CD8+ T cells are a key immune component for the eradication of many intracellular pathogens. This study aims to characterize the human CD8+ T cell response to naturally processed chlamydial Ags in individuals exposed to the intracellular pathogen Chlamydia trachomatis. By using C. trachomatis-infected autologous dendritic cells (DCs) as stimulators, Chlamydia-reactive CD8+ T cell responses were detected in all 10 individuals tested. The majority of the Chlamydia-reactive CD8+ T cells were non-MHC class Ia restricted in all three of the individuals tested. From one donor, three non-class Ia-restricted and two class Ia-restricted Chlamydia-specific CD8+ T cells were cloned and characterized further. All five T cell clones secreted IFN-γ in response to autologous DCs infected with viable Chlamydia, but not with DCs pulsed with inactivated chlamydial elementary bodies. MHC class Ia-restricted and non-class Ia-restricted responses were inhibited by DC treatment with a proteasomal inhibitor and an endoplasmic reticulum-Golgi transport inhibitor, suggesting that these T cells recognize a peptide Ag translocated to the host cell cytosol during infection that is processed via the classical class Ia Ag-processing pathway. Even though both restricted and nonrestricted CD8+ T cells produced IFN-γ in response to Chlamydia-infected fibroblasts, only the non-class Ia-restricted cells were lytic for these targets. The class Ia-restricted CTLs, however, were capable of cytolysis as measured by redirected killing. Collectively, these data demonstrate that both class Ia-restricted and non-classically restricted CD8+ T cells are elicited in C. trachomatis-exposed individuals. Their role in host immunity remains to be elucidated.

Chlamydia trachomatis is an obligate intracellular Gram-negative bacterium that is responsible for a wide spectrum of diseases in humans. C. trachomatis infection of the conjunctival epithelium is the most common cause of preventable blindness, with an estimated 146 million cases of active trachoma worldwide (1). C. trachomatis infection of the genital tract is now recognized to be the most common sexually transmitted disease in the United States (2). Approximately 75% of infected women will go undiagnosed and untreated due to a lack of symptoms. Without treatment, chlamydial infection can become chronic, producing a widespread upper genital tract disease that includes pelvic inflammatory disease, tubal scarring, infertility, and ectopic pregnancy. The pathological mechanisms by which C. trachomatis causes conjunctival scarring, fibrosis, or tubal scarring are not well understood.

With intracellular bacterial infections, effective immunity usually involves cell-mediated responses resulting in the recognition and destruction of the infected cells (3). Because C. trachomatis has a tropism for epithelial cells, one could speculate that immune control would involve MHC class Ia-restricted CD8+ T cells. In vivo experiments in murine chlamydial models, however, have suggested that lytic mechanisms are not involved in control of a C. trachomatis infection. Thus, mice with disrupted perforin, granzyme, Fas, or Fas ligand genes control chlamydial infections to the same extent as wild-type mice (for recent reviews, see Refs.4, 5, 6, 7). In adoptive transfer studies, however, mouse CD8+ T cells do contribute to immune protection, and this effect is mediated by IFN-γ production (8, 9).

Little is known about the human CD8+ T cell response to Chlamydia or the Ags recognized by these cells. Recently, several laboratories have begun to identify MHC class Ia-binding chlamydial peptides recognized by human CD8+ T cells. These peptides have been derived from the major outer membrane protein (MOMP) 3 using algorithms that predict binding to MHC class Ia molecules. MOMP-specific CTL responses have been preferentially found in children resolving infection or in adults, without scarring of the conjunctiva in a trachoma-endemic population (10), as well as in subjects who have acquired a genital tract infection (11, 12). Furthermore, these MOMP-specific T cells have been shown to lyse C. trachomatis-infected epithelial cells. One limitation of these studies is that these responses depend on predicted class Ia binding peptides derived from MOMP. As a result, it is unknown whether these peptides are immunodominant, naturally processed CD8+ T cell epitopes or what is the relative contribution of such T cells during natural infection in humans.

Whereas classical CD8+ T cells recognize antigenic peptides in the context of MHC class Ia (HLA A, B, and C), CD8+ T cells that recognize Ag presented by non-MHC class Ia molecules have recently emerged as an important response to intracellular pathogens such as Listeria, Salmonella, and Mycobacterium (13, 14, 15). These molecules have been collectively termed class Ib, and in humans include CD1a, b, c, and d and HLA E, F, and G. The contribution of non-classically restricted CD8+ T cells to chlamydial immunity has not yet been investigated. Vaccines targeting Ag presentation by these molecules would be advantageous because they are nonpolymorphic (unlike MHC class Ia and class II) in the general population. Very little is known about the function of the non-class Ia-restricted CD8+ T cells, and studies looking at their frequency and immune effector function are of great interest.

The present study was conducted to evaluate the relative contributions of both MHC class Ia- and non-class Ia-restricted CD8+ T cells in the human T cell response to C. trachomatis. Chlamydia-infected dendritic cells (DCs) were used as APCs to estimate the overall CD8+ T cell response to a potentially large repertoire of naturally processed chlamydial Ags. Further in vitro analysis of both classically and non-classically restricted CD8+ T cells allowed the comparison of their effector functions and the chlamydial Ag processing pathways. A better understanding of the Chlamydia-specific CD8+ T cell response in humans may aid in the design of vaccines to prevent diseases caused by Chlamydia.

Subjects were recruited from employees at Corixa Corporation, the Public Health-Seattle and King County Sexually Transmitted Disease Clinic at Harborview Medical Center, and the Hall Heath Clinic at the University of Washington. Protocols for venipuncture, apheresis, and skin biopsies were Institutional Review Board approved. HLA typing was performed by the Puget Sound Blood Center. Ligase chain reaction (LCR) detection of Chlamydia in urine, serological analysis by microimmunofluorescence, and past clinical history allowed determination of whether these individuals were currently infected, were previously exposed, and whether clearance required antibiotic treatment. PBMC proliferation and IFN-γ responses to chlamydial elementary bodies were used to determine whether these subjects had Chlamydia-reactive T cells.

Culture medium included Waymouth’s and RPMI 1640 media (Life Technologies, Gaithersburg, MD), FCS (HyClone Laboratories, Logan, UT), gentamicin (Life Technologies), and l-glutamine (Life Technologies). Recombinant human GM-CSF (rhGM-CSF) was generously provided by Amgen Corporation (Seattle, WA), rhIFN-γ was obtained from BD PharMingen (San Diego, CA), and rhIL-4 was produced in Escherichia coli and purified in house. mAbs were generated from hybridoma supernatants from the W6/32, B1.23.2, and BB7.2 cell lines obtained from the American Type Culture Collection (ATCC; Manassas, VA) using the Affi-Gel protein A MAPSII kit (Bio-Rad, Hercules CA) according to the manufacturer’s instructions. mAbs specific for CD1a–d, CD3, CD4, CD8, CD11a, CD14, CD16, CD19, CD40, CD54, CD80, CD86, HLA ABC, HLA-DP, HLA-DQ, HLA-DR, TCRαβ, TCRγδ, and mouse isotype controls (BD PharMingen) were used as direct conjugates to FITC or PE for flow cytometric analysis.

The human HeLa cervical epitheloid cell line (HeLa 229, CCL-2.1), the immortalized human fibroblast line VA-13 (CCL-75.1), and the murine P815 (TIB-64) cell line were purchased from ATCC. Human PBMCs were isolated by Ficoll-Hypaque 1.077 centrifugation (Nycomed, Oslo, Norway) of a leukapheresis product before cryopreservation. Previously frozen PBMCs were used for CD8+ T cell separations and generation of DCs. Primary human fibroblasts were grown from skin biopsies in Waymouth’s medium containing 15% FCS.

C. trachomatis lymphogranuloma venereum type II (L2/434/Bu, ATCC 902B-VR) was propagated in HeLa 229 cell monolayers for the most part as described (16). Briefly, HeLa cell monolayers were infected with C. trachomatis L2 by centrifugation. Elementary bodies (EB) were purified by Hypaque-70 two-step gradient ultracentrifugation (Nycomed) and frozen in sucrose-phosphate-glutamate buffer. Determination of inclusion forming units per milliliter of Chlamydia preparations was done by titration on HeLa cells. Chlamydial preparations were routinely negative for Mycoplasma, as shown by a Mycoplasma-specific PCR. Heat inactivation of Chlamydia was done by incubating Chlamydia twice at 56°C for 30 min followed by flash freezing in liquid nitrogen.

Monocyte-derived DCs were prepared from adherent PBMCs following the method of Romani et al. (17), as previously described (18). Phenotypic analysis of the cells revealed that >95% of the cells were large (forward angle light scatter) and positive for CD1a, CD4, CD11a, CD40, CD54, CD80, CD86, class I, and class II. DCs were negative for CD3, CD14, CD16, and CD19.

DCs and fibroblasts were plated at 2.5 × 105 and 1 × 105 per well in a 24-well plate (Costar, Cambridge, MA) in RPMI 1640 (10% FCS) and Waymouth’s medium (15% FCS), respectively. Cells were incubated overnight before infection. Medium was replaced with 0.3 ml of fresh medium containing the infection inoculum (multiplicity of infection of 10:1 for DCs and 1:1 for fibroblasts). Plates were centrifuged for 1 h at 1400 × g. Medium was aspirated after centrifugation and replaced with 1 ml of fresh medium, and plates were placed at 37°C, 7% CO2 for 24 h.

Chlamydia-specific CD8+ T cell responses were quantified by IFN-γ ELISPOT as described (19). Briefly, CD8+ T cells were purified by TCRγδ and CD4 depletion, followed by a CD8 enrichment using magnetic bead-conjugated Abs according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Purified CD8+ T cells were stimulated with 2 × 104 gamma-irradiated DCs (3500 rad with a 137Cs source; Chlamydia-infected, treated with heat-inactivated elementary bodies or negative control) in a previously coated and blocked 96-well nitrocellulose-backed plate (Multiscreen HA; Millipore, Bedford, MA). Plates were incubated overnight at 37°C, 5% CO2 before developing and quantifying IFN-γ spots.

Chlamydia-reactive CD8+ T cells were cloned by a limiting dilution approach described by Lewinsohn et al. (19). Briefly, CD8+ T cells were purified as described above and plated in a 96-well cloning plate (Nuclon; Nunc, Naperville, IL) at various cell numbers per well (100, 400, 1000, and 3500 per well) in RPMI 1640 (10% human serum). Chlamydia-infected DCs were harvested 24 h postinfection, gamma-irradiated, and plated at 104 cells per well. Feeder cells consisted of autologous gamma-irradiated PBMCs plated at 105 cells per well. Cultures were incubated at 37°C, 5% CO2 and rhIL-2 (10 U/ml) was added on days 1, 4, 7 and 10 after stimulation. After 10–14 days of culture, wells were screened for Chlamydia specificity by ELISPOT split well analysis. All wells were analyzed by seeding 25-μl aliquots of each clone into three separate ELISPOT plates. T cell clones were tested against autologous DCs, autologous C. trachomatis L2-infected DCs, and MHC mismatched heterologous C. trachomatis L2-infected DCs. The original T cell plates were placed at 37°C, 5% CO2. Clones exhibiting specific activity were expanded by splitting each clone into 96 wells of a cloning plate and seeding 1 × 104 gamma-irradiated B-lymphoblastoid cell line (B-LCL)/well, 7.5 × 104 gamma-irradiated PBMCs/well, and 30 ng/ml anti-CD3 Ab (OKT3; Ortho Biotech, Raritan, NJ) in 200 μl/well RPMI 1640 (10% human serum). Cultures were incubated at 37°C, 5% CO2, and 20 U/ml rhIL-2 was added on days 2, 6, and 10. After 14 days of culture, wells were retested for Chlamydia specificity.

T cell activity was measured by IFN-γ secretion. Gamma-irradiated APCs (DCs, VA-13, or fibroblasts) were seeded at 20,000–50,000 cells per well in 96-well flat-bottom plates (Costar). Cells were directly infected in these plates with C. trachomatis L2 (multiplicity of infection of 1:1 for fibroblasts and VA-13 and 10:1 for DCs) after an overnight adherence step. Twenty-four hours after infection, T cells were added to achieve a 1:1 ratio with the APCs, and 2 U/ml rIL-2 were added 24 h poststimulation. After 72 h, supernatants were collected and assayed for IFN-γ by ELISA.

One hour before and during infection of DCs in a 96-well flat-bottom plate, lactacystin (10 μM) or brefeldin A (10 μg/ml) (Sigma-Aldrich, St. Louis, MO) was added to the culture medium. Twenty-four hours after infection, DCs were washed, fixed with 2% paraformaldehyde (Sigma-Aldrich) in PBS, and washed extensively before adding T cells.

One hour before addition of T cells, 100 μg/ml of anti-class I Abs W6/32 (anti-HLA ABC), B1.23.2 (anti-HLA BC), or BB7.2 (anti-HLA A2) were added to the culture medium of APCs. After T cell addition, the final Ab concentration remained at 50 μg/ml throughout the assay.

After infection of fibroblasts in a 24-well plate, 150 μCi of 51Cr (NEN, Boston, MA) per well was added to the medium and placed at 37°C, 7% CO2 overnight. Fibroblasts were washed with PBS before detaching with trypsin. After further washes, fibroblasts were plated in a 96-well round-bottom plate at 5 × 104 cells per well. T cells were added to obtain the E:T ratios described. Plates were incubated at 37°C, 7% CO2 for 18 h before harvesting supernatants with the Skatron harvesting system (Sterling, VA). 51Cr was quantified in the supernatants with a Cobra Quantum gamma counter (Packard Instrument, Meriden, CT). The percentage of specific lysis was determined from the following equation: [(cpm experimental release − cpm medium release)/(cpm total release − cpm medium release)] × 100.

Cytolytic capacity of T cells was tested against the 51Cr-labeled murine mastocytoma FcγR+ P815 cell line by a CD3 mAb-redirected killing assay. Briefly, 10351Cr-labeled P815 were incubated with T cells, in the presence or absence of purified anti-CD3 mAb (0.05 μg/ml). After 4 h, supernatants were harvested for 51Cr release estimation.

HLA-A0101 and -B1501 were cloned from the original donor (D48) into pcDNA3 using the Gateway System (Life Technologies). For transfections, 2 × 104 VA-13 cells were plated in a 96-well flat-bottom plate 24 h before transfection. One hundred nanograms of DNA was mixed with OptiMem (Invitrogen, Carlsbad, CA) and 1 μl of the transfection reagent Fugene-6 (Roche, Basel, Switzerland) and added to plated VA-13 cells. Wells were supplemented with medium 0.5 h after transfection and incubated at 37°C. After 24 h, cells were infected with C. trachomatis as described above. After 48 h, 2–5 × 104 T cells/well were added to the transfected VA-13 cells.

We have demonstrated that human monocyte-derived DCs can be productively infected with C. trachomatis. 4 To determine whether C. trachomatis L2-infected DCs can be used to detect Chlamydia-specific CD8+ T cell responses from exposed individuals, an ELISPOT analysis was undertaken. Donors were considered to have been previously infected if their CD4+ T cells proliferated and secreted IFN-γ in response to heat-inactivated C. trachomatis elementary bodies, but not to Chlamydia pneumoniae elementary bodies (data not shown; P. Probst, S. Engardt, H. Fang, E. Stromberg, Y. Skeiky, J. Maisonneuve, J. Marrazzo, W. Stamm, and A. Bhatia, manuscript in preparation). Current infection was verified by clinical data and by detectable Ab titers. The IFN-γ ELISPOT analysis was performed by stimulating purified CD8+ T cells with C. trachomatis L2-infected autologous DCs. Effector frequencies were estimated by the number of IFN-γ spot-forming cells in response to L2-infected DCs after subtracting the number of spots in response to heat-inactivated L2-pulsed DCs and dividing this number by the total number of input CD8+ T cells. As seen in Table I, Chlamydia-specific CD8+ T cells were detected in all of the 10 individuals tested. The effector cell frequencies were within the reported sensitivity of the IFN-γ ELISPOT analysis (20). As a control, purified CD8+ T cells derived from cord blood were stimulated with autologous C. trachomatis L2-infected DCs. In neither of the two donors tested was a Chlamydia-specific CD8+ T cell response detected, whereas CD8+ T cells did respond to PHA (data not shown). There was no obvious relationship between current or past infection and CD8+ T cell effector frequencies.

Table I.

CD8+ T cell effector frequency analysis by IFN-γ ELISPOT

DonorSexClinical ManifestationaC. trachomatis IgG TiterabChlamydia-Specific CD8+ Effector Cell Frequencyc
CHH093 Past infectiond 1:128 1:410 
CHH035 Past infection 1:8 1:450 
D48 Past infection ND 1:1400 
CHH058 Past infection 1:8 1:2800 
D80 Past infection Seronegative 1:3300 
CT11 Symptomatic, PIDe 1:512 1:700 
CT4 Asymptomatic, LCRf positive 1:1024 1:3400 
CT12 Asymptomatic, LCR positive 1:64 1:4500 
CT7 Symptomatic 1:64 1:5300 
CT6 Symptomatic 1:64 1:9900 
DonorSexClinical ManifestationaC. trachomatis IgG TiterabChlamydia-Specific CD8+ Effector Cell Frequencyc
CHH093 Past infectiond 1:128 1:410 
CHH035 Past infection 1:8 1:450 
D48 Past infection ND 1:1400 
CHH058 Past infection 1:8 1:2800 
D80 Past infection Seronegative 1:3300 
CT11 Symptomatic, PIDe 1:512 1:700 
CT4 Asymptomatic, LCRf positive 1:1024 1:3400 
CT12 Asymptomatic, LCR positive 1:64 1:4500 
CT7 Symptomatic 1:64 1:5300 
CT6 Symptomatic 1:64 1:9900 
a

At the time of leukapheresis.

b

C. trachomatis IgG titers determined by microimmunofluorescence assay.

c

Effector frequencies calculated by number of spots in response to live L2-infected autologous DC − number of spots in response to autologous DC exposed to heat-inactivated EB/total number of purified CD8+ T cells. In all cases, the number of spots against heat-killed EB was 10% or less than the number of spots against viable EB.

d

Past infection determined by positive CD4+ proliferative and IFN-γ responses to C. trachomatis-inactivated elementary bodies as well as by clinical history.

e

Pelvic inflammatory disease.

f

Chlamydial shedding determined by ligase chain reaction (LCR).

g

M.R. Alderson, J. Maisonneuve, K.H. Grabstein, and P. Probst. Differential regulation of inflammatory cytokine secretion by human dendritic cells upon Chlamydia Trachomatic infection. Submitted for publication.

Non-class Ia-restricted CD8+ T cells have been found to be an important component of the T cell response to several intracellular pathogens. To characterize the relative frequencies of MHC class Ia-restricted and non-classically restricted Chlamydia-specific CD8+ T cells, a limiting dilution analysis was undertaken. This assay has been used to estimate the frequencies of classically and non-classically restricted Mycobacterium tuberculosis-specific CD8+ T cell precursors, without the need for repeated in vitro stimulations (19). Each CD8+ T cell clone was assayed for IFN-γ production after stimulation with noninfected autologous DCs, autologous DCs infected with C. trachomatis L2, and MHC-mismatched unrelated DCs infected with C. trachomatis L2 (Fig. 1). If a clone produced IFN-γ only in response to autologous L2-infected DCs, the clone was considered to be MHC class Ia restricted. If the clone secreted IFN-γ in response to both autologous and MHC-mismatched unrelated L2-infected DCs, the clone was considered to be non-class Ia restricted. Finally, clones that produced IFN-γ in response to both infected and noninfected DCs were disregarded. This analysis was undertaken using cells from three individuals (Table II). This assay yields a lower T cell frequency than did the ELISPOT analysis, probably due to the fact that in this assay the T cells have to expand in vitro. Thus, the frequency estimated by ELISPOT analysis is considered to be an effector cell frequency, whereas the frequency estimated by limiting dilution analysis is a precursor cell frequency. In all three individuals tested, the majority of the Chlamydia-reactive CD8+ T cells detected were non-MHC class Ia restricted.

FIGURE 1.

Strategy for the analysis of MHC-restricted and non-MHC-restricted CD8+ T cell responses. A clone is defined as nonspecific if it reacts with all three APCs. A clone is defined as MHC class Ia-restricted if it only responds to autologous infected DCs. A clone is defined as non-classically MHC-restricted if it responds to both autologous and allogeneic infected DCs, but not noninfected DC.

FIGURE 1.

Strategy for the analysis of MHC-restricted and non-MHC-restricted CD8+ T cell responses. A clone is defined as nonspecific if it reacts with all three APCs. A clone is defined as MHC class Ia-restricted if it only responds to autologous infected DCs. A clone is defined as non-classically MHC-restricted if it responds to both autologous and allogeneic infected DCs, but not noninfected DC.

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Table II.

CD8+ T cell precursor frequency analysis by limiting dilution and split well analysis

DonorMHC RestrictionaCD8+ T Cell Precursor Frequencyb
D48 MHC class Ia-restricted CD8+ 1:41,000 
 Non-MHC class Ia-restricted CD8+ 1:9,700 
CT7 MHC class Ia-restricted CD8+ 1:26,000 
 Non-MHC class Ia-restricted CD8+ 1:2,500 
CT6 MHC class Ia-restricted CD8+ 1:618,000 
 Non-MHC class Ia-restricted CD8+ 1:85,000 
DonorMHC RestrictionaCD8+ T Cell Precursor Frequencyb
D48 MHC class Ia-restricted CD8+ 1:41,000 
 Non-MHC class Ia-restricted CD8+ 1:9,700 
CT7 MHC class Ia-restricted CD8+ 1:26,000 
 Non-MHC class Ia-restricted CD8+ 1:2,500 
CT6 MHC class Ia-restricted CD8+ 1:618,000 
 Non-MHC class Ia-restricted CD8+ 1:85,000 
a

MHC restriction determined by responses to infected DC. If a CD8+ T cell clone responded to autologous infected DC only it was considered MHC-class Ia-restricted. If a CD8+ T cell clone responded to both autologous and allogeneic infected DC it was considered non-MHC class Ia-restricted.

b

Precursor frequency calculated by the zero term of the Poisson distribution.

Selected class Ia-restricted and non-class Ia-restricted Chlamydia-reactive CD8+ T cell clones were expanded from one of the individuals (D48, HLA-A0101, -A0201, -B0801, and -B1501) on anti-CD3 for further characterization. Of 30 clones picked, five retained specificity and expanded well. By flow cytometric analysis, all of the clones isolated were cell surface positive for TCRαβ, CD3, and CD8 and negative for TCRγδ and the NK marker CD16 (data not shown). All five clones secreted IFN-γ in response to autologous L2-infected DCs, but not to DCs pulsed with heat-inactivated Chlamydia (Fig. 2). The specific response of two clones (CD8-27 and CD8-30) was blocked by W6/32 Ab (anti-HLA-A, B, and C), whereas only the CD8-27 response was blocked by B1.23.2 Ab (anti-HLA-B and C). A blocking Ab to HLA-A2 failed to block the Chlamydia reactivity of either one of the T cell clones (Fig. 2). Collectively, these results suggested that the CD8-27 clone is most likely restricted by an HLA-B or HLA-C allele, whereas the CD8-30 clone is restricted by HLA-A0101.

FIGURE 2.

MHC restriction analysis of Chlamydia-specific CD8+ T cell clones by IFN-γ ELISPOT. Response to Chlamydia-infected (L2) or heat-inactivated Chlamydia exposed (HK L2) autologous DCs. Effect of anti-HLA-ABC (W6/32), anti-HLA-BC (B1.23.2), or anti-HLA-A2 (BB7.2) blocking Abs. Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8-11) of the three non-class Ia-restricted CD8+ T cell clones.

FIGURE 2.

MHC restriction analysis of Chlamydia-specific CD8+ T cell clones by IFN-γ ELISPOT. Response to Chlamydia-infected (L2) or heat-inactivated Chlamydia exposed (HK L2) autologous DCs. Effect of anti-HLA-ABC (W6/32), anti-HLA-BC (B1.23.2), or anti-HLA-A2 (BB7.2) blocking Abs. Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8-11) of the three non-class Ia-restricted CD8+ T cell clones.

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To further delineate the MHC allele responsible for presentation of chlamydial Ags to the two class-Ia MHC-restricted T cell clones, these T cells were tested against a panel of Chlamydia-infected HLA-typed DCs that share only a limited number of HLA molecules with the responding donor (Fig. 3). This analysis showed that infected DCs matching with D48 at HLA-B15 and HLA-A2 stimulated IFN-γ production by CD8-27, and this response was blocked by W6/32 Ab treatment. CD8-27 also secreted some IFN-γ in response to an HLA-A2-matched infected DC, however this response was considered to be nonspecific because it was not blocked by treatment with W6/32 Ab. Because treatment of autologous infected DCs with B1.23.2 Ab blocked the CD8-27 T cell-specific response (Fig. 2), CD8-27 is most likely restricted by HLA-B15. In contrast, CD8-30 only reacted to L2-infected DCs matching at HLA-A1, and this response was blocked by W6/32 Ab (Fig. 3). Thus, collectively these data suggest that recognition of Chlamydia-infected cells by CD8-30 T cells is restricted by HLA-A1.

FIGURE 3.

MHC restriction analysis of Chlamydia-specific CD8+ T cell clones by IFN-γ ELISPOT. Response to Chlamydia-infected (L2) autologous DCs and allele-matched allogeneic DCs (A2; A1; B8; or A2, B15). Effect of anti-HLA-ABC blocking Ab (W6/32). Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8-11) of the three non-class Ia-restricted CD8+ T cell clones.

FIGURE 3.

MHC restriction analysis of Chlamydia-specific CD8+ T cell clones by IFN-γ ELISPOT. Response to Chlamydia-infected (L2) autologous DCs and allele-matched allogeneic DCs (A2; A1; B8; or A2, B15). Effect of anti-HLA-ABC blocking Ab (W6/32). Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8-11) of the three non-class Ia-restricted CD8+ T cell clones.

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To confirm MHC restriction, DNA encoding for HLA-A0101 and -B1501 was cloned from D48 and transfected into an MHC class I mismatched fibroblast cell line (VA-13 and HLA-A68 and -B70). As shown in Fig. 4, CD8-27 T cells secreted IFN-γ in response to VA-13 cells transfected with HLA-B1501 and infected with Chlamydia, but not in response to noninfected VA-13 cells transfected with HLA-B1501 or nontransfected VA-13 cells infected with C. trachomatis L2. In addition, CD8-27 T cells did not respond to VA-13 cells infected with Chlamydia and transfected with HLA-A0101. Conversely, CD8-30 T cells secreted IFN-γ only when VA-13 cells were both transfected with HLA-A0101 and infected with Chlamydia and failed to respond to the L2-infected HLA-B1501 transfectants.

FIGURE 4.

Confirmation of HLA-B1501 or -A0101 restriction of MHC class Ia-restricted CD8-27 and CD8-30 T cell clones. Transfection of relevant alleles into VA-13 fibroblast cell line (HLA-A68, B70). Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8–11) of the three non-class Ia-restricted CD8+ T cell clones.

FIGURE 4.

Confirmation of HLA-B1501 or -A0101 restriction of MHC class Ia-restricted CD8-27 and CD8-30 T cell clones. Transfection of relevant alleles into VA-13 fibroblast cell line (HLA-A68, B70). Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8–11) of the three non-class Ia-restricted CD8+ T cell clones.

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Three CD8+ T cell clones (CD8-2, CD8-11, and CD8-21) that did not appear to be MHC class Ia restricted were also selected for further analysis. Specific responses of these T cell clones to C. trachomatis L2-infected DCs were not blocked by any of the anti-class I Abs tested (anti-HLA-ABC, anti-HLA-BC, or anti-HLA-A2; Fig. 2), and these T cells produced IFN-γ in response to all unrelated C. trachomatis L2-infected DCs, regardless of their MHC haplotype (Fig. 3). In addition, reactivity of these CD8+ T cells was not blocked by anti-class II Abs (data not shown), and the T cell clones failed to respond to DCs exposed to heat-inactivated elementary bodies (Fig. 2). These T cell clones were considered to be Chlamydia-specific, however, because they did not respond to autologous DCs infected with M. bovus, M. avium, or E. coli (data not shown).

Chlamydia resides in a membrane-bound vacuole throughout its developmental cycle, and thus it is of interest how chlamydial Ags gain access to the MHC class I Ag-processing pathway. To derive insights into the processing and presentation of chlamydial Ags to CD8+ T cells, inhibitors known to interfere with discrete stages of class I Ag processing were used. Lactacystin is a proteasome inhibitor and therefore blocks degradation of tagged cytosolic proteins to peptides for class I presentation. Brefeldin A induces two blocks in the secretory pathway: one at the endoplasmic reticulum-Golgi juncture and the second in the trans-Golgi network. Brefeldin A therefore will inhibit the transport of secretory proteins, including nascent MHC class I-peptide complexes derived from the endoplasmic reticulum to the cell surface. DCs were preincubated in the presence of inhibitor for 1 h before and during infection with Chlamydia. After Chlamydia infection for 24 h, cells were fixed with 0.2% paraformaldehyde and washed extensively before addition of T cells. A Chlamydia-specific CD4+ T cell clone derived from the same donor was used as a control, because lactacystin and brefeldin A have been documented to have no effect on the class II Ag-processing pathway. The CD4+ T cell clone produced IFN-γ in response to live infection as well as to DCs pulsed with heat-inactivated Chlamydia, and neither lactacystin nor brefeldin A blocked these responses. In contrast, both the MHC class Ia-restricted and the non-class Ia-restricted CD8+ T cell clones secreted IFN-γ only in response to live Chlamydia, and this response was blocked by both lactacystin and brefeldin A (Fig. 5). These blockers did not appear to be toxic to the DCs, because they did not inhibit Ag recognition by the CD4+ T cells and DCs were >90% viable after treatment, as assessed by trypan blue exclusion (data not shown). In addition, the inhibitors were not toxic to Chlamydia because similar infection rates and large Chlamydia-containing inclusions were obtained 48 h postinfection in the presence and in the absence of either lactacystin or brefeldin A (data not shown). Furthermore, C. trachomatis replication has been shown to be unaffected by brefeldin A (21).

FIGURE 5.

Effect of class I processing inhibitors on presentation of chlamydial Ags. IFN-γ production by the CD8+ class Ia-restricted, nonrestricted, and CD4+ T cell clones in response to autologous DCs noninfected (Medium), C. trachomatis infected (L2), or pulsed with heat-inactivated elementary bodies (HK L2). DCs were incubated with lactacystin (proteasomal degradation inhibitor, 1 μM) or brefeldin A (Golgi-endoplasmic reticulum transport inhibitor, 10 μg/ml) before and during infection with Chlamydia. Representative results for the two class Ia-restricted and three non-class Ia-restricted CD8+ T cell clones.

FIGURE 5.

Effect of class I processing inhibitors on presentation of chlamydial Ags. IFN-γ production by the CD8+ class Ia-restricted, nonrestricted, and CD4+ T cell clones in response to autologous DCs noninfected (Medium), C. trachomatis infected (L2), or pulsed with heat-inactivated elementary bodies (HK L2). DCs were incubated with lactacystin (proteasomal degradation inhibitor, 1 μM) or brefeldin A (Golgi-endoplasmic reticulum transport inhibitor, 10 μg/ml) before and during infection with Chlamydia. Representative results for the two class Ia-restricted and three non-class Ia-restricted CD8+ T cell clones.

Close modal

A potentially important role for CD8+ T cells during chlamydial infection is lysis of infected cells. To assess the lytic potential of both the class Ia-restricted and non-classically restricted CD8+ T cell clones, a 51Cr release assay was used. Interestingly, only the nonrestricted T cells were found to be capable of lysing autologous fibroblasts infected with Chlamydia in an 18-h 51Cr release assay (Fig. 6). Previous studies with murine CD8+ T cells specific for Chlamydia demonstrated that the presence of ICAM-I on the target cells greatly enhances the lytic capacity of CD8+ T cells (22). Therefore, autologous fibroblasts were retrovirally transduced with ICAM-1 and the assays were repeated. All transduced cells showed high levels of expression of ICAM-1 by flow cytometry (data not shown). However, ICAM-1 expression did not affect the lytic activity of either the class Ia-restricted or the nonrestricted T cells (Fig. 6). To address the possibility that Chlamydia-infected fibroblasts are not the optimal target cell for cytolysis, HeLa and U937 cells were stably transduced with HLA-A0101. CD8-30 secreted IFN-γ in response to A0101-transduced Chlamydia-infected cells but did not lyse these targets in a 51Cr release assay. As a control, the nonrestricted CD8-2 clone had lytic activity against both HeLa and U937 Chlamydia-infected cells (data not shown). Finally, the lytic capacity of the class Ia-restricted CD8+ T cell clones was further investigated by a redirected killing assay. As shown in Fig. 7, both of the class Ia-restricted CD8+ T cell clones were capable of lysing target cells, and thus the lack of class Ia-restricted lysis of a Chlamydia-infected cell does not appear to be due to a defect in the CD8+ T cell killing machinery.

FIGURE 6.

Lytic activity of non-class Ia-restricted and class Ia-restricted CTL clones. Targets are C. trachomatis L2-infected and noninfected autologous fibroblasts or ICAM-1 transduced fibroblasts in an 18-h 51Cr release assay. Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8-2) of the three non-class Ia-restricted CD8+ T cell clones.

FIGURE 6.

Lytic activity of non-class Ia-restricted and class Ia-restricted CTL clones. Targets are C. trachomatis L2-infected and noninfected autologous fibroblasts or ICAM-1 transduced fibroblasts in an 18-h 51Cr release assay. Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8-2) of the three non-class Ia-restricted CD8+ T cell clones.

Close modal
FIGURE 7.

Redirected killing of Fc receptor-expressing P815 cells by class Ia-restricted and non-class Ia-restricted CD8+ T cell clones with and without nonspecific stimulation by anti-CD3 Ab. Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8-2) of the three non-class Ia-restricted CD8+ T cell clones.

FIGURE 7.

Redirected killing of Fc receptor-expressing P815 cells by class Ia-restricted and non-class Ia-restricted CD8+ T cell clones with and without nonspecific stimulation by anti-CD3 Ab. Shown are results for the two class Ia-restricted CD8+ T cell clones and representative results for one (CD8-2) of the three non-class Ia-restricted CD8+ T cell clones.

Close modal

CD8+ T cells are an important component of immunity to a number of intracellular bacterial infections. Because murine studies have demonstrated that CD8+ T cells are involved in control of chlamydial infections by IFN-γ production rather than by lytic activity, we sought to characterize human Chlamydia-reactive CD8+ T cells and their in vitro effector functions. In this report, we have shown that 1) Chlamydia-infected monocyte-derived DCs can be used to stimulate CD8+ T cells and to estimate effector and precursor cell frequencies, 2) the majority of the CD8+ T cells detected by limiting dilution are not classically MHC class Ia restricted, 3) processing of chlamydial Ags that access both class Ia and class Ib is proteasome and Golgi dependent, and 4) non-classically restricted CD8+ T cells kill Chlamydia-infected fibroblasts, whereas class Ia-restricted T cells do not.

In the present study, C. trachomatis-infected DCs were used to estimate the contribution of the peripheral Chlamydia-specific CD8+ T cell responses in exposed individuals. During natural infection, DCs are likely the first professional APCs encountered by C. trachomatis. Chlamydia is capable of productively infecting DCs,4 and Chlamydia-infected DCs were recently shown to process and present Ags recognized by human T cells (23). Chlamydia-infected DCs should present a spectrum of chlamydial Ags expressed at different stages of the chlamydial developmental cycle. It is likely that Chlamydia-infected DCs are responsible for priming CD8+ T cell responses in vivo and that these CD8+ T cell responses to Ags presented by infected cells in vivo are the most relevant. It should be noted, however, that chlamydial Ags presented in vivo may differ from those presented under the in vitro culture conditions used in this study. Nevertheless, the use of infected DCs to analyze the CD8+ T cell response has a number of inherent advantages over other methods, such as preselection of Ags and subsequent prediction of MHC binding peptides.

Chlamydia-reactive CD8+ T cells were detected in all 10 individuals tested, with effector cell frequencies ranging from 100 to 2400 per 106 CD8+ T cells. These frequencies are comparable with published frequencies calculated from IFN-γ ELISPOT assays using whole PBMCs and stimulating with peptides derived from other infectious disease agents (20, 24, 25, 26, 27). In a study looking at measles-specific CD8+ T cell frequencies in which measles-infected B-lymphoblastoid cell line rather than peptides were used as stimulators, a mean frequency of 2400/106 CD8+ T cells was obtained (28). In a study similar to ours in which M. tuberculosis-infected DCs were used as stimulator cells, the frequencies obtained for M. tuberculosis-specific CD8+ T cells ranged from 34 to 344/106 CD8+ T cells (19). It should be noted that the frequencies of Ag-specific cells in this study were determined by a functional assay, that being measurement of IFN-γ production. We expect this approach to underestimate the frequency of Ag-specific cells, because it is unlikely that every Ag-specific T cell secretes IFN-γ. In this light, frequency estimates made by nonfunctional assays, such as tetramer staining, are typically higher than those made by functional assays, such as ELISPOT (26).

All individuals tested were either currently infected with C. trachomatis (as determined by chlamydial shedding as measured by LCR, high C. trachomatis IgG titers as measured by microimmunofluorescence assay, and clinical history) or had been exposed to C. trachomatis in the past (determined by C. trachomatis IgG titers, positive CD4+ T cell proliferation and IFN-γ production in response to C. trachomatis but not C. pneumoniae, and clinical history). No obvious differences in CD8+ T cell frequencies were observed between current and past exposure to C. trachomatis; however, such comparisons are difficult to make because of the small sample size and potentially undetected, asymptomatic, and/or persistent C. trachomatis or C. pneumoniae infections. The responses detected by IFN-γ ELISPOT are unlikely to be due to nonspecific activation of the DCs upon chlamydial infection because responses to heat-inactivated Chlamydia-pulsed DCs were <10% of the response to live-infected DCs, and these responses were subtracted as background when calculating frequencies. Furthermore, to determine that these responses were related to a past infection, CD8+ T cells derived from cord blood were tested against autologous infected DCs. Neither of the two donors tested had a detectable Chlamydia-specific CD8+ T cell response (data not shown).

A limiting dilution approach was used to distinguish between MHC class Ia and non-class Ia-restricted responses. This approach was used because it allows for the detection of CD8+ T cell responses after a single in vitro stimulation, thus minimizing the potential bias generated by multiple stimulations. In all three individuals tested, the majority of the Chlamydia-reactive CD8+ T cell responses were not restricted by MHC class Ia. Similar observations have been reported for the human CD8+ T cell response to M. tuberculosis, another intracellular bacterium (19). Although this suggests that the immunodominant CD8+ T cell response to C. trachomatis is not restricted by MHC class Ia, one has to take caution because such precursors may preferentially expand under the in vitro culture conditions used in this study.

To further characterize the CD8+ T cell response to C. trachomatis, both class Ia-restricted and non-class Ia-restricted T cell clones were expanded from a selected donor. The failure to demonstrate HLA-A, B, or C restriction for the non-classically restricted CD8+ T cell clones suggested that Ag presentation could be occurring through a nonpolymorphic class Ib molecule. Non-MHC class Ia-restricted CD8+ T cells have recently been found to be an important component of the CD8+ T cell response to other intracellular bacteria such as Listeria monocytogenes, Salmonella typhimurium, and M. tuberculosis (13, 14, 15). To date, there are no studies describing the presence of these cells in the CD8+ T cell response to Chlamydia. One possibility for Ag presentation to these T cells is via CD1 molecules, which have been reported to present lipid and glycolipid Ags to human CD8+ T cells. However, it is unlikely for a number of reasons that CD1 is the restricting allele for the three non-classically restricted CD8+ T cells described in this report. First, the T cells described herein do not recognize DCs pulsed with inactivated Chlamydia, whereas CD1 molecules (both group I and group II) sample the endocytic system and therefore present Ags derived from nonviable organisms (29, 30). Second, responses of these T cells to Chlamydia are proteasome dependent, suggesting that the Ag is a protein, not a lipid. Third, the non-classically restricted responses are sensitive to brefeldin A, whereas CD1 Ag processing is insensitive to brefeldin A (29). Finally, our Chlamydia-reactive T cells recognize Chlamydia-infected fibroblasts, despite the fact that human fibroblasts do not express either group I or group II CD1 molecules (Refs.31 and32 and data not shown). The other nonclassical class I molecules described to date are HLA-E, -F, and -G. In humans, M. tuberculosis-specific CD8+ T cells restricted by HLA-E have been described (14, 33). This processing pathway is not sensitive to brefeldin A and these responses are partially blocked by W6/32 treatment. In contrast, our Chlamydia-reactive T cells are not blocked by W6/32 and are brefeldin A sensitive and thus are unlikely to be restricted by HLA-E. Further studies are underway to characterize the restriction element.

All the CD8+ T cell clones derived in this study recognized Ags processed via the conventional class I processing pathway because Ag processing was blocked by treatment of DCs with either lactacystin or brefeldin A. These data suggest that the Ags recognized by these T cells are protein in nature and that these Ags access the host cell cytosol and require proteasomal degradation and transport of the assembled restriction element-Ag complex to the cell surface via the Golgi. In addition, the fact that neither the class Ia-restricted nor the nonrestricted CD8+ T cells recognize DCs pulsed with inactivated Chlamydia argues against an exogenous pathway of Ag presentation. Such Ags may have access to the host cell cytosol by translocation through a type III secretion system. Indeed, sequencing of the Chlamydia genome revealed the presence of orthologues of such a secretion system (34). Alternatively, Ags present in the Chlamydia inclusion membrane, such as Cap-1, a murine CD8+ T cell Ag, have been shown to gain access to the class I Ag-processing pathway (35).

All three non-classically restricted CD8+ T cell clones were capable of killing Chlamydia-infected autologous fibroblasts in an 18-h 51Cr release assay. In contrast, neither the HLA-A0101- nor the HLA-B1501-restricted T cell clone was capable of killing Chlamydia-infected target cells. Increased ICAM-1 expression has been shown to increase lysis of a Chlamydia-infected cell in the murine system (22). Stable transduction of ICAM-1 into our target cells, however, had no effect on the lytic activity of the T cells. HIV-specific (36) and prostate cancer-associated Ag-specific (37) CD8+ T cells that secrete IFN-γ but that do not kill have been recently described. The HIV-specific CD8+ T cells were found to be deficient in perforin. In contrast, both of our CD8+ T cell clones are capable of lytic activity as measured by a redirected killing assay. Thus, it is possible that the lack of killing is not due to a defect in the CD8+ T cell, but rather a defect in the target cell itself. One possibility is that a suboptimal target cell was used for cytotoxicity. This seems unlikely because primary human fibroblasts are sensitive CTL targets for Ags presented by MHC class Ia and are routinely used as CTL targets in vitro (M. Kalos, unpublished observation). Furthermore, Chlamydia-infected HeLa and U937 cells transduced with the relevant HLA allele were not efficiently lysed by the class Ia-restricted CTL, but were lysed by the nonrestricted CTL (data not shown). Another possibility for the lack of class Ia-restricted lytic activity could be the recently reported Chlamydia-induced inhibition of class Ia expression in infected target cells (38, 39). This possibility seems unlikely because we were unable to detect a decrease in expression of MHC class Ia in human fibroblasts 24 h after infection with C. trachomatis (data not shown). Furthermore, the class Ia-restricted CD8+ T cells do secrete IFN-γ in response to Chlamydia-infected fibroblasts. Because specific cytotoxicity requires lower Ag concentration than does IFN-γ production (40), it is unlikely that lack of cytotoxicity in the presence of IFN-γ secretion is due to a low antigenic stimulus. Finally, the lack of class Ia-restricted lytic activity could be attributed to an inhibition of apoptosis in the infected target cell. Indeed, Chlamydia has been shown to inhibit apoptosis induced by a wide range of proapoptotic agents (including perforin and Fas/CD95) (41). However, we do see killing of a Chlamydia-infected cell by the non-class Ia-restricted CD8+ T cells. Thus, it is possible that the cell death induced by the non-classically restricted clones follows a different pathway than the class Ia-restricted lytic pathway. We are currently exploring this hypothesis.

The role played by CD8+ T cells during chlamydial infection remains controversial. Although it is now well accepted that CD8+ T cells are primed during chlamydial infection of both mice and humans, their reported role ranges from no protection to IFN-γ-mediated protection in murine studies. However, there are potential limitations in interpreting murine models of chlamydial disease in that mice lack group 1 CD1 molecules (CD1 a, b, and c) as well as granulysin. Granulysin has been shown to directly kill many intracellular pathogens upon perforin-mediated lysis (42). In humans, the hypothesized role of CD8+ T cells ranges from protection against disease to associations with immunopathology. The Chlamydia-reactive CD8+ T cell clones described in this report secrete IFN-γ and have cytolytic machinery, but only the non-classically restricted clones were able to lyse Chlamydia-infected fibroblasts. These studies suggest that there may be a Chlamydia-specific mechanism that interferes with class Ia-restricted killing of an infected primary fibroblast. Non-class Ia-restricted CTLs therefore could play an important role in killing Chlamydia-infected cells and could represent a host mechanism for which Chlamydia has not yet been able to find an evasion strategy. The identification of the chlamydial Ags recognized by these CD8+ T cell clones and the definition of the restriction element(s) recognized by the non-classically restricted clones will be the focus of future studies.

We thank Jeremy Boynston for technical assistance in cloning the HLA molecules, Steven Fling and Bruce Hess for assistance with transfections, Anna Marie Beckmann for assistance in obtaining human apheresis products, and David Lewinsohn, Lee Ann Campbell, Sheila Lukehart, and Wesley Van Voorhis for fruitful discussions and suggestions.

1

This work was supported in part by Grant T32 AI07509 from the National Institutes of Health.

3

Abbreviations used in this paper: MOMP, major outer membrane protein; DC, dendritic cell; LCR, ligase chain reaction; rh, recombinant human; EB, elementary bodies.

4

M. R. Alderson, J. Maisonneuve, K. H. Grabstein, and P. Probst. Differential regulation of inflammatory cytokine secretion by human dendritic cells upon Chlamydia trachomatis infection. Submitted for publication.

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