Adaptive Immune Responses during Salmonella Infection

LISA A. CUMMINGS,¹ BROOKE L. DEATHERAGE,² AND BRAD T. COOKSON^1,2*

[SECTION EDITORS: FERRIC FANG AND MARTIN KAGNOFF]

Posted September 17, 2009

Department of Laboratory Medicine¹ and Department of Microbiology,² University of Washington, Seattle, WA 98195

*Corresponding author. Mailing address: Department of Microbiology, University of Washington, Seattle, WA 98195. Phone: (206) 598–6131, Fax: (206) 598–6189, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

INTRODUCTION

The significance of salmonellosis as a medical problem is readily appreciated by noting that vaccination efforts to protect against the disease began in the latter part of the 19th century (205). Six decades later, murine models of Salmonella infection, in which Salmonella enterica serovar Typhimurium infection in mice served as a model for Salmonella enterica serovar Typhi infection in humans, began to provide important new insights. Greater protection was afforded by sublethal infection with viable bacteria than by administration of heat-killed vaccines (62). Protection generated by live organisms did not correlate with anti-lipopolysaccharide (anti-LPS) antibody responses (62), and passive administration of antibody prolonged survival but did not afford complete protection. Subsequent studies indicated that T cells contributed to protective immunity (23, 80, 84). Thus, the principle that both cellular and humoral responses were required for protection was established (187). Importantly, this concept was confirmed in 1993 by the demonstration that adoptive transfer of immune serum and T cells provided protection to naïve, susceptible hosts (102).

Immunity requiring both T-cell responses and immunoglobulins (Igs) from B cells is consistent with our understanding of Salmonella as a facultatively intracellular pathogen. Bacteria encounter phagocytes either within the gastrointestinal tract (150), or during penetration of the mucosal epithelium (192). Salmonella-specific Ig produced by B cells can opsonize extracellular bacteria for phagocytic uptake. Salmonella then spread systemically within phagocytes (192) to colonize target organs such as liver and spleen (151, 157). Salmonella is able to survive and replicate within macrophages and dendritic cells (DCs) in vivo. Inside these professional antigen-presenting cells (APCs), major histocompatibility complexes (MHCs) serve as receptors surveying the host cellular cytosol and vacuolar compartments for the presence of foreign peptides. When bound by MHC, these peptides are presented on the APC surface for recognition by T-cell antigen receptors (TCRs). In addition to macrophages and DCs, B cells can also act as APCs. These cells also express MHC, and surface-expressed Ig promotes high-efficiency antigen (Ag) capture for Ag presentation. TCR recognition of Ag presented by APCs leads to activation and expansion of CD4+ T cells. In addition to Ig production, B cells express cytokines that appear to participate in the differentiation and function of effector CD4+ T cells (57). Activated CD4+ T cells in turn provide critical stimulation for B cells to undergo expansion and Ig class switching, as well as for activation and growth of cytotoxic CD8+ T cells and phagocytic cells such as macrophages (72). Thus, a pathogen whose virulence requires survival inside host phagocytic cells (38) provides a contextual framework for understanding adaptive immunity (Fig. 1).

Fig. 1Disease outcome is the cumulative result of Salmonella interactions with host adaptive immune functions. During Salmonella infection, there are several points at which pathogen and host immune cells interact. Multiple outcomes for each interaction are possible; the predominant outcome is determined not only by the virulence of the bacterium, but also the innate resistance or susceptibility of the host, and previous exposure of the host to the pathogen (host immune status). Uptake of bacteria by APCs (A) results in either APC elimination by pyroptosis (B) or APC survival (C). Pyroptosis leads to release of the inflammatory cytokines IL-1β and IL-18, and possibly releases bacteria or bacterial Ags for uptake by bystander APCs. Some APCs, such as macrophages, are capable of destroying intracellular bacteria (D). If APCs survive, they may be able to process and present Ag in the context of MHC (E). Salmonella interferes with this process via multiple mechanisms including repression of Ag expression, bacterial surface modifications that reduce Ag bioavailability and APC stimulation/maturation, and other SPI-2 -dependent processes that mediate bacterial survival within the phagosome (F). Protected from antibody detection, intracellular Salmonella can utilize APCs as vehicles for systemic dissemination and replication (G). If APCs are able to overcome bacterial interference to process and present Ag to T cells (E), Salmonella may still inhibit T-cell activation via stimulation of nitric oxide (NO) production and other direct, suppressive effects (H). Recognition of peptide-MHC on APCs by TCR-expressing naïve T cells leads to activation and expansion of Ag-specific effector T cells (I). Effector CD4+ T cells provide help for the activation of CD8+ CTLs (leading to cytokine production and lysis of infected host cells [J]), and B cells (K). B cells and T cells work synergistically: T cells provide help for antibody production, isotype class switching, and cytokine production by B cells, while B-cell cytokine production supports Th-1 T-cell differentiation, and Ig on B-cell surfaces mediate Ag capture for processing and presentation to T cells (K). Cytokines such as IFN-γ are produced by effector T cells to further enhance APC function and activate bacterial degradation by macrophages (L). In an immune host, previously primed Ag-specific memory T cells (M) may be activated by APCs that process and present Ag (N); activation of these cells and their effector functions (O) is much more rapid than for naïve T cells. In addition, circulating antibodies primed by previous immunization facilitate bacterial uptake via opsonization (P), accelerating the efficiency of Ag presentation up to 1,000-fold. Thus, the ultimate outcome of infection is the cumulative result of complex interactions between pathogen and host.

The generation of protective immunity in the naïve host relies on the reactive milieu of innate immune responses elicited at the initiation of bacterial infection by DCs, and to a lesser extent, macrophages. The inflammatory response critically benefits the host by helping to generate B- and T-cell responses, but recruitment of phagocytic cells to the site of initial bacterial invasion and replication may facilitate systemic bacterial spread (155). In addition, Salmonella are capable of destroying both macrophages and DCs by an inflammatory process called pyroptosis (see Chapter Host Cell Death) (13, 42, 190), which abrogates their important APC function(s) and thereby antagonizes initiation of the adaptive immune response (Fig. 1B and 2C). Thus, Salmonella infection readily kills the naïve susceptible murine host. In contrast, the observation that Salmonella also causes chronic colonization (87, 120), made famous by Typhoid Mary in the last century, indicates that Salmonella have evolved mechanisms to finely balance these seemingly opposing cellular reactions.

Fig. 2Development of adaptive immune responses during Salmonella infection. Interactions with Salmonella and its host are dynamic and complex. Flagellated and nonflagellated Salmonella are present in the gut lumen (A), where they must overcome initial barriers including the glycocalyx layer, antimicrobial peptides, and sIgA. Salmonella cross the epithelium (B) via M cells, by inducing endocytosis in epithelial cells, or following uptake by CX3CR1+ DCs. Bacteria in the gut lumen (A), within epithelial cells (B) and in the PP (C) express FliC protein; interaction of FliC with TLR5 initiates an inflammatory response characterized by production of IL-8 and CCL20 by epithelial cells. Release of inflammatory mediators triggers infiltration of macrophages, neutrophils, and CCR6+ DC. Interaction of these cells with Salmonella results in at least three possible outcomes: (1) Phagocytosis of bacteria by DC or macrophages, ultimately resulting in inflammatory cell death (flagellin-dependent pyroptosis). Pyroptosis eliminates potential APCs, leads to release of the inflammatory cytokines IL-1β and IL-18, and possibly releases Ags for uptake by bystander APCs (note that flagellin-negative bacteria have a reduced ability to trigger inflammation via TLR5 or pyroptosis). (2) Uptake of bacteria, which persist within the phagocyte. This interaction can lead to production of NO by APCs (inhibitory for T-cell activation) and upregulation of MHC and costimulatory molecules on DC. In addition, these cells could provide a means of transport to systemic sites such as the liver and spleen. (3) Bacteria are phagocytosed by neutrophils or macrophages and degraded. Mature CCR6+ DC in the PP (C) process and present Ags to naïve T cells; Ags acquired for processing and presentation are restricted to those expressed by bacteria in the gut lumen (A) or PP (C), or Ags that are present in gut lumen, and disassociated from the bacterial soma ([A] MVs, flagellin). Mature DCs that have processed and presented Ag on surface MHC enter into an “activation feedback” loop with naïve T cells: TNF-α and IL-12 produced by DCs enhance activation and expansion of Ag-specific T cells, while IFN-γ secretion by activated T cells further stimulates DC function. Memory T cells primed in the PP (C) express the α4β7-homing receptor as well as CCR6+, predisposing these cells to traffic to the inflamed gut during a secondary infection. Bacteria within APCs disseminate to MLN (D), where Ag presentation to T cells can also occur. Bacteria in the MLN have undergone complete adaptations to the intracellular environment: they no longer express FliC, actively reduce Ag bioavailability, and interfere with APC function (see Fig. 1). Dissemination to systemic sites such as liver and spleen (E) follows MLN colonization. Bacteria replicate within APCs at systemic sites. However, in naïve hosts, T-cell responses to Ags expressed by intracellular phase bacteria are generally not sufficient in magnitude or quality, or fail to develop rapidly enough, to combat infection. Further, T cells primed at early stages in the PP (C) will not recognize bacteria growing intracellularly at systemic sites (E), or that fail to express FliC in the PP (C, left).

Despite recent advances, many aspects of salmonellosis and immunity to Salmonella infection remain enigmatic. The mechanisms by which virulent Salmonella evades primary immune responses to mount lethal infections in naïve hosts are incompletely understood, as are the means by which live attenuated bacteria generate protective immunity. The identity of Ags recognized by the adaptive immune response, and the precise roles of T cells and B cells in providing immunity are likewise poorly defined. However, our view is that this system provides a rich model for defining significant and fundamental aspects of the immunopathogenesis of host-pathogen interactions. Both genetic (39, 95) and acquired (50) immune defects lead to increased susceptibility to salmonellosis, which is responsible for the annual loss of hundreds of thousands of lives (137). Salmonella infections therefore remain a contemporary scientific problem of substantial medical and economic significance measured annually in the billions of dollars worldwide (60, 154). Here, we highlight aspects of adaptive immune responses to Salmonella that shed critical light on our current state of understanding and provide a foundation to help address important questions in the future.

ANTIGEN-PRESENTING CELLS

APCs Are Required for the Development of Ag-Specific T-Cell Responses

The detection of bacterial Ags by T cells and B cells via their variable receptor molecules (TCR and BCR, respectively) is the cornerstone of adaptive immune recognition. Unlike B cells, T cells do not recognize bacterial proteins in their native state. Instead, bacterial Ags must be processed into peptides and presented by professional APCs on specialized surface display structures called MHCs. In general, peptides derived from extracellular pathogens internalized by the host cell or pathogens replicating in intracellular vacuoles are displayed on MHC-II molecules. Correspondingly, peptides derived from pathogens replicating in the host cell cytosol are displayed on MHC-I molecules. Presentation on MHC-II is required for activation of CD4+ T cells, while CD8+ T cells respond to peptides presented in the context of MHC-I molecules. Thus, the mammalian host has evolved mechanisms for immune surveillance of both extracellular and intracellular pathogens. The importance of APC function in the development of immunity to Salmonella infection, and the mechanisms by which Salmonella interfere with this function are discussed below.

DCs and Macrophages as Professional APCs during Salmonella Infection

Because of their ability to process and present Ag to T cells, professional APCs are critical for immunity to Salmonella infection. Macrophages and DCs are classified as professional APCs, although B cells may also serve this function (reference 185, and see “B-cell functions in addition to antibody production,” below). Upon Salmonella encounter in vitro, both macrophages and DCs are stimulated to upregulate the expression of molecules required for T-cell activation: costimulatory surface molecules CD40, CD80, and CD86 and immunomodulatory cytokines gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), and IL-18 (76, 141, 212). DCs are well established activators of naïve T cells (6), and recent advances have begun to reveal a complex and diverse array of functionally distinct subpopulations (46, 177, 200). Macrophages are also capable of priming naïve Salmonella-specific T-cell responses in vivo (142). Nevertheless, these two cell types may play different roles in the development of adaptive immunity (128, 141).

DCs are phagocytic cells, but bacterial uptake induces a morphological and functional transition from immature Ag-capturing cells to mature APCs (6). Intracellular Salmonella persist but do not replicate within bone marrow-derived DCs (73), and DCs express low levels of lysosomal proteases, degrading bacterial proteins more slowly than macrophages. (32). Therefore, the environment within the mature DC is optimized for Ag processing and presentation rather than killing bacteria. DCs reside in the subepithelial area of the Peyer's patches (PPs), ideally situated for an initial encounter with invading Salmonella. In contrast, relatively few macrophages reside in the PP (65). After oral infection, colocalization of Salmonella with DCs in the PP and mesenteric lymph nodes (MLNs) occurs rapidly (20, 65). In addition, CCR6+ DCs colocalize with Salmonella-specific T cells in the PP suggesting that DCs are the primary APCs at early stages of infection (156). DCs infected in vitro are capable of priming Salmonella-specific CD4+ and CD8+ T-cell responses after adoptive transfer to naïve mice (208, 212), demonstrating the potential of DCs to prime naïve T cells in vivo. Indeed, DCs are absolutely required for activation and expansion of Salmonella-specific T cells in the PP (156). Taken together these findings confirm that DCs play a critical role in the initiation of adaptive immunity to Salmonella.

Although macrophages are classified as professional APCs and are capable of presenting Salmonella-derived Ags to T cells (24, 76, 97, 201), it is unclear whether this is their primary function during Salmonella infection. Human macrophages appear to be more efficient in the production of proinflammatory chemokines (CCl5, CCL20, and CXCL10) and cytokines (IL-18, TNF-α) than DCs (141), whereas DCs are more efficient than macrophages in stimulating IFN-γ production by CD4+ T cells (76). This suggests that macrophages may be more important in mediating proinflammatory responses and that DCs contribute more to Th1-type adaptive responses. The effective generation of Salmonella-specific CD8+ T-cell responses and protection against subsequent challenge in macrophage-depleted mice (204) support the idea that APCs other than macrophages elicit T-cell-mediated protection. Importantly, depletion of macrophages from immune mice prior to challenge significantly impairs protection as measured by bacterial load in systemic organs (202). Thus, macrophages may mediate protection primarily via bacterial clearance rather than by APC function (128). It is also possible that Ag presentation by macrophages may be more important for stimulation of previously activated effector T cells rather than naïve T cells (142).

Bacterial Interference with APC Function

As described above, adaptive immune responses to Salmonella infection require the function of professional APCs. Surprisingly, Salmonella virulence depends on the ability of this pathogen to survive and replicate within the very cells required to mount immune responses against it. This can be explained by the fact that coordinated bacterial responses have evolved in the context of a host immune system in which a generalized innate response to bacteria leads to the development of a highly specific adaptive response and memory. This coevolutionary dynamic between pathogen and host has facilitated the development of multiple mechanisms by which Salmonella can subvert or evade APC function (references 16 and 182; also discussed below). The ability of Salmonella to simultaneously survive within APCs and interfere with the presentation of Ags to T cells contributes to virulence: activation of adaptive immunity is inhibited while APCs are exploited as protected reservoirs of replication and means for bacterial dissemination.

In order for bacterial Ags to be acquired, processed, and presented by APCs to T cells, they must be expressed by bacteria in accessible cellular compartments and at biologically significant levels. Together, these characteristics have been described as Ag bioavailability (1). Bacterial regulation of Ag bioavailability has been most extensively studied for the dominant Ag FliC, a flagellar subunit protein that is abundantly expressed on the bacterial surface. Serovar Typhimurium actively regulates FliC bioavailability during infection. The first level of this regulation exists at the level of nongenetic variation; even under conditions permissive for flagellar expression, serovar Typhimurium display a bistability phenotype in which a significant number of bacteria in the population are flagellin negative (27). Additional regulation of bioavailability occurs during the transition from extracellular to intracellular environments within the host, as serovar Typhimurium first restrict FliC expression to an intracellular bacterial compartment (1) and then repress FliC expression below the threshold required for T-cell activation (26). Notably, Ag sequestration alone is not sufficient to prevent processing and presentation of this Ag by DCs; modifications of the bacterial surface are also required (1). The global regulator PhoP, which is activated during intracellular growth (3) and required for bacterial survival in the phagosome (52), is one mediator of these modifications. PhoP regulates extensive remodeling of the bacterial surface which reduces its inflammatory properties, increases resistance to cationic antimicrobial peptides, and alters membrane permeability (36, 53, 79, 123). In the context of these modifications, the processing and presentation of bacterial Ags restricted to intracellular compartments is significantly reduced (1). Importantly, repression of FliC expression by intracellular-phase serovar Typhimurium is also PhoP dependent (10, 26). Therefore, at a time when Salmonella is residing and replicating within APCs, the organism coordinately regulates the expression of genes essential for both intracellular survival and the restriction of Ag bioavailability to APCs.

In addition to alterations in Ag bioavailability, Salmonella is able to directly interfere with APC function. For example, Salmonella causes caspase-1-mediated pro-inflammatory death (pyroptosis) of both macrophages (13, 42) and DCs (40, 190) (see Chapter Nitric Oxide in Salmonella and Escherichia coli Infections). Pyroptosis represents one potential mechanism (destruction of APCs) for avoiding the activation of adaptive immune responses, but may also facilitate host responses by providing a source of antigenic material for bystander APCs (34, 210). Salmonella also affects DC maturation: intracellular-phase Salmonella inefficiently stimulates MHC-II upregulation, costimulatory CD86 expression, and secretion of proinflammatory cytokines such as TNF-α and IL-12 (1). Virulent Salmonella may also inhibit Ag processing and presentation on MHC-II by delaying phagosome-lysosome fusion (183). This inhibition requires expression of genes encoded in the Salmonella Pathogenicity Island 2 (SPI2) and does not depend on Ag uptake, MHC-II upregulation, or APC viability (20, 182). The ability of Salmonella to interfere with APC function may at least partially account for host restriction, because only those serovars able to effectively prevent Ag processing and presentation by DCs within a specific host can limit activation of adaptive immune responses (15). Importantly, it appears that Salmonella actively inhibits APC function. SPI-2 mediated inhibition of Ag processing and presentation requires live infection (20), and live but not heat-killed PhoP^c mutant bacteria (which constitutively express PhoP in its activated state) inhibit Ag processing by activated macrophages (201). Therefore, at least some of the mechanisms that reduce Ag bioavailability (PhoP-regulated surface modifications and repressed Ag expression) also reduce the ability of APCs to process and present Ag to T cells. Finally, Salmonella is able to directly inhibit T-cell activation independent of Ag processing and presentation (188), possibly by downregulation of TCR β-chain expression (189).

Summary: APCs and Salmonella-Specific Immunity

The processing and presentation of Ags to T cells is a critical step in the development of adaptive immunity. Importantly, Salmonella bacteria are not simply passive collections of Ags, but rather actively modulate its interactions with host cells. Multiple bacterium-directed mechanisms, including altered Ag expression and bioavailability, and interference with APC activation and function, combine to modify Salmonella's “pathogenic signature” in order to minimize susceptibility to immune surveillance (Fig. 1B, F, and H).

T CELLS

T Cells Are a Critical Component of Immunity to Salmonella Infection

As a facultatively intracellular pathogen, Salmonella is capable of replicating within host cells (151, 157), protected from recognition by circulating antibodies. Therefore, a robust T-cell response is required for both clearance of primary infection and resistance to subsequent challenge (61, 101, 125, 198). This section discusses the relative contribution of various T-cell subsets to Salmonella-specific immunity, the potential mechanisms by which they mediate protection, and the identity of the Ags they recognize.

T-Cell Subsets and Salmonella Immunity

T lymphocytes recognize Ag via heterodimeric receptors (TCRs) expressed on their surface. The majority of T cells circulating throughout the peripheral lymphoid system express TCRs consisting of one α- and one β-chain. αβ T cells can be further grouped according to the expression of CD4 or CD8 coreceptors. CD4+ αβ T cells recognize bacterial peptides from endocytic or phagocytic vacuoles presented in the context of MHC-II molecules. They function as “helper” T cells by producing cytokines that stimulate B-cell antibody class switching, activation and growth of cytotoxic T cells, and activation of phagocytes such as macrophages (72). CD4+ T cells have been traditionally divided into Th1 and Th2 functional subsets based on expression ratios of various cytokines that lead to control of different classes of pathogens. However, additional CD4+ T-cell subsets have recently been identified (11, 55, 83, 197). CD8+ αβ T cells recognize bacterial peptides from the cytosol presented by MHC-I molecules. CD8+ T cells function as cytotoxic or “killer” T cells that lyse infected host cells (72), and like CD4+ T cells, are capable of secreting cytokines. In addition to αβ T cells, a small percentage of T cells express a TCR consisting of one γ- and one δ-chain and are concentrated in epithelial tissues, particularly in the gut mucosa (59). γδ T cells express TCRs of limited diversity compared with αβ T cells, do not appear to require MHC presentation of protein Ags for their activation, and may function in both innate and adaptive immune responses (59).

Because infection of susceptible hosts with virulent Salmonella is rapidly fatal, most studies of the dynamics of T-cell responses are performed with attenuated bacteria (149). Salmonella-specific CD4+ and CD8+ T cells are generated during infections with attenuated bacteria in mice (81, 91, 116) and humans (93, 158, 159, 160, 179). T-cell activation is quickly initiated after oral infection in mucosal lymphoid tissues (17, 106). This is followed by the expansion of a Salmonella-specific effector population in the lymphoid tissues that is capable of redistribution to nonlymphoid sites of infection (Fig. 2C) (81, 93, 116). IFN-γ-producing CD4+ and CD8+ T cells can persist for several months in mice and humans (24, 125, 159). In addition to αβ T cells, γδ T cells can also be detected after Salmonella infection; a subset is activated at early stages of infection via the MHC-Ib molecule Qa-1 (30, 119).

Many studies have confirmed that functional T cells are required for the resolution of both primary and secondary Salmonella infections. For example, mice lacking mature αβ T cells are severely impaired in their ability to control primary oral infection with attenuated aroA Salmonella (61, 119, 168, 198). Interestingly, T-cell function is not absolutely required in early stages of infection and it appears that T-cell-independent mechanisms largely mediate control of primary serovar Typhimurium infection for up to 21 days (61, 67, 198). Immunized mice that have been depleted of T cells are susceptible to secondary virulent challenge despite antibody production (61, 101, 125). Furthermore, transfer of immune T cells (with serum) (102, 125) or T-cell lines derived from immune mice (without serum) (138, 139) confers protection on naïve susceptible hosts.

The pathogenesis of Salmonella infections is reflected in the particular T-cell subsets most critical for host immunity. Because Salmonella replicates within phagocytic vacuoles of infected cells it is not surprising that there is an absolute requirement for CD4+ T cells in the control of both primary and secondary Salmonella infection (61, 101, 125). The importance of functional CD4+ T cells is highlighted by the increased frequency and severity of nontyphoidal Salmonella infections correlating with reduced CD4+ T-cell counts in patients suffering from HIV infection (19, 51, 63, 71, 77, 127). In addition, CD4+ T cells also appear to actively suppress serovar Typhimurium during chronic infection, as demonstrated by increased recurrence of serovar Typhimurium bacteremia in HIV patients (49, 50), or reactivation of infection in CD4+ T-cell-depleted mice (191).

CD8+ T cells are generated during Salmonella infection of mice and humans, and are restricted not only by MHC-Ia molecules, but also nonclassical MHC-Ib molecules (158, 170, 184). There is evidence to suggest that Salmonella-specific CD8+ T cells participate in protective immune responses. During primary infection with attenuated aroA Salmonella, MHC-I-deficient mice (lacking functional CD8+ T cells) have higher bacterial loads during primary oral infection than wild-type mice (30). Furthermore, T-cell receptor knockout mice (no functional CD4+ or CD8+ T cells) suffer from more severe infection than CD4+ T-cell-deficient MHC-II knockouts (61), suggesting that MHC-I-restricted CD8+ T cells participate in bacterial clearance. Additional studies indicate that CD8+ T cells may actually be more important in recall of immunity during secondary challenge. For example, although naive β2m^−/− mice (lacking functional MHC-I molecules) are eventually able to resolve an initial infection with attenuated bacteria, immune β2m^−/− mice are more susceptible than wild-type mice to virulent challenge (91). Moreover, adoptive transfer of CD8+ T-cell-depleted immune cells provides limited protection to naïve mice compared with CD8+ replete control cells (101, 125).

The contribution of γδ T cells to Salmonella immunity remains unclear. In one study, γδ knockout mice were as capable as wild-type mice of clearing infection with an aroA-attenuated serovar Typhimurium strain (61); similar results were observed after infection with Salmonella enterica serovar Dublin (198). Other groups detected slight defects in control of primary oral infection in γδ T-cell-deficient mice, and increased numbers of γδ T cells in the intestinal epithelium during infection (119). The latter observation, together with the fact that γδ T cells home to the gut mucosa (59), may indicate that γδ T cells have some function during early stages of Salmonella infection before systemic spread.

Mechanisms of T-Cell-Mediated Immunity to Salmonella Infection

Although T cells are required for successful resolution of Salmonella infections, the mechanisms whereby they contribute to immunity are poorly understood. It is well established that T cells generated in response to primary Salmonella infection are capable of producing IFN-γ and TNF-α (116) . Studies using IFN-γ or TNF-α gene knockout mice or antibody depletion of IFN-γ or TNF-α confirm the importance of these cytokines in control and resolution of Salmonella infection (7, 61, 101, 103, 122, 126), although the source of cytokines in these studies is not clearly defined. These data support the idea that cytokine production is the primary means by which T cells contribute to immunity, and substantial evidence supports a correlation between CD4+ T-cell function and IFN-γ production. For example, CD4+ T-cell knockout mice, which are unable to clear an infection with an aroA-attenuated serovar Typhimurium strain, produce 100-fold less IFN-γ than control mice (61). CD4+ T cells from mice deficient in the IFN-γ transcription factor Tbet do not produce IFN-γ or promote antibody isotype switching in B cells; these mice are also susceptible to infection with aroA-attenuated Salmonella (148). Interestingly, exposure to lead induces a shift from a Th1 response to a Th2 T-cell response (with a corresponding shift from high to low levels of IFN-γ and TNF-α production), thereby rendering resistant C3H/HeN mice susceptible to oral Salmonella infection (37). Others have demonstrated IFN-γ production by gut-associated lymphoid tissue and increased numbers of CD4+ and CD8+ T cells in the gut during Salmonella infection (7, 146), suggesting that localized expression of cytokines by T cells during primary infection may be important. Cytokine expression by T cells may account for the ability of immune mice to control a secondary infection with virulent Salmonella: rapid release of cytokines by T cells primed to preferentially home to the gut mucosa (121) facilitates rapid macrophage activation and recruitment to the site of initial host encounter with the pathogen (98). This idea is supported by studies in which administration of anti-TNF-α or IFN-γ antibodies exacerbated secondary virulent infection in previously immunized mice (101, 103). Finally, depletion of either IFN-γ or CD4+ T cells results in reactivation of latent Salmonella infection (120, 191), suggesting that IFN-γ may mediate CD4+ T-cell control of latent infections.

Despite this evidence for the importance of cytokine production by Ag-specific T cells in Salmonella infection, other studies suggest the existence of IFN-γ–independent T-cell actions. For example, it appears that cytokine production alone is insufficient to induce clearance in the absence of T cells: T-cell-depleted mice are susceptible to challenge with attenuated aroA Salmonella despite high serum levels of IFN-γ and TNF-α (101). Similarly, IL-12-null mice fail to control infection with attenuated serovar Typhimurium, despite equivalent IFN-γ and TNF-α production by T cells in mutant and wild-type mice. T cells in these mice did not proliferate in response to Ag, indicating the importance of IFN-γ-independent, proliferation-dependent T-cell responses in the control of Salmonella infection (143). Together with observations that NK-T cells and neutrophils have been identified as a significant source of IFN-γ production in response to serovar Typhimurium infection (145), and that T-cell function is required only in the later stages of primary sublethal infection (125), this finding suggests that T cells are not the primary source of IFN-γ, at least during early stages of infection (126).

Although the exact role of cytokine production by T cells remains to be defined, there are other possible mechanisms by which T cells could mediate immunity to Salmonella infection. For example, CD4+ T-cell help is required to establish and maintain a functional CD8+ memory response (Fig. 1J) (163, 176). Salmonella-specific CD8+ T cells are capable of lysing infected host cells both in vitro (91) and in vivo (94). Therefore, T-cell-mediated lysis of infected host cells could disrupt the protected intracellular niche to render intracellular bacteria accessible to phagocytes. In addition, CTL granules contain the protein granulysin that has been shown to have direct antimicrobial activity against Salmonella and other pathogens (173). Finally, CD4+ T-cell help is required for B-cell functions such as isotype switching and affinity maturation (Fig. 1K). For example, in athymic mice (lacking mature T cells), there is a dramatically reduced LPS-specific antibody response (168). CD28-deficient mice (with impaired T-cell activation) exhibit diminished production of IgG1, IgG2a, or IgG2b antibodies, and only low levels of IgM and IgG3 antibodies (117). It may be that a combination of all of these functions is required for optimal immunity to Salmonella infection (Fig. 1J, K, and L), and further work remains to define the exact importance of each contribution.

Antigenic Specificities of Salmonella-Specific T Cells

Despite the well-established role for T cells in Salmonella immunity, surprisingly little is known about the identity or relative importance of the specific Ags recognized. Perhaps the most thoroughly characterized Ag recognized by Salmonella-specific CD4+ T cells is the flagellar subunit protein FliC (24). This protein is abundant on the cell surface (a single bacterium can have up to 10 flagella, each composed of 30,000 or more flagellin molecules [207]) and is therefore an obvious target for host immune responses. Indeed, multiple epitopes have been identified within the FliC protein (9, 74, 75), and a significant fraction of Salmonella-specific T cells generated by sublethal infection recognize FliC (1, 107). Immunization with purified FliC protein is protective (107, 174). Significantly, the monomeric form of this protein is a ligand for TLR5 (47, 58) and presumably the Nod-like receptor Ipaf (112), and FliC is therefore a target of both innate and adaptive immune responses.

FliC regulation is complex (27, 85) and acts to limit adaptive immune responses: FliC is repressed below the T-cell activation threshold during systemic growth (26). The global regulator PhoP mediates this repression, and it may therefore seem surprising that PhoP^c bacteria (in which FliC expression is constitutively suppressed) are strongly immunogenic and prime protective immunity. However, given the large number of potential proteins encoded in the Salmonella genome, and a host T-cell compartment with the capacity of generating at least 10¹⁸ different receptors (25), it is likely that T cells responding to other specificities are primed during infection and capable of mediating protection. Indeed, analysis of proliferative responses to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-fractionated whole bacteria reveals that CD4+ T cells recovered from Salmonella-immune mice respond to multiple non-FliC specificities (26, 196). Despite this evidence, only a few additional proteins have been definitively established as immune T-cell targets: the Salmonella invasion protein SipC is recognized by CD4+ T cells (124), and peptides derived from GroEL are recognized by CD8+ cytotoxic T cells (92) induced after natural infection.

Although the exact identity of additional Ags remains to be determined, it appears that, like FliC, other surface-exposed proteins are good candidates for Salmonella-specific T-cell recognition. For example, bacterial extracts enriched for surface Ags can prime protective immunity, presumably mediated by IFN-γ production from immune splenocytes and the generation of IgG2a antibodies (133). T-cell lines derived from protectively immunized mice respond to fimbrial proteins (134, 135). Salmonella infection also primes T cells responding to outer membrane proteins such as OmpC, porins, and iron-regulated outer membrane proteins (IROMPS); immunization with these proteins is protective and associated with increased numbers of CD4+ and CD8+ T cells (33, 43, 54, 172). Finally, it has recently been shown that membrane vesicles (MVs) derived from the bacterial surface contain Ags recognized by Salmonella-immune T cells, and immunization with purified MVs primes a protective CD4+ T-cell response (2, 10).

T-Cell Contributions to Salmonella Immunity: Future Directions

Our understanding of T-cell responses to Salmonella infection has dramatically improved in recent years. Nonetheless, many questions remain to be answered. What is the exact mechanism by which immune T cells mediate protection? What is the complete antigenic profile of T cells generated by protective immunization? Do T and B cells recognize the same Ags? When and where are protective T-cell responses generated? There is a complex relationship between Salmonella and its hosts: immune recognition of microbial Ags selects for pathogens capable of exploiting, modulating, or evading that recognition in order to colonize and infect their hosts. Therefore, identification of additional Ags recognized by CD4+ T cells responding to infection will continue to provide insights into host functions required to resist Salmonella infection.

B CELLS

The Role of B Cells in Salmonellosis

Natural oral Salmonella infection progresses from penetration of the gastrointestinal epithelium to systemic dissemination, generating B-cell and T-cell immune responses that collectively promote protective immunity against virulent infection in susceptible hosts. While the cell-mediated and humoral arms of the adaptive immune response possess unique individual functions, interaction between B and T cells is also critical. Antibody isotype switching depends on T-cell help, and B cells contribute to the priming of T cells and generation of T-cell memory. Thus, B-cell functions, including but not limited to antibody production, are critical in the generation of protective immunity against Salmonella infection.

Studies in B-Cell-Deficient Mice

The importance of B cells in immunity to serovar Typhimurium has been explored by using mice devoid of B cells (Igh-6^−/−, Igμ^−/−) (82). Igμ^−/− mice possess a genetic defect that disrupts the gene encoding the constant region of the IgM heavy chain (μ-chain), rendering them B-cell-deficient due to arrest in the stage of pre-B-cell maturation (82). After intravenous (i.v.) or intraperitoneal (i.p.) infection, Igμ^−/− mice control and clear an attenuated serovar Typhimurium aroA strain from the liver and spleen (28, 100, 108, 118). However, unlike wild-type mice, Igμ^−/− mice experience sepsis during primary infection and are not protected against subsequent oral challenge with virulent serovar Typhimurium (100, 118). Transfer of immune serum from wild-type mice to Igμ^−/− mice confers immunity to virulent challenge, underscoring the importance of B cells in the generation of a protective immune response (108). Although B cells do not appear to contribute significantly to bacterial control during primary infection with aroA-attenuated Salmonella, Igμ^−/− mice are highly susceptible to primary infection with virulent serovar Typhimurium compared with wild-type mice (118). These data demonstrate that B cells, or the products of B cells, are necessary for both the initial control of primary virulent serovar Typhimurium, and the development of long-lived resistance against oral challenge with virulent Salmonella.

The Role of Secretory Antibodies in Protection against Serovar Typhimurium (See Also Chapter Mucosal Immune Responses to Escherichia coli and Salmonella Infections)

Secretory IgA (sIgA) is present in mucosal tissues and has been proposed to influence the pathogenesis of mucosal pathogens (22). Salmonella initiate infection via penetration of the mucosal epithelium, where colonization of Peyer's patches (PPs) is required for generation of sIgA (192). The role of Salmonella-specific sIgA, however, appears to be minimal in protective immunity. Salmonella-specific monoclonal sIgA reduces adherence of serovar Typhimurium to cells in vitro and confers protection to naïve mice against oral challenge when secreted by an implanted hybridoma tumor (113, 114). However, although the presence of a sIgA response does increase the dose necessary for initial PP colonization, mice lacking the ability to generate sIgA are able to generate a protective immune response equal to wild-type mice, suggesting that serovar Typhimurium-specific sIgA are not required for protective immunity (186). To reconcile these seemingly conflicting data, it is important to consider that the concentration of sIgA produced by the hybridoma in vivo is likely very high, and may significantly exceed the levels of Salmonella-specific sIgA generated during immunization with viable attenuated bacteria.

Innate sIgA antibodies are produced in response to commensal microbiota, and their generation does not depend on serovar Typhimurium infection. These antibodies are reactive against a wide range of nonspecific Ags, some of which likely cross-react with serovar Typhimurium Ags (140). Both conventional B2 B cells and CD5+ B1 B cells secrete innate sIgA antibodies (78). While B2 B cells require direct interaction with CD4+ T cells for stimulation of antibody secretion, B1 B cells do not. Secretion of innate antibodies by B1 B cells is thought to depend on the stimulating effects of bystander cytokines such as IL-5, IL-6, and IL-10 (199). Unlike Salmonella-specific sIgA (186), innate sIgA is protective against natural Salmonella infection (203). In mice that lack the ability to secrete IgA into their mucosa because of a defect in the polymeric immunoglobulin receptor (pIgR^−/− mice), sIgA destined for secretion is instead retained in the serum. pIgR^−/− mice are more susceptible to serovar Typhimurium infection via the natural oral route, resulting in a higher bacterial load and increased fecal shedding of Salmonella compared with wild-type mice. Fecal shedding of serovar Typhimurium by pIgR^−/− mice also increases the incidence of transmission to normally resistant wild-type mice, suggesting that innate sIgA may play a role in “coating” bacteria present in feces, resulting in decreased transmissibility of the organism (203).

The Influence of Salmonella-Specific Antibodies on Protection

Susceptibility of mice to Salmonella infection depends on several known genetic loci (66, 129, 194), and likely on other strain-specific genetic signatures yet to be determined. These unique host factors are as influential as pathogen-specific virulence factors in determining the outcome of host-pathogen interactions. The role that antibodies play in Salmonella immunity has been investigated in many different mouse strains, leading to a diverse data set. Eisenstein et al. (35) nicely demonstrated that, even though naturally resistant and naturally susceptible mice are equally able to generate antibodies, the resulting protective immunity against Salmonella was not equal. This study provides a helpful framework for interpretation of data exploring the role of Salmonella-specific antibodies in protective immunity. To examine the effects of host background on the generation of a protective response against serovar Typhimurium, four Salmonella-derived immunogens were administered i.p. to the inbred mouse strains C3H/HeJ, C3H/FeJ, and C3H/HeNCrlBR. Although each of these strains is derived from the C3H lineage, they vary in their natural susceptibility to Salmonella infection: C3H/FeJ mice are hypersusceptible, C3H/HeJ are susceptible, and C3H/HeNCrlBR mice are resistant. Whereas none of the vaccines protected C3H/HeJ mice, and only administration of acetone-killed Salmonella generated significant protection in C3H/FeJ mice, all four immunogens conferred significant protection on resistant C3H/HeNCrlBR mice. Transfer of immune sera from immunized C3H/HeNCrlBR donor mice to naïve C3H/HeNCrlBR recipients conferred complete protection against intraperitoneal challenge. However, the same serum transfer to naturally susceptible mouse strains C3H/HeJ and C3H/FeJ was unable to provide full protection against i.p. challenge. Thus, the ability of antibodies to provide protective immunity is strong when the host is naturally resistant to Salmonella infection, but naturally susceptible hosts appear to require additional immune factors to gain full protective immunity (see “T-cell subsets and Salmonella immunity,” above). Therefore, the role of antibodies in resistance to serovar Typhimurium infection depends on host susceptibility status.

In susceptible mouse strains, B cells appear to be most important for protection during virulent serovar Typhimurium challenge. In mice with a functional T-cell response, B cells are dispensable during primary i.v. infection with aroA-attenuated Salmonella but necessary for protection against subsequent i.p. or i.v. virulent challenge (108). Igμ^−/− and wild-type mice are equally capable of clearing primary infection with aroA-attenuated serovar Typhimurium, and mice of either background eliminate secondary i.v. challenge with attenuated bacteria. Wild-type mice with functional B and T cells clear secondary infection with attenuated organisms more rapidly than Igμ^−/− mice, indicating that optimal bacterial clearance requires both memory T cells and B-cell functions (108). However, Igμ^−/− mice are less able to control virulent Salmonella i.p. challenge, and Igμ^−/− mice immunized with attenuated bacteria become as resistant to virulent challenge as wild-type animals when wild-type immune serum is administered prior to challenge (108). Thus, in susceptible mouse strains with functional T-cell responses, B cells are dispensable during primary and secondary infection with aroA-attenuated Salmonella strains, but resistance to virulent serovar Typhimurium infection via the i.p. and i.v. routes depends on the presence of B cells and/or immune serum constituents. Consistent with these findings, Mastroeni et al. (100) demonstrated that protection of naturally susceptible mice against virulent oral Salmonella infection requires both antibodies and T-cell responses. Together with the data showing the necessity of antibody in protection against virulent organisms administered i.p. and i.v., these observations confirm the importance of antibodies in immunity against virulent serovar Typhimurium in naïve susceptible hosts. The mechanism by which antibodies provide protection remains unknown.

Naturally resistant mouse strains show a more variable but still important dependence upon antibody response for Salmonella-specific immunity. Administration of immune sera protects naïve Salmonella-resistant C3H/HeNCrlBR mice but not Salmonella-susceptible C3H/HeJ and C3H/FeJ mice from infection (35). Immune B cells and antibodies confer protection on otherwise susceptible male xid mice (harboring functionally defective B cells but functional T cells) when transferred from immunized Salmonella-resistant female littermates (130). Therefore, antibody production as well as other non-antibody-dependent B-cell functions (see below) aids in protection against serovar Typhimurium, even in naturally resistant hosts.

Salmonella-Specific Antibody Responses

It has long been recognized that antibody responses are generated against the abundant Salmonella surface Ag LPS. However, it has become clear that antibody responses to protein antigens also contribute to protection, although these responses have not been fully characterized. The complete repertoire of antibody specificities has not been defined in the murine response or in humans infected with serovar Typhi, although similarities likely exist (14). In addition, the contributions of antibodies with particular specificities in the generation of protective immunity have not been established. We focus below on antibody responses generated by infection with live Salmonella.

LPS, exposed on the surface of gram-negative bacteria, elicits a strong antibody response during serovar Typhimurium infection (14, 104, 206). The majority of the B-cell response to LPS targets the serotype-specific O-antigen polysaccharide chains on the outermost part of the molecule (166). The O-antigen likely obscures the more internal lipid A and core structures from antibody access. Conserved O-antigen residues, found across Salmonella serotypes, are less immunogenic than serotype-specific residues, which may account for the inability of anti-LPS antibodies to protect against challenge with different Salmonella serotypes (68).

By using two serotypes of Salmonella with unique O-antigens, Hormaeche et al. (68) elegantly demonstrated that protection against oral Salmonella challenge depends on antibody responses directed toward protein Ags in addition to those against LPS moieties. Mice that were i.v. immunized with either viable serovar Typhimurium (O-antigen type O4) or Salmonella enterica serovar Enteritidis (O-antigen type O9) and orally challenged with the immunizing strain were protected. Conversely, mice challenged with the Salmonella serotype not used for immunization (termed the “heterologous” strain) were not protected. The inability of heterologous strains, distinguished by O-antigen type, to confer immunity against one another indicates that LPS-specific immune responses are responsible for protection. These results suggested that immunization with serovar Enteritidis O4 would be fully protective against challenge with serovar Typhimurium O4, and vice versa. However, only partial protection is observed when mice are challenged with heterologous bacteria expressing the O-antigen type of the immunizing strain. These data are consistent with the presence of antibodies targeting non-LPS (protein) Ags, as well as the idea that non-B-cell responses, such as those from CD4+ T cells, also contribute to protective immunity. Importantly, generation of antibodies against protein Ags requires interaction with CD4+ T cells (see “Mechanisms of T-cell-mediated immunity to Salmonella infection,” above), and dependence on protein- and non-protein-specific antibodies for complete protection against Salmonella has been observed by several groups (90, 165, 178). Taken together, these data indicate that the Salmonella-specific antibody response generated during protective immunity depends on multiple Salmonella Ags, including both LPS and protein(s). The relative contribution of these specificities to protection remains to be determined.

Defining the specificity of antibody responses resulting from human serovar Typhi infection is likely to be complex, particularly in regions of the world afflicted with endemic typhoid fever. Anti-LPS antibody titers are already elevated in “naïve” subjects prior to immunization or infection, potentially obscuring the significance of anti-LPS antibody titers (193), and antibodies directed against LPS in humans appear to be minor compared with antibodies recognizing protein Ags. An inhibition enzyme-linked immunosorbent assay (ELISA) method, in which immune serum is preincubated with either outer membrane proteins or LPS, was used to assess the ability of these Ags to inhibit antibody binding to solid-phase bound Salmonella Ags. Greater than 60% inhibition of antibody binding to Salmonella Ags occurred after incubation with low concentrations of soluble outer membrane proteins, whereas preincubation with the highest concentrations of LPS inhibited binding by less than 40% (193). These data suggest that human anti-Salmonella antibodies may be directed primarily toward protein Ags.

As in human infections, B cells also generate antibody responses against a group of largely unknown protein Ags during natural oral infection of mice. Proposed identities of several B-cell antigens, based only on electrophoretic mobility in one-dimensional SDS-PAGE followed by Western blot analysis with immune sera, include OmpA, outer membrane porins, flagellar subunit protein FliC, outer membrane lipoprotein Lpp, and a cellular heat shock protein (14).

Antibody responses to the outer membrane protein OmpA resulting from infection have been experimentally confirmed, and the predominant response is directed against the C-terminal third of the protein (144). Surprisingly, this epitope is proposed to lie in the periplasm, indicating that the B-cell response is directed primarily against a region of the protein that is not surface exposed (144). However, OmpA has two structurally distinct membrane conformations (175). The major OmpA species is characterized by an N-terminal β-barrel in the outer membrane and an α-helix rich C terminus in the periplasm, whereas formation of the minor conformation likely results in surface exposure of parts of the C terminus (175). The C-terminal epitope is more immunodominant than the N terminus during infection (167), suggesting that surface exposure of the C terminus provides the opportunity for B-cell recognition and antibody production. The generation of anti-OmpA antibodies also occurs in humans following serovar Typhi infection, although the dominant epitope(s) have not been determined (14, 193).

Infection has also been confirmed to prime antibody responses to outer membrane porins OmpC and OmpF (165), and the dominant OmpC epitope is located on a surface-exposed loop (166). During natural serovar Typhi infection of humans, antiporin antibody titers increase 1,000-fold over control sera from uninfected subjects (18), a phenomenon that has been confirmed by additional studies by using both ELISA and immunoblot methods (136, 193). Any contribution of antiporin antibodies to protective immunity, however, remains unknown.

In addition to membrane proteins, abundant surface appendages such as flagella and fimbriae are also targets of the B-cell response. As an abundant extracellular Ag, Salmonella FliC elicits a strong antibody response in mice and humans, in addition to its role as an important T-cell Ag (see “Antigenic specificities of Salmonella-specific T cells,” above) (31, 161). The murine antibody response to FliC is generated following both oral and i.p. infection (31, 161). Flagellin also induces antibody production in humans infected with serovar Typhi (14, 18, 180). The antibody response to fimbrial Ags depends on which fimbrial subunit proteins are expressed during infection (70). Although serovar Typhimurium grown in laboratory culture prior to infection expresses only the FimA subunit, expression of ten different subunits occurs during murine infection (70). Expression of various combinations of these ten subunits occurs in vivo, with a corresponding antibody response to the expressed subunit(s), but expression of all ten in an individual mouse is not observed. These findings reinforce the concept that bacterial regulation of Ag expression influences adaptive immune responses (26), and that the most relevant bacterial Ags to investigate will not necessarily be obvious from the examination of bacteria grown in the laboratory setting.

Human B cells also generate an antibody response against the Vi capsular Ag, which is expressed by serovar Typhi and Salmonella enterica serovar Paratyphi C but not serovar Typhimurium or serovar Paratyphi A and B. Interestingly, whereas only 20% of patients with acute typhoid fever have a detectable anti-Vi antibody titer, approximately 90% of chronic gall bladder carriers demonstrate an antibody response to the Vi Ag (reviewed in reference 88). The dependence of temporal expression of Vi Ag in vivo on environmental signals suggests that, like FliC, altered Vi Ag expression may affect adaptive immune responses (see “Antigenic specificities of Salmonella-specific T cells,” above). Immunization with the Vi capsular Ag or live attenuated serovar Typhi Ty21a provides protection against typhoid fever caused by serovar Typhi (reviewed in reference 12). However, these licensed typhoid fever vaccines do not provide protection against serovar Paratyphi A infections (4, 132), and the coincident increase in incidence of Vi-negative serovar Paratyphi A outbreaks in endemic typhoid fever regions is consistent with the immunodominance of the Vi Ag (132, 171, 195).

Identification of Salmonella B-cell Ags has proven difficult. Ags that have been most frequently characterized are those expressed by bacteria grown in laboratory culture conditions, which may not accurately represent Ag expression and/or abundance in vivo. Selection of bacterial targets that approximate what may be seen by the host during infection is therefore a critical factor in Ag discovery. Investigating outer membrane proteins and surface appendages such as flagella and fimbriae has yielded information about B-cell Ags exposed on the bacterial cell surface. It is likely that important B-cell Ags will be revealed with further investigation of other surface organelles such as MVs (10), which are rich in outer membrane and periplasmic proteins and elicit protective responses when administered as an immunogen to both mice and humans (2, 162).

B-Cell Functions in Addition to Antibody Production

B cells contribute to the generation of protective immunity against serovar Typhimurium independent of antibody production. For example, B cells influence the immune response via secretion of cytokines (8, 56, 57, 169). Unlike splenocytes from immunized wild-type mice, splenocytes harvested from immune B-cell-deficient mice are unable to generate Th1-type cytokines (IFN-γ, IL-2) in response to soluble serovar Typhimurium Ags in vitro, despite the fact that T-cell populations in both murine backgrounds are capable of cytokine production (100). In the absence of B cells, the balance of the adaptive immune response shifts away from the characteristic Th1-type cytokine IFN-γ and toward the production of the Th2-type cytokine IL-4 (185). B-cell secretion of IFN-γ depends on combined stimulation of TLR2, 4, and 9 (8). These observations suggest that B cells support the generation of a Th1-type cytokine response, either directly through cytokine production and as APCs (see below) or indirectly through undefined mechanisms, thereby facilitating the downstream amplification of the immune responses necessary for protection against virulent serovar Typhimurium.

B cells also contribute to Ag presentation in vivo (21, 29, 96, 152). Early in infection, DCs prime CD4+ T cells through presentation of immunodominant peptides in the context of MHC-II molecules (see “DCs and macrophages as professional APCs during Salmonella infection,” above). However, once CD4+ T cells are primed, they are able to activate Ag-specific B cells, which internalize Ag 10,000-fold more efficiently than nonspecific cells such as DCs do (86). Activated B cells may, in turn, prime naïve T cells through presentation of a much wider range of peptides from the original protein Ag (96). Specifically, the ability of B cells to ingest, process, and present Salmonella Ags depends on the immune status of the host. For example, B cells harvested from naïve mice or mice immunized 2 weeks prior to harvest are refractory to infection in vitro, but B cells isolated from mice immunized 3 months prior to harvest can be infected by live serovar Typhimurium (185). B-cell populations exhibit upregulation of the costimulatory molecule CD86 and are able to present soluble serovar Typhimurium Ags, whether they are obtained from naïve, 2-week immune, or 3-month immune mice (209). Such Ags are available for capture independent of the bacteria from which they are derived, and may include surface organelles such as MVs released by Salmonella in vivo (45, 153). However, only B cells from 3-month immune mice are able to present Ags derived from intact heat-killed bacteria (185). Additionally, splenocytes harvested from wild-type mice are better able to present Salmonella Ags to Salmonella-specific CD4+ T-cell lines than splenocytes of Igμ^−/− mice or B-cell-depleted wild-type splenocytes, demonstrating that B cells are an important APC population in the spleen (185). These data suggest that B cells have unique Ag presentation capabilities at different times postinfection, supporting a model in which presentation of Ags to CD4+ T cells is shared between DCs and B cells.

B-Cell Contributions to Salmonella Immunity: Future Directions

It is clear that the generation of protective immunity depends on multiple B-cell functions, including antibody production, cytokine secretion, and/or Ag presentation (Fig. 1K). B-cell involvement in protection against primary serovar Typhimurium infection and the generation of protective immunity to secondary challenge may be manifest at many stages of pathogenesis: during initial migration to the intestinal epithelia, transition from the intestinal lumen to the PP, uptake by other immune cells such as macrophages, and/or progression to systemic organs. The exact stage(s) during which B cells and/or their products act, and how interactions occur between B cells and Salmonella, are largely unknown. In addition, the identity of Salmonella Ags that drive these responses will be a key area of interest in future studies, with the goal of determining the specificity of antibodies that induce protective immunity.

TEMPORAL AND ANATOMICAL ASPECTS OF SALMONELLA-SPECIFIC ADAPTIVE IMMUNITY: FliC AS A MODEL Ag

During natural infection, Salmonella must survive the intestinal environment (Fig. 2A), penetrate the mucosa of the small intestine (Fig. 2B), colonize the PP (Fig. 2C), and disseminate to systemic sites such as the liver and spleen to replicate within host cells (Fig. 2E). The gut mucosa presents a major initial barrier to Salmonella dissemination after oral infection. In addition to overcoming physical barriers such as the glycocalyx and mucus layers, Salmonella must withstand chemical attacks from antimicrobial peptides secreted by the intestinal mucosa (5, 44). Most Salmonella are contained by innate host defenses in the small intestine or are eliminated and do not progress farther than the intestinal lumen. Although high doses of Salmonella (many times the oral LD₅₀) have often been employed in experimental models of oral infection, only a few bacteria successfully enter the systemic circulation when doses near the oral LD₅₀ are used (110, 111).

Flagella (and the flagellar subunit FliC) are abundantly expressed on the surface of extracellular-phase Salmonella (Fig. 2A) and therefore serve as a large reservoir of ligands for both innate (47, 58) and adaptive (10, 24, 107) immune systems. After bacterial invasion or translocation of flagellin across the intestinal epithelial cell layer, TLR5 stimulation results in a strong proinflammatory response that is not induced by normal microbiota (Fig. 2B) (47, 48, 181, 211). Flagellin-stimulated secretion of chemokines such as IL-8 (47, 48, 69) and CCL20 (164) results in the recruitment of phagocytes, including DCs, to the small intestine (reviewed in reference 147). DCs are highly competent APCs that can directly sample the gut lumen for Ags to present to immune effector cells (150). In addition, flagellin stimulates the maturation of both DCs and macrophages (105, 109). Thus, the proinflammatory response to flagellin, in addition to the proinflammatory response evoked by Salmonella-induced cell death (41), results in intestinal localization and activation of cells capable of priming adaptive immune responses. APCs in the PP (Fig. 2C) therefore initially prime CD4+ T cells that respond to Ags expressed by bacteria invading the host intestinal epithelium or residing in the gut lumen.

Small numbers of bacteria escape the gut and disseminate by intracellular transport (192) to systemic sites such as the liver and spleen (110, 111). There, Salmonella replicate within macrophage phagosomes (151, 157), an intracellular niche providing a safe haven from host antibodies. Salmonella have evolved complex regulatory systems to precisely tailor gene expression to environmental context. One global regulator of Salmonella gene expression is PhoP, which is activated by intracellular growth conditions and mediates virulence gene expression, bacterial surface modifications, and repression of FliC, a dominant Ag for Salmonella-specific CD4+ T cells (3, 26, 36, 52, 123).

Regulation of FliC expression is complex. During later stages of Salmonella infection, when bacteria are replicating intracellularly at systemic sites, FliC expression is repressed below the T-cell activation threshold in a PhoP-dependent manner (26). In addition to reduced FliC expression, PhoP-mediated modifications of the bacterial surface restrict the access of this Ag to APCs for processing and presentation to T cells (1). The natural consequence of this repression is that CD4+ T cells primed to respond to Ags (such as FliC) that are expressed early in infection will fail to recognize intracellular bacteria growing at systemic sites during later stages of infection (Fig. 2E). Therefore, it appears that the coordinated control of Ag expression and accessibility to APCs (bioavailability) directly imposes temporal and anatomical limits on the corresponding T-cell response. This idea is supported by the findings that FliC-specific CD4+ T-cell activation is restricted to the gut, despite systemic bacterial colonization (106), and that bacteria present in the PP but not MLN or spleen express FliC protein. Importantly, Salmonella in the PP do not homogenously express FliC; up to 40% of the bacteria are FliC negative (27). Heterogeneous gene expression provides a mechanism whereby one bacterial population provides abundant Ags for T-cell priming, while a subpopulation already in “stealth mode” is poised to evade the developing T-cell responses (Fig. 2C left).

Repression of protective Ags like FliC, in concert with other genetically co-coordinated modifications of the bacterial surface as described above, could facilitate bacterial growth in vivo by affording Salmonella the opportunity for additional rounds of replication before the onset of effective adaptive immune recognition. This is a reasonable explanation for the different outcomes resulting from infection of naïve, susceptible mice with virulent versus attenuated bacteria. Attenuated aroA-mutant Salmonella grows more slowly in vitro and in vivo (64, 131) and is cleared by immunocompetent hosts, while virulent Salmonella rapidly overwhelms host defenses. The growth advantage of virulent bacteria is eliminated by prior immunization, as rapid activation of localized host T- (and B-) cell functions provides protection (Fig. 1 right) (99, 115). In this context, it is important to note that T cells from immune mice can detect intracellular (and therefore FliC) bacteria (26). However, it is not known when or where T cells responding to Ags expressed by intracellular phase (FliC) bacteria are primed, or whether these T cells are capable of mediating protection. Interestingly, it has been shown that systemic (spleen and liver) macrophages, although capable of processing and presenting Ags to T cells, do not affect the development of an adaptive T-cell response (204). This suggests that APCs at systemic sites (Fig. 2E) may not be capable of priming a T-cell response of sufficient speed or magnitude to protect a host from subsequent challenge with virulent Salmonella. From a host perspective, a T-cell response localized to the gut and directed toward Ags expressed by extracellular bacteria at early stages of infection (when the risk of systemic dissemination is greatest) appears to be an effective strategy. This idea is supported by the high rate of nontyphoidal Salmonella bacteremia in AIDS patients compared with immunocompetent hosts (63, 71, 77). Studies using a model peptide Ag indicate that systemic Salmonella-specific CD8+ memory responses appear to be restricted to Ags encountered in the gut mucosa (33). CD4+ and CD8+ T-cell levels in the gut increase threefold during secondary challenge of protectively immunized mice (7). This is likely because DCs in the PP (the most likely site of T-cell priming during primary infection) are specifically able to imprint gut-homing specificity on T cells (Fig. 2C) (121). Indeed, after oral immunization in humans with serovar Typhi, most of the effector (IFN-γ-producing) CD4+ and CD8+ T cells express the α4β7 gut-homing receptor (93). Finally, memory T cells express CCR6, the only known receptor for CCL20, a chemokine secreted by intestinal epithelial cells after stimulation by flagellin (89). Therefore, in an immunized host, it appears that memory T cells primed to respond to early-phase Ags expressed by extracellular bacteria are recruited to the intestine where their resulting activation could facilitate the containment of bacterial replication and dissemination. In addition, it seems likely that opsonic Ig would beneficially augment the host response by facilitating Ag uptake, processing, and presentation (Fig. 1P).

CONCLUDING REMARKS

As a facultatively intracellular pathogen, Salmonella is an effective model organism for the study of adaptive immune responses. Antibodies, the products of B cells, opsonize extracellular bacteria for phagocytic uptake (Fig. 1P), and Ig on the B-cell surface mediates high-efficiency Ag capture for Ag presentation (Fig. 1K). CD4+ and CD8+ T cells generated in response to Salmonella infection detect intracellular bacteria and are required for bacterial clearance. Accordingly, full immunity to Salmonella infection in naïve, susceptible hosts requires both T- and B-cell functions.

The requirement of both sera and T cells for immunity suggests that, in addition to their individual functions, the interaction between T and B cells is important. Although there has traditionally been a differentiation between “cellular’” and “humoral” immunity, in reality these responses are tightly interconnected. T cells are required to activate B-cell isotype class switching and affinity maturation. B cells may present Ags to T cells and produce cytokines that support the development of a Th1-type T-cell response (Fig. 1K). CD4+ T cells are required to initiate activation of cytotoxic CD8+ T cells (Fig. 1J). In addition, cytokines released by T cells may activate APCs to more efficiently process and present Ags, while cytokines released by activated APCs facilitate T-cell activation and proliferation (Fig. 1E and L).

The interaction between Salmonella and its host is complex and dynamic. The host mounts an immune defense against the pathogen, which in turn acts to reduce, evade, or exploit these responses to successfully colonize the host. Correspondingly, a greater understanding of adaptive immune responses provides tools and reagents for gaining additional insights into mechanisms of bacterial pathogenesis. Salmonella bacteria are not just passive collections of Ags, but rather actively participate in their encounters with host cells. Multiple bacteria-directed mechanisms, including altered Ag expression and bioavailability, interference with APC activation and function, and direct inhibition of T-cell activation (188), combine to modify Salmonella's “pathogenic signature” to minimize susceptibility to immune surveillance (Fig. 1B, F, and H).

Although our understanding of Salmonella-specific immunity has dramatically improved in recent years, many unanswered questions remain. For example, the details of intestinal DC function, T-cell priming, T-cell migration, and the generation of T-cell memory in human serovar Typhi infection remain incompletely understood. The exact mechanisms by which T and B cells act to clear Salmonella are unknown. Surprisingly little is known about the identity, regulation, and relative contributions to immunity of the individual bacterial Ags recognized by Salmonella-specific B and T cells (the antigenisome). As discussed above, emerging data suggest that bacterial regulation of Ag expression and localization is critically important: temporal and anatomical restrictions imposed by bacterial regulation of Ag expression affect the development of FliC-specific T-cell responses (1, 26). MVs have recently been identified as bacterial organelles capable of priming protective immune responses in the absence of infection (2). As nonreplicating structures derived from the surface of genetically malleable organisms such as Salmonella, MVs could provide a powerful new tool for both the analysis and delivery of immune targets. Collectively, these data suggest that continued identification of adaptive immune targets will guide rational vaccine development, provide insights into the host functions required to resist Salmonella infection, and correspondingly provide valuable reagents for defining the critical pathogenic capabilities of Salmonella that contribute to its ability to cause acute and chronic infections.