B Cells Enhance Antigen-Specific CD4 T Cell Priming and Prevent Bacteria Dissemination following Chlamydia muridarum Genital Tract Infection

B cells can contribute to acquired immunity against intracellular bacteria, but do not usually participate in primary clearance. Here, we examined the endogenous CD4 T cell response to genital infection with Chlamydia muridarum using MHC class-II tetramers. Chlamydia-specific CD4 T cells expanded rapidly and persisted as a stable memory pool for several months after infection. While most lymph node Chlamydia-specific CD4 T cells expressed T-bet, a small percentage co-expressed Foxp3, and RORγt-expressing T cells were enriched within the reproductive tract. Local Chlamydia-specific CD4 T cell priming was markedly reduced in mice lacking B cells, and bacteria were able to disseminate to the peritoneal cavity, initiating a cellular infiltrate and ascites. However, bacterial dissemination also coincided with elevated systemic Chlamydia-specific CD4 T cell responses and resolution of primary infection. Together, these data reveal heterogeneity in pathogen-specific CD4 T cell responses within the genital tract and an unexpected requirement for B cells in regulating local T cell activation and bacterial dissemination during genital infection.


Introduction
Chlamydia trachomatis is an obligate intracellular pathogen that causes the most prevalent bacterial sexual transmitted infection worldwide [1]. In the US, Chlamydia is now the most common notifiable disease reported to the US Centers for Disease Control (CDC). The 1.4 million cases of Chlamydia infection reported in 2011 represent an 8% increase over the previous year and is the largest number of annual infections ever reported to the CDC for any condition [2]. The introduction of a Chlamydia screening and control program in the mid-1990s has not prevented annual increases in infection, although a portion of this increase is due to improved disease surveillance [3]. Overall, the CDC reports a median 8.3% Chlamydia positivity test among women aged 15-24, making this one of the most prevalent bacterial infections in the US.
Most Chlamydia infections are initially asymptomatic and therefore unlikely to be treated. However, 5-15% of females with untreated infection will eventually develop serious pelvic inflammatory disease (PID) as a consequence. Furthermore, 1 in 6 women who develop PID will become infertile, and many others will develop chronic pelvic inflammation and pain, or suffer from ectopic pregnancy [4][5][6]. The combination of an extraordinarily high number of infections, the asymptomatic nature of initial disease, and the potential for serious reproductive pathology in young women, means that Chlamydia is now recognized as a growing health care problem in the US. The current consensus among scientists and clinicians is that an effective Chlamydia vaccine is urgently needed [7].
The development of an effective Chlamydia vaccine would likely alleviate the burden of Chlamydia on the public health care system. However, the rational design of a Chlamydia vaccine would be aided by improved understanding of the cellular immune response to infection of the female reproductive tract. As Chlamydia is an obligate intracellular pathogen, IFN-c production by CD4 Th1 cells is essential for protective immunity to primary and secondary infection [8][9][10][11][12][13]. Unfortunately, we have at present only a rudimentary understanding of the development of protective Th1 responses in the context of the female upper reproductive tract and the extent of T helper heterogeneity is unclear. One of the major roadblocks to improving this situation is the lack of antigen-specific reagents that would allow detailed investigation of Chlamydiaspecific CD4 T cell responses using a relevant genital challenge model.
Previous studies have demonstrated that antibody production by B cells can assist protective immunity during secondary Chlamydia infection [14][15][16]. In contrast, B cells are thought to be dispensable for resolving primary Chlamydia infection, and B cell-deficient and wild type mice shed similar numbers of Chlamydia, as measured by vaginal swabs [17,18]. However, another study using the respiratory route of infection demonstrated that intranasal infection with Chlamydia requires B cells for efficient CD4 T cell activation [19]. Therefore, the issue of whether B cells contribute to initial CD4 T cell priming during vaginal infection requires additional analysis.
In this study, we generated MHC class-II tetramers to visualize the endogenous CD4 T cell response to systemic and genital tract Chlamydia infection. We show that, unlike intravenous infection, reproductive tract infection is associated with a short delay in the clonal expansion of Chlamydia-specific CD4 T cells in the local draining lymph node. While almost all expanded CD4 T cells expressed the Th1 marker, T-bet, we detected an expanded pool of Chlamydia-specific Tregs that co-expressed T-bet and Foxp3, and a population of Chlamydia-specific Th17 cells that were specifically enriched in the reproductive tract. In addition, we noted a surprising requirement for B cells in Chlamydia-specific CD4 cell priming within local draining lymph nodes. Loss of local priming in the absence of B cells coincided with bacterial dissemination to the peritoneal cavity inducing inflammatory infiltrate and ascites. Together, these data demonstrate heterogeneity in Chlamydia-specific T helper responses and an unexpected role for B cells in local CD4 T cell priming and bacterial containment within the upper reproductive tract.

Visualization of Chlamydia-specific CD4 T cell expansion after intravenous infection
In order to develop an overview of the adaptive response to Chlamydia infection, we initially examined the kinetics of bacterial growth and Chlamydia-specific CD4 T cell expansion during systemic infection with Chlamydia. When C57BL/6 female mice were infected intravenously (i.v.) with 1610 5 inclusion-forming units (IFUs) of C. muridarum, the bacterial burden in the spleen peaked around day 4 post-infection and decreased quickly thereafter (Fig. 1A). At day 20, no infectious Chlamydia was detected in the spleen (Fig. 1A). Consistent with previous findings [20], a small number of Chlamydia were found in the lung during the first week of systemic infection, but no bacteria were detected in kidney or heart at any time point (data not shown).
Numerous C. muridarum MHC class-II epitopes have been uncovered by Immunoproteomic analysis of infected APCs [21]. We used an ELISPOT assay to monitor the frequency of CD4 T cells responding to multiple C. muridarum epitopes after systemic infection. A population of IFN-c-secreting CD4 T cells responding to RplF 51-59 , Aasf 24-32 , and PmpG-1 303-311 was detected as early as 4 days after infection ( Fig. 1B and C). Expansion of IFNc-secreting CD4 T cells peaked around day 4-7, and was followed by a slow contraction of the population over the next 90 days, before a plateau was reached that lasted for at least 352 days ( Fig. 1B and 1D). Thus, peak expansion of IFN-c-secreting CD4 cells closely mirrored peak bacterial burdens in vivo, and stable Chlamydia-specific CD4 T cell memory frequencies were maintained in the absence of active Chlamydia infection.

Construction of Chlamydia-specific pMHCII tetramers
Previous studies have demonstrated that pMHC class-II tetramers can be used in conjunction with tetramer enrichment, to visualize low frequency endogenous antigen-specific CD4 T cells in infected and immunized mice [22,23]. We constructed three distinct pMHC class-II tetramers, containing I-A b with a Chlamydia-specific epitope (RplF 51-59 , Aasf 24-32 , or PmpG-1 303-311 ) bound to the MHC class-II b chain. Uninfected C57BL/6 mice contained low frequency antigen-specific CD4 T cell population specific for each Chlamydia epitope ( Fig. 2A). However, in mice immunized subcutaneously with peptide/CFA, or infected intravenously with C. muridarum, an expanded population of CD44 hi CD4 T cells was detectable 7 days post immunization or infection ( Fig. 2A). Tetramer staining was specific, as infection with Salmonella Typhimurium did not induce expansion of tetramerspecific CD4 T cells ( Fig. 2A). Furthermore, no CD8 T cells were detected that bound to Chlamydia tetramers ( Fig. 2A) Together, these results demonstrate that all three tetramers, RplF 51-59 :I-A b , Aasf 24-32 :I-A b , and PmpG-1 303-311 :I-A b can be used to detect endogenous C. muridarum-CD4 T cells in vivo.

Visualization of Chlamydia-specific CD4 T cell expansion to intravaginal infection
We next used the PmpG-1 303-311 :I-A b tetramer to visualize clonal expansion of antigen-specific CD4 T cells during intravaginal (i.vag.) infection. We focused on PmpG-1 because CD4 T cell clonal expansion against RplF, Aasf and PmpG-1 is similar (Fig. 1B, 1C and 1D) yet PmpG-1 is a promising Chlamydia vaccine candidate [24]. Consistent with previous findings, C. muridarum bacterial loads measured by vaginal swab peaked around day 4 post-infection and steadily decreased until clearance around day 35 (Fig. 2B). To visualize the primary site of endogenous T cell priming to Chlamydia infection, we examined PmpG-1-specific T cell activation in multiple secondary lymphoid tissues. One week after infection, PmpG-1-specific CD4 T cells expanded in iliac lymph nodes and spleen, but were barely detectable in other nondraining lymph nodes ( Fig. 2C and 2D). At day 14, endogenous PmpG-1-specific CD4 T cell expansion peaked in all secondary lymphoid tissues, and was followed by a notable contraction phase (Fig. 2D). The kinetics of the CD4 T cell response to local Chlamydia genital tract infection was therefore markedly delayed in comparison to systemic infection with the same pathogen ( Fig. 1).

Identification of Th1/Treg/Th17 cell subsets in Chlamydia-specific CD4 T cells
To examine CD4 T helper differentiation, the expression of lineage-specific transcription factors was examined in expanded CD44 hi PmpG-1 303-311 :I-A b+ CD4 T cells. Following either systemic or intravaginal infection, almost all PmpG-1-specific CD4 T cells expressed T-bet while no GATA3 expression was detected (Fig. 3A). This is consistent with previous reports that Th1 CD4 T cells are the dominant helper subset following Chlamydia infection [10,11]. However, a distinct population of PmpG-1-specific CD4 T cells that co-expressed Foxp3 and T-bet was also detected after i.v. and i.vag. infection (Fig. 3B), suggesting that induced Chlamydia-specific Treg cells are also contained within the expanded CD4 pool. We also utilized RORct-GFP reporter

Author Summary
Sexually transmitted infections caused by Chlamydia are increasing every year in the US and an effective vaccine is urgently required. Unfortunately, we currently only have a rudimentary understanding of the natural host immune response to Chlamydia infection, especially in the context of the female genital tract. Here, we have developed new reagents that allow direct visualization of the host T cell responses to vaginal Chlamydia infection using a mouse model of infection. These new tools reveal an unexpectedly complex CD4 T cell response to infection and a surprising role for B cells in preventing the spread of bacteria to multiple host tissues. This greater understanding of the host response to infection may eventually allow the construction of an effective vaccine.
mice and combined staining with all three Chlamydia tetramers to examine the potential development of Chlamydia-specific Th17 cells after vaginal infection. While GFP-positive cells were undetectable among expanded tetramer-positive cells in the spleen or draining lymph nodes of infected mice, approximately 7% of CD4 + CD44 hi tetramer + T cells in infected non-lymphoid tissues expressed GFP (Fig. 3C). Furthermore, stimulation of lymphocytes purified from the genital tract confirmed the presence of T cells producing both IL-17A and IFN-c (Fig. 3D). Thus, Chlamydia infection of the reproductive tract induces a heterogenous T helper response that comprises expanded T-bet + Th1 cells, T-bet + Foxp3 + Tregs, and Th17 cells that are enriched in infected non-lymphoid tissues.

Reduced local CD4 expansion and Chlamydia dissemination in mice lacking B cells
Next, we examined bacterial shedding after vaginal infection of WT and mMT mice with Chlamydia. Consistent with previous reports [17,18], bacterial shedding was unaffected by the absence of B cells (Fig. 4A). However, more detailed analysis of the local draining lymph nodes of mMT mice suggested significantly reduced activation of CD4 T cells (Fig. 4B). Indeed, using the PmpG-1 303-311 :I-A b tetramer, we detected much lower clonal expansion of Chlamydia-specific CD4 T cells in the local draining lymph node of infected mMT mice compared to WT mice (60612 in mMT mice vs 166636 in WT mice, p,0.01; Fig. 4C). This reduced local response was also accompanied by dissemination of Chlamydia to the spleen and peritoneal cavity, where ascites was noted 14 days post infection ( Fig. 5A and 5B). Analysis of ascites fluid from mMT mice revealed a large proportion of macrophages (F4/80 + ), monocytes (Gr-1 + ) and T lymphocytes (Fig. 5C). In addition, PmpG-1-specific CD4 T cells were abundant in ascites, demonstrating that much of the lymphocyte infiltrate into the peritoneal cavity is likely to be Chlamydia-specific (Fig. 5C). Thus, the absence of B cells reduces local CD4 T cell priming and allows bacterial dissemination.

Enhanced systemic CD4 T cell priming in B cell deficient mice
In contrast to the local response, polyclonal CD4 T cells in the spleen of mMT mice displayed evidence of increased activation (Fig. 4B). Consistent with increased systemic activation, expansion of PmpG-1-specific CD4 T cells was markedly increased in the spleen of mMT mice (3360066044 in mMT mice vs 549461164 in WT mice, p,0.001; Fig. 4C). Furthermore, Chlamydia-specific     Fig. 4D  and E). A greater percentage of multifunctional CD4 T cells producing IFN-c and TNF-a was also detected (Fig. 4F). Consistent with an enhanced effector response, the percentage of Chlamydia-specific CD4 T cells expressing CCR7 was considerably lower in mMT mice (Fig. 4D). These data suggest a compensatory systemic T cell response is induced to clear the disseminated bacteria that accompany B cell deficiency.

Discussion
In vivo visualization studies of pathogen-specific CD4 T cell responses have typically involved adoptive transfer of monoclonal TCR transgenic T cells and have rarely focused on sexually transmitted infections [25,26]. Here, we describe the generation of peptide-MHC class II tetramers that allow direct visualization of endogenous, polyclonal antigen-specific CD4 T cell responses to Chlamydia infection. Using Chlamydia tetramers and ELISPOT assays, we detected differences in the tempo of the initial CD4 T cell responses to systemic or vaginal challenge with Chlamydia. Unlike other mucosal tissues, the female genital mucosa lacks defined lymphoid structures [7], which may explain the delayed clonal expansion of CD4 T cells after Chlamydia genital tract infection. Alternatively, this delay may represent an unappreciated virulence mechanism of Chlamydia to delay early T cell priming, as has been noted during respiratory infection with MTB [27]. Thus, delayed CD4 T cell priming in the draining lymph node may be due to limited access to bacterial antigen by tissue dendritic cells or impeded migration to the local lymph nodes. Further studies will be required to examine this issue directly.
Our studies show that Chlamydia-specific memory CD4 T cells persist for at least one year after infection, which may suggest the presence of persistent antigen and/or persistent Chlamydia without brefeldin A. IFNc and TNFa production by activated CD4 T cells (CD44 hi CD4 + ) were detected by flow cytometry. The percentages of IFNc + , TNFa + and IFNc + TNFa + CD4 T cells were summarized in the graph. Error bars show mean number 6 SEM. doi:10.1371/journal.ppat.1003707.g004 culturable Chlamydia organisms [28], after the clearance of infectious bacteria. Interestingly, a recent study by Johnson et al suggested that the PmpG-1 303-311 epitope can be detected on splenic APC for at least 6 months after the clearance of primary genital tract infection [29]. Our data, together with others, also support the potential role of PmpG-1 specific CD4 T cells in Chlamydia protective immunity [24,29].
Our data confirm directly the previous notion that T-bet expressing Th1 cells are the predominant CD4 effector lineage among Chlamydia-specific CD4 T cells. However, we also identify small populations with the phenotypic marker of Tregs in the lymph node and spleen following both systemic and mucosal infection, as well as the enrichment of Chlamydia-specific Th17 cells at the genital mucosa itself. These data suggest that Chlamydiaspecific Tregs are expanded to regulate the large Th1 response that develops during infection [30]. The enrichment of Th17 cells at mucosal surfaces has also been detected in other infection models [23,[31][32][33]. For example, flagellin-specific Th17 cells are predominantly enriched in the gut mucosal sites after Salmonella oral infection [23]. Further experimentation is required to determine whether these pathogen-specific Th17 cells contribute to Chlamydia clearance and/or the induction of upper genital tract pathology.
The presence of large numbers of B cells in the lower and upper genital tract during Chlamydia infection, as shown by both immunohistochemical staining and flow cytometry [34] (and data not shown), led us to speculate that B cells play an important role during primary Chlamydia infection. Indeed, our data show that in the absence of B cells, there is a marked reduction in antigen-specific CD4 T cell priming within the draining iliac lymph nodes. Given this effect of early CD4 T cell expansion it is possible that B cells participate directly in antigen presentation during the early stages of primary infection. Although this has not previously been observed during genital tract infection, a similar finding was reported after C. muridarum lung infection [19]. A recent study has suggested that macrophage deficiency could also account for protective defects of mMT mice against viral infections [35]. However, our observations that ascites did not occur at early time point (7 days post infection, data not shown) suggested that the phenotype we observed is mediated by altered adaptive immune mechanisms.
Although mild Chlamydia dissemination to other mucosal sites has been reported previously in C57BL/6 mice [36], a large number of disseminated Chlamydia has only previously been found in IFN-c-deficient and SCID mice and largely attributed to deficient Th1 development [8]. B cell deficient mice therefore provide an unexpected additional model where Chlamydia also disseminate to non-mucosa tissues. Notably, our data provides a clear example where the marked pathology of Chlamydia infection does not always correlate with the inability of host to clear the infection. A likely explanation for highly efficient bacteria clearance in mMT mice is the robust systemic CD4 T cell response that may compensate for the loss of initial CD4 T cell priming within the local draining lymph nodes of the genital tract. The fact that Chlamydia genital tract infection can lead to ascites in the absence of B cells is also clinically relevant: Chlamydia infection induces ascites in patients with salpingitis and peritonitis [37][38][39][40], although it is unclear whether Chlamydia dissemination in mouse models reflects the same mechanism that leads to symptoms in human such as PID and Fitz-Hugh-Curtis syndrome. Further pathological studies are needed to understand differences of mouse and human infections.
Overall, our data demonstrate unappreciated heterogeneity of the CD4 T cell response to genital tract infection in a model where Th1 cells are essential for protective immunity. In addition, our data uncover a surprising involvement of B cells in local expansion of effector Chlamydia-specific T cells in the genital tract and prevention of bacterial dissemination. Greater understanding of the mechanism of Chlamydia dissemination from the genital tract and the T and B cell responses that restrain this spread of bacteria may reduce the risk of sequelae after Chlamydia genital infection in infected women.

Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory

Bacteria
Chlamydia muridarum strain Nigg II was purchased from ATCC (Manassas, VA). The organism was cultured in HeLa 229 cells in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS). Elementary bodies (EBs) were purified by discontinuous density gradient centrifugation as previously described and stored at 280 degree [41]. The number of IFUs of purified EBs was determined by infection of HeLa 229 cells and enumeration of inclusions that were stained with anti-Chlamydia MOMP antibody. Heat-killed EBs (HKEBs) were prepared by heating at 56uC for 30 min. A fresh aliquot was thawed and used for every infection experiment. Salmonella enterica serovar Typhimurium strains BRD509 (AroA 2 D 2 ) were kindly provided by Dr. D. Xu (University of Glasgow, Glasgow, U.K).

Chlamydia infection and enumeration
For systemic infection, mice were injected intravenously in the lateral tail vein with 1610 5 C. muridarum. To enumerate the bacteria burden in tissues, the spleen, liver and kidney, were crushed in 5 mL SPG buffer and tissue homogenate was placed in a tube with glass beads to disrupt cells. After shaking for 5 min, and centrifugation at 500 g for 10 minutes, supernatants were collected and serial dilutions were plated on HeLa 229 cells. For intravaginal infections, estrus was synchronized by subcutaneous injection of 2.5 mg medroxyprogesterone acetate (Greenstone, NJ) 7 days before infection. 1610 5 C. muridarum in 5 mL SPG buffer were then deposited into vaginal vaults. To enumerate bacteria, vaginal swabs were collected, shaken with glass beads, and serial dilutions were plated on HeLa 229 cells.

ELISPOT assay
Spleen and lymph nodes (axillary, brachial, inguinal and mesenteric) were harvested and a single-cell suspension prepared.

Construction of pMHCII tetramers
The methodology for construction of pMHCII tetramers has been described in detail [22]. Briefly, biotinylated I-A b monomers containing a covalently linked C. muridarum peptide (RplF 51-59 , Aasf 24-32 or PmpG-1 303-311 ) were expressed by S2 Drosophila insect cell lines and cultured in a Wave Bioreactor (GE Healthcare Biosciences, Pittsburgh, PA). After purification, I-A b monomers were tetramerized by co-incubated with fluorochrome-conjugated streptavidin at a pre-determined optimal ratio at room temperature for 30 min [22]. To test for tetramer specificity, C57BL/6 mice were immunized with each of the three peptides and CFA (Sigma-Aldrich, St. Louis, MO). Seven days post-immunization, draining lymph nodes were isolated and tetramer positive cells enriched using the methodology described below.

Statistical analysis
All data sets were analyzed by unpaired Student's t-test using Prism (GraphPad Software, La Jolla, CA). A p value,0.05 were considered statistically significant.