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Infection and Immunity, November 2005, p. 7226-7235, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7226-7235.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

CD4+-T-Cell Responses Generated during Murine Salmonella enterica Serovar Typhimurium Infection Are Directed towards Multiple Epitopes within the Natural Antigen FliC

Molly A. Bergman,1,{dagger} Lisa A. Cummings,2 Robert C. Alaniz,2 Laura Mayeda,2 Ivana Fellnerova,2 and Brad T. Cookson1,2*

Departments of Microbiology,1 Laboratory Medicine, University of Washington, Seattle, Washington2

Received 29 March 2005/ Returned for modification 5 July 2005/ Accepted 1 August 2005


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ABSTRACT
 
The flagellar filament protein FliC is a natural antigen recognized by memory CD4+ T cells recovered from Salmonella enterica serovar Typhimurium-infected humans and mice. To further investigate T-cell responses to FliC, we derived FliC-specific CD4+-T-cell clones from mice of two different haplotypes following oral S. enterica serovar Typhimurium infection. Using C-terminal truncations of MalE-FliC recombinant fusion proteins, we mapped antigenic activity to four different regions of FliC; three of the four epitope-containing regions were present in both FliC and the alternate flagellin subunit FljB. We determined that two novel FliC epitopes were also present in flagellins from several gram-negative enteric bacterial species: Ek-restricted FliC 80-94 (amino acids 80 to 94) and Ab-restricted FliC 455-469. Further mapping confirmed the presence of two previously identified FliC epitopes: Ak-restricted FliC 339-350 and Ab-restricted FliC 428-442. Therefore, like the recognition site of the innate immune receptor Toll-like receptor 5, three of four FliC epitopes recognized by CD4+ T cells colocalize in the D0/D1 domains of FliC. Salmonella-infected macrophages and dendritic cells stimulated epitope-specific CD4+-T-cell proliferation; infected dendritic cells also activated T cells to produce gamma interferon. These data demonstrate that Salmonella infection generates murine CD4+-T-cell responses to multiple epitopes in the natural antigen FliC and that recognition of infected phagocytes by FliC-specific CD4+ T cells triggers effector functions known to be essential for protective immunity. Together, these data suggest that FliC-specific CD4+ T cells may contribute to cell-mediated host defenses against Salmonella.


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INTRODUCTION
 
Salmonella spp. are facultative intracellular pathogens capable of causing localized and systemic disease of significant morbidity and mortality. Salmonella enterica serovar Typhi causes typhoid fever in humans, and a similar systemic illness develops during murine infection with S. enterica serovar Typhimurium. Natural acquisition of Salmonella via contaminated food or water introduces bacteria to the gastrointestinal tract, where Salmonella invades M cells to colonize underlying mucosal tissue (22). Salmonella disseminates to deeper tissues of spleen and liver (6) where bacteria preferentially replicate within macrophage phagosomes there (48); unchecked bacterial replication is fatal for the infected host. Early infection can be controlled by natural killer cells and gamma interferon (IFN-{gamma}) (42, 44, 45, 50), the latter being a cytokine that activates the intrinsic antimicrobial functions of macrophages and other professional phagocytes. Mice lacking the IFN-{gamma} receptor fail to control Salmonella infection, including the attenuated vaccine strain (18), highlighting the importance of IFN-{gamma} during immune responses to Salmonella.

Oral infection with viable, attenuated bacteria generates protective immunity against virulent Salmonella infection and requires both humoral and cellular immune functions (32). B cells produce antibodies that mediate clearance of extracellular Salmonella from infected tissue and are required for immunity (12); mice lacking B-cell functions demonstrate increased susceptibility to Salmonella infection (37, 40). Antibodies are largely directed towards bacterial surface antigens such as lipopolysaccharide and flagellin (4), the major subunit of the bacterial flagella. Robust CD4+-T-cell responses result from the phagosomal localization of Salmonella or Salmonella antigens; phagocytes acquire, process, and present pathogen-derived peptides in the context of major histocompatibility complex (MHC) class II, thus engaging and activating CD4+ T cells via the T-cell receptor (TCR) (8). CD4+ T cells are required for immunity to Salmonella, as mice lacking these T cells due to knockout mutations (18, 58) or antibody depletion (33, 41) are highly susceptible to Salmonella infection. The mechanisms by which Salmonella-specific CD4+ T cells contribute to protective immunity are incompletely understood (39), but T-cell proliferation, the sine qua non of CD4+-T-cell activation, and the production of IFN-{gamma} can be regarded as in vitro indicators of these essential elements of protective immunity. Although Salmonella-specific CD4+ T cells probably also provide essential help for B-cell function, it is likely that, as observed in studies of Listeria monocytogenes infection (2, 5), secretion of IFN-{gamma} by T cells promotes clearance of Salmonella from infected tissues via activation of professional phagocytes. Salmonella exploits phagocyte infection in vivo, as Salmonella mutants that fail to survive in macrophages are avirulent (15). Host recognition of infected phagocytes such as macrophages or dendritic cells occurs when T-cell receptors engage MHC-peptide complexes displayed on the phagocyte surface. Dendritic cells specifically function to prime naïve antigen-specific T cells, licensing T-cell proliferation and activating effector functions such as cytokine secretion (38); macrophages function to clear bacteria (16, 43), presumably after activation by antigen-specific T cells.

Although the complete repertoire of Salmonella antigens recognized by CD4+ T cells during bacterial infection is unknown, FliC flagellin is one natural antigen recognized by CD4+ T cells from both humans and mice orally immunized with attenuated Salmonella (7, 35, 57). Macrophages are capable of processing and presenting FliC to activate FliC-specific CD4+ T cells (7), illustrating the importance of these cells for facilitating anti-Salmonella immune responses. FliC contains at least two epitopes for CD4+ T cells from infected mice: the Ak-restricted FliC epitope at residues 339 to 350 (FliC 339-350) from C3H/HeJ (H-2k) mice (7) and the Ab-restricted FliC 428-442 from C57BL/6 (H-2b) mice (35).

To better understand the host response to Salmonella flagellin, we investigated FliC-specific CD4+-T-cell responses in detail. We identified four epitopes within FliC that are recognized by CD4+ T cells from Salmonella-immune mice, two epitopes in each haplotype examined (H-2k and H-2b). Three epitopes localized within the N- or C-terminal regions of flagellin, which are the same regions conserved among flagellins expressed by multiple gram-negative bacterial species. The same conserved regions comprise the domains recognized by Toll-like receptor 5 (TLR5), the innate immune receptor for flagellin. T-cell clones specific to each of the four epitopes responded to Salmonella-infected macrophages and dendritic cells by proliferating and secreting the effector cytokine IFN-{gamma}. Our results demonstrate that the natural antigen FliC contains multiple epitopes recognized by CD4+ T cells and that T-cell recognition of FliC-peptide-MHC complexes on infected host cells results in IFN-{gamma} production. Collectively, these data suggest that IFN-{gamma} production by FliC-specific CD4+ T cells in vivo may contribute to protective immunity against Salmonella.


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MATERIALS AND METHODS
 
Mice and immunizations. Six- to 8-week-old female C3H/HeJ (Jackson Laboratory, Bar Harbor, ME) and C57BL/6 (National Cancer Institute, Bethesda, MA) mice were used for immunizations and splenocyte antigen-presenting cells (APC). Mice were inoculated by oral gavage (feeding needle no. 7920; Popper & Sons, Inc., New Hyde Park, NY) with 109 viable SL3261 bacteria (see below). Mice were housed in specific-pathogen-free conditions, and studies were performed according to the University of Washington institutional guidelines for animal use and care.

Bacterial strains. See Table 1 for a full list of bacterial strains. Salmonella enterica serovar Typhimurium strain SL3261 (SL1344 {Delta}aroA) was used for oral immunization of mice. S. enterica serovar Typhimurium SL1344 strains carrying both, one, or no flagellin genes were used for antigen preparations and in vitro phagocyte infections. To generate {Delta}fliC/{Delta}fljB mutant strains, S. enterica serovar Typhimurium LT2 strains encoding the kanamycin resistance (Kanr) gene in place of either the fliC or fljB open reading frame (constructed according to the method of Datsenko and Wanner [10]) were kindly provided by Heather Bonifield and Kelly Hughes. Kanr, flanked by FLP (FRT) recognition sites, was transferred from HB686 (fliC::FRT · Kan · FRT) or JG368 (fljB::FRT · Kan · FRT) to SL1344 by P22-mediated generalized transduction. The gene encoding Kanr was excised following the introduction of plasmid pCP20 (temperature-sensitive replicon, ampicillin resistant [Ampr]), which expresses the flp recombinase gene after thermal induction; resultant Kans Amps colonies were confirmed to be {Delta}fliC or {Delta}fljB by PCR screening. The same procedure was repeated to create strains in which both fliC and fljB were deleted. Motility (or lack thereof) of all strains was confirmed in soft agar (data not shown). Generation of malE fliC alleles lacking sequentially greater amounts of fliC DNA from the 3' end of the gene has been described previously (7); sequencing of the 3' end of individual malE fliC open reading frames coding for stimulatory or nonstimulatory antigen identified the fliC deletion endpoints and the remaining FliC amino acids present in each mutant. Mutant alleles encoding FliC with in-frame 31-amino-acid insertion mutations will be described elsewhere (S. L. R. Barrett and B. T. Cookson, unpublished data).


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  		TABLE 1. Bacterial strains

Bacterial antigens. Heat-killed bacterial antigen (HKAg) was prepared by heating stationary-phase bacteria at 65°C for 1 h. To purify FliC and FljB flagellins, flagella from logarithmic-phase bacteria expressing only FljB or FliC were mechanically sheared from bacterial cells (56) (Waring, East Windsor, NJ), depolymerized at 60°C for 20 min, and passed through a Centricon 100,000-molecular-weight-cutoff filtration unit (Millipore, Bedford, MA) to remove high-molecular-weight lipopolysaccharide. The resulting monomeric flagellin preparations were confirmed to be free of contaminating antigens by failure to stimulate CD4+ T cells specific for antigens other than FliC (data not shown). Construction of vectors encoding malE fliC alleles downstream of an IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducible promoter was described previously (7); vectors were transformed into nonmotile Escherichia coli DH5{alpha} or flagellin-negative BC696 (Table 1), and recombinant MalE-FliC proteins were expressed following IPTG induction. HKAg from salmonellae and other gram-negative bacteria was made from bacteria confirmed as flagellin positive (motility in soft agar or Western blot) (data not shown). Synthetic peptides were purchased from Global Peptide Services (Ft. Collins, CO).

Eukaryotic cell culture and infected phagocytes. All eukaryotic cells were maintained in RPMI 1640 medium supplemented with L-glutamine, 50 µM 2-mercaptoethanol, and 10% fetal calf serum (HyClone, Logan, UT), with penicillin, streptomycin, and gentamicin (all reagents were from Invitrogen, Carlsbad, CA, except for serum) and incubated at 37°C in 5% CO2. Every 14 to 17 days, T cells were restimulated with irradiated syngeneic splenocytes plus antigen for 48 h, followed by dilution into supplemented medium containing interleukin-2 (T-STIM; BD Discovery Labware, San Diego, CA) and methyl-{alpha}-D-mannopyranoside (Calbiochem, San Diego, CA). Elicited peritoneal macrophages were obtained 3 days after intraperitoneal injection of 0.1 ml sterile Brewer's thioglycolate (BD Diagnostic Systems, Sparks, MD) and 48 h of culture in medium containing 50 U/ml IFN-{gamma} (R&D Systems, Minneapolis, MN). Dendritic cells were derived from in vitro culture of bone marrow cells with 20 ng/ml granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN) for 6 days according to standard methods (3). To generate infected phagocyte APC, bacteria from stationary-phase cultures were added to phagocytes at a multiplicity of infection of 50:1 in antibiotic-free media, incubated for 15 min (dendritic cells) or 2 h (macrophages), washed, and incubated an additional 2 h in medium containing 15 µg/ml gentamicin to kill extracellular bacteria. After 2 h of further incubation, infected phagocytes were fixed with 0.2% paraformaldehyde-Hanks balanced salt solution for 20 min and washed extensively, followed by replacement of culture medium and addition of T cells.

Generation of FliC-specific CD4+-T-cell clones. Salmonella-specific CD4+ T cells, which require immunization to be generated, were isolated as described previously (1, 7). Briefly, mice were sacrificed ≥90 days after immunization, and nylon wool-purified splenic lymphocytes were stimulated with irradiated syngeneic splenocyte APC and 5 x 106 CFU/ml heat-killed S. enterica serovar Typhimurium antigen to generate Salmonella-specific CD4+-T-cell lines. No Salmonella-specific T cells could be isolated from naïve mice. FliC-specific T-cell clones were isolated by limiting the dilution with 10 µg/ml purified FliC as a stimulatory antigen (see above). All clones responded to purified FliC antigen and expressed the CD4 coreceptor as determined by flow cytometry (data not shown). Clones from C3H/HeJ mice were restricted to MHC molecule Ak or Ek, as determined by proliferative responses to antigen presented by Ak-expressing splenocyte APC from B10.4R mice (Jackson Laboratory, Bar Harbor, ME) or inhibition of proliferative responses in the presence of anti-Ek blocking antibody (clone 14-4-4S; BD Biosciences Pharmingen, San Diego, CA) (data not shown), while clones from C57BL/6 mice were restricted to MHC molecule Ab (the gene encoding the {alpha} chain of the Eb molecule is inactivated in the C57BL/6 genome [11]). TCR Vß expression was identified by flow cytometry with the Mouse TCR Vß Screening Panel (BD Biosciences Pharmingen, San Diego, CA).

T-cell stimulation assays. T-cell proliferation in response to APC plus antigen was assayed as previously described (7). Briefly, 105 T cells and 106 irradiated syngeneic splenocytes plus 5 x 106 HKAg or 0.1 to 1.0 µg/ml antigen were combined in triplicate, [3H]thymidine was added after 48 h, DNA was harvested after 16 h, and incorporated 3H was measured using liquid scintillation spectrophotometry. All standard errors were <10% of the means, and for clarity of presentation, the error bars are not shown. Alternatively, 104 T cells were cocultured with 105 infected dendritic cells, and IFN-{gamma} in culture supernatant was measured after 48 h by sandwich enzyme-linked immunosorbent assay (BD Biosciences, Pharmingen, San Diego, CA).


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RESULTS
 
Isolation and characterization of FliC-specific CD4+ T cells from Salmonella-immune mice. We orally immunized C3H/HeJ (H-2k) and C57BL/6 (H-2b) mice with S. enterica serovar Typhimurium strain SL3261 (19) and generated Salmonella-specific CD4+ T cells as described previously (1, 7). Using purified FliC as a stimulatory antigen, we isolated 37 CD4+ FliC-specific T-cell clones restricted to Ek, Ak, or Ab MHC class II molecules with variable TCR Vß usage (each clone expressed a single TCR Vß chain) (Table 2). Recombinant MalE-FliC fusion proteins with sequential C-terminal truncations of FliC (7) allowed the identification of four FliC regions containing stimulatory antigen for the T-cell clones: amino acids 89 to 344, 345 to 400, 401 to 460, and 461 to 494 (Table 2) stimulated clones correspondingly categorized as groups I, II, III, and IV. As FliC amino acid residues 1 to 170 and 404 to 494 are nearly identical between FliC and FljB (the alternate flagellin subunit protein expressed by S. enterica serovar Typhimurium) (49), we predicted that some FliC-specific T-cell clones from each group would be cross-reactive against FljB. T-cell proliferation in response to Salmonella bacteria expressing either FliC or FljB, or purified FliC and FljB flagellins, revealed that group I, III, and IV clones recognized both Salmonella flagellins (Fig. 1A and C). Group II clones responded only to FliC (Fig. 1B and D). No T-cell clones responded to flagellin-negative Salmonella (Fig. 1A and B), confirming that T-cell responses were specific to flagellin proteins. Collectively, these results demonstrate that FliC-specific CD4+ T cells from Salmonella-immune mice respond to multiple regions of the stimulatory antigen FliC and that some T cells demonstrate cross-reactivity to the alternate Salmonella flagellin protein, FljB.


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  		TABLE 2. Classification of 37 FliC-specific CD4+-T-cell clones isolated from Salmonella-immune C3H/HeJ and C57BL/6 mice



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  		FIG. 1. FliC-specific CD4+-T-cell clones recognize epitopes present in one or both Salmonella flagellins. Proliferative responses of group I (A, C) and group II (B, D) clones (represented by HeJ.F8 [I] and HeJ.3A7 [II]) to HKAg from Salmonella expressing only FliC, only FljB, or neither flagellin protein (A, B) or purified FliC or FljB protein (C, D) were measured (see Materials and Methods). Responses of group III and IV Ab-restricted clones (represented by B6.2D4 [III] and B6.2A4 [IV]) to HKAg (A) and purified FliC and FljB (C) were similar to those of group I clones (data not shown). Data are representative of three experiments.

Mapping and identification of FliC epitopes. We observed that the Ak-restricted group II T-cell clones responded to stimulatory antigen present in the 345- to 400-amino-acid region of FliC (Table 2) that contains residues of the previously identified Ak-restricted epitope FliC 339-350 (7). Indeed, all the group II clones we isolated, like the previously described Ak-restricted 7.4.8 clone (Table 2), responded to MalE-FliC1-351 but not MalE-FliC1-344 antigen and proliferated in response to synthetic FliC 339-350 peptide (Fig. 2A). Therefore, we concluded that the group II clones described here are specific for FliC 339-350. However, unlike clone 7.4.8, group II clones responded to MalE-FliC1-347 (Fig. 2A), which lacks FliC amino acids 348 to 350 of the previously identified stimulatory FliC 339-350 peptide. Consistent with these observations, group II clones express different TCR Vß chains compared with 7.4.8 (Table 2); we therefore concluded that the native amino acids in positions 348, 349, and 350 are dispensable for recognition by most group II clones from immunized C3H/ HeJ mice.



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  		FIG. 2. FliC-specific CD4+-T-cell clones respond to previously unknown FliC epitopes. Representative FliC-specific CD4+-T-cell clones from groups I and II (A) and groups III and IV (B) were assayed for proliferative responses to flagellin-negative Salmonella (BC696) expressing recombinant MalE-FliC fusion proteins (HKAg), purified FliC protein, and synthetic peptides containing the previously identified FliC epitope Ak-restricted FliC 339-350 (7) (A) or Ab-restricted FliC 428-442 (35) (B). A single experiment performed in triplicate is shown; data are representative of three experiments.

The Ab-restricted group III clones responded to stimulatory antigen within the 401- to 460-amino-acid region of FliC (Table 2) that encompasses the known Ab-restricted epitope FliC 428-442 (35). Group III clones also proliferated in response to the FliC 428-442 peptide (Fig. 2B). These results indicate that group III clones are identical or highly similar to the previously described FliC 428-442-specific CD4+-T-cell clones from C57BL/6 mice (Table 2) (35), with two notable exceptions: group III clones expressed different Vß chains in their TCRs and only comprised 25% (Table 2) of the total FliC-specific clones isolated from immunized C57BL/6 mice. In a previous study using the same strain of mice, 100% of the FliC-specific T-cell clones recognized FliC 428-442 in the context of Ab (35).

The remaining two groups of clones, Ek-restricted group I clones and Ab-restricted group IV clones, comprised 24% and 75%, respectively, of the total FliC-specific CD4+ T cells derived from Salmonella-immune mice (Table 2). Neither group responded to either the FliC 339-350 or FliC 428-442 peptides (Fig. 2A and B). To map the FliC epitopes recognized by these clones, we measured proliferative responses to additional flagellin antigens (MalE-FliC truncated fusion proteins, FliC with in-frame 31-amino-acid insertion mutations and flagellins from different bacterial species) (Fig. 3A to F). Stimulatory antigen for group I clones mapped between FliC residues 79 and 93 (Fig. 3A) was present in flagellins expressed by some Enterobacteriaceae but was absent from Pseudomonas aeruginosa and Serratia marcescens flagellin (Fig. 3B). These data confirmed that the stimulatory region of Salmonella FliC mapped to the conserved N-terminal amino acids. Sequence analysis of different flagellin proteins containing or lacking stimulatory activity for group I clones, and a consensus peptide sequence defining the Ek-binding motif derived from 27-peptide antigens recognized in the context of Ek (46), allowed us to predict a potential Ek-binding motif within the 77- to 92-amino-acid sequence (FliC 81-89) (Fig. 3C). Overlapping synthetic peptides FliC 78-92, FliC 80-94, and FliC 82-96 all contained stimulatory antigen for group I clones while the FliC 77-91 peptide did not (Fig. 3C), thus identifying FliC 82-92 as the minimal Ek-restricted epitope. As the FliC 80-94 peptide was the most potent stimulatory antigen for the group I clones (Fig. 3C), we have termed these cells FliC 80-94-specific CD4+-T-cell clones.



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  		FIG. 3. Identification of new FliC epitopes recognized by FliC-specific CD4+ T cells. Shown are sequences of flagellin proteins and responses of FliC-specific CD4+-T-cell clones representative of the Ek-restricted group I (A to C) and the Ab-restricted group IV (D to F) to different flagellins; responses are shown as ++ (≥20 kcpm), + (≥10 kcpm), ± (≥5 kcpm), or ø (≤0.2 kcpm, same as background). Antigens used for stimulation assays included HKAg prepared from flagellin-negative Salmonella (BC696) expressing either recombinant MalE-FliC proteins (vector-encoded amino acids [aa], differing from Salmonella FliC residues, are shown in italics) (A, D) or FliC containing in-frame 31-amino-acid insertion mutations (insertion sites indicated in A), HKAg prepared from other flagellin-expressing bacteria (amino acids different from S. enterica serovar Typhimurium FliC are marked in boldface type) (B, E), or synthetic peptides (C, F). Data are representative of three experiments (A to F). NA, not applicable. (G) Location of T-cell-stimulatory FliC epitopes within the primary amino acid sequence of FliC. Residues marked in boldface type indicate sequences conserved between different bacterial flagellins (D0/D1 domains) flanking the hypervariable regions (D2/D3 domains) (49). Epitope-containing sequences are highlighted; clone group, MHC restriction, and amino acid residue numbers are given for each epitope. The asterisk indicates a stop codon.

For group IV clones, stimulatory antigen localized to the C-terminal FliC residues 461 to 494 (Table 2), and further mapping with truncated MalE-FliC fusion proteins revealed that FliC residues 465 to 468 were required for T-cell stimulation (Fig. 3D). Similar to the Ek-restricted group I clones (Fig. 3B), the Ab-restricted group IV clones also showed cross-reactive responses to flagellins expressed by some Enterobacteriaceae (but not P. aeruginosa flagellin) (Fig. 3E), demonstrating that T cells generated to FliC during Salmonella infection recognize antigen(s) from several bacteria among the Enterobacteriaceae. From sequence analysis of those bacterial flagellins containing or lacking stimulatory activity for group IV clones, and the recently identified Ab-binding motif (28) (Fig. 3F), we predicted that FliC residues 458 to 466 may comprise part of the stimulatory epitope. Indeed, synthetic peptides FliC 452-471 and 455-469 both contained stimulatory antigen for group IV clones (Fig. 3F) and identify FliC 455-469 as the second epitope recognized by 75% of the Ab-restricted FliC-specific T cells (Table 2A) derived from Salmonella-infected C57BL/6 mice.

The four epitopes discussed here map to discrete locations within the FliC amino acid sequence. FliC 80-94, FliC 428-442, and FliC 455-469 localize within the highly conserved N- and C-terminal regions of the FliC monomer, defined as the D0/D1 domains (49) and containing residues required for monomer secretion and polymerization into filaments (Barrett and Cookson, unpublished) (Fig. 3G). FliC 339-350 maps to the D2 domain (Fig. 3G); the D2/D3 domains, comprising the hypervariable portion of FliC, are dispensable for flagellar function (motility) (20).

FliC epitopes are presented by Salmonella-infected phagocytes. To determine if Salmonella-infected phagocytes can process and present FliC epitopes to stimulate epitope-specific CD4+ T cells, we infected primary murine macrophages or dendritic cells with Salmonella in vitro for use as APC in T-cell proliferation assays. Macrophages (Fig. 4A) and dendritic cells (Fig. 4B) infected with FliC+ Salmonella processed and presented all four FliC epitopes and stimulated epitope-specific CD4+-T-cell proliferation (groups I to IV) in a dose-dependent manner. T-cell clones failed to respond to macrophages or dendritic cells infected with FliC-negative Salmonella (Fig. 4A and B), indicating that T-cell recognition of infected phagocytes was FliC specific. These results demonstrate that infected macrophages and dendritic cells are capable of processing and presenting FliC epitopes from viable FliC+ Salmonella to stimulate epitope-specific CD4+-T-cell proliferation.



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  		FIG. 4. FliC epitopes are presented by Salmonella-infected macrophages and dendritic cells. Syngeneic peritoneal macrophages or bone marrow-derived dendritic cells (DC) were used as antigen-presenting cells and infected with live FliC+ or FliC– Salmonella (both bacterial strains were FljB negative) (see Materials and Methods). Effector responses of FliC-specific CD4+-T-cell clones from groups I, II, III, and IV to epitopes FliC 80-94, 339-350, 428-444, and 455-469 presented by infected macrophages (A) or infected dendritic cells (B, C) were detected by proliferation (A, B) or IFN-{gamma} secretion (C). Data are representative of three experiments.

Because production of IFN-{gamma} by antigen-specific T cells is one indicator of effector T-cell function, we investigated whether FliC-specific CD4+ T cells secreted IFN-{gamma} after coculture with infected phagocytes. Dendritic cells infected with viable FliC+ Salmonella stimulated all four groups of FliC-specific T-cell clones to secrete IFN-{gamma} (Fig. 4C). Cytokine production was comparable to that observed when the same T-cell clones were incubated with uninfected splenocyte APC pulsed with nonviable FliC+ bacteria as an antigen (data not shown). FliC-specific CD4+ T cells did not secrete IFN-{gamma} in response to dendritic cells infected with FliC-negative Salmonella (Fig. 4C), indicating that effector responses were antigen specific. Taken together, these results demonstrate that FliC-specific CD4+ T cells derived from Salmonella-infected mice respond to infected phagocytes with characteristic effector T-cell responses, i.e., production of IFN-{gamma}.


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DISCUSSION
 
Here, we demonstrate that CD4+ T cells isolated from mice orally immunized with attenuated bacteria recognize four epitopes from the Salmonella enterica serovar Typhimurium FliC protein: Ek-restricted FliC 80-94, Ak-restricted FliC 339-350, and Ab-restricted FliC 428-442 and FliC 455-469. Three of the four epitopes localized to FliC amino acid sequences conserved among different bacterial flagellins, and correspondingly, CD4+-T-cell clones specific to conserved flagellin peptides cross-reacted with flagellins from different bacterial species. Finally, Salmonella-infected macrophages and dendritic cells were capable of processing and presenting FliC epitopes to stimulate epitope-specific CD4+-T-cell proliferation and IFN-{gamma} production.

Our observations extend the repertoire of known natural FliC epitopes recognized by CD4+ T cells from Salmonella-immune hosts by identifying novel FliC epitopes for each of the two murine haplotypes examined in this study. The frequency of T-cell clones generated for specific epitopes differed, with approximately 75% of the clones from each haplotype recognizing a single epitope. This suggests a possible hierarchy of specificities in the starting pool of Salmonella-specific CD4+ T cells primed by infection, i.e., epitope immunodominance. For example, it may be that T-cell responses to FliC 339-350 (recognized by 76% of the FliC-specific clones from immune C3H/HeJ mice) dominate responses to FliC 80-94 (recognized by 24% of the isolated clones) in H-2k haplotype hosts and that more T cells respond to FliC 455-469 than to 428-442 (recognized by 75% and 25% of the FliC-specific clones derived from immune C57BL/6 mice, respectively) in H-2b haplotype hosts. Supporting this hypothesis is the previous observation that responses to FliC 428-442 constitute only a small fraction of the total FliC-specific CD4+-T-cell response during primary and secondary Salmonella infection (35).

Crystal structure analysis of S. enterica serovar Typhimurium FliC confirmed previous observations that the flagellin monomer has four domains (D0, D1, D2, and D3) and revealed the monomer to be shaped like a bent hairpin, with the D3 domain forming the looped end of the hairpin and the D1 and D2 regions comprising the arms (49) (Fig. 5). The D1 domain (and D0, by prediction) is buried in the center of the filament and mediates intermolecular interactions between adjacent monomers, while many residues in the D2 and D3 domains are exposed on the filament outer surface (49). The D2 and D3 regions are dispensable for FliC function and are highly variable in amino acid sequence (20, 49). This variability is exploited for serological discrimination of different bacterial flagellins, as humoral immune responses generally target D2/D3 residues exposed on the outer surface of the polymerized flagellar filament (17, 23, 25, 26, 51) (Fig. 5). In contrast, the D0 and D1 domains are highly conserved (49), are required for secretion and polymerization (Barrett and Cookson, unpublished), and contain residues recognized by the innate immune receptor TLR5 that are exposed in monomeric, but not polymeric, flagellin (53). Most FliC epitopes stimulatory for CD4+ T cells (described here and elsewhere) (24, 35), including those generated by hyperimmunization with purified FliC protein, also map to the conserved domains of flagellin: FliC 80-94 and FliC 428-442 are in D1, FliC 455-469 is within D0, and seven of the eight described epitopes in S. enterica serovar Muenchen FliC (H-2d restricted; T cells recovered from BALB/c mice immunized with purified FliC) (24) are scattered throughout the D0/D1 regions (Fig. 5). Thus, both innate (TLR5) and adaptive (T-cell receptor) immune responses target the most highly conserved flagellin domains, the same domains that are also required for protein function. The observation that monomeric flagellin is more stimulatory for TLR5 than polymeric flagellin (53) suggests that biochemical and/or structural information in the monomer may bias certain epitopes in D0/D1 domains for antigen processing and presentation in the context of MHC class II. Alternatively, B-cell recognition of D2/D3 domains may actually inhibit T-cell responses directed towards D2/D3 epitopes, as antibodies complexed to model antigens have been shown to modulate the processing and presentation of peptide-MHC complexes to T cells, such that T-cell responses to particular determinants can be enhanced or suppressed (30, 31, 52). The inherent adjuvanticity of flagellin (36) may also contribute to adaptive immune recognition of FliC.



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  		FIG. 5. Mapping of FliC epitopes within the tertiary structure of flagellin. Shown is the C{alpha} backbone trace of full-length S. enterica serovar Typhimurium FliC flagellin (prepared with Swiss-Pdb Viewer); the four globular domains (49) D0 (A1-A55 and S451-R494), D1 (N56-Q176 and W402-R450), D2 (K177-G189 and A284-A401), and D3 (Y190-V283) are indicated. T-cell epitopes predominantly localize to conserved D0/D1 regions. The four FliC epitopes recognized by CD4+ T cells recovered from S. enterica serovar Typhimurium-infected mice are shown in purple: FliC 80-94 (D1), 339-350 (D2), 428-442 (D1), and 455-469 (D0). Five of the eight regions containing residues stimulatory for T cells recovered from mice hyperimmunized with S. enterica serovar Muenchen-purified flagellin (24) are shown in gold (minimal residues in eight sets of overlapping dodecameric peptides, identified here using the numbering of the mature 494-amino-acid FliC from S. enterica serovar Typhimurium): FliC 33-39 and 45-50 (D0) and 131-138, 155-163, and 416-420 (D1). The three remaining epitope-containing regions from S. enterica serovar Muenchen FliC are not shown for clarity, as S. enterica serovar Muenchen FliC epitopes 81-88 and 91-98 (both in D1) overlap with the S. enterica serovar Typhimurium FliC epitope 80-94, and S. muenchen FliC epitope 293-301 maps to the hypervariable D3 region, which differs in amino acid sequence from that of S. enterica serovar Typhimurium FliC. B-cell epitopes for multiple bacterial flagellins localize to the hypervariable D2/D3 regions (17, 23, 25, 26, 51).

Innate and adaptive immune recognition of conserved antigens like flagellin can lead to protective immune responses against flagellated pathogens; paradoxically, these responses can also cause pathology in gastrointestinal and mucosal diseases. Indeed, novel flagellins expressed by normal intestinal microbiota were recently identified as the bacterial antigens driving antibody responses in both experimental models of intestinal colitis and clinical cases of inflammatory bowel disease (29). Adoptive transfer of CD4+ T cells capable of recognizing flagellin transferred disease to noncolitic mice, suggesting that the pathogenesis of intestinal inflammation results from inappropriate or poorly regulated immune responses to flagellin expressed by intestinal microbiota previously considered innocuous. Those authors also observed that flagellin-specific CD4+-T-cell responses occurred in several experimental models of intestinal colitis, suggesting that both inappropriate immune recognition of flagellin and intestinal pathology result from the distinct underlying genetic deficiencies present in each mouse model (29). Initial flagellin recognition is likely mediated by mucosal epithelial cells, which express TLR5 and are exquisitely sensitive to flagellin monomer, capable of responding to femptomolar concentrations in vitro (27). In vivo administration of flagellin to mucosal surfaces, for example, in the respiratory tract, stimulates lung epithelial cells and other recruited cells to produce massive amounts of cytokines and inflammatory molecules (21). Indeed, flagellin is a required virulence factor of Pseudomonas aeruginosa (13), the major causative agent of lung infections in cystic fibrosis patients, suggesting the possibility that this pathogen exploits TLR5 recognition to enhance pathogenesis and cause disease. Many normal mutualistic/commensal bacteria associated with humans, and myriad environmental microbes, express flagellin (47); the conserved nature and abundance of this antigen relative to other bacterial antigens may thus bias host recognition of flagellin during both inappropriate self-reactive/destructive and appropriate protective immune responses.

The observation that professional phagocytes can process and present FliC epitopes from viable Salmonella to stimulate epitope-specific CD4+-T-cell effector responses highlights the importance of the tripartite interaction between bacteria, phagocytes, and T cells from the point of view of both the pathogen and the host. Salmonella infection of macrophages in vivo is crucial for bacterial virulence, as bacterial mutants that cannot survive in phagocytes are also avirulent (14). Conversely, phagocyte and T-cell recognition of bacterial infection is critical for host immune responses, as hosts with defects in phagocyte or T-cell functions are more susceptible to bacterial infection (39). The role of FliC-specific CD4+ T cells during in vivo Salmonella infection is beginning to emerge. In vivo tracking of adoptively transferred transgenic FliC-specific CD4+ T cells after oral Salmonella infection revealed activation of T cells only in lymphoid organs immediately downstream of the intestine, i.e., Peyer's patches and mesenteric lymph nodes, but not in spleen (34). These data demonstrate that activation of FliC-specific T cells is compartmentalized by organ and that either Salmonella-infected phagocytes or phagocytes that have captured FliC antigen are present exclusively in intestinal lymphoid tissue after oral infection. Recent studies using the same adoptive transfer system found that suppression of FliC-specific T-cell responses in Salmonella-infected peripheral lymphoid tissue may result from the massive expansion of CD4+ T cells specific to other Salmonella antigens during infection (54) and possibly by sequestration of FliC-containing antigen-presenting cells away from transferred FliC-specific CD4+ T cells (55). More likely, bacterial regulation of antigen expression in vivo prevents effective antigen-specific T-cell recognition of Salmonella during infection (1). Our studies found that Salmonella downregulates FliC production during intracellular replication, as demonstrated by reduced FliC expression by salmonellae genetically altered to resemble their physiological state during intraphagosomal growth (1) and by direct examination of bacteria growing inside host phagocytes both in vitro and in vivo (9). Our observations are consistent with the hypothesis that FliC-specific CD4+ T cells are activated only in the intestinal lymphoid organs where FliC+ bacteria in the original population of orally delivered Salmonella are either captured by or infect host cells; subsequent FliC-specific T-cell responses in the peripheral lymphoid tissue are not activated due to bacterial repression of FliC antigen production (9). Thus, the proinflammatory responses to FliC, and FliC epitope-specific T-cell effector responses, would be predicted to function during the early stages of Salmonella infection. Future studies addressing the functional consequences of aberrant FliC expression during oral immunization, and the attendant alterations of FliC-specific immune responses, hold promise for shedding additional light on the interaction of Salmonella with the host immune system.


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ACKNOWLEDGMENTS
 
This work supported by NIH grant AI47242.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departments of Laboratory Medicine and Microbiology, University of Washington Medical Center, Box 357110, 1959 NE Pacific Street, Seattle, WA 98195. Phone: (206) 598-6131. Fax: (206) 598-6189. E-mail: cookson{at}u.washington.edu. Back

Editor: V. J. DiRita

{dagger} Present address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02112. Back


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Infection and Immunity, November 2005, p. 7226-7235, Vol. 73, No. 11
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