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,
* Center for Pulmonary and Infectious Disease Control, Departments of
Microbiology and Immunology,
Biochemistry, and
Medicine, University of Texas Health Center, Tyler, TX 75708;
¶ National Cancer Institute, Bethesda, MD 20892; and
|| Epimmune Corp., San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting
CD8+ T cells were present in local and systemic
compartments after primary infection, and these cells expanded after
pathogen re-exposure. CD8+ T cell lines to the 18
Cpn epitope-bearing peptides were cytotoxic, displayed a
memory phenotype, and secreted IFN- and TNF-
, but not IL-4. These
CTL lines lysed Cpn-infected macrophages, and the lytic
activity was inhibited by brefeldin A, indicating endogenous processing
of CTL Ags. Finally, Cpn peptide-specific
CD8+ CTL suppressed chlamydial growth in vitro by direct
lysis of infected cells and by secretion of IFN- and other soluble
factors. These studies provide information on the mechanisms by which
CD8+ CTL protect against Cpn, furnish the
tools to investigate their possible role in immunopathology, and lay
the foundation for future work to develop vaccines against acute and
chronic Cpn infections. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD8+ T cells play a critical role in protection against most intracellular pathogens, including Chlamydia. Pathogen-derived Ags from organisms that replicate in the host cell cytosol, such as Listeria monocytogenes and Trypanosoma cruzi, readily induce a CD8+ T cell response, as microbial proteins are directly accessible to the MHC class I Ag-processing machinery. In contrast, for Chlamydia, which resides in a membrane-bound vacuole termed an inclusion, and for other intravacuolar pathogens, such as Mycobacterium tuberculosis, Ags need to traffic into the cytosol for CD8+ CTL induction. Nevertheless, CD8+ CTL responses are induced to mycobacterial Ags (10), and CD8+ T cells primed during C. trachomatis infection lyse chlamydia-infected cells (11, 12). Moreover, depletion and adoptive transfer of CD8+ T cells have, respectively, abrogated and conferred protection to C. psittaci- and C. trachomatis-challenged mice (12, 13, 14). Despite the clear role of CD8+ T cells in resistance to chlamydial pathogens, only two C. trachomatis CD8+ CTL target Ags have been identified to date (15, 16).
Information on immunity to Cpn is sparse, but studies using
a mouse model that faithfully mimics important aspects of human
Cpn infection (17, 18, 19) indicate that
CD8+ T cells and IFN- are critical for
protection (20, 21, 22). In the absence of
CD8+ T cells, Cpn-infected mice have
increased bacterial burdens and disease severity (20, 21),
and in animals lacking IFN- signaling, bacterial loads are higher,
and clearance of organisms is greatly hampered (22).
Nevertheless, it is uncertain whether CD8+ T
cells recognize Cpn-infected cells and whether this T cell
subset contributes to protection through cytokine production or a lytic
mechanism, as cytokine-producing CD8+ CTL have
not been documented during Cpn infection. Furthermore, the
Cpn-derived Ags contributing to MHC class I-restricted
CD8+ T cell responses remain unidentified.
We report in this work that Cpn-infected mice generate pathogen-specific CD8+ CTL with a type 1 cytokine secretion pattern and that these effector cells recognize multiple MHC class I-restricted epitopes from Cpn Ags endogenously processed by productively infected macrophages. We also show that Cpn peptide-specific CD8+ CTL and their soluble factors significantly inhibit chlamydial growth in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies used Mycoplasma-free stocks of the Cpn Kajaani 6 (K6) (obtained from Dr. M. Puolakkainen, University of Helsinki, Helsinki, Finland), AR39 (University of Washington, Seattle, WA), and CWL029 (obtained from Dr. C. M. Black, Centers for Disease Control, Atlanta, GA) strains. For propagation to high titers, each bacterial strain diluted in Chlamydia medium was centrifuged (500 x g, 1 h, 35°C) onto monolayers of HL cells (23) grown in 12-well plates (BD Biosciences, Franklin Lakes, NJ). Plates were incubated for 1 h at 37°C in 6% CO2 before replacing the inocula with cycloheximide-containing medium that was then used to incubate infected cultures for 72 h. Infected monolayers were harvested with glass beads and sonicated on ice for 20 s. After removing cell debris by low speed centrifugation (200 x g, 10 min, 4°C), bacteria were pelleted (33,100 x g, 35 min, 4°C), resuspended in cold sucrose-phosphate-glutamate solution, and aliquoted for titration and storage at -70°C. Similarly processed uninfected HL cell monolayers were used to prepare control material.
Mice and infections
Six- to 10-wk-old female C57BL/6J (B6) mice (H-2b) (The Jackson Laboratory, Bar Harbor, ME) were used in all experiments. Mice were kept in microisolator cages and housed in a pathogen-free environment. B6 mice were infected by intranasal (i.n.) inoculation with 106 inclusion-forming units (IFU) of Cpn K6 in 40 µl PBS under methoxyflurane anesthesia. In most experiments animals were reinfected i.n. with the same infectious dose 35–100 days after the initial infection. Control mice were inoculated with material prepared from uninfected cells. To induce T. cruzi trypomastigote surface Ag (TSA)-1-specific CTL, B6 mice were infected with this parasite as previously described (24). The institutional animal care and use committee approved all procedures involving animals.
Cell lines and culture media
RMA-S (H-2b; TAP2-, T cell lymphoma; provided by Dr. H.-G. Ljundggren, Karolinska Institute, Stockholm, Sweden) (25), mAM (H-2b; murine alveolar macrophage cell line; Z. Chroneos, unpublished observations), HL (University of Washington) (23), and HEp-2 (ATCC CCL 23, American Type Culture Collection, Manassas, VA) were maintained in complete RPMI 1640 medium containing 10% heat-inactivated FBS (HyClone, Logan, UT), 20 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 20 µg/ml gentamicin (all from Invitrogen-Life Technologies, Gaithersburg, MD). T2Kb and T2Db (T2 cells transfected with the Kb and Db genes; supplied by Dr. P. Cresswell, Yale University, New Haven, CT) (26) were maintained in complete IMDM with 0.4 mg/ml Geneticin (Invitrogen). T cell medium (TCM) was prepared by supplementing complete RPMI 1640 with 50 µM 2-ME (Invitrogen). TCM-RCAS was made by addition of 5% supernatant from Con A-stimulated rat splenocytes (T-STIM without Con A; Collaborative Biomedical Products, Bedford, MA). Chlamydia medium consisted of complete DMEM/F-12 medium (Invitrogen) with an additional 0.5 mg/ml glucose and 0.26 mg/ml sodium bicarbonate. Cycloheximide (1 µg/ml; Sigma-Aldrich, St. Louis, MO) was included when indicated.
Histology and immunohistochemistry
Lungs collected at various time points between 2 and 165 days after primary and secondary Cpn or mock infections (three to five mice per time point) were perfused with 10 ml PBS via the right ventricle and inflated by intratracheal instillation with Excell fixative (American MasterTech Scientific, Lodi, CA). After postfixation, tissues were embedded in paraffin, and sections (6 µm) were stained with H&E for histological analysis. To detect Cpn, deparaffinized tissue sections were treated for 15 min with 3% H2O2, blocked for 20 min with 5% BSA, and then incubated overnight at 4°C with a 1/2000 dilution of the Cpn major outer membrane protein (MOMP)-specific mAb RR-402 (University of Washington) (27, 28). A 1/1000 dilution of biotinylated goat anti-mouse IgG (ICN, Costa Mesa, CA) was then applied to the sections for 30 min, followed by 15 min with a 1/10 dilution of streptavidin-HRP (Innovex Biosciences, Richmond, CA). Color development and counterstaining was achieved using Turbo AEC (Innovex) and Contrast Blue (KPL, Gaithersburg, MD), respectively. After each staining step, sections were rinsed with wash solution (KPL) and signal enhancing buffer (Innovex).
Flow cytometric analysis of cell surface markers
mAbs used for cell surface staining were FITC and PE
anti-CD8 (53-6.7), FITC anti-CD4 (H129.19), PE anti-CD44
(IM7), PE anti-TCR
(H57-597), FITC anti-CD3
(145-2C11),
FITC- and PE isotype-matched control mAbs (all from BD PharMingen, San
Diego, CA), purified anti-Db (28-14-8S; ATCC
HB176; ATCC), and purified anti-Kb (Y3; ATCC
HB176; ATCC). Cells (5 x 105–1 x
106) were washed with cold FACS buffer (1%
BSA/0.05% NaN3 in PBS) and stained for 45 min at
4°C in 100 µl buffer with saturating concentrations of Abs. For
purified mAbs, cells were then stained for 30 min on ice with a 1/50
dilution of FITC-F(ab')2 goat anti-mouse IgG
(Southern Biotechnology, Birmingham, AL). Cells were washed twice after
each staining step and then analyzed on an EPICS C flow cytometer
(Beckman Coulter, Hialeah, FL).
Intracellular IFN- staining of lung mononuclear cells
Single-cell suspensions of perfused lungs from Cpn-
and mock-infected mice were prepared by homogenizing the organs in
100-µm pore size mesh cell strainers (BD Biosciences). After lysing
RBC, cells were washed and resuspended at 107/ml
in TCM. Isolated lung mononuclear cells (LMNC) were plated
(106/100 µl/well) into TCM-washed, 40-h
cultures of Cpn K6-infected and uninfected mAM monolayers
prepared in 96-well, flat-bottom plates (Costar, Cambridge, MA). The
mAM (2 x 104/well) were infected at 4
IFU/cell, then incubated in medium without cycloheximide. Parallel
Cpn-infected mAM monolayers, fixed for 10 min in methanol
and stained with an FITC-conjugated Chlamydia genus-specific
mAb (Pathfinder Chlamydia Culture Confirmation System;
Bio-Rad, Hercules, CA), indicated that 
60–70% of mAM had
Cpn inclusions. After 1.5 h of coculture, 100 µl TCM
with 2 µl/ml GolgiPlug (brefeldin A (BFA); BD PharMingen) was added
to each well. LMNC were harvested 3.5 h later, washed once in FACS
buffer, incubated for 15 min on ice with a 1/100 dilution of
anti-CD16/CD32 (2.4G2) mAb (BD PharMingen), and then
surface-stained with FITC anti-CD8. Cells were washed, fixed,
and permeabilized (Cytofix/Cytoperm kit, BD PharMingen), then incubated
for 30 min on ice with a 1/100 dilution of PE-conjugated
anti-IFN- (XMG1.2) before analysis by flow cytometry. An
isotype-matched mAb (rat IgG1) was used to control for the specificity
of intracellular cytokine staining.
Peptides
H-2b motif-bearing Cpn and
control peptides (Table I
) were
synthesized by F-moc-based solid phase chemistry using an ABI 430A
peptide synthesizer (Applied Biosystems, Foster City, CA) and were
purified by reverse phase HPLC. To confirm their purity (95%) and
identity, peptides were analyzed by mass spectrometry. The control
peptides used were Kb-restricted T.
cruzi TSA-1515 epitope VDYNFTIV
(TcTSA) (24),
Kb-restricted OVA257
epitope SIINFEKL (OVA) (29), and
Db-restricted influenza A
NP366 epitope ASNENMETM (FLUnp)
(30). Lyophilized peptides were dissolved in DMSO (Sigma)
at 20 mg/ml and stored at -70°C. Before use, peptides were further
diluted with RPMI 1640. No cell toxicity was associated with any
peptide.
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Peptide binding to Kb or
Db was measured by the stabilization of class I
molecules on the surface of RMA-S cells (31) and by a
quantitative molecular binding assay that measures the inhibition of
binding of a radiolabeled probe peptide to soluble
Kb or Db molecules
(32). For the MHC class I stabilization assay, RMA-S cells
(106/ml) were cultured at 26°C in 6%
CO2 for 24 h, followed by 1 h in the
presence of peptide (0.1–50 µM). Cells were then transferred to
37°C for 2 h and washed with FACS buffer, and up-regulated cell
surface expression of Kb and
Db was detected by FACS analysis after staining
for immunofluorescence. Results were expressed as the mean fluorescence
intensity (MFI) ratio: MFI of peptide-treated cells/MFI of
untreated cells. For the quantitative binding assay, test peptides (1
nM to 100 µM) were coincubated with radiolabeled probe peptides
(1–10 nM; SGPSNTYPEI for Db; RGYVFQGL for
Kb), purified soluble Db or
Kb H chain (5–500 nM), and
2-microglobulin (1 µM; Scripps Laboratories,
San Diego, CA) for 48 h at room temperature in the presence of
protease inhibitors. The percentage of MHC-bound radioactivity was
determined by gel filtration, and the concentration required to inhibit
50% (IC50) of the binding of radiolabeled
peptide was calculated.
Generation of effector cells
To generate Cpn peptide-specific CTL, mice were killed 2 wk to 6 mo after the first or second Cpn infection. Immune spleen cells (SC) were washed, resuspended in TCM, and seeded in 24-well plates (Costar) at 5 x 106 cells/well. Individual peptides (2 µM) were included in each 2-ml culture. After 2 days of incubation at 37°C, 6% CO2, cultures were made to 5% RCAS and incubated for 4 additional days. Peptide-stimulated effectors were also generated from LMNC removed 1 wk to 45 days after primary or secondary infection.
Preparation of peptide-pulsed and Cpn-infected target cells
To prepare RMA-S targets, cells preincubated for 24 h at
26°C in 6% CO2, were seeded into 24-well
plates (106/2 ml/well) and incubated overnight in
the presence of peptide (0.1 µM) and 100 µCi
Na251CrO4
(51Cr; Amersham, Arlington Heights, IL). Cells
were shifted to 37°C for 2 h before processing for CTL assays.
T2Kb and T2Db targets were
prepared by overnight incubation at 37°C with
51Cr and peptide. To prepare
Cpn-infected targets, 24-h mAM monolayers growing in 12-well
plates (3 x 105/well) were centrifuged with
0.4 ml/well Chlamydia medium containing live or heat-killed
Cpn (4 IFU/cell). Heat-killed Cpn was prepared by
incubating bacteria at 60°C for 1 h. Control mAM targets were
inoculated with HL cell-derived material. After 1 h of incubation
at 37°C, the inocula were removed, and 3 ml medium with 100 µCi
51Cr was added to each well. The plates were
incubated at 37°C for 20 h, at which time BFA (10 µg/ml) was
added to a subset of wells containing Cpn-infected mAM. Two
hours later, monolayers were washed with RPMI 1640 and treated with
Cell Dissociation Buffer (Invitrogen) to prepare single-cell
suspensions. Chlamydia-specific immunofluorescent staining
of infected mAM incubated for 24 h more indicated that 60–70%
of the cells were infected. Similar rates of infection were achieved
for the K6, AR39, and CWL029 Cpn strains. BFA treatment did
not affect Cpn growth, as determined by subculture of 5-h
BFA-treated infected mAM monolayers.
CTL assay
Cytotoxic activity of effector cells on target cells was
assessed by 51Cr release assays, as previously
described (24). Briefly, peptide-sensitized,
Cpn-infected, and control 51Cr-labeled
target cells (5 x 103/well) were incubated
for 5-h with effector cells at various E:T cell ratios in 96-well,
round-bottom plates (Corning, Corning, NY). BFA (10 µg/ml) was
included in those wells containing Cpn-infected BFA-treated
target cells. Effectors depleted of CD8+ and
CD4+ T cells were only tested at the highest E:T
cell ratio. Depletions were conducted with magnetic beads coated with
anti-CD8 or anti-CD4 mAbs (Miltenyi Biotec, Auburn, CA) or by
incubating cells on ice for 30 min with anti-CD8 (3.155; ATCC TIB
211, ATCC) or anti-CD4 (GK1.5; ATCC TIB 207; ATCC), followed by 30
min at 37°C with rabbit complement. Supernatants were harvested
(Skatron SCS System; Molecular Devices, Sunnyvale, CA), and released
51Cr was counted on a gamma counter. The percent
specific lysis was calculated from the mean of triplicates as 100
x (experimental release - spontaneous release)/(maximum
release - spontaneous release). Spontaneous release did not
exceed 26% of the maximum release. SEs were <6% of the mean. A CTL
response was considered positive when, at the highest E:T ratio, a
difference of 
10% lysis was obtained by subtracting the percentage
lysis for control peptide from the percentage lysis for the test
peptide.
Generation and maintenance of Cpn-specific CD8+ T cell lines
Peptide-specific CD8+ T cell lines were generated as previously described (24). In brief, 4 x 107 immune SC from Cpn-infected mice were incubated with CTL peptides (2 µM) for 6 days at 37°C in 6% CO2 in 10 ml TCM using T25 flasks (Corning). RCAS (5%) was added on day 2 of culture. In each of two to four subsequent 6-day cycles of restimulation, 4 x 106 viable effector cells were cultured with 4 x 107 peptide-pulsed irradiated (3000 rad) syngeneic SC in 15 ml TCM-RCAS. A similar protocol was used to generate CD8+ T cell lines from immune LMNC.
Cytokine determinations
Culture supernatants of peptide-stimulated SC were harvested
48 h into the first and second cycles of stimulation, and levels
of IFN-, IL-4, and TNF- were determined by sandwich ELISA.
Capture and detection Ab pairs were R4-6A2/XMG1.2 (BD PharMingen) for
IFN-, BVD4-1D11/BVD6-24G2 (Caltag, Burlingame, CA) for IL-4, and Ag
affinity-purified goat polyclonal Ab/MP6-XT3 (R&D Systems and BD
PharMingen) for TNF-. The lower detection limits for IFN-, IL-4,
and TNF- were 40, 8, and 40 pg/ml, respectively.
ELISPOT assay for IFN--secreting cells
Nitrocellulose-backed 96-well plates (MultiScreen MAHA S4510;
Millipore, Bedford, MA) were coated overnight at 4°C with 75 µl PBS
containing 10 µg/ml anti-IFN- mAb R4-6A2. After washing with
PBS, the wells were blocked for 2 h at 37°C with 100 µl TCM.
Two-fold serial dilutions of freshly isolated LMNC or SC starting at
107 cells/ml were added in TCM-RCAS in triplicate
wells (100 µl/well) containing peptide-pulsed (1 µM) irradiated (16
krad) RMA-S cells (105/100 µl/well). Irradiated
unpulsed RMA-S cells were used as a control for Ag-independent IFN-
secretion. As positive controls, LMNC and SC containing unpulsed RMA-S
cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml;
Sigma). After incubation at 37°C in 5% CO2 for
24 h, plates were washed three times with PBS, followed by three
times with PBS/0.05% Tween 20 (PBS/T). Wells then received 75 µl of
a solution of 3 µg/ml biotinylated anti-IFN- mAb XMG1.2 in
PBS/T/0.5% FBS. After a 16-h incubation at 4°C, plates were washed
six times with PBS/T, and 100 µl alkaline phosphatase-ExtrAvidin
(Sigma; 1/800 dilution) was added to each well. Following 1 h at
26°C wells were washed four times with PBS/T and twice with PBS.
Spots were developed with 75 µl/well
5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate
(Sigma). After 20 min the plates were rinsed with water and dried, and
spots in each well were counted under a stereomicroscope.
Inhibition of chlamydial inclusion development assay
To evaluate the inhibition of Cpn growth by
CD8+ T cell-derived soluble factors, 24-h HEp-2
cell monolayers prepared in 48-well plates (5 x
104/well) were treated with supernatants from CTL
lines for 18 h before Cpn infection. Supernatants
preincubated with anti-IFN- XMG1.2 or IgG1 control mAbs (20
µg/ml; BD PharMingen) were also used for HEp-2 cell pretreatment.
After Cpn infection (3 IFU/cell), plates were incubated for
1 h at 37°C, the inocula were removed, and monolayers were
incubated for 24 h more with new samples of the same supernatants.
Following 48 h of incubation in Chlamydia medium,
cultures were stained with the Pathfinder Chlamydia
genus-specific mAb, and the number of inclusions in each well was
counted in 10 x400 high powered fields (HPF). Supernatants were tested
in triplicate. The numbers of inclusions in Cpn-infected
HEp-2 cells treated with supernatants from cultures lacking
CD8+ T cells, peptide, or both were also
assessed. All results were compared with those obtained using
supernatants from immunomagnetically purified naive
CD8+ T cells stimulated with Cpn CTL
peptides.
To test the chlamydial growth-inhibiting activity of Cpn peptide-specific CTL, mAM monolayers prepared 1 day earlier in 48-well plates (4 x 104/well) were infected with Cpn (4 IFU/cell) and incubated for 20 h before adding graded numbers of CD8+ T cells (2.5 x 104–4 x 105/ml/well). Cultures with or without effectors were washed 4 h later, and 1 ml Chlamydia medium with cycloheximide was added to each well. After 48 h at 37°C, mAM monolayers were scraped, plates were frozen and thawed once, and serial dilutions of cleared pooled material from triplicate wells were used to infect fresh HEp-2 cell monolayers. Total numbers of mAM recovered from parallel cultures were similar for all effector cell types and densities tested, ranging between 1–1.4 x 105/triplicate. Inoculated HEp-2 cells were incubated for 72 h in cycloheximide-containing medium and processed for immunofluorescence, and chlamydial inclusions were counted in 10 HPF. Control effector cells were T. cruzi TSA-1515-specific CD8+ CTL and purified naive CD8+ T cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-producing CD8+ T cells
Mouse models of Cpn infection indicate that
CD8+ T cells and IFN- play important roles in
protective immunity (20, 21, 22). However, the induction of
Cpn-specific CD8+ T cells during
infection has not been reported. Therefore, we first determined whether
the pulmonary inflammatory response of B6 mice during primary and
secondary Cpn infections included pathogen-specific
CD8+ T cells capable of IFN- production. Lung
sections from mock-infected animals showed no evidence of pulmonary
inflammation up to 100 days after each of two inoculations (Fig. 1A). In contrast, a mild
interstitial mononuclear cell infiltration with a perivascular and
peribronchiolar lymphoid cuffing was present in the lungs from
Cpn-infected mice from days 5–60 after infection (Fig. 1, C and D). During reinfection, a moderate to
marked pneumonia and lymphoid infiltrate was detected 2 days
postinoculation, peaked on days 8–12 (Fig. 1B), and
gradually declined, but remained present for up to 2 mo. Pulmonary
bacterial loads commonly peaked 2–4 days before maximal inflammatory
responses with mean IFUs per lung of 3 x
105 ± 9 x 104 after
primary infection and 2.9 x 104 ± 8
x 103 after reinfection. Staining with a
Cpn MOMP-specific mAb localized chlamydial Ag within
epithelial and mononuclear cells throughout the interstitium,
especially in the perivascular and peribronchiolar inflammatory foci
(Fig. 1D). No staining was detected using an IgG3 control
mAb (Fig. 1C). The MOMP-specific mAb did not stain lung
tissue from mock-infected mice (data not shown).
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, G and H vs E and F). When
LMNC were cocultured for 5 h with mock-infected mAM, the
percentage of CD8+ T cells that were
IFN-+ was 6.5% in Cpn-infected
animals ((1.1/17) x 100), but only 1.1% in mock-inoculated mice
((0.1/8.9) x 100; Fig. 1, G vs E). This
level of IFN- production in pulmonary CD8+ T
cells from infected mice may reflect specific activation in vivo. After
coculture with Cpn-infected mAM, the percentage of
CD8+ T cells expressing IFN- was 27.1% in
cells from infected mice ((5/18.4) x 100), and only 2.2% in
cells from mock-infected animals ((0.2/9.3) x 100; Fig. 1, H vs F). Coculture with Cpn-infected
mAM elicited 4-fold increase in the percentage of
CD8+ IFN-+ T cells (Fig. 1, H vs G). These data establish that
Cpn-specific CD8+ T cells are present
in the lungs from infected B6 mice and suggest that they may
participate in the immune response against this pathogen by producing
IFN-. Mice infected with Cpn generate a CTL response that recognizes peptides from multiple chlamydial Ags
Based on the foregoing results, we asked: Which Cpn Ags
reach the cytosol of infected cells and are recognized as MHC class
I-peptide complexes by CD8+ T cells? From the
available Cpn genome sequence databases
(33, 34, 35) we selected 35 proteins that included outer and
inclusion membrane proteins, chaperones, and selected proteins unique
to Chlamydia or Cpn. In the sequences of these
proteins we identified 461 segments of 8–9 aa conforming to the murine
H-2Kb and Db class I
binding motifs (36). Of these, 39 sequences from 16
proteins were selected for peptide synthesis (Table I). When the
ability to bind to Kb and
Db was examined by the RMA-S stabilization assay
(31), 22 peptides were classified as high or intermediate
binders (MFI 2.0). Using a quantitative molecular binding assay
(32), which provides precise measurements of binding
affinity that better correlate with CD8+ T cell
immunogenicity (37), 17 peptides bound with high or
intermediate affinity (IC50 
500) to purified
Kb or Db molecules. Four
additional peptides with affinities of 500 nM but 1000 nM were
considered good binders, as CD8+ T cell
immunogenicity has also been reported in this affinity range
(38). Overall, 20 peptides were identified as high to
intermediate binders by both binding assays (Table I).
All synthetic peptides were next assayed for their ability to target
H-2b-bearing cells for lysis by SC from
Cpn-infected mice obtained after culture with each
individual peptide. Of the 39 peptides, 18 generated effector cells
that specifically lysed RMA-S cells (H-2b; MHC
class II-) pulsed with the respective peptide,
but not RMA-S cells pulsed with control OVA (Kb)
and FLUnp (Db) CTL peptides (Fig. 2). The 18 peptides represent sequences
in 12 Cpn Ags: five outer membrane proteins (omp), a 76-kDa
protein, a family of four hypothetical 43-kDa proteins, heat shock
protein-70, and an inclusion membrane protein (33, 34, 35, 39, 40, 41, 42, 43, 44) (Table I and Fig. 2). At the highest E:T cell ratio, net
peptide-specific lysis for effectors stimulated with the 18 CTL
peptides ranged from 10–27.9% after primary infection and from
10.4–34.2% after reinfection. As illustrated for a subset of
peptides, CTL activity was detectable after primary infection (Fig. 2A). For most of the 18 positive peptides, the in vitro
recall CTL responses were slightly enhanced in reinfected mice, and
absent in mock-infected animals (Fig. 2, B and
C). Thus, most peptides capable of binding to
H-2b molecules with high to intermediate affinity
elicit functional CTL, and the polyclonal multi-Ag-specific CTL
response is induced as a result of infection.
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To further characterize the lytic response to the positive
Cpn CTL peptides, cell depletion and MHC class I restriction
analyses were conducted. As illustrated for a subset of positive
peptides, effector cells generated from SC of Cpn-reinfected
mice lysed RMA-S cells pulsed with Cpn peptide but not with
irrelevant peptides, and target cell lysis was eliminated by depleting
effectors of CD8+, but not of
CD4+ T cells (Fig. 3). T2 target cells pulsed with
Kb-binding CTL peptides (e.g., peptides 19, 20,
and 39) were lysed by the respective peptide-specific effectors only if
the targets expressed Kb but not
Db molecules. In contrast, effectors generated
with Db-binding CTL peptides (e.g., peptides 27,
30, 31, and 37) lysed T2Db but not
T2Kb cells sensitized with the homologous peptide
(Fig. 3). These results demonstrate that SC from
Cpn-infected B6 mice recognize pathogen-derived peptides in
an Ag-specific, Kb- or
Db-restricted, and CD8+ T
cell-dependent manner.
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To further characterize Cpn peptide-specific T cells,
SC from Cpn-infected mice were expanded by three
weekly cycles of stimulation with each peptide and cell lines
were evaluated for cytokine secretion profiles and surface phenotypes
(Table II). During the first cycle,
supernatants contained IFN- but not IL-4. After the second cycle,
IFN- levels were 4- to 9-fold higher, but IL-4 levels remained
undetectable. All the CTL peptides elicited significant TNF-
production. IFN- and TNF- levels varied for each CTL peptide, but
were significantly higher than the levels produced by stimulation with
four Cpn peptides without CTL activity (Table II). All T
cell lines were >90%
CD3+CD8+TCR+CD44high
(Table II) and highly lytic to Cpn peptide-pulsed targets
(data not shown). These results indicate that the 18 Cpn
peptides stimulate memory CD8+ T cells in the
spleens of infected mice displaying cytolytic function and a Tc1
cytokine secretion pattern.
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To ascertain whether the 18 Cpn peptides
represent CTL epitopes that are presented on the surface of infected
cells, we used Cpn K6-infected mAM as targets for
peptide-stimulated effectors. As shown for seven Cpn CTL
peptides, effector cells generated after two cycles of stimulation with
individual peptides lysed Cpn-infected but not mock-infected
or BFA-treated infected mAM (Fig. 4).
Short term CTL lines did not lyse mAM inoculated with heat-killed
Cpn (Fig. 4). Similar results were obtained when mAM targets
were treated with the Cpn strains AR39 and CWL029 (data not
shown). Thus, all 12 Cpn CTL Ags are processed by the
endogenous pathway, and the 18 epitope-bearing peptides are presented
to CTL only in productively infected cells.
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To determine whether Cpn peptide-specific
CD8+ CTL were present in the lungs of infected
mice, we purified LMNC 12 days after reinfection and stimulated them
with Cpn CTL peptides. Six days later, the lytic activity of
undepleted and CD8+ T cell-depleted effectors was
tested against peptide-pulsed target cells. For five representative
peptides, each from a different Cpn CTL target Ag, resulting
effectors displayed CD8+ T cell-dependent CTL
activity against RMA-S cells sensitized with homologous peptide, but
not against the same cells pulsed with control CTL peptides or with no
peptide (Fig. 5A). As shown
for a Db- and a
Kb-restricted Cpn CTL peptide, T cell
effectors generated after four cycles of stimulation were highly
enriched for peptide-specific, H-2b-restricted
lytic activity and a CD8+
CD44high memory phenotype. Moreover, these
LMNC-derived CTL lines lysed mAM inoculated with live AR39
Cpn bacteria, but not with heat-killed organisms unless they
were also sensitized with peptide (Fig. 5B). These results
demonstrate that Cpn-specific CD8+ CTL
are present in the lungs of infected animals and further support the
natural presentation of identified epitopes by Cpn-infected
cells.
|
-secreting CD8+
T cells
We initially observed that LMNC from Cpn-infected B6
mice contained CD8+ T cells capable of rapidly
producing IFN- in response to Cpn-infected cells (Fig. 1, E–H). Once Cpn CTL determinants
became defined, we asked whether epitope-specific
CD8+ T cells were detectable in the lungs and
spleens of infected mice without stimulation, and if so, we determined
precursor frequencies of these cells during primary and secondary
Cpn infections. Immune LMNC and SC from B6 mice were
obtained 11 days after infection or reinfection and were stimulated for
24 h with peptide-pulsed and unpulsed RMA-S cells, followed by
enumeration of IFN- secreting CD8+ T cells by
ELISPOT. We measured the CD8+ T cell precursor
frequencies for peptides 13, 37, and 39, which were associated
with the highest IFN- production (Table II). The mean number of LMNC
isolated at 11 days postinfection (5.3 x
106 ± 8 x 105) or
reinfection (6.7 x 106 ± 1.4 x
106) was 6- to 7-fold higher than the yields
obtained for mock-infected animals (9 x 105
± 3 x 105). Based on these data and the
frequencies of IFN- spot-forming cells (SFC) obtained by ELISPOT,
the lungs of infected animals contained an estimated 165, 145, and 250
peptide 13-, 37-, and 39-specific IFN--producing
CD8+ T cells, respectively. For each peptide the
number of IFN- SFCs was 2- to 2.5-fold higher after reinfection
(Fig. 6A). Parallel studies
with SC showed that the precursor frequencies of peptide 13-, 37-, and
39-specific CD8+ T cells were 2- to 5-fold higher
after reinfection compared with primary infection (69–172 vs 20–39
IFN- SFCs/106 cells; Fig. 6B).
IFN- secretion was specific, as spots were rarely observed when
immune cells were incubated with unpulsed RMA-S cells. Furthermore,
peptide-specific IFN- SFCs were not detected using cells from
mock-infected animals. Thus, Cpn CTL peptide-specific,
IFN--secreting CD8+ T cells are present in
local and systemic compartments after primary chlamydial infection, and
these cells expand after reinfection.
|
IFN- contributes to protection against Cpn infection
(21, 22) and inhibits chlamydial growth in vitro
(45). Our results suggested that Cpn-specific
CD8+ T cells could participate in protective
immunity through secretion of IFN- and/or killing of infected cells.
To evaluate the ability of CD8+ T cell-derived
soluble factors to inhibit bacterial growth, we counted the number of
Cpn inclusions in infected HEp-2 cells that had been treated
with supernatants collected from CD8+ T cell
cultures (Fig. 7A). As shown
for peptides 13, 20, 37, and 39, undiluted culture supernatants from
peptide-specific CD8+ T cells inhibited
inclusion development by 95–100% compared with undiluted culture
supernatant from Cpn peptide-stimulated naive
CD8+ T cells. Serial dilutions of the
supernatants resulted in 70–92% (1:5) and 8–51% (1:25) inhibition,
indicating a dose-dependent effect on chlamydial growth. Addition of
anti-IFN- mAb to the undiluted supernatants from
peptide-specific CD8+ T cells reduced inhibition
by 18–65%. No reversal of inhibition was observed using control mAb
(data not shown). Supernatants from cultures containing peptide alone
or peptide-stimulated naive CD8+ T cells failed
to inhibit chlamydial multiplication. Minimal inhibition was detected
using supernatant from unstimulated peptide-specific
CD8+ T cells, perhaps due to accumulation of
inhibitory factors during the 48 h before collection of the
supernatants for testing. Thus, Cpn-specific
CD8+ CTL suppress chlamydial growth by production
of IFN- and other unidentified soluble factors.
|
B). At high
CD8+ T cell densities, Cpn peptide
13-, 20-, 37-, and 39-specific CTL inhibited 94–100% the number of
Cpn inclusions that developed in the presence of naive
CD8+ T cells, and the extent of inhibition
decreased with decreasing numbers of Cpn peptide-specific
CD8+ T cells. Significant inhibition was observed
for all Cpn peptide-specific CD8+ CTL
lines compared with studies in the absence of
CD8+ T cells or in the presence of irrelevant
T. cruzi TSA-specific CD8+ T cells.
These data indicate that Cpn-specific
CD8+ CTL decrease the output of viable organisms
from infected cells. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. We also report the
identification of 18 H-2b-restricted Tc1
epitope-bearing sequences in the first 12 bona fide
Cpn target Ags of the CD8+ T
cell response induced in infected mice. Development of vaccines that induce CD8+ T cell responses against Cpn requires knowledge of the mechanisms by which these cells mediate resistance to infection. Equally important for the design of anti-Cpn vaccines is to determine the response kinetics and functional attributes of CD8+ T cells found in the lungs and systemic compartments of Cpn-infected hosts and whether these cells associate exclusively or collectively with protection, persistence, or immunopathology. However, understanding Cpn-specific CD8+ T cell responses and designing vaccines to maximize protection and minimize tissue damage are contingent upon the ability to assess such responses and on the identification of the Cpn Ags or epitopes that are recognized by CD8+ T cells. As a first step to characterize the anti-Cpn CD8+ T cell response, we structured a strategy based on three components: 1) the mouse model of Cpn infection, which is an excellent system to study the immune mechanisms thought to control this pathogen in humans; 2) the Cpn genome sequence databases, from which putative target Ags of CD8+ T cells were selected; and 3) the Cpn-competent mAM cell line, which served to stimulate and detect the activity of Cpn-specific CD8+ T cells. We selected the macrophage because Cpn disseminates from the respiratory tract by infecting this cell (48), and a similar mechanism may permit this pathogen to spread systemically in humans.
We first asked whether chlamydial infection primes
Cpn-specific CD8+ T cells in the lungs
of infected B6 mice. To select a time point when we could detect T
cells, we conducted a kinetic analysis of histopathologic changes and
bacterial loads in the lungs of Cpn-infected mice. From this
analysis we determined that mononuclear cells are the main inflammatory
cell type during infection, peak inflammatory responses are stronger
and emerge faster after reinfection than after primary infection, and
fewer pulmonary bacteria are recovered from Cpn-reinfected
mice than from infected animals. These results indicated that
Cpn infection induces immunoprotective responses. Because
Cpn MOMP+ foamy mononuclear cells were
seen adjacent to lymphoid cells composing the inflammatory foci, we
postulate that these consist of macrophages priming pathogen-specific
CD8+ T cells. Indeed, LMNC from 12-day infected
mice contained Cpn-specific CD8+ T
cells, and nearly one-fourth of the infiltrating
CD8+ T cells produced IFN- upon stimulation
with Cpn-infected mAM. Furthermore,
CD8+ T cells play a key role in early and late
resistance to Cpn infection (20, 21). Early in
the infection, CD8+ T cells are thought to
contribute to protection by modifying the CD4+ T
cell cytokine pattern from a Th2 to a protective Th1 phenotype
(20). We speculate that Cpn-specific
IFN--producing CD8+ T cells contribute to the
type 1 cytokine milieu required to control early Cpn growth
in the lungs of infected B6 mice.
We next conducted a genome-wide search for sequences encoding potential target Ags of murine anti-Cpn CD8+ T cells, selecting Cpn proteins that we believed were most likely to reach the cytosol of infected cells for processing and MHC class I presentation. These included membrane proteins specific to Chlamydia or with relatives in other organisms, chaperones, and Ags unique to Cpn, or with orthologs in other chlamydial species. Within the primary sequences of 16 Cpn Ags, 39 of 146 Kb- or Db motif-fitting segments were synthesized as peptides and tested for the capacity to sensitize target cells for lysis by peptide-stimulated SC and LMNC from Cpn-infected mice. Remarkably, a specific, H-2b-restricted, and CD8+ T cell-dependent CTL response was detected for 18 peptides in 12 Cpn Ags. Of the 18 CD8+ CTL epitope-bearing peptides, seven represent sequences within five outer membrane protein complex Ags, namely, Omp2, MOMP, OmpB, Omp85 homolog, and Omp5 (Pmp10) (33, 34, 35, 39, 43, 44). In the group of four chaperones, only a peptide from DnaK (41) elicited recall CD8+ CTL responses. Interestingly, DnaK is associated with the outer membranes of C. trachomatis (49). Finally, in the group of Cpn Ags with or without chlamydial orthologs, eight CTL peptides represent sequences within a family of four 43-kDa protein homologs, one within a C. trachomatis-like 76-kDa protein and another within an inclusion membrane protein similar to C. psittaci IncA (33, 34, 35, 42). The 43- and 76-kDa proteins may also be surface exposed, as Cpn Ags of these molecular masses are recognized by human and rabbit immune sera, the 43-kDa primary sequences revealed potential transmembrane domains, and a specific anti-76-kDa antisera neutralizes Cpn infectivity in vitro (42, 50, 51).
Aside from macrophages, Cpn can infect and multiply in nonprofessional APC, including airway epithelial, endothelial, and smooth muscle cells (52, 53). However, macrophages and dendritic cells are the APC most likely responsible for priming a Cpn-specific CD8+ CTL response. Because in addition to the endogenous MHC class I pathway, professional APC can process and present exogenous Ag to CD8+ T cells (54), detected Cpn-specific CTL could have been primed by processed Ags from phagocytosed bacteria and not from chlamydial Ags accessing the cytosol of infected APC. Arguing against this possibility was the finding that all 18 peptide-specific CD8+ CTL effectors killed mAM only when inoculated with live Cpn bacteria. Moreover, the fact that BFA inhibited this lytic activity strongly suggested that the 12 Cpn Ags access the endogenous MHC class I processing machinery. The cytotoxic mechanism that prevailed in the killing of Cpn-infected mAM was probably not Fas/Fas ligand dependent, as BFA inhibits this pathway in CTL effectors (55). BFA did not affect Cpn growth, a finding consistent with that described in a study in which the endogenous Ag processing pathway was shown to target C. trachomatis-infected fibroblasts for lysis by pathogen-specific CD8+ CTL (11). In support of our findings, C. trachomatis MOMP and the inclusion membrane protein Cap1 are the targets of infection-primed CD8+ CTL that kill C. trachomatis-infected nonprofessional APC (15, 16). Thus, although our results do not rule out the contribution of alternative pathways to the processing of Cpn Ags by professional APC, they imply that CD8+ CTL primed by endogenously processed Ags are the most likely to recognize all Cpn-infected cells.
Despite their intravacuolar location, chlamydiae interact with multiple host cell processes to ensure that the inclusion is a safe niche for their survival and replication. These interactions are needed to acquire nutrients, avoid fusion with lysosomes, obtain membrane components from Golgi-derived exocytic vesicles, and modify host cell functions (56, 57). The chlamydial products that control these processes are thought to be proteins translocated through or inserted into the inclusion membrane via a type III secretion apparatus (58). However, of the 12 identified Cpn CTL Ags, none is known to be secreted into the host cell cytosol, and only one, the Ag similar to C. psittaci IncA, is likely to belong to the Inc family of inclusion membrane proteins. Identified Inc proteins have a unique 50- to 60-aa hydrophobic region and domains that localize to the cytoplasmic face of the inclusion (59, 60, 61). These exposed domains may be cleaved by proteases in the cytosol or on the membranes of interacting vesicles, and after proteasomal processing, Inc-derived CTL epitopes, such as peptide 39, may be generated. How do the Cpn CTL target Ags located or associated with the envelopes of developing chlamydiae become accessible to the endogenous MHC class I presentation pathway? As no report has localized Cpn envelope Ags in the cytosol of infected cells, we speculate that these and other envelope Ags may reach the cytosol in a denatured or preprocessed form. Unfolded envelope Ags or fragments thereof may arise during the extensive membrane remodeling that occurs during chlamydial replication and differentiation, from the "ghost-like" membranous material present alongside organisms within a typical inclusion (47), or from a small fraction of developing chlamydiae undergoing autolysis.
There is little variation in the sequences of various genes from multiple Cpn isolates, and nearly identical sequences were reported for the genomes of Cpn strains CWL029 and AR39 from the United States and J138 from Japan (33, 34, 35). Our CTL data are in line with these findings, as all 18 peptide-specific CD8+ T cell effectors were generated from mice infected with the Finnish K6 Cpn strain, and these CTL lysed mAM infected with Cpn strains K6, AR39, or CWL029. These results are encouraging, as the CTL Ags or epitopes included in vaccines against Cpn will require this level of sequence conservation among strains from different geographic locations. Moreover, because 80% of the predicted coding sequences for Cpn have an ortholog in C. trachomatis, and, on average, orthologs from the two species share 62% aa identity (33), some CTL epitopes may be conserved between orthologous chlamydial Ags. Indeed, the sequence of Cpn Omp2-derived peptide 3 is identical with its ortholog in C. trachomatis. In contrast, the six Cpn CTL peptides derived from the 76-kDa protein, Omp85 homolog, DnaK, OmpB, and MOMP differed by one to eight residues from the respective sequences in the orthologous C. trachomatis Ags. Nevertheless, these Ags could still be the targets of CTL responses during human or experimental C. trachomatis infections, as recently shown for C. trachomatis MOMP (16).
Lung-derived Cpn peptide-specific CD8+
T cells lysed Cpn-infected cells, expressed a
CD44high memory phenotype and produced Tc1
cytokines. IFN- is crucial for the control of chlamydial infections
(21, 22, 62), and IFN--producing
CD44high CD8+ CTL mediate
protection in murine models of M. tuberculosis and T.
cruzi infection (24, 63). Similarly, we found that
peptide-specific CD8+ T cells can inhibit
Cpn inclusion development by producing soluble factors that
limit Cpn growth and by direct effects of
CD8+ T cells themselves on the infectious titers
produced by Cpn-infected macrophages. The capacity of
supernatants to suppress Cpn growth correlated with the
levels of IFN-. However, neutralization of IFN- only partially
restored inclusion development, suggesting that other factors inhibit
Cpn multiplication. Cpn peptide-specific
CD8+ T cells produce TNF-, which may
contribute to the suppressive effect, as it synergizes with IFN- in
inhibiting Cpn growth (45). Cpn
multiplication may also be influenced by other
CD8+ T cell-derived factors, including RANTES,
macrophage inflammatory proteins-1 and -1, and IL-16, which
suppress HIV replication (64, 65). The anti-chlamydial
activity of purified Cpn peptide-specific
CD8+ T cells was probably due to direct lysis of
Cpn-infected mAM. Because most organisms released by lysed
mAM were most likely in the form of noninfectious reticulate bodies,
Cpn viability may have been affected by the release of
molecules with antimicrobial activity, similar to the human
CD8+ T cell-derived granulysin (66).
This possibility is being evaluated using Cpn-infected
cells, where organisms have differentiated into infectious elementary
bodies. Altogether these results suggest that Cpn-specific
CD8+ T cells may also inhibit Cpn
growth in vivo through the combined actions of both effector functions.
We are conducting adoptive transfer experiments to determine whether
CD8+ T cells to each of the identified
Cpn CTL epitopes can reduce pulmonary chlamydial loads in
Cpn-challenged mice and to define the mechanisms by which
CTL protect against infection.
Although CD8+ CTL might protect against
Cpn, they may also be partially responsible for inducing
persistent infection and immunopathology. IFN- may have a dual role
in controlling the outcome of chlamydial infections in vivo, as
exposure of infected cells to high IFN- concentrations irreversibly
inhibits chlamydial replication, while lower concentrations induce the
formation of persistent forms (45). Thus, production of
IFN- by Cpn-specific CD8+ T cells
may be critical to achieve the local threshold concentration required
to inhibit Cpn growth and prevent persistence. Persistent
Cpn infection in humans and rodents (3, 19) may
be due to a suboptimal CD8+ T cell response
during the course of an active Cpn infection. Nevertheless,
we believe that an efficacious anti-Cpn
CD8+ CTL response can be induced through
vaccination. Vaccines against Cpn will most likely include
multiple determinants from various chlamydial Ags that induce
CD8+ CTL capable of eliminating productively and
possibly persistently infected cells without causing serious tissue
inflammation. In the current studies we provide direction for the
development of such vaccines, as we have demonstrated the generation of
Cpn-specific MHC class I-restricted
CD8+ CTL with anti-chlamydial growth activity
in infected mice and have identified 12 CTL target molecules. Work is
in progress to further delineate the dynamics of the murine
CD8+ T cell response to the Cpn CTL
epitope-bearing sequences described in this study and to determine
whether Cpn-infected humans also generate CTL responses
against the Ags identified using the murine model. Such information is
important to validate the use of the mouse model to provide insight
into the aspects of the CTL response that contribute to protection and
those that mediate immunopathology, and to identify and test CTL target
Ags that may ultimately be used to develop vaccines against
Cpn.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Benjamin Wizel, Department of Microbiology and Immunology, Center for Pulmonary and Infectious Disease Control, University of Texas Health Center, 11937 U.S. Highway 271, Tyler, TX 75708. E-mail address: bwizel{at}uthct.edu
3 Abbreviations used in this paper: Cpn, Chlamydia pneumoniae; BFA, brefeldin A; HPF, high powered field; IFU, inclusion-forming units; i.n., intranasal; LMNC, lung mononuclear cell; mAM, murine alveolar macrophage; MFI, mean fluorescence intensity; MOMP, major outer membrane protein; RCAS, rat Con A supernatant; SC, spleen cell; SFC, spot-forming cell; TCM, T cell medium; TSA, trypomastigote surface Ag.
Received for publication April 3, 2002. Accepted for publication July 1, 2002.
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References |
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), HL cell-derived material (
), or
heat-killed bacteria (
). Lytic activity of effector cells was also
tested against BFA-treated Cpn-infected mAM (
). BFA
(10 µg/ml) was added for the last 2 h of infection and during
the CTL assay. Results for seven of 18 Cpn
peptide-specific, short term CTL lines are shown.
