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Departments of
*
Medical Microbiology and Immunology,
Medicine, and
Pathology, University of Wisconsin Medical School, Madison, WI 53706; and
§
Laboratory of Genetics, University of Wisconsin, Madison, WI 53706
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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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Available data suggest that past infections of humans with Ct may confer partial protection against subsequent infections (5, 6, 7). Such immunity appeared to be of relatively short duration, and recurrent infections were commonly observed. Partial immunity to reinfection was also observed in monkeys genitally infected with Ct (8); less severe infections of a shorter duration developed after reinoculation with either the same or a different serotype, indicating that cross-protective immune responses could be elicited upon primary Ct infection. In humans, serovars isolated from secondary infections were often different from those in primary infections (7). This has suggested that neutralizing Ab responses to variable segment (VS) epitopes in the major outer membrane protein (MOMP) might be important in protective immunity. Indeed, it was demonstrated in mice that MOMP-specific neutralizing Abs reduced susceptibility to reinfection in vivo (9, 10). This kind of evidence and the detailed genetic and immunochemical knowledge of MOMP have stimulated multiple attempts to design a MOMP-based subunit vaccine (reviewed in 11). Encouragingly, MOMP-based vaccines induced acquired immunity in mice and conferred partial protection against a subsequent challenge (12, 13, 14). However, the nature of MOMP-specific immune responses generated in immunized animals was not thoroughly characterized with regard to the types of protective responses and their antigenic specificity. Nonetheless, these studies in mice suggested that MOMP might be the potentially important target of protective immunity in humans as well.
Our laboratory investigates MOMP-specific human T cell responses generated upon genital tract infections with Ct. We have previously reported isolation of HLA class II-restricted T cells from infected humans that recognized multiple Th cell epitopes in MOMP (15, 16)5. The study demonstrated that MOMP is a highly immunogenic Ct Ag in humans and produced potentially useful information with regard to developing a subunit vaccine based on human B cell (17) and Th cell epitopes derived from MOMP.
In the present study, we examined whether HLA class I-restricted CD8+ CTL responses are elicited to MOMP in human genital tract infections with Ct. The ability of CTLs to lyse cells infected with intracellular bacterial pathogens has been established as an important surveillance mechanism (reviewed in 18). However, the induction of CTL responses in human chlamydial infection has not been reported, and their potential role in immune protection is unassessed. Here we present the first demonstration that HLA class I-restricted CD8+ CTLs specific for MOMP are regularly found in the peripheral blood of humans who acquired genital tract infections with Ct. Utilizing synthetic peptides, we identified five HLA class I-restricted CTL epitopes in MOMP that are commonly recognized by infected humans. Significantly, we show that these epitopes can be naturally processed and presented by Ct-infected ME180 human cervical epithelial cells, which are lysed by peptide-elicited CTLs in a way that is specific for both eliciting peptides and HLA class I restriction elements.
Our ability to obtain Ct MOMP peptide-specific CTLs from infected people with relative ease will facilitate studies of immunologically important questions about chlamydial infections that could not be addressed in the past due to the lack of appropriate tools (specific CTLs) and information (epitopes). The knowledge presented here will prompt future investigations of the role of CTLs at the site of infection as well as HLA class I pathways of chlamydial Ag processing and presentation in immunologically relevant human APCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human subjects who had recent symptomatic genital tract infections with Ct were recruited through the Blue Bus Clinic at the University of Wisconsin (Madison, WI) with the exception of STD116, who was recruited at the Indiana University STD Research Center (Indianapolis, IN). Diagnosis of Ct infection and determination of infecting serovars was performed as previously described (15). All the infected STD subjects were treated with an oral dose of azythromycin upon confirmation of Ct infection. HLA-A2+-uninfected control subjects were recruited from the similar age group at the University of Wisconsin (Madison, WI). Control subjects had been sexually active but lacked previous history of genital tract infections with Ct.
HLA class I typing was performed at the Tissue Typing Laboratory of the University of Wisconsin (Madison, WI) by PCR-sequence specific primer amplification, using Class I ABC SSP Unitray kit (Pel-Freez Clinical Systems, Brown Deer, WI).
Cell lines and culture media
B lymphoblastoid cell lines (LCLs) were established from human
subjects by transformation of PBLs with EBV (15). The HLA class I
Ag-loss mutant cell lines used as targets in CTL assays were derived
from LCL 721 (19) (Fig. 1
). Mutants
LCL.45 and LCL.19 were derived by mutagenizing LCL 721 with gamma rays
and by using complement plus appropriate Abs to select for HLA deletion
mutants (19). Further mutagenesis produced mutant LCL.144, which is
HLA-A null due to a homozygous deletion at the locus (20). Similarly,
HLA-B-null mutant LCL.53 (21) was derived from LCL.19 as a result of
intragenic deletion at the locus (our unpublished results). LCLs were
cultured at 37°C in humidified 5% CO2 in "2/1 RPMI"
(RPMI 1640 (85%) supplemented with FCS (5%), defined/supplemented
calf serum (10%), 25 mM HEPES, 44 mM NaHCO3, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin sulfate).
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Human T cells were grown at 37°C in humidified 5% CO2 using DMEM containing 4.5 g/L glucose and supplemented with 10% pooled AB-negative human serum, 100 µM nonessential amino acids, 25 mM HEPES, 44 mM NaHCO3, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. When needed, recombinant human IL-2 (rhIL-2) was added at 25 U/ml.
Human serum was purchased from Pel Freez, and bovine sera were purchased from HyClone Laboratories (Logan, UT). All tissue culture media and reagents were purchased from Life Technologies (Grand Island, NY). All other chemicals were purchased from Sigma (St. Louis, MO).
Peptide synthesis
Nine-mer peptides (Table I)
possessing the known binding motifs for HLA-A2 or HLA-B51 (24) were
identified in the mature Ct serovar-E MOMP sequence (15).
Chlamydia pneumoniae (Cpn) MOMP peptides (see Fig. 4) were
made according to the published amino acid sequences (25).
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90% according to reverse phase HPLC analysis. Identities
of peptides were confirmed by amino acid analysis and matrix-assisted
laser desorption/ionization mass spectrometry. Lyophilized peptides
were dissolved in DMSO at 20 mM, aliquotted, and stored at -80°C.
Peptides were diluted to 4 mM with serum-free culture medium and used
at desired final concentrations. Identification of HLA-A2-binding peptides
Ct MOMP peptides that can bind to HLA-A2 molecules were identified by their ability to increase the expression of HLA-A2 on the surface of TAP-deficient mutant cell line LCL.174 (20) as previously described (26). Briefly, LCL.174 was plated in a round-bottom, 96-well plate at 2 x 105 cells/well in 200 µl of 2/1 RPMI together with 50 µM of peptide and incubated overnight at 37°C. The cells were then stained with HLA-A2-specific mAb BB7.2 (American Type Culture Collection (ATCC), Manassas, VA), followed by FITC-conjugated goat anti-mouse IgG. Fluorescence intensity was analyzed by flow cytometry. Influenza virus matrix M1 protein peptide, FluMP58, known as an HLA-A2-restricted CTL epitope (27), was used as a positive control. Hepatitis B virus envelope Ag peptide, HBenvAg125, does not bind to HLA-A2 (28) and was used as a negative control.
In vitro stimulation of peptide-specific CTLs
PBMCs were prepared from 
30 ml of heparinized peripheral
blood obtained from human subjects by centrifugation over
Ficoll-Hypaque (Sigma) (29). CD8+ T cells were positively
selected from freshly isolated PBMCs, or sometimes from PBMCs frozen in
liquid N2, using anti-CD8 magnetic microbeads according
to the manufacturer’s instructions (Milteny Biotec, Auburn, CA).
Negatively selected cells were resuspended in serum-free DMEM and
plated in 500-µl aliquots into 48-well plates at 3 x
106 cells/well. After 2 h at 37°C, 5%
CO2, nonadherent cells were removed by repeated washing,
and adherent monocytes were incubated for 4 h with 50 µM peptide
and 5 µg/ml human ß2-microglobulin (Sigma). After being
washed with serum-free DMEM, each well received 1.5 x
106 CD8+ cells (>90% pure by flow cytometry)
in 500 µl of DMEM containing 10% human serum supplemented with
rhIL-7 (0.5 ng/ml; R&D Systems, Minneapolis, MN) (30, 31). rhIL-2 was
given at 25 U/ml after 2 days and twice a week thereafter by replacing
half of the culture medium. On day 10, CTL cultures were restimulated
at a responder to stimulator ratio of 5 with irradiated (5000 rad),
autologous LCLs incubated with 20 µM peptide. Alternatively, LCL.174
incubated with 50 µM peptide was used to restimulate CTL cultures
obtained from HLA-A2+ subjects. CTL assays were performed a
week after restimulation as described below. Peptide-stimulated CTLs
(indicated as CTL-peptide' in Figures) could be frozen after initial
characterization in medium that consisted of 30% human serum, 10%
DMSO, and 60% DMEM, and then thawed and restimulated for further
analysis.
Influenza virus matrix M1 protein peptide, FluMP58, was used as a positive control for in vitro stimulation of peptide-specific CTLs.
CTL assays
Cytolytic activity of peptide-stimulated CTL cultures was
assessed in [3H]thymidine release assays (32, 33) or in
[3H]uridine release assays (34). Target LCLs (3 x
105 cells/ml) were labeled overnight with
[3H]thymidine (2.0 Ci/mmol; New England Nuclear, Boston,
MA) or with [3H]uridine (2530 Ci/mmol; Amersham,
Arlington Heights, IL) at 10 µCi/ml, while in growth phase. After
1 h incubation with or without 10 µM peptide, the target cells
were washed three times to remove excess peptides. Target cells (5,000)
were then plated in round-bottom wells of 96-well plates along with
different numbers of CTLs in a total volume of 200 µl of 2/1 RPMI to
give desired E:T ratios. After 6 h at 37°C, 100 µl of
supernatant was harvested from each well, air-dried on glass fiber
filters, and counted in a liquid scintillation counter. Spontaneous
release was determined for target cells in medium alone. Maximal
labeling was determined from the equivalent wells by taking 100 µl
after thoroughly mixing the contents of the wells. Maximal labeling was
30005000 cpm for [3H]thymidine-labeled LCLs, and
60008000 cpm for [3H]uridine-labeled LCLs. Spontaneous
release was typically 510% of maximal labeling.
When ME180 cells were used as targets, adherent cells were incubated
overnight with radioactive labels as described above. Cells were then
trypsinized and incubated for 1 h with or without 10 µM peptide
before being plated together with CTLs. Maximal labeling was
5,0006,000 cpm for [3H]thymidine-labeled ME180 cells,
and 10,000 cpm for [3H]uridine-labeled ME180 cells.
Spontaneous release was usually 510% of maximal labeling.
Percent lysis was calculated as 100 x (release in the presence of CTLs - spontaneous release)/(maximal labeling - spontaneous release). Percent peptide-specific lysis was defined as % lysis in the presence of peptide - % lysis in the absence of peptide. The degree of peptide-dependent lysis of targets by CTLs increased with increasing E:T ratio (data not shown); an E:T ratio of 50 was adopted for most of our CTL assays. Data presented are the means of duplicate determinations.
Preparation of C. trachomatis-infected target cells for CTL assays
C. trachomatis serovar E/UW-5 genital strain was obtained from Dr. Gerald Byrne (University of Wisconsin, Madison, WI), grown in HeLa cells, and purified by density gradient centrifugation as previously described (35). The purified elementary bodies (EBs) were resuspended in SPG (sucrose-phosphate-glutamic acid buffer) (36) and stored at -80°C until use. Inclusion forming units of purified organisms were assayed on HeLa cells by indirect fluorescent-Ab staining as previously described (36).
ME180 and ME180[A2] cells were maintained without antibiotics until they were inoculated with C. trachomatis. Cells were seeded at 3 x 105 cells/well in a 12-well plate (Costar, New York, NY) together with 10 µCi/ml [3H]uridine. 24 h later, the subconfluent monolayers were washed twice with PBS and inoculated with live, heat-killed or UV-killed EBs at a multiplicity of infection of 10 (i.e., 10 inclusion forming units per cell) in 500 µl of SPG for 2 h at 37°C. Heat-killed EBs were prepared by incubating them in a 56°C water bath for 30 min, and UV light-inactivated EBs by exposing the organisms to a 30 W UV source (10 erg/s, General Electric, Fairfield, CT) at a distance of 10 cm for 30 min. Live EBs and killed EBs derived from them were used at equal dilutions. Inoculum was removed by washing, and infected cells were cultured for 24 h or for 48 h in antibiotic-free RPMI 1640 containing 10% FCS before use in CTL assays. Uninfected cells were treated with SPG alone, incubated for the same amount of time, and used as a control in CTL assays.
CTL assays were performed with 5000 infected cells per well at an E:T
ratio of 50 as described above. Spontaneous release from infected cells
was 10% of maximum labeling at 24 h postinfection and
1520% at 48 h postinfection. At 72 h postinfection,
6070% of infected cells spontaneously lysed and was excluded from
our experiments. Essentially all cells were lysed by 96 h after
infection with live EBs. Spontaneous release from cells incubated
with killed organisms remained similar (10% of maximal labeling) up
to 96 h postinoculation.
Statistical analysis
Data obtained from infected HLA-A2+ subjects (Table II) and uninfected HLA-A2+
control subjects (Table IV) were compared by Fisher’s exact
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We chose to examine Ct MOMP-specific CTL responses restricted by HLA-A2 and HLA-B51, which are among the most common HLA alleles found in various ethnic populations (37). Out of 16 Ct-infected subjects who enrolled in our research program, nine were typed to be HLA-A2+, and three were typed to be HLA-B51+. Tables II and III summarize relevant information about the HLA-A2+ and HLA-B51+ subjects who participated in the present study.
Synthetic peptides derived from MOMP of Ct serovar E were used to
stimulate outgrowth of CD8+ T cells obtained from
peripheral blood of Ct-infected human subjects. Serovar E was chosen
for the study, because it is one of the most common causes of human
genital tract infections. Fourteen MOMP peptides possessing a proposed
HLA-A2-binding motif (Table I
) were tested for their ability to bind to
HLA-A2 molecules as described in Materials and Methods.
Peptides MOMP155, MOMP258, and MOMP260 were identified as binders (data
not shown) and were subsequently used for in vitro stimulation of
CD8+ T cells obtained from HLA-A2+ subjects.
Four peptides possessing an HLA-B51-binding motif were used in in vitro
stimulation of CD8+ T cells from HLA-B51+
subjects without performing preliminary peptide binding assays.
With the working protocol described in Materials and
Methods, we routinely detected influenza virus FluMP58-specific
CTLs in almost all of our HLA-A2+-infected subjects (data
not shown) and uninfected control subjects (Table IV). Moreover, MOMP peptide-specific CTL
populations could be obtained from all of our Ct-infected subjects
with relative ease (Tables II and III; see below). Initial CTL
assays performed in 1997 produced some relatively low peptide-specific
lysis values that probably resulted from culturing CTLs in RPMI-based
medium. DMEM, used for all CTL cultures in 1998, supported growth of
rapidly expanding CTLs better than did RPMI and allowed us to detect
much greater and unquestionable MOMP peptide-specific CTL activity from
the same subjects. The data presented in all Figures and in Tables IV
and V were obtained with CTLs cultured in
DMEM.
Characterization of HLA class I-restricted, MOMP peptide-specific CD8+ CTLs obtained from humans infected with C. trachomatis
Three HLA-A2-binding MOMP peptides and four MOMP peptides
containing an HLA-B51-binding motif (Table I) were pooled or used
individually to stimulate the outgrowth of CD8+ T cells
obtained from peripheral blood of Ct-infected subjects. Cytolytic
activity of each CTL culture was assessed in
[3H]thymidine release assays using peptide-pulsed,
HLA-matched LCLs as targets. Representative CTL assays performed with
HLA-A2+ and HLA-B51+ individuals are summarized
in Tables II and III, respectively.
Notably, all of our infected subjects had CTL populations specific for
one or more MOMP peptides tested. As shown in Table II, all nine
HLA-A2+ subjects had CTLs specific for MOMP260, and seven
of them had CTLs specific for MOMP258. Additionally, MOMP155 was
recognized by one HLA-A2+ subject (STD215). Furthermore,
all three HLA-B51+ subjects tested had CTLs that recognized
MOMP99, one of the four peptides possessing an HLA-B51-binding motif
(Table III). Two of these individuals also had CTLs specific for
MOMP345. As shown in Tables II and III, many of our infected subjects
were tested more than once over a period of more than 1 yr, and the
MOMP peptide-specific CTLs were reproducibly detected in their
peripheral blood. In case of STD75 (Table II), we could still detect
MOMP peptide-specific CTLs almost 2 yr after infection. During the
interval of our study, none of our STD subjects acquired symptomatic
infections with Ct since their first clinic visits shown in Tables II
and III.
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illustrates detailed
characterization of CTLs stimulated with HLA-A2-binding MOMP peptides
with regard to their specificity of target cell recognition. The CTLs
lysed HLA-A2+ target LCLs only when the peptide used for in
vitro stimulation was present. For example, CD8+ T cells
stimulated with MOMP155 specifically recognized MOMP155 pulsed onto
HLA-A2+ LCL.45 targets, and the same targets incubated with
other HLA-A2-binding peptides were not lysed (Fig. 2A). A
similar pattern of peptide-specific target cell recognition was
observed with CTLs stimulated with MOMP258 or with MOMP260 (Fig. 2, B and C).
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) as
targets in CTL assays. As shown in Fig. 2B, the lysis of
LCL.53 by CTLs stimulated with MOMP258 eliminates the possibility of
peptide presentation by HLA-B51, because LCL.53 is HLA-A2+
and HLA-B-null due to a homozygous deletion at the HLA-B locus. Thus,
HLA-A2 is identified as the probable restriction element; this was
confirmed by the failure of the same CTLs to lyse mutant LCL.144, which
is HLA-B51+ but HLA-A-null due to a homozygous deletion of
the HLA-A locus. HLA-C is eliminated as a restriction element for
MOMP258, because the same HLA-C allele is expressed in LCL.53 and
LCL.144. The results shown in Fig. 2 demonstrate that all three
peptides, MOMP155, MOMP258 and MOMP260, are presented to the CTLs in
association with HLA-A2. Notably, peptides MOMP258 and MOMP260,
overlapping by seven amino acids, contain two distinct CTL epitopes, as
CTLs specific for one peptide did not recognize the other peptide.
The CTLs stimulated with peptides MOMP99 and MOMP345, containing an
HLA-B51-binding motif, were also examined for the specificity of their
target cell recognition (Fig. 3). The
CTLs lysed HLA-B51+ target cells only when the peptide used
for in vitro stimulation was present. Target cell recognition required
peptide presentation by HLA-B51; LCL.144 (HLA-A-null,
HLA-B51+), but not LCL.53 (HLA-A2+,
HLA-B-null), was able to present MOMP99 and MOMP345 to the CTLs.
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). MOMP155 spans VS2 and CS3 of serovar E-MOMP, where the amino acid
sequence in the VS2 portion (SLDQS) varies significantly among
different Ct serovars and other chlamydial species. Although further
studies are needed for confirmation, MOMP155 is likely a Ct serovar
E-specific epitope. MOMP peptide-specific CTLs indeed resulted from genital tract infections with C. trachomatis
To determine whether the MOMP peptide-specific CTLs described above were indeed elicited by genital tract infections with Ct, six HLA-A2+ uninfected subjects were recruited, and their CD8+ T cells were stimulated in vitro with peptides FluMP58, MOMP258, and MOMP260, following the same protocol used for infected subjects. The cytolytic activity of CTL cultures was assessed in [3H]thymidine release and [3H]uridine release assays performed in parallel, using HLA-A2+ LCL.53 as targets.
As shown in Table IV, five of six
uninfected control subjects had no detectable CTL activity against
peptide MOMP258 or MOMP260, while one (subject #2) of them had CTL
populations specific for both peptides. The basic CTL stimulation
protocol was functional in this experiment, as we detected
FluMP58-specific CTLs in all six control subjects. Unusually, CTLs of
subject #5 that were stimulated with MOMP258 and MOMP260 showed
FluMP58-specific cytolytic activity. We found out that this subject had
acquired influenza infection shortly before a blood sample was taken
for this experiment. Culturing CD8+ T cells in
rhIL-2-supplemented medium might have resulted in expansion of
FluMP58-specific CTLs that had already been activated in vivo.
The difference in frequency of MOMP peptide-specific CTL responses
between infected (9/9; Table II) and uninfected (1/6; Table IV)
HLA-A2+ subjects was significant (p
= 0.002 at the significance level of 0.01, according to Fisher’s exact
test). Therefore, the MOMP peptide-specific CTLs detected in infected
subjects most likely descended from CTLs generated in vivo as a result
of genital tract infections with Ct. In a separate study, an additional
control subject (HLA-A2+ and HLA-B51+) was
tested with HLA-B51-restricted CTL epitopes, MOMP99 and MOMP345, as
well as with HLA-A2-restricted CTL epitopes, FluMP58, MOMP258, and
MOMP260. None of these peptides, except for FluMP58, stimulated CTLs in
this subject (data not shown).
The CTLs obtained from Ct-infected mice in previous studies recognized cross-reactive, probably genus-specific epitopes (39, 40). The possibility of cross-reacting CTLs pertains especially to MOMP peptide epitopes, because the gene encoding MOMP is nearly 70% identical among different chlamydial species (38). In addition, the four CTL epitopes located in CSs of MOMP were recognized by almost all of our Ct-infected subjects, regardless of infecting serovars. Therefore, we examined whether these four Ct MOMP peptides would cross-react with corresponding MOMP peptides derived from a related chlamydial species, C. pneumoniae (Cpn), to which a majority of adults have been exposed (41).
Fig. 4 shows that CTLs stimulated with Ct
MOMP peptides specifically recognized target cells only in the presence
of the Ct MOMP peptides used for stimulation but did not recognize the
same targets incubated with other peptides, including the Cpn MOMP
peptides. Thus, it is unlikely that CTLs recognizing Ct MOMP peptides
are cross-reacting CTLs that were primed during infections with related
chlamydial species. Instead, the data shown in Fig. 4 suggest that the
CTL epitopes we identified in CSs of Ct MOMP are Ct species specific
and further verify that the CTLs specific for these epitopes must have
been elicited by exposure to Ct during genital tract infections.
ME180 human cervical epithelial cells are susceptible to lysis by C. trachomatis MOMP-peptide specific CTLs
We next investigated whether human genital tract epithelial cells
presenting appropriate MOMP peptide epitopes would be susceptible to
lysis by MOMP peptide-specific CTLs. The CTLs specific for MOMP
peptides were obtained from HLA-A2+ and
HLA-B51+ subjects identified in Table V, and their
cytolytic activity was first assessed using LCLs as targets in
[3H]thymidine release assays (data not shown). ME180
human cervical epithelial cell and its transferent cells expressing an
HLA-A2 (ME180[A2]) or a HLA-B51 (ME180[B51]) transgene were
subsequently used as targets. Interestingly, the lysis of ME180 cells
by the CTLs was not detectable with [3H]thymidine release
(data not shown) but was detectable with [3H]uridine
release, as summarized in Table V.
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). As expected, ME180 and
ME180[A1] cells were not recognized by these CTLs. Again, the lack of
lysis of ME180[A2] cells by HLA-B51-restricted and MOMP99- or
MOMP345-specific CTLs verifies that epitopes MOMP155, MOMP258, and
MOMP260 are presented by HLA-A2. Similarly, CTLs stimulated with an
HLA-B51-restricted epitope, MOMP99 or MOMP345, specifically recognized
the stimulating peptide when it was presented by ME180[B51] cells,
but not by ME180 or ME180[A2] cells (Table V).
It is interesting and of potential methodological importance that
mechanisms of cytotoxicity caused by CTLs in cervical epithelial cells
and B cells are different. CTLs appear to induce the death of cervical
epithelial cells via cell membrane disintegration (detectable by
[3H]uridine release) without causing prelytic DNA
fragmentation (detectable by [3H]thymidine release). In
contrast, B cells are susceptible to both cell death pathways; the
lysis of target LCLs by FluMP58-specific CTLs detected by
[3H]uridine release was comparable to that assessed by
[3H]thymidine release in 6 h CTL assays (Table IV).
C. trachomatis-infected ME180 human cervical epithelial cells are killed by MOMP peptide-specific CTLs
To examine whether the MOMP CTL epitopes are naturally processed and presented by Ct-infected cells and capable of targeting the infected cells for lysis by the MOMP peptide-specific CTLs, ME180 cells infected with Ct serovar E were prepared as described in Materials and Methods and used as targets in CTL assays.
The growth of Ct in ME180 cells was monitored microscopically; all
cells of infected cultures were lysed after approximately 96 h.
Spontaneous lysis of many infected cells during the CTL assays was
detected at 72 h postinfection; thus CTL assays were performed
only at 24 and 48 h postinfection. HLA-A2-restricted CTLs specific
for peptide MOMP258 or MOMP260 were prepared from five different STD
subjects and tested in preliminary CTL assays to confirm their peptide-
and HLA allele-specificity of target cell recognition (data not shown).
The CTLs were then tested for their ability to lyse Ct-infected ME180
cells. Similar results were obtained with all five subjects, and
representative data are shown in Fig. 5.
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A) and MOMP260-specific CTLs
(Fig. 5B) killed ME180 cells expressing an HLA-A2 transgene,
ME180[A2] cells, infected with Ct. Killing by the CTLs was detectable
at 24 h postinfection and was significantly higher at 48 h
postinfection. Uninfected ME180[A2] cells were not lysed by the CTLs
unless incubated with exogenous peptide epitopes before the CTL assays.
The lack of lysis of infected ME180 parent cells indicates that killing
of infected targets by the CTLs stimulated with MOMP258 or MOMP260
depends on the presentation of a specific peptide epitope in
association with HLA-A2. Collectively, these results demonstrate that
both peptide epitopes, MOMP258 and MOMP260, can be processed and
presented by Ct-infected cells, thereby targeting the infected cells
for killing by the CTLs specific for these epitopes.
Furthermore, as also shown in Fig. 5, ME180[A2] cells were
susceptible to lysis by MOMP peptide-specific CTLs when infected with
viable Ct but not when inoculated with equal numbers of heat-killed or
UV-inactivated organisms. Therefore, the sensitivity of genital tract
epithelial cells to lysis by the CTLs requires a productive infection
of host epithelial cells and is not due to the presentation
of nonviable antigenic material present in the inoculum.
Studies with HLA-B51-restricted MOMP CTL epitopes could not be performed at this time because our HLA-B51+ subjects graduated from the university and were no longer available to participate in our experiments with Ct-infected target cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is highly likely that the MOMP peptide-specific CTLs derived from
infected subjects had indeed been primed in vivo as a result of recent
genital tract infections with Ct. Combining all the HLA-A2+
and HLA-B51+ subjects tested, we detected one or more MOMP
peptide-specific CTL populations in all 10 infected subjects (100%
positive; Tables II and III). In contrast, such CTLs were detectable
only in one of seven uninfected subjects tested (14% positive; Table IV and accompanying text). The possibility of in vitro activation of
naive, primary CTLs present in most people, infected or not, is ruled
out because, with just one exception, the MOMP peptide-specific CTL
stimulation predominantly occurred with subjects known to have been
infected. In addition, the lack of cross-reactivity with peptides
derived from Cpn MOMP indicates that the CTL epitopes we identified in
Ct MOMP are species specific. Collectively, the data presented in this
report suggest that the MOMP peptide-specific CTLs found in our STD
subjects must have resulted from the exposure to Ct during genital
tract infections. The CTLs found in one of our control subjects
(subject #2; Table IV) probably reflect the asymptomatic nature of many
genital tract infections caused by Ct, which remains to be confirmed by
further examination.
More conclusive evidence for the induction of MOMP-specific CTLs as a result of genital tract infections would be provided by studies with truly naive blood samples; e.g., cord blood of newborns and blood samples from specific groups of adults, such as nuns, who are unlikely to have been exposed to Ct. Considering the high frequency of asymptomatic Ct infections, our control subjects were recruited rather crudely from the sexually active adult population and were presumed to be uninfected based on the lack of previous history of symptomatic Ct infections. Nonetheless, the prevalent absence of MOMP peptide-specific CTL activity in these control subjects was clearly distinguishable from their ubiquitous presence in STD subjects who had recent Ct infections. In addition, it is noteworthy that asymptomatic Ct infection might be identified in a seemingly uninfected subject, as was in our subject 2, by monitoring the MOMP peptide-specific CTL activity in vitro.
The knowledge of HLA class I-restricted CTL epitopes in MOMP and the ability to obtain MOMP-specific human CTLs with relative ease is a critical first step toward establishing a system for investigating some intriguing aspects of antichlamydial CTL responses in humans who have genital tract infections. In most previous studies with mice, Ct-specific CTLs were obtained from mice infected i.p. or i.v., rather than in the genital tract. However, the impact of such artificial routes of inoculation on interpretations related to genital tract infections may be important in light of the recent findings that suggested differential processing and presentation of a given Ag by various types of APCs (42, 43, 44). APCs utilized in studies with mice might have resulted in immunological events that are distinct from those triggered by APCs present in human genital tract.
Moreover, cytolytic activities of murine CTLs were assessed using infected mouse fibroblast L cells as targets in CTL assays. Mouse fibroblast cells and human genital tract epithelial cells are nonprofessional phagocytic cells that can support the growth of genital strains of Ct, but the two cell types may differ in certain respects with regard to the mechanism of Ag processing and presentation. Besides, fibroblasts are not known to be natural host cells for Ct during infections of either humans or mice. Our demonstration that Ct-infected ME180 human cervical epithelial cells are lysed by MOMP-specific CTLs isolated from humans genitally infected with the organism will help design an in vitro model that is physiologically and immunologically relevant to studies of human Ct infections.
The role of HLA class I-restricted CD8+ CTL responses in
human chlamydial infections is poorly understood but has largely been
implicated in pathology because of an association of certain HLA class
I alleles with long-term inflammatory diseases caused by Ct (45). In
contrast, a potentially important role of MHC class I-restricted
CD8+ CTLs for in vivo resolution of chlamydial infection
and for immune protection has been demonstrated in mice: Ct-specific
CTLs obtained from infected mice lysed infected target cells in vitro
(39, 40, 46, 47, 48), and adoptive transfer of the CTLs protected against a
challenge (48). In addition, ß2-microglobulin-deficient
mice defective in CD8+ T cell function were unable to
resolve chlamydial infection as efficiently as wild-type mice and
showed greater mortality (49). However, none of these studies with mice
identified the Ct Ags recognized by protective CTLs. In contrast, we
identified five CTL epitopes in Ct MOMP and demonstrated that the
MOMP-specific CTLs could lyse infected ME180 cells relatively early in
the chlamydial growth cycle (at 24 and 48 h postinfection). This
suggests that the CTLs might be capable of killing infected cells in
vivo while most organisms are replicating as reticulate bodies, unable
to infect neighboring cells. We also observed that the lysis by CTLs
occurred when host epithelial cells were infected with viable organisms
but not when inoculated with equal numbers of killed organisms (Fig. 5). This implies that the CTLs are capable of selectively and
specifically destroying epithelial cells productively infected with Ct
but not the bystander cells that might have taken up nonviable
antigenic material. Therefore, direct lysis of infected cells early in
the chlamydial growth cycle could provide protection against Ct by
clearing infected cells from the genital tract and limiting the
replication of Ct. On the other hand, since MOMP is constitutively
expressed throughout the chlamydial growth cycle (50), the
MOMP-specific CTLs might facilitate the spread of infectious organisms
if the lysis of infected cells is delayed for some reason until late in
the chlamydial growth cycle after most organisms have converted into
infectious forms.
Further investigations are needed to define mechanisms by which CTLs
might protect against Ct and to examine whether CTLs are in part
responsible for immunopathology. Notably, a majority of lymphocytes
infiltrating into the site of Ct-caused salpingitis in rhesus macaques
were CD8+ T cells that produced perforin and IFN-
mRNA
(51). This is in stark contrast to the observation made in mice that
recruitment of CD4+ T cells and MHC class II APCs to the
genital tract is associated with protection against Ct infection (52, 53). It will be interesting and important to further study the
functions of infiltrating CD8+ T cells at inflamed sites of
Ct infection and examine whether the findings made with a monkey model
better reflect the events of immune responses occurring in human
genital tract infection. Taking advantage of the knowledge of MOMP CTL
epitopes, we can now attempt to look for MOMP peptide-specific CTLs at
the inflamed sites in the genital tract of Ct-infected humans. In
addition, whether such CTLs are indeed capable of killing normal,
primary genital tract epithelial cells infected with Ct can also be
examined. Initially, it will be difficult to carry out such
characterizations in humans. However, in recent years, several groups
have reported that various species of nonhuman primate, such as rhesus
macaques described above, are susceptible to experimental chlamydial
infection of the genital tract and develop chlamydial diseases similar
to those in humans (54, 55, 56, 57). Studies in such monkey models may provide
an important clue and basis for future investigations of the protective
and/or pathological role of CTLs in human chlamydial infections.
We presented convincing evidence that MOMP-specific CTLs are primed in vivo upon genital tract infection with Ct and that such CTLs are capable of killing Ct-infected epithelial cells. How does MOMP gain access to HLA class I pathways of Ag processing and presentation in infected cells and in APCs such as macrophages or dendritic cells that are most likely responsible for initiating primary CTL response?
After entering a host epithelial cell, Ct multiplies while sequestered within an inclusion body throughout its life cycle. No report to this date has localized MOMP in the cytosol of infected cells, and it is not obvious how MOMP might gain access to endogenous MHC class I pathway of Ag processing and presentation. Alternatively to the endogenous pathway, evidence suggests that phagocytosed intact organisms might be processed and presented to CTLs via an exogenous MHC class I pathway (58, 59). It is not known whether genital tract epithelial cells are capable of presenting phagocytosed Ags via an exogenous pathway. In contrast, professional phagocytic cells, such as dendritic cells and macrophages, have been shown to efficiently prime CTLs by the exogenous MHC class I pathway and to play a major role in the initiation of CD8+ CTL responses to certain intracellular bacteria (60, 61, 62). A recent finding that dendritic cells, upon internalization of Ct, stimulated proliferation of Ct-specific CD4+ T cells (63) suggests a potentially important role for such APCs in initiating Ct-specific T cell responses in vivo. Genital strains of Ct are not known to replicate in dendritic cells (63) or in macrophages (64). It will be interesting to examine whether such professional APCs can process phagocytosed Ct organisms by an exogenous HLA class I pathway for presentation to CD8+ CTLs. The MOMP CTL epitopes we report here are valuable tools for dissecting HLA class I pathways of processing and presentation of Ct MOMP in infected cells as well as in professional phagocytic APCs. These studies are under way in our laboratory.
Other aspects of chlamydial research may also benefit from the knowledge of MOMP CTL epitopes. For example, CTL responses to the MOMP epitopes can be assessed to evaluate correspondences between studies of Ct-specific immune responses in animals and in humans. In addition, the induction of MOMP peptide-specific CTLs can be monitored to evaluate the efficacy of a vaccine in inducing acquired cell-mediated immunity to Ct. Cross-reactive CTLs, as demonstrated in mice, could contribute to perceived immune responses to Ct and might confuse interpretations of monitored results in certain vaccine trials. In contrast, the CTLs that recognize Ct species-specific epitopes in MOMP define parameters that can be assessed as potential correlates of protection in controlled vaccine trials with a Ct MOMP-based vaccine. In this respect, it is noteworthy that four out of the five MOMP CTL epitopes we identified are Ct species specific and located in CSs of MOMP, where sequence variation is highly limited. Furthermore, both of the HLA-A2-restricted CTL epitopes we described, MOMP258–266 and MOMP260–268, largely overlap with MOMP peptide 249–265, in which we previously identified at least six different Th cell epitopes recognized by 83% of infected subjects studied (15). The close association of Th and CTL epitopes that are commonly recognized by infected humans may make peptide MOMP249–268, which includes these epitopes, an especially interesting candidate vaccine component. In addition, HLA-A2 and HLA-B51, which present the four CS CTL epitopes, are among the most common HLA allotypes in the general population (37). Moreover, our experiments with Ct-infected epithelial cells suggest that MOMP-specific CTLs, if elicited locally at the genital mucosa by immunization, might help efficiently resolve Ct infection by limiting replication of infectious organisms. Our present study with MOMP-specific CTLs raises the prospect of using the CTL epitopes in protective immunization of a large number of individuals with a subunit vaccine against STDs caused by Ct.
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Acknowledgments |
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Footnotes |
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2 This is publication No. 3521 from the Laboratory of Genetics of the University of Wisconsin-Madison.
3 Address correspondence and reprint requests to Seon-Kyeong Kim or Dr. Robert DeMars, Laboratory of Genetics, University of Wisconsin, 445 Henry Mall, Madison, WI 53706. E-mail addresses:
4 Abbreviations used in this paper: Ct, Chlamydia trachomatis; STD, sexually transmitted disease; MOMP, major outer membrane protein; LCL, B lymphoblastoid cell line; rhIL-2, recombinant human IL-2; CS, constant segment; VS, variable segment; Cpn, Chlamydia pneumoniae; EB, elementary body; SPG, sucrose-phosphate-glutamic acid buffer.
5 L. Ortiz, D. Watkins, M. Angevine, and R. DeMars. T cell epitopes in variable segments of Chlamydia trachomatis major outer membrane protein (MOMP) elicit serovar-specific immune responses by infected humans. Submitted for publication.
Received for publication November 16, 1998. Accepted for publication March 9, 1999.
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