Ags. Understanding how different determinants of bacterial Ags influence the nature and magnitude of host defense is critical for designing effective vaccines that induce potent responses and for selecting candidate Ags that can serve as protective targets.
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2 Address correspondence and reprint requests to Dr. Hao Shen, Department of Microbiology, University of Pennsylvania School of Medicine, 3610 Hamilton Walk, Philadelphia, PA 19104. E-mail address: hshen{at}mail.med.upenn.edu
3 Abbreviations used in this paper: NP, nucleoprotein; BHI, brain heart infusion; DHFR, dihydrofolate reductase; LCMV, lymphocytic choriomeningitis virus; p.i., postinfection; rLM-nsAg, recombinant L. monocytogenes expressing nonsecreted Ag; rLM-sAg, recombinant L. monocytogenes expressing secreted Ag; LLO, listeriolysin O.
Received for publication April 8, 2002. Accepted for publication September 11, 2002.
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) or rLM-nsAg (
) at
a multiplicity of infection of 
), and
the bacterial load in the spleens on days 2 and 3 p.i. were
determined. Bacterial loads for rLM-sAg and rLM-nsAg were similar in
the spleen and liver of BALB/c mice (data not shown). Bars represent
the mean ± SD from three mice. Data are representative of three
independent experiments with similar results.
