MAIT cells and pathogen defense

Abstract

Mucosa-associated invariant T (MAIT) cells are a unique population of innate T cells that are abundant in humans. These cells possess an evolutionarily conserved invariant T cell receptor α chain restricted by the nonpolymorphic class Ib major histocompatibility (MHC) molecule, MHC class I-related protein (MR1). The recent discovery that MAIT cells are activated by MR1-bound riboflavin metabolite derivatives distinguishes MAIT cells from all other αβ T cells in the immune system. Since mammals lack the capacity to synthesize riboflavin, intermediates from the riboflavin biosynthetic pathway are distinct microbial molecular patterns that provide a unique signal to the immune system. Multiple lines of evidence suggest that MAIT cells, which produce important cytokines such as IFN-γ, TNF, and IL-17A, have the potential to influence immune responses to a broad range of pathogens. Here we will discuss our current understanding of MAIT cell biology and their role in pathogen defense.

Introduction

The primary purpose of the immune system is to quickly detect invading pathogens and eliminate them from the host. This requires an elaborate network of cells and receptors that have the capacity to discriminate self from non-self [1]. Classically, the immune system has been divided into two separate ‘arms’ that broadly reflect their specificity, speed, and capacity to develop memory, termed innate and adaptive immunity [2]. Innate immunity relies primarily on germline-encoded ‘pattern recognition receptors’ that function as an early surveillance system [1]. Following detection of unique molecular patterns that are foreign to the host, cells expressing these innate immune receptors quickly elaborate anti-microbial effector molecules, cytokines, and chemokines that act as an initial defense, but also influence the ensuing adaptive immune response.

In contrast, adaptive immunity is slower to develop, but frequently required to fully eradicate the pathogen. Adaptive immune cells (composed primarily of conventional B and T lymphocytes) express a highly diverse collection of antigen-recognition receptors that have undergone somatic gene rearrangements [3]. Following selection in the thymus, the emerging adaptive immune cells are generally naïve, with each cell expressing a unique receptor and present in low frequencies in the circulation. Importantly, conventional adaptive CD8⁺ and CD4⁺ T cells recognize only peptide antigens presented by highly polymorphic major histocompatibility complex (MHC) class I or class II molecules, respectively [4]. It is only after recognition of their cognate antigen that these low frequency naive T cells undergo clonal expansion and acquire effector functions, a crucial process that requires several days.

Innate T cells have the capacity to act during this critical lag time while adaptive immune responses develop. Similar to conventional T cells, innate T cells possess somatically rearranged T cell receptors (TCR) that have undergone thymic selection, but unlike their conventional counterparts, innate T cells recognize non-peptide microbial molecular patterns (or danger signals) and rapidly produce effector functions after activation [5]. This property, coupled with their relatively large clonal sizes, allows innate T cells to mount effector immune responses much earlier than conventional T cells, and potentially act as a ‘bridge’ between innate and adaptive immune responses.

Conventional CD4⁺ and CD8⁺ T cells recognize a wide range of peptide antigens through their expression of a TCR heterodimer composed of highly variable α-chains and β-chains. In contrast, two distinct innate-like αβ TCR T cell populations have been identified that have restricted T cell repertoires characterized by the expression of an invariant or semi-invariant TCRVα chain (Table 1). Mucosa-associated invariant T (MAIT) cells were first shown to express the canonical Vα7.2-Jα33 chain in humans (and the orthologous Vα19-Jα33 in mice), while invariant natural killer (iNKT) cells express the invariant Vα24-Jα18 chain in humans (Vα14-Jα18 in mice) [6–8]. Subsequent studies have identified additional human MAIT cell populations characterized by TCRα chains with alternative TRAJ gene segments, which include Vα7.2-Jα12 and Vα7.2-Jα20 rearrangements [9, 10]. Pairing of these specific TCRα chains with one of several Vβ chains results in a TCR with limited ligand discrimination [10]. For iNKT cells, the resulting semi-invariant TCR recognizes lipid-based ligands presented by CD1d molecules [7]. Interestingly, MAIT cells recognize a novel class of microbial molecular pattern, recently shown to be derived from vitamin B-based metabolites, which are presented by the nonpolymorphic class Ib molecule MHC-related protein 1 (MR1) [11, 12].

Table 1.

Key differences between MAIT cells and iNKT cells

	iNKT cells	MAIT cells
MHC restriction	CD1d	MR1
Antigen	Glycolipids	Metabolic intermediates derived from the riboflavin biosynthetic pathway
T cell receptor	TCRVα14-Jα18 (mouse) or Vα24-Jα18 (human) paired with limited Vβ chains	TCRVα19-Jα33 (mouse) or Vα7.2-Jα33/12/20 (human) paired with several Vβ chains
Activation	CD1d-bound microbial ligands; endogenous CD1d ligands; cytokines (IL-12 and IL-18)	MR1-bound microbial ligands; endogenous MR1 ligands unknown; cytokines (IL-12 and IL-18)
Acquisition of effector phenotype	Exit the thymus expressing PLZF and an effector memory phenotype	Exit the thymus naïve; acquire PLZF expression and effector memory phenotype in the periphery, prior to birth
Abundance	<1 % of human peripheral blood T cells (CD3+)	1–10 % of human peripheral blood T cells (CD3+)
Response kinetics	Peak in numbers early, prior to conventional CD4+ and CD8+ T cell responses (murine studies)	Early impact on immune responses, but peak in numbers concomitant with conventional CD4+ and CD8+ T cells (murine studies)
Microbiota influence	iNKT cell numbers increase in GF mice [52]	MAIT cells undetectable in GF mice

iNKT innate natural killer T, MAIT mucosa-associated T, GF germ-free, MR1 MHC class I-related protein, TCR T cell receptor

The MR1 gene is highly conserved among both placental and marsupial mammals, with a predicted amino acid sequence identity of ~90 % in the mouse and human α1/α2 MR1 domains that contact antigen [13–15]. In comparison, the mouse and human CD1d α1/α2 domains exhibit only 60/63 % sequence identity [16]. The extremely high degree of sequence conservation of MR1, coupled with the high amino acid sequence identity of the human, mouse, and cow MAIT TCR Vα chains (~72–75 %) [8], indicates that strong evolutionary selective pressures have maintained these genes, and further suggests an important physiological role for MAIT cells.

Since mammals lack the capacity to synthesize vitamin B, the unique ability of MAIT cells to recognize metabolites derived from bacterial and yeast vitamin B biosynthesis pathways suggests that one of the primary functions of MAIT cells is to detect microbial infection. Here we will review the current state of understanding of the biology of MAIT cells, with emphasis on their role in microbial detection and pathogen defense.

MAIT cell activation by microbes

Although MAIT cells were initially identified in 1993 by Porcelli et al., and further characterized as a distinct T cell subpopulation by Tilloy et al. [8], a role for MAIT cells in pathogen defense only recently became evident when it was observed that both human and mouse MAIT cells were activated by infected host cells [6, 8, 17, 18]. Gold et al. demonstrated that human MAIT cell clones produced IFN-γ in response to Mycobacterium tuberculosis-infected alveolar epithelial cells and dendritic cells [17]. Similarly, Le Bourhis et al. [18] showed that human MAIT cells produced IFN-γ and expressed the activation marker CD69 following co-culture with E. coli-infected monocytes. In both studies, MAIT cell activation was blocked by addition of anti-MR1 antibodies. Subsequent studies have further demonstrated that MAIT cells possess innate anti-microbial activity; MAIT cells purified from pathogen-naïve TCR-transgenic mice expressing the canonical MAIT TCR Vα chain (Vα19i-transgenic mice) exhibited an IFN-γ-dependent ability to inhibit the growth of the intracellular pathogens Mycobacterium bovis BCG and Francisella tularensis in murine macrophages [19, 20].

In vitro studies have demonstrated that MAIT cells are activated by a wide variety of microorganisms, including several bacteria (Lactobacillus acidophilus, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus and epidermidis) and yeast (Candida albicans, Candida galbrata, and Saccaromyces cerivisiae) (Table 2). Consistent with the discovery that microbe-derived riboflavin metabolites activate MAIT cells, several studies indicate that MAIT cells are not activated by microbes lacking the riboflavin biosynthetic pathway [17, 18]. Importantly, this includes all viruses tested to date and some bacteria (Enterococcus faecalis, Listeria monocytogenes, Streptococcus group A). Recent work by Corbett et al. [11] confirmed that the riboflavin biosynthetic pathway is the primary source of microbial ligands that bind the MR1 antigenic cleft; culture supernatants derived from Lactococcus lactis and S. typhimurium mutants with deletions in the early genes for riboflavin synthesis were—unlike supernatants derived from their intact parent strains—unable to activate MAIT cells. Overall, this cumulative data suggests that while MAIT cells are broadly reactive to a wide range of microbes, organisms that lack key genes in the riboflavin biosynthetic pathway are unable to activate MAIT cells via TCR recognition of MR1.

Table 2.

Summary of studies examining microbial activation of MAIT cells in vitro

Microbe	APC type	Activation measure	MAIT cell activation?	References
M. tuberculosis	Human monocyte-derived DCs	IFN-γ	Yes	[17]
	A549 human epithelial cell line	IFN-γ/TNF	Yes	[17]
	Human large airway epithelial cells derived from tracheal brushings	IFN-γ	Yes	[17]
M. smegmatis	Human monocyte-derived DCs	IFN-γ	Yes	[17]
	A549 human epithelial cell line	IFN-γ/TNF	Yes	[9]
E. coli	Human monocyte-derived DCs	CD69 and IFN-γ	Yes	[18]
	Murine BMDCs	CD69 and CD25	Yes	[18]
	Murine fibroblasts	CD69 and CD25	Yes	[18]
	HeLa cells	CD69 and CD25	No	[25]
	Human B cell line and primary human B cells	CD69 and cytokine production	Yes	[26]
	Human monocytic cell line (THP-1)	IFN-γ	Yes	[24]
S. typhimurium	Human monocyte-derived DCs	IFN-γ	Yes	[17]
	HeLa cells	CD69 and CD25	No	[25]
	A549 human epithelial cell line	TNF	Yes	[9]
L. monocytogenes	Human monocyte-derived DCs	IFN-γ	No	[17]
P. aeruginosa	Murine BMDCs	CD69	Yes	[18]
K. pneumoniae	Murine BMDCs	CD69	Yes	[18]
L. acidophilus	Murine BMDCs	CD69	Yes	[18]
S. aureus	Murine BMDCs	CD69	Yes	[18]
	Human monocyte-derived DCs	IFN-γ	Yes	[17]
S. epidermidis	Murine BMDCs	CD69	Yes	[18]
S. cerivisiae	Murine BMDCs	CD69	Yes	[18]
C. galbrata	Murine BMDCs	CD69	Yes	[18]
C. albicans	Murine BMDCs	CD69	Yes	[18]
	A549 human epithelial cell line	IFN-γ/TNF	Yes	[9]
E. faecalis	Murine BMDCs	CD69	No	[18]
	Human monocytic cell line (THP-1)	IFN-γ	Yes	[24]
Strep group A	Murine BMDCs	CD69	No	[18]
M. abscessus	Murine BMDCs	CD69	Yes	[18]
F. tularensis LVS	Murine BMDMs	IFN-γ	Yes	[20]
M. bovis BCG	Murine BMDMs	IFN-γ	Yes	[19]
S. flexneri	HeLa cells	CD69 and CD25	Yes	[25]
S. typhi	Human B cell line	CD69 and cytokine production	Yes	[26]
L. lactis (culture supernatant)	Human B cell line	CD69	Yes	[11]
L. lactis Δ RibA or RibG (culture supernatant)	Human B cell line	CD69	No	[11]

MAIT mucosa-associated T, BMDC bone marrow-derived dendritic cells, BMDM bone marrow-derived macrophages

Despite a clear role for riboflavin metabolites as microbial antigens that stimulate MAIT cell activation, it is important to note that MAIT cells may possess other pathways for activation. Other innate T cell types, such as iNKT cells, do not rely solely on recognition of cognate antigen for activation, but instead can be activated via cytokine-driven, microbial antigen-driven, or self-antigen-driven pathways, with the potential for one pathway to override or influence the other [21, 22]. Indeed, iNKT cells constitutively express the IL-12 receptor, and several in vivo pathogen infections were shown to activate iNKT cells primarily via IL-12, rather than through recognition of cognate antigen [22]. Importantly, recent data suggest that both human and mouse MAIT cells are similarly activated by innate cytokines in the absence of cognate antigen. Billerbeck et al. [23] observed that human peripheral blood MAIT cells expressed high levels of the IL-18 receptor; correspondingly, Ussher et al. [24] demonstrated that human MAIT cells produced IFN-γ in a MR1-independent manner following treatment with recombinant IL-12 and IL-18. MR1-independent MAIT cell activation by innate cytokines was further demonstrated by the ability of human monocytes cultured with Enterococcus faecalis, which lacks the riboflavin biosynthetic pathway, to stimulate MAIT cell production of IFN-γ in an MR1-independent, but IL-12 + IL-18-dependent, manner [24]. Similarly, Vα19i-transgenic murine MAIT cells relied almost entirely on IL-12 but not MR1 to produce IFN-γ and control M. bovis BCG intramacrophage growth [19].

Importantly, the fact that BCG possesses the riboflavin biosynthetic pathway suggests that a cytokine-driven MAIT cell activation pathway may dominate under certain conditions even in the presence of microbes that can synthesize an MR1-binding antigen. This phenomenon is similar to that observed for iNKT cells, which also exhibited predominantly cytokine-driven activation in response to microbes that expressed CD1d-binding antigens [22]. Although these recent MAIT cell data appear to conflict with several previous studies, multiple factors such as the intracellular location of the pathogen, microbial virulence factors, the type of antigen presenting cell, and the time of co-culture may influence the ability of a microbe to stimulate MAIT cells through MR1 [24–26]. In support of this possibility, recent data demonstrated that Shigella dysenteriae, which escapes into the cytoplasm, but not Salmonella enterica typhimurium, which blocks endosome-lysosome fusion, activated MAIT cells [25]. Since Salmonella clearly produces MAIT cell-activating ligand(s) [11, 25], it is likely that Salmonella’s capacity to impede normal endosomal maturation and/or express other inhibitory virulence factors may prevent efficient ligand loading onto MR1; indeed, it has been shown that MR1 must traffic through a late endosomal compartment for optimal cell surface expression and efficient MAIT cell activation [27].

Collectively, the data presented above point to a model whereby iNKT cells and MAIT cells, which both express high levels of innate cytokine receptors, possess the capacity to become activated upon encounter of an inflammatory environment rather than a specific antigen, allowing them to respond to a diverse set of microbes and disease states. Indeed, MAIT cells have been implicated in a variety of inflammatory diseases that lack any apparent involvement of riboflavin-producing microbes. The demonstration that MAIT cells produced IFN-γ in response to human PBMCs treated with the TLR8 agonist ssRNA₄₀ suggests they may have the capacity to respond to viral infections [24]. In support of this, CD161^hi CD8⁺ T cells—the majority of which are MAIT cells—were enriched in the liver of patients with chronic hepatitis C virus [23]. Similarly, CD161^hi CD8⁺ T cells were present in demyelinating lesions of patients with multiple sclerosis [28]. In the murine model, MAIT cells were shown to provide protection in autoimmune encephalomyelitis, and to exacerbate collagen antibody-induced arthritis [29, 30]. Overall, the combined evidence suggests that MAIT cells may have a broader role in immunity than pathogen defense, and possess the potential to provide both protective and immunopathogenic functions.

MAIT cells in pathogen defense

Despite the above evidence indicating that MAIT cells likely contribute to defense against infectious disease, studies examining their in vivo functionality during pathogen infection have thus far been limited. The demonstration that a panel of MAIT cell clones derived from human peripheral blood all expressed the mucosal homing integrin α4β7 points to a role for these cells in mucosal tissue surveillance [17, 31]. Correspondingly, human peripheral blood MAIT cells were found to express chemokine receptors involved in trafficking to the intestine and liver (CCR9⁺ and CXCR6⁺), but not the lymph nodes (CCR7⁻) [23, 32]. This hypothesis is supported by the fact that MAIT cells have been detected in a number of peripheral tissues, including the lungs of M. tuberculosis-infected patients and human intestinal samples, but not human lymph nodes [31, 32]. Further, MAIT cells have been shown to comprise as much as 50 % of T cells in human liver [32]. Thus, MAIT cells clearly have the capacity to traffic to mucosal tissues such as the digestive tract and the lungs.

However, the demonstration that MR1 mRNA is ubiquitous in all human cell types [13] and MR1 protein is detectable in all mouse tissues [27] indicates that MAIT cells—contrary to their name—are likely active in more than just mucosal tissues. A recent study by Lepore et al. [33] demonstrated that MAIT cells were present in a number of healthy non-mucosal human tissues, such as the kidneys, prostate, and ovaries, although the functionality of MAIT cells at these sites remains to be defined. Importantly, since adult peripheral blood MAIT cells express CCR2, they have the potential to be recruited to multiple inflammatory locations [23, 27]. Indeed, a number of important human studies suggest that MAIT cells traffic to the site of both mucosal and non-mucosal infections. In support of their role in mucosal defense, it was observed that patients with active pulmonary M. tuberculosis infection exhibited lower frequencies of MAIT cells in peripheral blood as compared to uninfected individuals [17, 18]. Similar results were seen following vaccination with a live attenuated oral Shigella vaccine [25]. Interestingly, however, MAIT cell frequencies in the blood also diminished following a variety of pathogen infections, including non-mucosal skin and soft tissue infections [34]. Overall, human studies are naturally limited in the extent of the information that can be obtained about MAIT cell activities in vivo, but these results are generally interpreted as evidence that MAIT cells migrate from the periphery to infected tissues.

Although MAIT cells are abundant in human peripheral blood and liver, early studies failed to detect large numbers of MAIT cells in mice, which comprised less than 2 % of DN αβT cells in murine mesenteric lymph nodes [8, 31]. However, many of the early murine studies examined naïve, uninfected mice and were hampered by the lack of a definitive marker for identification of MAIT cells. Further, as noted above, MAIT cell frequencies are likely low in lymph nodes. Interestingly, recent work by Meierovics et al. [20] demonstrated that pulmonary infection of naïve, wild type C57BL/6 mice with F. tualrensis live vaccine strain (LVS) resulted in accumulation of large numbers of MAIT cells in the lungs; pulmonary MAIT cell numbers increased as much as 25-fold after infection and produced critical cytokines such as IFN-γ, TNF, and IL-17A. Thus, although initial MAIT cell numbers are difficult to detect in mice, these cells clearly expand and/or are recruited to the site of infection. Further, MAIT cells actively contributed to in vivo defense through the production of important anti-microbial cytokines.

The importance of the contribution of MAIT cells to pathogen defense has been demonstrated using MR1-deficient mice, which lack MAIT cells. MR1-deficient mice displayed increased susceptibility to pulmonary infections with F. tularensis LVS and M. bovis BCG, as well as intraperitoneal infection with K. pneumonia [19, 20, 35]. Additionally, Vα19i-transgenic and Vβ6-transgenic mice, which are highly enriched for MAIT cells and lack conventional CD4⁺ and CD8⁺ T cells, exhibited higher M. abscessus and E. coli organ burdens after infection as compared to their MR1-deficient counterparts [18]. Thus, MAIT cells clearly have non-redundant in vivo functions that either directly or indirectly result in control of pathogen growth.

Although the ability of MAIT cells to directly inhibit pathogen growth is difficult to definitively measure in vivo, in vitro studies have clearly shown that MAIT cells possess a number of antibacterial mechanisms. As noted above, IFN-γ production by Vα19i-transgenic murine MAIT cells inhibited intramacrophage growth of both M. bovis BCG and F. tularensis; in both cases, control of bacterial growth was dependent upon macrophage production of the cytotoxic effector molecule nitric oxide [19, 20]. Although macrophage production of nitric oxide (via the enzyme inducible nitric oxide synthase) is commonly used by mice to control intracellular pathogen growth, this mechanism is not used by human macrophages [36], raising the question of how MAIT cells inhibit pathogen growth in human macrophages and other infected cell types. Interestingly, both Lepore et al. and Dusseaux et al. demonstrated that human MAIT cells produced high levels of Granzyme B in response to E.coli-infected antigen presenting cells [32, 33]; correspondingly, Lepore et al. [33] determined that MAIT cells induced apoptosis of the infected cells. Further, Le Bourhis et al. [25] noted that human MAIT cells possessed intracellular stores of perforin and granulysin, and displayed cytotoxic activity against epithelial cells infected with Shigella flexneri. Collectively, these data demonstrate that MAIT cells can lyse infected cells, possibly as a mechanism of preventing further propagation of intracellular bacteria. Thus, MAIT cells possess a number of classic T cell mechanisms that can affect pathogen growth in both hematopoetic and non-hematopoetic cells.

The kinetics of MAIT cell responses during in vivo infection are only beginning to be elucidated. Adult human MAIT cells express a constitutive ‘effector memory’ phenotype (CCR7⁻ CD62L^lo CD45RO⁺ CD45RA⁻ CD95^hi) and possess the capacity to quickly release cytokines in response to pathogen stimulation [32, 37, 38]. These features implicate MAIT cells in early pathogen detection, similar to iNKT cells. The ability of MAIT cells to respond to several types of infected hematopoetic and non-hematopoetic cell types (macrophages, B cells, dendritic cells, epithelial cells, fibroblasts; Table 2) suggests they have multiple targets through which they can detect the early stages of infection and influence subsequent immune responses. Indeed, MR1-deficient mice infected with K. pneumoniae exhibited increased organ burdens and significantly decreased production of multiple cytokines very early (day 2) after infection [35], suggesting that initiation of immune responses was impaired in the absence of MAIT cells. Similarly, early production of important cytokines such as IFN-γ and IL-17A was impaired in the lungs of MR1-deficient mice following F. tularensis LVS pulmonary infection [20]. These results are reminiscent of those observed for iNKT cells, whose extremely early production of IFN-γ and/or other cytokines is thought to influence other innate immune cells and the progression of adaptive immune responses [21]. Indeed, MR1-deficient mice exhibited delayed recruitment of activated IFN-γ-producing CD4⁺ and CD8⁺ T cells to the lungs following F. tularensis LVS pulmonary infection [20]. Thus, early MAIT cell activities clearly influence the initiation of adaptive immune responses.

However, unlike other non-conventional TCRαβ⁺ innate T cells, recent evidence suggests that MAIT cell activities are not limited to the early phases of infection. Murine studies of iNKT cells and H2-M3-restricted T cells demonstrated that these two innate cell types are early effectors that acted prior to the peak of conventional T cell responses [21, 39]. In contrast, murine MAIT cells continued to accumulate in the lungs of mice during pulmonary F. tularensis LVS infection, reaching maximum numbers at the same time as conventional CD4⁺ and CD8⁺ T cells [20]. Additionally, MAIT cells continued to actively secrete critical cytokines such as IFN-γ and TNF throughout pulmonary F. tularensis LVS infection. Therefore, although MAIT cells appeared to have an early role in the initiation of immune responses, they also actively contributed to immunity throughout the later stages of infection. Unfortunately human studies to confirm the unique kinetics of MAIT cell expansion/recruitment to infected tissues are not realistic. However, these murine studies mark a potentially important difference between MAIT cells and our current understanding of the functions of other innate TCRαβ⁺ T cells, and suggests that MAIT cells have the capacity to augment and influence adaptive immune responses throughout all phases of infection.

Interestingly, MAIT cells are uniquely affected by HIV infection, an observation that has recently been reviewed elsewhere [40]. Several studies have shown that MAIT cell numbers declined in the peripheral blood of patients with HIV [41–44], whereas their numbers in intestinal tissues were relatively preserved in some studies [41, 42], and significantly depleted in another [43]. Further investigation revealed that the residual MAIT cells present in the blood were activated and exhausted, with an impaired ability to up-regulate CD69 and produce cytokines in response to E. coli in vitro [41, 42]. Studies by Leeansyah et al. [41] suggested that some MAIT cells may persist in the blood as hyper-activated CD161⁻ Vα7.2⁺ T cells, although their definitive identification as MAIT cells by tetramer or other means remains to be determined. Since MAIT cells were found to be relatively resistant to HIV infection, it appears unlikely that their loss is a direct result of virus infection [42]. Instead, it has been proposed that impaired immune responses at mucosal sites impose a heightened burden on MAIT cell activities, which leads to their exhaustion. Indeed, Cosgrove et al. [42] demonstrated the presence of LPS in the lamina propria of HIV patients, suggesting that the intestinal epithelial integrity of HIV⁺ individuals is compromised. This has the potential to produce a high concentration of MAIT cell-activating riboflavin metabolites and inflammatory cytokines in these tissues. Since exposure of MAIT cells to E. coli in vitro triggered apoptosis [42], the continued microbial assaults encountered at mucosal surfaces in HIV patients may deplete MAIT cells through activation-induced cell death.

Since MAIT cells are likely an important first line of defense against a wide range of pathogens in mucosal tissues, these findings have important implications regarding treatment of HIV-infected individuals. In particular, HIV infection is well known to predispose individuals to M. tuberculosis (TB) infection and/or reactivation; the risk of TB increases quickly after HIV infection (<1 year), prior to the emergence of other AIDS-related conditions [45]. Similarly, significant depletion of circulating MAIT cells has been observed early after HIV infection (2–3 weeks), although whether the observed low circulating MAIT cell numbers in these individuals was present prior to HIV infection is unknown [44]. Regardless, it is possible that MAIT cell exhaustion and depletion increases the susceptibility of HIV-infected individuals to TB infection and/or reactivation. Indeed, as previously observed in HIV-infected individuals, patients with HIV-TB co-infection also possessed low levels of circulating MAIT cells, although unfortunately the functional exhaustion of these cells remains to be determined [44]. Overall, it is reasonable to speculate that development of immunotherapies to restore the MAIT cell compartment may help alleviate the susceptibility of HIV patients to infectious agents such as TB. This is particularly important given that MAIT cell numbers in the peripheral blood of HIV patients did not recover after long-term highly active anti-retroviral therapy, despite recovery of CD4⁺ T cells and suppression of viral load [41–43].

MAIT cell development: a role for the microbiota?

Studies in germ-free mice were the first to demonstrate that MAIT cells may be critically dependent upon microbial exposure for their development. MAIT cells were undetectable in the mesenteric lymph nodes (MLN) and lamina propria of germ-free mice [18, 31]. Importantly, reconstitution of germ-free mice with commensal bacteria that possess the riboflavin biosynthetic pathway (such as Enterobacter cloacae or Lactobacillus casei), but not one that lacks this pathway (E. faecalis), resulted in a significant increase in MAIT cells in the MLN [18].

The development and maturation of MAIT cells appear to be different from conventional CD4⁺ and CD8⁺ T cells, as well as innate iNKT cells, and has been reviewed in detail elsewhere [46]. One of the hallmarks of iNKT cell development is the acquisition of an effector/memory phenotype during thymic development that is critically dependent upon their expression of the transcription factor PLZF [47]. Thus, ‘naïve’ iNKT cells express an effector/memory phenotype (CD44^hi CD62L^lo) in the periphery that directs them away from lymph nodes and into the tissues. In contrast, conventional T cells lack expression of PLZF and exit the thymus with a naïve phenotype (CD44^lo CD62L^hi). Conventional T cells maintain this naïve phenotype in the periphery until an antigen-specific infectious assault directs their activation and expansion. In contrast, MAIT cells appear to follow a unique developmental pathway. Similar to iNKT cells, MAIT cells exhibited a PLZF⁺ effector/memory phenotype in the periphery of adult humans [47, 48]. However, unlike iNKT cells, MAIT cells did not acquire PLZF expression during thymic development and exited the thymus bearing a naive phenotype [38]. Thus, unique signals occur after thymic development that drives MAIT cell acquisition of PLZF expression and maturation into effector cells. Interestingly, a recent study by Leeansyah et al. [48] demonstrated that MAIT cells in the intestine, lung and liver (but not the spleen and MLN) of human fetuses expressed PLZF and a memory phenotype—strong evidence that MAIT cells functionally mature prior to establishment of commensal microflora.

Overall, taking evidence from the murine model into account, it has been suggested that the final expansion of MAIT cells in the periphery is dependent upon commensal microflora. Thus far, the precise signals that drive the final expansion of MAIT cells in the periphery of humans remain unresolved, but B cells appear to be critical for this process [31]. Dusseaux et al. [32] observed that MAIT cell numbers increased steadily in the peripheral blood of infants to reach adult levels 1–2 years after birth. Further, Gold et al. [38] observed that M. tuberculosis-reactive MAIT cells were much more abundant in the peripheral blood of naïve adults as compared to cord blood. Thus, unlike iNKT cells, which maintain constant abundance in human peripheral blood throughout life [49], MAIT cells appear to undergo a population expansion that begins shortly after birth. Interestingly, MAIT cells in second trimester human fetuses possessed high levels of Ki67 (associated with an active cell cycle) and robust in vitro proliferative responses in response to microbial stimulation [48], a feature that was initially found to be lost in adult peripheral blood MAIT cells [32, 37]. Surprisingly, Leeansyah et al. [48] found that despite marginal Ki67 expression in adult peripheral blood MAIT cells, they proliferated in vitro in response to E. coli and IL-2 stimulation. One of the primary differences between this study and previous studies was the inclusion of exogenous IL-2 in the stimulation conditions, demonstrating that adult MAIT cells possess the capacity to proliferate in the presence of certain cytokines. Regardless, the collective data suggest that microbial exposure after birth stimulates the first robust expansion of a population of highly proliferation-sensitive MAIT cells during the first few years of life.

MAIT cell subsets

Although MAIT cells were initially identified as a CD4⁻ CD8⁻ double negative (DN) T cell subset [6], Walker et al. [37] found that MAIT cells in human peripheral blood (as defined by CD161^hi Vα7.2 expression) were 90 % CD8α⁺, with the majority of the remaining cells being DN. Similarly, Tilloy et al. [8] determined that MAIT cell frequencies in human peripheral blood were approximately 1/10 in DN, 1/50 in CD8⁺, and 1/6,000 in the CD4⁺ T cell subsets. This has recently been confirmed through the use of a specific MR1 tetramer; Reantragoon et al. [10] determined that tetramer-positive MAIT cells in human peripheral blood were mostly CD8⁺ and DN, with only a minor CD4⁺ subset. Accordingly, most human studies to date have focused on MAIT cells in the CD8⁺ T cell subset, and found these cells respond to in vitro stimulation via secretion of IFN-γ, TNF, IL-22, and IL-17A [17, 37]. Interestingly, CD8⁺ MAIT cells in human peripheral blood are approximately equally divided between the αβ and αα CD8 isoforms. Since Walker et al. [37] found that human CD161^hi CD8αβ cells could quickly convert to CD161^hi CD8αα cells in vitro in the absence of specific stimulation; the ability of MAIT cells to switch expression of CD8 isoforms is an intriguing phenomenon that warrants further study. Investigation of the functional differences between the two CD161^hi CD8⁺ isoform subsets revealed a small but significant increase in IL-17 production by the CD8αα subset as compared to the CD8αβ population after in vitro stimulation with PMA/ionomycin; no differences were observed in any other parameters examined, including their global gene expression profiles as measured via microarray [37]. In mice, expression of CD8αα on other T cell types has been suggested to act as a corepressor, with a protective role in an adoptive transfer model of experimental colitis [50]. Thus, far it remains to be determined whether differences in MAIT cell coreceptor expression reflect functionally different subsets in vivo.

Of note, studies by Kawachi et al. in Vα19i-Tg mice found that DN MAIT cells differed from CD4⁺ and CD8⁺ MAIT cells in their production of high levels of IL-4, IL-5, and IL-10 after anti-CD3/CD28 stimulation [51]. However, since DN MAIT cells responding to pulmonary F. tularensis LVS infection in the lungs of wild type (non-transgenic) mice produced IFN-γ, TNF, and IL-17A [20], it is reasonable to speculate that the local environment during MAIT cell activation influences their subsequent phenotype.

Importantly, recent studies have identified human MAIT cell subpopulations that lack expression of the canonical Vα7.2-Jα33 chain, but instead express one of several alternative Vα7.2-TRAJ rearrangements [9, 10, 33]. Pathogen-reactive MAIT cells were most frequently found to express the TRAJ33, TRAJ12, and TRAJ20 gene transcripts [9, 10, 33]. Further analyses of the Vα7.2-Jα12 MAIT cell population by Lepore et al. [33] revealed that it possesses MR1-restricted anti-microbial activity, but is less abundant in human peripheral blood than ‘classical’ Vα7.2-Jα33 MAIT cells. Surprisingly, however, the Vα7.2-Jα12 MAIT cell subset was more abundant than Vα7.2-Jα33 cells in the tissues of some donors, including the liver, kidneys, and intestines [33]. Whether these differences are indicative of different tissue-homing capacities between the two subsets and/or different infection histories of the individuals remain to be investigated. However, it is clear that different MAIT cell subsets are differentially represented in the tissues of some individuals. Correspondingly, recent work by Gold et al. [9] demonstrated that peripheral blood MAIT cells reactive to different microbes displayed different TCR profiles both within and between individuals, suggesting that MAIT cell clonotype usage is complex and specific for each individual. Moreover, MAIT cell clones with distinct TCRs exhibited differential responses to a riboflavin metabolite, raising the intriguing possibility that different MAIT cell clonotypes may selectively emerge in the periphery as a result of exposure to different pathogens expressing diverse MAIT cell-activating antigens.

Concluding remarks

MAIT cells are a unique T cell subset with a number of features that distinguish them from other T cell types, including their closest ‘relative’, iNKT cells (Table 1). While both cell types express invariant TCRs that act as microbial pattern recognition receptors, MAIT cells recognize a unique microbial antigen derived from vitamin B metabolites and develop via an unusual ontogenic pathway that requires microbiota. The surprisingly high abundance of MAIT cells in human peripheral blood, coupled with their capacity to home to mucosal tissues and detect a common microbial metabolite, suggests that these cells are uniquely poised to act at the forefront of microbial detection. Indeed, the studies performed to date suggest that MAIT cells perform an important microbial surveillance function that influences subsequent innate and adaptive immune responses. However, in addition to this early role in immunity, MAIT cells contribute to immune responses throughout the later stages of infection via the active production of important cytokines. This implicates MAIT cells in an immunomodulatory role that may be harnessed to augment vaccine-induced immune responses at mucosal surfaces through the development of novel vaccine adjuvants and immunotherapeutics.