Chlamydia muridarum-Speciﬁc CD4 T-Cell Clones Recognize ... · recognize the infected epithelial...

INFECTION AND IMMUNITY, Oct. 2009, p. 4469–4479 Vol. 77, No. 100019-9567/09/$08.00�0 doi:10.1128/IAI.00491-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Chlamydia muridarum-Specific CD4 T-Cell Clones RecognizeInfected Reproductive Tract Epithelial Cells in an

Interferon-Dependent Fashion�

Krupakar Jayarapu,1 Micah S. Kerr,1 Adrian Katschke,2 and Raymond M. Johnson1*Department of Medicine1 and Department of Biostatistics,2 Indiana University School of Medicine, Indianapolis, Indiana 46202

Received 4 May 2009/Returned for modification 20 July 2009/Accepted 3 August 2009

During natural infections Chlamydia trachomatis urogenital serovars replicate predominantly in the epithe-lial cells lining the reproductive tract. This tissue tropism poses a unique challenge to host cellar immunity andfuture vaccine development. In the experimental mouse model, CD4 T cells are necessary and sufficient to clearChlamydia muridarum genital tract infections. This implies that resolution of genital tract infection depends onCD4 T-cell interactions with infected epithelial cells. However, no laboratory has shown that Chlamydia-specificCD4 T cells can recognize Chlamydia antigens presented by major histocompatibility complex class II (MHC-I)molecules on epithelial cells. In this report we show that MHC-II-restricted Chlamydia-specific CD4 T-cellclones recognize infected upper reproductive tract epithelial cells as early as 12 h postinfection. The timing ofrecognition and degree of T-cell activation are dependent on the interferon (IFN) milieu. Beta IFN (IFN-�) andIFN-� have different effects on T-cell activation, with IFN-� blunting IFN-�-induced upregulation of epithelialcell surface MHC-II and T-cell activation. Individual CD4 T-cell clones differed in their degrees of dependenceon IFN-�-regulated MHC-II for controlling Chlamydia replication in epithelial cells in vitro. We discuss ourdata as they relate to published studies with IFN knockout mice, proposing a straightforward interpretationof the existing literature based on CD4 T-cell interactions with the infected reproductive tract epithelium.

Chlamydia trachomatis is the most common bacterial sexu-ally transmitted infection in the developed world, with 2 to 3million actively infected individuals in the United States (3)and similar numbers in Europe (17). In women C. trachomatisinfections can ascend into the upper reproductive tract, caus-ing pelvic inflammatory disease and scarring with resultinginfertility and ectopic pregnancies.

Histopathology studies show that C. trachomatis replicatespredominantly in the reproductive tract epithelium during nat-ural human infections (16, 36) and experimental murine C.muridarum infections (21). Inclusions are not seen in other celltypes even though Chlamydia can undergo limited replicationin macrophages and dendritic cells (33). It is unlikely thatreplication in non-epithelial cell lineages makes a major con-tribution to genital tract shedding. The mouse model for Chla-mydia genital tract infections supports a critical role for CD4 Tcells in protective immunity, as mice deficient in major histo-compatibility complex class II (MHC-II) cannot control C.muridarum genital tract infections (22), and CD4 T-cell deple-tion is detrimental to resolution of primary genital tract infec-tions (23). Because C. muridarum replicates in epithelial cellslining the reproductive tract, the most straightforward mecha-nism for clearing the genital tract would involve Chlamydia-specific CD4 T-cell interactions with infected epithelial cells.However in the absence of any data supporting this specificinteraction, other indirect mechanisms based on CD4 T-cellproduction of gamma interferon (IFN-�) and provision of help

to B cells and CD8 T cells have been proposed as the mech-anism for clearance (29).

C. muridarum-specific CD4 T-cell lines protective in adop-tive-transfer studies were shown to control C. muridarum rep-lication in polarized epithelial monolayers (14). The mecha-nism of control was dependent on IFN-� and physicalinteraction of T cells with the infected epithelial cells viaLFA-1. In the presence of IFN-�, T-cell engagement of epi-thelial cells via LFA-1 was shown to augment epithelial nitricoxide production above that induced by IFN-� alone, and nitricoxide was shown to be the effector molecule responsible forcontrolling Chlamydia replication (13). This anti-Chlamydiaeffector mechanism did not require that the CD4 T-cell clonerecognize the infected epithelial monolayer in an antigen-spe-cific fashion, as the same preactivated CD4 T-cell clone con-trolled Chlamydia psittaci replication in polarized epithelialmonolayers even though it does not recognize a C. psittaciantigen.

Epithelial cells are semiprofessional antigen-presenting cells(APCs) and, in their unperturbed state, likely play a role inimmunotolerance at mucosal surfaces (19). However in inflam-matory environments, such as those resulting from transplantrejection and graft-versus-host disease, epithelial cells changetheir immunophenotype by upregulation of MHC-II (5, 25). Intrachoma, an eye infection caused by Chlamydia trachomatisserovars A to C, conjunctival epithelial cells from human clin-ical specimens showed upregulated cell surface MHC-II andwere presumably competent to present antigens to CD4 T cells(11, 12). In vitro studies have shown that rat and murineuterine epithelial cells process and present exogenous ovalbu-min to OVA-specific CD4 T cells (28, 37). However in vitroprocessing and presentation of concentrated extracellularovalbumin to CD4 T cells by uterine epithelial cells do not

* Corresponding author. Mailing address: Division of InfectiousDiseases, Department of Medicine, Indiana University School of Med-icine, 545 Barnhill Drive, #435, Indianapolis, IN 46202. Phone: (317)278-6968. Fax: (317) 274-1587. E-mail: [email protected].

� Published ahead of print on 10 August 2009.

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directly address whether Chlamydia antigens sequestered inmembrane-bound inclusions get processed and presented toChlamydia-specific CD4 T cells in vivo. The mechanics of CD4T-cell contributions to resolution of genital tract infectionsremain unclear.

For this study we derived an epithelial cell line from theupper reproductive tract of a female C57BL/6 mouse and apanel of 10 Chlamydia-specific CD4 T-cell clones from im-mune C57BL/6 mice that previously self-cleared C. muridarumgenital tract infections. These reagents gave us the opportunityto directly investigate (i) whether Chlamydia-specific CD4 Tcells can recognize C. muridarum-infected reproductive tractepithelial cells, (ii) when during the time course of infectionrecognition occurs, and (iii) the role of IFNs in modulatingepithelial interactions with CD4 T cells. We present the resultsof those investigations here.

(These data were presented in part at the 2009 ChlamydiaBasic Research Conference.)

MATERIALS AND METHODS

Mice. Female C57BL/6 and BALB/c mice were purchased from Harlan Lab-oratories (Indianapolis, IN). Female C57BL/6J, B6.C-H2bm12/KhEg, and B6.C-H2bm1/ByJ female mice were purchased from The Jackson Laboratory (BarHarbor, ME). All mice were housed in Indiana University Purdue University-Indianapolis (IUPUI) specific-pathogen-free facilities. The IUPUI InstitutionalAnimal Care and Utilization Committee approved all experimental protocols.

Cells, bacteria, and culture reagents. C57epi.1 is a cloned oviduct epithelialcell line derived from a C57BL/6 mouse (H-2b) using the methodology previouslydescribed (9, 10, 15) except for initial ex vivo culture in serum-free mediummedia supplemented with bovine pituitary gland extract (Gibco/Invitrogen,Carlsbad, CA) for several passages prior to switching to epithelial cell mediasupplemented as described below. The cultured C57epi.1 cells were grown at37°C in a 5% CO2 humidified incubator in epithelial cell medium (1:1 Dulbecco’smodified Eagle medium-F12K; Sigma, St. Louis, MO), supplemented with 10%characterized fetal bovine serum (HyClone, Logan, UT), 2 mM L-alanyl-L-glu-tamine (Glutamax I; Gibco/Invitrogen), 5 �g of bovine insulin/ml, and 12.5 ng/mlof recombinant human fibroblast growth factor 7 (keratinocyte growth factor;Sigma). C57epi.1 cells were fixed in 1:1 acetone-methanol and stained withanticytokeratin monoclonal antibody AE1/AE3 (Cappel ICN, Irvine, CA) orcontrol antibody 36-5-7 (anti-H-2Kk; BD Biosciences, San Diego, CA) and coun-terstained with DAPI (4��,6-diamidino-2-phenylindole; nuclear stain) to con-firm an epithelial lineage as previously described (15).

Mycoplasma-free Chlamydia muridarum (Nigg isolate), previously known asthe C. trachomatis strain mouse pneumonitis biovar (MoPn), was grown inMcCoy cells (American Type Culture Collection no. CRL-1696). The titers ofmycoplasma-free C. muridarum stocks were determined on McCoy cells withcentrifugation as previously described (15). UV-inactivated C. muridarum stockswere made by diluting concentrated stocks in sucrose-phosphate-glutamic acid(SPG) buffer and then exposing 3 to 4 ml of diluted stock in a sterile petri dishto 1,200 J twice in a UV-cross-linking cabinet (Spectralinker; Spectronics Cor-poration, Westbury, NY). No viable C. muridarum inclusions were detectable ininoculated McCoy cell monolayers after UV inactivation.

Soluble C. muridarum antigen was prepared by infecting C57epi.1 epithelialcell monolayers in four 175-cm2 tissue culture flasks with C. muridarum at 3inclusion-forming units (IFU) per cell. At 32 h the monolayers were harvestedwith glass bead agitation in 15 ml of residual medium per flask. Debris waspelleted with a low-speed spin (1,400 rpm [464 � g] for 10 min), and supernatantwas collected; then elementary bodies were pelleted out of medium (depleted)with a high-speed spin (16,000 rpm [25,000 � g] for 30 min). The resultingsupernatant was concentrated with a 10,000-kDa-molecular-mass-cutoff centrif-ugal filter (Amicon-15; Millipore, Bilerica, MA), aliquoted, and stored at �80°C.

Infection of mice. C57BL/6 mice were treated with 2.5 mg of depoprogester-one (Depo-Provera; Pfizer, New York, NY) injected subdermally 1 week prior toinfection. Vaginal infections were accomplished with 5 � 104 IFU of C. muri-darum in 10 �l of SPG buffer. Mice were swabbed 7 days later to confirminfection. Vaginal swab IFU were recovered in SPG buffer and quantified usingMcCoy cell monolayers as previously described (15).

Generation of CD4 T-cell clones. T-cell cultures were grown in RPMI 1640(Sigma) supplemented with 10% characterized fetal bovine serum (HyClone), 2mM L-alanyl-L-glutamine (Glutamax I; Gibco/Invitrogen), 25 �g/ml gentamicin(Sigma), and 5 � 10�5 M 2-mercaptoethanol (Sigma). This supplemented me-dium is referred to as T-cell medium hereafter. Secondary mixed lymphocyteculture (MLC) supernatants were prepared by combining 25 � 106 C57BL/6splenocytes with 25 � 106 UV-irradiated (1,500 rads) BALB/c splenocytes in anupright 25-cm2 flask containing 20 ml of Dulbecco’s modified Eagle mediumsupplemented with 10 mM HEPES, 10% characterized fetal bovine serum (Hy-Clone), 2 mM L-alanyl-L-glutamine (Gibco/Invitrogen), 25 �g/ml gentamicin(Sigma), and 5 � 10�5 M 2-mercaptoethanol (Sigma; DME CM). Ten days laterthe viable C57BL/6 T cells were recovered and stimulated with irradiatedBALB/c splenocytes (10 � 106 C57BL/6 T cells plus 25 � 106 irradiated BALB/csplenocytes in 20 ml of DME CM in an upright 25-cm2 flask) for 20 h. Super-natants were collected, filtered through 0.22-�m filters, aliquoted, and stored at�80°C until use.

Chlamydia-specific CD4 T-cell clones were derived from immune C57BL/6(H-2b) female mice that had cleared a primary C. muridarum genital tractinfection and were 7 days into clearing a secondary vaginal challenge with C.muridarum. Immune splenocytes harvested from mice were plated at 12.5 � 106

cells per well in tissue culture-treated 12-well plates, in T-cell medium containingmurine recombinant interleukin-1� (IL-1�; 2 ng/ml), IL-6 (2 ng/ml), IL-7 (3ng/ml), IL-15 (4 ng/ml), human recombinant IL-2 (100 units/ml), 20% 2° MLC,and 10 �g of UV-inactivated C. muridarum (�2.5 IFU equivalents per spleno-cyte) or 15 �l of soluble C. muridarum antigen (�1.5 cm2 infected monolayerequivalents). The resulting polyclonal T-cell populations were serially passagedand limiting diluted to obtain CD4 T-cell clones. CD4 T-cell clones designateduvmo-1, uvmo-2, uvmo-3, and uvmo-4 were derived from four independentpolyclonal T-cell lines originating from immune splenocytes of four differentmice using UV-inactivated C. muridarum as the antigen. These T-cell clones alsorecognize irradiated splenocytes pulsed with UV-inactivated C. muridarumgrown in C57epi.1 (H-2b) epithelial cells, ruling out specificity for McCoy al-loantigens originating from the McCoy fibroblasts used to propagate C. murida-rum (data not shown). CD4 clones designated LN4-10, LN4-11, LN4-12, andLN4-13 were derived from the lymph nodes draining the reproductive tract(inguinal, iliac, and para-aortic), and Spl4-10 and Spl-11 were from immunesplenocytes of a fifth mouse using soluble C. muridarum antigen. With theexception of IL-2, the recombinant T-cell growth factors used reflect thosesecreted by infected epithelial cells (15) and bone marrow-derived dendritic cellspulsed with heat-killed C. muridarum (32), which are remarkably similar. AllT-cell clones listed above were CD4� CD8� by flow cytometry (data not shown).

For routine passage of clones uvmo-1, -2, and -3, 1 � 105 CD4 clone cells wereplated in 24-well tissue culture-treated wells containing 1.5 ml of T-cell medium–15% MLC supernatant supplemented with murine IL-1� (2 ng/ml), IL-6 (2ng/ml), IL-7 (2 ng/ml), IL-15 (4 ng/ml), and human recombinant IL-2 (75 units/ml) plus 5 � 106 gamma-irradiated C57BL/6 splenocytes (1,200 rads) that hadbeen prepulsed at 37°C with 2.5 IFU equivalent of UV-irradiated C. muridarumper splenocyte for 30 min. The remaining clones (uvmo-4; LN4-10, -11, -12, and-13; and Spl4-10 and -11) were passaged using the same conditions except thatthe irradiated splenocytes came from female mice that had self-cleared a C.muridarum genital tract infection (i.e., immune-irradiated splenocytes) and theantigen was 1.5 cm2 equivalent soluble C. muridarum antigen per 5 � 106

irradiated splenocytes. Immune-irradiated splenocytes are likely better able toefficiently process antigens present in low concentrations in the soluble antigenpreparation (30). Chlamydia-specific CD4 T-cell clones were passaged every 6 to8 days under these conditions. For the experiments in this study T cells were usedon day 7 of their culture cycle. Recombinant murine cytokines were purchasedfrom a commercial vendor (R&D Systems, Minneapolis, MN). Human recom-binant IL-2 was obtained from Chiron Corporation (Emeryville, CA).

Epithelial cell infections. C57epi.1 cells were plated in 6-, 12-, 24-, or 48-welltissue culture plates and were used when confluent. Cells were infected with 3IFU of C. muridarum per cell in 0.25 to 2 ml of culture medium depending on theculture plate format. The plates were centrifuged at 1,200 rpm (300 � g) in atabletop centrifuge for 30 min and then incubated at 37°C in a 5% CO2 humid-ified incubator without change of medium for 3 to 21 h, depending upon theassay. Mock-infected wells received an equivalent volume of SPG buffer lackingC. muridarum.

Antibodies and flow cytometry. T cells were dislodged from tissue cultureplastic by removal of medium and incubation for 5 min in phosphate-bufferedsaline–EDTA. C57epi.1 cells were dislodged from tissue culture plastic using anEDTA wash followed by Hanks’ salt-based enzyme-free cell dissociation buffer(Sigma). Cells were stained for 20 min on ice in phosphate-buffered saline–2%bovine serum albumin with phycoerythrin (PE)-coupled 53-5.8 (CD8), PE-

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coupled YTS191.1 (CD4) (Cedarlane Laboratories, Burlington, NC), fluoresceinisothiocyanate-coupled mouse immunoglobulin G2a (IgG2a) (control antibody),PE-coupled rat IgG2b (control antibody), PE-coupled M5/114.15.2 (MHC-II),GK1.5 (CD4; low endotoxin/no azide), and rat IgG2a (control; low endotoxin/noazide) (Ebioscience, San Diego, CA). Cells were fixed with 1% paraformalde-hyde after staining. Cells were analyzed at the Indiana University Cancer CenterFlow Cytometry Facility using a FACScan cytometer (BD Biosciences).

ELISA determination of IFN-�. Relative IFN-� levels were determined byenzyme-linked immunosorbent assay (ELISA) using monoclonal antibodyXMG1.2, according to the manufacturer’s protocol (Pierce-Endogen, Rockford,IL). Recombinant murine IFN-� (R&D Systems) was used as the standard.

T-cell proliferation assays. Epithelial cell targets were treated with 50 �g/ml ofmitomycin C for 20 min at 37°C, washed twice with EDTA, dislodged withenzyme-free cell dissociation buffer, filtered through a 40-�M nylon filter, andcounted. T cells (5 � 104) with 5 � 104 epithelial cells were cocultured in 200 �lof T-cell medium. At 36 h culture supernatants were harvested (50 �l) forcytokine analysis and wells were pulsed with 0.5 �Ci of [3H]thymidine per wellfor 12 h. Proliferation assay products were harvested on glass fiber filters andcounted using a Packard Matrix 9600 direct beta counter.

Replication. To test whether the CD4 clones could control Chlamydia repli-cation in vitro, C57epi.1 monolayers in 48-well plates were untreated or treatedwith IFN-� (10 ng/ml) for 14 h prior to infection or at the time of infection with3 IFU of C. muridarum per cell. After addition of C. muridarum the plates werespun at 1,200 rpm (300 � g) for 30 min. Four hours after infection the inoculumwas removed and CD4 T-cell clones were added in T-cell medium. Thirty-sixhours postinfection, the cells and medium in each well were harvested by scrap-ing and stored at �80°C until C. muridarum titers were determined on McCoymonolayers as previously described (15). Recombinant murine IFN-� at allconcentrations tested (up to 1,000 pg/ml) had no effect on C. muridarum titra-tions done on the McCoy monolayers; maximum IFN-� carryover in dilutionsused for quantifying C. muridarum was �50 pg/ml.

Statistical analysis. Summary figures for each experimental investigation arepresented as “pooled” means with their associated standard errors of the means(SEM). Figure legends indicate the number of independent experiments pooledto generate each figure. Student’s two-tailed t test was used to assess significanceof pooled experimental data. P values that were �0.05 were considered statisti-cally significant.

RESULTS

Derivation and characterization of the C57epi.1 oviduct ep-ithelial cell line and Chlamydia-specific CD4 T-cell clones. Anupper reproductive tract epithelial cell line was derived from aC57BL/6 (H-2b) mouse by limiting dilution cloning as de-scribed in Materials and Methods. C57epi.1 epithelial cellmonolayers in chamber slides were fixed and stained with con-trol antibody (Fig. 1A) and antibody specific for cytokeratins(Fig. 1B). C57epi.1 cells express cytokeratins, consistent withan epithelial lineage. Also consistent with an epithelial lineage,they also have IFN-�-inducible MHC-II expression (Fig. 1C).

There are no published murine CD4 or CD8 Chlamydia-specific T-cell clones derived from mice that self-cleared pri-mary genital tract infections. We derived a panel of 10 C.

muridarum-specific CD4 T-cell clones from five C57BL/6 (H-2b) female mice that cleared primary genital infections. Im-mune lymphocytes were harvested from spleens and lymphnodes draining the genital tract 1 week into a second vaginalchallenge. The Chlamydia antigens used to activate T cells invitro were crude preparations of UV-irradiated C. muridarumelementary bodies and soluble C. muridarum antigens; theAPCs for routine passage were naive irradiated C57BL/6splenocytes or immune-irradiated splenocytes. C57epi.1 cellswere pretreated with IFN-� and then mock infected or infectedwith C. muridarum for 12 and 18 h prior to harvest for use astargets. The 10 C. muridarum-specific CD4 T-cell clones weretested for their ability to recognize mock-infected versus C.muridarum-infected C57epi.1 epithelial cells at 12 and 18 hpostinfection. They were also tested for their ability to recog-nize mock-pulsed syngeneic irradiated naive splenocytes (au-toreactivity control) versus immune syngeneic irradiatedsplenocytes pulsed with UV-inactivated C. muridarum (specificantigen) (Table 1). The T-cell assays were done in the presenceof 10 �g/ml tetracycline to block synthesis of additional Chla-mydia polypeptides and progression of infection. Immune ir-radiated splenocytes pulsed with C. muridarum secreted amodest amount of IFN-� without proliferating, while naiveirradiated splenocytes make no detectable IFN-� under iden-tical conditions (data not shown). For the splenocyte APC datain Table 1, the IFN-� produced by immune irradiated spleno-cytes pulsed with UV-inactivated C. muridarum in control wellslacking CD4 T-cell clones was subtracted from IFN-� pro-duced in experimental wells containing antigen-pulsed im-mune irradiated splenocytes plus CD4 T-cell clones. ThisIFN-� accounting procedure had no effect on the experimentalconclusions.

As seen in Table 1, all CD4 T-cell clones, regardless ofderivation strategy, were able to recognize infected epithelialcells and immune splenocytes pulsed with UV-inactivated C.muridarum. CD4 T-cell clones differed in their abilities torecognize infected epithelial cells at 12 h and 18 h postinfec-tion, and T-cell activation as determined by measuring IFN-�production was significantly less for infected epithelial cellsthan for antigen-pulsed immune splenocytes for all CD4 T-cellclones. The limited numbers of T-cell clones derived using thedifferent strategies are too small to draw conclusions aboutderivation-specific differences in relative activation by antigen-pulsed splenocytes versus infected epithelial cells. However, itis clear from comparing each clone’s activation by infectedepithelial cells to its activation by antigen-pulsed irradiated

FIG. 1. Characterization of oviduct epithelial cell line C57epi.1 (H-2Kb). (A) Control staining with an irrelevant antibody specific for H-2Kk.Nuclei were visualized with DAPI counterstain. (B) Staining for cytokeratins to confirm an epithelial lineage. (C) C57epi.1 cells were exposed to10 ng/ml IFN-� for 29 h and then stained for MHC-II and analyzed by flow cytometry.

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splenocytes that Chlamydia-specific CD4 T-cell activation byinfected epithelial cells was submaximal for all clones tested.

C. muridarum-specific CD4 T cells control C. muridarumreplication in epithelial cells in vitro with variable dependenceon IFN-�. Igietseme et al. (14) showed that T-cell lines thatprotected mice from vaginal infections with C. muridarum inadoptive-transfer experiments were also able to control C.muridarum replication in a polarized epithelial tumor cell linein vitro. We tested the ability of our panel of CD4 T-cell clonesto control C. muridarum replication in C57epi.1 epithelial cells(Fig. 2). Monolayers of C57epi.1 cells in 48-well plates(�200,000 epithelial cells per well) were untreated or treatedwith IFN-�, either 14 h prior to infection or at the time ofinfection, and then infected with C. muridarum. Four hourspostinfection the inoculating medium was replaced with T-cellmedium containing CD4 T-cell clones; 150,000 T cells wereadded per well, for an effector-to-target ratio of �0.75:1. Thirty-two hours later the wells were harvested with additional SPGbuffer and recovered C. muridarum was titered on McCoymonolayers to score replication. Pretreatment of C57epi.1 cellswith IFN-� had a modest effect on C. muridarum replication inthe control wells (medium, [15 10] � 106 IFU/well; IFN-�treatment, [4 2] � 106 IFU/well; pooled means from twoexperiments; P � 0.001). IFU recovered from experimentalwells were compared with IFU from identically treated (un-treated or IFN-�-treated) parallel control wells (no T cells) tocalculate % control replication. This normalization controls forthe difference in C. muridarum replication in the untreatedversus IFN-�-treated C57epi.1 cells.

Eight of the 10 CD4 T-cell clones were able to block �90%of C. muridarum replication when epithelial cells were treatedwith IFN-� prior to infection (Fig. 2A). The two clones thatcould not were LN4-11 and uvmo-4. Control of replicationonly loosely correlated with each CD4 T-cell clone’s ability tomake IFN-� when activated by infected epithelial cells (Table

1). The “ineffective” clone uvmo-4 (77 pg/ml) was one of theT-cell clones that was least activated by infected epithelialcells, while the other “ineffective” clone, LN4-11 (335 pg/ml),was in the middle. CD4 T-cell clones that made less IFN-� thanLN4-11 (LN4-10 and -13 and Spl4-10 and -11) were still able tocontrol C. muridarum replication. Seven of the 10 clonesshowed improved control of C. muridarum replication withIFN-� pretreatment of the epithelial monolayers (Fig. 2A). Ofnote, three CD4 T-cell clones (uvmo-1, uvmo-2, and uvmo-3)were able to block �80% C. muridarum replication withoutIFN-� pretreatment of the epithelial monolayers.

A previous study with human epithelial tumor cell lines andC. trachomatis serovar L2 showed that Chlamydia infectionprior to IFN-� exposure blocked IFN-�-mediated upregulationof epithelial MHC-II by degrading an MHC-II transcriptionfactor (38). To test whether C. muridarum could avoid cell-mediated immunity via this mechanism in vitro, the experi-ments shown in Fig. 2A were repeated except that IFN-� wasadded at the time of C. muridarum infection. Addition ofIFN-� at the time of infection had no effect on C. muridarumreplication (medium, [5.6 0.8] � 106 IFU/well; IFN-� treat-ment, [5 1] � 106 IFU/well). When IFN-� was added at thetime of infection only three CD4 clones were able to block�90% of C. muridarum replication (uvmo-1, uvmo-2, anduvmo-3) and only 4 of the 10 clones showed improved controlof C. muridarum replication with IFN-� treatment of the epi-thelial monolayers (Fig. 2B).

In summary we found that 2 of 10 Chlamydia-specific CD4T-cell clones were ineffective even though they recognizedinfected epithelial cells, 5 clones effectively controlled C. muri-darum in an IFN-�-dependent fashion, and 3 clones efficientlycontrolled C. muridarum replication even without exogenousIFN-� treatment of the epithelial monolayers. In addition, wefound that C. muridarum infection interfered with the ability ofthe five IFN-�-dependent CD4 clones to control replication

TABLE 1. IFN production by C. muridarum-specific CD4 T-cell clonesa

T-cell clone

IFN production (pg/ml) by:

Epithelial cells Immune splenocytes

Mock infected Infected for 12 h Infected for 18 hb Mock infected Pulsedc

uvmo-1d,f 420 180 590 100 2,820 640* 1,212 867 414,000 70,000*uvmo-2d,f 29 5 111 48* 2,800 360* 0 146,000 44,000*uvmo-3d,f 45 20 170 24* 3,630 200* 0 323,000 67,000*uvmo-4d,g 12 6 56 58 77 53* 0 176,000 17,000*LN4-10e,g 4 26 26 26 235 56* 0 86,700 7,400*LN4-11e,g 26 17 35 11 335 90* 0 86,000 5,500*LN4-12e,g 0 4 0 3 96 38* 0 125,000 21,000*LN4-13e,g 0 5 0 2 216 100* 0 97,000 11,000*Spl4-10e,g 0 5 20 25 273 99* 0 125,000 21,000*Spl4-11e,g 47 7 71 13* 204 45* 0 39,000 9,000*

a C57epi.1 epithelial cells were pretreated with IFN-� (10 ng/ml) for 12 h, mock infected or infected with 3 IFU C. muridarum per cell for 12 h or 18 h, treated withmitomycin C just prior to harvest, and used as targets. CD4 T-cell clone cells (50,000) were cocultured with 50,000 epithelial cells in the presence of tetracycline (10�g/ml). Culture supernatants were collected at 48 h. Irradiated splenocytes were mock pulsed or pulsed with UV-inactivated C. muridarum using passage conditions(Materials and Methods). T cells (50,000) were cocultured with 1 � 106 irradiated splenocyte APCs. Supernatants were collected at 36 h and analyzed for IFN-� contentby ELISA. Aggregate data from two independent experiments SEM are shown.

b �, P � 0.05, comparing experimental wells to mock controls for each APC type.c Pulsed, pulsed with UV-inactivated C. muridarum.d Derivation antigen, UV-inactivated C. muridarum.e Derivation antigen, soluble C. muridarum antigen.f Maintenance APCs, irradiated naive C57BL/6 splenocytes.g Maintenance APCs, irradiated immune C57BL/6 splenocytes.



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when IFN-� was added at the time of infection but not when itwas added 14 h prior to infection. Based on the existing liter-ature, these data strongly suggested that C. muridarum infec-tion had a negative effect on IFN-�-mediated upregulation ofMHC-II and that the level of epithelial MHC-II expression wasa limiting factor for the IFN-�-dependent CD4 T-cell clones(LN4-10, -12, and -13 and Spl4-10 and -11). There was no clearcorrelation between lymphoid organ of origin (spleen versusdraining lymph node) or antigen used ex vivo to activate T-celllines (UV-inactivated C. muridarum versus soluble antigen)and the ability of the resulting T-cell clones to control in vitroC. muridarum replication.

We investigated the effect of C. muridarum on inducibleepithelial cell surface MHC-II expression using the same ex-perimental protocol used for the replication control experi-ments. C57epi.1 cells pretreated with IFN-� for 14 h prior toinfection (Fig. 2C) were compared to C57epi.1 cells treatedwith IFN-� at the time of infection (Fig. 2D). Eighteen hourspostinfection the epithelial monolayers were harvested, stained

for MHC-II, and analyzed by flow cytometry. Consistent withthe C. trachomatis serovar L2 data and the hypothesis that therole of IFN-� for the IFN-�-dependent CD4 T-cell clones is toupregulate epithelial MHC-II, C. muridarum infection mod-estly but reproducibly blocked MHC-II upregulation by IFN-�added at the time of infection (Fig. 2D) but not 14 h prior toinfection (Fig. 2C). Because of the difference in duration ofIFN-� exposure, the absolute amount of cell surface MHC-IIwas higher with 14-h IFN-� pretreatment (Fig. 2C) than withIFN-� addition at the time of infection (Fig. 2D). The threemost effective CD4 clones, uvmo-1, -2, and -3, achieved nearlymaximal inhibition of replication with the modest increase inMHC-II induced by addition of IFN-� at the time of infection,while the IFN-�-dependent CD4 clones (LN4-10, -12, and -13and Spl4-10 and -11) could not control C. muridarum with thislower level of MHC-II. These data, combined with the data inTable 1 showing that uvmo-1, -2, and -3 are better activated byinfected epithelial cells as measured by IFN-� production, areconsistent with the hypothesis that the IFN-�-independent

FIG. 2. CD4 T-cell clones control C. muridarum replication in vitro: correlation with IFN-�-inducible MHC-II expression. (A) C57epi.1monolayers were treated with 10 ng/ml IFN-� for 14 h prior to infection. Monolayers were then infected with 3 IFU per cell. CD4 T-cell cloneswere added 4 h later. Wells were harvested 36 h postinfection, and IFU were quantified. (B) C57epi.1 monolayers were simultaneously exposedto IFN-� and infected with C. muridarum. Wells were harvested 36 h later and IFU were quantified. Aggregate data from two independentexperiments are shown. * (A and B), P � 0.05, comparing the untreated monolayer to the IFN-� treated monolayer for an individual clone.(C) Flow cytometric analysis of MHC-II expression with 14 h of IFN-� pretreatment plus 18 h of infection (approximating conditions for panelA). (D) MHC-II expression with simultaneous IFN-� exposure and infection. Cells were stained 18 h postinfection (approximating the conditionsfor panel B). Flow cytometry data shown are representative of two independent experiments. Numbers in parentheses (C and D) are the meanfluorescence values in arbitrary units for each condition.



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CD4 clones are better able to control Chlamydia replicationbecause they are better activated at lower levels of epithelialMHC-II. At the higher levels of epithelial MHC-II induced by14 h of IFN-� pretreatment, there was little difference betweenuvmo-1, -2, and -3, LN4-10, -12, and -13, and Spl4-10 and -11in their abilities to control C. muridarum replication (Fig. 2A).

Chlamydia-specific CD4 T-cell clones are MHC-II restrictedand utilize the CD4 coreceptor when activated by infectedepithelial cells. For logistical reasons we chose to focus onthree CD4 T-cell clones (uvmo-1, uvmo-2, and uvmo-3) tofurther investigate CD4 T-cell interactions with infected repro-ductive tract epithelial cells. These three CD4 T-cell cloneswere derived from independent mice and were the most effec-tive at controlling C. muridarum in vitro.

We investigated the role of the CD4 coreceptor during ac-tivation by infected epithelial cells because this directly ad-dresses the role of MHC-II in T-cell activation. All the CD4T-cell clones were CD4� CD8�. A representative CD4 stain-ing for T-cell clone uvmo-2 is shown in Fig. 3A. We mappedthe MHC restriction element for uvmo-1, -2, and -3 usingC57BL/6J (H-2b), bm1 (H-2IabKbm1), and bm12 (H-2Iabm12)mouse naive splenocytes mock pulsed or pulsed with UV-inactivated C. muridarum. These C57BL/6-derived mousestrains have a single MHC-II � heterodimer; C57BL/6Jsplenocytes are syngeneic with the CD4 T-cell clones, bm1splenocytes are mismatched at the MHC class I K locus, andbm12 splenocytes are mismatched at the MHC-II locus. Allthree clones recognized C57BL/6J splenocytes and bm1splenocytes pulsed with UV-inactivated C. muridarum, buttheir recognition of bm12 splenocytes (MHC-II mismatch)pulsed with C. muridarum was negligible or markedly attenu-ated (Fig. 3B). The bm12 MHC-II heterodimer differs fromthat of the C57BL/6J heterodimer by 3 amino acids in the betachain (20). This small change likely accounts for the residualpartial activation of the uvmo-3 clone by UV-inactivated C.muridarum-pulsed bm12 splenocytes. With that small caveat,all three CD4 T-cell clones clearly recognize Chlamydia anti-gens presented by an MHC-II molecule.

Having demonstrated that the CD4 T-cell clones wereMHC-II restricted, we asked whether the CD4 coreceptor wasimportant during activation of CD4 T cells by infected epithe-lial cells. Infected epithelial cell targets were prepared by pre-treating C57epi.1 monolayers with IFN-� and then mock in-fecting them or infecting them with C. muridarum for 18 h.Eighteen hours postinfection the cell monolayers were har-vested and cocultured with the CD4 T-cell clones in the pres-ence of an anti-CD4 monoclonal antibody (GK1.5) or controlantibody. Twenty-four hours later culture supernatants wereharvested and assayed for IFN-� content by ELISA to measureT-cell activation. Engagement of the CD4 coreceptor by epi-thelial MHC-II was critical for activation of all three CD4T-cell clones (Fig. 4).

Chlamydia-specific CD4 T-cell clones do not recognize in-fected epithelial cells until 12 or more hours postinfection.The timing of CD4 T-cell recognition of infected epithelialcells during the course of infection is unknown. Bone marrow-derived macrophages pulsed with heat-killed C. muridarumand fixed at �2-h intervals showed nearly maximal activationof Chlamydia-specific CD4 T cells by 4 h post-antigenic pulse(34). To determine when CD4 T cells could recognize infectedepithelial cells over the time course of infection, C57epi.1monolayers were untreated or pretreated with IFN- plusIFN-� prior to infection (mock infection was time zero).C57epi.1 cells were pretreated with both IFN- and IFN-�because the reproductive tract epithelium of a wild-type mouseis likely exposed to IFN- and IFN-� during C. muridarumgenital tract infections (addressed in more detail in next sec-tion). IFN treatments and infections were staggered such thatall targets were ready at the same time. Monolayers wereharvested and cocultured with CD4 T-cell clones in the pres-ence of tetracycline. Tetracycline served to block progressionof the C. muridarum infection and additional protein synthesis,the source of the Chlamydia polypeptides that serve as T-cellantigens. Thirty-six hours later culture supernatants were har-vested to measure IFN-�, and experimental wells were pulsedwith [3H]thymidine to measure proliferation. CD4 T-cell

FIG. 3. MHC-II restriction of CD4 T-cell clones. (A) Representative staining of CD4 T-cell clone uvmo-2 with an isotype control antibody anda monoclonal antibody specific for CD4. (B) MHC restriction mapping with inbred mouse strains. CD4 T-cell clone cells (50,000) were coculturedwith 1 � 106 UV-inactivated C. muridarum-pulsed or mock-pulsed irradiated naive splenocytes for 36 h in the presence of tetracycline; then culturesupernatants were harvested and analyzed for IFN-� content by ELISA. Black bars, control C57BL/6J irradiated splenocytes mock pulsed(syngeneic); white bars, C57BL/6J irradiated splenocytes pulsed with 2.5 IFU equivalents of UV-inactivated C. muridarum per cell (syngeneic); graybars, bm1 irradiated splenocytes pulsed with UV-inactivated C. muridarum (MHC-I mismatch); light gray hatched bars, bm12 irradiatedsplenocytes pulsed with UV-inactivated C. muridarum (MHC-II mismatch). Aggregate data from two independent experiments SEM are shown.**, P �0.01; ***, P � 0.001, comparing C57BL/6J cells to bm1 and bm12 cells; NS, not significant.



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clones were not stimulated to proliferate by infected epithelialcells at any time point during the course of infection; all cloneswere stimulated to proliferate by Chlamydia-pulsed irradiatedsplenocytes (data not shown). uvmo-1, -2, and -3 were acti-vated by infected epithelial cells, as determined by measuringproduction of IFN-� (Fig. 5).

For two of the three CD4 clones, pretreatment of the epi-thelial monolayer with IFN-/� improved T-cell activationcompared to that in untreated monolayers, as determined bymeasuring IFN-� production. Improved T-cell activation couldrepresent either increased engagement of the T-cell receptor(TCR) due to more MHC-II antigen complexes on the epithe-lial cell surface or changes in epithelial accessory moleculesthat augment the TCR signal. Plotting the data with a smallerIFN-� scale allows visualization of the earliest recognitionevents (Fig. 5). For CD4 T-cell clone uvmo-1, pretreatment ofthe epithelial monolayers with IFN-/� moved recognitionfrom �18 h in the untreated state to �12 h with IFN pretreat-ment; for CD4 clones uvmo-2 and uvmo-3, recognition ad-vanced from �18 h to �15 h postinfection. These experimentsshow that input antigen alone was not sufficient for CD4 T-cellrecognition, as infection had to progress for at least 12 h(before addition of tetracycline) to generate a CD4 T-cell-recognizable target. IFN pretreatment improved both CD4T-cell activation and recognition, though the magnitude ofthese effects varied by CD4 T-cell clone.

IFN-� augments epithelial activation of CD4 T cell clonesbut antagonizes IFN-� by blunting upregulation of MHC-II.The Chlamydia pathogenesis knockout mouse literature showscontrasting roles for type 1 and type 2 IFNs during clearance ofgenital tract infections. As a broad generalization, type 2 IFN(IFN-�) makes a positive contribution to clearance (7, 8), while

type 1 IFNs (IFN-�/) have a negative effect, recently docu-mented in the IFNAR1 knockout mouse (24). Physiologic lev-els of IFN-� have been documented in the genital secretions ofChlamydia-infected humans (2) and mice (7). IFN- is se-creted by infected epithelial cells (9, 10, 15) and is detectablein the genital secretions of mice infected vaginally with C.muridarum (W. A. Derbigny, personal communication). Thepresence of type 1 and type 2 IFNs in genital secretions duringinfection makes it likely that many reproductive tract epithelialcells are exposed to IFNs prior to becoming infected withChlamydia. We examined the role of type 1 and type 2 IFNs invitro using our reproductive tract epithelial cell line and CD4T-cell clones. We propose that untreated epithelial cells mimicthe IFN milieu of early infection (low IFN levels), IFN--pretreated cells mimic IFN-� knockout mice (IFN- with noIFN-�), IFN-�-pretreated cells mimic IFNAR1 knockout mice(IFN-� with no functional IFN-�/), and IFN-/�-pretreatedcells mimic wild-type mice.

C57epi.1 cells were untreated or pretreated for 14 h withIFN-, IFN-�, or IFN-/� and then infected with C. murida-rum. Eighteen hours postinfection the epithelial monolayerswere harvested and cocultured with CD4 T-cell clones in thepresence of tetracycline. Twenty-four hours later culture su-pernatants were harvested and analyzed for IFN-� to scoreT-cell activation (Fig. 6).

For all three CD4 T-cell clones, IFN- pretreatment ofepithelial cells prior to infection augmented CD4 T-cell acti-vation compared with that in untreated infected epithelialcells, with the caveat that the comparison for uvmo-3 had a Pvalue of 0.050 (Fig. 6A). IFN-� pretreatment impressively aug-mented T-cell activation beyond that seen with IFN- for allthree CD4 clones; interestingly, copretreatment with IFN-�and IFN- blunted IFN-� augmentation, though copretreat-ment was still better at activating the CD4 T-cell clones thanwas IFN- pretreatment alone. We hypothesized that theseresults could be explained by effects of type 1 and type 2 IFNson expression of epithelial MHC-II.

To look at the effects of type 1 and 2 IFNs on inducibleepithelial cell surface MHC-II expression in the setting of C.muridarum infection, we used the same protocol that was usedfor preparing the infected epithelial targets described above.C57epi.1 cells were untreated or pretreated with IFN-,IFN-�, or IFN-/� prior to infection. Eighteen hours postin-fection the epithelial monolayers were harvested, stained forMHC-II, and analyzed by flow cytometry (Fig. 6B). As hypoth-esized, the levels of CD4 T-cell activation correlated with therelative amounts of epithelial cell surface MHC-II induced bythe different IFN pretreatments: untreated � IFN- pre-treated � IFN-/� pretreated �� IFN-� pretreated. The rela-tive cell surface levels of MHC-II correlate with the publishedC. muridarum clearance rates from IFN knockout mice: IFN-�knockout (IFN- pretreatment) � wild type (IFN-/� pre-treatment) �� IFNAR1 knockout (IFN-� pretreatment).

DISCUSSION

Derivation of biologically intact upper reproductive tractepithelial cell lines and Chlamydia-specific CD4 T-cell clonesfrom mice that self-cleared primary C. muridarum genital tractinfections gave us the opportunity to investigate basic Chla-

FIG. 4. Chlamydia-specific CD4 T-cell clones use the CD4 core-ceptor during recognition of infected C57epi.1 epithelial cells.C57epi.1 epithelial cells were pretreated with 10 ng/ml IFN-� for 14 h,infected with 3 IFU/cell for 18 h, treated with mitomycin C, harvested,and used as T-cell targets. CD4 T-cell clone cells (50,000) were cocul-tured with 50,000 mock-infected or infected epithelial cells in thepresence of tetracycline and either 10 �g/ml isotype control antibodyor 10 �g/ml anti-CD4 monoclonal antibody GK1.5. Culture superna-tants were collected after 24 h and analyzed for IFN-� content byELISA. Aggregate data from two independent experiments SEMare shown. ***, P � 0.001, comparing anti-CD4 treatment to theantibody control.



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mydia pathogenesis questions related to CD4 T-cell interac-tions with infected epithelial cells. The first question addressedwas whether Chlamydia-specific CD4 T cells could recognizeinfected reproductive tract epithelial cells. Compared with pro-fessional APCs, epithelial cells express low levels of MHC-II,lack critical costimulatory molecules, and lack an obvious an-tigen presentation pathway for Chlamydia antigens. Therehave been doubts about whether CD4 T cells directly mediateclearance through interactions with infected reproductive tractepithelial cells (29), though depletion studies show a dominantrole for CD4 T cells in primary infection (23) and knockoutmice show an absolute dependence on MHC-II for clearanceof C. muridarum from the genital tract (22). Data presented

here clearly show that Chlamydia-specific CD4 T cells canrecognize infected epithelial cells in antigen-specific fashion,including engagement of the CD4 coreceptor by epithelialMHC-II molecules.

We investigated the timing of Chlamydia-specific CD4 T-cellrecognition of infected epithelial cells over the course of in-fection. When T cells recognize infected targets is an importantmechanistic consideration, as delayed recognition narrows thewindow for an effector mechanism to act before noninfectiousreticulate bodies transition to infectious elementary bodies.We found that CD4 T-cell recognition of infected epithelialcells occurred roughly 12 to 18 h postinfection and that recog-nition was influenced by type 1 and 2 IFNs. This window of

FIG. 5. CD4 T-cell clone recognition of infected epithelial cells over the time course of infection, showing the influence of IFNs on recognition.C57epi.1 epithelial monolayers were untreated (squares) or pretreated with IFN-/� (100 units per ml or 10 ng per ml; circles) for 14 h and theninfected with 3 IFU C. muridarum per cell in staggered fashion over a time course of infection going from 0 h (mock infected) to 21 h. Infectedmonolayers were treated with mitomycin C just prior to harvest. CD4 T-cell clone cells (50,000) were cocultured with 50,000 epithelial targets for36 h in the presence of tetracycline (10 �g/ml); culture supernatants were collected and analyzed for IFN-� content by ELISA. Complete,complete time course from 0 h to 21 h; early events, time course from 0 h to 15 h plotted on a more sensitive IFN-� scale to visualize earlylow-level IFN-� production. Aggregate data from two experiments SEM are shown. *, P � 0.05, comparing each time point within atreatment (untreated or IFN-/�) to its time zero (mock infection); #, P � 0.05, comparing IFN-/�-pretreated to untreated epithelial cellsat each time point that was �0 h.



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time is relatively late during the replication cycle of C. muri-darum in oviduct epithelial cells (26) but is theoretically earlyenough to allow T-cell-mediated disruption of replication byeither disabling the epithelial host cell (incubator) or by di-rectly attacking the inclusion by, for example, production ofnitric oxide (13).

We stringently tested whether our CD4 T-cell clones couldcontrol Chlamydia replication in vitro by using them at aneffector-to-target ratio of �1 in flat-bottom tissue cultureplates, which provide an additional surface area challenge forphysically small lymphocytes. Eight of our 10 CD4 T-cellclones controlled replication of C. muridarum in the oviductepithelial cell line even though T-cell activation by infectedepithelial cells was clearly submaximal, as reflected by lowerIFN-� production (50- to 100-fold) and lack of proliferationcompared with activation by antigen-pulsed irradiated spleno-cytes. Interestingly the CD4 T cells that could control replica-tion in vitro had two phenotypes. One set of CD4 clones(uvmo-1, -2, and -3) was able to control C. muridarum repli-cation with or without IFN-� pretreatment of epithelial cells;the other set (LN4-10, -12, and -13 and Spl4-10 and -11) wasdependent on IFN-� pretreatment of the epithelial cells priorto infection. We propose that the existence of Chlamydia-specific CD4 T-cell clones that recognize and control C. muri-darum replication in epithelial monolayers without IFN-� invitro explains the in vivo observation that 99.9% of C. murida-rum is cleared from the genital tracts of IFN-� knockout micewith nearly normal kinetics (6, 27). We hypothesize that thisIFN-�-independent subset of CD4 T cells recognize Chlamydiaantigens that are efficiently processed, making it to the cellsurface bound to MHC-II molecules at very low levels of

MHC-II expression. Alternatively, this T-cell subset may havehigh-affinity TCRs that allow activation with relatively few an-tigen-MHC complexes on the epithelial cell surface. While it isnot possible to draw conclusions based on the limited numbersof T-cell clones from each derivation strategy, it is interestingthat the three most effective CD4 clones came from a culturesystem in which the APC was a naive splenocyte pulsed withUV-inactivated C. muridarum. Because the inoculum was low,�2.5 IFU per naive splenocyte, this suggests either very effi-cient processing of select C. muridarum antigens or a bias ofthe culture system toward selection of T cells with high-affinityTCRs. Either scenario suggests that development of a success-ful subunit vaccine could depend on choosing a specific Chla-mydia antigen(s).

The source of the Chlamydia-specific CD4 T-cell clones didnot appear to be predictive of their ability to control in vitroreplication of C. muridarum in epithelial cells. The most effec-tive and least effective CD4 T-cell clones came from thespleens of immune mice. CD4 clones derived from lymphnodes draining the reproductive tract were not more effectivethat those from splenocytes. The finding that Chlamydia-spe-cific CD4 T cells (uvmo-4 and LN4-11) could be activated yetineffective for controlling replication raises the possibility thatineffective CD4 T-cell responses contribute to inflammatorydamage without contributing to clearance.

It is tempting to attribute T-cell-mediated control of Chla-mydia replication in vitro to an indirect IFN-�-based mecha-nism such as induction of nitric oxide because the three mosteffective CD4 T-cell clones (uvmo-1, -2, and -3) make the mostIFN-� in response to infected epithelial cells. However pro-duction of IFN-� in response to infected epithelial cells only

FIG. 6. IFN- and IFN-� pretreatment of epithelial cells had different effects on epithelial MHC-II expression and activation of CD4 T-cellclones. (A) C57epi.1 epithelial cells were untreated or pretreated with IFN- (100 units/ml), IFN-� (10 ng/ml), or IFN-/� (100 units or 10 ng perml) for 14 h, infected with 3 IFU C. muridarum per cell for 18 h, treated with mitomycin C just prior to harvest, and used as targets. CD4 T-cellclone cells (50,000) were cocultured with 50,000 epithelial cells in the presence of tetracycline (10 �g/ml); culture supernatants were collected at24 h and analyzed for IFN-� content by ELISA. Black bars, no pretreatment; white bars, IFN- pretreatment; gray solid bars, IFN-� pretreatment;gray hatched bars, IFN-/� pretreatment. Aggregate data from three independent experiments SEM are shown. *, P � 0.05, comparing eachcondition to untreated cells and IFN-/�-pretreated cells to IFN--pretreated cells (bracket). (B) MHC-II expression in untreated 18 h-infectedC57epi.1 cells versus that in cells pretreated for 14 h with IFN-, IFN-�, or IFN-/� followed by infection for 18 h. Eighteen hours postinfectionthe cells were harvested and stained for MHC-II (approximating the conditions for panel A). Numbers in parentheses are the mean fluorescencevalues in arbitrary units for each experimental condition. Data shown are representative of two independent experiments.



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loosely correlated with the ability of individual CD4 T-cellclones to control C. muridarum replication, and none of thefive CD4 T-cell clones dependent on IFN-� pretreatment wereable to control C. muridarum replication when exogenousIFN-� was added to epithelial monolayers at the time of in-fection, arguing against a direct effect of IFN-�. In addition, C.muridarum replicating in murine epithelial cells (this report;26, 31) and mouse embryonic fibroblasts (1, 4) is largely indif-ferent to recombinant IFN-�. This makes it highly unlikely thatIFN-� of T-cell origin, which would not be present until severalhours after recognition (12 to 18 h postinfection), could di-rectly contribute to the anti-Chlamydia effector mechanism ofthese clones. Inhibition of nitric oxide production does notinterfere with the ability of CD4 T-cell clones (uvmo-1, -2, and-3) to control C. muridarum replication in vitro (our unpub-lished data). Our data support the hypothesis that the majorrole of IFN-� at the epithelial interface during infection is toupregulate MHC-II on epithelial cells in order to efficientlyactivate Chlamydia-specific CD4 T cells.

We showed that infection modestly blunted upregulation ofcell surface MHC-II when IFN-� was added at the time ofinfection but not when added 14 h prior to infection, consistentwith data published by Zhong et al. for C. trachomatis serovarL2 (38). We hypothesize that, for the CD4 T-cell clones de-pendent on IFN-� pretreatment, IFN-� is needed to upregu-late MHC-II in order to increase the availability of MHC-IImolecules to present Chlamydia antigens at the cell surface,though we cannot rule out IFN-� effects on antigen processing.At the low levels of epithelial cell surface MHC-II seen withsimultaneous IFN-� addition and infection, the modest infec-tion-associated blunting of MHC-II upregulation resulted insignificant attenuation of CD4 T-cell-mediated control of C.muridarum replication for the five IFN-�-dependent CD4 T-cell clones, presumably through decreasing T-cell recognitionand/or activation. The in vivo importance of infection interfer-ence with IFN-�-mediated upregulation of MHC-II, however,is unclear, as Darville et al. (7) have shown physiologic IFN-�levels in genital secretions by day 3 post-C. muridarum infec-tion and El-Asrar et al. have shown upregulated epithelialMHC-II in human trachoma clinical specimens (11, 12). Thesedata combined with our in vitro IFN-� pretreatment data sug-gest that the host may be able to overcome this evasion tacticby “pretreating” the vulnerable reproductive tract epitheliumwith IFN-� prior to impending infection of individual epithelialcells.

Type 1 and 2 IFNs modulated T-cell activation by infectedepithelial cells in this report, and C. muridarum infections ofIFN knockout mice have yielded interesting results in the ex-isting literature. In wild-type mice both type 1 (IFN-�/) andtype 2 (IFN-�) IFNs are part of the inflammatory milieu duringChlamydia genital tract infections. Nagarajan et al. have shownthat IFNAR1 knockout mice lacking the receptor for IFN-�/clear C. muridarum from the genital tract more efficiently andwith less oviduct pathology than wild-type mice (24). Weshowed here that IFN-� pretreatment of epithelial cells dra-matically improved CD4 T-cell activation by infected epithelialcells and that this effect was greatly attenuated when IFN-was included during pretreatment. The rate of C. muridarumgenital tract clearance by different mouse strains has beencorrelated with the timing and magnitude of IFN-� responses

(7, 8), which occur before demonstrable recall cellular immu-nity has developed (35). It is possible that the balance of IFN-versus IFN-� within an individual may be an important param-eter affecting resolution of genital tract infections.

IFN- attenuation of IFN-�-enhanced T-cell activation cor-related with marked attenuation of IFN-�-induced MHC-IIexpression on C57epi.1 epithelial cells. This finding is consis-tent with previously described IFN- blockade of IFN-�-induc-ible MHC-II expression (18). IFNAR1 knockout mice likelyclear C. muridarum from the genital tract faster because thereproductive tract epithelium expresses higher levels of MHC-II, and on that basis infected epithelial cells more efficientlyactivate the CD4 T cells, allowing a larger fraction of Chlamy-dia-specific CD4 T cells to participate in clearance. TheMHC-II level on IFN-/�-pretreated C57epi.1 cells was onlyslightly higher than that on IFN--pretreated cells, and bothwere higher than levels on untreated cells. That is, IFN-�knockout mice (type 1 only IFN milieu) likely have some ep-ithelial MHC-II, and that level is sufficient for IFN-�-indepen-dent CD4 T-cell clones like uvmo-1, -2, and -3, described inthis report, to mediate clearance, albeit with a lower efficiency,consistent with the observed low-level residual genital tractshedding of C. muridarum in IFN-� knockout mice.

Based on the demonstrated ability of C. muridarum-specificCD4 T-cell clones to recognize infected upper reproductivetract epithelial cells and control Chlamydia replication in them,it is reasonable to interpret the CD4 depletion data (23) andMHC-II knockout mouse study (22) as showing a critical rolefor CD4 T cells in directly clearing Chlamydia from the genitaltract. These results do not address the controversial role ofCD8 T cells and, in our view, do not rule out a redundantnoncritical role for CD8 T cells in genital tract clearance. Ourdata suggest that not all Chlamydia-specific Th1 cells, eventhose generated during the course of a genital tract infection,are capable of contributing to clearance. Therefore, identifi-cation of the protective CD4 T-cell subsubset(s) and the anti-gens that they recognize may be critical for development of aneffective Chlamydia vaccine.

ACKNOWLEDGMENTS

We have no conflicts of interest related to this study.We thank Wilbert Derbigny for his thoughtful critique of the manu-

script as it was being developed.This research was funded by NIH T32-AI060519, 1R01AI070514-

01A1, and the Showalter Trust.

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