shown to out-compete the resident microbiota in a mouse colitis model (20). One explanation for this phenomenon is that inflammation provides *S. typhimurium* with a respiratory electron acceptor that members of the resident microbiota are unable to utilize. In particular, reactive oxygen species generated by neutrophils (PMNs) during inflammation can react with endogenous thiosulfate to form tetrathionate, a respiratory electron acceptor (21). The ability to respire tetrathionate has been mapped to the *ttrRSBCA* locus, which is located in SPI-2 (22). Under anaerobic conditions in which thiosulphate was oxidized to tetrathionate, *S. typhimurium* displays a growth advantage in comparison to resident microbiota under the same conditions (21).

Both resident microbiota and *S. typhimurium* compete for resources available for fermentation at the mucosal layer; however, resident microbiota are incapable of using the fermentation end products (21). By reducing the tetrathionate made available by the inflammatory response to infection, *S. typhimurium* is capable of respiring the fermentation end products in anaerobic conditions, thereby providing it with an advantage over the resident microbiota (21). Remarkably, the growth benefit is conferred to *S. typhimurium* only in the presence of inflammation, and it has been suggested that a reason *S. typhimurium* has evolutionarily maintained its inflammation-inducing virulence mechanisms could be to provide it with an ecological advantage at the mucosal surface of the intestine (21).

#### **PANETH CELLS**

Paneth cells are specialized epithelial cells located at the base of crypts of Lieberkuhn that generate and secrete antimicrobial peptides of ~20–40 amino acids in length (**Figure 1**). There are four families of antimicrobial peptides: defensins, cathelicidins, histatins, and lactoferrin (**Figure 1**, **Table 1**). Defensins are positively charged and directly interact with the negatively charged membrane of pathogenic microorganisms resulting in membrane destabilization and pore formation. Cathelicidins are also positively charged, and they function in binding and neutralizing lipopolysaccharides (LPS), ultimately resulting in pore formation. Unlike defensins and cathelicidins, histatins do not interact with the membranes of pathogenic bacteria. Instead, histatins are ingested by the bacteria, inhibit mitochondrial respiration, and kill the microorganism by generating reactive oxygen species [for review, see Ref. (23)]. Lactoferrin is a cationic protein that sequesters iron, an essential nutrient for pathogenic

**Table 1 | Summary of antimicrobial peptides**.

microorganisms. Additionally, lactoferrin can bind LPS and destabilize bacterial membranes similar to defensins and cathelicidins (23, 24). The antimicrobial activities of these peptides are nonspecific, as their activity provides a first line of defense against both Gram-positive and Gram-negative bacteria, fungi, and enveloped viruses.

All antimicrobial peptides are produced in an inactive, prepropeptide form and must be processed (i.e., enzymatically) either intracellularly or extracellularly to become active (10). For example, the alpha-defensin HD-5 is stored in Paneth cells in an inactive, pre-propeptide form and is processed by trypsin into its active form (29). Antimicrobial peptide production has been shown to be upregulated in response to bacteria (30). However, pathogenic microorganisms have developed methods to counteract the effectiveness of the antimicrobial peptides. Examples of these methods include covalently modifying the bacterial cell membrane to reduce its net negative charge, using bacterial proteases to catalytically inactivate the antimicrobial peptides, and using ATP-driven pumps to physically remove the antimicrobial peptides from the bacterial cytoplasm [for review, see Ref. (31)]. Certain pathogens have developed resistance to the antimicrobial activities of the peptides secreted by Paneth cells in order to promote their intracellular survival.

Antimicrobial peptides that provide protection against *S. typhimurium* infection have been identified using transgenic mouse models. Alpha-defensin HD-5 transgenic mice were shown to consistently have a significant reduction in the *S. typhimurium* burden in the distal intestine and spleen in comparison to wildtype mice that do not express this antimicrobial peptide, indicating the antimicrobial activity of HD-5 conferred the transgenic mice with an enhanced ability to kill *S. typhimurium* in the intestinal lumen (26). Another alpha-defensin shown to provide increased defense against *S. typhimurium* infection is HD-6, which binds bacterial membrane proteins, thereby inhibiting contact of *S. typhimurium* with epithelial cells (25). Since HD-6 does not kill *S. typhimurium*, HD-6 transgenic mice do not display the decrease in bacterial burden seen with HD-5 transgenic mice; however, HD-6 transgenic mice display a profound increase in survival rate in comparison to wild-type mice that do not express this antimicrobial peptide, indicating the antimicrobial activity of HD-6 must act in concert with another defense mechanisms at the mucosal barrier to eliminate *S. typhimurium* (25).

In addition to defensins, mouse models have also identified the significance of cathelicidins and lactoferrin. The sole murine cathelicidin called cathelin-related antimicrobial peptide (CRAMP) has been shown to impair intracellular replication of *S. typhimurium in vivo* and *in vitro* (27). Additionally, *S. typhimurium* displayed enhanced survival in macrophages derived from CRAMP-deficient mice (27). CRAMP is similar in structure and antimicrobial properties to the only human cathelicidin called LL-37, which has been shown to display a broad spectrum of activity against bacteria including *S. typhimurium* (28). A recent study identified the *in vivo* effect of lactoferrin on *S. typhimurium*. In this study, mice treated with bovine lactoferrin displayed a reduction in severity, mortality, and inflammation during infection, indicating the antimicrobial properties of lactoferrin are significant for defense against *S. typhimurium* (24).

#### **THE EPITHELIAL BARRIER**

In addition to mucosal defenses described above, interactions between cells of the epithelial cells in the monolayer also provide a barrier against bacterial entry. Tight junctions are dynamic structures composed of zonula occludens (ZO) and junctional adhesion molecules that effectively adhere the cells of the epithelial monolayer to each other (**Figure 1**) (8). The integrity of this seal is maintained by the interaction of tight junction components with the actin cytoskeleton. However, the permeability of this seal is regulated by physiological conditions, and it therefore can be manipulated. For example, treating epithelial monolayers with inflammatory cytokines, such as IL-1β, increases the permeability of the tight junctions (32). The increase in tight junction permeability can facilitate the translocation of bacteria from the lumen to the subepithelial region, making them a target for pathogenic manipulation. Pathogenic microorganisms can accomplish the manipulation of tight junctions by usurping signaling pathways, such as the Rho-GTPase pathway, which regulates actin cytoskeleton rearrangement (8).

*Salmonella typhimurium* infection has been shown to regulate certain tight junction proteins, which ultimately promotes translocation of the bacteria through the epithelial cell monolayer (33). Upon infection with *S. typhimurium*, occludin becomes dephosphorylated and subsequently removed from epithelial tight junctions (33). Additionally, ZO-2 is recruited from the cytosol to membrane, indicating *S. typhimurium* alters the intracellular distribution of this tight junction protein (33). Surprisingly, ZO-1, which is normally regulated by pathogens in a similar manner to ZO-2, appears to be degraded during *S. typhimurium* infection (33). Manipulation of tight junction proteins serves to disrupt the epithelial barrier by increasing its permeability, thereby allowing *S. typhimurium* to more effectively invade the basolateral side of the epithelial cell monolayer.

In order to mount a successful infection, *S. typhimurium* must disrupt some aspects of the protective mechanisms employed by the mucosal epithelium. As mentioned previously, the two T3SS and the secreted bacterial effector proteins promote entry, inflammation, and intracellular survival. In addition, in order to subvert the action of antimicrobial peptides, *S. typhimurium* uses the twocomponent system PhoQ/PhoP, which regulates the expression of SPI-2 encoded genes as well. Specifically, PhoP/PhoQ regulators promote remodeling of the bacterial envelope, resulting in increased resistance to antimicrobial peptides that recognize LPS. Furthermore, the PhoP/PhoQ regulators repress transcription of genes for the T3SS, in attempt to avoid detection, and induce protective mechanisms against hydrogen peroxide (10). In addition to rearranging the actin cytoskeleton and targeting specific tight junction proteins, *S. typhimurium* also manipulates tight junctions via the action of SipA, SopE, SopE2, and SopB (8). These effector proteins induce Rho-GTPase activation, and inhibition of this effector-induced Rho-GTPase activation prevents tight junction disruption (8).

The manipulation of tight junctions has also recently been shown to facilitate the transmigration of PMNs across the epithelial cell monolayer (33). The primary mechanism of PMN migration in *S. typhimurium* infection involves the recruitment of neutrophils into the subepithelium and the formation of a chemoattractant gradient that directs the neutrophils into the lumen (**Figure 2**, discussed in detail later). However, recent research implicates the disruption of tight junctions in facilitating PMN migration even in the absence of the chemoattractant gradient (33). Thus, *S. typhimurium* not only modulates the release of neutrophil chemoattractants that induce PMN migration, but also directly influences the tight junctions that maintain the fidelity of the epithelial cell monolayer in order to promote bacterial translocation and PMN transepithelial migration.

#### **LAMINA PROPRIA**

The lamina propria is the connective tissue underlying the epithelial cell monolayer. It contains multiple immune effector cells including B cells, T-cells, dendritic cells, natural killer (NK) cells, macrophages, eosinophils, and mast cells. If enteric pathogens are capable of surmounting the barriers described above and penetrate the intestinal epithelium, a coordinated immune response utilizing these immune effector cells is activated. Sampling of luminal antigens occurs in specialized cells called M cells, which transport

the antigens to a subepithelial region where the antigen comes in contact with dendritic cells. Dendritic cells bound to antigen then migrate to the mesenteric lymph node to present the luminal antigens to naïve T-cells and B cells. These naïve lymphocytes then differentiate into several effector cells including CD8 cytotoxic T-cells, CD4 helper T-cells, regulatory T-cells, and antibody secreting B cells. Although this marks the beginning of a coordinated immune response to pathogenic bacteria, the resting lamina propria does have protective functions that provide an added layer of defense prior to the full activation of the mucosal immune system.

The most abundant B cell found in the lamina propria is the IgA-secreting B cell (**Figure 1**). Secreted IgA is also the primary secreted immunoglobulin found in the thick mucus layer. One of the main roles of secreted IgA is a process called immune exclusion, which includes prevention of pathogens from adhering to the mucosal surface on the luminal side of the intestinal epithelium and removal of antigens from the basolateral side of the intestinal epithelium. On the luminal side, secreted IgA primarily interferes

#### **FIGURE 2 | Mechanism of PMN recruitment and PMN transmigration**.

S. typhimurium utilizes its T3SS to secrete effector proteins into epithelial cells to activate inflammatory signaling pathways. In particular, the activation of Rho-GTPases by SopE, SopE2, and SopB result in the induction of mitogen-activated protein kinase (MAPK) pathways. The stimulated pathways include ERK, JNK, and p38, resulting in the terminal activation of major inflammatory regulator NF-κB. Activation of NF-κB results in the basolateral secretion of IL-8 producing a chemoattractant gradient that recruits neutrophils to the subepithelial region from the underlying microvasculature. Th17 cells are also present in the subepithelial region, and function to recruit and activate neutrophils in the subepithelium. PMN transmigration is facilitated by another chemattractant HXA3. HXA3 is a bioactive eicosanoid that is synthesized from arachidonic acid via the 12/15-lipoxygenase pathway in epithelial cells. It is secreted into the lumen via the action of an ATP-binding cassette transporter called MRP2. S. typhimurium effector protein SipA stimulates the recruitment of PKC-α to the apical membrane, which in addition to the ERM protein ezrin, modulate the localization of MRP2 to the apical membrane, thereby allowing secretion of HXA3 into the lumen and production of the chemoattractant gradient that induces PMN transmigration.

with microbial adhesins, whereas on the basolateral side secreted IgA functions in an export mechanism by binding antigens and shuttling them back across the epithelial monolayer into the lumen (34). Secreted IgA does not activate an inflammatory immune response when it neutralizes pathogenic microorganisms, thereby upholding the integrity while preventing inflammation-induced damage of the mucosal epithelium (35).

Secreted IgA also has a direct effect on the virulence mechanisms of certain pathogens. In *S. typhimurium* infection, it has been shown that a monoclonal, polymeric IgA antibody Sal4 binds the O-antigen (O-Ag) component of LPS on the bacterial membrane, resulting in its destabilization [Ref. (36); for review, see Ref. (37)]. Recent evidence indicates that the bacterial membrane destabilization results in impaired T3SS translocon formation, decrease in effector protein delivery, and decrease in flagellumbased motility (36). *S. typhimurium* responds to the binding of Sal4 to O-Ag by triggering exopolysaccharide (EPS) production and biofilm formation, though this response renders the bacteria non-invasive and avirulent (38). The mechanism of EPS production and biofilm formation has been attributed to the activation of a cyclic dimeric guanosine monophosphate-dependent pathway via an inner membrane diguanylate cyclase YeaJ (38). Furthermore, it has been suggested that the triggering of this pathway by *S. typhimurium* could be a mechanism to restore membrane stability, as EPS production could serve to shed IgA antibody or increase resistance to other luminal insults (38).

The resting lamina propria contains a heterogeneous population of CD8 cytotoxic T-cells, CD4 helper T-cells, and regulator T-cells even in the absence of pathogenic infection. The number of effector T-cells in the resting lamina propria would in any other tissue indicate an inflammatory response; however, the amount of T-cells present in the mucosal tissue of the gut is more indicative of constant immune surveillance and recognition than chronic inflammation. The cytokines produced by these T-cells maintain the mutualistic response to resident microbiota, stimulate production of IgA, induce secretion of antimicrobial peptides, and promote epithelial repair. Additionally, in the absence of proinflammatory cytokines (which are usually produced by innate immune cells in the presence of pathogen), the dendritic cells in the resting lamina propria contribute to maintaining tolerance to non-pathogenic antigens by promoting the production of CD4 regulatory T-cells. Regulatory T-cells produce immunosuppressive cytokines that inhibit T-cell proliferation and dendritic cell differentiation, which prevents unnecessary immune response to innocuous antigens (35).

In addition to a heterogeneous population of T-cells, the macrophages in the resting lamina propria play a key role in host defense against *S. typhimurium* infection, as well. The macrophages of the resting lamina propria can be divided into two different classes: M1 ("classically activated"), and M2 ("alternatively activated") polarized macrophages. M1 polarized macrophages are pro-inflammatory and display high phagocytic and antimicrobial activity, whereas M2 polarized macrophages are anti-inflammatory and display low phagocytic and antimicrobial activity. Hence, with this type of opposing macrophage regulation it is considered that M1 macrophages function in the clearance of infection versus M2 macrophages that assist in wound healing and suppression of T-cell function. Manipulation of macrophage polarization by *S. typhimurium* has become increasingly realized as a defense mechanism against bacterial clearance [for review, see Ref. (39, 40)]. A recent study demonstrated that the SPI-1 T3SS enables *S. typhimurium* to guide macrophage toward the M2 polarization (40). This type of control permits *S. typhimurium* to escape the more hostile environment of M1 polarized macrophages, resulting in a macrophage-specific decrease in pro-inflammatory signaling (40).

The mechanism of immunity to invasive *Salmonella* is still disputed, specifically the relevance of cell-mediated versus humoral immunity. The debate is complicated by attempts to compare different experimental models, which vary in route of *Salmonella* administration and/or susceptibility of mouse strains to *Salmonella*. In terms of cellular immunity, mice deficient for TCR α/β, MHC class II, or interferon-γ (IFN-γ) receptor fail to clear a primary *Salmonella* infection that can be resolved in normal mice (41, 42). Recently, it has also been shown that Thy1+ NK cells are essential for the early production of IFN-γ during control of *Salmonella* infection (43). CD8+ T-cells seem to also play a role in *Salmonella* clearance (44). It has also been documented that *Salmonella* infection promotes the expansion of intestinal intraepithelial lymphocytes (iIELs) and the activation of particularly, CD8+ TCRγδ+ iIELs, which in turn trigger cytolytic activity against *Salmonella-*infected epithelial cells (44).

The role of antibody-producing immune cells or B cells against *Salmonella* is still controversial (45–47). Some reports have shown the importance of antibody production and T-cell activation for protection from virulent *Salmonella* (45, 46). However, another study reported that the protective immunity provided by an attenuated *S. typhimurium* strain required B cells independently of antibody production, proposing that they confer protective immunity by presenting antigen to T-cells and acting as a source of inflammatory cytokines (47). It has also been demonstrated that transfer of immune serum into B cell-deficient mice can partially but not completely provide protective immunity (48).

#### **THE TYPE III SECRETION SYSTEM: CO-OPTING HOST PATHWAYS TO PROMOTE ENTRY AND IMMUNE EVASION**

Upon contact of *S. typhimurium* with the epithelial cell monolayer, the SPI-1 effector proteins SopE, SopE2, and SopB initiate the process of bacterial entry by activating host cell Rho-GTPases resulting in actin rearrangements (49, 50). SipA is another SPI-1 effector protein that antagonizes actin depolymerizing agents and tethers actin monomers together to form membrane ruffles, which promotes bacterial internalization (51). *S. typhimurium* is engulfed by epithelial cells through a macropinocytosis event termed bacterial-mediated endocytosis, and is ultimately contained within in a membrane-bound vesicle called a macropinosome [more commonly termed the *Salmonella* containing vacuole (SCV)]. Although prior studies thought that SopB was the sole mediator of macropinosome formation, a cooperative interaction regulated by the phosphatase activity of SopB has implicated SopD as another mediator of this process (49).

*Salmonella typhimurium* also targets antigen-sampling microfold (M) cells to translocate across the gut epithelium. M cells constitute a small subset of highly specialized follicle-associated epithelium (FAE) enterocytes overlying lymphoid follicles in the gut, and are characterized by an irregular brush border, a reduced glycocalyx and lysosomal apparatus, and are programed to efficiently transcytose a wide variety of macromolecules and microorganisms from the gut lumen to the underlying immune inductive Peyer's patches (PPs) (52). Recent evidence shows the *S. typhimurium* type III effector protein SopB also induces an epithelial–mesenchymal transition of the FAE into M cells. This cellular transdifferentiation is a result of SopB-dependent activation of Wnt/β-catenin signaling leading to induction of both receptor activator of NF-κB ligand (RANKL) and its receptor RANK. The autocrine activation of RelB-expressing FAE enterocytes by RANKL/RANK induces the EMT-regulating transcription factor Slug that marks epithelial transdifferentiation into M cells. Thus, *S. typhimurium* may also transform primed epithelial cells into M cells to promote host colonization and invasion (52, 53).

Following bacterial entry of mucosal epithelia, *S. typhimurium* employs a second set of SPI-1 effector proteins to ensure repair of the actin cytoskeleton. SptP is one such effector that is directly responsible for reversing the affects of SopE and SopE2. SptP promotes restoration of the epithelial cell membrane by functioning as a GTPase-activating protein for the Rho-GTPase proteins Rac-1 and Cdc42 (54). Similarly, as several of the early SPI-1 effectors induce inflammation of the mucosal epithelium, there are effector proteins that have an anti-inflammatory function, providing *S. typhimurium* with a form of regulatory control over the inflammatory state of the mucosal tissue (inflammation induced by *S. typhimurium* will be discussed later).

After successful entry into epithelial cells and restoration of the epithelial cell membrane, *S. typhimurium* relies primarily on the T3SS encoded by SPI-2 to survive and replicate intracellularly by translocating SPI-2 effector proteins across the membrane of the SCV into the epithelial cell cytoplasm. SPI-2 effector proteins that appear to be necessary for survival and virulence of *S. typhimurium* inside the SCV are SifA, SseJ, SseF, SseG, SopD2, and PipB2 (50, 55, 56). SifA has been shown to promote tubulation of the SCV through correlation with another effector protein SseJ (57). SCV tubulation in conjunction with the effects of SseF and SseG localize the SCV to the perinuclear region in close proximity of the Golgi apparatus (58). The localization of the SCV is important for intracellular survival because vesicular trafficking through the Golgi network allows for the acquisition of nutrients, thereby allowing the establishment of a replication niche for *Salmonella* (58, 59).

An additional means by which SPI-2 promotes intracellular survival of *S. typhimurium* is by encoding factors that mediate the evasion of immune responses. SPI-2 promotes protection from reactive oxygen intermediates produced by macrophages, specifically nitric oxide (NO) and NADPH oxidase [for review, see Ref. (60–62)]. *S. typhimurium* has been shown to evade NO-mediated killing in macrophages by inhibiting IFN-γ-induced NO production in a SPI-2-dependent manner (61). SPI-2 is also involved in avoiding NADPH oxidase-dependent killing by interfering with the trafficking of NADPH oxidase (62). Although the specific SPI-2 effector proteins involved in the evasion of both NO-dependent and NADPH oxidase-dependent killing of *S. typhimurium* have yet to be identified, the established role of SPI-2 in evasion of both immune responses suggests a possible role for one or more encoded effector proteins in promoting resistance to reactive oxygen intermediates in macrophages.

#### **SALMONELLA-INDUCED INFLAMMATION**

#### **IMMUNE RECOGNITION**

Pathogen-associated molecular patterns (PAMPs) are recognized by pattern-recognition receptors (PRRs), namely toll-like receptors (TLRs), located on inflammatory cells and epithelial cells. TLRs can recognize a wide range of PAMPs, though some TLRs do show some specificity for particular PAMPs. For example, TLR4 is mostly involved in recognition of LPS and TLR5 is mostly involved in the recognition of bacterial flagellin. TLRs in epithelial cells are localized to the basolateral or apical membrane, as well as in intracellular vesicles. Thus, TLRs can recognize pathogens on either side of the epithelial cell monolayer and endocytosed extracellular pathogens. Additionally, inflammatory cells, such as macrophages, expressing TLRs can also recognize PAMPs. The importance of some TLRs, specifically TLR4 and TLR5, in *S. typhimurium* infection have been established, as mutating them has been shown to increase susceptibility to infection and inflammation (35). Intracellular recognition of bacteria or their products in the cytoplasm is also mediated by nucleotide-binding oligomerization domain proteins NOD1 and NOD2. NOD1 recognizes peptides containing diaminopimelic acid, which is a component of Gram-negative bacterial cell walls, whereas NOD2 recognizes a muramyl dipeptide present in the peptidoclycan layers of both Gram-positive and Gram-negative bacteria. Similar to TLRs, mutations in NOD1 and NOD2 proteins increase susceptibility to disease and infection caused by intracellular bacteria [for review, see Ref. (63, 64)].

#### **RECRUITMENT OF IMMUNE CELLS**

The host immune system also activates inflammatory pathways in response to infection with *S. typhimurium*. The binding of TLRs and NOD1/NOD2 proteins to their respective ligands activates the NF-kB pathway leading to production of pro-inflammatory cytokines and chemokines. Basolateral secretion of the cytokine IL-8 recruits neutrophils and is necessary for PMN migration into the subepithelium. Additional chemokines, such as CCL20, play a role in attracting immature dendritic cells, which upon exposure to antigen, can mature and present antigenic peptides to naïve B and T-cells in the mesenteric lymph nodes (35). *S. typhimurium* can also react with TLRs on macrophages in the subepithelial region after being transcytosed through M cells, thereby activating and inducing them to also produce cytokines and chemokines. Cytokines produced by these activated macrophages include IL-1, IL-6, and IL-23, all of which drive the differentiation of TH17 cells whose primary function in the subepithelium is recruiting and activating neutrophils (**Figure 2**) (35, 65). Other cytokines produced by these activated macrophages include IL-18 and IL-12, both of which drive the IFN-gamma-dependent production of antigen-specific TH1 cells (35).

#### **MECHANISM OF NEUTROPHIL RECRUITMENT**

A hallmark of *S. typhimurium*-induced inflammation is the recruitment of PMNs from the underlying microvasculature to the subepithelial region of the epithelial cell monolayer (**Figure 2**). The neutrophils then migrate across the monolayer into the lumen, resulting in the inflammatory pathology of Salmonellosis. New information is shedding light on the molecular mechanisms and signaling pathways involved in neutrophil recruitment across the intestinal epithelium. As discussed above, it is becoming increasingly appreciated how inflammation induced by *S. typhimurium* increases its pathogenic bacterial fitness.

In addition to promoting bacterial entry, many effector proteins encoded by SPI-1 also activate inflammatory signaling pathways. The activation of Rho-GTPases by SopE, SopE2, and SopB result in the induction of mitogen-activated protein kinase (MAPK) pathways (**Figure 2**). In particular, the ERK, JNK, and p38 pathways are stimulated, resulting in the terminal activation of inflammatory regulators AP-1 and NF-κB (**Figure 2**) [Ref. (66); for review, see Ref. (50)]. Furthermore, the activation of NF-κB and AP-1 stimulates the secretion of the cytokine IL-8 on the serosal side of the epithelial cell monolayer, a requirement for the recruitment of neutrophils to the subepithelial region (**Figure 2**) (67). Although IL-8 is necessary for PMN migration into the lumen, it has been shown that IL-8 alone is not sufficient enough to drive the migration across the epithelial cell monolayer [Ref. (67, 68); for review, see Ref. (69)].

The migration of neutrophils from the basolateral side to the luminal side of the epithelial cell monolayer is driven by another PMN chemoattractant, hepoxilin A3 (HXA3) (**Figure 2**) (68, 70). HXA3 is a bioactive eicosanoid that is synthesized from arachidonic acid via the 12/15-lipoxygenase pathway in epithelial cells (**Figure 2**) (70). After synthesis, HXA3 is secreted from the apical surface of epithelial cells by an ATP-binding cassette transporter called multidrug resistant protein 2 (MRP2) (**Figure 2**) (71). Secretion of HXA3 into the lumen forms a chemoattractant gradient that causes neutrophils to migrate from the region underlying the epithelial cell monolayer into the lumen (**Figure 2**) (70).

Activation of the effector protein SipA has been shown to be necessary for induction of HXA3 synthesis and the resulting PMN migration (**Figure 2**) (72). Remarkably, the mechanism for activating SipA was recently shown to require processing by the host enzyme caspase-3 at a particular cleavage site, resulting in two distinct effector domains (73). Furthermore, the two domains were shown to be functionally different. The ability to promote PMN migration is confined to the SipA N-terminal domain, whereas the C-terminal domain has been shown to be involved in actin rearrangement (72, 73). The current understanding of the mechanism of SipA-dependent synthesis of HXA3 is that SipA induces the recruitment of ADP-ribosylation factor 6 (ARF6) to the apical membrane of the epithelial cells. ARF6 activates phospholipase D, which generates phosphatidic acid. Phosphatidic acid is then converted to diacylglycerol (DAG), which recruits protein kinase C-α (PKC-α) to the apical membrane (**Figure 2**). PKC-α, in addition to an ERM protein ezrin, modulate the localization of MRP2 to the apical membrane of epithelial cells, thereby allowing the secretion of HXA3 into the lumen and production of the chemattractant gradient that induces neutrophil transmigration (**Figure 2**) (72, 74, 75).

#### **CONCLUSION**

The architecture of the mucosal immune system, including mucins, antimicrobial peptides, resident microbiota, paracellular junctions, and effector cells of the lamina propia, functions to prevent pathogenic bacteria from disrupting the epithelial cell monolayer and causing disease. If enteric pathogens are able to penetrate these barriers, then it results in a host inflammatory response and eventually activation of an adaptive immune response, designed to eradicate the intruding pathogen. However, *S. typhimurium* has evolved systems, namely the SPI-1 and SPI-2 T3SS, to manipulate the defensive mechanisms of the mucosal immune system in order to develop a replication niche in the mucosal epithelium. Additionally, the ability of *S. typhimurium* to exploit inflammation allows it to penetrate the epithelial barriers, a condition in which activation of the adaptive immune response would be required for pathogenic clearance. Investigating how *S. typhimurium* exploits host cell signaling pathways will allow for increased understanding in its pathogenesis, and consequently provide further insight into how inflammation can seemingly result in both increased bacterial fitness and increased pathogenic clearance.

#### **ACKNOWLEDGMENTS**

The authors would like to thank Erik J. Boll for critical reading of the manuscript.

#### **REFERENCES**

colonization of intestinal epithelia by *Salmonella typhimurium*. *J Immunol* (1998) **160**(1):455–66.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 26 February 2014; accepted: 20 June 2014; published online: 04 July 2014. Citation: Patel S and McCormick BA (2014) Mucosal inflammatory response to Salmonella typhimurium infection. Front. Immunol. 5:311. doi: 10.3389/fimmu.2014.00311 This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Patel and McCormick. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Mucosal-associated invariant T-cells: new players in anti-bacterial immunity

## **James E. Ussher 1,2, Paul Klenerman1,3 and Chris B.Willberg1,3\***

1 Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK

2 Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand

3 Oxford NIHR Biomedical Research Centre, John Radcliffe Hospital, Oxford, UK

#### **Edited by:**

Constantino López-Macías, Mexican Social Security Institute, Mexico; University of Oxford, UK

#### **Reviewed by:**

Heinrich Korner, Menzies Research Institute Tasmania, Australia Siobhan Cowley, Food and Drug Administration, USA Leslie Saurer, University of Bern, Switzerland

#### **\*Correspondence:**

Chris B. Willberg, Peter Medawar Building for Pathogen Research, University of Oxford, Oxford OX3 1SY, UK

e-mail<EMAIL_ADDRESS>
Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population involved in anti-bacterial immunity. In human beings, MAIT cells are abundant, comprising ~10% of the CD8+ T-cell compartment in blood. They are enriched at mucosal sites and are particularly prevalent within the liver. MAIT cells are defined by the expression of a semi-invariantT-cell receptor (Vα7.2-Jα33/12/20) and are restricted by the non-polymorphic, highly evolutionarily conserved MHC class Ib molecule, MHC-related protein (MR)1. MR1 has recently been shown to present an unstable pyrimidine intermediate derived from a biosynthetic precursor of riboflavin; riboflavin biosynthesis occurs in many bacteria but not in human beings. Consistent with this, MAIT cells are responsive to riboflavin-metabolizing bacteria, including Salmonella. In mouse models, MAIT cells have been shown to play a non-redundant role in anti-bacterial immunity, including against Escherichia coli, Klebsiella pneumoniae, and Mycobacterium bovis BCG. In human beings, MAIT cells are decreased in frequency in the blood of patients with tuberculosis or pneumonia, and their frequency has been inversely correlated with the risk of subsequent systemic bacterial infection in patients in intensive care. Intriguingly, MAIT cells are also depleted from the blood early in HIV infection and fail to recover with anti-retroviral therapy, which may contribute to the susceptibility of patients infected with HIV to certain bacterial infections, including non-typhoidal Salmonella. In this review, we will discuss what is currently known about MAIT cells, the role that Salmonella has played in elucidating MAIT cell restriction and function, and the role MAIT cells might play in the control of Salmonella infection.

#### **Keywords: MAIT cells, anti-bacterial, inflammation, CD161,TCR, MR1**

## **INTRODUCTION**

In 1999, Tilloy et al. first described mucosal-associated invariant T (MAIT) cells (1). Interest in this unique subset of innatelike T-cells has increased rapidly over the last 5 years as novel findings have revealed their unique anti-bacterial function and phenotype (2, 3). MAIT cells represent the most abundant innate-like T-cell population within human beings, comprising up to ~5% of the total T-cell population; this compares with just ~0.1% for invariant natural killer T (iNKT) cells (4, 5). They are characterized by the expression of a semi-invariant TCR (Vα7.2-Jα33/12/20) that recognizes the evolutionarily conserved MHC-like protein 1 (MR1), which presents a bacterialderived ligand (6–11). Although they can be activated through their TCR, they are also readily stimulated by innate cytokines, either leading to the expression of pro-inflammatory cytokines or the release of cytotoxic and pro-inflammatory granzymes (**Figure 1**) (12, 13). Furthermore, MAIT cells have been associated with a number of disease settings, including bacterial infections (14), and pro-inflammatory diseases such as multiple sclerosis (15) and psoriasis (16). Thus, this large T lymphocyte population is likely to have an important role in human health.

This review will explore what is currently known about MAIT cells in human beings. Comparisons between human and murine MAIT cells have been made elsewhere (4). Furthermore, we will discuss the role that *Salmonella* has played in identifying the functions of this cell type, and the potential role MAIT cells may have in controlling *Salmonella* infections.

## **MAIT CELL PHENOTYPE**

In addition to possessing the Vα7.2-Jα33/12/20 TCR, MAIT cells can be identified in human beings by the expression of a characteristic phenotypic signature composed of a number of additional surface and transcriptional markers.

## **MEMORY PHENOTYPE**

In adults, MAIT cells typically express an effector memory phenotype: CD45RO+, CCR7−, CD62L−, CD27+, and CD28+ (17–19). However, in cord blood, MAIT cells possess a naïve phenotype (CD45RA+, CCR7+, CD62L+), but still retain a phenotypic signature characteristic of adult MAIT cells, including the expression of CD161, interleukin (IL)-18Rα, CD8αα, and CCR6 (3, 5, 17, 20). A recent study demonstrated that MAIT cells in the thymus, spleen, and mesenteric lymph nodes of aborted second trimester fetuses also had a naïve phenotype and expressed only low levels of the characteristic MAIT cell markers, such as IL-18Rα and CD8αα, while MAIT cells in the fetal intestine, liver, and lung had a more memory phenotype (21).

**FIGURE 1 | Potential MAIT cell response to Salmonella infected cells**. **(1)** Internalization of Salmonella by an antigen-presenting cell, either through infection or actively by phagocytosis. **(2)** Lysis of the bacteria, within endocytic compartments, releases 5-A-RU, which is converted to 5-OE-RU or 5-OP-RU and binds to and stabilizes MR1. **(3)** The stable MR1 translocates to the cell surface, where it is presented along with other co-stimulatory molecules, e.g., CD80 or CD86. **(4)** Bacterial components trigger pathogen recognition receptors (PRR), such as TLR8. **(5)** PRR triggering drives cytokine expression, such as IL-12, and the activation of the inflammosome, resulting

## **CD161**

CD161 is a C-type lectin-like receptor originally identified by Lanier et al. (22). It is found on a broad range of lymphocytes, including CD4+, CD8+, γδ+ T-cells, and NK cells. The majority of NK cells express CD161 (>90%), while in the CD4+, CD8+, and γδ+ T-cell subsets, CD161 expression is limited to ~30% of cells (19, 23). However, within the CD8+ and CD8− CD4− Tcell population, CD161 expression can distinguish three separate subsets, CD161−, CD161intermediate/+, and CD161high/++; MAIT cells populate the CD161++ subset (17, 18). In adult peripheral blood, MAIT cells represent ~85% of the CD161++ subset (24). However, in cord blood, the MAIT cells make up a much smaller proportion of this subset, averaging ~15% of the CD161++ CD8+ T-cell population (21, 25, 26). During early childhood, this population expands so that by the age of 24 months the MAIT cell population already represents ~50% of the CD161++ CD8+ T-cell population (25).

The function of CD161 on MAIT cells is yet to be fully elucidated. On NK cells, binding of CD161 to its ligand [lectin-like transcript (LLT) 1] leads to an inhibition of cytotoxicity (27– 29). Two studies explored the role of CD161 on CD8+ T-cells in the release of active-IL-18. **(6)** MAIT cells are activated either by TCR recognition of MR1 in combination with co-stimulatory receptors, e.g., CD28, and/or by cytokines, e.g., IL-12 and IL18. **(7)** Activated MAIT cells express pro-inflammatory cytokines, e.g., IFNγ, TNFα, and IL-17. **(8)** These cytokines can directly act anti-bacterially, or recruit and stimulate other immune cells, e.g., neutrophils by IL-17. **(9)** Activation of MAIT cells upregulates perforin and granzyme B expression. **(10)** Theoretically, the degranulation of cytotoxic granules into infected cells (target cells), via recognition of MR1, could induce cell death and, thus, the potential clearance of infected cells.

and reached opposing conclusions (27, 29). Rosen et al. found that cross-linking CD161 had no effect on anti-CD3/CD28 stimulated CD8+T-cells in terms of IFNγ expression and inhibited TNFα expression, whereas Aldemir demonstrated increased IFNγ expression after CD161 signaling. Le Bourhis et al. recently reported that ligation of CD161 on MAIT cells inhibited cytokine production but had no effect on cytotoxicity (30). One explanation for these differences could be due to the different CD161 cross-linking antibody clones used.

CD161 surface expression has recently been reported to be downregulated after long-term activation *in vitro*; downregulation was associated with greater rounds of proliferation (13). In HIV infection, loss of expression of CD161 by MAIT cells has been suggested (31). Given that MR1-tetramer-positive cells are not found in the CD161-negative population in healthy individuals (8), this requires further investigation.

## **IL-18R**α

CD161 expression is associated with high levels of IL-18Rα expression. Of all human T lymphocytes, MAIT cells express the highest level of IL-18Rα (12, 17). Naïve MAIT cells, derived from cord blood, have also been shown to be IL-18Rα + (5, 24). Recent observations of tissue from second trimester fetuses suggest that IL18Ra expression is low upon egress from the thymus but increases as MAIT cells mature in the tissues (21). Expression of IL-18Rα conveys an ability to respond robustly to cytokine stimulation (discussed below), which is limited in the other human T-cell populations (12).

## **ZBTB16**

The development of MAIT cells parallels that of NKT cells. Both innate-like T-cell subsets are selected for by non-classical MHC molecules, MR1 and CD1d, respectively, expressed on double negative thymocytes (32), and both subsets also express the transcription factor ZBTB16 (33). However, while NKT cells exit the thymus as an expanded and mature population, MAIT cells do not. Instead, murine models have shown that MAIT cells within the periphery have a naïve and immature phenotype, lacking expression of ZBTB16, CD25, CD69, or ICOS and further require an established microbiota and B-cells to expand into a memory phenotype (33). In contrast, MAIT cells found in the periphery of human fetuses have already acquired a more mature, memory phenotype, expressing ZBTB16, IL-18Rα and, upon bacterial stimulation, the capacity to express IFNγ and, in cells from the small intestines, IL-22 (5, 21). What drives selection and maturation *in utero* is unclear, but suggests that an endogenous ligand for MR1 may exist. Alternatively, this may reflect *in utero* exposure to commensal microflora, as has recently been suggested (34–36).

#### **ROR**γ**T**

CD161 expression is also a key phenotypic maker of IL-17 secreting cells (19, 37). Within the CD4+T-cell subset, Th17 cells represent a novel helper cell population that can secrete IL-17A under the control of the master transcription factor retinoic acid-related orphan receptor (ROR) γt (38, 39). Within the CD8+T-cell subset, the expression of RORγt and the secretion of IL-17 are restricted to Tc17 cells that are CD161++. Thus, MAIT cells represent the vast majority of Tc17 cells (17, 24). The ability of MAIT cells to express IL-17A has recently been shown to depend on their preexposure to cytokines IL-1beta, IL-23, and IL-7 (40). Interestingly, hepatic stromal cells constitutively express IL-7, emphasizing the link between MAIT cells and the liver (41, 42).

#### **CO-RECEPTOR USAGE**

CD161++/MAIT cells also encompass the CD8alpha/alpha population in adult blood, small bowel, and liver (5). Interestingly, this population is not seen in cord blood, or fetal thymus, but is seen at low levels in fetal intestine, liver, and lung (21), suggesting that it is derived from the CD8alpha/beta population through the regulation of CD8beta. Functionally and phenotypically, there is no difference between the CD8alpha/alpha and CD8alpha/beta subsets of the CD161++/MAIT cell population (5). However, Walker et al. have also described the expression of CD8alpha/alpha as the marker of terminally differentiated CD8+ T-cells that can be found in a number of chronic viral infections (26).

#### **MULTIDRUG RESISTANCE TRANSPORTER ABCB1**

Mucosal-associated invariant T-cells express the ATP-binding cassette (ABC) B1 drug resistance transporter, which can also be seen on CD161+T-cells to a more limited degree (43). The ability to efflux drugs has been shown to allow MAIT cells to persist during chemotherapy for the treatment of acute myeloid leukemia (AML) or breast cancer (24, 43). The expression of the ABCB1 transporter possibly reflects the diverse environmental toxins MAIT cells are exposed to in their niche, although its overall role is not defined.

## **CHEMOKINE RECEPTOR EXPRESSION**

Mucosal-associated invariant T-cells express a range of chemokine receptors (CCR6, CCR5, CCR9, and CXCR6) that localize them to the gut, but more prominently to the lungs and liver (24, 44). The liver receives 25% of its blood supply from the hepatic artery and 75% from the portal vein. The portal vein delivers blood direct from the gastrointestinal track and spleen, placing the liver in the front line in the defense against microbial infection. Moreover, Balmer et al. recently described the liver as a firewall against infection from commensal bacteria that have invaded the body through either the intestines or systemic vascular circuits (45). Therefore, given their anti-bacterial function, it is unsurprising that MAIT cells represent up to 45% of all liver lymphocytes (24, 40). Furthermore, both CD161+ CD4+ and CD161+CD8+ T-cells are selectively recruited to the liver during inflammation (17, 23, 40).

## **ANTI-BACTERIAL ACTIVITY OF MAIT CELLS**

Two studies in 2010, by Gold et al. and Le Bourhis et al., observed that MAIT cells could recognize antigens derived from a range of bacteria presented on antigen presenting cells (APCs) (2, 25). Gold et al. observed that a large population of CD8+ Tcells able to respond to *Mycobacterium tuberculosis* (Mtb) was present even in unexposed individuals. Functional and phenotypic analysis showed that this population was MR1 restricted and expressed the TCR Vα7.2 chain, i.e., MAIT cells. Furthermore, they showed that MAIT cells responded to *Salmonella enterica* serovar Typhimurium-infected dendritic cells, as well as *Escherichia coli* and *Staphylococcus aureus*. Le Bourhis et al. demonstrated the ability of MAIT cells to recognize and be activated by monocytes exposed to *E. coli* in an MR1-dependent manner. Furthermore, they observed that MAIT cells are activated by a wide range of bacteria (*E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Lactobacillus acidophilus, Staphylococcus aureus, Staphylococcus epidermidis,* and *Mycobacterium abscessus*) but not by all (*Enterococcus faecalis, Streptococcus pyogenes*). In addition, they showed activation in response to some yeasts (*Candida albicans, Candida glabrata,* and *Sacchromyces cerevisiae*). Moreover, MAIT cells were not observed in germ-free mice or in germ-free mice repopulated with *E. faecalis*, but were in germ-free mice repopulated with *Enterobacter cloacae* or *L. acidophilus*, underscoring the important role of certain bacterial species in MAIT cell development.

The ability of MAIT cells to control bacterial infections was elegantly demonstrated by Georgel et al. through the use of luminescent-*K. pneumoniae* (46). Despite the low frequency of MAIT cells in common laboratory strains of mice, they showed that in the absence of MR1, and, therefore, MAIT cells, mice succumbed to disseminated infection, while wild-type mice cleared the infection within 2 days. No defect in clearance was seen with *E. coli*, *Shigella dysenteriae,* or *Yersinia enterocolitica* suggesting that redundancy in the immune response is sufficient to control the dissemination of these organisms. In contrast, enhanced control of *E. coli* infection and *M. abscessus* infection was demonstrated in Vα19 or Vβ6 transgenic mice, which have an increased frequency of MAIT cells, on a MR1 replete background compared with a MR1−/− background. Subsequent studies by Chua et al. and Meierovics et al*.* have demonstrated the need for MAIT cells in the early control of *Mycobacterium bovis,*BCG,and *Francisella tularensis* following a mucosal challenge (47, 48). Moreover, the presence of MAIT cells had a strong influence on the timing of recruitment and activation of conventional T-cells, and provided long-term protection alongside a conventional T-cell response (48).

## **THE MHC-RELATED PROTEIN (MR)1 AND ITS LIGAND**

The broad range of bacteria MAIT cells can respond to was recently explained with the identification of the ligand that binds MR1 (49). Kjer-Nielsen et al. discovered the structure of MR1 and the nature of the ligand that it binds after their serendipitous finding that 6-formyl pterin (6-FP), a photodegradation product of folic acid that was present in tissue culture media, was able to bind to MR1 and allowed its refolding. However, while 6-FP was able to stabilize the MR1 molecule, it was unable to activate MAIT cells. Given that the culture supernatant of *S. enterica* serovar Typhimurium was able to activate MAIT cells, they reasoned that it must contain the activating ligand. Therefore, they refolded MR1 with the culture supernatant of *S. enterica* serovar Typhimurium grown in minimal media that lacked vitamins (M9 minimal media), and analyzed the refolded MR1 by mass spectrometry. They observed a single ligand with a mass to charge ratio (*m/z*) of 329.11. This was consistent with the ligand being reduced 6-hydroxymethyl-8-d-ribityllumazine (rRL-6-CH2OH), a metabolic by-product of riboflavin metabolism. Chemical synthesis of reduced rRL-6-CH2OH confirmed that it had the same *m/z* as the ligand identified in the culture supernatant. Furthermore, synthetic reduced rRL-6-CH2OH, as well as related compounds derived from riboflavin metabolism, 7-hydroxy-6 methyl-8-d-ribityllumazine (RL-6-Me-7-OH) and 6,7-dimethyl-8-d-ribityllumazine (RL-6,7-diMe), were able to activate primary MAIT cells. This pivotal discovery was consistent with the prior observation that MAIT cells could be activated by *Salmonella*, along with other *Enterobacteriaceae*, *P. aeruginosa, S. aureus*, and some yeast species, which all contain the riboflavin synthetic pathway, but not by *S. pyogenes* or *E. faecalis*, which lack the riboflavin synthetic pathway (2, 25).

As the origin of the previously identified ligand, rRL-6- CH2OH, was not clear, Corbett et al. derived various strains of *Lactococcus lactis* with different mutations in the riboflavin synthesis operon and assessed their ability to activate MAIT cells (50). Mutants lacking genes involved in the riboflavin synthesis pathway (*ribA* and *ribG*) were unable to activate MAIT cells, and the MR1-binding ligand (*m/z* 329.11) was undetectable. By contrast, no defect in MAIT cell activation was seen with *ribB* and *ribH* mutants, pinpointing the production of 5-amino-6-dribitylaminouracil (5-A-RU), an early intermediate in riboflavin synthesis. The importance of 5-A-RU was confirmed by the lack of MAIT cell activation and absence of the MR1-binding ligand (*m/z* 329.11) in the culture supernatant of *S. enterica* serovar Typhimurium SL1344 with mutated *ribD* and *ribH* genes; furthermore, complementation restored reactivity. Despite the necessity for 5-A-RU, it was unable to bind MR1 or activate MAIT cells directly. However, upon non-enzymatic condensation with glyoxal or methylglyoxal, byproducts of other metabolic pathways (including glycolysis), 5-A-RU formed unstable intermediates [5- (2-oxoethylideneamino)6-d-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU)], which were able to covalently bind to MR1. Furthermore, these unstable intermediates formed reversible covalent Schiff base complexes with Lys43, analogous to 6-FP (49). Moreover, 5-OE-RU and 5-OP-RU could be demonstrated in the culture supernatant of activating bacteria, but not of non-activating bacteria, and could be captured by MR1. Therefore, MR1 captures unstable intermediates that would otherwise convert to lumazines. MR1 refolded with 5-A-RU and methylgyoxal (5-OP-RU) produced a mass spectrometry peak of *m/z* 329.11, consistent with what was originally found in *Salmonella* supernatant (49). This suggested that 5-OP-RU, and not reduced rRL-6-CH2OH, was the true ligand for MR1. In support of this hypothesis, they also found a 315.09 *m/z* species (corresponding to 5-OE-RU) bound to MR1 refolded in the presence of *E. coli* DH5α; the 315.09 *m/z* species was also detected with *Salmonella,* but was much less abundant. This interesting finding demonstrates that different bacteria may produce different MAIT-activating ligands.

Overall, our current understanding of this complex process is that bacterially derived 5-A-RU is converted to 5-OE-RU or 5- OP-RU by reaction with bacterial or host-derived metabolites. These unstable intermediates covalently bind to, and stabilize MR1, which can then be recognized by the MAIT cell TCR.

Recently, Eckle et al. published that MAIT cells activation can be competitively inhibited by a novel MR1 ligand, acetyl-6 formylpterin (Ac-6-FP) (51). Ac-6-FP was identified in a search for analogs of 6-FP that were stronger antagonists of MAIT cell activation. MR1 efficiently refolded in the presence of Ac-6-FP and increased surface expression was seen in an MR1-transduced cell line treated with Ac-6-FP. Neither Ac-6-FP-MR1 tetramers nor 6-FP-MR1 tetramers were able to stain PBMCs. Consistent with antagonist activity, Ac-6-FP strongly inhibited the activation of the Jurkat.MAIT cell line in response to the agonist ligands, rRL-6-CH2OH and 5-OP-RU. Therefore, Ac-6-FP will be a useful tool in future studies of MAIT cell function.

## **THE MAIT CELL TCR**

The defining feature of MAIT cells is the expression of a semiinvariant TCR, Vα7.2Jα33/12/20, which restricts MAIT cells to the evolutionarily conserved, non-polymorphic MHC-related protein 1 (MR1) (1, 6, 9, 11, 44). Vbeta chain usage does vary; however, it is dominated by Vβ2 and Vβ13.2 (1, 9, 11). Homologous TCRs have also been identified in mice (Vα19Jα33, paired with Vβ6 or Vβ8) as well as cattle (1, 5, 52).

The structure of the MAIT cell TCR and the residues that are critical for interacting with ligand-bound MR1 were determined prior to identification of the MAIT cell ligand (9, 11). Analysis of the MAIT cell TCR structure demonstrated that the CDR3α region is composed of the Vα7.2 and Jα33 of the alpha chain. *S. enterica* serovar Typhimurium was used as the source of the MR1-binding ligand to show individual residues in Vα7.2 (in CDR1α Gly28α and Asn30α, in CDR2α Tyr48α, and in CDR3α Asp92α, Asn94α, and Tyr95α), but not the Vβ chain, made critical contacts with MR1. However, switching the entire CDR3β loop with one from a non-MAIT cell TCR abolished recognition of MR1. A similar pattern was observed with other bacteria (*E. coli, K. pneumoniae, P. aeruginosa,* and *S. epidermidis*). Given the wide specificity, the authors suggested that the MAIT cell TCR was like a pattern recognition receptor. By mutational analysis, they identified residues Leu65 and Glu158 on MR1 as critical for the interaction with the TCR. These two residues mapped centrally to opposite sides of the antigen-binding cleft of MR1.

The identification of the ligand paved the way for the development of MR1 tetramers for the identification of MR1-restricted T-cells (8) . Reantragoon et al. used MR1 with a K43A mutation to make tetramers. MR1 K43A could be refolded in the absence of ligand and once refolded was able to be loaded with reduced rRL-6-CH2OH. To further prove that the MR1-tetramer was specific to MAIT cells, PBMCs were depleted of MR1-tetramer binding cells and tested for reactivity to *S. enterica* serovar Typhimurium supernatant or to reduced rRL-6-CH2OH.

Using the loaded tetramers, Reantragoon et al. confirmed that the majority of MR1-restricted T-cells express the canonical semiinvariant T-cell receptor, Vα7.2-Jα33, but also identified minor (8–31%) MAIT cell populations exist that express Vα7.2-Jα12 and Vα7.2-Jα20. Interestingly, all alpha chains contained the conserved Tyr95α within CDR3α loop, which forms a hydrogen bond with RL-6-Me-7-OH bound by MR1 and is crucial for MAIT cell activation (8, 53). Interestingly, non-canonical TCRs showed a bias in Vβ usage toward TRBV6-4. Both canonical and non-canonical TCRs appeared to have the same ligand specificities. MR1-tetramer staining of lymphocytes isolated from the jejunal mucosa confirmed that MAIT cells are enriched at this site, with ~60% of CD3+ CD4-cells being MAIT cells. Furthermore, the majority of the tetramer positive cells in the jejunum were shown to be Vα7.2-Jα33, as in blood. This suggests that frequency of MAIT cells varies in different anatomical locations within the gastrointestinal tract, with higher frequencies in jejunum (8) than in ileum (54), colon, and rectum (31, 55). Alternatively, the variations observed in MAIT cell frequency in different tissues may reflect differences in methodology between studies.

Using a mouse MR1 tetramer in a MAIT-enriched mouse model (transgenic for Vα19 on Cα−/− background), the differences between mouse MAIT cells and human MAIT cells were highlighted (8). While human MAIT cells were predominantly CD8+ with some double negative cells and only a small CD4+ subset, in Vα19-transgenic mice >40% of MAIT cells were CD4+, with the remainder mostly DN rather than CD8+. Whether this reflects functional differences between human and mouse MAIT cells remains to be determined.

A recent study by Gold et al. investigated the TCR usage of MAIT cells that were responsive to different microbes (56). CD8+ T-cells from healthy donors were stimulated *ex vivo* with A549 cells infected with *Mycobacterium smegmatis, Salmonella typhimurium,* or *C. albicans.* Vα7.2 + CD8 + T-cells that produced TNFα were sorted and their TCR usage determined. They found greater diversity in the *TRAJ* gene usage than previously reported; while *TRAJ33* dominated, a range of other *TRAJ* genes were identified, including some (*TRAJ9* and *TRAJ39*) that do not encode for the Tyr95a residue that has been reported to be critical for MAIT cell activation (8, 11, 52). However, as the authors note, there is no allelic exclusion at the *TRA* locus, so the non-canonical *TRAJ* genes identified may not contribute to the functional MAIT cell TCR. *TRAJ* usage and CDR3α sequence of the responding MAIT cells differed between microbes; there was more similarity in the CDR3α sequence in MAIT cells activated by the same microbe than with those activated by a different microbe. Furthermore, there was significant diversity in *TRBV* gene sequences of MAIT cells responding to different microbes, although there was minimal overlap in the CDR3β sequence across donors and microbes. Overall, this suggested that different MAIT cells respond to different microbe-derived ligands. In support of this, of four MAIT cell clones that were robustly activated by *M. smegmatis*, only two were activated by RL-6,7-diMe. The authors speculate that MR1 ligand diversity drives MAIT cell TCR diversity and that that the Vα chain primarily mediates contact with MR1, while the CDR3β chain, positioned above the MR1 ligand-binding groove, contributes to ligand discrimination, as previously described by López-Sagaseta et al. (57, 58). This is consistent with the findings of Eckle et al., who showed that the novel inhibitory MR1 ligand, Ac-6-FP, induced structural alterations in MR1, which prevented the MAIT cell TCR CDR3β chain from binding (51). These two studies open up the exciting possibility that a wide range of novel MR1 ligands exists, which could modulate the MAIT cell response.

## **MAIT CELL ACTIVATION**

A number of studies have suggested adult MAIT cells to be terminally differentiated and, as a result, less responsive to TCR signaling, showing low IFNγ production and little proliferation compared to stimulation that bypasses the TCR (such as activation by PMA and ionomycin, or PHA) (24, 33, 43). In contrast, MAIT cells derived from cord blood or fetal tissues readily proliferate with TCR stimulation (21, 33). This lack of responsiveness by adult MAIT cells to TCR stimulation can, however, be overcome. Turtle et al. demonstrated that TCR signaling required the addition of an innate signal/s from either the co-receptor CD28 or the cytokine IL-12 in order to induce high levels of IFNγ and strong proliferation (20). Using transcriptional profiling, they went on to show that the mechanisms controlling TCR signaling in MAIT cells were distinct from those seen in anergic or exhausted T-cells.

A study by Chua et al. demonstrated that in a murine model of bacterial infection using *M. bovis* BCG, IL-12 signaling, but not TCR signaling, was required for the control of infection; blockade of IL-12 but not MR1, inhibited the anti-bacterial activity of MAIT cells (47). This regulation of activation is similar to that seen in iNKT cells, where IL-12 signaling dominates over CD1d-induced TCR signaling (59, 60). In human models of MAIT cell activation, there is a dual role for TCR- and cytokine-induced activation of MAIT cells. At early time points, 5 hours after a MAIT cell encounters an antigen-presenting cell (APC) presenting its cognate antigen on MR1, TCR signaling dominates activation (12). However, at later time points (20 hours), cytokine-mediated activation is equally important and MR1 blockade has a more limited effect. In contrast to the murine models, IL-12 alone is not sufficient to induce IFNγ expression, but requires the addition of other innate cytokines, such as IL-18 (12). The ability to respond to IL-12 plus IL-18 is similar to that of NK cells (61), and implicates MAIT cells in a range of infectious and non-infectious inflammatory diseases. Furthermore, signaling via toll-like receptors (TLR) is able to drive the expression of a range of pro-inflammatory cytokines from professional APCs, which can activate MAIT cells (**Figure 1**) (12, 62). We have shown that TLR8 agonists are particularly potent stimulators of IL-12 and IL-18 secretion, and therefore capable of driving MAIT cell IFNγ expression (12, 62). Thus, this suggests that in addition to their anti-bacterial role, MAIT cells may play an important role in anti-viral responses, and this may also provide a mechanism that explains their involvement in other pro-inflammatory settings such as experimental autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, psoriasis, and arthritis (15, 16, 54, 63, 64).

## **MAIT CELL CYTOTOXICITY**

How MAIT cells affect their anti-bacterial function remains poorly defined. Upon activation, MAIT cells are able to produce several cytokines, including IFNγ, TNFα, and IL-17 (17, 24). In addition, it has recently been demonstrated that MAIT cells are also cytotoxic. Le Bourhis et al. reported that MAIT cells can recognize epithelial cells (HeLa cells) infected with *Shigella flexneri* but not *S. enterica* serovar Typhimurium (30). Consistent with this, MAIT cells were able to kill HeLa cells infected with *S. flexneri* but not *S. enterica* serovar Typhimurium. This cytotoxicity was dependent upon MR1 and was evident in HeLa cells expressing endogenous levels of MR1. No reactivity was seen with a *Salmonella* pathogenicity island 1 (SpI-1)-deleted strain of *S. enterica* serovar Typhimurium (SpI-1 is required for invasion). Similarly, MAIT cell activation and cytotoxicity was not seen with a SpI2 deleted strain of *S. enterica* serovar Typhimurium. Therefore, the virulence factors that prevent MR1 loading remain to be defined. The authors' suggestion was that *Salmonella* avoids detection as it resides in vacuoles and prevents fusion with the lysosome, while *Shigella* escapes to the cytoplasm. Indeed, invasion by *Shigella* was important as HeLa cells infected with a DMxiD strain were unable to efficiently activate MAIT cells. *In vivo* activation (determined by HLA-DR expression) and decreased frequency of MAIT cells in blood was observed in human beings after oral challenge with an attenuated strain of *S. dysenteriae* suggesting that MAIT cells responded to *S. dysenteriae in vivo.*

Recently, Kurioka et al. described increased cytotoxic potential of MAIT cells after activation (13). These cytotoxic, or "licensed," MAIT cells displayed upregulation of granzyme B, normally not expressed in resting MAIT cells, and enhanced perforin expression, which is low in resting MAIT cells (13). The licensed MAIT cells displayed an increased capacity to kill cognate-target cells, and maintained this phenotype even after several rounds of proliferation.

### **A ROLE FOR MAIT CELLS IN THE CONTROL OF SALMONELLA INFECTION**

The role of MAIT cells in *Salmonella* infection remains to be defined. As discussed above, MAIT cells can be activated by *Salmonella* sp. *in vitro* (8, 11, 25). MAIT cells activated by *Salmonella* sp. produce IFNγ and TNFα; these cytokines have been shown to be important in the control of *Salmonella* infection (65). In addition, MAIT cells can secrete IL-17 in response to stimulation with *E. coli* (55). IL-17 has recently been suggested to be critical in preventing the dissemination of *Salmonella.* In IL-17 receptor-deficient mice, increased systemic dissemination of *S. enterica* serovar Typhimurium was seen (66). Furthermore, in SIVinfected rhesus macaques, increased dissemination of *S. enterica* serovar Typhimurium was seen, which was associated with loss of Th17 cells and the IL-17 response from the ileum (66). Therefore, along with the loss of Th17 cells (67), the loss of MAIT cells in HIV infection may contribute to the increased susceptibility to disseminated non-typhoidal *Salmonella* infection (68).

Mucosal-associated invariant T-cells may also contribute to the control of *Salmonella* infection through cytotoxic activity. Cytotoxic T-cells are important in the clearance of *Salmonella* infection (69). However, MAIT cells are unable to kill HeLa cells, an epithelial cell line, infected with *Salmonella* (69). This may be because live *Salmonella* sp. is able to avoid MR1 containing compartments by preventing phagosome-lysome fusion (70). Future investigations with live *Salmonella* sp. may help elucidate the MR1 loading pathway/s, and subsequent activation of MAIT, revealing potential therapeutic targets.

The low numbers of MAIT cells in common laboratory mouse strains has hampered the study of the role MAIT cells in response to *Salmonella* infection. Therefore, a robust murine model is required to investigate the role of MAIT cells in the control of *Salmonella* infection.

## **MAIT CELLS IN INFECTIOUS DISEASE**

The role of MAIT cells in human disease has not been fully assessed due to the difficulties of obtaining tissue samples. However, there are a number of interesting associations between the frequency of MAIT cells within peripheral blood and disease. During active *M. tuberculosis* infection, MAIT cells numbers are lower in peripheral blood compared to healthy controls (2, 25, 71, 72). Consistent with these findings, Grimaldi et al. looked at MAIT cell numbers in critically ill septic and non-septic patients (14). They observed that all critically ill patients studied, including those with severe bacterial or viral infections and those with non-infective illness, had low MAIT cell counts compared to healthy controls. This was least pronounced in individuals with severe viral infections, and most striking in individuals infected with bacteria other than *Streptococcus* species (14). This suggests that the loss of MAIT cells from the periphery could be due, in part, to compartmentalization during disease (2, 25). Interestingly, however, the authors also observed that those individuals with persistent MAIT cell depletion at day 4 post-admission were at increased risk for subsequent nosocomial infections. Therefore, MAIT cell exhaustion or death, two mechanisms proposed to occur in HIV (see below), may contribute to this phenotype.

In both HCV and HIV, MAIT cells are depleted from the blood (17, 31, 55). During HCV infection, the loss of peripheral MAIT cells is potentially due to their relocation to the liver (17). Moreover, the frequency of IFNγ and IL-17 dual-expressing CD8+ T-cells in the liver, a proxy for MAIT cells, was inversely correlated with the fibrosis score, suggesting that they either play a protective role during HCV infections, or that they are progressively lost from the liver with increasing fibrosis. This might contribute to the higher rates of bacteremia seen in individuals with cirrhosis (45, 73, 74).

In HIV infection, the loss of CD161++ MAIT cells from blood occurs early during infection and persists despite otherwise successful anti-retroviral therapy (31, 55, 71, 75, 76), although the nature of this perturbation is unclear. Cosgrove et al. reported that MAIT cells, defined as CD161++ CD8+ T-cells by flow cytometry, were depleted as a proportion of the CD8+ T-cell population in blood (55). They proposed that this depletion was due, at least in part, to activation induced cell death from overstimulation secondary to microbial translocation. While Leeansyah et al. also observed a decrease in size of the CD161++Vα7.2+ population, they suggested that this was due to downregulation of CD161 and functional exhaustion of MAIT cells (31). In support of this, they noted an increase in frequency of CD161-Vα7.2+ T-cells as a proportion of CD3+ T-cells. Importantly, the antibody against Vα7.2 used in these studies is not specific for the canonical MAIT cell TCR (33). Therefore, the recently described MR1 tetramer will be useful to determine whether the CD161-Vα7.2+ T-cells seen in HIV infection are MAIT cells or not (8). Interestingly, in healthy donors, MR1 tetramer does not bind the CD161-Vα7.2+ T-cell population.

## **MAIT CELLS IN INFLAMMATORY DISEASE**

In addition to their anti-microbial activity, MAIT cells have also been implicated in a range of pro-inflammatory settings. Serriari et al. observed that individuals with inflammatory bowel diseases had lower numbers of circulating MAIT cells compared to controls (54), as seen during bacterial and viral infections (2, 17, 25, 54, 71). However, within individuals, the frequencies of MAIT cells were increased within inflamed tissues compared to healthy tissue, suggesting recruitment of MAIT cells from the blood to sites of inflammation. This is a theme consistent in other inflammatory diseases such as psoriasis, rheumatoid arthritis, multiple sclerosis, and experimental autoimmune encephalomyelitis (15–17, 63, 77). Interestingly, CD56− MAIT cells have been observed infiltrating kidney and brain tumors, implying a potential role in tumor immunity (78).

Overall, these studies in human disease demonstrate that MAIT cells are a population of innate-like cells that rapidly translocate to sites of inflammation, regardless of whether the inflammation is due to bacterial infection or to other pro-inflammatory stimuli.

## **WHAT THE FUTURE HOLDS FOR MAIT CELL RESEARCH**

Through the use of tools such as *Salmonella*, our understanding of MAIT cell functions has increased rapidly over the last 5 years. However, there are still a number of important questions to be answered. Are there other ligands presented by MR1? Is there an endogenous ligand within the thymus for MAIT cell selection? How is MR1 regulated? What are the relative roles of TCR-dependent and TCR-independent triggering of MAIT cells in host defense? Understanding what MR1 presents and how it is regulated will be critical for understanding where and when MAIT cells have a definitive role in disease. What role MAIT cells play during human disease, in both infectious and autoimmune settings also needs to be addressed. Although it is important to study human disease, much will be learnt from animal models. Understanding how MAIT cells are regulated will potentially allow their *in vivo* manipulation for a positive outcome. Given the rise of antibiotic resistant bacteria, as highlighted by the recent WHO report (April 2014) future prophylactic and therapeutic strategies that harness the anti-bacterial potential of MAIT cells may be particularly important (79, 80).

## **ACKNOWLEDGMENTS**

The authors would like to thank Joannah Fergusson, Ayako Kurioka, Bonnie van Wilgenburg, Parbhjeet Phalora, and Alba Llibre for their comments and critical reading of this manuscript. This research was supported by the NIHR Biomedical Research Program (Oxford) and the Wellcome Trust (WT091663MA).

## **REFERENCES**

MR1 proteins. *Proc Natl Acad Sci U S A* (2013) **110**:E1771–8. doi:10.1073/pnas.<PHONE_NUMBER>

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 02 July 2014; paper pending published: 27 July 2014; accepted: 05 September 2014; published online: 08 October 2014.*

*Citation: Ussher JE, Klenerman P and Willberg CB (2014) Mucosal-associated invariant T-cells: new players in anti-bacterial immunity. Front. Immunol. 5:450. doi: 10.3389/fimmu.2014.00450*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Ussher, Klenerman and Willberg . This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Salmonella as a model for non-cognateTh1 cell stimulation

## **Hope O'Donnell 1,2 and Stephen J. McSorley 1\***

1 Department of Anatomy, Physiology and Cell Biology, Center for Comparative Medicine, School of Veterinary Medicine, University of California Davis, Davis, CA, USA

2 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota Medical School – Twin Cities, Minneapolis, MN, USA

#### **Edited by:**

Adam Cunningham, University of Birmingham, UK

#### **Reviewed by:**

Adrianus Wilhelmus Maria Van Der Velden, Stony Brook University, USA Prosper N. Boyaka, The Ohio State University, USA

#### **\*Correspondence:**

Stephen J. McSorley, Center for Comparative Medicine, University of California Davis, County Road 98 and Hutchison Drive, Davis, CA 95616, USA

e-mail<EMAIL_ADDRESS>
Salmonella has been a model pathogen for examining CD4 T cell activation and effector functions for many years due to the strength of the Th1 cell response observed during Salmonella infections, the relative ease of use of Salmonella, the availability of Salmonella-specific T cell reagents, and the well-characterized nature of the model system, the pathogen, and the immune response elicited. Herein, we discuss the use of Salmonella as a model pathogen to explore the complex interaction of T cells with their inflammatory environment. In particular, we address the issue of bystander activation of naïveT cells and non-cognate stimulation of activated and memoryT cells. Further, we compare and contrast our current knowledge of these non-cognate responses in CD8 versus CD4 T cells. Finally, we make a case for Salmonella as a particularly appropriate model pathogen in the study of non-cognate CD4 T cell responses based on the strength of the Th1 response during infection, the requirement for CD4 T cells in bacterial clearance, and the well-characterized inflammatory response to conserved molecular patterns induced by Salmonella infection.

**Keywords: Salmonella, CD4 T cell,Th1 cell, innate stimulation,TLR, NLR, IFN-**γ

## **INTRODUCTION**

T cell activation and effector functions have been extensively studied *in vitro*, allowing for controlled interactions within a defined environment. However, studying T cells *in vitro* inherently limits interactions to those that have been previously defined. To explore more complex systems of interactions beyond known parameters requires using an *in vivo* model system. One common technique for studying T cell responses *in vivo* is to examine a population of T cells with known antigen specificity. This includes the use of T cell receptor (TCR) transgenic mice, model antigens like ovalbumin, and major histocompatibility complex (MHC) class I and II tetramers presenting defined peptide sequences, which allows for the detection of T cells specifically recognizing that peptide. These reagents have greatly facilitated the tracking of antigen-specific T cells and the study of monoclonal T cell responses. Together with *in vitro* studies, the examination of antigen-specific T cells *in vivo* has been essential in defining much of what we know about T cell immunology.

When trying to understand the diverse polyclonal responses that are induced by infections, *in vivo* techniques that examine individual antigen-specific responses are likely to be limited. The natural breadth of the naïve TCR repertoire is an important strength of the adaptive immune response and can only be maintained by having pools of individual clones at very low frequency. Recent evidence has shown that altering the frequency of a given T cell clone can impact the activation strength, kinetics, and memory formation of the resulting T cell response (1–4). This issue complicates TCR transgenic mouse studies, which focus on a monoclonal population, generally used at unnaturally highfrequency. Studying the natural endogenous precursor population is therefore important and also complex since the frequency of individual clones also varies within the naïve repertoire (5).

Furthermore, individual TCR specificities may be predisposed toward different fates (6) and may also be regulated by temporal and anatomical antigen expression by the pathogen, factors that might significantly affect some clonal populations differently than the overall polyclonal T cell response (7, 8). These issues affect the use of TCR transgenic mice, MHC tetramer studies, and model antigens, because it may lead to a situation where the T cell response under study may not be representative of the overall T cell response to the pathogen. Likewise, studies that attempt to activate T cells with model antigens in the absence of infection are unlikely to accurately reflect the complex interactions that occur between T cells and the rest of the immune system in the context of a strong inflammatory response. Thus, to examine the full range of T cell functions and interactions within the larger immune network, it is necessary to study them in the context of a natural polyclonal response that includes a broad range of antigens and the inflammatory milieu that differentiates infection from other surrogate means of activation.

When exploring the responses of CD4 T cells, in particular, it is critical to examine their functions under circumstances in which they are naturally induced and required. In other words, it makes very little sense to study the effector function of Th1 cells using models where these Th1 cells do not contribute to pathogen clearance. The role of the Th1 subset of CD4 T cells and its effector cytokine IFN-γ in *Salmonella* infections has been very well established (9–11), making *Salmonella* model systems particularly appropriate for characterizing Th1 cell functions. Additionally, the innate immune response and inflammatory responses occurring during *Salmonella* infections are relatively well-defined (12–16), making it an ideal model to characterize the influence of natural inflammatory conditions on these Th1 cell responses.

In this review, we will highlight the unique advantage of the *Salmonella* model system for studying Th1 responses to innate stimuli. First, in Section "Armed and Ready: T Cell Responses to Innate Signals," we discuss and compare conventional cognate T cell stimulation, non-cognate stimulation of activated conventional T cells, and the responses of innate-like T cells. Thus far, most studies examining non-cognate T cell responses have focused on CD8 T cells, primarily in viral infection models. It is likely that the rules governing non-cognate CD8 T cell responses differ in certain aspects to those governing non-cognate responses in CD4 T cells. However, comparing these responses in infection models that generate overall weak CD4 T cell responses due to poor activation does not allow accurate comparison of the capacity of the non-cognate CD4 T cell response. In Section "A Complicated Relationship: The Dynamic Interactions of *Salmonella* and Th1 Cells," we discuss the dynamic interaction of *Salmonella* and the Th1 response, focusing on the important role of Th1 cells in the resolution of *Salmonella* infection, and potential ways that *Salmonella* might be able to subvert cognate T cell recognition and thus increase the requirement for non-cognate recognition pathways.

## **ARMED AND READY: T CELL RESPONSES TO INNATE SIGNALS**

#### **CONVENTIONAL T CELL RESPONSES**

Before discussing innate stimulation of T cells, we will first briefly review initial T cell activation, differentiation, secondary stimulation at sites of infection, and formation of memory. Both CD4 and CD8 T cells are activated upon recognition of a specific peptide sequence in host MHC by the TCR. CD8 T cells recognize this peptide presented within MHC-I expressed on most cell surfaces, while CD4 T cells interact with antigen presented in MHC-II only by antigen presenting cells (APCs). These APCs, often dendritic cells (DCs), collect and process antigen in the periphery and return to the lymph nodes and other secondary lymphoid organs with the antigens presented on their surface, where they can be recognized by interacting T cells. This first antigen-specific interaction is referred to as signal 1 of T cell activation (**Figure 1**) because, although it is required, the TCR stimulation alone is not sufficient to functionally activate the T cell. On its own this TCR interaction will ultimately lead to anergy, a state of unresponsiveness that maintains peripheral tolerance.

During the initial TCR:MHC interaction, the T cell requires a second signal for the priming process. This second signal is referred to as costimulation (**Figure 1**), and can be achieved by a number of different interactions, although the most common activating signal is between CD28 on CD4 T cells and B7 molecules on DCs. The expression of these costimulatory molecules are upregulated on DCs after DC activation by inflammation, increasing the likelihood that T cells will be activated only when the antigen is encountered by the DC under appropriate conditions. While this second signal prevents anergy, a third signal is required to complete CD4 T cell priming and instruct the differentiation pathway.

Differentiation is a critical step in CD4 T cell priming because of the eclectic capacities of CD4 T cells. Once a CD4 T cell has recognized antigen presented by MHC-II and a costimulation signal, a third cytokine signal (**Figure 1**) will instruct the CD4 T cell to differentiate into a subset trained for a particular function. In

co-receptor shown stabilizes the T cell interaction with MHC-II. Signal 2: activated DCs (dendritic cells) upregulate the costimulatory molecules B7.1 and B7.2 (also called CD80 and CD86). CD28 on T cells recognize these costimulatory molecules as a second signal for activation. In the absence of costimulation T cells undergo anergy or death. Signal 3: the cytokine environment instructs the final stage of T cell priming by determining the differentiation pathway undergone by the activated T cell.

this review, we focus on the Th1 subset of CD4 T cells, in which interleukin-12 (IL-12) allows sustained upregulation of the transcription factor T-bet and, upon re-stimulation, production of effector cytokines such as IFN-γ, TNFα, and IL-2. Additional CD4 subsets include Th2 (which respond to IL-4 and are regulated by GATA3),Th17 (combinations of TGF-β, IL-6, IL-21, and IL-23 lead to RORγt expression), Tfh (T follicular helper, respond to IL-6 and IL-21 to upregulate Bcl-6), and iTreg cells (induced T regulatory, TGF-β and IL-2 lead to expression of Foxp3), as well as other, less well-characterized subsets of CD4 T cells. It is important to note, however, that substantial evidence now supports the impermanence of some of these differentiation pathways, a concept known as effector plasticity. Thus, while CD4 T cells require these initial differentiation instructions, they often retain the capacity to acquire new functions under sufficient alternative stimulation.

The initial process of T cell activation dramatically alters the cell, causing upregulation of cascades of transcription factors, as well as altering the miRNA regulation, epigenetic modifications, and post-translational pathways. These changes program the cell with the capacity to rapidly respond upon re-stimulation in a specialized manner. However, the actual secretion of effector cytokines still requires some regulation to prevent unnecessary inflammation. For this reason, activated T cells arrive at sites of infection primed for rapid response, but not constitutively secreting cytokines. Traditionally, the secondary interactions of activated T cells at sites of infection are believed to consist of additional antigen-dependent interactions of the TCR and peptide-MHC

complexes, which triggers a transient robust effector response in the appropriate location (17). Unlike the initial activation process, antigen-specific interaction alone is sufficient to induce cytokine production, because of the T cell's activated state (18). However, much of this work has been conducted in low-inflammatory conditions and focused on the requirement for TCR interactions. Such work does not rule out a role for inflammatory stimulation of T cells during infections. Indeed, the ability of inflammatory cytokines to either activate naïve T cells or stimulate cytokine secretion from effector T cells will be discussed in the next section.

After initial activation, T cells undergo massive clonal expansion. This expansion of specific effector Th1 cells typically takes a few days, and in prolonged infections like *Salmonella* this T cell response can take a few weeks to reach the peak of expansion. During this time, T cells are responding to a complex network of signals, including IL-7 to survive, IL-2 to proliferate, pro-inflammatory cytokines, anti-inflammatory cytokines, and potentially secondary TCR signals. The combination of these encounters does more than just stimulate T cells to produce effector cytokines – it establishes their fate. While T cell responses are critical to pathogen clearance in many cases, they also have the potential to damage host tissues and for this reason, most T cells will ultimately be instructed to die. Thus, after the peak of clonal expansion T cells undergo a contraction phase in which most T cells receive apoptotic signals and are removed from circulation.

However, some T cells receive just the right combination of stimuli and survival signals to transition from an "armed and ready" effector state to a quiescent memory state. CD4 T cells can exist in a number of different memory states, which may ultimately affect their longevity, the areas in which they circulate, and the requirements for re-activation. The best described examples of CD4 memory subsets are the central versus effector memory T cells, which circulate in lymphoid tissue or non-lymphoid tissue, respectively. CD8 T cells are also believed to form these subsets, as well as memory populations called short-lived effector cells (SLECs) or memory precursor effector cells (MPECs) whose formation depends heavily on the inflammatory signals received, but which have not been described for CD4 T cells. Memory T cells are an important component of the rapid response to re-challenge with previously encountered pathogens because of their lowered activation threshold, pre-differentiated state, and extensive epigenetic modifications that allow for rapid relay of the signals needed to elicit effector function. Understanding how these memory T cells are formed and are able to respond is especially crucial to vaccine design.

While the above mechanisms comprise a very basic understanding of conventional T cell activation, there are a number of caveats and exceptions that are worth discussing. In Section "Bystander Activation and Non-Cognate Stimulation,"we will discuss the ability of T cells to respond to non-TCR stimuli, focusing on what has been referred to in the literature alternatively as bystander activation or non-cognate stimulation. A partial mechanism for non-cognate stimulation of Th1 cells is illustrated in **Figure 2**. In Section "Innate-Like T Cells and ILCs," we will outline some of the non-conventional T cell subsets that are able to respond to non-cognate stimuli to draw parallels between these "innatelike" cell types and the innate-like functions of classically activated

T cells. **Figure 3** compares the interactions that occur in each of these cell responses and highlights areas that require further elaboration.

non-cognate elicitation of IFN-γ. CD4 T cell stimulation at sites of infection likely involves additional cytokine pathways, including IL-12, which can act

synergistically with IL-18 to stimulate Th1 cells.

**FIGURE 3 | Elicitation of CD4 and "innate-like"T cell effector functions**. T cell effector functions can result from interactions with APCs or cytokines at various stages. In the conventional T cell response (first column, blue), naïve T cells are activated by direct interactions with APCs in the secondary lymphoid tissues, proliferate, migrate to sites of infection, and then are stimulated by secondary direct APC contact to produce effector cytokines. Some of these effector T cells will go on to become memory T cells. T central memory (TCM) cells circulate in secondary lymphoid tissues, and upon re-activation will once again proliferate and differentiate into effector cells. T effector memory (TEM) cells can either migrate in the periphery or be resident in tissue, and respond more rapidly than the TCM cells because upon re-activation they can secrete effector cytokines directly. The signals that result in the TEM or TCM fate decision are still unclear, as are the interactions required by each for re-activation, although it is presumed that re-activation occurs after direct interaction with an APC. Non-cognate stimulation (second column, indigo) occurs at sites of infection in T cells that have already undergone conventional activation in the secondary lymphoid tissues. Instead of being stimulated by secondary direct APC contact, these cells receive stimulatory signals from cytokines that induce IFN-γ production. Whether these cells go on to join the memory pool and how this different stimulation signal affects the fate decision of CD4 T cells is unknown. Bystander activation is a term that has been used loosely to mean any TCR-independent T cell stimulatory interaction. In the third column (purple), we focus on the idea of bystander activation as a mechanism to prime a CD4 T cell in a TCR-independent manner. While the effect of cytokines on naïve T cells have been studied at length in vitro, there is limited evidence that a naïve CD4 T cell can be activated by cytokine signals alone, and no evidence that TCR-independent activation (Continued)

#### **FIGURE 3 | Continued**

can produce a fully functional effector T cell. The last column (pink) provides a general representation of innate-like T cell subsets. Although the initial priming signals for the different innate-like T cell populations vary and are still unclear in some cases, they include alternative activation mechanisms such as restricted TCRs or constitutive priming. Stimulation of effector responses at sites of infection in innate-like T cells is known to occur rapidly in response to cytokine stimulation, hence the name "innate-like" T cells. However, it is possible that all T cells have the capacity, once activated, to respond rapidly to cytokine stimulation, and that what really separates these innate-like cell types are their unique priming mechanisms.

#### **BYSTANDER ACTIVATION AND NON-COGNATE STIMULATION**

The strict processes governing conventional T cell activation are important to avoid the uncontrolled activation of effector T cell responses. However, under some circumstances, such as during a rapidly dividing or systemic infection, these may become a hindrance to achieving the necessary strength and rapidity of the effector response. Thus, non-cognate stimulation of conventional T cells has been described in a number of model systems. Noncognate interactions are defined negatively as any stimuli without TCR recognition of cognate peptide-MHC complexes presented on APCs. This type of T cell activation has often been referred to as "bystander activation" (19–22). This name seemingly refers to the idea that these are T cells which just happen to be in proximity to inflammatory stimuli that is perhaps intended for other cognate T cells, thus, assuming that non-cognate stimulation is an accidental "bystander" to the conventional response. However, it is equally possible that this "bystander" response is not incidental, but instead represents an integral functional capacity of T cells to respond and recognize inflammatory stimuli that are produced under extreme stress.

The earliest descriptions of bystander activation focus on cytokine or innate stimuli that drive T cell proliferation in the absence of antigen (20, 21, 23). These innate stimuli are also referred to as microbe-associated molecular patterns (MAMPs), and include toll-like receptor (TLR) and NOD-like receptor (NLR) ligands among others. However, it should be noted that most of this early work was completed in viral infection models, focuses largely on CD8 T cells, and does not differentiate between naïve and previously activated T cells. Thus, it is difficult to conclude from these studies whether naïve T cells can actually be primed by non-cognate interactions, particularly naïve CD4 T cells. Further, these data must be interpreted with caution, since cytokineinduced proliferation may not lead to an effector T cell state, and especially given the evidence that these same signals can induce apoptosis (20). Transient expression of the early activation marker CD69 was observed in naïve CD8 after Type I IFN stimulation, but neither this activation was not maintained nor was it shown to induce effector functional capacity (21).

In addition to non-cognate proliferation or upregulation of activation markers, there is also considerable work describing elicitation of effector functions from CD8 T cells by non-cognate stimuli, which is also confusingly referred to as bystander activation or stimulation (19, 24, 25). However, it is important to note that this work generally describes stimulation of previously activated T cells, or makes no distinction between activated and naive T cells, thus, this is not activation in the sense of initial T cell priming. For clarity, in **Figure 3**, a non-cognate primary activation interaction is illustrated as "bystander activation," while a non-cognate interaction following (but separate from) a cognate primary activation interaction is referred to as "non-cognate stimulation." This stimulation of activated T cells is discussed further below.

While most of the work described above examines CD8 T cells, less examination of TCR-independent effector responses exists specific to CD4 T cells, and most shows that bystander responses occur only in previously activated or memory CD4 T cells (26– 28). There are, however, limited *in vitro* data showing that very high doses of IL-2 can make naïve CD4 T cells responsive to IL-12 or IL-18 without TCR signaling (29). Considering these distinctions, as well as the inability of many of these studies to rule out innate-like populations that were not yet identified at the time the studies were conducted, the question of whether naive T cells can be primed in a non-cognate manner under sufficient inflammatory stimulation, and whether these cells would be functional as effectors *in vivo*, lacks a definitive answer. However, non-cognate stimulation of effector and memory T cells has been shown, and some of the mechanisms are beginning to be understood.

As described in the previous section, an activated Th1 cell is primed to produce effector cytokines rapidly. Here,we focus on the effector cytokine IFN-γ, secreted by both CD8 T cells and Th1 cells. This priming means that the IFN-γ gene locus is modified to be open for faster transcription, IFN-γ mRNA has been transcribed and is ready for rapid translation, and IFN-γ protein has been translated and awaits the signals necessary for post-transcriptional modifications and secretion. Despite all of this preparation, Th1 do not constitutively secrete IFN-γ. When they receive antigenspecific TCR signals, the signaling cascade downstream of the TCR allows rapid release of the prepared IFN-γ transcripts and protein from the regulatory mechanisms that otherwise keep this production in check. However, other signaling pathways can also stimulate IFN-γ production in activated Th1.

The signals eliciting production of IFN-γ in various cell types have been studied extensively, and yet the complexity of the regulation of this cytokine continues to unfold. For example, while T-bet is considered to be a master transcriptional regulator of IFNγ production, in natural killer (NK) cells and CD8 T cells Eomes is able to make up for the loss of T-bet, while in CD4 T cells T-bet signaling is required (30). Further, it was shown that the promoter region of IFN-γ that is utilized after TCR signaling is different than the promoter region required for IFN-γ production in response to IL-12 and IL-18 (31). In addition to transcriptional regulation, extensive post-transcriptional regulation of IFN-γ mRNA has been shown to take place, and varies between naïve, effector, and memory T cell populations (32).

While some evidence has suggested that an ability for innate ligands to interact directly with CD4 T cells to stimulate their proliferation or function (33), it is also likely that innate ligands can stimulate T cells through a second messenger that would allow amplification of the signal. Many cells are capable of responding to TLR or NLR ligands to produce inflammatory cytokines,including those cytokines known to stimulate IFN-γ production from Th1 cells. Thus, it is easy to imagine a mechanism whereby Th1 cells respond indirectly to innate stimuli by responding to inflammation. In fact, this has been shown to occur during viral infections in response to TLR ligands, and during *Salmonella* infection in response to NLR ligands, both in CD8 T cells (24, 25, 34).

Recently, we described a mechanism for innate Th1 stimulation that relies on the convergence of both TLR and NLR signaling pathways to elicit IL-18 production, which can then be recognized by activated Th1 to result in IFN-γ secretion. This mechanism is illustrated in **Figure 2**. Previous work has typically focused on very small populations of innately responding T cells, particularly for CD4 cells, consequently supporting the concept of the "unintended bystander." However, the small numbers of T cells that can be seen responding at any given time during the normal course of infection do not necessarily represent a small subset of cells capable of innate response. Nor does this imply that innate stimulation of T cells occurs too infrequently to have a significant impact. Using *Salmonella* infection, we showed that Th1 cells in an infectious model of strong Th1 activation are highly susceptible to innate stimulation, with a large proportion of the Th1 capable of secreting IFN-γ in response to LPS stimulation. Finally, we demonstrated that mice whose T cells lack the capacity to be innately stimulated by IL-18 suffer a reduced capacity to clear *Salmonella* infection (35). Together, this suggests that a pathway of innate T cell response that not only can occur but must also occur for normal immune function.

#### **INNATE-LIKE T CELLS AND ILCs**

While the previous section focused on the non-cognate interactions of conventional αβ T cells, numerous cell types have been described that have overlapping surface markers, developmental lineages, transcription factor profiles, or effector functions to conventional T cell subsets, but which respond in a non-conventional manner (36). These cells are often thought of as innate-like cells with adaptive-like functions that can provide critical assistance in the early immune response. Examples include natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells, γδ T cells, and innate lymphoid cells (ILCs).

Innate lymphoid cells are a rapidly expanding group of cells defined predominantly by their lack of lymphocyte antigen receptors (TCR or BCR, B cell receptor) or lineage-specific markers (36). Recently, a uniform nomenclature for ILCs was proposed that divides the various cells into three main groups. Much like the CD4 T helper subsets, Group 1 ILCs can be characterized by the expression of T-bet and IFN-γ, Group 2 express GATA3, and Group 3 express RORγt and produce IL-17 or IL-22 (37). Most ILCs require IL-7R signaling and express the surface marker CD90 and the transcription factor ID2, although it has been proposed in mice that IL-7 may inhibit transition of ILC3 to ILC1 (38). Further elaboration will center on the Group 1 ILC subset, given its similarity to the Th1 subset of CD4 T cells.

Natural killer cells have been included within this nomenclature as a Group 1 ILC, alongside ILC1. While NK cells have been very well-described as an early source of IFN-γ and TNFα, in addition to their cytotoxic functions, very little is known about ILC1. Although there is evidence that they develop from ILC3s after IL-12 stimulation, it was recently shown that ILC1 can also develop independently from a common innate lymphoid progenitor (39). However, whether they arise separately or as a consequence of functional plasticity, it remains that there is a group of T-bet+ ILCs, which can respond to IL-12 and IL-18 signals to produce IFN-γ, but which are not NK cells and do not exhibit cytotoxicity (37, 38). As a whole, the early innate effector responses of the Group 1 ILCs during intracellular infections play a key role in host protection, inflammation, and initiation of adaptive responses.

While ILCs lack a TCR, several other cell types express unique TCRs that allow for a non-conventional response, known collectively as "innate-like T cells" due to their ability to respond rapidly to innate stimulation. Among these innate-like T cells, one common method allowing for a non-cognate-antigen response is an invariant or semi-invariant TCR. While conventional TCRs undergo recombination activating gene (RAG) dependent rearrangement of their α and β chains during development in the thymus to allow for a broader repertoire with improved specificity, some innate-like cell populations possess TCRs with single α-chain and restricted β-chain specificities. Particular examples of cells with these alternative TCRs include invariant NKT cells (iNKT) and MAIT cells (36).

Natural killer T cells are perhaps the best described of these innate-like T cell subsets. NKT cells are innate-like T cells in the sense that they develop in the thymus and express a TCR, but they also express NK1.1 and several innate activating or inhibitory receptors typically found on NK cells (40). Two types of NKT cells exist: NKT I are the well-described, iNKT cells known to respond to lipids and especially with high affinity to α-galactosylceramide presented by the MHC-related molecule CD1d, while NKT II are less studied, to date, but have a diverse TCR repertoire and fail to respond to α-galactosylceramide (41). While iNKT can be activated by CD1d presentation of foreign lipid antigens, they may also be activated by CD1d presentation of lipid self-antigens and require inflammatory cytokine signals, allowing for more rapid and innate-like responses (40–42). Further, it was recently shown that iNKT can be activated in an antigen-independent manner by cytokine alone during some infections, like *Salmonella*, but not others (43).

Another semi-invariant T cell population are the recently described MAIT cells, characterized by their localization to mucosal tissue and their recognition of the MHC-related molecule, MR1, which binds the metabolites of B vitamins generated by bacteria and fungi (36). MAIT cells develop and are pre-programed in the thymus, but quickly acquire an activated phenotype in the periphery. There is now evidence to suggest that this activation occurs in response to microbiota; in particular, the observation that germ-free mice have diminished numbers of MAIT cells, which can be recovered upon monoculture reconstitution with many bacteria or yeast, but not *Enterococcus faecalis*, which lacks the riboflavin metabolic pathway. Unlike NKT, they seem to respond predominantly to TCR ligation and do not require cytokine stimulation to elicit effector functions, which consist mostly of IFN-γ and TNFα, although they can also express IL-17 (44, 45).

γδ T cells are a unique exception, in that they possess recombined TCRs, but can respond in a rapid, innate-like manner to inflammatory cytokines. Thus, these T cells are technically a component of the adaptive immune response, but are often discussed in the context of early innate responses (46). Differentiation programing of γδ T cells occurs during thymic development, determining either an IFN-γ, IL-17, or IL-4 producing phenotype (47), but peripheral activation is still required before effector functions can be elicited. The relative contributions of TCR, costimulation and cytokine signals to this activation still seem to be a matter of some debate, and may be partially dependent on the subset, but whatever the mechanism these cells respond far more rapidly than their αβ T cell counterparts (46).

All of these innate-like T cells (iNKT,MAIT, and γδT), although possessing different TCRs and recognizing different antigen repertoires, share some common features. For one thing, in each cell type the ability to generate or maintain immunological memory is poorly defined, as are the required signals for survival and proliferation (36). For both iNKT and γδ T cells functional subsets have now been described analogous to the CD4 T helper subsets (40, 47), although unlike CD4 T cells these subsets are pre-determined during development in the thymus. Although subsets have not yet been defined as such for MAIT cells, and they typically respond to IL-12 to produce IFN-γ in a T-betdependent manner, they also express RORγt and can express IL-17 and IL-22 under appropriate stimulation. Further, there is now some evidence for an immunoregulatory function of MAIT cells. Thus, whether MAIT cells have functional subsets or are simply functionally promiscuous remains to be determined (44, 45).

In further similarity, each is described as "innate-like" due to an ability to rapidly respond to innate stimulation – that is, they respond to the inflammatory cytokines that result from innate stimuli. However, for each cell subset the specific requirements of initial priming, and in particular whether this priming can occur without any peripheral TCR stimulation, is still a matter of debate within their respective fields. While earlier literature suggested that these cells respond rapidly because they are able to respond to cytokine alone, other work shows that these cells require TCR interactions (48), and for iNKT at least this requirement can be met by self-antigens under inflammatory conditions to allow more rapid responses (40–42). More recent evidence suggests that these requirements may differ under varying circumstances (43). Herein, we make an argument that conventional αβ T cells can also respond rapidly to inflammatory cytokines in an innate-like manner once they have been primed. The parallels between these responses and their mechanisms suggest that a conservation of these stimulatory mechanisms between conventional and innate-like T cells, and highlight the need for a better understanding of the activation requirements of non-conventional T cells.

### **A COMPLICATED RELATIONSHIP: THE DYNAMIC INTERACTIONS OF SALMONELLA AND Th1 CELLS SALMONELLA: A PERSISTENT GLOBAL CHALLENGE**

While many bacteria live and replicate extracellularly, entering the host cell only when engulfed and destroyed by phagocytes, some bacteria have adapted unique survival strategies to allow a protected life cycle within host cells. Some of these bacteria are obligate intracellular pathogens, like *Chlamydia*, that cannot replicate outside of the cell, but many intracellular bacteria are capable of occupying either space. The immune system has, in turn, developed a number of ways to recognize pathogens within cells, pathways which the pathogen actively attempts to thwart (49).

In this review, we focus on *Salmonella*, a Gram-negative enteric pathogen that resides predominantly within the phagosomes of macrophages located in the spleen, liver, and bone marrow. In human beings, there are two forms of systemic salmonelloses: typhoid fever and non-typhoidal salmonellosis, or NTS. Typhoid and paratyphoid fevers are caused by the human-specific pathogens *Salmonella enterica* serovar Typhi and Paratyphi, still occur endemically in developing countries, and can cause severe systemic disease even in healthy individuals. Estimates range as high as 27 million annual infections with either Typhi or the clinically indistinguishable Paratyphi. In contrast, NTS occurs only in immunocompromised individuals, but can originate from any of the >2000 *Salmonella* serovars capable of causing foodborne illness in human beings and harbored in a wide variety of animal reservoirs (50). Thus, both systemic infections remain a source of concern for public health officials worldwide.

Although antibiotics effective against *Salmonella* are available, the options are relatively limited for intracellular pathogens as compared to more accessible extracellular pathogens. Additionally, among those antibiotics currently available, there is a growing incidence of drug resistance, including multi-drug resistance to the first-line treatments, and resistance to the now standard fluoroquinolones. Further, decreased susceptibility to the fluoroquinolone ciprofloxacin has been associated in enteric fever patients with prolonged fever and increased rates of treatment failure (51). Finally, an analysis of the *Salmonella* metabolic pathways has suggested that most of the major or non-redundant pathways have already been targeted or considered for drug inhibition, suggesting that a limitation to prospective future development of new antibiotic treatments (52). Together, these studies emphasize the need for alternative treatment options and for improved vaccination strategies that could lessen the need for, and consequently the selective pressure upon, traditional antibiotic therapy.

Currently, two vaccinations are commercially available in the U.S. for travelers to typhoid endemic countries. One is a Vi capsular polysaccharide (ViCPS) vaccine administered intramuscularly as one dose at least 1 week prior to exposure. The second is an oral, attenuated Ty21a vaccine available under several formulations, typically administered every other day as three separate doses 2 weeks prior to exposure. Both vaccinations suffer from limitations that impair their practicality in typhoid endemic regions, not the least of which is the need for regular re-vaccination, and the low-reported efficacy at 3 years of 51–55% (53). The ViCPS vaccine is approved in children over the age of 2 years old, and the oral vaccine for children over the age of 5 years, while repeated exposure before the age of 5 in endemic areas has been shown. This suggests that the vaccines available miss a key population.

In addition, while evidence suggests that Ty21a may be crossprotective for paratyphoid, the ViCPS vaccine targets an antigen that does not exist in Paratyphi and even some strains of Typhi (51, 54). Further, because the oral vaccine is a live, attenuated *Salmonella* strain, it is not suitable for use in immune-compromised patients, posing a challenge to widespread use in areas co-endemic for HIV. Thus, currently available vaccination strategies are not adequate to allow control of systemic typhoidal disease (53). Whether currently available vaccines mediate any protection to non-typhoidal systemic diseases has not been thoroughly characterized. These data emphasize a need to better understand the immune response during systemic *Salmonella* infections, to inform better vaccine design.

#### **IMPORTANCE OF Th1 CELLS AND IFN-**γ **IN INTRACELLULAR INFECTIONS**

As mentioned earlier, some bacteria and other pathogens have developed the capacity to reside within cells and effectively hide from extracellular immune recognition. Often, these pathogens enter the cells initially using the cells' own phagocytic capacity, but then are able to escape phagolysosomal degradation or escape the phagosome entirely, by a wide range of different mechanisms (49, 55). Given this unique lifestyle,intracellular pathogens require a special type of immune response designed to recognize infected phagocytes and mediate either killing of the infected cell or internal pathogen killing mechanisms. CD8 T cells have cell-specific cytotoxic capacity, allowing directed killing of infected cells, while both CD8 and CD4 T cells can secrete pro-inflammatory cytokines that activate phagocytes to initiate internal mechanisms of pathogen destruction.

CD8 T cells have multiple cytolytic capacities initiated by TCR interactions, including release of secretory granules and death receptor-mediated apoptosis. However, while these responses have a critical role in anti-viral defenses, their role against other intracellular pathogens is limited (56). Of more importance to intracellular bacterial infections, CD8 can produce pro-inflammatory cytokines such as IFN-γ, which activates macrophages to undergo changes that alter the intracellular environment to become less hospitable to the invading pathogen. While CD8 T cells respond to MHC-I complexes that mostly present antigen processed from the cytosol of nearly all cell types, CD4 T cells respond to MHC-II presented antigen on special APCs derived from the endocytic pathway. This allows CD4 T cells to recognize antigens from pathogens hiding inside of cells within endosomes, as well as antigens taken up from outside of the cell (57). Reliance on different antigen processing pathways and consequent MHC presentation allows a partial division of labor between the CD8 and CD4 T cells, although they have retained some redundancy in critical functions, such as production of IFN-γ.

Among the various CD4 T cell subsets, the CD4 Th1 cells provide the primary response to intracellular pathogens. As mentioned earlier, after CD4 T cells are activated they receive a differentiation signal that determines their cytokine profile. In Th1 cells, IL-12 upregulates the transcription factor T-bet, which is required by CD4 T cellsfor IFN-γ production (58). Once activated,Th1 cells are programed to secrete pro-inflammatory cytokines that include IFN-γ, TNFα, and IL-2 upon re-stimulation. In contrast, CD8 T cell priming is not usually thought of in terms of differentiation of particular subsets with distinct functions, although functional subsets have been described. Termed Tc1, Tc2, and Tc17 in reference to their Th counterparts, these cells are found in relatively low-frequency under normal circumstances, and the mechanisms driving the development of these alternative CD8 T cells remain poorly understood (58). Signal 3 was initially identified in CD4 and CD8 T cells simply as the inflammatory cytokine(s) required to induce proliferation and differentiation to effector capacity (59).

Type I interferons and IL-12 have both extensively been shown to result in the survival, expansion, and differentiation of CD8 T cells, and are critical for the conventionally described CD8 T cell effector responses, including cytolytic activity and IFN-γ production (59–62).

Additional differences between CD4 and CD8 T cell activation include, but are not limited to a shorter required duration of antigenic stimulation in CD8 T cells (63–65), different transcriptional regulation, including partial redundancy for the transcription factors T-bet and Eomes in IFN-γ production in CD8, but not CD4 (30), and differences in cellular trafficking and antigen surveillance (66). Further, evidence suggests that CD4 T cells can help to initiate CD8 T cell priming (67, 68), and may be required for optimal CD8 memory formation, a regulatory interaction that argues against mechanistic redundancy in the activation process. Thus, while T cells have evolved to share many similar pathways, CD4 and CD8 T cells are distinct cell types with different functions and rules to govern them. When studying T cell functions it is critical to keep these differences in mind and to choose a model system capable of demonstrating the full potential of the cell of interest. Accordingly, the strong requirement for Th1 cell functions in *Salmonella* infections makes this model system ideal for study of Th1 responses (9–11).

The cytokine IFN-γ is especially important during intracellular infection because of its critical capacity to activate macrophages to become M1, or classically activated, macrophages. M1 macrophages modify their internal environment to become as inhospitable as possible, including production of anti-microbial compounds like reactive oxygen and nitrogen species, as well as themselves secreting pro-inflammatory cytokines (69). The importance of IFN-γ-mediated macrophage activation is highlighted by the effects, resulting from the loss of IFN-γ or IFNγ-inducing cytokines and transcription factors (11, 70, 71). In both mice and human beings, loss of IFN-γ results in an inability to effectively clear intracellular pathogens (72). Further, in human beings with chronic granulomatous disease or mycobacterial granulomas, IFN-γ is an effective, albeit toxic, therapeutic (73–75). Combined, these studies clearly demonstrate the requirement of IFN-γ for effective clearance of intracellular pathogens.

### **PLAYING HARD TO GET: HOW SALMONELLA SUBVERTS THE Th1 CELL RESPONSE**

The critical role of Th1 cells in *Salmonella* clearance makes them an obvious target for immune evasion strategies. While there is extensive information available on the ways that *Salmonella* has found to manipulate the system (76, 77), we focus here particularly on immune evasion strategies that impair the ability for T cells to recognize their specific antigen. Three of the ways that *Salmonella* have developed to achieve this include downregulation of antigens that may be recognized by T cells, effects on TCR expression or function, and impairment of MHC processing or presentation of peptides (**Figure 4A**). The active avoidance of TCR recognition employed by *Salmonella* provides one possible explanation for the maintenance of a TCR-independent T cell stimulatory pathway.

When *Salmonella* change environments, from contaminated source to intestine to myeloid cells, their expression of antigens recognized by T cells rapidly changes (8). In addition to responding to a change in needs, this antigenic shift acts as a highly effective immune evasion strategy, resulting in the activation and expansion of large numbers of T cells that specifically recognize antigens that will not be present at the site of infection. The T cell response to FliC was shown to be inefficient at resolving infection over a decade ago, and both SipC and FliC elicit an early T cell response despite rapid downregulation of these antigens by *Salmonella* (7, 78, 79). Thus, much of the early T cell response may develop to antigens that are not available at the site of infection, preventing cognate activation for these cells.

Additionally, a number of mechanisms have been demonstrated by which *Salmonella* causes downregulation of the TCR on T cells (80). Flagellin stimulation has been shown to result in upregulation of SOCS1, which impairs TCR expression in T cells (81). Further, direct contact of *Salmonella* with T cells results in secretion of the enzyme l-asparaginase II by *Salmonella*, which breaks down l-asparagine and consequently impairs T cell blastogenesis, proliferation, and cytokine secretion by downregulating the TCR β chain (82, 83). While these mechanisms may impair the initial priming of T cells, the requirement for direct contact between T cells and bacteria in some of these studies suggests that the importance of these evasion techniques at sites of infection.

Finally, many strategies have been demonstrated by which *Salmonella* is able to inhibit either the processing of antigens into peptides or the presentation of these antigenic peptides on the surface of APCs within the MHCs (80, 84). Nearly 20 years ago, the twocomponent regulatory system member PhoP was shown to impair processing and presentation of antigens in macrophages (85). Further, numerous *Salmonella* pathogenicity island-2 (SPI-2) effector proteins have been implicated in impaired MHC function, including impaired loading of peptides onto MHC, prevention of lysosomal degradation that results in decreased peptide availability, and polyubiquitination of MHC that results in degradation rather than surface expression (86–89). Each of these interactions targets a step in the antigen presentation pathway that ultimately results in an impaired ability for infected cells to signal to T cells.

Given the hindrance of cognate T cell stimulation, the ability for T cells to be stimulated by non-cognate interactions as well could play an important role in *Salmonella* clearance. *Salmonella* induces a number of different non-cognate responses via PRRs, as outlined in **Figure 4B**. These include well-characterized TLR and NLR interactions, as well as other, less well-defined PRR interactions (90). Recognition of these various non-cognate ligands results in inflammation, including production of inflammatory cytokines that can stimulate T cells. This indirect stimulation of T cells in response to non-cognate *Salmonella* products provides a complementary mechanism for T cell stimulation at sites of infection with a broad array of conserved triggers. While the multitude of Th1 evasive mechanisms accentuates the need for innate signaling pathways in the elicitation of Th1 cell functions, it is important to note that innate immune pathways are not exempt from evasion strategies (91, 92). Thus, in order to provide T cells the best chance to encounter and respond to signs of infection, redundant mechanisms for stimulation that rely on either cognate antigen or MAMP-driven inflammation have developed.

of the TCR. These strategies aim to block cognate antigen recognition each step of the way, making additional non-cognate mechanisms for T cell stimulation crucial. **(B)** Many innate immune recognition pathways respond to

downregulation of MHC itself, and (3) effects upon the expression or function

Salmonella is unknown. NLRP3 recognizes a number of nuclear components, which act as danger signals when present in the cytosol, but other ligands have been proposed. (3) Although less well-characterized, other classes of PRRs have the potential to recognize Salmonella, including the various cytosolic nucleic acid sensing receptors.

## **DISCUSSION**

Herein, we have discussed a number of advantages to studying Th1 cell responses in a *Salmonella* model. The strong understanding of both CD4 T cell and innate immune pathways during the response to *Salmonella* infection provides a strong foundation to further explore the potential interaction between these pathways. Moreover, the tools for these studies are readily available in such a well-studied and easily manipulated model pathogen. Together, these factors make *Salmonella* an ideal model pathogen for the study of non-cognate Th1 cell responses.