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*Substance via MeSH
Medline Plus Health Information
*Chlamydia Infections
The Journal of Immunology, 2006, 177: 7974-7979.
Copyright © 2006 by The American Association of Immunologists, Inc.

Antigen-Specific CD8+ T Cells Respond to Chlamydia trachomatis in the Genital Mucosa1

Nadia R. Roan and Michael N. Starnbach2

Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Following sexual transmission, Chlamydia trachomatis specifically targets genital tract epithelial cells. Because epithelial cells are readily recognized by CD8+ T cells, the response of CD8+ T cells to Chlamydia infection has been explored in a number of studies. It has been shown that CD8+ T cells are present in the genital tracts of mice following C. trachomatis infection, but the specificity of these T cells has remained undefined. To determine whether Chlamydia-specific CD8+ T cells migrate to the genital tract in response to Chlamydia infection, we generated retrogenic mice that express a TCR specific for a Chlamydia-specific T cell Ag CrpA. T cells from the retrogenic mice were transferred into naive recipient animals to increase the frequency of Chlamydia-specific T cells to a level at which they could be tracked during primary infection. We observed that the Chlamydia-specific retrogenic T cells proliferated in lymph nodes draining the genital tract in response to genital infection with C. trachomatis. Furthermore, we found that these cells acquired the ability to produce IFN-{gamma} and migrated into the genital mucosa of the infected mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The bacterium Chlamydia trachomatis is an obligate intracellular pathogen that is the most common cause of bacterial sexually transmitted disease in the United States and a major public health concern. In mouse models of Chlamydia infection, CD8+ T cells can enhance the clearance of the bacteria. CD8+ T cells can be detected at the site of infection (1, 2) and adoptive transfer of immune CD8+ T cells can protect against Chlamydia challenge (3, 4, 5).

Interestingly, Ags recognized by CD8+ T cells are typically derived from cytosolic proteins, yet C. trachomatis is strictly confined to a vacuole during the intracellular stages of development. Because the majority of C. trachomatis proteins also are confined to the vacuole, they are not typically processed for recognition by CD8+ T cells. However, during replication there appears to be a limited subset of Chlamydia proteins that do have access to the cytoplasm, subjecting them to possible recognition by CD8+ T cells. These include membrane proteins that lie at the interface between the Chlamydia vacuole and the host cytosol (6). Two of these proteins, Cap1 and CrpA, are recognized by CD8+ T cells following systemic Chlamydia infection (7, 8). Of the two Ags, CrpA elicits the more dominant CD8+ T cell response following Chlamydia infection. Immunization of mice with recombinant vaccinia expressing CrpA provides immunity against systemic Chlamydia challenge, suggesting that CrpA-specific CD8+ T cells can play a role in limiting infection with C. trachomatis (8).

Infection with C. trachomatis occurs naturally in the genital tract, where the organisms have a strict tropism for epithelial cells (9). Although CD4+ T cells contribute significantly in controlling Chlamydia infection (10, 11), they may not recognize Chlamydia-infected epithelial cells in the genital mucosa because epithelial cells typically express low levels of MHC class II. Because epithelial cells express abundant levels of MHC class I, we sought to determine whether Chlamydia-specific CD8+ T cells are recruited to the genital epithelium in response to Chlamydia infection of the genital tract. Specifically, we wanted to track the localization of CrpA-specific T cells, their proliferation, and their effector functions during the early stages of primary Chlamydia infection. However, one difficulty in studying Ag-specific T cell responses in vivo is the low frequency of these cells in a naive mouse. To increase the number of Ag-specific T cells so that the initial events following infection can be monitored, investigators have transferred T cells from TCR transgenic mice into naive mice (12, 13, 14). Alternatively, Ag-specific T cells can be obtained from TCR retrogenic mice generated using a newly developed technique that combines retroviral transduction and bone marrow reconstitution (15). Retrogenic mice offer a number of advantages over traditional TCR transgenic mice, including a faster production time. We have used this new approach to generate retrogenic mice expressing a TCR specific for CrpA. T cells from these mice have allowed us to examine the priming and trafficking of Chlamydia-specific CD8+ T cells following genital infection with C. trachomatis. We discovered that CrpA-specific retrogenic T cells proliferate in the draining lymph nodes (DLN)3 following genital infection with C. trachomatis. These T cells then develop effector function and migrate into the infected genital mucosa. We describe in this study a new tool for studying Ag-specific CD8+ T cells in the genital mucosa following infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female C57BL/6 mice (H-2b) and RAG1–/– mice (H-2b) were obtained from The Jackson Laboratory and used at 7–9 wk of age. All experiments were approved by the Institutional Animal Care and Use Committee.

Tissue culture and cell lines

Cell line 1308.1 was maintained in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FBS, L-glutamine, 50 µM 2-ME, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cell lines 293T and GP plus E86 (16) were maintained in complete DMEM (Invitrogen Life Technologies) supplemented with 10% FBS, L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Bone marrow derived macrophages (BMM) were cultured from C57BL/6 mice by culturing bone marrow cells in RPMI 1640 (Invitrogen Life Technologies) supplemented with 20% FBS, L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 20% supernatant from L929 cells (American Type Culture Collection).

Growth, isolation, and detection of C. trachomatis

Elementary bodies of C. trachomatis serovar L2 434/Bu were propagated within McCoy cell monolayers grown in MEM (Invitrogen Life Technologies) supplemented with 10% FCS, 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate and purified by density gradient centrifugation as described (17). Aliquots were stored at –70°C in a medium containing 250 mM sucrose, 10 mM sodium phosphate, and 5 mM L-glutamic acid (SPG medium) (18) and thawed immediately before use. UV-inactivated C. trachomatis organisms were generated by subjecting purified elementary bodies to 10 min of UV irradiation.

Stimulation and maintenance of CD8+ T cells

C57BL/6 females were immunized with a recombinant vaccinia construct expressing CrpA (8). Splenocytes from mice harvested 21 day after immunization were cultured with irradiated (2000 rad) Chlamydia-infected BMM (multiplicity of infection 5:1) and naive, syngeneic splenocytes in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FBS, L-glutamine, 50 µM 2-ME, 50 U/ml penicillin, 50 µg/ml streptomycin, {alpha}-methyl mannoside, and 5% supernatant from Con A-stimulated rat spleen cells. CD4+ T cells were depleted using Dynabeads Mouse CD4 according to the manufacturer’s protocol (Invitrogen Life Technologies). The CD8+ T cells were restimulated with fresh Chlamydia-infected BMM every 7 days. The resulting T cell line was cloned by limiting dilution to generate an individual T cell clone we designated NR23.4. Chromium release assays were conducted as previously described (3).

Protection assay

Ten days following restimulation, 5 x 106 NR23.4 T cells were transferred into naive C57BL/6 recipient mice. Control immune mice were infected 3 wk earlier with 107 inclusion forming units (IFU) of C. trachomatis L2. All mice were then challenged i.v. with 107 IFU of C. trachomatis L2 and spleens were harvested 3 days later. To quantitate C. trachomatis, spleens from infected mice were homogenized, sonicated, diluted in SPG medium, and applied to McCoy cell monolayers. Inclusions were enumerated by immunofluorescent microscopy 36 h postinfection and reported as IFU per spleen.

Flow cytometry and Abs

FITC, PE, PerCP, or allophycocyanin conjugated Abs specific for CD4, CD8, CD90.2, and IFN-{gamma} were purchased from BD Biosciences. The Db/CrpA63–71 tetramer was obtained from the National Institute of Allergy and Infectious Diseases tetramer facility, whereas the Db/GP33–41 tetramer was purchased from Immunomics. Data were collected on a BD Biosciences FACSCalibur flow cytometer and analyzed using CellQuest software. Intracellular cytokine staining of NR23.4 was performed by stimulating cells for 5 h in the presence of GolgiPlug (BD Biosciences) and untreated, peptide-pulsed (100 nM of either CrpA63–71 or YopH249–257) or Chlamydia-infected (multiplicity of infection 5:1) BMM. Intracellular cytokine staining of retrogenic cells was performed by stimulating cells for 4 h in the presence of PMA (50 ng/ml; MP Biomedicals), ionomycin (1 µg/ml; Sigma-Aldrich), and GolgiPlug (BD Biosciences). Cells were permeabilized with the Cytofix/Cytoperm Plus kit (BD Biosciences). PE-conjugated rat IgG1 (BD Biosciences) was used as an isotype control Ab. In experiments with SNARF, the SNARF-labeled retrogenic cells injected into the flow cytometer were live and identified by gating on GFP+ cells. In experiments with CFSE, retrogenic cells needed to be fixed and permeabilized following cell surface staining to eliminate GFP fluorescence, which masks CFSE fluorescence when analyzed by flow cytometry. The CFSE-labeled retrogenic T cells were identified by staining for CD90.2 instead of gating on GFP+ cells.

Retroviral constructs and producer cell lines

The rearranged TCR from NR23.4 used the V{alpha}4JTA13 and Vbeta8.2Jbeta2.5 receptor chains. Total RNA was isolated from NR23.4 using Ultraspec RNA (Biotecx), treated with RNase-free DNase (Promega), and converted into cDNA using MMLV Reverse Transcriptase (Invitrogen Life Technologies). A TCR construct was produced by recombinant PCR and cloned into a 2A peptide-linked, GFP-labeled retroviral vector previously described (15, 19, 20). Expression of the construct was verified using TransIT-293 (Mirus) to transiently transfect 293T cells with each construct along with a multicistronic CD3 vector provided by D. Vignali (St. Jude Children’s Research Hospital, Memphis, TN). A retroviral producer cell line was generated as described (19, 20). Briefly, 293T cells were transiently transfected using TransIT-293 (Mirus) and a retroviral producer cell line was generated by repeated transduction of GP plus E86 cells with retroviral supernatant. The OTI retroviral producer cell line was also provided by D. Vignali, St. Jude Children’s Research Hospital.

Retroviral-mediated stem cell gene transfer

Retroviral transduction of murine bone marrow cells was performed as described (15, 19, 20). Briefly, 8- to 10-wk-old RAG1–/– donor mice were injected with 5 mg of 5-fluoracil (InvivoGen). Two days later, bone marrow cells were harvested and cultured in complete DMEM (Invitrogen Life Technologies) with 20% FBS, 20 ng/ml IL-4, 50 ng/ml IL-6, and 50 ng/ml stem cell factor (PeproTech). One day later, bone marrow cells were added to the retroviral producer cell lines described earlier and transduced for another 2 days. Approximately 3 x 106 transduced bone marrow cells were injected i.v. with 2 U/ml heparin (Sigma-Aldrich) into irradiated (450 rad) RAG1–/– recipient mice. Mice were analyzed for TCR expression by orbital bleeding 6–10 wk after transplantation.

Adoptive transfer and tissue preparation

A single cell suspension of spleens and peripheral lymph nodes was harvested from retrogenic mice and stained with the fluorescent dyes SNARF (3 µg/ml; Molecular Probes) or CFSE (5 µM; Molecular Probes). Recipient mice were injected i.v. with 1–2 x 106 labeled retrogenic cells. Where indicated, mice were infected i.v. with 107 IFU of C. trachomatis L2 1 day after the transfer of cells. For genital tract infections, mice were treated with 2.5 mg of medroxyprogesterone acetate s.c. 1 wk before infection to synchronize the mice into a diestrus state (21, 22). Intrauterine infection was conducted by inoculating the uterus with 106 IFU of C. trachomatis serovar L2 in 5 µl of SPG medium. At various times postinfection, single cell suspensions of splenocytes or DLN taken from the iliac lymph nodes were prepared and analyzed by flow cytometry as described earlier. To isolate cells from the genital mucosa, genital tracts (oviduct, uterus, and cervix) were removed from mice and digested with collagenase (type XI; Sigma-Aldrich) for 1 h before staining and flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation and characterization of a CrpA63–71-specific CD8+ T cell clone

To generate the retrogenic mice used in this study, we first identified and cloned a CrpA-specific TCR that the mice would be engineered to express. This TCR was obtained from a T cell clone designated NR23.4 that was generated by stimulating splenocytes from a CrpA immune mouse on Chlamydia-infected BMM. NR23.4 bound a tetramer consisting of H2-Db complexed to the CrpA epitope CrpA63–71 but not a control tetramer consisting of H2-Db complexed to GP33–41 from lymphocytic choriomeningitis virus (Fig. 1A), suggesting that the TCR expressed by NR23.4 was CrpA63–71-specific.


Figure 1
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  		FIGURE 1. CD8+ T cell clone NR23.4 is CrpA63–72-specific and recognizes Chlamydia-infected cells in vitro. A, NR23.4 was stained with a control GP33–41-specific (thin line histogram) or a CrpA63–71-specific (thick line histogram) tetramer. Results were gated on live, CD8+ T cells. B, NR23.4 was incubated with BMMs that were untreated, CrpA63–71-pulsed, YopH249–257-pulsed, or Chlamydia-infected as indicated. Intracellular IFN-{gamma} levels were determined after 5 h in the presence of brefeldin A. The values shown in the gated region correspond to the percentage of cells that are CD8+IFN{gamma}+. C, NR23.4 was tested on uninfected or Chlamydia-infected 1308.1 epithelial cells in a 51Cr release assay.

 
To further verify that NR23.4 was specific for CrpA63–71, we tested whether the clone produced IFN-{gamma} in response to CrpA63–71. Previous work from our laboratory has shown that the protective ability of Chlamydia-specific CD8+ T cells depends on the ability of these cells to secrete IFN-{gamma} (23). We incubated NR23.4 with CrpA63–71-pulsed BMM and measured IFN-{gamma} production by intracellular cytokine staining. As shown in Fig. 1B, NR23.4 produced significant levels of IFN-{gamma} in response to CrpA63–71-pulsed BMM. The response was specific for CrpA63–71 because BMM pulsed with the H2-Db-restricted peptide epitope YopH249–257 from Yersinia enterocolitica did not stimulate NR23.4 to secrete IFN-{gamma}. Furthermore, NR23.4 produced IFN-{gamma} in response to BMM infected with C. trachomatis, demonstrating that NR23.4 recognized CrpA63–71 during Chlamydia infection of cells in vitro. Lastly, NR23.4 also recognized and lysed Chlamydia-infected 1308.1 epithelial cells, demonstrating that this T cell clone recognized Chlamydia-infected cells in the context of an epithelial cell infection (Fig. 1C).

Adoptive transfer of NR23.4 protects against C. trachomatis challenge

To determine whether T cell clone NR23.4 conferred protection against C. trachomatis infection, NR23.4 T cells were transferred into naive C57BL/6 mice. The mice were then challenged i.v. with 107 IFU of C. trachomatis L2, and spleens were titered 3 days later. As shown in Fig. 2, mice that had received NR23.4 had ~10-fold fewer C. trachomatis IFU in their spleen relative to mice that had not received NR23.4. The level of protection observed in naive mice that had received NR23.4 appeared to be comparable to the level of protection observed in immune mice that had recovered from C. trachomatis infection.


Figure 2
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  		FIGURE 2. Adoptive transfer of NR23.4 protects against C. trachomatis infection. Naive mice, immune mice that had recovered from a prior infection, or naive mice transferred with 5 x 106 NR23.4 T cells were infected with 107 IFU of C. trachomatis L2. The number of infectious organisms present in the spleens of these mice was determined 3 days later.

 
Generation of CrpA63–71-specific retrogenic mice

Although we were able to use NR23.4 to demonstrate the specificity and protective ability of CrpA63–71-specific T cells, the NR23.4 clone described has been restimulated extensively in vitro. In vitro restimulation renders the T cells less useful in trafficking studies in vivo because cells that have been cultured extensively do not retain properties of cells that have never encountered Ag (24). To generate an abundant source of CrpA63–71-specific T cells that have not previously encountered Ag, we used a recently described technique to generate retrogenic mice (15, 20). We isolated cDNA from the NR23.4 T cell clone and cloned the CrpA63–71-specific TCR into a retroviral vector marked by an internal ribosomal entry site-encoded GFP (19). Retroviral particles encoding the NR23.4 TCR were packaged and used to transduce RAG1–/– bone marrow cells. We then reconstituted irradiated RAG–/– recipient mice with the transduced bone marrow cells to produce retrogenic mice expressing the NR23.4 TCR. By ~10 wk following bone marrow transplantation, CD8+ T cells, but not CD4+ T cells, were efficiently selected in the thymus of the retrogenic mice (Fig. 3A). This result was in contrast to a C57BL/6 thymus, where both CD4+ and CD8+ cells were efficiently selected. In the peripheral blood of the retrogenic mice, there was a significant population of H2-Db/CrpA63–71 tetramer-binding GFP+ cells that did not bind a control H2-Db/GP33–41 tetramer (Fig. 3B). Together, these results showed that we successfully generated retrogenic mice that can be exploited as an abundant source of CrpA63–71-specific T cells for use in analyzing Chlamydia-specific T cell responses in vivo.


Figure 3
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  		FIGURE 3. CrpA63–71-specific retrogenic mice have a high frequency of CrpA63–71-specific T cells. A, Thymocytes from C57BL/6 mice or the retrogenic (Rg) mice expressing the NR23.4 TCR were analyzed for CD4 and CD8 expression. Results for retrogenic thymocytes were gated on live GFP+ cells to identify cells that had been transduced. B, Peripheral blood from the retrogenic mice was stained with a CrpA63–71-specific or a GP33–41-specific tetramer.

 
CrpA63–71-specific CD8+ retrogenic T cells proliferate following systemic Chlamydia infection in vivo

To test whether the CrpA63–71-specific retrogenic T cells responded to infection in vivo, we tested whether they proliferated following systemic Chlamydia infection. We labeled the CrpA63–71-specific retrogenic T cells with the fluorescent dye SNARF and adoptively transferred 1–2 x 106 labeled cells into C57BL/6 recipients. SNARF is a fluorescent dye that partitions equally among daughter cells as they divide, so cells that have proliferated have a lower fluorescence intensity than cells that have not proliferated. One day following the transfer, recipient mice were infected i.v. with 107 IFU of C. trachomatis L2. As shown in Fig. 4, infected mice had a significant population of retrogenic cells with low levels of SNARF, suggesting that these cells had proliferated in response to infection. In addition, proliferation of the CrpA63–71 retrogenic T cells appeared to be Ag-specific as OVA257–264-specific retrogenic T cells failed to proliferate following Chlamydia infection. Lastly, UV-inactivated C. trachomatis did not stimulate the CrpA63–71-specific retrogenic cells to proliferate significantly. These results are consistent with the inability of UV-inactivated C. trachomatis to develop into the replicating developmental form, thereby limiting production of CrpA and/or access of CrpA to the MHC class I processing pathway.


Figure 4
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  		FIGURE 4. CrpA63–71-specific retrogenic T cells proliferate in response to systemic C. trachomatis infection in vivo. One to two million of the CrpA63–71- or OVA257–264-specific SNARF-labeled retrogenic (Rg) T cells were transferred into C57BL/6 recipients. One day later, groups of mice were infected i.v. with 107 IFU of live C. trachomatis or with an equivalent dose of UV-inactivated C. trachomatis. Splenocytes were harvested 3 days later to determine the extent to which retrogenic T cells had proliferated. Retrogenic T cells were identified by gating on live GFP+ cells.

 
Following genital infection with C. trachomatis, CrpA63–71-specific CD8+ T cells proliferate, produce IFN-{gamma}, and migrate to the infected genital tract

Because C. trachomatis is primarily a pathogen of the genital mucosa, our goal was to determine whether CrpA63–71-specific CD8+ T cells were stimulated during genital infection with C. trachomatis. We therefore tested whether intrauterine inoculation with C. trachomatis induced CrpA63–71-specific retrogenic T cells to proliferate. To monitor proliferation, we labeled CrpA63–71-specific retrogenic T cells with the fluorescent dye CFSE that, like SNARF, also is diluted in cell populations as they proliferate. CFSE-labeled retrogenic T cells, which expressed CD90.2, were transferred into CD90.1 congenic recipients so that transferred cells could be readily identified by staining for CD90.2. Recipients were then either mock infected or infected in the uterus with 106 IFU of C. trachomatis. At various times postinfection, cells from the DLN were harvested and analyzed by flow cytometry. No proliferation was observed 3 and 4 days postinfection, whereas by 5 days postinfection retrogenic T cells in the DLN began proliferating (data not shown). By 6 days postinfection, extensive proliferation had occurred in the DLN of infected but not mock-infected mice (Fig. 5A). These results suggested that the CrpA63–71-specific retrogenic T cells were stimulated in the regional lymph nodes following infection of the genital tract with C. trachomatis.


Figure 5
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  		FIGURE 5. CrpA63–71-specific retrogenic T cells proliferate, produce IFN-{gamma}, and migrate to the genital mucosa of mice infected in the uterus with C. trachomatis. One to two million CFSE-labeled CD90.2+ CrpA63–71-specific retrogenic T cells were transferred into CD90.1+ female recipients, which were then mock infected or infected in the uterus with 106 IFU of C. trachomatis L2. A, Retrogenic cells from the DLNs of mock infected or infected recipients were examined for CFSE fluorescence. The dashed line marks the average CFSE fluorescence intensity of cells that did not proliferate. Results were gated on live, CD90.2+CD8+ cells to identify the CrpA63–71-specific retrogenic T cells. B, Genital tract tissue from mock-infected (top left) or infected (top right) recipients was examined for the presence of the retrogenic cells. Results were gated on live cells. Retrogenic T cells from the infected genital tract (bottom) were examined for CFSE fluorescence. The dashed line marks the average CFSE fluorescence intensity of cells in the DLNs that did not proliferate. Results were gated on live, CD90.2+ CD8+ cells to identify the CrpA63–71-specific retrogenic T cells. C, Retrogenic T cells from the DLNs or genital tract (GT) tissue of mock infected or infected recipients were examined for IFN-{gamma} production following PMA/ionomycin stimulation. IFN-{gamma} production in the genital tract of uninfected recipients was not determined due to the absence of retrogenic T cells in this tissue. Results were gated on live, CD90.2+ CD8+ cells.

 
To determine whether Chlamydia-specific CD8+ T cells that had proliferated in the DLN migrated into the genital mucosa, we examined whether the CrpA63–71-specific retrogenic T cells were present in the genital epithelium of infected mice. Retrogenic T cells were first observed in the genital mucosa 5 days postinfection (data not shown). By 6 days postinfection, significant numbers of the retrogenic T cells were present in the genital mucosa of the infected but not the mock-infected mice (Fig. 5B, top panels). Furthermore, although both cells that had not proliferated and cells that had proliferated were present in the DLN of infected mice (Fig. 5A), only cells that had proliferated were present in the infected genital mucosa (Fig. 5B, bottom panel). These results suggested that only activated CrpA63–71-specific CD8+ T cells migrated to the site of infection.

Because IFN-{gamma} is a crucial effector cytokine in the control of Chlamydia infection (23, 25, 26), we tested whether the proliferating CrpA63–71-specific T cells produced IFN-{gamma}. CFSE-labeled CrpA63–71-specific T cells were isolated from the DLN or genital tracts of mock infected or infected recipients and then stimulated ex vivo with PMA and ionomycin. Proliferating IFN-{gamma}-producing cells were first observed in the DLN and genital tract 5 days postinfection (data not shown) and further accumulated by 6 days postinfection (Fig. 5C). In summary, following introduction of C. trachomatis into the genital epithelia, Chlamydia-specific CD8+ T cells were primed, developed effector function, and migrated into the infected genital mucosa.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although CD8+ T cells can contribute to controlling Chlamydia genital infection (4), it has been unclear whether Chlamydia-specific CD8+ T cells actually migrate into the genital tract in response to infection. CD8+ T cells have been identified in the genital mucosa following C. trachomatis infection (1, 2), but it has been unclear whether these cells were specific for Chlamydia Ags or whether they were nonspecifically recruited by the inflammatory environment. To develop a system to identify and track the activity of Chlamydia-specific CD8+ T cells in vivo, we focused on CrpA63–71, a Chlamydia-specific CD8+ T cell Ag that we recently identified (8). We first generated the CrpA63–71-specific T cell clone NR23.4 and demonstrated its specificity in vitro. We then generated retrogenic mice expressing the CrpA63–71-specific TCR from NR23.4. By transferring the retrogenic cells into recipient mice, we determined that these CrpA63–71-specific T cells proliferated in the DLN in response to genital infection with C. trachomatis. Furthermore, the cells that had proliferated were readily detected in the infected genital tract, demonstrating that Chlamydia-specific CD8+ T cells migrate into the genital mucosa in response to infection.

CrpA63–71-specific CD8+ T cells were not detected in the uninfected genital tract, consistent with the notion that the genital mucosa lacks organized lymphoid elements and that naive T cells do not reside in this tissue (27). Naive T cells are, however, present in regional lymph nodes, and it is likely that Ags from the genital tract must drain to these nodes to activate T cells against genital pathogens. The activated T cells must subsequently migrate back to the site of infection. This appears to contrast with the intestinal mucosa, where Peyer’s patches situated within the intestinal wall can serve as a reservoir of naive T cells. It has been demonstrated that following oral infection with Salmonella, Salmonella-specific CD4+ T cells in the Peyer’s patches are activated within hours (28). Although the Salmonella study examined the response of CD4+ T cells while our study examined Chlamydia-specific CD8+ T cells, we have also recently demonstrated that activation of Chlamydia-specific CD4+ T cells appears to occur outside of the genital tract (29). Therefore, architectural differences between the genital and intestinal mucosa may result in distinct mechanisms for inducing immunity against pathogens that infect these tissues.

The CrpA63–71-specific CD8+ T cells that were recruited to the infected genital tract were able to produce the effector cytokine IFN-{gamma}. Several groups have demonstrated that IFN-{gamma} is required for resolution of Chlamydia infection (25, 30), and work in our laboratory suggests that IFN-{gamma} secretion is the key mechanism by which CD8+ T cells control Chlamydia replication (23). One way that IFN-{gamma} can control Chlamydia infection in vivo is by enhancing the ability of phagocytes to eliminate the organisms. In addition, depending on the type of host cell, IFN-{gamma} can up-regulate a number of proteins that can lead to inhibition of Chlamydia replication within infected cells. In human cells, the induction of IDO may deprive Chlamydia of intracellular tryptophan (31). In contrast, studies in mice and murine cell lines have suggested that induction of inducible NO synthase may restrict Chlamydia growth (32, 33, 34). More recent evidence suggests that following infection of murine cells with human serovars of C. trachomatis, IFN-{gamma} may control infection primarily through the induction of a p47 GTPase Iigp1 (35). IFN-{gamma} secretion by Chlamydia-specific CD8+ T cells in the genital mucosa may affect clearance of Chlamydia through a combination of these pathways.

Although retrogenic mice have been used to study autoimmunity (20) and thymic selection (15), this technology has not been applied to study T cell responses to pathogens. The retrogenic mice described in this study have proven useful as we can now study the initial encounter of CD8+ T cells with Chlamydia in vivo. Because retrogenic mice require fewer resources to produce relative to conventional TCR transgenic mice, we envision that retrogenic mice will be useful for studying and comparing T cell responses to a variety of Chlamydia Ags. Because many Chlamydia proteins are developmentally regulated (36), T cell responses against different Ags may differ depending on the expression pattern of the particular Ag. By comparing T cell responses to multiple Ags we will obtain a better understanding of the overall Chlamydia-specific CD8+ T cell response following genital infection.


   Acknowledgments
 
We thank L. Du for technical assistance, A. van der Velden for critical reading of the manuscript, and D. Vignali and the members of his laboratory for their generosity in guiding us in the techniques used for generating retrogenic mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants AI039558 and AI055900 from the National Institute of Allergy and Infectious Diseases. Back

2 Address correspondence and reprint requests to Dr. Michael N. Starnbach, Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. E-mail address: starnbach{at}hms.harvard.edu Back

3 Abbreviations used in this paper: DLN, draining lymph node; BMM, bone marrow-derived macrophage; IFU, inclusion forming unit. Back

Received for publication June 20, 2006. Accepted for publication September 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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