Temporal Expression of Bacterial Proteins Instructs Host CD4 T Cell Expansion and Th17 Development

Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS) effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.


Introduction
Generating vaccines for current and emerging infectious diseases remains an important goal of immunological research [1,2]. An ideal vaccine will induce the expansion and maturation of naïve pathogen-specific lymphocyte clones that exist at low frequency in uninfected or unimmunized individuals [3]. During infection or immunization, pathogen-specific T cells expand within secondary lymphoid tissues after recognition of foreign peptides in the context of host MHC molecules. These dividing pathogen-specific T cells acquire effector capabilities that are tailored to combat different classes of microbial pathogen [4,5]. Following the resolution of primary infection, a cohort of these expanded pathogen-specific T cells persists to provide robust secondary immunity against another encounter with the same pathogen [6].
Naïve CD4 T cells can differentiate into T helper 1 (Th1), Th2, or Th17 effector lineages depending on the instructional cues delivered during initial activation [7]. Th1 cells express the transcription factor T-bet, secrete IFN-c, and protect the host against infection with intra-macrophage pathogens [8,9]. In contrast, Th2 cells express GATA-3, secrete IL-4, and combat large extracellular pathogens [10,11]. Th17 cells are a recently described effector cell lineage that express RORct, secrete IL-17, and contribute to clearance of extracellular bacterial and fungal infections [5,12]. A number of variables can influence CD4 T helper cell differentiation, including TCR affinity for peptide/ MHC, antigen dose, costimulatory signals, and the local concentration of inflammatory cytokines [7].
In vivo analysis of CD4 T cell differentiation during infection has often involved visualization of adoptively transferred TCR transgenic T cells [13][14][15][16][17][18][19]. Although this experimental approach allows detection of pathogen-specific T cells using conventional methodologies, it can also introduce experimental variables that are not present in a natural host [20][21][22]. The development of peptide-MHC tetramer enrichment methodologies now allows direct visualization of low frequency antigen-specific CD4 T cell populations without requiring adoptive transfer of TCR transgenic cells [23]. Using this approach, infection via the intra-nasal route was shown to enhance development of Listeria-specific Th17 cells while systemic infection encouraged Th1 development [24]. Most pathogens differentially regulate their protein expression in mucosal or systemic tissues depending on local metabolic or virulence requirements [25,26], but it is not yet clear how this might affect CD4 T cell expansion and differentiation to stagespecific antigens expressed in different tissues.
Here, we examined this issue using peptide-MHC tetramers and ELISPOTs that allow simultaneous tracking of CD4 T cell responses to Salmonella flagellin and type-III-secretion system (TTSS) effector proteins. We found that flagellin-specific CD4 T cells undergo early expansion and contraction and after oral infection generate Th17 cells that localize to infected mucosal tissues. In contrast, CD4 T cells responding to TTSS effector proteins expanded and accumulated over several weeks and primarily differentiated to a Th1 lineage localized in systemic tissues. Thus, an infected host simultaneously develops distinct populations of CD4 effector lineages that detect stage-specific antigens and have divergent anatomical localization.

Results
Identification of novel class-II Salmonella epitopes I-A b epitopes have been identified within Salmonella flagellin [27,28], and these remain the only confirmed class-II epitopes for examining CD4 response to Salmonella in C57BL/6 mice [29]. However, flagellin is an unusual antigen that can be directly detected by the innate immune system, and is also rapidly downregulated as bacteria transition to intracellular growth [30][31][32]. In order to uncover antigens that are recognized by CD4 T cells during intracellular bacterial growth, we used a bioinformatic approach to identify novel Salmonella I-A b epitopes. Based on the published structure of I-A b in complex with human class-II invariant chain peptide (CLIP) [33], we used a positional-specific scoring matrix (PSSM) and Hidden Markov Model (HMM) to interrogate all open reading frames in the Salmonella genome for I-A b epitopes. Although this yielded numerous epitopes that were then confirmed by peptide immunization (Table 1 and Figure 1A), none were found to be natural epitopes during murine Salmonella infection or after immunization with heat-killed Salmonella (data not shown). Since many of these candidate peptides were derived from cytoplasmic proteins that are unlikely to enter the class-II presentation pathway, we modified our approach to focus on outer membrane or secreted Salmonella proteins, ( Table 2), many of which are required for bacterial persistence in vivo [34]. Using this strategy we identified natural I-A b epitopes in two effector proteins that are encoded by Salmonella Pathogenicity Island 2 (SPI2) TTSS, SseI and SseJ ( Figure 1B and Table 3).
In marked contrast to flagellin, SseI and SseJ are though to be expressed during intra-macrophage replication [35]. We confirmed the differential regulation of flagellin (FliC) and SseJ by examining bacterial mRNA expression in vitro. While FliC mRNA was highly expressed under SPI1-inducing conditions, it was down-regulated under SPI2-inducing conditions which simulate the intra-macrophage environment ( Figure 2). In contrast, SseJ was expressed under SPI2-inducing conditions but down-regulated under SPI1-inducing conditions ( Figure 2). Thus, Salmonella differentially regulate the production of flagellin and SseJ, depending on local environmental cues. Although it was technically challenging to measure Salmonella mRNA expression in vivo, we were able to confirm the differential regulation of FliC and SseJ at early time points after infection. FliC mRNA was highly expressed at 30 minutes after infection but significantly reduced at 5 hours, while in contrast, SseJ mRNA was low at 5 minutes but expression increased at 30 minutes ( Figure 2). Thus, flagellin (FliC) and SseJ represent antigens that are differentially regulated during the transition from extracellular to intracellular growth.

Construction of Salmonella-specific class-II tetramers
We constructed class-II I-A b tetramers containing flagellin 427-441 and SseJ 329-341 epitopes and visualized endogenous flagellin 427-441 -, SseJ 329-341 -specific CD4 T cells using a sensitive tetramer enrichment methodology [36]. C57BL/6 mice contained low numbers of naive flagellin-and SseJ-specific CD4 T cells, but a population of expanded CD44 Hi tetramer positive cells was readily detected in the draining lymph nodes after immunization with flagellin 427-441 or SseJ 329-341 and CFA (Figure 3). Similarly, infection with Salmonella (BRD509) allowed detection of an expanded population of flagellin-and SseJ-specific CD4 T cells ( Figure 3). Salmonella-specific T cells were not detected in unbound column fractions or in CD8 T cells bound to enrichment columns ( Figure 3), demonstrating the specificity of these detection regents and the efficiency of enrichment. We chose to focus on visualizing CD4 T cells responding to Salmonella strain BRD509 since this strain has been widely studied previously [37][38][39], and use of more virulent strains precludes analysis at late time points after infection of C57BL/6 mice.

Distinct kinetics of flagellin-and SseJ-specific CD4 T cell expansion following infection
We examined the kinetics of flagellin 427-441 -specific and SseJ 329-341 -specific CD4 T cell expansion after intravenous (IV) infection with Salmonella. The pooled secondary lymphoid tissues of a C57BL/6 mouse contained approximately 32 flagellin 427-441 -specific and 30 SseJ 329-341 -specific CD4 T cells ( Figure 4A). After Salmonella infection, flagellin 427-441 -specific CD4 T cells expanded to a peak of 410 cells at day 7 (12.9 fold expansion over naïve frequency), before contracting to 88 cells by day 160 ( Figure 4A). At the peak of clonal expansion, flagellin 427-441 -specific CD4 T cells decreased surface expression of CCR7 and CD27, indicating the development of T effector cells ( Figure 4B and C). Flagellin 427-441 -specific T cells started to contract as early as day 10 following infection, and this coincided with a gradual increase in the percentage of cells expressing CCR7 and CD27 ( Figure 4A-C). Importantly, this CD4 contraction phase occurred while bacterial burdens remained high in vivo ( Figure 4D). In marked contrast, SseJ 329-341 -specific CD4 T cells expanded after infection and continued to accumulate in secondary lymphoid tissues until day 52 post-infection, eventually reaching a peak of 5,900 cells (210fold expansion over naïve frequency), before decreasing to 1,800 cells by day 160 ( Figure 4A). In contrast to the flagellin-specific response, SseJ 329-341 -specific CD4 T cells maintained low expression of CCR7 and CD27 until bacterial clearance had been achieved ( Figure 4B-D). We also examined intracellular expression of transcription factors associated with T helper lineage commitment in these two populations at time points close to peak expansion. The vast majority of expanded flagellin 427-441 -specific and SseJ 329-341 -specific CD4 T cells expressed T-bet, while no staining above background levels was detected using antibodies specific for GATA-3, FoxP3, or RORct ( Figure 4E and data not shown).
In order to confirm the distinct tempo of CD4 responses to Salmonella flagellin and TTSS effector proteins, we also examined

Author Summary
Pathogens alter protein expression in an infected host, depending on metabolic or virulence requirements, but the effect of these changes on the immune response is unclear. We identified new class-II epitopes within Salmonella type-III secretion system effector proteins and generated a methodology to visualize endogenous T cells responding to these epitopes. Our study shows that Salmonella flagellin generates a mixed Th1 and Th17 response that contracts early and is enriched in mucosal tissues. In contrast, we found that Salmonella T3SS effectors generate a sustained Th1 response that requires a persisting infection and is enriched in systemic tissues. These data demonstrate that in vivo antigen regulation substantially alters the antigen specificity, helper differentiation, and anatomical location of pathogen-specific CD4 T cells. CD4 T cell responses by ELISPOT. IFN-c-producing CD4 T cells were detected seven days after Salmonella infection and were found to predominantly focus on flagellin, rather than TTSS effector epitopes ( Figure 5). In marked contrast, 40 days after infection, larger pools of IFN-c-producing CD4 T cells were detected responding to SseI and SseJ epitopes rather than flagellin  Table 1 at 5 mg/peptide in CFA. Eight days later, draining lymph nodes were isolated and purified CD4 T cells restimulated with individual peptides (Table 1) in the presence of irradiated splenocytes. IFN-c production was measured 24-36 hours later by ELISPOT. (B) 129Sv mice were orally infected with virulent 5x10 7 Salmonella (SL1344) and spleens harvested 11 weeks later. Purified CD4 T cells were restimulated with peptides from Table 2

Bacterial persistence is required for accumulation of SseJspecific T cells
We hypothesized that the temporal difference in the CD4 T cell response to flagellin and SseJ was due to the maintenance of SseJ expression in vivo by persistent bacteria. However, it was also possible that these different kinetics reflected T cell-intrinsic variables that were completely unrelated to antigen persistence. We therefore examined the clonal expansion of SseJ-specific T cells in mice that were administered antibiotics to clear bacteria, beginning five days after infection. Although SseJ-specific T cells were clearly detected in both treated and untreated mice, clonal expansion was significantly reduced in mice that had been treated with antibiotics ( Figure 6). Thus, the continued accumulation of SseJ-specific CD4 T cells at later time points after infection required bacterial persistence.

Enrichment of flagellin-specific Th17 CD4 T cells in mucosal tissues after oral infection
Next, we used ELISPOTs to examine the CD4 T cell response to oral Salmonella infection and closely monitored Th1, Th2, and Th17 responses to flagellin and TTSS effector proteins in mucosal and systemic tissues. We used three oral doses of Salmonella (BRD509) as this was required for optimal priming of effector CD4 T cell responses. Consistent with the absence of Th2 responses after IV infection, IL-4 production was not detected in either mucosal or systemic tissues (data not shown). In contrast, IFN-cproducing CD4 T cells were detected in intestinal (mesenteric lymph nodes, MLNs, and lamina propria, LP) and systemic (spleen and liver) tissues ( Figure 7A). SseI and J-specific CD4 Th1 cells were more numerous in the spleen and liver, while flagellinspecific CD4 Th1 cells were predominantly detected in intestinal tissues ( Figure 7A). Furthermore, IL-17A-producing CD4 T cells were also detected and this response was notably focused on the flagellin epitopes and occurred in mucosal tissues ( Figure 7B). Indeed, flagellin-specific CD4 T cells in intestinal tissues had a higher ratio of Th17:Th1 cells, while in contrast SseI and J-specific CD4 T cells in systemic tissues had a higher ratio of Th1:Th17 cells ( Figure 7C). In order to confirm the preferential generation of flagellin-specific Th17 cells in the intestine, we also examined IL-22 production by CD4 T cells recovered from the spleen or MLN of Salmonella-infected mice. Consistent with the ELISPOT data, IL-22 was detected after stimulation of CD4 T cells from the MLN but not the spleen of infected mice, and was directed towards flagellin 427-441 rather than T3SS effectors ( Figure 8).

Discussion
This study describes the first examination of endogenous CD4 T cell responses to multiple Salmonella epitopes using tetramer and ELISPOT assays. Previous analysis of Salmonella-specific CD4 T cells has been severely limited by the lack of defined epitopes and has largely focused on the response to a single I-A b epitope within flagellin [13,40,41]. Our ability to track multiple epitope-specific CD4 responses in this study has revealed that in vivo regulation of bacterial antigen expression is a critical factor in orchestrating both the tempo and effector maturation of Salmonella-specific CD4 T cells at mucosal and systemic sites. High expression of flagellin is reported from extracellular bacteria but this is rapidly decreased after infection of macrophages, and flagelin cannot be detected in the spleen of infected mice [32,42]. In contrast, expression of SPI-2 genes is initiated within the phagolysosome of infected macrophages and is required for bacterial survival in vivo [35]. Our data confirm these findings and demonstrate differential expression of FliC and SseJ mRNA in response to environmental cues, both in vitro and in vivo. Thus, naïve Salmonella-specific CD4 T cells will encounter flagellin only during the early stage of infection within the intestine, whereas SPI-2 TTSS effector  proteins accumulate as more intracellular bacteria replicate in the systemic tissues of the spleen and liver. Interestingly, our tetramer data reveal a pattern of CD4 T cell expansion and contraction to flagellin and SseJ that corresponds closely to their relative temporal abundance during in vivo growth. In particular, we detected early contraction of flagellin-specific T cells, while SseJ-specific T cell responses remained elevated for several weeks. There may be T cell-intrinsic factors that determine the peak clonal expansion of FliC-and SseJ-specific T cells since SseJ-specific T cells exhibit greater clonal expansion. However, this issue does not explain the maintenance of the SseJ response for several weeks after infection. Furthermore, the elimination of bacteria using antibiotics severely dampened the CD4 T cell response to SseJ, indicating that antigen persistence is essential for sustained clonal expansion of this population. Although antibiotics can affect adaptive response indirectly via effects on commensal flora, it is more likely that the reduction in the SseJ response is due to a direct effect on bacterial persistence. Together, these data suggest that CD4 T cells are surprisingly dependent upon local antigen availability even after clonal expansion has occurred and indicate that clearer definition of in vivo protein expression by pathogens might be critical for identification of protective antigens, especially for pathogens that reside in multiple tissues or have distinct life-cycle stages. Indeed, a correlation between temporal antigen expression and in vivo activation of protective CD4 T cell responses has recently been observed during M. tuberculosis infection [43,44]. Furthermore, a requirement for sustained antigen expression for the generation of optimal CD4 T cell responses has also been identified in other model systems [45,46].
Our data also demonstrate divergent CD4 T cell helper development to flagellin and TTSS effector proteins. After intravenous infection, CD4 T cells responding to each of these antigens expressed T-bet and produced IFN-c following in vitro restimulation, thus Salmonella generates distinct antigen-specific Th1 cell populations that appear at different stages of infection. This finding may help explain why Th1 cells can be detected within days of Salmonella infection but do not appear to actively participate in bacterial clearance until several week later [47]. If the majority of the early Salmonella-specific Th1 response is largely focused on antigens that are transiently expressed, such as flagellin or SPI-1 gene products, then intracellular bacteria can evade detection by the initial wave of Th1 effector cells. In contrast, a second wave of Th1 cells that are specific for highly expressed intracellular antigens such as T3SS effectors would be more likely to participate in the later stages of bacterial clearance from systemic tissues. A similar model of bacterial antigen regulation and effector T cell evasion has been proposed in mycobacterial infection [43,48], and may be a common feature of persistent bacterial infection.
After oral infection with Salmonella, we detected Th17 cells specific for flagellin epitopes and these were particularly enriched in the intestine. TGF-b is abundant in mucosal tissues and plays an important role in the generation of both Treg and Th17 cells [5]. Intestinal IL-6 production is induced by Salmonella infection and is likely to drive Salmonella-specific differentiation towards a Th17 lineage in the intestine [49,50]. Indeed, flagellin is a ligand for TLR5 and can directly induce IL-6 production in vitro and in vivo [38,51]. This may explain why Th17 cells specific for flagellin epitopes found only in infected intestinal tissues while Th17 responses to T3SS effector proteins are largely absent. Th17 cells are thought to provide protective immunity during extracellular bacterial infections via upregulation of anti-bacterial mediators and neutrophil recruitment [5,52]. Since Salmonella grow intracellularly in vivo [53], a protective role for Salmonella-specific Th17 CD4 T cells during primary infection is not immediately obvious. However, Th17 cells may be vital for mobilizing local innate defenses against secondary infection after bacteria penetrate intestinal epithelium. Indeed, it may be particularly important that these mucosal Th17 cells recognize bacterial antigens such as flagellin that are expressed by the initial bacterial burden.
In summary, this study has identified new Salmonella I-A b epitopes in T3SS effectors and used peptide-MHC tetramers and ELIPOT assays to simultaneously track multiple CD4 T cell responses during Salmonella infection for the first time. These data show that an infected host develops distinct CD4 effector lineages that are tightly regulated by bacterial antigen expression patterns in vivo, a finding that may assist the generation of vaccines directed against microbial pathogens that replicate in mucosal and systemic tissues.

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

Salmonella infection and antibiotic treatment
S. Typhimurium were cultured overnight in Luria-Bertani (LB) broth without shaking, and diluted in PBS after estimation of bacterial concentration using a spectrophotometer. Mice were infected intravenously in the lateral tail vein with 5X10 5 BRD509. For oral infection, mice were administered 0.1ml of 5% sodium bicarbonate to neutralize stomach pH before oral infection with 5x10 9 BRD509. The actual bacterial dose administered was confirmed by plating serial dilutions of the original culture onto MacConkey agar plates counting the number of colonies that grew after overnight culture at 37uC. In some experiments mice were administered Enrofloxacin (Baytril) at 2mg/ml in their drinking water, beginning on 5 days post-infection, as previously described [54].

Salmonella I-A b epitope discovery
The crystal structure of the murine I-A b molecule in complex with human class II invariant chain-associated peptide (CLIP) [33], was used to identify potential I-A b epitopes from S. Typhimurium. Based on the proposed alignment, the likelihood of a particular amino acid residue appearing in a specific position in the MHC class-II binding groove was assigned a numerical score. We focused on binding positions 1, 4, 6, 7, and 9 as these were the most likely to contribute to MHC class-II binding. For example, a tyrosine at position 1 was found in 30 out of 75 core residues examined and was assigned a score of 30. An overall score total was calculated by adding individual scores from each potential binding residue, with 109 being the highest possible score. This scoring approach was applied bioinformatically to each open reading frame of Salmonella using 9-mers overlapping by one (a total of 995,592 peptides). Two algorithms were then applied to this data; the first was a position-specific scoring matrix (PSSM) [55], and the second a hidden Markov model (HMM) [56]. The epitopes with highest PSSM (25 epitopes from 105 to 84) and HMM scores (19 epitopes from 82 to 43) were tested for immunogenicity using an IFN-c ELISPOT assay. Briefly, four groups of eleven peptides were mixed in equal amounts (5 mg/ peptide) emulsified in complete Freund's adjuvant (CFA) and injected subcutaneously into groups of C57BL/6 mice. Draining lymph nodes were harvested eight days later, CD4 T cells enriched by negative selection, and mixed 1:1 with irradiated splenocytes in IFN-c ELISPOT plates. Duplicate wells were stimulated for 36 hours with individual peptides and spot forming cells (SFC) per 1x10 6 total cells enumerated. Although 88% (PSSM) and 74% (HMM) of peptides induced a CD4 T cell response in HKST/ CFA-immunized mice, no response to these epitopes was detected after infection with Salmonella. The approach was therefore modified using PSSM to identify the two highest scoring candidate peptides from a combination of 13 outer membrane and secreted proteins. These epitopes were tested using purified CD4 T cells from the spleen and mesenteric lymph nodes of 129Sv mice orally infected 11 weeks previously with 5x10 7 Salmonella (SL1344). In these peptide identification experiments, genetically resistant 129Sv mice were used so that CD4 responses to virulent (SL1344) bacteria could be examined at late time points when C57BL/6 mice would normally succumb to infection.

Quantifying Flagellin (FliC) and SseJ mRNA
For SPI1 or SPI2-inducing cultures, Salmonella BRD509 were grown overnight at 37uC in LB broth, diluted in either LB broth for SPI1-inducing conditions or modified N minimal medium [57] for SPI2-inducing conditions, and grown for either 3 hours for SPI1 or 6 hours for SPI2 conditions. For in vivo detection of mRNA expression, C57BL/6 mice were infected intravenously with 5X10 5 BRD509 and spleens were harvested at either 30 minutes or 5 hours after infection. Total RNA was isolated using Aurum Total RNA Mini Kit (Bio-Rad, Hercules, CA) or TRI reagent (Applied Biosystems/Ambion, Austin, TX) according to the manufacturer's protocol. To enrich bacterial RNA from infected splenocytes, the MICROBEnrich kit (Ambion) was used, and traces of genomic DNA eliminated using DNA-free DNase treatment kit (Ambion) according to manufacturer's recommendations. To quantitate expression of Salmonella Flagellin (FliC) or SseJ by real-time qPCR, 1 mg of bacterial RNA was used as a template and cDNA synthesis by random hexamers was performed using TaqMan reverse transcription reagents (Applied Biosystems). SYBR Green (Applied Biosystems) based real-time qPCR was carried out based on published procedures [58]. The following primers were used in this study; 16S rRNA, 59tgttgtggttaataaccgca-39 and 59-gactaccagggtatctaatcc-39 [59]; FliC, 59-gtaacgctaacgacggtatc-39 and 59-atttcagcctggatggagtc-39 [58]; SseJ, 59-tattacgagactgccgatgc-39 and 59-gcccgtggtgagtataagggt-39 (GeneScript's real-time PCR primer design tool). Data was acquired using a ViiA 7 Real-time PCR system (Applied Biosystems) and analyzed using comparative Ct method (Applied Tracking Salmonella specific endogenous CD4 T cell response using class-II tetramers Endogenous Salmonella specific flagellin 427-441 -or SseJ 329-341 -CD4 T cell responses were monitored using a previously described methodology. Biotinylated I-A b monomers with an attached peptide derived from Salmonella flagellin 427-441 (Flagellin:I-A b ) or SseJ 329-341 (SseJ:I-A b ) were produced in S2 Drosophila insect cells cultured using a Wave Bioreactor (GE Healthcare Biosciences, Pittsburgh, PA). Purified monomers were tetramerized using fluorochrome-conjugated streptavidin and batch-tested for optimal binding to CD4 T cells from day 7 peptide-immunized mice, as previously described [36]. Briefly, C57BL/6 mice were immunized with either 100 mg of flagellin 427-441 or SseJ 329-341 peptide in the presence of CFA (Sigma-Aldrich, St. Louis, MO). On day 7, the inguinal, axillary, and brachial lymph nodes were harvested from naïve or peptide-immunized mice and stained with respective class-II tetramers in the presence of Fc block (culture supernatant from the 24G2 hybridoma, 2% mouse serum, 2% rat serum, and 0.01% sodium azide). Cells were incubated with anti-fluorochrome microbeads (Stem cell technologies, Vancouver, Canada) and tetramer-specific cells enriched using a magnet. Bound and unbound fractions were stained with fluorochrome-conjugated Abs specific for CD3, CD4, CD8, CD11b, CD11c, B220, F4/80, CCR7, and CD27 (eBioscience, or BD Bioscience, San Diego, CA). For transcription factor staining, cells were surface stained, and then treated with Foxp3 staining buffer set (eBioscience). Permeabilized cells were stained with fluorochrome-conjugated Abs specific for T-bet, GATA3, RORcT, or Foxp3. Cells were then analyzed by flow cytometry using an LSR II (BD Bioscience) or FACS Canto (BD Bioscience). Endogenous tetramer specific CD4 T cells were identified using a previously described gating strategy [36]. Tetramer specificity was confirmed by the absence of tetramer binding CD8 T cells in the bound fraction and in CD4 T cells within the unbound fraction. All the data were analyzed using FlowJo software (Tree Star, San Carlos, CA).

Cell processing for ELISPOT assay
Spleen and MLN, including Peyer's patches, were crushed through nylon mesh, and then RBC in spleen was lysed with ACK lysis buffer (Lonza, Walkersville, MD). For nonlymphoid tissue processing, we followed published methods [60]. Livers were perfused, crushed through a cell strainer (BD Biosciences), and resuspended in 35% Percoll (Sigma-Aldrich). After centrifugation, pelleted cells were treated with ACK lysis buffer and then washed with 2% FBS/PBS. For Lamina propria (LP) lymphocytes, small intestine was dissected, and washed with 2% FBS/CMF (Ca 2+ and Mg 2+ free HBSS with 1mM HEPES, and 2.5mM NaHCO 3 ). The intestine pieces were then stirred at 37uC for 30 min in CMF containing 10% FBS and 1mM dithioerythritol. The epithelial cells were removed by stirring the pieces in 1.3 mM EDTA/HBSS solution at 37uC for 30 min. The intestinal tissues were then treated with collagenase (Invitrogen, Carlsbad, CA) in RPMI 1640 (with 1mM CaCl 2 , 1mM MgCl 2 , and 5% FBS) at 37uC for 1 h. Cells were washed in 2% FBS/PBS, and then centrifugated on a 44%/67% Percoll gradient (GE Healthcare Biosciences). Viable cells at the interface were collected, and these cells are LP lymphocytes.
Examining Salmonella specific endogenous CD4 T cell response using ELISPOT assay Multiscreen HTS ELISPOT plate (Millipore, Billerica, MA) was coated with either purified anti-mouse IFN-c (BD Bioscience) or IL-17A (eBioscience). Total CD4 T cells from each tissue were isolated using EasySep Mouse CD4 T cell enrichment kit (Stem cell technologies) according to the manufacturer's protocol. The purity of CD4 T cells were measured by flow cytometry and was typically .90% for splenocytes and MLN, and .70% for nonlymphoid tissues. Purified CD4 T cells (10 5 cells/well) were added to ELISPOT plates in the presence of 4610 5 cells irradiated splenocytes. Cells were restimulated with 10 mM peptides (Table 3) at 37uC overnight. Bound IFN-c or IL-17A was detected by biotin-conjugated anti-mouse IFN-c (BD Bioscience) or IL-17A Figure 7. Detection of flagellin-specific CD4 Th17 cells in intestinal tissues. C57BL/6 mice were infected three times orally with 5610 9 Salmonella (BRD509) at one-month intervals. On day 7 after the third infection, CD4 T cells from the spleens, MLN, including Peyer's patches, livers (LV), and laminar propria (LP) were purified. Isolated CD4 T cells (1610 5 ) were restimulated with 10 mM of various peptides (Table 3)  (eBioscience), followed by AP-conjugated streptavidin (BD Bioscience). Bound antibodies were visualized using One-step NBT-BCIP substrate (Thermo, Rockford, IL), and plates analyzed using an ELISPOT reader ImmunoSpot (Cellular Technology, Shaker Heights, OH).

IL-22 ELISA
Purified CD4 T cells (1610 5 cells/well) from spleens and MLN, including Peyer's patches, were restimulated with 10 mM peptides (Table 3) in the presence of 4610 5 irradiated splenocytes for 16 h. IL-22 secretion was measured from culture supernatant using mouse IL-22 ELISA Ready-SET-Go kit (eBioscience). Briefly, High-protein binding plates (Costar, Corning, NY) were coated overnight with capture IL-22 antibody. After incubation in Assay Diluent for 1 hour at room temperature, plates were washed twice before culture supernatant was added. After incubation for 2 hours, plates were washed and biotin-conjugated detection IL-22 antibody added. After 1 hour, plates were washed and incubated for 30 min with avidin-HRP. Bound antibody was visualized using TMB substrate and after 15 min, the reaction stopped by adding 50 ml of 2N H 2 SO 4 . Plates were analyzed using a spectrophotometer (SpectraMax M2, Molecular Devices, Sunnyvale, CA).

Statistical analysis
Statistical differences between groups of normally distributed data were examined using Prism (GraphPad Software, La Jolla, CA). Data in each group were compared using an unpaired t test and were considered significantly different with a p,0.05. Figure 8. Mucosal flagellin-specific CD4 T cells produce IL-22 upon restimulation. C57BL/6 mice were infected three times orally with 5610 9 Salmonella (BRD509) at one-month intervals. On day 7 after the third infection, CD4 T cells from spleen and MLN, including Peyer's patches, were isolated. Purified CD4 T cells were restimulated with 10 mM peptide (Table 3) for 16 h in the presence of irradiated splenocytes. Culture supernatants were collected and IL-22 production measured by ELISA. Data show IL-22 production in culture supernatants as scatter plots representing individual mice. Data are pooled from three separate experiments. Numbers above each group indicate statistical significance and show the p value of a comparison between media alone (no stimulation) and peptide stimulation group. doi:10.1371/journal.ppat.1002499.g008