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Division of Molecular and Clinical Rheumatology, Department of Medicine, and Graduate Program in Immunology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
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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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Salmonella species are intracellular Gram-negative bacterial
pathogens that infect both phagocytic and non-phagocytic cells. These
pathogens cause a range of diseases including enteric fever and
gastroenteritis (14, 15, 16). Athymic mice and TCR-
ß-deficient mice
have impaired abilities to clear Salmonella infection
(17, 18, 19). In studies that utilized a natural oral challenge model,
Mastroeni et al. demonstrated that depletion of either CD8+
or CD4+ T cells impaired the ability to transfer protective
immunity to virulent S. typhimurium (20, 21). Similarly,
using an adoptive transfer model, other investigators demonstrated that
removal of both CD4+ and CD8+ T cells
completely abrogated transfer of protective immunity to systemic
infection with virulent S. abortusovis, while depletion of
either CD4+ or CD8+ T cells, in particular,
impaired the protective effect (22). Collectively, these studies
indicate that CD4+ and CD8+ T cells act
synergistically to control infection with virulent
Salmonella species.
Limited information is available on the properties of CTLs induced following infection with Salmonella. Studies that analyzed the capacity of recombinant S. typhimurium expressing a foreign protein, e.g., OVA or Plasmodium berghei circumsporozoite gene, to serve as a vaccine carrier, demonstrated that target protein-specific CD8+ T cells were induced (23, 24, 25). Moreover, CD8+ effectors capable of mediating lectin-dependent cytotoxicity and delayed-type hypersensitivity were elicited in mice challenged with Salmonella (26, 27). Also, when S. enteritidis-immunized mice were challenged with the virulent S. typhimurium strain C5, CD8+ CTLs capable of specifically killing P815 cells infected with S. enteritidis were detected. Taken together, these results indicate that Salmonella-derived Ags can be processed and presented to CD8+ CTLs and suggest that such cells may be an important defense mechanism to virulent pathogens.
In the present study, we assessed the contribution of CTL response to host defense against S. typhimurium. The finding that ß2-microglobulin (ß2m)-/-4 mice are more susceptible to infection with both virulent and avirulent S. typhimurium illustrates a role for CTLs in the eradication of intracellularly localized Gram-negative bacteria. CD8+ CTLs that recognize Salmonella-infected cells can only be readily detected in mice that have been challenged with virulent S. typhimurium. Furthermore, our data prove that a significant fraction of the Salmonella-specific CTLs elicited in vivo recognize infected targets in an MHC class Ib-restricted fashion and that the nonclassical class Ib molecule Qa-1b is a dominant restricting element. Finally, our findings demonstrate that processing of the Qa-1b-epitope is TAP- and proteasome-dependent. Taken together, these results reveal a novel mechanism for Ag presentation by H2-T23-encoded Qa-1 molecules and expand the range of pathogens for which class Ib molecules are relevant Ag-presenting structures to include the Gram-negative bacteria.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type virulent S. typhimurium strain C5 (C5), S. typhimurium strain SL3235 Aro- (SL3235), S. dublin strain Lane, S. enteritidis strain 11RX, and Escherichia coli strain HB101 were grown in Antibiotics Medium 3 (Difco Laboratories, Detroit, MI) broth or on Luria-Bertani (LB) agar plates. Listeria monocytogenes (ATCC 43251) was grown in bovine heart infusion medium (Difco). The number of bacteria was calculated from a standard curve showing CFUs vs OD at 600 nm (A600). Bacterial strains were grown overnight in LB medium at 37°C in a shaking water bath. The following day, bacterial cultures in the saturation density were diluted 1:10 and grown to midlogarithmic phase (A600, -0.5 to 0.6). Bacterial cultures were centrifuged and washed in PBS twice before use.
Mouse strains and cell lines
C57BL/6 (B6, H-2b/Qa-1b) and CB6F1 (BDF1, H-2b/d/Qa-1b) mice were obtained from the National Cancer Institute, National Institute of Health, (Bethesda, MD). BALB/cJ (H-2d, Qa-1b) and C57BL/6J-B2mtm1Unc (B6-ß2m-/-, H-2b/Qa-1b) mice were obtained from The Jackson Laboratory (Bar Harbor, ME).
L cells transfected with H2-Kd and -Dd were a gift from Dr. Iwona Stroynowski (University of Texas Southwestern Medical Center, Dallas, TX). P815 (H-2d), J774 (H-2d), and L cells were cultured in DMEM supplemented with 10% FCS (DMEM-10). IC-21 (H-2b), RMA (H-2b) and RMA-S, (H-2b) were maintained in RPMI 1640 supplemented with 10% FCS (RP-10). L-Qa-1b (L-g37) (28), L-Ld, and L cells transfected with the pSV2-neo vector alone (LV) were cultured in the same medium as parent cell lines, except supplemented with G418 (Sigma, St. Louis, MO) at 600 µg/ml. L-Kd and L-Dd were grown in DMEM-10 with 1x hypoxanthine, aminopterin, thymidine (Sigma). A20 cells transfected with human B7.1 (A20-B7.1) were a gift from Dr. Hyam Levitsky (Johns Hopkins Oncology Center, Baltimore, MD) and cultured in RP-10 supplemented with G418 at 600 µg/ml. T cell medium (TCM) is composed of RPMI 1640 supplemented with all the ingredients as described previously (29), except gentamicin (50 µg/ml) was substituted for Pen-strep (MediaTech, Herndon, VA). Cell lines to be used for infection were maintained at 5% CO2 in the absence of antibiotics, except T cell cultures, which were incubated in 7.5% CO2 in the presence of gentamicin.
Measurement of susceptibility to in vivo bacterial infection
C57BL/6 and B6-ß2m-/- mice were
injected i.p. with 1 x 106 SL3235 in 0.2 ml PBS.
Survival of infected mice was scored every 2–3 days until day 65. On
day 65, survivors of C57BL/6 and ß2m-/-
were divided into subgroups and received an i.p. challenge with 1
x 106, 1 x 104, or 1 x
102 virulent C5 (9, 5, and 5 animals per subgroup,
respectively, for the dose specified above). Survival was recorded for
another 4 wk. Four naïve animals were each challenged with
1 x 106 C5 as controls. Where indicated, the 
square test was used to assess levels of significance.
In vivo infection regimens
Female 8- to 10-wk-old mice were injected i.p. with 1 x 106 SL3235 in 0.2 ml PBS, followed by an i.p. challenge of 105 C5 in 0.2 ml PBS given 3 wk after the SL3235 inoculation (27). Immune splenocytes were harvested on day 7 following C5 challenge and used for subsequent experiments.
In vitro infection of tumor cell lines
A modification of the procedure of Pope et al. (27) was utilized for infection of tumor cells. Briefly, 1 x 106 log-phase tumor cells and C5 were mixed at the multiplicity of infection of 100, dispensed in 1-ml aliquots into 15-ml conical tubes or in 0.2-ml aliquots into each well of 96-well U-bottomed plates for large-scale preparation. The bacteria-tumor cell suspensions were then centrifuged at 2000 rpm for 10 min, incubated at 37°C for 30 min, harvested, and washed in complete RP-10 three times. Cell pellets were resuspended at the density of 1 x 106/ml in RP-10, and the infected cells were incubated for 1 h at 37°C to allow processing of Ags. Infection of cells with S. dublin, S. enteritidis, and E. coli utilized the same protocol as described above. Generation of L. monocytogenes-infected J774 macrophages has been described (30). The efficiency of infection was assessed 2 h after the last wash step by an invasion assay as described previously (31) and by staining intracellular bacteria with the Diff-quik stain set (Baxter Scientific, Edison, NJ) per manufacturer’s instructions.
Establishment of Salmonella-specific CTLs
Immune splenocytes derived from BALB/c and from C57BL/6 mice were harvested from SL3235-infected animals 7 days after C5 booster. Suspensions of splenocytes were adjusted to 1 x 106/ml in TCM containing Con A (1 µg/ml), 2-ml aliquots were dispensed into the wells of 24-well flat-bottom plates (Corning Costar, Cambridge, MA) and incubated at 37°C for 3 days. On day 3, Con A blasts were harvested and pooled. Viable cells were counted, and the cell density adjusted according to the E:T ratios needed in 51Cr-release assays.
For in vitro stimulation, 5 x 106 BALB/c and 5
x 106 C57BL/6 immune splenocytes were stimulated with
2 x 105 irradiated (20,000 rads) C5-infected J774 and
IC-21 cells, respectively, in each well of 24-well plates for 5 days
before use as effector cells in 51Cr-release assays. For
establishment of the CTL line SalT3, 5 x 106 CB6F1
immune splenocytes were coincubated with 2 x 105
irradiated C5-infected J774 cells in 2-ml aliquots in the wells of
24-well trays for 7 days. A week later, 2 x 105
irradiated C5-infected IC-21 cells were added to each well, and
incubation continued for another week. At the end of the second week,
the contents of each well were aspirated, centrifuged through
lympholyte cell separation medium (Accurate Chemical, Westbury, NY) at
1500 rpm, and washed three times in DMEM. Beginning the third week of
restimulation, 2 x 105 viable cells per well that
recovered from the last wash were cocultured, first with C5-infected
J774, and then with C5-infected IC-21 cells at 1-wk intervals. Included
in each coculture were 5 x 106 irradiated (2000 rads)
CB6F1 splenocytes per well in TCM supplemented with 10% rat Con A
supernatant (Collaborative Biomedical, Bedford, MA) and
-methylmannoside (Sigma) at 10 g/L. SalTCTL3 was cloned by limiting
dilution in the presence of C5-infected A20-B7.1 to achieve higher
cloning efficiency. A microcytotoxicity assay was performed using
C5-infected L-Qa-1b for those wells with evident cell
growth. One of the positive CTL clones established using these
conditions was further subcloned sequentially at 1 cell and 0.3
cells/well to generate SalTc 1.69. SalTc 1.69 was maintained by weekly
restimulation with infected A20-B7.1 cells. The cellular composition
was analyzed by flow cytometry with anti-Lyt2.2 MAb 2.43-FITC (1
µg) vs anti-L3T4 MAb GK1.5-PE (1 µg) and by
anti-TCR-ß-FITC (1 µg) vs anti-TCR-
-PE (1 µg).
All Abs used for flow cytometry were purchased from PharMingen (San
Diego, CA).
CTL assays and determination of bacteria-specific CTL frequencies
Infected and uninfected target cells (2 x 106)
were labeled with 51Cr for 1 h. Labeled cells were
washed with prewarmed (37°C) medium, resuspended in 2 ml TCM,
centrifuged through 1.5 ml warm FCS, resuspended in 5 ml TCM, and
allowed to incubate at 37°C for 1 h. Suspensions of labeled
cells were then spun through FCS again and counted (27). The density of
the labeled target cells was adjusted to 5 x 104/ml.
A total of 100 µl (5 x 103 cells) of the labeled
targets were dispensed into each well of 96-well U-bottomed plates and
mixed with an equal volume of effector cells at the indicated E:T
ratios. In Ab-blocking experiments using anti-T cell reagents,
effectors were preincubated with either 10 µl anti-Lyt2.2 MAb
2.43 ascites (a gift from Dr. Hyam Levitsky) or 20 µg anti-L3T4
MAb GK1.5 (Becton Dickinson, San Jose, CA) per well for 1 h before
the addition of target cells. In blocking studies using anti-class
Ia/Ib mAbs, targets cells were preincubated with isotype-matched
control Abs or mAbs reactive with H-2Kd/Dd
(34-1-2S) (32), H-2Ld (30-5-7S) (33), or Qa-1b
(6F10). The mAb 6F10 was produced by immunizing B6-Tlaa
mice with a peptide from the unique -2 domain of the
Qa-1b molecule. The hybridoma derived from these mice is
specific for Qa-1b as determined by flow cytometric
analysis of spleen cell from Tla-region congenic mice and
analysis of Qa-1b-transfected cell lines. A detailed
description of the generation and characterization of this reagent is
in preparation (H.O. and M.J.S., manuscript in preparation).
For measurement of bacteria-specific CTL frequencies by limiting dilution, immune splenocytes harvested from C5-infected BALB/c mice depleted of RBC by ammonium chloride were cultured in 11 graded doses (48 wells per dose) with 6 x 105 irradiated (2000 rads) syngeneic uninfected splenocytes and 5 x 104 C5-infected J774 (20,000 rads) in the presence of 20% rat Con A supernatant. A week later, half of the contents (100 µl) of each well was assayed for cytolytic activity against 51Cr-labeled 5 x 103 C5-infected P815 and C5-infected L-Qa-1b, respectively. Percent specific lysis >3 SDs of the mean taken from control wells without responders were scored positive. Fraction negative wells for each data point represent a set of 24 wells for a single dose of responders.
For cold target inhibition experiments, 5 x 10351Cr-labeled C5-infected P815 cells per well were mixed thoroughly with 2.5 x 105, 1 x 105, and 5 x 104 each of unlabeled C5-infected L-Qa-1b, uninfected L-Qa-1b, C5-infected L, and uninfected L cells before the addition of SalT3 at 2.5 x 104 per well. For experiments using metabolic inhibitors, P815 cells were preexposed to 50 µM Z-L3VS (a gift from Drs. Hidde Plough and Matthew Bogyo, Massachusetts Institute of Technology, Cambridge, MA) (34) or 1 µg/ml brefeldin A (Calbiochem, La Jolla, CA) for 2 h, washed three times with complete medium, and infected with C5 as described above. Coincubation of these labeled target cells with T cells was limited to 2 h to avoid recovery from inhibitory effects.
Following 4 h of incubation, the plates containing CTLs and
labeled targets were centrifuged at 1500 rpm for 5 min. The amount of
51Cr released was determined by counting the radioactivity
of 100 µl of culture supernatant from each well on a gamma counter
(LKB Instruments, Gaitherburg, MD). The percent specific release was
calculated as 100% x (cpm of experimental release - cpm of
spontaneous release)/(cpm of total release in the presence of 1%
Nonidet P-40 - cpm of spontaneous release). All CTL assays were
performed in TCM supplemented with 10 g/L -methylmannoside, and each
data point represents the mean of triplicate samples. Percent SE of the
raw counts representing each set of triplicate samples is < 10%.
Spontaneous release was 5–15% of total release in the presence of
Nonidet P-40, except for C5-infected J774 targets, in which spontaneous
release was as high as 30% of total release, presumably due to
apoptosis induced by invasive Salmonella (35).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the contribution of CD8+ T cells to
anti-Salmonella immunity, we evaluated the
susceptibility of B6-ß2m-/- (on C57BL/6
background, B6-ß2m-/-) and wild-type
C57BL/6 (B6) mice to S. typhimurium infections. B6 mice are
Itys (36) and, hence, highly
susceptible to infection with virulent strains of S.
typhimurium (LD50 < 101 in naive animals)
(37). However, prior infection with avirulent strains of S.
typhimurium induced protection against challenge with virulent
organisms (38). To determine the contribution of class I-restricted T
cell immunity to the development of protective immunity to S.
typhimurium, B6 and B6-ß2m-/- mice
were infected with the avirulent SL3235 Aro-
strain of S. typhimurium (SL3235) and then challenged with
the virulent C5 strain (C5). Infection with SL3235 caused mortality in
B6-ß2m-/- mice as soon as day 16 (8%,
p < 0.05), and, by day 65, 30%
(p < 0.001) of
B6-ß2m-/- had died (Fig. 1
A). In contrast, all
wild-type B6 mice survived without any mortality or significant
morbidity. These results suggest a role for class I-restricted
presentation of bacterial Ags to T cells in the initial resistance to
avirulent S. typhimurium.
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B).
Naive B6 and B6-ß2m-/- rapidly succumbed to
infection with 106 C5 with 100% mortality within 3 days.
However, SL3235-inoculated B6 and B6-ß2m-/-
showed increased resistance to strain C5, surviving inoculation with
102 organisms, a dose that is lethal (LD50
<101 C5) to naïve animals (data not shown, and
37). However, this protective effect is more pronounced for B6
mice than for B6-ß2m-/- (Fig. 1B), since B6-ß2m-/- mice are
consistently more susceptible to larger inocula of C5
(104-106 organisms). The increased
susceptibility of B6-ß2m-/- to C5 challenge
indicates a role for class I-restricted T cells in the generation of
protective immunity to infection with virulent S.
typhimurium. Potent CTL responses can be elicited following challenge with a virulent S. typhimurium C5 strain
The studies using class I-deficient
B6-ß2m-/- mice suggest a role for
CD8+ T cells in immunity to S. typhimurium. To
identify and characterize these cells, our initial studies focused on
the detection of Salmonella-specific CD8+ CTLs
in the spleens of infected mice. BALB/c (H-2d) or C57BL/6
(H-2b) mice were vaccinated with an avirulent strain of
S. typhimurium SL3235 and challenged with 1 x
105 C5. Cytotoxic activity against C5-infected MHC-matched
(H-2d) P815 targets was detected in BALB/c splenocyte
populations recovered 1 wk after challenge with C5 (Fig. 2A). Restimulation of BALB/c
splenocytes from C5-challenged (Fig. 2B), but not
unchallenged, BALB/c mice (Fig. 2C) in vitro with Con A,
yielded a population with enhanced cytotoxicity. This cytotoxicity
could be completely blocked by anti-CD8 MAb (2.43) but not
anti-CD4 MAb (GK1.5)(Fig. 2B), which suggests the major
effectors as CD8+ T cells. Comparable enhancement of lytic
activities could be achieved with in vitro restimulation with
irradiated C5-infected syngeneic J774 macrophages (H-2d)
(Fig. 2D). Analogous results were obtained using splenocytes
derived from C57BL/6 mice (data not shown). Based on these
observations, we concluded that, following infection with virulent
S. typhimurium, CD8+ CTLs that recognize
bacterial epitopes presented on class I molecules are readily detected
in the spleens of Salmonella-infected mice.
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, effectors from C5-infected C57BL/6
mice (H-2b) recognized C5-infected syngeneic IC-21
(H-2b) macrophage cells as well as allogeneic C5-infected
P815 (H-2d) targets. In addition, effectors from BALB/c
(H-2d) mice recognized C5-infected syngeneic P815 cells and
allogeneic C5-infected IC-21 targets. This recognition was
CD8-dependent, and effectors from normal spleens are not cytotoxic
(Fig. 3C). Collectively, these observations indicate that a
significant fraction of Salmonella-specific
CD8+ CTLs recognize Ags presented by shared class Ib
molecules.
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To characterize the role of class Ib Ag presentation structures, a
Salmonella-specific CTL line was generated by in vitro
restimulation of immune splenocytes with alternating MHC-disparate
C5-infected cells. Immune splenocytes derived from BDF1 mice were
restimulated in vitro with C5-infected J774 cells (H-2d,
Qa-1b) followed by a second restimulation with IC-21 cells
(H-2b, Qa-1b) 1 wk later. By alternation of
H-2-disparate stimulators, T cells were selected for the capacity to
recognize epitopes presented on shared Ag-presenting structures. Fig. 4A displays analyses of a T
cell line SalCTL3, which demonstrate that this cell line recognizes
Salmonella-infected P815 as well as IC-21 targets. This cell
line is 
85–90% CD8+, TCR ß+ (data not
shown), and its CTL activity is completely blocked by anti-CD8 Abs
(data not shown). To determine the involvement of class Ia vs class Ib
Ag-presenting structures, a panel of class Ia and
Qa-1b-transfected (L-Qa-1b) murine L cells were
used as targets in CTL assays. Following exposure to S.
typhimurium C5, only the L-Qa-1b cells were recognized
by the T cell line SalCTL3 (Fig. 4B).
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C, only C5-infected P815 or
L-Qa-1b blocked the recognition of 51Cr-labeled
Salmonella-infected P815 cells. No significant inhibition
was observed with uninfected targets or infected targets that lacked
Qa-1 expression. Interestingly, the infected L-Qa-1b cold
targets were 80–90% as effective as the infected syngeneic P815
targets. These results suggest that CTLs that recognize
Salmonella epitopes presented on Qa-1 represent a
significant fraction of the Salmonella-specific CTL activity
within the T cell line SalCTL3.
We next examined whether a dominant role for Qa-1b as an
Ag-presenting structure is also observed when unselected
Salmonella-specific CTL populations are examined. In this
study, immune splenocytes from C5-challenged BALB/c mice were
restimulated in vitro with MHC-matched C5-infected J774 macrophages and
assayed for cytolytic activity against C5-infected P815 cells. In
parallel, various class Ia- and Ib-specific mAbs were included to test
their capacity to block this recognition (Fig. 5A). The
Qa-1b-specific mAb 6F10 blocked target cell recognition up
to 50%, while the class Ia-specific reagents 34-1-2S
(anti-H-2Kd, Dd) and 30-5-7S
(anti-H-2Ld) displayed significantly less blocking
activity. To confirm this observation, restimulated immune splenocytes
from C5-challenged BALB/c mice were also tested for their ability to
recognize a panel of class Ia/Qa-1b-expressing L cells
(Fig. 5B). Consistent with the above results, CTL effectors
recovered from C5-challenged animals recognized only C5-infected
Qa-1b-expressing L cell fibroblasts.
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C, the calculated frequency of CTLs that
recognize C5-infected P815 targets was 1:20,683, while the frequency of
CTLs that display Qa-1b-restricted
Salmonella-specific recognition was 1:39,655. If adjusted
for the fraction of total immune splenocytes that were CD8+
(13.3%), 1:2,751 CD8+ T cells were
Salmonella-specific, while 1:5,274 displayed
Qa-1b-restricted recognition. Comparison of the two
frequencies reveals that 56% of the Salmonella-specific
CTLs are Qa-1b-restricted (Fig. 5C),
corroborating the Ab-blocking studies and identifying Qa-1b
as a major restricting element for Salmonella-specific CTLs. Characterization of Qa-1b-restricted recognition of Salmonella epitopes
A panel of Salmonella-specific CTL clones was
established by limited dilution. Phenotypic analysis of these clones
indicated that they were all CD8+, TCR-ß+
and that the majority exhibited Salmonella-specific class
Ib-restricted recognition, defined as the ability to recognize
C5-infected P815 and IC-21 targets (data not shown). A representative
clone, SalTc 1.69, was selected for further characterization and shown
to specifically recognize C5-infected L-Qa-1b (Fig. 6A) and, therefore, was
defined as Qa-1b-restricted.
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B demonstrate that the Salmonella-specific T
cell clone SalTc 1.69 recognized C5-infected RMA lymphoma cells, but
not C5-infected TAP-defective RMA-S cells, suggesting that
Qa-1b presents a TAP-dependent
Salmonella-specific epitope. Pretreatment of target cells
with brefeldin A, an inhibitor that has been shown to block transport
from the endoplasmic reticulum (ER) to the cis-Golgi, and
Z-L3VS, a potent irreversible proteasomal inhibitor (34)
dramatically diminished the level of killing against C5-infected cells
(Fig. 6C). Collectively, these data are consistent with
characteristics of a "classical" class I pathway that includes
proteasomal degradation, TAP-dependent translocation of epitopes across
ER membranes, and ER-to-Golgi transport as key features.
To examine the epitope specificity of the Qa-1b-restricted
T cell clone SalTc1.69, we tested its capacity to recognize J774 target
cells infected with Gram-positive as well as several related
Gram-negative bacteria. As expected, clone SalTc1.69 failed to
recognize J774 macrophages infected with L. monocytogenes
(Fig. 7B). Unexpectedly,
SalTc1.69 recognized J774 cells infected with S. enteritidis, S.
dublin, and E. coli (Fig. 7A). Taken
together, these data suggest that clone SalTc1.69 recognizes a shared
epitope presented by Qa-1b molecules on target cells
infected with Gram-negative enteric bacteria closely related to
S. typhimurium.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Examination of spleen cells from mice inoculated with attenuated S. typhimurium followed by challenge with the virulent C5 strain demonstrated a strong Salmonella-specific CD8-dependent CTL response. However, this CTL activity was not evident following infection with the Aro- strain SL3235 alone and was only detectable following challenge with virulent Salmonella. This observation is consistent with previous reports but is surprising, given that B6-ß2m-/- were more susceptible to infection with the vaccine strain SL3235 than the wild-type B6 mice (27). Several studies have shown that avirulent strains of Salmonella have a limited capacity to invade and replicate intracellularly (42, 43). Thus, infection with avirulent strains could lead to a weak CTL response not detected in our assays. Alternatively, the increased sensitivity of B6-ß2m-/- to avirulent Salmonella could reflect a role for noncytolytic functions of class I-restricted CD8 T cells or NK1.1+ T cells that are also deficient in ß2m-/- (44). Interestingly, the levels of splenic NK1.1+ T cells increase in a time dependent fashion following infection with S. typhimurium, SL3235 (W.-F.L. and M.J.S., unpublished observation), which is suggestive of such a possibility.
CD1, H2-M3, and Qa-1 have all been implicated as Ag-presentation structures in the CTL responses to Mycobacterium and Listeria, but their relative contribution to the entire CTL response has not been measured. The results of our Ab-blocking studies and the bacteria-specific CTL frequency analysis, clearly demonstrate that Qa-1b restriction is a dominant feature of the CTL response to S. typhimurium. To our knowledge, this is the first demonstration that a class Ib Ag-presenting structure plays such a major role in the generation of an effector T cell response. These results indicate that, in addition to binding leader peptides derived from class I molecules and serving as a target for NK cells (45, 46), Qa-1 can also bind and present other peptide ligands relevant to protective immunity to intracellular bacterial pathogens.
The dominance of Qa-1 in this response was surprising given that the class Ib molecule, H2-M3, has been shown to present N-formyl-methionine peptides of prokaryotic origin to Listeria-specific CTLs. Nevertheless, L cells that express endogenous H2-M3 are only recognized by Salmonella-specific CTLs when transfected with Qa-1b. Thus, H2-M3 does not play a major role in presenting bacterial epitopes in the CTL response to this Gram-negative pathogen.
Class Ia-restricted CTLs specific for epitopes derived from recombinant foreign Ags expressed in Salmonella has been detected (23, 24, 47). Our results, showing that anti-H-2 K/D/L mAbs partially block the recognition of C5-infected targets by Salmonella-specific CTLs, indicate that a minor component of the CTL response generated following infection with S. typhimurium includes epitopes presented by class Ia molecules, although the class Ib response appears to dominate. It is noteworthy that, using a panel of transfected L cells, only Qa-1b-restricted Salmonella-specific CTLs could be identified. Previous studies examining recombinant plasmid-encoded epitopes presented on class Ia molecules found that such epitopes are efficiently presented by macrophages but not by epithelial cells (25, 48). In our studies, the class Ib-restricted CTLs characterized to date have the ability to recognize Salmonella-infected macrophages, as well as P815 plasmacytoma, L cells, and RMA T cell lymphoma targets. Thus, while class Ia- and Ib-restricted CTLs are evoked following virulent Salmonella infection, the class Ia-restricted CTL activity may be directed toward infected phagocytes, while the class Ib-restricted component of the CTL response may broadly target all Salmonella-infected cells. Given that Salmonella can infect phagocytic and nonphagocytic cells, the class Ib-restricted CTL population may be relevant to the efficient clearance of virulent infection and avoidance of a carrier state.
The finding that Qa-1 serves a dominant role in presenting Salmonella epitopes to murine CTLs may be relevant to the human CTL response to Salmonella infections in humans. Similar to murine Qa-1, the human class Ib molecule, HLA-E, can bind hydrophobic nonameric class I molecule-derived leader sequences, and the anchor residues relevant to binding HLA-E and Qa-1 are largely identical (49, 50). Based on these properties, Qa-1 and HLA-E are considered cross-species orthologues with overlapping peptide binding specificities. Thus, it is reasonable to speculate that HLA-E can bind and present Salmonella epitopes to Salmonella-specific human CTLs. Interestingly, studies that examined cellular immune responses generated following oral infection with an attenuated Salmonella vaccine-detected Salmonella-specific CTL activity that was blockable by the class I Ab W6/32 (51). Given that W6/32 can bind to HLA-A/B/C and to HLA-E molecules, such a population of cells may contain CTLs that recognize epitopes presented by both class Ia and Ib molecules. Recent studies suggest that HLA-E, like class Ia molecules, is capable of binding hydrophilic viral peptides (52), despite the tight specificity of the peptide-binding cleft (53). Accordingly, HLA-E and, likewise, Qa-1 may be fully functional as Ag presenting molecules.
Studies using alloreactive Qa-1b-specific CTL clones specific for the peptide AMAPRTLLL (Qdm), derived from the leader sequence of D region class I molecules, revealed that this peptide was presented in a TAP-dependent, but proteasome-independent fashion (29, 54), suggesting that peptides presented by Qa-1 might derive from a novel processing pathway. Our studies using a TAP-defective cell line, together with specific metabolic inhibitors of proteasome and class I biogenesis, indicate that Qa-1 can also present antigenic peptides processed through a proteasome/TAP-dependent classical class I pathway. It is uncertain if the requirement for functional TAP in our system reveals the generation of Qa-1b-bound peptide from cytosolic degradation or reflects the reduced proportion of peptide-loaded class I molecules on the cell surface (55). However, the inhibitory effect exerted by Z-L3VS favors the former possibility and indicates that Qa-1b can load and present peptides derived from multiple processing pathways.
It remains unclear how the Salmonella-derived Ags gain access to the cytosol for proteasome-dependent degradation. Salmonella infects host cells and remains predominantly, if not exclusively, in the endosomal compartments. Several mechanisms have been proposed for the loading of Salmonella-derived peptide onto class I molecules. In one, antigenic proteins are transported into the cytosol via a "leakage" mechanism where they intersect the MHC class I pathway (10, 12, 13). This class I pathway has been demonstrated to operate in a subset of professional APCs (56), although there are other examples of phagocytic uptake of foreign Ags by keratinocytes and fibroblasts (12). An alternative pathway, or peptide regurgitation model, has been described where peptides generated within an endosomal compartment are transported extracellularly and load mature class I molecules on the cell surface (57, 58). This latter pathway has been shown to be both TAP- and proteasome-independent and is exclusively associated with phagocytic cell types. Our observations that phagocytic and nonphagocytic cell types are capable of presenting the Qa-1b-restricted Salmonella epitopes imply a third model where bacterial proteins can gain access to class I processing pathways in a variety of cell types. Recently it has been demonstrated that S. typhimurium can direct the translocation of selected bacterial proteins into nonphagocytic host cells through type III protein secretion system (59). This mode of Ag delivery has recently been exploited to transfer viral epitopes into host cytosol for class I-restricted recognition (60).
It has been argued that epitopes presented by class Ib molecules would be novel targets for vaccines since their conserved nature would facilitate the generation of protective immunity that is independent of MHC polymorphism (61). The finding that Qa-1-restricted CTLs are a significant component of the CD8+ CTL response to virulent Salmonella infection suggests that bacterial epitopes presented by Qa-1 may be of use in the design of vaccines to induce protective immunity to bacterial infections. Our data indicate that epitopes presented by Qa-1b are shared among Salmonella species, as well as the closely related Gram-negative bacterium E. coli. Thus, such a vaccine may also provoke immunity that is cross-reactive with other Gram-negative pathogens. It is interesting to note that following infection of host macrophages, the two dominant proteins expressed by Salmonella are GroEL and DnaK (62). These and perhaps other highly conserved bacterial proteins may prove to be targets for presentation by Qa-1 and recognition by CTLs.
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Footnotes |
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2 Under an agreement between PharMingen and Johns Hopkins University, the authors are entitled to a share of sales royalty received by the University from PharMingen, as a result of sales of anti-Qa-1b mAbs. The terms of this arrangement are being made by the University in accordance with its conflict of interest policies.
3 Address correspondence and reprint requests to Dr. Mark J. Soloski, Department of Medicine Ross Research Building, Room 1042, Johns Hopkins University School of Medicine 720 Rutland Avenue, Baltimore, MD 21205. E-mail address:
4 Abbreviations used in this paper: ß2m, ß2-microglobulin; C5, Salmonella typhimurium strain C5; SL3235, S. typhimurium strain SL3235 Aro-; TCM, T cell medium; B6-ß2m-/-, ß2m knock-out on the C57BL/6 background.
Received for publication November 23, 1998. Accepted for publication February 2, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß and T cells in the host response to Salmonella infection as demonstrated in T-cell-receptor-deficient mice of defined Ity genotypes. Infect. Immun. 65:2306.[Abstract]
and IFN in recall of immunity to oral challenge with virulent salmonellae in mice vaccinated with live attenuated Aro- Salmonella vaccines. Microb. Pathog. 13:477.[Medline]
aroA aroD mutants expressing a foreign recombinant protein induce specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes in mice. Infect. Immun. 61:5374.ß cells and IFN- in bacterial clearance independent of intracellular location. J. Immunol. 156:3321.[Abstract]
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|
|
|
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|
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|
|
|
|
|
|
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|
|
|
|
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) were
included for Ab-blocking studies. C, Immune splenocytes
harvested from BALB/c mice infected with SL3235 without a rechallenge
with C5 (or mock challenge) were activated with Con A in vitro for 3
days and used as effectors. The percent specific lysis at the indicated
E:T ratios shown here represents the mean of triplicate samples. All
data were generated from at least two separate experiments with similar
results. The number of viable bacteria/P815 cells recovered from
infected cells was 1.8 by averaging CFUs from three separate
samples.
) or presence of 20 µg/ml
of control IgG1 myeloma protein (
),
anti-H-2Kd/Dd (34-1-2S,
),
anti-H-2Ld (30-5-7S,
), or anti-Qa-1b
(6F10 
) or uninfected P815 cells (
). B, BALB/c
immune splenocytes restimulated in vitro with C5-infected J774 cells
were assayed using a panel of L cells expressing Qa-1b,
H-2Kd, H-2Dd, or H-2Ld at an E:T
ratio of 50:1. C, Measurement of
Salmonella-specific CTL frequencies by limiting dilution
analysis in immune BALB/c splenocytes against C5-infected P815 (
) or infected with C5 (
), S. enteritidis strain
11RX (
), or S. dublin strain lane (
),
respectively, at an multiplicity of infection of 100 and used as
targets for SalTc 1.69 recognition. B,
Listeria-specific CTLs generated from BALB/c mice by in
vitro stimulation with Listeria-infected J774 cells (63)
were assayed for cytotoxic activity against
Listeria-infected (
) or -uninfected (