Naturally Occurring Altered Peptide Ligands Control Salmonella-Specific CD4⁺ T Cell Proliferation, IFN-γ Production, and Protective Potency

Three distinct, yet interrelated, stimulatory signals are required for the activation and differentiation of naive T cells into cytokine-producing effector T cells (1, 2). Although naive T cells likely receive these stimulation signals concurrently during contact with APCs in a coordinated fashion, each signal also has been shown to individually control unique facets of T cell activation, proliferation, and differentiation. For example, Ag specificity controlled by TCR signaling dictates which subset of T cells becomes initially activated (3, 4), whereas costimulation signals primarily mediated by CD28 signaling prevent these newly activated T cells from becoming anergic (5, 6). In this regard, a growing list of specific cytokines that includes IL-6, IL-12, IL-21, type I IFNs, and TGF-β have each been demonstrated to provide additional stimulation signals that control the expansion, survival, and differentiation program of newly activated CD4⁺ and CD8⁺ T cells (7–15). Therefore, coordinated stimulation through the TCR, costimulation receptors, and specific cytokine receptors each has the potential to play unique and defined roles required for synchronized T cell activation, proliferation, and differentiation.

Although there is ample evidence supporting the abilities of specific cytokines to control CD4⁺ T cell differentiation into each distinct Th lineage, other T cell stimulation signals also have been implicated to play important roles in this process. Altered peptide ligands (APLs) containing amino acid substitutions in TCR contact residues from defined MHC class II peptide Ags have been used to characterize how TCR stimulation can also control CD4⁺ T cell differentiation regardless and independent of exogenous cytokines. For example, stimulation of CD4⁺ T cells from TCR transgenic mice specific for an I-A^b restricted peptide within the Mycobacterium tuberculosis Ag 85B protein with wild-type Ag85B_244–252 peptide primes robust IFN-γ with minimal IL-4 production, whereas stimulation with a peptide variant containing a single glycine to alanine substitution within the TCR contact residue at position 248 abolishes IFN-γ production and is replaced by reciprocal IL-4 production (16). In agreement with these results where T cells are stimulated in vitro, stimulation with APLs also can have profound effects on CD4⁺ T cell differentiation in vivo. For example, CD4⁺ T cell IFN-γ production that normally occurs after “immunization” with the human collagen IV protein is abolished and replaced with IL-4 production when a peptide variant containing a single amino acid substitution within a defined TCR contact residue is used instead (17). Recently, the impact of TCR stimulation also has been extended to play important roles in controlling the Ag-specific CD8⁺ T cell response after in vivo infection. With recombinant Listeria monocytogenes that express either the parental H-2K^b OVA_257–264 peptide or defined APLs derived from this peptide, TCR stimulation was found to dictate the kinetics of CD8⁺ T cell contraction and migration within lymphoid organs (18). Interestingly, despite these differences, OVA_257–264-specific CD8⁺ T cells were activated and formed functional memory cells similarly regardless of differences in TCR stimulation (18). Taken together, these results indicate that differences in TCR stimulation may control critical and unanticipated features in T cell differentiation and the Ag-specific T cell response during infection.

In this study, we sought to explore how differences in TCR stimulation may impact proliferation, differentiation, and protective potency for pathogen-specific CD4⁺ T cells. Given the importance and protective effects of CD4⁺ T cells in host defense against Salmonella typhimurium, a defined MHC class II peptide that spans aa 431–439 within the protective FliC Ag of Salmonella was used in this study (19–24). This FliC_431–439 peptide is presented to CD4⁺ T cells by the murine MHC class II molecule, I-A^b, because CD4⁺ T cells from FliC-specific TCR transgenic mice derived from C57BL/6 mice expand in an Ag-specific manner after adoptive transfer into syngeneic recipient mice (21, 25). Furthermore, given the highly conserved nature of FliC and other flagellum components among diverse bacterial species, we examined the prevalence and explored the potential use of altered TCR stimulation for CD4⁺ T cells specific to this Ag by other bacteria. This led to the identification of four naturally occurring APLs containing single amino acid substitutions in putative TCR contact residues within the FliC_431–439 peptide. When compared with the parental FliC_431–439 peptide, naturally occurring APLs that prime proliferation of FliC-specific CD4⁺ T cells either more or less potently were identified. Remarkably, despite these differences in proliferation, each APL compared with the parental FliC peptide primed reduced IFN-γ production in vivo and conferred diminished protection against subsequent challenge with virulent Salmonella.

C57BL/6 (I-A^b) mice were purchased from The National Cancer Institute and used between 6 and 8 wk of age. FliC_431–439-specific (SM1) CD4⁺ TCR transgenic mice were intercrossed with CD45.1⁺ mice and maintained on a Rag-1–deficient background as described (25). All of the mice were housed within University of Minnesota specific pathogen-free facilities, and experiments were conducted under Institutional Animal Care and Use Committee-approved protocols.

The parental I-A^b–restricted wild-type FliC_431–439 peptide and each APL derived from this peptide were purchased from United Biochemical Research (≥90% purity; Seattle, WA): RFNSAITNLGN (wild-type FliC_431–439 [WT FliC]), RFNSAITNIGN (FliC_L438I), RFDSAITNLGN (FliC_N432D), RFESAITNLGN (FliC_N432E), and RFNFAITNLGN (FliC_S433F). The I-A^b‑restricted Ag85B_244–252 peptide (AYNAAGGHNAV) was used as an irrelevant stimulation control. All of the peptides were dissolved in DMSO (100 mM) and further diluted in sterile saline to the indicated concentration used for in vitro stimulation. For in vivo stimulation, 50 μg of each peptide was diluted with saline (200 μl) and i.v. injected into mice.

For in vitro stimulation, splenocytes from SM1 TCR transgenic mice were cultured in 96-well round-bottom plates (1 × 10⁶ cells per milliliter) containing the indicated concentration of each peptide in DMEM supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine, 50 μM 2-ME, 1% nonessential amino acids, and penicillin (100 U/ml) and streptomycin (100 U/ml). For some experiments, CD4⁺ T cells from SM1 TCR transgenic mice were labeled with CFSE (Invitrogen, Carlsbad, CA) prior to stimulation using standard labeling conditions (5 μM for 10 min at room temperature). For adoptive transfer, 2 × 10⁴ CD4⁺ T cells from SM1 TCR transgenic mice were i.v. inoculated into recipient mice 1 d prior to peptide inoculation or recombinant L. monocytogenes infection. Abs and other reagents for cell surface, intracellular, or intranuclear staining were purchased from BD Biosciences (San Jose, CA) or eBioscience (San Diego, CA). To measure cytokine production by cells stimulated in vitro, brefeldin A was added to cultures for the final 5 h prior to intracellular cytokine staining. To measure cytokine production by CD4⁺ T cells ex vivo, splenocytes were stimulated with WT FliC peptide or each FliC-derived APL in the presence of brefeldin A for 5 h prior to intracellular cytokine staining.

L. monocytogenes ΔActA strain DPL-1942 and methods for L. monocytogenes transformation have been described previously (26–28). Briefly, recombinant L. monocytogenes expressing either the parental wild-type FliC_431–439 peptide or each APL derived from this peptide were generated by cloning the coding sequence for each into the “open” pAM401-based expression construct that allows transcription behind the L. monocytogenes-specific hly promoter and secretion based on the LLO-specific signal sequence (28, 29). Specifically, the coding and noncoding sequences that correspond to each peptide (Supplemental Table I) were annealed together and ligated in-frame into the PstI and StuI sites within the coding sequence for hen egg OVA. Peptide Ags introduced in this site form a recombinant fusion protein containing the desired Ag (N terminus) and a truncated form of OVA (C terminus) that primes Ag-specific CD4⁺ or CD8⁺ T cells (28, 29). Relevant portions of each construct were verified by DNA sequencing. L. monocytogenes protein preparation, SDS gel electrophoresis, and blotting using anti-hemagglutinin Ab (clone HA-11; Covance, Emeryville, CA) were performed as described (28, 29). For infection, L. monocytogenes was grown to log phase in brain heart infusion media (BD Biosciences) containing chloramphenicol (20 μg/ml) at 37°C, washed, and diluted with saline to a final concentration of 1 × 10⁶ CFUs per 200 μl and injected i.v. into mice as described (28, 29). L. monocytogenes ΔActA cannot spread from infected cells into adjacent noninfected cells, is rapidly cleared postinfection, and therefore allows the use of higher inocula to optimally prime the expansion of pathogen-specific CD4⁺ T cells (29). The virulent S. typhimurium strain SL1344 has been described (21, 25) and was grown to early log phase in brain heart infusion broth at 37°C. For infections, 1 × 10² CFUs of S. typhimurium were washed and diluted in saline (200 μl) and injected i.v. into mice.

The differences in mean numbers of recoverable bacterial CFUs between groups of mice were evaluated using the Student t test with p < 0.05 taken as statistically significant (GraphPad, San Diego, CA).

The flagellum structural protein, FliC, is an immune-dominant Ag that confers protection against S. typhimurium infection (21). The peptide that spans aa 431–439 within FliC is presented by MHC class II I-A^b because CD4⁺ T cells with specificity for this peptide are readily identified in naive C57BL/6 mice and cell lines derived from these mice after stimulation with heat-killed Salmonella (25, 30). Alignment with other well-characterized I-A^b peptides reveals the putative MHC class II anchor and TCR contact residues within the FliC_431–439 peptide (Fig. 1) (31–34). An essential role for amino acid positions P1, P4, P6, and P9 in direct contact and anchoring the peptide to I-A^b MHC has been demonstrated through mutational analysis and biochemical-binding/affinity assays (31, 32), and these results have been confirmed by the resolved crystal structure of I-A^b–restricted peptides bound to this MHC molecule (33, 34). These studies reveal that amino acids containing large, hydrophobic aromatic side chains are predominantly found at position P1, whereas amino acids with small, uncharged side chains are frequently present at positions P4, P6, and P9. Accordingly, the phenylalanine corresponding to residue 431 within FliC corresponds to position P1, and Ala⁴³⁴, Thr⁴³⁶, and Gly⁴³⁹ correspond to positions P4, P6, and P9, respectively (Fig. 1). The importance of positions P2, P3, P5, P7, and P8 for direct contact with the TCR also has been confirmed by both biochemical and crystallographic experimental approaches (31–34). In turn, residues Asn⁴³², Ser⁴³³, Iso⁴³⁵, Asn⁴³⁷, and Leu₄₃₈ within FliC_431–439 correspond to the TCR contact sites at positions P2, P3, P5, P7, and P8, respectively (Fig. 1). Therefore, to identify potential naturally occurring APLs within the FliC_431–439 peptide Ag, a directed basic local alignment search tool search of the National Center for Biotechnology Information peptide database was performed for known proteins containing substitutions in these TCR contact sites. The parental FliC_431–439 peptide sequence containing substitutions incorporating each of the other 19 possible amino acids at each TCR contact residue was used for this database search. This analysis revealed four naturally occurring FliC_431–439-derived APLs that were each identified within the flagellar FliC protein homologues of various Salmonella serovars and other Gram-negative bacteria known to colonize or invade through the gastrointestinal tract (Fig. 1). Interestingly, all of these FliC_431–439-derived APLs were identified from human clinical isolates of virulent bacteria (35). Among these FliC_431–439-derived APLs, one APL contains a conserved leucine to isoleucine substitution at residue 438 (FliC_L438I), which retains an uncharged, hydrophobic residue at position P8, whereas the other three APLs each contain nonconservative substitutions at either position P2 (FliC_N432D and FliC_N432E) or position P3 (FliC_S433F). In the case of APLs FliC_N432D and FliC_N432E, an uncharged asparagine residue is replaced by a negatively charged aspartate or glutamate residue, respectively. For APL FliC_S433F, the small, hydrophilic serine residue is replaced by a large, hydrophobic phenylalanine residue. The presence of these APLs within the immune-dominant epitope of FliC isolated from clinical samples of Salmonella and other Gram-negative bacterial pathogens suggests that altered TCR stimulation may play an important role in controlling the CD4⁺ T cell response to this Ag.

Salmonella FliC_431–439-specific CD4⁺ T cells derived from SM1 TCR transgenic mice were used to characterize potential differences in how each naturally occurring APL compared with the parental WT FliC peptide primes CD4⁺ T cell activation, proliferation, and differentiation (25). As expected, WT FliC readily primes the proliferation of CD4⁺ T cells from SM1 TCR transgenic mice. More than 95% of these CD4⁺ T cells were CFSE dilute by day 5 after stimulation with this peptide at 10 and 100 μM, whereas progressive reductions in percent CFSE dilute cells were found when lower concentrations of WT FliC peptide were used (Fig. 2A). Interestingly, although the degree of CFSE dilution for cells stimulated with APL FliC_L438I was comparable to those for cells stimulated with WT FliC at higher peptide concentrations (10 and 100 μM), the reduction in CFSE dilution after stimulation with APL FliC_L438I did not become apparent until much lower concentrations compared with that of WT FliC peptide (Fig. 2A). In contrast, although stimulation with APLs FliC_N432D, FliC_N432E, and FliC_S433F each triggered some CFSE dilution among FliC-specific CD4⁺ T cells at the highest peptide concentration, this level of CFSE dilution was significantly reduced compared with that of the cells stimulated with either WT FliC or APL FliC_L438I and became rapidly extinguished when a 10-fold reduction in concentration was used for stimulation. Collectively, these results indicate that naturally occurring FliC_431–439-derived APLs stimulate CD4⁺ T cell proliferation either more potently (APL FliC_L438I) or less potently (APLs FliC_N432D, FliC_N432E, and FliC_S433F) compared with the parental WT FliC peptide.

On the basis of these differences in proliferation, the impact of stimulation with each APL compared with that of the parental WT FliC peptide on T cell activation by FliC-specific CD4⁺ T cells was quantified. Consistent with their ability to readily prime proliferation, CD4⁺ T cells stimulated with WT FliC and APL FliC_L438I dramatically upregulated CD25 and CD44 expression and downregulated CD62L expression (Fig. 2B). Conversely and consistent with the weak levels of proliferation, stimulation with APLs FliC_N432E or FliC_S433F did not cause appreciable changes in CD25, CD44, or CD62L expression, and the expression levels for each were essentially identical to those of cells stimulated with an irrelevant control peptide. Interestingly, increased CD25 and CD44 expression was found after stimulation with APL FliC_N432D, albeit to a lesser extent than that in cells stimulated with either WT FliC or APL FliC_L438I peptides (Fig. 2B). Importantly, these differences in CD4⁺ T cell proliferation and activation triggered by WT FliC peptide compared with those of each APL are not due to differences in apoptotic cell death because no significant differences in the levels of annexin V expression were observed (Fig. 2B). Therefore, the expression of cell surface markers associated with T cell activation (CD25 and CD44 upregulation and CD62L downregulation) directly correlates with the robust proliferation of FliC-specific CD4⁺ T cells after stimulation with WT FliC and APL FliC_L438I, whereas reduced or minimal changes in the expression of each marker are associated with only limited proliferation after stimulation with APLs FliC_N432D, FliC_N432E, or FliC_S433F.

These differences in proliferation and expression of T cell activation markers were extended to explore potential differences in CD4⁺ T cell differentiation by measuring the production of Th lineage-defining cytokines such as IFN-γ, IL-4, and IL-17 representative of Th1, Th2, and Th17 lineages, respectively. Stimulation with WT FliC peptide primed IFN-γ–producing CD4⁺ T cells at higher peptide concentrations (10 and 1 μM), and the level of IFN-γ production was dramatically diminished as the concentration of WT FliC peptide was reduced to 0.1 μM (Fig. 3A). Interestingly, although APL FliC_L438I compared with WT FliC primed proliferation to a similar or greater extent at all of the peptide concentrations tested, IFN-γ production was markedly reduced at relatively high peptide concentrations (10 and 1 μM) but was only marginally reduced and exceeded the level present in WT FliC-stimulated cells when lower peptide concentrations were used (0.1 μM) (Fig. 3A). Furthermore, the level of IFN-γ production by cells stimulated with APLs FliC_N432D, FliC_N432E, and FliC_S433F was essentially at background levels and comparable to that of cells stimulated with an irrelevant peptide control. Importantly, the reduced IFN-γ production by cells stimulated with each FliC_431–439-derived APL compared with that of the WT FliC peptide was not associated with a reciprocal increase in the production of either IL-4 or IL-17, because the levels for both of these cytokines remained at the limits of detection for all of the stimulated cells quantified using both intracellular cytokine staining and ELISA.

Given the importance of the T-box transcription factor T-bet in CD4⁺ T cell IFN-γ production and Th1 differentiation, additional experiments quantified relative T-bet expression in FliC-specific CD4⁺ T cells stimulated with WT FliC or each APL peptide (36, 37). Levels of T-bet expression were found to be directly correlated with the production of IFN-γ. The mean fluorescent intensity for T-bet expression in cells stimulated with WT FliC peptide at the higher peptide concentration (10 μM) was dramatically increased (∼7-fold) compared with that of cells stimulated with an irrelevant control peptide, and this level of T-bet expression progressively diminished to background levels when the peptide concentration was reduced. In contrast, but completely consistent with the level of IFN-γ production, T-bet expression in cells stimulated with APL FliC_L438I compared with that in cells stimulated with WT FliC peptide was reduced 20% at the 10 μM peptide concentration but did not return to levels comparable to that of cells stimulated with an irrelevant control peptide even after dilution to 0.1 μM (Fig. 3B). As expected, T-bet expression for FliC-specific CD4⁺ T cells stimulated with APLs FliC_N432D, FliC_N432E, or FliC_S433F were each only at background levels consistent with the absence of IFN-γ production. Taken together, these results demonstrate that although APL FliC_L438I and WT FliC both efficiently prime CD4⁺ T cell proliferation, CD25 and CD44 upregulation, and CD62L downregulation at relatively high peptide concentrations (10 μM), stimulation with WT FliC compared with that with APL FliC_L438I primes significantly more IFN-γ production, which is associated with increased T-bet expression. However, at lower peptide concentrations (0.1 μM), IFN-γ production and T-bet expression are maintained for cells stimulated with APL FliC_L438I but not WT FliC peptide. These results reflect important differences in CD4⁺ T cell differentiation following stimulation with each of these naturally occurring Ags.

On the basis of these stark contrasts in degree of T cell activation, proliferation, and differentiation between each naturally occurring FliC_431–439-derived APL and WT FliC peptide after stimulation in vitro, additional experiments sought to characterize potential differences in the expansion and differentiation of FliC_431–439-specific CD4⁺ T cells after in vivo stimulation. For these experiments, purified WT FliC peptide, APL FliC_L438I, APL FliC_N432D (representative of results with APL_N432E and APL_S433F), and an irrelevant control peptide were each i.v. inoculated into mice adoptively transferred with congenically marked (CD45.1⁺) FliC-specific CD4⁺ T cells from SM1 TCR transgenic mice 1 d prior. Both purified WT FliC peptide and APL FliC_L438I primed robust expansion of FliC-specific CD45.1⁺CD4⁺ T cells; a >100-fold increase in percentages and total numbers of CD45.1⁺CD4⁺ T cells was present in mice inoculated with WT FliC or APL FliC_L438I compared with those of mice inoculated with an irrelevant control peptide (Fig. 4A, 4B). In contrast, but in complete agreement with the weak levels of proliferation after stimulation in vitro, i.v. inoculation with APL FliC_N432D did not prime significant CD4⁺ T cell expansion, because the percentages and total numbers of CD45.1⁺CD4⁺ T cells recovered from these mice were not significantly different from those of mice inoculated with an irrelevant control peptide (Fig. 4A, 4B). When cytokine production by FliC-specific CD4⁺ T cells stimulated in vivo was quantified, the overall number of IFN-γ–producing CD4⁺ T cells was markedly reduced for FliC-specific CD45.1⁺CD4⁺ cells primed with APL FliC_L438I compared with that for those primed with WT FliC peptide following ex vivo stimulation with WT FliC peptide (Fig. 4C). Importantly, this reduction in IFN-γ production by FliC-specific CD45.1⁺CD4⁺ cells primed with APL FliC_L438I compared with that in cells primed with WT FliC peptide reflects intrinsic differences due to in vivo priming, because similar reductions were found when APL FliC_L438I was used for ex vivo stimulation (Fig. 4C). Furthermore, FliC-specific CD45.1⁺CD4⁺ T cells produced no detectable IFN-γ when primed in vivo with APL FliC_N432D or an irrelevant control peptide after stimulation ex vivo with either WT FliC peptide or each APL (Fig. 4C). Therefore, despite either enhanced or reduced levels of proliferation, markedly reduced IFN-γ production after stimulation with each APL compared with that after stimulation with the WT FliC peptide is observed under noninfection conditions in vivo.

Additional experiments sought to characterize how stimulation with WT FliC peptide and each APL would impact CD4⁺ T cell priming during experimental infection in vivo because infection triggers complex cascades of immune cytokines and signaling molecules not reproduced by stimulation with purified peptide alone. Because even aflagellated Salmonella retains a high degree of virulence in C57BL/6 mice (38), this natural vector could not be used for priming FliC-specific CD4⁺ T cells postinfection in vivo. Therefore, recombinant L. monocytogenes was engineered to express either WT FliC or each FliC-derived APL. For these experiments, the attenuated L. monocytogenes ΔActA parental strain was used, thus allowing a relatively high bacterial inoculum to be used that optimizes the priming and expansion of Ag-specific CD4⁺ T cells after recombinant L. monocytogenes infection (29). The coding sequence for either the WT FliC peptide or each APL was cloned behind the L. monocytogenes-specific hly promoter with an N terminus listeriolysin O-specific signal sequence that allows each recombinant protein to be secreted by the bacterium (Fig. 5A). The uniform expression and secretion of the recombinant proteins that contain either the WT FliC peptide, each APL, or control peptide Ag were verified by protein immune blotting using an Ab against the hemagglutinin tag (Fig. 5B). Although protective immunity to L. monocytogenes infection is predominantly mediated by CD8⁺ T cells, we and others have shown that infection with either virulent or L. monocytogenes ΔActA also primes the robust expansion of CD4⁺ T cells with specificity for both endogenous L. monocytogenes or recombinant Ags secreted by L. monocytogenes (11, 27, 29). Similar to results after stimulation with purified peptide, mice infected with L. monocytogenes expressing either wild-type FliC (rLM-WT) or APL FliC_L438I (rLM-L438I) each contained robust FliC-specific CD45.1⁺CD4⁺ T cell expansion, whereas mice infected with L. monocytogenes expressing APL FliC_N432D (rLM-N432D) contained only background levels of CD45.1⁺CD4⁺ T cells comparable to levels found in mice infected with L. monocytogenes expressing an irrelevant control Ag (rLM-Control) (Fig. 5C).

Related experiments quantified IFN-γ production by FliC-specific CD45.1⁺CD4⁺ T cells after stimulation with WT FliC or each APL administered in the context of recombinant L. monocytogenes infection. Because L. monocytogenes ΔActA at an inocula of 10⁶ CFUs triggers the robust production of cytokines such as IL-12 and type I IFN that together synergistically prime IFN-γ production by CD4⁺ T cells (39), we hypothesized that the observed differences in IFN-γ production relative to the degree of proliferation for FliC-specific CD4⁺ T cells primed with WT FliC compared with that for those primed with APL FliC_L438I would be normalized in this highly polarizing Th1 cytokine environment. Alternatively, persistent reductions in IFN-γ production relative to the degree of proliferation for FliC-specific CD4⁺ T cells primed with APL FliC_L438I compared with that for those primed with WT FliC peptide would demonstrate a critical role for TCR stimulation in controlling CD4⁺ T cell IFN-γ production even in the cytokine milieu triggered by L. monocytogenes ΔActA infection. Remarkably and in complete agreement with stimulation studies using purified peptide, IFN-γ–producing FliC-specific CD45.1⁺CD4⁺ T cells were drastically reduced in mice primed with rLM-L438I compared with those from mice primed with rLM-WT (∼60% reduction, p < 0.05) (Fig. 5D, 5E). As expected, the few cells that did not appreciably expand postinfection with rLM-N432D also did not produce detectable amounts of IFN-γ relative to mice inoculated with recombinant rLM-Control (Fig. 5D, 5E). Additional experiments extended the time course for these experiments to characterize the survival of CD4⁺ T cells at later time points postinfection with each recombinant L. monocytogenes. By day 30 postinfection, the numbers of CD45.1⁺CD4⁺ T cells for mice infected with either rLM-WT or rLM-L438I had contracted ∼20-fold compared with day 3 levels (Fig. 5C). Interestingly, despite this large degree of contraction, the ∼60% reduction in the number of IFN-γ–producing CD45.1⁺CD4⁺ T cells was maintained for rLM-L438I–infected mice compared with that for rLM-WT–infected mice through this later time point (Fig. 5E).

Given the overall importance of CD4⁺ T cells and IFN-γ in host defense against intracellular bacterial pathogens such as Salmonella (19–24), additional experiments sought to characterize the role of IFN-γ production by Salmonella-specific CD4⁺ T cells in immunity against this infection. Mice primed initially with recombinant L. monocytogenes expressing WT FliC or each APL were challenged with a lethal dose of virulent S. typhimurium 30 d after L. monocytogenes infection, and the degree of protection was quantified by enumerating the number of recoverable CFUs of S. typhimurium from each group of mice in the spleen and liver. Compared with mice transferred with FliC-specific CD4⁺ T cells alone, significant differences in CFUs of S. typhimurium were found only for mice primed with rLM-WT where an ∼10-fold reduction (p < 0.05) in S. typhimurium bacterial burden was found day 5 after challenge (Fig. 6). In contrast, mice primed with rLM-L438I, rLM-N432D, or rLM-Control each had significantly increased CFUs of S. typhimurium compared with those of mice primed with rLM-WT and at levels not significantly different from those of control mice that only received FliC-specific CD4⁺ T cells without L. monocytogenes infection (Fig. 6). This observed reduction in CFUs of S. typhimurium conferred by infection with rLM-WT cannot be attributed to nonspecific immune activation secondary to L. monocytogenes ΔActA infection because mice primed with rLM-L438I, rLM-N432D, and rLM-Control were each infected with the same inocula of L. monocytogenes ΔActA. Furthermore, because similar numbers of FliC-specific CD4⁺ T cells are present both during expansion and after T cell contraction (Fig. 5B) for mice primed with WT FliC and APL FliC_L438I, differences in the absolute number of FliC-specific CD4⁺ T cells alone also cannot account for reductions in CFUs of S. typhimurium conferred by rLM-WT compared with those conferred by rLM-L438I. Despite the significant reduction in CFUs of S. typhimurium observed in mice primed with rLM-WT compared with those observed in mice primed with rLM-L438I, rLM-N432D, or rLM-Control, the overall survival and time to death for these groups of mice were not significantly different. Regardless of the initial priming condition, the majority of mice in each group became moribund or died by day 7 postchallenge. Taken together, these results indicate a critical role for IFN-γ production by pathogen-specific CD4⁺ T cells in host defense against Salmonella infection and demonstrate how alterations in TCR stimulation can have dramatic impacts on CD4⁺ T cell differentiation and protective potency.

CD4⁺ T cell activation and differentiation require at least three distinct yet interrelated stimulatory signals mediated through the TCR, costimulation receptors, and receptors for specific “inflammatory” cytokines (7–15). Although these signals together uniformly stimulate T cell activation, proliferation, and differentiation, each signal has been proposed to play specific and defined roles in this process. For example, the Ag specificity for the T cell response is controlled by stimulation through the TCR, whereas lineage differentiation is believed to be largely dictated by the presence or absence of specific cytokines. Unfortunately, because these roles have been primarily characterized after T cell stimulation in vitro or during noninfection conditions in vivo, the precise role for each stimulatory signal in controlling the specific incremental steps required for T cell activation during infection when the expression levels for all of the T cell stimulatory signals are drastically altered remains largely undefined. Moreover, the impact of changes in each stimulatory signal on CD4⁺ T cell-mediated protection from infection is unknown. Accordingly, the importance of TCR stimulation in CD4⁺ T cell activation was experimentally examined by measuring potential differences in proliferation, expansion, and differentiation after stimulation under noninfection and infection conditions with the Salmonella-derived FliC_431–439 MHC class II peptide or APLs containing substitutions in TCR contact residues derived from this peptide. In these experiments, naturally occurring APLs containing amino acid substitutions in TCR contact residues within the FliC_431–439 peptide were identified and used for stimulation to explore how natural ligands from other bacterial pathogens may control the CD4⁺ T cell response to this Ag. Among these naturally occurring APLs, one primed FliC_431–439-specific CD4⁺ T cell proliferation more potently, whereas others primed CD4⁺ T cell proliferation less potently compared with the parental wild-type FliC peptide following stimulation in vitro. Despite differences in proliferation, these APLs primed dramatically reduced IFN-γ production following stimulation in vivo. Importantly, the protective potency of these CD4⁺ T cells after challenge with virulent Salmonella was directly correlated with IFN-γ production regardless of expansion magnitude, and this increased protective potency was reflected in the significant reduction in Salmonella bacterial burden after secondary challenge. Interestingly, however, these reductions in Salmonella CFUs were not associated with detectable difference in survival. This observation may be due to the inherent defects in innate host defense against virulent S. typhimurium infection present in C57BL/6 mice. Nevertheless, these results collectively demonstrate a critical and previously unanticipated role for TCR stimulation in controlling CD4⁺ T cell differentiation into protective, IFN-γ–producing effector T cells.

A two-step model for the initial upregulation and eventual stabilization of T-bet expression in naive CD4⁺ T cells required for IFN-γ production and Th1 differentiation has been proposed (40, 41). In this model, the initial expression of T-bet is dependent on TCR stimulation and IFN-γ production, whereas the latter phase of T-bet stabilization is dependent on IL-12 receptor stimulation. Interestingly, termination of TCR stimulation permitted upregulation of IL-12 receptor expression required for maintaining T-bet expression (40). Our demonstration that APL FliC_L438I is a more potent inducer of FliC-specific CD4⁺ T cell proliferation compared with WT FliC yet only weakly upregulates IFN-γ production at high concentrations is consistent with this hypothesis. The discordant expression of T-bet with proliferation potency after APL FliC_L438I stimulation may be due to stronger TCR stimulation that inhibits the latter phase of T-bet expression required for the stabilization of IFN-γ production by FliC-specific CD4⁺ T cells. This notion that APL FliC_L438I compared with WT FliC stimulates stronger TCR signaling is supported by our results demonstrating that T-bet expression and IFN-γ production by FliC-specific CD4⁺ T cells stimulated with APL FliC_L438I are less perturbed by reductions in peptide concentrations (Fig. 3A). Additional experiments that more specifically characterize the kinetics of T-bet, IFN-γ, and IL-12 receptor expression are needed to elucidate the mechanism underlying this observed discordance between CD4⁺ T cell proliferation and IFN-γ production.

The identification of naturally occurring APLs within protective immune-dominant Ags suggests that alterations in TCR stimulation may be used by pathogenic bacteria to modulate or avoid the pathogen-specific T cell response. This notion is bolstered by the identification of naturally occurring APLs derived from Salmonella FliC_431–439 among human clinical isolates of various invasive bacterial pathogens that we describe in this study (Fig. 1). For example, APL FliC_N432D has been described in over 100 unique Salmonella clinical isolates derived from serovars that have the potential to cause typhoidal or nontyphoidal disease in humans (35, 42). Moreover, this APL also has been identified in numerous clinical isolates of other invasive Gram-negative human pathogens such as Shigella sonnei, Yersinia intermedia, and both toxigenic and hemorrhagic forms of Escherichia coli (43–49). Collectively, the identification of APLs within FliC_431–439 from this diverse range of bacterial pathogens that cause clinical disease indicates that pathogens containing these mutations not only retain virulence but also may be more capable of enhanced pathogenesis associated with immune evasion of protective T cells. Although our experiments examined the response for CD4⁺ T cells with unique specificity for one Salmonella-specific immune-dominant peptide Ag, these results nevertheless provide experimental evidence implicating alterations in CD4⁺ T cell TCR stimulation as a potential immune evasion strategy used by bacterial pathogens. Therefore, further investigation characterizing the differences in virulence properties and immune response to pathogens that contain APLs in immune-dominant T cell Ags is needed and will likely reveal novel and important information on the pathogenesis and immune response to these infections.

We thank Drs. Marc Jenkins, Stephen McSorley, David Masopust, and Vaiva Vezys for helpful discussions and Dr. Stephen McSorley for the gift of SM1 TCR transgenic mice and critical review of this manuscript.

Disclosures The authors have no financial conflicts of interest.