Chlamydia has been shown to evade host-specific IFN-γ-mediated bacterial killing; however, IFN-γ-deficient mice exhibit suboptimal late phase vaginal Chlamydia muridarum clearance, greater dissemination, and oviduct pathology. These findings introduce constraints in understanding results from murine chlamydial vaccination studies in context of potential implications to humans. In this study, we used mice deficient in either IFN-γ or the IFN-γ receptor for intranasal vaccination with a defined secreted chlamydial Ag, chlamydial protease-like activity factor (CPAF), plus CpG and examined the role of IFN-γ derived from adoptively transferred Ag-specific CD4+ T cells in protective immunity against genital C. muridarum infection. We found that early Ag-specific IFN-γ induction and CD4+ T cell infiltration correlates with the onset of genital chlamydial clearance. Adoptively transferred IFN-γ competent CPAF-specific CD4+ T cells failed to enhance the resolution of genital chlamydial infection within recipient IFN-γ receptor-deficient mice. Conversely, IFN-γ production from adoptively transferred CPAF-specific CD4+ T cells was sufficient in IFN-γ-deficient mice to induce early resolution of infection and reduction of subsequent pathology. These results provide the first direct evidence that enhanced anti-C. muridarum protective immunity induced by Ag-specific CD4+ T cells is dependent upon IFN-γ signaling and that such cells produce sufficient IFN-γ to mediate the protective effects. Additionally, MHC class II pathway was sufficient for induction of robust protective anti-C. muridarum immunity. Thus, targeting soluble candidate Ags via MHC class II to CD4+ T cells may be a viable vaccine strategy to induce optimal IFN-γ production for effective protective immunity against human genital chlamydial infection.

Chlamydia trachomatis continues to be the leading cause of sexually transmitted bacterial disease worldwide, despite availability of effective antimicrobial treatment (1, 2). Within the United States, ∼2 billion dollars are incurred in health care costs due to complications of genital chlamydial infections, including pelvic inflammatory disease and infertility. Additionally, accumulating evidence suggests that chlamydial screening and treatment programs lead to an increase in the number of reinfections over time, possibly by abbreviating development of antichlamydial immunity (3). Therefore, an efficacious vaccine to generate antichlamydial immunity in vivo is highly desirable for prevention of Chlamydia-induced morbidity.

The murine model of genital chlamydial infection using C. trachomatis and Chlamydia muridarum has been used extensively for the study of pathogenesis and immunity against this disease (2). The general consensus from these studies is that CD4+ T cells are important for inducing optimal protective antichlamydial immunity (2, 4, 5, 6, 7, 8). Moreover, Th1-type CD4+ T cells that secrete IFN-γ have been shown to be present in large numbers within Chlamydia-infected genital tracts (9, 10, 11, 12, 13, 14). IFN-γ from CD4+ T cells appears to be important for clearance of C. trachomatis, but not C. muridarum, infections from the murine genital tract (12, 15, 16). Specifically, human and murine strains of Chlamydia have evolved mechanisms to evade the effects of IFN-γ in host-specific fashion, and thus C. trachomatis, but not C. muridarum, has been shown to be highly sensitive to the bactericidal effects of murine IFN-γ in vitro (15). The early phase of C. muridarum clearance in mice also appears to be independent of the effects of IFN-γ (12). However, mice deficient in IFN-γ production (IFN-γ−/− mice) have been shown to exhibit suboptimal late C. muridarum clearance, increased dissemination of the bacterium, and enhanced oviduct pathology (12, 14, 17, 18, 19, 20, 21, 22). Based on these findings, the role of IFN-γ in C. muridarum clearance induced by Ag-specific CD4+ T cells remains unclear. Given the importance of understanding the mechanisms of protective immunity for effective vaccine design and the widespread usage of C. muridarum infection in mice for vaccination studies, we sought to resolve the role of IFN-γ production from Ag-specific CD4+ T cells in protective immunity against genital C. muridarum infection in the murine host.

In this study, we used chlamydial protease-like activity factor (CPAF)4 (23, 24, 25, 26, 27, 28, 29), a chlamydial vaccine Ag that induces protective immunity in a CD4+ T cell-dependent fashion (17, 30, 31, 32), to examine the requirement and sufficiency of IFN-γ production from Ag-specific CD4+ T cells to protection against C. muridarum. Using a two-step approach, including vaccination and adoptive transfer of CPAF-specific CD4+ T cells involving IFN-γ−/− mice and IFN-γR−/− mice, we have found that onset of genital C. muridarum clearance correlates with local IFN-γ secretion and Ag-specific CD4+ T cell infiltration into the genital tract. Importantly, we also found that IFN-γ secretion from Ag-specific CD4+ T cells is necessary and that sufficient IFN-γ is produced by these cells, in an otherwise IFN-γ-deficient environment, to induce accelerated genital C. muridarum clearance and reduction of oviduct pathology. These results are supported by the observation that Ag presentation via the MHC II pathway, in the absence of the MHC I pathway, is sufficient for the induction of protective anti-C. muridarum immunity afforded by rCPAF plus CpG vaccination.

The rCPAF was purified as described previously (17, 24, 30, 31, 32). Briefly, rCPAF constructs from C. trachomatis L2 genome with a 6X histidine tag (His) were cloned into pBAD vectors. The fusion proteins were expressed in Escherichia coli and purified using Ni-NTA agarose beads (Amersham Biosciences). The purity of the protein preparation was evaluated by SDS-PAGE and by Western blot. The endotoxin levels in the purified protein samples were determined to be <1 endotoxin unit (EU)/mg protein (1 endotoxin unit = 0.2 ng) using the Limulus amebocyte assay (Sigma-Aldrich). CpG oligodeoxynucleotides (5′-TCC ATG ACG TTC CTG ACG TT-3′, designated CpG in this article) were synthesized and obtained from Sigma Genosys (31).

C. muridarum was grown on confluent HeLa cell monolayers as described previously (33). Cells were lysed using a sonicator and elementary bodies purified on Renograffin gradients. Aliquots of bacteria were stored at −70°C in sucrose-phosphate-glutamine buffer.

Four- to 6-wk-old female C57BL/6, BALB/c, C57BL/6 IFN-γR-deficient mice (IFN-γR−/− mice), BALB/c IFN-γ-deficient mice (IFN-γ−/− mice), and C57BL/6 β2-microglobulin-deficient mice (β2m−/− mice) were purchased from The Jackson Laboratory. Mice were housed and bred at the University of Texas. Animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee guidelines.

Animals were immunized as described previously (17, 30, 31, 32). Groups of mice were anesthetized and immunized i.n. on day 0 with 15 μg of rCPAF plus 10 μg of CpG dissolved in 25 μl of sterile PBS. Mice were boosted i.n. with the same doses on days 14 and 28. Groups of mice that received PBS (mock) alone served as controls.

CD4+ T cells were purified as described previously (30). Briefly, animals were euthanized and single-cell suspensions of splenocytes were layered over a Ficoll density gradient to collect mononuclear cells. CD4+ T cell populations were isolated using magnetic particles (Stem Cell Technologies) and the purity was determined to be at least >98% CD4+ T cells by flow cytometry using an allophycocyanin-conjugated anti-CD4 mAb (BD Biosciences).

Spleens were removed and single cells prepared. Genital tracts were removed and treated with collagenase-DNase solution for 20 min at 37°C and single cells were prepared. The Ag-specific cytokine production was measured as described previously (17). Briefly, splenocytes (106 cells/well) or genital tract cells (105 cells/well) collected before chlamydial challenge were stimulated for 72 h in vitro with rCPAF (1 μg/ml) or with the unrelated Ag hen egg lysozyme (HEL; 1 μg/ml), UV-inactivated C. muridarum (105 inclusion-forming units (IFU)/well), or medium alone in culture plates. The spleens and genital tracts removed on days 3 and 6 after challenge were cultured for 72 h in medium alone because these cells should have been stimulated by CPAF produced during the chlamydial infection in vivo. Supernatants from the culture wells were analyzed for IFN-γ, IL-2, and IL-4 production using BD OptEIA kits (BD Pharmingen) as described previously (17).

Mice were vaccinated i.n. with three doses of rCPAF plus CpG or PBS (mock). Ten days following the last boost, splenic CD4+ T cells were purified and labeled with CFSE (2 μM/107 cells; Molecular Probes) (34, 35). The CFSE-labeled cells were injected (107 cells/mouse) i.p. into female C57BL/6 mice 2 h after they were challenged intravaginally (i. vag.) with 105 IFU of C. muridarum or PBS (mock). On days 0, 3, or 6 after challenge, CFSE+ cells within genital tract cell suspensions were enumerated by flow cytometry (BD Biosciences LSR II). In some experiments, unlabeled purified CD4+ T cells (107 cells/mouse) were transferred into recipient Chlamydia-challenged mice and bacterial shedding was monitored. For vaccination/challenge experiments, animals were challenged 1 mo after the final vaccination. In all experiments, the estrous cycle of animals was synchronized using two s.c. injections of Depo-Provera (Pharmacia Upjohn) on days 10 and 3 before chlamydial challenge.

Vaginal swab material collected at the indicated days after challenge was plated onto HeLa cell monolayers. Chlamydial inclusions were probed using an anti-Chlamydia murine mAb (17) and Cy3-conjugated anti-mouse IgG secondary Ab plus Hoescht nuclear stain, and counted using a Zeiss Axioskop microscope. The average number of inclusions in 10 midline microscopic fields was calculated for each animal for earlier time points (until day 9 after challenge) and entire coverslips for later time points (days 12–30 after challenge), and results are expressed as mean ± SEM of inclusions per animal group. The percentage of animals shedding Chlamydia at each time point is also reported. On day 80 after challenge, animals were euthanized, dissected, and the number with bilateral or unilateral hydrosalpinx enumerated. Briefly, the genital tracts were placed on a flat white surface next to a standard metric ruler, and image was acquired using a Fuji film F10 camera at a fixed distance of 15 cm from the surface. Dilated oviducts measuring ≥2 mm in diameter were used as an indicator of hydrosalpinx. The enumeration of chlamydial counts and hydrosalpinx was conducted in a blinded fashion.

Sigma Stat (Systat Software) was used to perform all tests of significance. ANOVA was used for comparisons between multiple groups. The Kaplan-Meier test was used for comparisons of time to bacterial clearance. A value of p < 0.05 was considered statistically significant. All data are representative of two independent experiments and each experiment was analyzed independently.

We previously have shown that animals vaccinated i.n. with rCPAF plus IL-12 or CpG display significant reduction in vaginal chlamydial shedding, compared with mock-vaccinated mice, beginning around day 6 after challenge (17, 30, 31, 32). Local IFN-γ production has been shown to be important for optimal resolution of genital chlamydial infection (1, 2, 12, 18, 19, 20, 21), and the protective effects of CPAF vaccination were highly dependent upon endogenous IFN-γ production (17). Therefore, we examined the temporal induction of genital IFN-γ production in vaccinated/challenged BALB/c mice according to the schedule shown in Fig. 1. As shown in Fig. 2 A, splenocytes, not genital tract cells, from rCPAF plus CpG- or rCPAF-alone-vaccinated animals displayed elevated levels of CPAF-specific IFN-γ production before challenge (day 60 after initial immunization). Additionally, the splenocytes and genital tract cells from rCPAF plus CpG-vaccinated mice displayed elevated levels of IFN-γ production only on day 6, not day 3, after chlamydial challenge. Interestingly, rCPAF-alone- vaccinated animals displayed IFN-γ production from spleens, but not genital tracts, on day 6 after challenge. Animals treated with CpG or PBS (mock) did not display Ag-specific cytokine production, even at day 6 after challenge. There was minimal IL-4 production in all cultures (data not shown). As expected, incubation with HEL or medium alone did not induce Ag-specific cytokine production. Collectively, these results suggest that vaccinated animals displayed genital Ag-specific IFN-γ production after a lag following chlamydial challenge and that rCPAF plus CpG immunization induced Ag-specific IFN-γ production within the genital tracts as early as day 6 after challenge. Moreover, rCPAF plus CpG-vaccinated animals, but not other groups of animals, displayed early reduction in bacterial shedding at day 6 after challenge (17, 32), suggesting a strong correlation between bacterial clearance and local, not splenic, Ag-specific IFN-γ production in the genital tracts.

FIGURE 1.

Outline of the experimental design. A, Time line after immunization. Groups of mice (as indicated) were vaccinated i.n. with rCPAF plus CpG on day 0 and boosted on days 14 and 28. Ten days after the last booster immunization, spleens were removed and CD4+ T cells were purified and injected i.p. into recipient mice (as indicated). The recipient mice were pretreated with two doses of s.c. Depo-Progesterone (days 28 and 35, respectively) and challenged i.vag. with C. muridarum (day 38) on the day of cellular transfers. In vaccination/challenge experiments involving β2m−/− and β2m+/+ mice, vaccinated animals were treated with s.c. Depo-Progesterone on days 50 and 57 and challenged i.vag. with C. muridarum on day 60 after initial immunization. B, Time line after bacterial challenge. After i.vag. C. muridarum challenge, the genital Ag-specific CD4+ T cell infiltration and IFN-γ production were analyzed on days 3 and 6. In experiments to determine protective immunity, the bacterial shedding was monitored every third day for a period of 30 days and oviduct pathology was analyzed on day 80 after challenge.

FIGURE 1.

Outline of the experimental design. A, Time line after immunization. Groups of mice (as indicated) were vaccinated i.n. with rCPAF plus CpG on day 0 and boosted on days 14 and 28. Ten days after the last booster immunization, spleens were removed and CD4+ T cells were purified and injected i.p. into recipient mice (as indicated). The recipient mice were pretreated with two doses of s.c. Depo-Progesterone (days 28 and 35, respectively) and challenged i.vag. with C. muridarum (day 38) on the day of cellular transfers. In vaccination/challenge experiments involving β2m−/− and β2m+/+ mice, vaccinated animals were treated with s.c. Depo-Progesterone on days 50 and 57 and challenged i.vag. with C. muridarum on day 60 after initial immunization. B, Time line after bacterial challenge. After i.vag. C. muridarum challenge, the genital Ag-specific CD4+ T cell infiltration and IFN-γ production were analyzed on days 3 and 6. In experiments to determine protective immunity, the bacterial shedding was monitored every third day for a period of 30 days and oviduct pathology was analyzed on day 80 after challenge.

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FIGURE 2.

Genital Ag-specific responses after rCPAF plus CpG vaccination and i.vag. C. muridarum challenge. Groups of BALB/c mice (three mice per group) were immunized with three doses of rCPAF + CpG, rCPAF alone, CpG alone, or PBS (mock). A, The animals were rested for 1 mo and splenocytes or genital tract cells were removed before challenge (day 0) and stimulated in vitro for 72 h with rCPAF, HEL, or medium alone. Some mice were challenged i.vag. with C. muridarum. Splenocytes and genital tract cells were collected on days 3 or 6 after challenge and cultured in medium for 72 h. The supernatants were analyzed for IFN-γ production. Results are expressed as mean ± SD of IFN-γ from each group. B and C, Ten days after the last immunization, splenic CD4+ T cells were purified. B, Percentage of CD4+ T cells in cell preparations before and after the purification procedure. C, The purified CD4+ T cells were labeled with CFSE and transferred into C. muridarum- or PBS (mock)-challenged recipient mice. On days 3 or 6 after challenge, the population of CFSE+ cells infiltrating the genital tract was analyzed by flow cytometry. All results are representative of two independent experiments.

FIGURE 2.

Genital Ag-specific responses after rCPAF plus CpG vaccination and i.vag. C. muridarum challenge. Groups of BALB/c mice (three mice per group) were immunized with three doses of rCPAF + CpG, rCPAF alone, CpG alone, or PBS (mock). A, The animals were rested for 1 mo and splenocytes or genital tract cells were removed before challenge (day 0) and stimulated in vitro for 72 h with rCPAF, HEL, or medium alone. Some mice were challenged i.vag. with C. muridarum. Splenocytes and genital tract cells were collected on days 3 or 6 after challenge and cultured in medium for 72 h. The supernatants were analyzed for IFN-γ production. Results are expressed as mean ± SD of IFN-γ from each group. B and C, Ten days after the last immunization, splenic CD4+ T cells were purified. B, Percentage of CD4+ T cells in cell preparations before and after the purification procedure. C, The purified CD4+ T cells were labeled with CFSE and transferred into C. muridarum- or PBS (mock)-challenged recipient mice. On days 3 or 6 after challenge, the population of CFSE+ cells infiltrating the genital tract was analyzed by flow cytometry. All results are representative of two independent experiments.

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Because CPAF-specific CD4+ T cells are a significant source of IFN-γ production and rCPAF-mediated immunity was strongly dependent upon Ag-specific CD4+ T cells (30), we examined the infiltration of adoptively transferred CFSE-labeled CPAF-specific CD4+ T cells into the genital tract after challenge. The transferred cells contained at least 98% CD4+ T cells as determined by flow cytometry using an allophycocyanin-conjugated anti-CD4 mAb (BD Biosciences) (Fig. 2,B). As shown in Fig. 2 C, minimal CFSE+ cells were observed in the genital tract of each group of animals on day 3 after C. muridarum or mock (PBS) challenge. However, on day 6 after challenge, genital tracts of C. muridarum-challenged mice that received CPAF-specific CD4+ T cells displayed a higher frequency of CFSE+ cells (28.3%), as compared with animals receiving mock (PBS) CD4+ T cells (9.8%). Mock-challenged mice receiving either group of CD4+ T cells displayed minimal (3.9–4.0%) influx of CFSE+ cells into the genital tracts. These results suggest that: 1) Ag-specific, not mock (PBS), CD4+ T cells respond early to the infection site and 2) initiation of an infection is required for homing of primed CD4+ T cells into the genital tract. Moreover, the temporal kinetics of the cellular infiltration correlates with local IFN-γ production and onset of bacterial clearance from the genital tract. Together, these results suggest the importance of genital CPAF-specific IFN-γ-inducing CD4+ T cells in the enhanced resolution of genital chlamydial infection following CPAF vaccination.

Given the pivotal importance of IFN-γ for chlamydial clearance (1, 2, 12, 18, 19, 20, 21) and the correlation of genital Ag-specific CD4+ T cell infiltration to IFN-γ production, we evaluated the dependency of CPAF-specific CD4+ T cells on this cytokine for C. muridarum clearance according to the schedule shown in Fig. 1. Purified CD4+ T cells from rCPAF plus CpG- or mock (PBS)-vaccinated C57BL/6 mice were transferred (107 cells/mouse) into recipient C. muridarum-challenged IFN-γR−/− or C57BL/6 IFN-γR+/+ mice, and bacterial shedding was measured at the indicated time periods. As shown in Fig. 3,A, CPAF-specific CD4+ T cell -transferred IFN-γR+/+ mice, but not IFN-γR−/− mice, displayed significantly reduced vaginal bacterial shedding as early as day 9, and at later time-periods, after vaginal C. muridarum challenge, when compared with mock groups. Additionally, IFN-γR+/+ mice that received CPAF-specific CD4+ T cells completely resolved the infection by day 18 postchallenge (Fig. 3 B). In contrast, 100% of the IFN-γR−/− mice receiving CPAF-specific or mock (PBS) CD4+ T cells were shedding C. muridarum at day 18, with only 50% of animals in each group resolving the infection by day 30 after challenge. As expected, IFN-γR+/+ mice receiving mock (PBS) CD4+ T cells completely resolved the infection by day 27 after challenge. These results clearly demonstrate that CPAF-specific CD4+ T cells induce resolution of genital C. muridarum infection in a highly IFN-γ-dependent fashion and that other cytokines or components from these cells may not contribute significantly to C. muridarum clearance in the absence of IFN-γ signaling.

FIGURE 3.

CPAF-specific CD4+ T cells induce antichlamydial immunity in an IFN-γ-dependent fashion. Groups of C57BL/6 mice were vaccinated with three doses of rCPAF+CpG or PBS (mock). Ten days after the last immunization, CD4+ T cells were purified and transferred (107 cells/mouse) i.p. into C. muridarum-challenged recipient IFN-γR−/− or IFN-γR+/+ animals (six mice per group). Vaginal chlamydial shedding was measured every third day after challenge. A, Numbers of chlamydial IFU recovered from vaginal swabs at the indicated days after genital challenge. Results are expressed as means ± SEM of the bacterial recovery from all animals in a group. *, p < 0.05 (ANOVA) between C. muridarum-challenged IFN-γR+/+ mice receiving CPAF-specific CD4+ T cells and other experimental groups. B, Percentage of animals shedding Chlamydia after genital challenge. *, p = 0.0001 (Kaplan-Meier test) for resolution time between C. muridarum-challenged IFN-γR+/+ mice receiving CPAF-specific CD4+ T cells and other groups. Results are representative of two independent experiments.

FIGURE 3.

CPAF-specific CD4+ T cells induce antichlamydial immunity in an IFN-γ-dependent fashion. Groups of C57BL/6 mice were vaccinated with three doses of rCPAF+CpG or PBS (mock). Ten days after the last immunization, CD4+ T cells were purified and transferred (107 cells/mouse) i.p. into C. muridarum-challenged recipient IFN-γR−/− or IFN-γR+/+ animals (six mice per group). Vaginal chlamydial shedding was measured every third day after challenge. A, Numbers of chlamydial IFU recovered from vaginal swabs at the indicated days after genital challenge. Results are expressed as means ± SEM of the bacterial recovery from all animals in a group. *, p < 0.05 (ANOVA) between C. muridarum-challenged IFN-γR+/+ mice receiving CPAF-specific CD4+ T cells and other experimental groups. B, Percentage of animals shedding Chlamydia after genital challenge. *, p = 0.0001 (Kaplan-Meier test) for resolution time between C. muridarum-challenged IFN-γR+/+ mice receiving CPAF-specific CD4+ T cells and other groups. Results are representative of two independent experiments.

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Although the importance of IFN-γ in antichlamydial immunity has been known (1, 2, 12, 18, 19, 20, 21), the specific cell types required for optimal induction of this cytokine have not been specifically demonstrated. To address this issue, groups of IFN-γ−/− mice and corresponding wild-type IFN-γ+/+ mice were immunized with three doses of CPAF plus CpG on days 0, 14, and 28. Ten days after the last boost, splenocytes isolated from vaccinated IFN-γ−/− mice, as expected, did not exhibit IFN-γ production, but displayed levels of Ag-specific IL-2 production comparable to similarly treated IFN-γ+/+ mice, suggesting that CD4+ T cells did get primed in an IFN-γ-deficient environment (Fig. 4,A). The splenic CD4+ T cells were purified from these animals and transferred i.p. into recipient C. muridarum-challenged IFN-γ−/− mice according to the schedule in Fig. 1. One group of naive IFN-γ−/− mice that were challenged with the bacterium, but did not receive cellular transfer, served as baseline control for effects of chlamydial infection. The only source of IFN-γ production in the recipient animals was the adoptively transferred IFN-γ+/+ CD4+ T cells. As shown in Fig. 4,B, C. muridarum-challenged IFN-γ−/− mice that received IFN-γ+/+ CPAF-specific CD4+ T cells displayed significantly reduced vaginal bacterial shedding when compared with other recipient groups, as early as day 9, and at later time periods until day 30 after challenge. All (100%) recipient IFN-γ−/− mice that received CPAF-specific CD4+ T cells from IFN-γ+/+ mice, but not IFN-γ−/− mice, resolved the infection by day 18 after challenge (Fig. 4,C). The majority of the recipient IFN-γ−/− mice (83 and 67%) that received IFN-γ+/+ mock (PBS) CD4+ T cells or IFN-γ−/− CPAF-specific CD4+ T cells, respectively, were still shedding C. muridarum as late as day 30 after challenge. We further examined the development of hydrosalpinx in these groups of animals. All (100%) of the IFN-γ−/− mice that received IFN-γ−/− CPAF-specific CD4+ T cells, or those that did not receive any cells, developed bilateral hydrosalpinx at day 80 after chlamydial challenge (Fig. 4 D). In contrast, IFN-γ−/− mice that received IFN-γ+/+ CPAF-specific CD4+ T cells were highly protected, with hydrosalpinx evident in significantly fewer animals (33% bilateral). These results demonstrate that CPAF-specific CD4+ T cells produce IFN-γ, in an otherwise IFN-γ-deficient environment, at a level that is sufficient to mediate the enhanced resolution of murine genital C. muridarum infection and reduction of oviduct pathology.

FIGURE 4.

CPAF-specific CD4+ T cells are a sufficient source of IFN-γ for resolution of genital chlamydial infection and reduction of oviduct pathology. Groups of IFN-γ−/− mice and IFN-γ+/+ mice were immunized with three doses of rCPAF plus CpG or PBS (mock). A, Ag-specific cytokine production. Ten days after the last immunization, some animals (three mice per group) in each group were euthanized and splenocytes were stimulated in vitro with rCPAF or medium alone. After a 72-h incubation, the supernatants were analyzed for IFN-γ and IL-2 production. Results are expressed as mean ± SD of cytokine production in each group. B–D, Ten days after the final immunization, CD4+ T cells were purified from the spleens of vaccinated animals and transferred into C. muridarum-challenged recipient IFN-γ−/− mice (six mice per group). One group of IFN-γ−/− mice (six mice per group) that did not receive cellular transfers were challenged as controls. Vaginal chlamydial shedding was measured every third day after challenge. B, Numbers of chlamydial IFU recovered from vaginal swabs at the indicated days after genital challenge. Results are expressed as means ± SEM of the bacterial recovery from all animals in a group. *, p < 0.05 (ANOVA) between C. muridarum-challenged IFN-γ−/− mice receiving CPAF-specific IFN-γ+/+ CD4+ T cells and other experimental groups. C, Percentage of mice shedding bacteria at each time period is reported. *, p = 0.0009 (Kaplan-Meier test) for resolution time between mice receiving IFN-γ+/+ CPAF-specific CD4+ T cells and other groups. D, On day 80 after chlamydial challenge, the percentage of animals exhibiting hydrosalpinx is reported. *, p < 0.05 (ANOVA) between mice receiving IFN-γ+/+ CPAF-specific CD4+ T cells and other groups. B, Bilateral; U, unilateral. All results are representative of two independent experiments.

FIGURE 4.

CPAF-specific CD4+ T cells are a sufficient source of IFN-γ for resolution of genital chlamydial infection and reduction of oviduct pathology. Groups of IFN-γ−/− mice and IFN-γ+/+ mice were immunized with three doses of rCPAF plus CpG or PBS (mock). A, Ag-specific cytokine production. Ten days after the last immunization, some animals (three mice per group) in each group were euthanized and splenocytes were stimulated in vitro with rCPAF or medium alone. After a 72-h incubation, the supernatants were analyzed for IFN-γ and IL-2 production. Results are expressed as mean ± SD of cytokine production in each group. B–D, Ten days after the final immunization, CD4+ T cells were purified from the spleens of vaccinated animals and transferred into C. muridarum-challenged recipient IFN-γ−/− mice (six mice per group). One group of IFN-γ−/− mice (six mice per group) that did not receive cellular transfers were challenged as controls. Vaginal chlamydial shedding was measured every third day after challenge. B, Numbers of chlamydial IFU recovered from vaginal swabs at the indicated days after genital challenge. Results are expressed as means ± SEM of the bacterial recovery from all animals in a group. *, p < 0.05 (ANOVA) between C. muridarum-challenged IFN-γ−/− mice receiving CPAF-specific IFN-γ+/+ CD4+ T cells and other experimental groups. C, Percentage of mice shedding bacteria at each time period is reported. *, p = 0.0009 (Kaplan-Meier test) for resolution time between mice receiving IFN-γ+/+ CPAF-specific CD4+ T cells and other groups. D, On day 80 after chlamydial challenge, the percentage of animals exhibiting hydrosalpinx is reported. *, p < 0.05 (ANOVA) between mice receiving IFN-γ+/+ CPAF-specific CD4+ T cells and other groups. B, Bilateral; U, unilateral. All results are representative of two independent experiments.

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Given that the MHC II pathway presents Ags to CD4+ T cells and has been shown previously to be important for induction of protective immunity after rCPAF vaccination (32), we further examined whether the MHC II pathway was sufficient to induce the complete range of protective effects after rCPAF plus CpG vaccination. To address this issue, we specifically examined whether absence of the MHC I pathway altogether, but with an intact MHC II pathway, would alter the effects of rCPAF plus CpG vaccination. Groups of mice deficient in β2-microglobulin (β2m−/− mice) and therefore in the MHC I-loading complex and the corresponding wild-type C57BL/6 mice (β2m+/+ mice) were vaccinated with three doses of rCPAF plus CpG, rested for 30 days, and challenged i.vag. with 105 IFU of C. muridarum according to the schedule described in Fig. 1. The complete absence of MHC I complexes would exclude the priming of CD8+ T cells in these experiments. As shown in Fig. 5,A, rCPAF plus CpG-vaccinated β2m−/− and β2m+/+ mice displayed significantly reduced vaginal bacterial shedding when compared with the corresponding PBS (mock)-immunized animals, as early as day 9 and at later time periods after challenge. rCPAF plus CpG-vaccinated β2m−/− mice completely resolved the infection by day 18 postchallenge, which was comparable to resolution in vaccinated wild-type β2m+/+ mice. Mock (PBS)- vaccinated β2m−/− mice and β2m+/+ mice displayed comparable reduced resolution kinetics, with chlamydial clearance occurring by day 30 after challenge in each group (Fig. 5,B). On day 80 after challenge, significant reduction in hydrosalpinx was observed in rCPAF plus CpG-vaccinated β2m−/− mice (20% bilateral, 20% unilateral) and β2m+/+ mice (20% bilateral), when compared with PBS (mock)-vaccinated β2m−/− mice (100% bilateral) and β2m+/+ mice (80% bilateral, 20% unilateral), respectively; Fig. 5 C). These data provide additional support that presentation of rCPAF via the MHC II pathway to CD4+ T cells is sufficient to induce a protective response against genital C. muridarum infection.

FIGURE 5.

MHC II pathway is sufficient for rCPAF + CpG-induced antichlamydial immunity. Groups of β2m−/− mice and C57BL/6 β2m+/+ mice (five mice per group) were immunized with three doses of rCPAF + CpG or PBS (mock) and rested for 1 mo. The animals were then challenged i.vag. with C. muridarum (105 IFU) and chlamydial shedding was measured at the indicated time periods (A and B). A, Numbers of chlamydial IFU recovered from vaginal swabs at the indicated days after genital challenge. Results are expressed as means ± SEM of the bacterial recovery from all animals in a group. *, p < 0.05 (ANOVA) between rCPAF+CpG-vaccinated and PBS (mock)-immunized β2m+/+ mice. ♣, p < 0.05 (ANOVA) between rCPAF + CpG- vaccinated and PBS (mock)-immunized β2m−/− mice. B, Percentage of mice shedding bacteria at each time period is reported. *, p = 0.015 (Kaplan-Meier test) for resolution time between mice receiving rCPAF+CpG-vaccinated and PBS (mock)-immunized β2m+/+ mice; ♣, p = 0.015 (Kaplan-Meier test) between rCPAF + CpG-vaccinated and PBS (mock)- immunized β2m−/− mice. C, On day 80 after chlamydial challenge, the percentage of animals exhibiting hydrosalpinx is reported. B, Bilateral; U, unilateral. *, p < 0.05 (ANOVA) for resolution time between mice receiving rCPAF + CpG-vaccinated and PBS (mock)-immunized β2m+/+ mice; ♣, p < 0.05 (ANOVA) between rCPAF + CpG-vaccinated and PBS (mock)-immunized β2m−/− mice. Results are representative of two independent experiments.

FIGURE 5.

MHC II pathway is sufficient for rCPAF + CpG-induced antichlamydial immunity. Groups of β2m−/− mice and C57BL/6 β2m+/+ mice (five mice per group) were immunized with three doses of rCPAF + CpG or PBS (mock) and rested for 1 mo. The animals were then challenged i.vag. with C. muridarum (105 IFU) and chlamydial shedding was measured at the indicated time periods (A and B). A, Numbers of chlamydial IFU recovered from vaginal swabs at the indicated days after genital challenge. Results are expressed as means ± SEM of the bacterial recovery from all animals in a group. *, p < 0.05 (ANOVA) between rCPAF+CpG-vaccinated and PBS (mock)-immunized β2m+/+ mice. ♣, p < 0.05 (ANOVA) between rCPAF + CpG- vaccinated and PBS (mock)-immunized β2m−/− mice. B, Percentage of mice shedding bacteria at each time period is reported. *, p = 0.015 (Kaplan-Meier test) for resolution time between mice receiving rCPAF+CpG-vaccinated and PBS (mock)-immunized β2m+/+ mice; ♣, p = 0.015 (Kaplan-Meier test) between rCPAF + CpG-vaccinated and PBS (mock)- immunized β2m−/− mice. C, On day 80 after chlamydial challenge, the percentage of animals exhibiting hydrosalpinx is reported. B, Bilateral; U, unilateral. *, p < 0.05 (ANOVA) for resolution time between mice receiving rCPAF + CpG-vaccinated and PBS (mock)-immunized β2m+/+ mice; ♣, p < 0.05 (ANOVA) between rCPAF + CpG-vaccinated and PBS (mock)-immunized β2m−/− mice. Results are representative of two independent experiments.

Close modal

The direct contribution of IFN-γ production from Ag-specific CD4+ T cells to antichlamydial protective immunity has not been fully determined. In this study, we used IFN-γ−/− and IFN-γR−/− mice in an i.n. rCPAF plus CpG-vaccination model and examined the role of adoptively transferred purified CD4+ T cells in conferring protective immunity against i.vag. C. muridarum challenge. We found that rCPAF plus CpG-vaccinated animals displayed enhanced numbers of Ag-specific CD4+ T cells and elevated levels of Ag-specific IFN-γ production locally in the genital tract, at early intervals after i.vag. chlamydial challenge. Moreover, Ag-specific CD4+ T cells 1) induced early chlamydial clearance and reduced oviduct pathology via production of IFN-γ; 2) produced sufficient amounts of IFN-γ, in an otherwise IFN-γ deficient environment, to induce antichlamydial immunity, and 3) the MHC II pathway was sufficient, in the absence of MHC I, to induce early bacterial clearance and reduction in oviduct pathology.

We previously have shown that vaccination with rCPAF plus a suitable Th1 adjuvant such as CpG or IL-12 induced bacterial clearance significantly earlier than rCPAF alone or mock (PBS) immunization (17, 30, 31, 32). In this study, the animals vaccinated with rCPAF plus CpG displayed elevated levels of Ag-specific CD4+ T cells and IFN-γ production within the genital tract at early intervals when compared with those immunized with rCPAF alone. Interestingly, the lag of at least 3–6 days in the onset of chlamydial clearance also correlates with a paucity of Ag-specific CD4+ T cell infiltration and IFN-γ production in the genital tract of rCPAF plus CpG-vaccinated animals during this interval following chlamydial challenge. These results are in agreement with the previous report demonstrating high levels of memory CD4+ T cell infiltration around day 7 following secondary genital chlamydial challenge (10). This delay in the onset of Ag-specific response may be due in part to the fact that CPAF is not present on the surface of the bacterium at the time of infection and is made only during the later stages of the chlamydial developmental cycle (23). Thus, CPAF-specific CD4+ T cells may not be recruited to the genital tract immediately after infection and the progesterone pretreatment, a component of the murine genital chlamydial infection model, may induce immunosuppression during early infection (1). Collectively, these results suggest a strong correlation between the onset of bacterial clearance and infiltration of Ag-specific CD4+ T cells and IFN-γ production locally in the genital tract.

Importantly, murine Ag-specific CD4+ T cells failed to induce protective anti-C. muridarum immunity in an IFN-γR-deficient environment. Ag-specific Th1 CD4+ T cells are capable of inducing protective immunity against infectious agents via the production of cytokines such as IFN-γ (18) and TNF-α (36, 37) or by induction of apoptosis via the Fas-Fas ligand pathway (16, 38). In the context of chlamydial infections, it has been shown previously that mice depleted of TNF-α or mice deficient in TNFR, Fas, or Fas ligand, each clear a primary genital C. muridarum infection in a manner comparable to that of wild-type animals (14, 16, 39, 40). IFN-γ has been shown to be effective for clearance of C. trachomatis; however, C. muridarum has been shown to be less sensitive to the direct microbicidal effects of murine IFN-γ (15). Additionally, vaginal C. muridarum clearance during early phases of primary genital infection has been shown to be largely independent of the effects of this cytokine (12). In our study, the protective immunity induced by CPAF-specific CD4+ T cells was completely abrogated in the absence of the IFN-γR, providing direct evidence that Ag-specific CD4+ T cells mediate C. muridarum clearance in an IFN-γ-dependent fashion. These results are not contradictory because 1) IFN-γ production from Ag-specific CD4+ T cells during later phases of infection, as opposed to innate immune cells during early infection, may be optimal to overcome the relative resistance of C. muridarum to the effects of this cytokine and/or 2) IFN-γ signaling may induce the production of other cytokines downstream which, in turn, may act as effectors for C. muridarum clearance.

Our results have clearly defined for the first time that IFN-γ secretion from Ag-specific CD4+ T cells is necessary and that sufficient IFN-γ is produced by these cells to mediate antichlamydial immunity. Specifically, Ag-specific CD4+ T cells from IFN-γ+/+ mice, but not IFN-γ−/− animals, were capable of inducing early chlamydial clearance and reduction of oviduct pathology in an otherwise IFN-γ-deficient environment. CD8+ T cells and NK cells also are considerable sources of IFN-γ and may contribute to its production (41) during primary and secondary chlamydial infections. However, the finding of the sufficiency of IFN-γ produced by Ag-specific CD4+ T cells has significant relevance to antichlamydial vaccine development. Given that an IFN-γ response can be induced by multiple adaptive immune cells, predominantly CD4+ T cells and CD8+ T cells, the targeting of the candidate Ag to MHC I or II pathway may be crucial in eliciting a dominant CD8+ T cell or CD4+ T cell response, respectively. Soluble Ags (e.g., rCPAF) are taken up by endosomes and processed primarily via the MHC II pathway. Ags delivered into the extracellular space may also get processed and cross-presented via the MHC I pathway to CD8+ T cells (42, 43). Thus, it may appear that soluble recombinant Ags use both MHC I and II pathways for induction of optimal protective immunity. However, in the context of rCPAF plus CpG vaccination, the complete absence of the MHC I pathway did not adversely affect the genital chlamydial clearance or reduction of oviduct pathology. This suggests that Ag presentation of soluble CPAF via the MHC II endosomal pathway to CD4+ T cells is sufficient to induce robust IFN-γ production for protective antichlamydial immunity. It is to be noted that a role for the MHC I pathway in induction of protective Ag-specific immunity cannot be excluded for Ags (for e.g., DNA vaccines using viral vectors) targeted specifically to the cytosolic compartment. Additionally, although the vaginal bacterial clearance in both rCPAF plus CpG-vaccinated (or PBS (mock)-immunized) β2m+/+ and β2m−/− mice were identical, there were minor, but not statistically significant, differences in the number of mice developing hydrosalpinx between the corresponding groups. This may be explained by the fact that vaginal chlamydial shedding is not a direct indicator of infection in the upper genital tract (the site of pathology). Therefore, the determination of upper genital tract pathology in addition to vaginal chlamydial shedding assumes importance for appropriate interpretation of the results from chlamydial-protective vaccination studies.

Collectively, these studies provide compelling evidence to suggest that murine Ag-specific CD4+ T cells induce enhanced chlamydial clearance and reduce oviduct pathology in an IFN-γ-dependent fashion and produce sufficient amounts of IFN-γ to induce anti-C. muridarum immunity in an otherwise IFN-γ-deficient environment. Therefore, targeting of recombinant soluble vaccine Ags to the MHC II pathway to elicit robust IFN-γ production from Ag-specific CD4+ T cells may be a viable vaccination strategy to achieve efficacious immunity against human genital chlamydial infections.

The authors have no financial conflict of interest.

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 National Institutes of Health Grant SO6GM008194-24.

4

Abbreviations used in this paper: CPAF, chlamydial protease-like activity factor; MHC I/II, MHC class I/II; i.n., intranasal; HEL, hen egg lysozyme; IFU, inclusion-forming unit; i.vag., intravaginal(ly).

1
Brunham, R. C., J. Rey-Ladino.
2005
. Immunology of Chlamydia infection: implications for a Chlamydia trachomatis vaccine.
Nat. Rev. Immunol.
5
:
149
-161.
2
Morrison, R. P., H. D. Caldwell.
2002
. Immunity to murine chlamydial genital infection.
Infect. Immun.
70
:
2741
-2751.
3
Brunham, R. C., B. Pourbohloul, S. Mak, R. White, M. L. Rekart.
2005
. The unexpected impact of a Chlamydia trachomatis infection control program on susceptibility to reinfection.
J. Infect. Dis.
192
:
1836
-1844.
4
Landers, D. V., K. Erlich, M. Sung, J. Schachter.
1991
. Role of L3T4-bearing T-cell populations in experimental murine chlamydial salpingitis.
Infect. Immun.
59
:
3774
-3777.
5
Su, H., H. D. Caldwell.
1995
. CD4+ T cells play a significant role in adoptive immunity to Chlamydia trachomatis infection of the mouse genital tract.
Infect. Immun.
63
:
3302
-3308.
6
Morrison, S. G., H. Su, H. D. Caldwell, R. P. Morrison.
2000
. Immunity to murine Chlamydia trachomatis genital tract reinfection involves B cells and CD4+ T cells but not CD8+ T cells.
Infect. Immun.
68
:
6979
-6987.
7
Igietseme, J. U., K. H. Ramsey, D. M. Magee, D. M. Williams, T. J. Kincy, R. G. Rank.
1993
. Resolution of murine chlamydial genital infection by the adoptive transfer of a biovar-specific, Th1 lymphocyte clone.
Reg. Immunol.
5
:
317
-324.
8
Ramsey, K. H., R. G. Rank.
1991
. Resolution of chlamydial genital infection with antigen-specific T-lymphocyte lines.
Infect. Immun.
59
:
925
-931.
9
Morrison, S. G., R. P. Morrison.
2000
. In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection.
Infect. Immun.
68
:
2870
-2879.
10
Kelly, K. A., R. G. Rank.
1997
. Identification of homing receptors that mediate the recruitment of CD4+ T cells to the genital tract following intravaginal infection with Chlamydia trachomatis.
Infect. Immun.
65
:
5198
-5208.
11
Cain, T. K., R. G. Rank.
1995
. Local Th1-like responses are induced by intravaginal infection of mice with the mouse pneumonitis biovar of Chlamydia trachomatis.
Infect. Immun.
63
:
1784
-1789.
12
Perry, L. L., K. Feilzer, H. D. Caldwell.
1997
. Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-γ-dependent and -independent pathways.
J. Immunol.
158
:
3344
-3352.
13
Perry, L. L., K. Feilzer, J. L. Portis, H. D. Caldwell.
1998
. Distinct homing pathways direct T lymphocytes to the genital and intestinal mucosae in Chlamydia-infected mice.
J. Immunol.
160
:
2905
-2914.
14
Perry, L. L., H. Su, K. Feilzer, R. Messer, S. Hughes, W. Whitmire, H. D. Caldwell.
1999
. Differential sensitivity of distinct Chlamydia trachomatis isolates to IFN-γ-mediated inhibition.
J. Immunol.
162
:
3541
-3548.
15
Nelson, D. E., D. P. Virok, H. Wood, C. Roshick, R. M. Johnson, W. M. Whitmire, D. D. Crane, O. Steele-Mortimer, L. Kari, G. McClarty, H. D. Caldwell.
2005
. Chlamydial IFN-γ immune evasion is linked to host infection tropism.
Proc. Natl. Acad. Sci. USA
102
:
10658
-10663.
16
Perry, L. L., K. Feilzer, S. Hughes, H. D. Caldwell.
1999
. Clearance of Chlamydia trachomatis from the murine genital mucosa does not require perforin-mediated cytolysis or Fas-mediated apoptosis.
Infect. Immun.
67
:
1379
-1385.
17
Murthy, A. K., J. P. Chambers, P. A. Meier, G. Zhong, B. P. Arulanandam.
2007
. Intranasal vaccination with a secreted chlamydial protein enhances resolution of genital Chlamydia muridarum infection, protects against oviduct pathology, and is highly dependent upon endogenous γ interferon production.
Infect. Immun.
75
:
666
-676.
18
Cotter, T. W., K. H. Ramsey, G. S. Miranpuri, C. E. Poulsen, G. I. Byrne.
1997
. Dissemination of Chlamydia trachomatis chronic genital tract infection in γ interferon gene knockout mice.
Infect. Immun.
65
:
2145
-2152.
19
Ito, J. I., J. M. Lyons.
1999
. Role of γ interferon in controlling murine chlamydial genital tract infection.
Infect. Immun.
67
:
5518
-5521.
20
Johansson, M., K. Schon, M. Ward, N. Lycke.
1997
. Genital tract infection with Chlamydia trachomatis fails to induce protective immunity in γ interferon receptor-deficient mice despite a strong local immunoglobulin A response.
Infect. Immun.
65
:
1032
-1044.
21
Rank, R. G., K. H. Ramsey, E. A. Pack, D. M. Williams.
1992
. Effect of γ interferon on resolution of murine chlamydial genital infection.
Infect. Immun.
60
:
4427
-4429.
22
Wang, S., Y. Fan, R. C. Brunham, X. Yang.
1999
. IFN-γ knockout mice show Th2-associated delayed-type hypersensitivity and the inflammatory cells fail to localize and control chlamydial infection.
Eur. J. Immunol.
29
:
3782
-3792.
23
Zhong, G., P. Fan, H. Ji, F. Dong, Y. Huang.
2001
. Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors.
J. Exp. Med.
193
:
935
-942.
24
Dong, F., H. Su, Y. Huang, Y. Zhong, G. Zhong.
2004
. Cleavage of host keratin 8 by a Chlamydia-secreted protease.
Infect. Immun.
72
:
3863
-3868.
25
Dong, F., Y. Zhong, B. Arulanandam, G. Zhong.
2005
. Production of a proteolytically active protein, chlamydial protease/proteasome-like activity factor, by five different Chlamydia species.
Infect. Immun.
73
:
1868
-1872.
26
Dong, F., M. Pirbhai, Y. Zhong, G. Zhong.
2004
. Cleavage-dependent activation of a Chlamydia-secreted protease.
Mol. Microbiol.
52
:
1487
-1494.
27
Sharma, J., Y. Zhong, F. Dong, J. M. Piper, G. Wang, G. Zhong.
2006
. Profiling of human antibody responses to Chlamydia trachomatis urogenital tract infection using microplates arrayed with 156 chlamydial fusion proteins.
Infect. Immun.
74
:
1490
-1499.
28
Sharma, J., F. Dong, M. Pirbhai, G. Zhong.
2005
. Inhibition of proteolytic activity of a chlamydial proteasome/protease-like activity factor by antibodies from humans infected with Chlamydia trachomatis.
Infect. Immun.
73
:
4414
-4419.
29
Sharma, J., A. M. Bosnic, J. M. Piper, G. Zhong.
2004
. Human antibody responses to a Chlamydia-secreted protease factor.
Infect. Immun.
72
:
7164
-7171.
30
Murphey, C., A. K. Murthy, P. A. Meier, G. M. Neal, G. Zhong, B. P. Arulanandam.
2006
. The protective efficacy of chlamydial protease-like activity factor vaccination is dependent upon CD4+ T cells.
Cell. Immunol.
242
:
110
-117.
31
Cong, Y., M. Jupelli, M. N. Guentzel, G. Zhong, A. K. Murthy, B. P. Arulanandam.
2007
. Intranasal immunization with chlamydial protease-like activity factor and CpG deoxynucleotides enhances protective immunity against genital Chlamydia muridarum infection.
Vaccine
25
:
3773
-3780.
32
Murthy, A. K., Y. Cong, C. Murphey, M. N. Guentzel, T. G. Forsthuber, G. Zhong, B. P. Arulanandam.
2006
. Chlamydial protease-like activity factor induces protective immunity against genital chlamydial infection in transgenic mice that express the human HLA-DR4 allele.
Infect. Immun.
74
:
6722
-6729.
33
Murthy, A. K., J. Sharma, J. J. Coalson, G. Zhong, B. P. Arulanandam.
2004
. Chlamydia trachomatis pulmonary infection induces greater inflammatory pathology in immunoglobulin A deficient mice.
Cell Immunol.
230
:
56
-64.
34
Asquith, B., C. Debacq, A. Florins, N. Gillet, T. Sanchez-Alcaraz, A. Mosley, L. Willems.
2006
. Quantifying lymphocyte kinetics in vivo using carboxyfluorescein diacetate succinimidyl ester (CFSE).
Proc. Biol. Sci.
273
:
1165
-1171.
35
Lyons, A. B..
2000
. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution.
J. Immunol. Methods
243
:
147
-154.
36
Deepe, G. S..
2007
. Tumor necrosis factor-α antagonism by the murine tumor necrosis factor-α receptor 2-Fc fusion protein exacerbates histoplasmosis in mice.
J. Interferon Cytokine Res.
27
:
471
-480.
37
Ritter, U., A. Lechner, K. Scharl, Z. Kiafard, J. Zwirner, H. Korner.
2008
. TNF controls the infiltration of dendritic cells into the site of Leishmania major infection.
Med. Microbiol. Immunol.
197
:
29
-37.
38
Shustov, A., P. Nguyen, F. Finkelman, K. B. Elkon, C. S. Via.
1998
. Differential expression of Fas and Fas ligand in acute and chronic graft-versus-host disease: up-regulation of Fas and Fas ligand requires CD8+ T cell activation and IFN-γ production.
J. Immunol.
161
:
2848
-2855.
39
Perry, L. L., K. Feilzer, H. D. Caldwell.
1998
. Neither interleukin-6 nor inducible nitric oxide synthase is required for clearance of Chlamydia trachomatis from the murine genital tract epithelium.
Infect. Immun.
66
:
1265
-1269.
40
Darville, T., C. W. Andrews, Jr, R. G. Rank.
2000
. Does inhibition of tumor necrosis factor α affect chlamydial genital tract infection in mice and guinea pigs?.
Infect. Immun.
68
:
5299
-5305.
41
Nammous, A. H., M. Pietruczuk, D. Zubacki, I. Dobrzycki.
2005
. Structure and biologic function of IFNγ.
Przegl. Lek.
62
:
890
-893.
42
Heath, W. R., G. T. Belz, G. M. Behrens, C. M. Smith, S. P. Forehan, I. A. Parish, G. M. Davey, N. S. Wilson, F. R. Carbone, J. A. Villadangos.
2004
. Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens.
Immunol. Rev.
199
:
9
-26.
43
Belz, G. T., F. R. Carbone, W. R. Heath.
2002
. Cross-presentation of antigens by dendritic cells.
Crit. Rev. Immunol.
22
:
439
-448.