Chlamydia is responsible for millions of new infections annually, and current efforts focus on understanding cellular immunity for targeted vaccine development. The Chlamydia-specific CD4 T cell response is characterized by the production of IFN-γ, and polyfunctional Th1 responses are associated with enhanced protection. A major limitation in studying these responses is the paucity of tools available for detection, quantification, and characterization of polyfunctional Ag-specific T cells. We addressed this problem by developing a TCR-transgenic (Tg) mouse with CD4 T cells that respond to a common Ag in Chlamydia muridarum and Chlamydia trachomatis. Using an adoptive-transfer approach, we show that naive Tg CD4 T cells become activated, proliferate, migrate to the infected tissue, and acquire a polyfunctional Th1 phenotype in infected mice. Polyfunctional Tg Th1 effectors demonstrated enhanced IFN-γ production compared with polyclonal cells, protected immune-deficient mice against lethality, mediated bacterial clearance, and orchestrated an anamnestic response. Adoptive transfer of Chlamydia-specific CD4 TCR-Tg T cells with polyfunctional capacity offers a powerful approach for analysis of protective effector and memory responses against chlamydial infection and demonstrates that an effective monoclonal CD4 T cell response may successfully guide subunit vaccination strategies.
CD4 T cells contribute to cell-mediated immunity through effector functions mediated by the production of cytokines. Polyfunctional Th1 cells can sequentially produce IFN-γ, IL-2, and TNF in response to TCR stimulation (1). This phenotype has been reported in a variety of infectious disease models, including Leishmania (2). tuberculosis (3), HIV (4), Plasmodium (5), and Chlamydia (6). Polyfunctional Th1 cells demonstrate enhanced protective efficacy in comparison with IFN-γ monofunctional cells (3), potentially by producing higher levels of Th1 cytokines (7, 8). Th1 polyfunctionality represents a measure of immunogenicity in vaccine studies (9), and generation of durable polyfunctional Th1 memory will likely be critical for Chlamydia vaccine development (6).
Protective immunity against Chlamydia is primarily mediated through Th1 cells (10, 11), and the importance of Chlamydia-specific CD4 T cells has been demonstrated by adoptive transfer (12, 13) and depletion studies (14). Despite the importance of CD4 T cells in controlling chlamydial infection, little is known about the generation of polyfunctional Th1 cells and how they contribute to cell-mediated immunity. Previous studies showed that a Chlamydia-specific IFN-γ monofunctional Th1 clone was not protective, whereas a clone producing IFN-γ and TNF cleared Chlamydia muridarum infection in nude mice (15). Vaccine models have shown that Ags and adjuvants generating polyfunctional (IFN-γ+ TNF+) Th1 cells were more protective than monofunctional IFN-γ Th1 cells (16, 17), and this protection has been observed in immunogenicity studies investigating single (18, 19) or multiple chlamydial Ags (20–23).
To our knowledge, we recently developed the first TCR-transgenic (Tg) mouse specific for a conserved Ag in C. muridarum and Chlamydia trachomatis to investigate the polyfunctional Th1 response in vivo. Identification of a polyfunctional Th1 clone allowed us to isolate and clone the Chlamydia-specific TCR for Tg mouse generation. After adoptive transfer, naive TCR-Tg CD4 T cells proliferated in the iliac lymph nodes (ILNs), migrated to the infected genital tract, and primarily differentiated into polyfunctional Th1 cells. Compared with polyclonal, predominantly monofunctional, Th1 cells, polyfunctional Tg Th1 cells exhibited enhanced effector function characterized by increased IFN-γ production associated with improved bacterial clearance. To our knowledge, these studies demonstrate the first Tg TCR to protect against C. muridarum genital infection and exhibit C. trachomatis cross-reactivity and further define Ag-specific enhanced effector function afforded by Th1 polyfunctionality.
Materials and Methods
Strains, cell lines, and culture conditions
C. muridarum Nigg stock (AR Nigg) was obtained from Roger Rank (University of Arkansas for Medical Sciences) and has been described previously (24). C. trachomatis D/UW-3/Cx (25) was obtained from the American Type Culture Collection (Manassas, VA) and plaque purified before use (24). Plaque-purified C. trachomatis D/UW-3/Cx, Nigg strain CM001 (26), and plasmid-deficient CM3.1 (27) were propagated in mycoplasma-free L929 cells (28) and titrated by plaque assay or as inclusion-forming units (29), using a fluorescently tagged anti-chlamydial LPS mAb (Bio-Rad). UV-inactivated bacteria were prepared, as described (30).
Generation of Chlamydia-specific T cell hybridoma and Tg mice
Two 8-wk-old female C57BL/6J mice were intravaginally infected with 3 × 105 inclusion-forming units (IFU) of wild-type C. muridarum Nigg. Infected mice were allowed to resolve primary infection and were rechallenged 2 mo later. The spleen and lymph nodes were collected 1 wk postsecondary challenge, and single-cell suspensions were stimulated ex vivo with reticulate body (RB)-enriched Nigg (1 μg/ml) (31) for 5 d prior to fusion with murine BW5147 T cell lymphoma cells (32) in 50% polyethylene glycol solution. Fused cells were cultured in hypoxanthine-aminopterin-thymidine medium for an additional 7–9 d. Hybridomas were screened and sorted based on CD3, CD4, CD8, and TCRβ expression. Sorted CD4 T cell hybridomas underwent limiting dilution and were cocultured with irradiated syngeneic splenocytes in the presence of Nigg elementary bodies (EBs; 1 μg/ml) or RBs (1 μg/ml) for 24–48 h at 37°C. Harvested supernatants were tested for IL-2 and IFN-γ levels by ELISA (R&D Systems). The CD4 T cell clone with the highest coproduction of IL-2 and IFN-γ in the presence of Nigg EBs was harvested and cultured for cloning of TCRα and TCRβ cDNA.
RNA from the CD4 T cell clone was isolated using an RNeasy Mini Kit (QIAGEN), and TCRα and TCRβ cDNA was obtained using the SuperTCRExpress Mouse T Cell Receptor Vα/Vβ Repertoire Clone Screening Assay Kit (BioMed Immunotech), which contains 5′ RACE primers for all TCR Vα/Vβ. The cDNAs were cloned into the TOPO vector (Promega), sequenced, and identified as Vα6 and Vβ10. Genomic sequences corresponding to the mRNA sequences were used to map the variable, joining, and constant regions in the sequence. Primers with a flanking XmaI site and a NotI site, 5′-GATCCCGGGCAGAGCTGCAGCCTTCCCAAGGCTC-3′ and 5′-CATGCGGCCGCAGTGCTAGGAAGGGCGGCCTGGAC-3′, were generated for amplifying the V region of Vα6 from genomic DNA. Primers with a flanking XhoI site and a SacII site, 5′-TCCGCTCGAGCCTTGACCCAACTATGGGCTGT-3′ and 5′-ATTCCCGCGGCTGGTCTACTCCAAACTACTCCAGG-3′, were generated to amplify the V region of Vβ10. Vα6 amplicon was cloned into the pTαcass and Vβ10 amplicon into pTβcass vectors (33), which contain the respective promoters for Vα and Vβ expression and provided the joining and C region, as a genomic clone (Supplemental Figs. 1, 2). DNA constructs were sequenced for confirmation, linearized at SalI (Vα6) and KpnI (Vβ10) sites, respectively, purified, and injected into the pronuclei of (C57BL/6J × SJL/J) F2 fertilized eggs.
Female C57BL/6J mice (stock number 000664), B6.SJL-Ptprca Pepcb/BoyJ mice (CD45.1+; stock number 002014), B6.129S7-Rag1tm1Mom/J mice (Rag1−/−; stock number 002216), and B6.129S2-Tcratm1Mom/J mice (Tcra−/−; stock number 002116) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were given food and water ad libitum in an environmentally controlled pathogen-free room, with a cycle of 12 h of light and 12 h of darkness. TCR-Tg mice, generated as described above at the University of Pittsburgh, were backcrossed onto the C57BL/6J background for >10 generations. Tg mice were screened for expression of Vα6 and Vβ10 on CD4+ T cells from peripheral blood by PCR and FACS. Experimental mice were matched for age and used between 8 and 12 wk of age. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh and the University of North Carolina.
Generation of bone marrow–derived dendritic cells
Dendritic cells were generated from the tibias/femurs of C57BL/6J mice, as previously described (34). Briefly, erythrocytes were lysed with ammonium-chloride-potassium lysis buffer, and bone marrow precursors were cultured for 7 d in complete media (RPMI 1640 containing 10% FBS, 2 mM glutamine, 10 mM HEPES [pH 7.4], 100 μM nonessential amino acids, 1 mM sodium pyruvate, 50 μM 2-ME, and 50 μg/ml gentamicin) supplemented with 1000 U/ml recombinant murine GM-CSF and 1000 U/ml recombinant murine IL-4 (both from PeproTech). CD11c+ dendritic cells were isolated using specific beads (Miltenyi Biotec), according to the manufacturer’s protocol.
Ag-specific T cell proliferation, activation, and cytokine analysis
The spleens of littermate or TCR-Tg mice were processed into a single-cell suspension, as described previously (35). Splenocytes (1 × 105 cells per well) were seeded in a 96-well flat-bottom tissue culture plate in complete media with 5 μg/ml C. muridarum AR Nigg, plasmid-deficient CM3.1, C. trachomatis D/UW-3/Cx, or rOVA (Sigma) for 6 d. Splenocytes were treated with 20 U/ml murine IL-2 (PeproTech) for the final 48 h. Cells were treated with 20 μl of alamarBlue (MyBioSource) 6 h before the end of the 6-d culture, and proliferation was measured at 530 nm excitation/590 nm emission with a BioTek fluorescence microplate reader.
Alternatively, Tg or polyclonal CD4+ T cells were isolated from the spleens of naive TCR-Tg or wild-type C57BL/6J mice by negative magnetic selection (EasySep Mouse CD4+ T Cell Isolation Kit). Isolated CD4+ T cells were cocultured at a 1:5 ratio with bone marrow-derived dendritic cells (BMDCs) for 3 d in the presence or absence of C. muridarum AR Nigg (5 μg/ml). Expression of CD69 and Ki67 was determined by FACS surface and nuclear staining, respectively, as described previously (36). Supernatants from dendritic cell–stimulated CD4+ T cells were collected, and IL-2 concentrations were determined by ELISA.
Murine Chlamydia infection and monitoring
For genital tract infection, female mice ≥8 wk of age were injected s.c. with 2.5 mg of medroxyprogesterone (Depo-Provera; Upjohn) 5–7 d prior to infection to induce a state of anestrous (37). Mice were intravaginally inoculated with 3 × 105 IFU CM001 diluted in 30 μl of sucrose–sodium phosphate–glutamic acid buffer. Mice were monitored for cervicovaginal shedding via endocervical swabs (29), and IFU were calculated, as described previously (35). Prior to reinfection, mice were treated i.p. with 0.3 mg doxycycline in 100 μl of PBS for 5 d (38). Animal welfare was monitored daily. Genital tract gross pathology, including the presence of hydrosalpinx, was examined and recorded at sacrifice.
Lymphocyte isolation and flow cytometry
Spleen, ILNs, oviducts, uterine horns, and cervical tissues were isolated from sacrificed mice. Cervical tissue and uterine horns were minced separately and incubated with 1 ml of collagenase I (Sigma) for 20 min at 37°C before neutralization with EDTA (10 μM). Single-cell suspensions were prepared by dispersing tissues through a 70-μm tissue strainer (Falcon). Cell suspensions were treated with erythrocyte lysis buffer (VitaLyse; BioE), incubated in Fc block (5 μg/ml) for 10 min, and stained with LIVE/DEAD Fixable Yellow (Life Technologies) plus various combinations of the following fluorochrome-labeled Abs: anti-CD3 (clone 17A2) anti-CD3e (145-C211), anti-CD4 (GK1.5, RM4-5, H129.19), anti-CD8a (53-6.7), anti-TCRVβ10 (V21.5), anti-TCRβ (H57-597), anti-CD45 (30-F11) anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD44 (IM7), anti-CD62L (MEL-14), and anti-CD69 (1H.2F3) (all from BD Biosciences). The samples were analyzed on a CyAN ADP (Beckman Coulter) or LSR II (BD Biosciences) flow cytometer, and data were analyzed with FlowJo software.
CFSE labeling and adoptive transfer
Tg or polyclonal CD4+ T cells were negatively separated, and a sample of isolated cells was analyzed by flow cytometry to confirm >93% purity. The indicated numbers of Tg or wild-type CD4+ T cells were injected i.v. into Depo-Provera–treated CD45.1+, Rag1−/−, or Tcra−/− mice 5–6 d prior to intravaginal infection. In some experiments, Tg CD4+ T cells were labeled with 1 μM CFSE (Thermo Fisher Scientific) for 5 min at 37°C prior to i.v. transfer and analysis.
Intracellular cytokine detection
Lymphocytes isolated from infected mice, as described above, were incubated in a 96-well plate at a concentration of 1 × 106 cells per well in the presence of UV-irradiated C. muridarum AR Nigg (5 μg/ml) or media alone for 6 h at 37°C; GolgiPlug (BD Biosciences) was added, and incubation was continued for an additional 12–16 h. Control samples were stimulated for 4–6 h in the presence of PMA/ionomycin (Cell Stimulation Cocktail; eBioscience) and GolgiPlug. Surface staining was performed as described above, and the cells were fixed in BD Bioscience Cytofix/Cytoperm for 20 min. For detection of intracellular cytokines, cells were incubated for 30 min in BD Bioscience Perm/Wash with various combinations of the following fluorochrome-labeled Abs: anti–IL-2 (JES6-5H4), anti–TNF-α (MP6-XT22), and anti–IFN-γ (XMG1.2) (all from BD Biosciences).
Differences between the means of experimental groups postinfection were calculated using repeated-measures (RM) two-way ANOVA. Significant differences in flow cytometric data were determined by one-way and two-way ANOVA. Comparisons of animal survival were performed by an exact log-rank test. Prism software (GraphPad) was used for statistical analyses, and p ≤ 0.05 was considered significant.
Generation of a Chlamydia-specific TCR-Tg mouse
Previous studies demonstrate that the frequency of IFN-γ–producing CD4 Th1 cells correlates with enhanced chlamydial clearance from the genital tract (10, 11, 13, 39). To directly monitor CD4 T cell responses during murine infection, we generated a Tg mouse strain with a TCR reactive with C. muridarum, which demonstrated cross-reactivity with C. trachomatis (Fig. 1A). The TCR genes were isolated from a hybridoma expressing Vα6 and Vβ10 chains specific for C. muridarum EBs and RBs. These genes (Supplemental Figs. 1, 2) were cloned into an expression vector used to generate germline Tg mice. Founder Tg mice almost uniformly express Vβ10 on the surface of autologous CD4 T cells (Fig. 1B), and they were backcrossed to C57BL/6J mice for >10 generations. Tg mouse splenocytes demonstrated a 6–8-fold increase in Chlamydia-specific proliferation compared with littermates, with minimal proliferation induced by OVA (Fig. 1A). C. muridarum Nigg plasmid-competent and -deficient (CM3.1) strains stimulated TCR-Tg splenocytes equally. To further confirm the specificity of Tg CD4 T cells, we analyzed their ability to activate and proliferate in comparison with wild-type polyclonal CD4 T cells in vitro. Tg CD4 T cells demonstrated the ability to coexpress high levels of CD69 and Ki67 when cultured with BMDCs and C. muridarum EBs, with >40% being double-positive and ∼50% expressing CD69 (Fig. 1C). In contrast, minimal activation was observed with polyclonal CD4 T cells. This enhanced proliferation was associated with significantly increased levels of IL-2 in the supernatants of stimulated TCR-Tg CD4 T cells that were 29 times higher than polyclonal CD4 T cells (Fig. 1D). We next examined the ability of Tg CD4 T cells to become activated and proliferate in vivo.
C. muridarum genital infection initiates TCR-Tg CD4 T cell proliferation and activation in vivo
To determine the ability of TCR-Tg CD4 T cells to proliferate and become activated in response to intravaginal C. muridarum infection, we used an adoptive transfer approach. To first test whether these cells proliferate in vivo, we labeled naive, wild-type CD45.2+ TCR-Tg CD4 T cells with CFSE and transferred 1 × 106 cells i.v. into congenic CD45.1+ mice. An increased percentage of CD45.2+ TCR-Tg CD4 T cells was detectable on day 5 postinfection in the ILNs compared with mock-infected controls (Fig. 2A), and infection resulted in their loss of CFSE expression consistent with proliferation (Fig. 2B). We then compared the activation state of endogenous and Tg CD4 T cells postinfection by examining expression of the activation markers CD44, CD69, and CD62L on CD45.2+ TCR-Tg and endogenous CD45.1+ CD4 T cells in the spleen, ILNs, and oviducts. The gating strategy is shown in Fig. 2C. Comparison of the surface marker frequency between all CD45.1+ CD4 T cells and CD45.1+ CD4 T cells expressing the Vβ10 chain did not significantly alter the frequency of surface marker–positive endogenous cells (data not shown). TCR-Tg CD4 T cells upregulated CD44 and CD69 concomitantly with downregulation of CD62L by day 5 in the ILNs and exhibited higher percentages of activated cells in peripheral and secondary lymphoid organs compared with the endogenous pool by day 8 postinfection (Fig. 2D, 2E). TCR-Tg cells expressed significantly higher levels of CD69 in the ILN on each day analyzed after primary infection compared with endogenous cells. This was further associated with significantly decreased percentages of CD62Lhi TCR-Tg cells on days 5, 8, 22, and 44 postprimary infection. Similar CD62L kinetics was observed in the spleen.
CD44hi expression is used as a marker of T cell activation and Th1 memory (40, 41), and this memory phenotype was significantly increased among TCR-Tg CD4 T cells in the ILNs throughout the course of primary infection, as well as on day 13 of secondary infection, compared with endogenous cells. Similar CD44 expression was observed for splenic TCR-Tg cells. On day 13 postsecondary challenge, 85–98% of splenic Tg T cells were CD44hi, and these Tg cells make up ∼7% of the total splenic CD44hi CD4 T cell pool (Fig. 2F). The kinetics reflect the enhanced ability of Tg cells to adopt an activated effector and/or effector memory phenotype in the lymphoid tissues throughout infection compared with the endogenous T cell repertoire, as well as by day 8 in the infected peripheral tissues. These data collectively support other studies demonstrating Chlamydia-specific CD4 T cell priming and proliferation in the ILN (42, 43) and the presence of activated cells in the genital tract 1 wk postinfection (44).
Naive TCR-Tg CD4 T cells confer protection, acquire effector function, and demonstrate a recall response
After demonstrating that TCR-Tg CD4 T cells become activated, proliferate, and migrate to the infected genital tract, we investigated whether they would provide protection and produce IFN-γ upon challenge. Prior data from the Darville laboratory had revealed that the plaque-purified strain of C. muridarum Nigg, CM001, resulted in disseminated lethal infection in Rag1−/− mice after intravaginal inoculation (26). Rag1−/− mice received adoptive transfers of 103 Tg or 103, 104, or 106 polyclonal CD4 T cells 5 d prior to infection (Fig. 3A). A precursor frequency of 106 polyclonal CD4 T cells was used as a positive control, based on previous observations that this dose conferred protection (data not shown). Mice receiving 103 Tg CD4 T cells survived infection, whereas 103 and 104 polyclonal CD4 T cells were not protective. TCR-Tg CD4 T cells demonstrated a recall response characterized by the production of inflammatory cytokines. Tg CD4 T cells isolated from the uterine horns and oviducts on day 7 postsecondary challenge produced IFN-γ and TNF in response to in vitro restimulation with UV-irradiated C. muridarum (Fig. 3B). These data indicate that TCR-Tg CD4 T cells prevent death from disseminating infection, migrate to infected tissues, and acquire Th1 effector functions postinfection.
TCR-Tg CD4 T cells can mediate bacterial clearance during primary and secondary infection
We next investigated whether adoptively transferring TCR-Tg CD4 T cells to αβ TCR-deficient mice would lead to clearance of primary genital tract infection and enable resistance to challenge infection. Mice that did not receive T cells failed to clear infection, whereas adoptive transfer of 103 or 106 TCR-Tg CD4 T cells to Tcra−/− mice resulted in equivalent rates of infection clearance, with a 3.5-log reduction in shedding being detected by day 10 postinfection (Fig. 3C). In addition, infection clearance after adoptive transfer of 103 or 106 TCR-Tg CD4 T cells was accelerated compared with groups that received 103 or 106 polyclonal CD4 T cells, indicating that the Tg CD4 T cells are more efficient effectors (Fig. 3C).
We also investigated whether TCR-Tg CD4 T cells would contribute to a recall response upon secondary challenge. Immune mice that had received 103 or 106 TCR-Tg CD4 T cells prior to primary infection exhibited a 4.5- and 3-log reduction in shedding, respectively, on day 3 postchallenge compared with primary infection (Fig. 3C, 3D). Mice that received 103 Tg CD4 T cells prior to primary infection were more resistant to challenge compared with mice that received 106 Tg CD4 T cells (Fig. 3D). This was consistent with findings in other TCR-Tg models, in which lower numbers of adoptively transferred naive Tg CD4 T cells induce better memory development. It is possible that decreased interclonal competition for Ag leads to enhanced differentiation of the fittest effectors into memory cells (45). In contrast, infectious burden during secondary infection was lowered significantly in mice receiving 106, but not 103, polyclonal CD4 T cells. In this instance, a broad array of Ag-specific cells avoids interclonal competition for peptide–MHC class II stimulation.
Although mice that were reinfected without prior receipt of adoptive T cells failed to exhibit any decline in infectious burden up to 2 wk postinoculation, on day 3 postchallenge, their infectious burden was 2-log lower than that observed during primary infection. This transient protection may be a result of circulating T cell–independent Ab or memory γδ T cells that are not capable of clearing infection independently of conventional CD4 T cells.
TCR-Tg CD4 T cells preferentially adopt a polyfunctional Th1 phenotype with increased IFN-γ production
The TCR-Tg mouse was developed using a TCR that induced a Th1 response after chlamydial stimulation in vitro. We hypothesized that TCR-Tg CD4 T cells would differentiate into polyfunctional Th1 cells in vivo, because adoptive transfer of these cells led to enhanced chlamydial clearance during primary infection (Fig. 3C). On day 13 postinfection, we detected significantly increased percentages of IFN-γ+TNF+ double-positive cells in the spleen and genital tissues of Tcra−/− mice receiving Tg CD4 T cells compared with mice receiving polyclonal CD4 T cells (Fig. 4A, 4B). Additionally, Tg polyfunctional IFN-γ+TNF+ CD4 T cells expressed significantly higher amounts of IFN-γ compared with polyfunctional polyclonal populations. TCR-Tg polyfunctionality for TNF and IFN-γ was associated with increased IFN-γ production compared with cells positive for only IFN-γ (Fig. 4C). These data indicate that TCR-Tg CD4 T cells preferentially adopt a polyfunctional phenotype that is characterized by high levels of IFN-γ production. Furthermore, the percentage of triple-positive (IFN-γ+TNF+IL-2+) CD4 T cells in the spleen was increased among the Tg CD4 T cell population compared with polyclonal CD4 T cells on day 48 postinfection (Fig. 4D, 4E). Triple-positive Tg cells expressed significantly higher levels of IFN-γ per cell compared with single- or double-positive cells (Fig. 4F), as previously described for pathogen-specific polyfunctional T cells in other models of infection (4, 8, 46). Triple-positive cells also expressed significantly higher levels of TNF per cell compared with TNF monofunctional cells, but at a reduced magnitude (60% increase), and no difference was observed in IL-2 geometric mean fluorescent intensity (GMFI; data not shown). These data collectively show that Tg CD4 T cells have superior functional capacity with enhanced cytokine production, and this polyfunctional effector response is associated with enhanced bacterial clearance.
Pathogen-specific TCR-Tg mice have been used in a variety of infectious disease models (47–54), including NR1 mice that recognize C. trachomatis (44). Adoptive transfer of naive TCR-Tg cells is a superior approach to transfer of in vitro–maintained T cell lines, because naive TCR-Tg cells allow analysis of the initial Ag encounter and phenotypic differences between in vivo–derived effector and memory populations. We developed a TCR-Tg mouse that recognizes a conserved Ag between C. trachomatis and C. muridarum. The TCR-Tg cells of this mouse react to C. trachomatis serovars D, F, H, and L2 (data not shown), and preliminary biochemical analyses reveal that they recognize a soluble secreted protein enriched in RBs (data not shown). We have excluded commonly studied immunogenic Ags, such as major outer membrane porin, outer membrane complex protein B, heat shock protein 60, and polymorphic membrane protein G, based on their inability to stimulate TCR-Tg cellular proliferation in vitro. To our knowledge, this article reports the first TCR to protect against C. muridarum genital infection, which allowed us to analyze enhanced effector function afforded by Th1 polyfunctionality at a level that had not been previously attainable. Generation of this mouse has allowed for the unique ability to adoptively transfer TCR-Tg cells for investigation of Ag-specific T cell responses to mouse and human chlamydial strains in the murine model of genital tract infection.
Development of the Chlamydia-specific TCR-Tg mouse was based on selection of a Th1 clone specific for C. muridarum EBs and RBs. We used a nonbiased approach, by analyzing T cell clones demonstrating the strongest IFN-γ and IL-2 production, which has been shown to be effective in TCR-Tg mouse development. Selecting clones reactive against the whole organism or crude Ag preparations has resulted in Tg CD4 T cells with the capacity to mount robust effector and memory responses following infection and vaccination (53) compared with model Ags (55). Our studies reveal that Tg CD4 T cells possess a TCR that confers protection against intravaginal C. muridarum infection.
These Tg CD4 T cells become activated, proliferate extensively, and produce high levels of IL-2 when stimulated with C. muridarum. Naive and memory CD4 T cells require TCR stimulation in combination with IL-2 signaling to proliferate (56), and TCR engagement upregulates IL-2R subunits (57). The strength of IL-2 signaling also correlates with the magnitude of proliferation in Th1 cells (58), and IFN-γ expression increases with successive cell divisions (59). Furthermore, IL-2 signaling during priming enhances differentiation of the effector pool into memory (60).
Based on the ability of these cells to recognize Chlamydia in vitro, we used an adoptive-transfer approach to analyze the proliferation and activation kinetics in vivo. Similar to the C. trachomatis model, our approach revealed that C. muridarum infection induced significant TCR-Tg CD4 T cell activation and expansion in the ILNs by day 5, and Tg cells expressed an activated phenotype (CD44hiCD69+CD62Llo). The CD44hiCD62Llo phenotype was also observed in the infected oviducts. Expression of CD69 on Tg cells in the spleen and oviducts on day 8, in our model, is likely due to the ability of CM001 to quickly disseminate to the distal organs and rapidly ascend the genital tract. Increased CD69 expression on CD4 T cells early in CM001 infection may be a result of local priming events, prior to tissue infiltration of activated T cells primed in the ILN. At later time points, CD44 expression in secondary lymphoid organs steadily increased, particularly during reinfection, consistent with the formation of memory T cells. These kinetics are similar to other infectious disease models of CD4 T cell activation and memory (44, 49, 54, 61). After priming in the ILN, Tg cells made up ∼7% of all CD44hi CD4 T cells in the spleen, which consistently decreased through infection, until mice received a secondary challenge. This is consistent with other systems, demonstrating that peak CD4 T cell expansion typically occurs after 1 wk (62) and is followed by CD4 T cell contraction over 1–2 wk, during which 90–95% of the expanded population undergoes cell death (63, 64). Late in the course of C. muridarum infection and during reinfection, a majority of Tg cells expressed high levels of the memory marker CD44. Additional phenotyping experiments are required to determine the proportions of Tg T cells in the terminal effector, effector memory, and central memory pools.
TCR-Tg cells prevented death in immunocompromised mice infected with CM001, and these cells were recalled to the infected tissues and produced IFN-γ and TNF upon secondary challenge. These results parallel other infectious disease models, demonstrating that adoptive transfer of Ag-specific naive CD4 T cells can protect against lethality (54). Adoptive transfer to T and B cell–deficient Rag1−/− hosts illustrates the CD4 Th-independent protective function of TCR-Tg cells, which is likely mediated through their production of IFN-γ (65). Furthermore, transfer of these cells into αβ TCR-deficient mice led to enhanced protection against primary infection and equivalent protection against a secondary challenge compared with the polyclonal response. Comparable levels of oviduct gross pathology were observed between the TCR-Tg and polyclonal groups (100 and 95% hydrosalpinx, respectively), which was not surprising given the ability of CM001 to induce severe pathology in wild-type mice (27). Future studies using vaccination or adoptive transfer of in vitro primed TCR-Tg cells should help to reveal their capacity to protect against oviduct pathology.
Reduced bacterial burden mediated by TCR-Tg cells was associated with increased frequencies of double- and triple-positive Th1 populations producing higher levels of IFN-γ compared with polyclonal CD4 T cells. IFN-γ is a critical effector molecule for controlling chlamydial replication (13, 66–69), and enhanced frequencies of polyfunctional Tg cells producing IL-2 could allow for enhanced Th1 effector proliferation. Our TCR-Tg cells clearly recognize an Ag that drives a favorable response that leads to enhanced bacterial clearance and resistance to challenge infection. Persistent Ag and Ag depots reduce the memory pool, leading to nonprotective responses from terminally differentiated exhausted T cells. Removal of Ag drives T cell transition to memory (70), and these cells remain plastic and heterogeneous (71, 72). Thus, triple-positive Th1 Tg cells could be a consequence of improved effector function leading to lower bacterial load (73, 74). In addition, Tg cells may demonstrate greater functional avidity, which has been linked with improved disease outcomes (75) and expression of decreased levels of inhibitory receptors (76). High-avidity T cells are less susceptible to activation-induced cell death (77) and demonstrate increased polyfunctionality (78, 79).
Our analyses were limited to the study of CD4 T cells and focused on profiling three major Th1 cytokines. A comprehensive analysis of TCR-Tg cell production of cytokines, chemokines, and cytotoxic effectors, as well as their helper function for Ab production by B cells, is needed to fully delineate their protective or pathological mechanisms. Alternative effectors (80) and Ab (81, 82) have been shown to play a significant role in mediating chlamydial clearance. Additional analysis of the recall response is needed to determine the mechanisms by which equivalent protection from reinfection occurred in Tcra−/− mice that had received 103 polyclonal T cells or 103 monoclonal TCR-Tg T cells. Potentially, polyclonal polyfunctional T cells were maintained, and IFN-γ monofunctional cells were culled, or monofunctional cells responding to a variety of Ags elicit similar protection to polyfunctional TCR-Tg cells recognizing a single Ag.
The TCR-Tg C57BL/6 background presents a caveat. Some TCR-Tg mice are backcrossed to Rag backgrounds to eliminate endogenous TCR subunit expression; however, this can result in deletion of TCR-Tg cells as a result of the requirement of a second endogenous TCRα-chain for thymic emigration (54). Endogenously activated CD4 T cells expressing a second TCR can be present in TCR-Tg mice. However, the spleen population exists at a small frequency (83) and at similar numbers in wild-type mice (84). This phenomenon led us to use equal and greater numbers of wild-type CD4 T cells as controls for adoptive-transfer experiments.
The finding that a single immunogenic Ag that elicits polyfunctional T cells can successfully induce a protective response is encouraging from a subunit vaccinology perspective. Viral models demonstrate that primary and secondary effectors share organ-specific expression patterns, but secondary effectors are more polyfunctional (triple positive); polyfunctional cells also express higher levels of genes associated with survival and migration (85). Whether CD4 T cell polyfunctionality can predict memory generation and subsequent enhanced secondary effector functions is an important area to be addressed. Once we have identified the Ag recognized by TCR-Tg T cells, we can determine whether vaccination with this Ag drives induction of a protective polyfunctional response and whether adoptive transfer of in vitro Ag-primed Tg T cells can protect from infection and disease.
In conclusion, we have demonstrated that adoptive transfer of Tg CD4 T cells specific for a single Chlamydia Ag induces polyfunctional CD4 T cells that provide enhanced immunity against Chlamydia. Tg CD4 T cells specific for Chlamydia can be further used to directly monitor differentiation of Ag-specific effector and memory responses during infection and to better delineate protective responses upon challenge. The development of a successful vaccine will be facilitated by better understanding of how CD4 T cell polyfunctionality is generated, sustained, and provides immunity at the mucosal surface.
We thank the UNC Flow Cytometry Core Facility for technical assistance and Dr. Chunming Bi and the staff of the University of Pittsburgh Transgenic and Gene Targeting Core Facility for assistance with Tg mouse development.
This work was supported by National Institutes of Health–National Institute of Allergy and Infectious Diseases Grants R01 A105624 and U19 A1084024 (to T.D.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
The authors have no financial conflicts of interest.