Induction of HLA Class I-Restricted CD8⁺ CTLs Specific for the Major Outer Membrane Protein of Chlamydia trachomatis in Human Genital Tract Infections

Human subjects

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 Similarly, HLA-B-null mutant LCL.53 (21) was derived from LCL.19 as a result of intragenic deletion at the locus (our unpublished results).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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FIGURE 1.

Origin of HLA class I Ag loss mutants used as targets in CTL assays. LCL 721 was derived from a normal human female (19). 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). Each mutant shown has a large deletion of the short arm of one chromosome 6 and has only one copy of the MHC class I region. LCL.144 was produced from LCL.45 and is HLA-A-null due to a homozygous deletion at the locus (20). HLA-B-null LCL.53 (21) was derived from LCL.19 as a result of intragenic deletion at the locus (our unpublished results). Typing for HLA-C was not definitive, but several serological tests support the allelic assignments shown.

ME180 human epithelioid cervical carcinoma cells (HLA-A1, -A32, -B8, and -B44 according to PCR-based typing at the Tissue Typing Laboratory, Madison, WI) (22) were used as a model for female genital tract epithelial cells. ME180 cells were cultured in MEM containing 10% FCS, 100 μM nonessential amino acids, 25 mM HEPES, 44 mM NaHCO₃, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. ME180 cells expressing an HLA-A1 (ME180[A1]), HLA-A2 (ME180[A2]), or HLA-B51 transgene (ME180[B51]) were prepared by introducing into ME180 cells the RSV.5neo vector carrying the genomic HLA-A*0101 (23), HLA-A*0201 (23), or HLA-B51*01 gene (our unpublished results). Stable transferent cells were selected for resistance to G-418 sulfate (500 μg/ml).

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).

Peptides were synthesized at the University of Wisconsin Biotechnology Center (Madison, WI) by F-moc chemistry. Purity of crude peptides was routinely ≥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 × 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 × 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 β₂-microglobulin (Sigma). After being washed with serum-free DMEM, each well received 1.5 × 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 × 10⁵ cells/ml) were labeled overnight with [³H]thymidine (2.0 Ci/mmol; New England Nuclear, Boston, MA) or with [³H]uridine (25∼30 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 3000∼5000 cpm for [³H]thymidine-labeled LCLs, and 6000∼8000 cpm for [³H]uridine-labeled LCLs. Spontaneous release was typically 5∼10% 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 5,000∼6,000 cpm for [³H]thymidine-labeled ME180 cells, and ∼10,000 cpm for [³H]uridine-labeled ME180 cells. Spontaneous release was usually 5∼10% of maximal labeling.

Percent lysis was calculated as 100 × (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 × 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 15∼20% at 48 h postinfection. At 72 h postinfection, 60∼70% 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.

Protocol for in vitro stimulation of peptide-specific CTLs

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.

Fig. 2⇓ 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. 2⇓A). 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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FIGURE 2.

HLA-A2-restricted, MOMP peptide-specific CD8⁺ CTLs. CD8⁺ T cells were isolated from the peripheral blood of HLA-A2⁺ STD subjects, and outgrowth was stimulated with, individually, peptide MOMP155, MOMP258, or MOMP260. After restimulation with peptide-pulsed LCL.174 (A) or autologous LCLs (B and C), cytolytic activity of CTL cultures was analyzed in [³H]thymidine release assays for peptide specificity and HLA allele specificity at E:T ratio of 50 against a panel of target cells shown. LCL.45 is HLA-A2⁺ and B51⁺, LCL.53 is HLA-A2⁺ and B-null, and LCL.144 is HLA-A-null and B51⁺ (see Fig. 1⇑). FluMP58 is a known HLA-A2-restricted CTL epitope and was used as a control for nonspecific killing.

HLA class I allotypes presenting given MOMP peptide epitopes to the CTLs were defined using HLA class I Ag-loss mutant LCLs (Fig. 1⇑) as targets in CTL assays. As shown in Fig. 2⇑B, 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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FIGURE 3.

HLA-B51-restricted, MOMP peptide-specific CD8⁺ CTLs. Outgrowth of CD8⁺ T cells isolated from the peripheral blood of an HLA-B51⁺ STD subject was stimulated with peptides, MOMP99 or MOMP345. After restimulation with peptide-pulsed autologous LCLs, cytolytic activity of CTL cultures was analyzed in [³H]thymidine release assays for peptide specificity and HLA allele specificity at E:T ratio of 50 against a panel of target cells shown. LCL.144 is HLA-A-null and B51⁺, and LCL.53 is HLA-A2⁺ and B-null (see Fig. 1⇑).

Collectively, the above data demonstrate that MOMP155, MOMP258, and MOMP260 are HLA-A2-restricted CTL epitopes, and MOMP99 and MOMP345 are HLA-B51-restricted CTL epitopes. Four of these epitopes are localized in CSs of serovar E MOMP, where their corresponding amino acid sequences are identical among different Ct serovars (38): MOMP258 and MOMP260 in CS4, MOMP99 in CS2, and MOMP345 in CS5. Not surprisingly, these four peptides were recognized by almost all of infected subjects tested, even though some of the responders were infected with serovars other than E (Tables II and III), suggesting that they are probably Ct species-specific epitopes (see below). In contrast, MOMP155 stimulated CTLs in only one (STD215) of four HLA-A2⁺ subjects infected with serovar E, and not in subjects infected with other serovars (Table II⇑). 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.

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FIGURE 4.

C. trachomatis MOMP-derived CTL epitopes are not cross-reactive with C. pneumoniae MOMP peptides. Peptides derived from Cpn MOMP that correspond to the four CTL epitopes in CSs of Ct MOMP were synthesized. Cpn sequence deviations from Ct sequences are underlined. A, HLA-A2-restricted CTLs stimulated with Ct versions of the peptides MOMP258 or MOMP260 were tested for their ability to recognize Ct and Cpn peptides. FluMP58 was used as a control for nonspecific killing. B, HLA-B51-restricted CTLs stimulated with Ct MOMP99 or MOMP345 were examined for cross-reactivity with Cpn peptides. LCL.45 cells (HLA-A2⁺ and B51⁺) pulsed with indicated peptides were used as targets in [³H]thymidine release assays. E:T ratio was 50 for all experiments.

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⇓.

When incubated with appropriate peptides, ME180[A2] cells were clearly susceptible to lysis by HLA-A2-restricted CTLs specific for MOMP155, MOMP258, or MOMP260 (Table V⇑). 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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FIGURE 5.

C. trachomatis MOMP-specific CTLs can kill infected ME180 cervical epithelial cells. HLA-A2-restricted CTLs stimulated with peptide MOMP258 (A) or MOMP260 (B) were tested for their ability to kill Ct-infected cells. ME180[A2] cells were inoculated with live, heat-killed, or UV-killed organisms as described (see Materials and Methods) and used as targets in [³H]uridine release assays 24 h and 48 h later. Uninfected ME180[A2] cells were cultured for the same amount of time and used as targets in the presence or absence of specific peptide epitopes. HLA-A2-negative ME180 cells were uninfected or infected with live Ct and used as targets to demonstrate HLA-A2-specificity of lysis by MOMP258- and MOMP260-specific CTLs. E:T ratio was 50 for all assays.

Both MOMP258-specific (Fig. 5⇑A) and MOMP260-specific CTLs (Fig. 5⇑B) 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.