Immune regulatory activity of NKT cells can be mediated by cytokines secreted by NKT cells themselves or following their interaction with other immune cells, including DCs, NK cells, Tregs, monocytes, and B cells. Activation of NKT cell subsets can result in the deviation of a cytokine secretion profile in MHCrestricted CD4+/CD8+ T cells toward either a pronounced Th1-, Th2-, or Th17-like response.

It is noteworthy that in inflamed target tissues, such as in pancreas in non-obese diabetic (NOD) mice that spontaneously develop type 1 diabetes (T1D) and in the CNS during experimental autoimmune encephalomyelitis (EAE), both type I and type II NKT cells accumulate (13, 27). However, activation of type II NKT cells following sulfatide or lyso-PC administration leads to a rapid accumulation of type I NKT cells into liver in an IL-12 and macrophage inflammatory protein 2 (MIP2)-dependent fashion. But these recruited type I NKT cells are neither activated nor do they secrete cytokines, and consequently they are anergic, leading to decreased levels of IFNγ followed by reduced recruitment of myeloid cells, NK cells, and protection from liver damage (28, 29). In contrast to the activation of lyso-PE-reactive type II NKT cells in an infectious model of HBV, hepatic type I NKT cells are not anergized but stimulated to secrete cytokines (16). This difference in type I NKT stimulation may relate to the differential milieu in liver during sterile versus infectious immunity.

### A Novel Type II NKT Cell-Mediated Immune Regulatory Pathway

Sulfatide-mediated type II NKT cell stimulation *in vivo* results in the activation of predominantly hepatic plasmacytoid DCs (pDC) but not conventional DC (cDC) and ultimately induction of anergy in hepatic type I NKT cells. This unique immune regulatory pathway not only involves cross-regulation of type I NKT cells but also inhibition of pathogenic Th1/Th17 cells through tolerization of hepatic cDC and tissue-resident antigen-presenting cells (APCs), such as microglia in the CNS (28, 30). By contrast, activation of type I NKT cells following αGalCer administration predominantly activates hepatic cDC (28, 29). We are currently investigating the molecular mechanism of these NKT–DCs interactions.

It has been shown that this immune regulatory pathway effectively controls EAE, T1D, inflammatory liver diseases, and systemic lupus erythematosus (SLE) (17, 27, 28, 30–32) (Halder, unpublished). A recent study has suggested that the ICOS and PD-1 ligand pathways are required for the regulation of T1D in NOD mice by CD4+ type II NKT cells (33). Sulfatide-mediated type II NKT cell activation can also result in IL-10 secretion and, consequently, inhibition of type I NKT cells and diabetogenic or encephalitogenic CD4+ and CD8+ T cells (13, 27). Furthermore, activation of type II NKT cells also induces alterations in other innate cells, including myeloid-derived suppressor cells (MDSCs), CD11b+Gr-1+ cells, and neutrophils (17, 28, 30–32). Accordingly, MDSCs have been shown to protect mice from EAE (34). CD11b+Gr-1+ cells and neutrophil alterations can also protect from inflammatory liver diseases (31, 32, 35). Additionally, activation of type II NKT cells by PD-L1-deficient DCs increases the IL-4 and IL-13 levels and, consequently, decreases the numbers of IFNγ and IL-17-secreting pathogenic T cells (36). Thus, targeting type II NKT-mediated inhibition of the effector functions of Th1/Th17 cells and APCs in peripheral organs as well as in affected target tissues offers a potent strategy for intervention in autoimmune and inflammatory diseases (30).

## Type II NKT Cells in Autoimmune and Inflammatory Diseases

The activation of type II NKT cells with sulfatide controls both antigen-induced and spontaneously arising autoimmune diseases. Additionally, sulfatide-mediated immune regulation inhibits inflammatory liver diseases elicited by type I NKT cells (13, 17, 27, 28, 30–32, 35). Sulfatide-reactive type II NKT cells also have been shown to abrogate ischemic–reperfusion injury in mice and in patients with acute tubular necrosis (37). Interestingly, during EAE and T1D progression, sulfatide-reactive type II NKT cells accumulate in the target tissue and in the draining of lymph nodes, respectively. This greater abundance of type II NKT cells in the CNS inverts the usual ratio of type II/type I NKT cells (type II NKT cells, 3–4%, and type I NKT cells, 0.6–0.9%) (13, 27). Thus, administration of brain-derived sulfatides or synthetic cic-tetracosenoyl or tetracosenoyl sulfatide affords protection from EAE and diabetes (13, 27, 30). In both cases, it is the sulfatide with the longer fatty acid chain that is able to efficiently activate type II NKT cells and prevent autoimmunity. These data suggest that sulfatide analogs should be examined in clinical studies in multiple sclerosis (MS) and T1D. Our preliminary studies also suggest that activation of type II NKT cells following administration of sulfatide significantly inhibits development of lupus nephritis in (NZB X NZW) F1 mice, further indicating a regulatory role of type II NKT cells (5) (Halder, unpublished).

Recent studies have indicated key mutual interactions among NKT cells, CD1d+ cells, and commensal microbiota in the intestine (38). Evidence from several animal models of inflammatory bowel disease (IBD) demonstrates that type I NKT cells can be either protective or pathogenic (39). Interestingly, type II NKT cells seem to promote intestinal inflammation and mediate a pathogenic response when both CD1d expression and the frequency of IL-13 producing type II NKT cells are increased in mice as well as patients with ulcerative colitis (40–42). It is noteworthy that type II NKT cells involved in ulcerative colitis in humans are also reactive to lysosulfatide, but in contrast to the liver type II NKT cells, they secrete IL-13 and not IFNγ (41). This suggest that there are subsets of type II NKT cells that may have different TCR repertoires as well as different cytokine secretion patterns in different tissues, just as there are different subsets of type I NKT cells.

## Type II NKT Cells in Metabolic and Liver Disorders

Both NKT cell subsets have been shown to be involved in adipose-tissue inflammation, diet-induced obesity, and glucose metabolism (43). Roles for eosinophils, macrophages, and innate lymphoid type 2 cells (ILC2) have also been suggested in metabolic disorders (44, 45). More recently, type II NKT cells induced by both IL-25 and sulfatide treatments have been shown to be involved in the regulation of inflammation in adipose tissue and prevention of high fat diet-induced obesity in mice. Transfer of type II NKT cells into obese mice induced a greater and prolonged weight loss and improved glucose tolerance (44).

In inflammatory liver diseases, type I and type II NKT cells have been shown to play opposing roles (35). Earlier, it was shown that following liver injury after ischemic–reperfusion or ConA administration, a rapid activation of IFNγ-secreting type I but not type II NKT cells takes place (28, 31). Activation of type I NKT cells generates a cascade of events that contributes to liver inflammation and damage. The secretion of pro-inflammatory cytokines, such as IFNα, and chemokines leads to accumulation of CD11b+ Gr-1+ cells as well as other myeloid cells resulting in the destruction of hepatocytes. By contrast, sulfatide-activated type II NKT cells inhibit the cascade of pro-inflammatory events through a mechanism that includes activation of pDC resulting in tolerization of cDC, anergy in type I NKT cells and consequently protection from liver injury (28, 31, 35). In a mouse model of chronic alcohol liver disease (ALD), we have found that type I, but not type II, NKT cells are activated, leading to recruitment of inflammatory neutrophils and liver damage (46, 47). Inhibition of type I NKT cells following a novel direct mechanism involving all-trans retinoic acid (48) and its receptor (RAR-γ) signaling, or an indirect mechanism mediated by sulfatide-activated type II NKT cells significantly blunts ALD (46). Consistent with this, accumulation of activated type I NKT cells in patients with NAFLD has recently been shown (49–51). Currently, clinical studies are being carried out to examine the potential use of a RAR-γ analog for the treatment of alcohol- and non-alcoholic liver disease. We are presently investigating in humans the role of both type I and type II NKT cells in the promotion as well as regulation of inflammatory immune responses in liver and gut. The identification of the role of these cell subsets in liver disorders could potentially lead to the development of novel therapeutics.

## Type II NKT Cells in Infectious Diseases

Natural killer T cells contribute to the early immune response against a broad range of microbial pathogens, playing either a beneficial role in some infections or a negative role in others (52). Frequently, type I and type II NKT cells can have opposing roles in microbial immunity. For example, in the case of *Trypanosoma cruzi* infections, type II NKT cells were shown to promote inflammation and mortality and reduced titers of pathogen-specific antibodies, whereas type I NKT cells led to reduced inflammation and improved mortality and antibody titers (53). By contrast, during murine *Schistosoma mansoni* infection, type II NKT cells led to increased Th2 cytokine secretion and decreased IFNγ production, while type I NKT cells reinstated IFNγ levels (54).

Sulfatide-activated type II NKT cells have also been shown to affect the course of infectious diseases. Previously, we showed that sulfatide-activated type II NKT cells inhibit HIV-1 replication and enhance multi-lineage hematopoiesis in a SCID-Hu (Thy/Liv) HIV model (55). We hypothesized that sulfatide-mediated activation of type II NKT cells and pDC results in the induction of anergy in type I NKT cells. It was shown that peripheral CD4+ type I NKT cells are depleted in early HIV infection and that the remaining cells in the circulation during HIV infection are functionally impaired in IFNγ expression (56). Sulfatide-mediated activation of type II NKT cells also has a protective effect in a *Staphylococcus aureus* murine model of sepsis and is associated with a decrease in pro-inflammatory cytokines, such as TNFα and IL-6 (57). This beneficial outcome was found to depend on CD1d but not on type I NKT cells.

Recent studies in HBV infection in animal models and humans have shown that NKT cells contribute to the initiation of antiviral immune responses against HBV. An early activation of type I and type II NKT cells was found following infection, mainly in the liver, which correlated with IFNγ-dependent suppression of viral replication; but also NKT cells contribute to HBV-induced hepatitis (16, 58). Using a mouse model of infection with HBV-expressing adenoviral particles (Ad-HBV), it was demonstrated that Jα18-deficient (lacking type I NKT cells) and CD1d-deficient (lacking all NKT cells) mice exhibited a significant decrease in NK, B, CD4+, and CD8+ T cell activation and hepatic immune infiltration, supporting the idea that NKT cells play a role in the immune response to HBV (16). More importantly, it was shown that HBV infection induces production of modified ER self-lipids, including phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (lyso-PE), direct activation of liver type II NKT cells, and downstream cytokine-dependent activation of type I NKT cells. Type II NKT activation required hepatocyte expression of microsomal triglyceride transfer protein (MTP) and CD1d (16). In a murine acute hepatitis B transgenic model, NKG2D-dependent activation of type II NKT cells has been shown to result in liver damage (59).

### Type II NKT Cells in Tumor Immunity

Similar to the immune responses in liver, type I and type II NKT cells have been shown to play opposing roles in tumor immunity

### References

(10, 60). Type I NKT cells are usually associated with the promotion of tumor immunity, whereas type II NKT cells are associated with its suppression (61). Thus, type I NKT cells were found to induce lysis of tumor cells directly via a perforin/ granzyme-dependent mechanism or indirectly by induction of Th1 cytokine secretion and activation of NK and DC cells. By contrast, type II NKT cells have shown immunosuppressive activity down-regulating tumor immunosurveillance (60, 62–64). Type II NKT cell-induced tumor suppression can be mediated by IL-13 secretion resulting in the activation of TGF-β-secreting MDSCs that inhibit tumor-specific CD8+ T cells or type I NKT cells. In humans, Chang et al. have also shown an increase in IL-13-secreting lyso-PC-reactive type II NKT cells in multiple myeloma patients (18). Interestingly, type I NKT cells are decreased in these patients, suggesting opposing roles, as their increased frequency is associated with better prognosis.

### Future Studies and Challenges

Availability of stable reagents for analysis of type I NKT cells has resulted in characterization of changes in their frequency and phenotype in humans in different disease conditions, including autoimmune and infectious diseases and cancer. A detailed characterization of type II NKT cell repertoires and their ligands in humans is required for a broader understanding of their physiological role in health and in disease. A recent study suggesting a role for lyso-glucosylsphingosine (lyso-GL1)-reactive type II NKT cells in Gaucher disease is an important development (19). Together, all of these observations indicate that it may be possible in the future to differentially activate or inhibit type I and type II NKT cells for the development of novel immunotherapeutic protocols in altering the course of cancer and both infectious and autoimmune diseases.

### Acknowledgments

This work was supported by grants from the National Institutes of Health, USA, R01 CA100660 and R01 AA020864 (VK) and from the Juvenile Diabetes Research Foundation (JDRF) grants 24-2007-362 (VK).

self-glycolipid. *Proc Natl Acad Sci U S A* (2010) **107**(24):10984–9. doi:10.1073/ pnas.1000576107

II NKT cells attenuates alcoholic liver disease. *Hepatology* (2015) **61**(4):1357–69. doi:10.1002/hep.27632

a new immunoregulatory axis. *J Immunol* (2007) **179**(8):5126–36. doi:10.4049/ jimmunol.179.8.5126

**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.

*Copyright © 2015 Marrero, Ware and Kumar. 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.*

# The extended family of CD1drestricted NKT cells: sifting through a mixed bag of TCRs, antigens, and functions

#### *Elodie Macho-Fernandez and Manfred Brigl\**

*Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA*

Natural killer T (NKT) cells comprise a family of specialized T cells that recognize lipid antigens presented by CD1d. Based on their T cell receptor (TCR) usage and antigen specificities, CD1d-restricted NKT cells have been divided into two main subsets: type I NKT cells that use a canonical invariant TCR α-chain and recognize α-galactosylceramide (α-GalCer), and type II NKT cells that use a more diverse αβ TCR repertoire and do not recognize α-GalCer. In addition, α-GalCer-reactive NKT cells that use non-canonical αβ TCRs and CD1d-restricted T cells that use γδ or δ/αβ TCRs have recently been identified, revealing further diversity among CD1d-restricted T cells. Importantly, in addition to their distinct antigen specificities, functional differences are beginning to emerge between the different members of the CD1d-restricted T cell family. In this review, while using type I NKT cells as comparison, we will focus on type II NKT cells and the other non-invariant CD1d-restricted T cell subsets, and discuss our current understanding of the antigens they recognize, the formation of stimulatory CD1d/antigen complexes, the modes of TCR-mediated antigen recognition, and the mechanisms and consequences of their activation that underlie their function in antimicrobial responses, anti-tumor immunity, and autoimmunity.

Keywords: CD1d, antigen presentation, lipid antigens, glycosphingolipids, phospholipids, inflammation, infection, cancer

### Introduction

Traditionally, the immune system has been separated into innate and adaptive immunity. However, in an unconventional way, innate T cells such as CD1d-restricted natural killer T (NKT) cells, MR1 restricted mucosal-associated invariant T (MAIT) cells, γδ T cells, and some CD1a, b, c-restricted T cells share features of both innate and adaptive immune cells, allowing them to form a critical bridge between the two arms of the immune system (1–4). NKT cells recognize lipid antigens presented by the monomorphic MHC class-I-like molecule CD1d and are highly conserved in humans and mice. In response to a wide range of self- and foreign antigens, NKT cells are activated rapidly and exhibit both pro-inflammatory and immunoregulatory characteristics, resulting in either protective or harmful roles in numerous pathological states in mice and humans, including microbial infection, autoimmune disease, allergic disease, and cancer (5–10). Therefore, NKT cells represent an immunotherapeutic target with broad clinical potential. CD1d-restricted T cells can be divided into four main subsets based on their T cell receptor (TCR) usage and antigen specificities (**Table 1**). Type I (or invariant) NKT cells constitute the first and best characterized subset, and use an invariant

#### *Edited by:*

*Thierry Mallevaey, University of Toronto, Canada*

#### *Reviewed by:*

*Vipin Kumar, University of California San Diego, USA Sebastian Joyce, Vanderbilt University School of Medicine, USA*

#### *\*Correspondence:*

 *Manfred Brigl, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Harvard New Research Building, Boston, MA 02115, USA<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

> *Received: 21 May 2015 Accepted: 04 July 2015 Published: 28 July 2015*

#### *Citation:*

*Macho-Fernandez E and Brigl M (2015) The extended family of CD1d-restricted NKT cells: sifting through a mixed bag of TCRs, antigens, and functions. Front. Immunol. 6:362. doi: 10.3389/fimmu.2015.00362*

#### TABLE 1 | Classification of CD1d-restricted T cells.

α*-GalCer,* α*-galacotsylceramide;* α*-GSL,* α*-glycosphingolipid;* α*-GDAG,* α*-glyco(Gal/Glc)diacylglycerol;* β*-GlcSph,*β*-glucosylsphingosine; DPG, diphosphatidylglycerol; h, human; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LSM, lysosphingomyelin; m, mouse; PG, phosphatidylglycerol; NKT, natural killer T cell; TCR, T cell receptor.*

TCRα chain (Vα14-Jα18 in mice and Vα24-Jα18 in humans). In addition, there are CD1d-restricted T cells that use a more varied TCR repertoire, including Vα24− and Vα10+ NKT cells in mice and humans, respectively, diverse or type II NKT cells, as well as T cells expressing γδ or δ/αβ TCRs. In this review, while using type I NKT cells as comparison, we will focus on type II NKT cells and the other non-invariant CD1d-restricted T cell populations and discuss our emerging understanding of their TCR usage, antigen specificities, mode of antigen/CD1d recognition, innate-like mechanisms of activation, and their immunological functions.

### The CD1d-Restricted T Cell Family

#### Type I NKT Cells

The discovery of the first CD1d-presented antigen, α-galactosylceramide (α-GalCer), by Kawano and colleagues in 1997 enabled several important steps forward in our understanding of NKT cell biology, particularly of type I or invariant NKT (iNKT) cells (11). Type I NKT cells express an invariant Vα14Jα18 TCR α-chain in mice and Vα24Jα18 in humans, paired with a limited repertoire of TCR β-chains (Vβ8, Vβ7, Vβ2 in mice and exclusively Vβ11 in humans) (**Table 1**). Type I NKT cells are highly autoreactive even at steady state and display an activated/memory phenotype with high surface levels of the activation markers CD69, CD44, and CD122 (IL-2R β-chain) and low expression of CD62L, a marker expressed by naïve T cells that home to lymph nodes (12, 13). The use of mice that are deficient in CD1d (lack type I and type II NKT cells) or selectively deficient in type I NKT cells (Jα18−/−), administration of α-GalCer to activate type I NKT cells *in vivo*, and the ability to track type I NKT cells with fluorescent CD1d/α-GalCer tetramers has allowed the elucidation of many functions of type I NKT cells *in vivo*. Type I NKT cells play critical roles in local and systemic immune responses and are essential for controlling tumor development and antimicrobial immune responses. They can also exert detrimental effects in the pathogenesis of autoimmune and allergic disorders. The biology of type I NKT cells has been extensively covered in several excellent recent reviews (2, 3, 14–16), including their roles in microbial infections (2, 7, 17–20), autoimmunity and inflammation (9, 21, 22), and tumor immunity (23). This broad range of type I NKT cell functions relies on their rapid secretion of copious amounts of various cytokines, including IFN-γ, IL-2, IL-4, IL-9, IL-10, IL-13, IL-17, IL-21, and GM-CSF (24–26), and their interactions with other immune cells (2, 16). Based on the differential expression of cytokines, transcription factors and surface markers, several functionally distinct type I NKT cell subsets have been described in humans and mice (**Table 1**). Human type I NKT cells expressing CD4 produce Th2-type cytokines whereas both CD4+ and CD4− subsets can generate Th1-type cytokines and secrete cytotoxic molecules such as perforin and granzyme (24, 27). In C57BL/6 mice, Th1-like type I NKT cells (also referred to as NKT1 cells) represent the majority of type I NKT cells in liver and spleen, are characterized by the Th1-associated transcription factor T-bet, mostly express NK1.1 and IL-12 receptor (IL-12R) and their pronounced production of IFN-γ is critical for their function during various immune responses, including anti-tumor immunity (24, 28, 29). Th2-like type I NKT cells (NKT2 cells) are the most abundant type I NKT cell subset in BALB/c mice and are enriched in lung and intestine of C57BL/6 mice. NKT2 cells have been reported to play an important role in Th2-mediated diseases through the secretion of IL-4, IL-9, IL-10, and IL-13 (30, 31). Th17-like type I NKT cells (NKT17 cells) are primarily found in lung, skin and peripheral lymph nodes, produce IL-17A and IL-22 (26, 32, 33), and express the retinoic acid receptor-related orphan receptor γt (RORγt) transcription factor (34). Finally, a small number of type I NKT cells with developmental, phenotypical, and functional features of follicular helper (35) T cells secrete IL-21, support the formation of germinal centers (36–38) and require the transcription factor Bcl-6 for their development (36). Despite their varied functional differentiation, most type I NKT cell subsets express the transcription factors PLZF, which is fundamental for their development, and GATA3 (39). However, instead of PLZF, a distinct type I NKT cell population, newly referred to as NKT10 cells, express the transcription factor E4BP4 which confers their regulatory properties (40, 41). Thus, the remarkable functional versatility of type I NKT cells during various immune responses can at least in part be explained by the existence of functionally distinct subsets, while organ-specific functions and plasticity of type I NKT cell subsets have not been adequately investigated.

#### **α**-GalCer-Reactive V**α**24**−** and V**α**10**+** NKT Cells

Recognition of α-GalCer by type I NKT cells was thought to be highly correlated with expression of the invariant Vα14-Jα18 or Vα24-Jα18 TCR α-chains in mice and humans, respectively. However, α-GalCer-reactive, CD1d-restricted NKT cells that use different TCR α-chains have subsequently been described in both mice and humans. In humans, one study using α-GalCerloaded CD1d tetramers found Vα24-negative T cell populations expressing Vα10, Vα2, or Vα3 joined to Jα18 and paired with Vβ11 (42) whereas another study found a diversity of Vα24− Jα18−/Vβ11− TCRs that stained with α-GalCer/CD1d tetramers (**Table 1**) (42–44). Despite their equivalent reactivity to α-GalCer or bacterial α-linked glycosphingolipids (GSLs), Vα24-negative subset displayed preferential antigen specificities for α-GlcCer (42). Surprisingly, a Vα24−/CD1d-α-GalCer+ population was found to predominantly display a naïve phenotype and low or intermediate expression level of PLZF (45). So far, data regarding the function of these NKT cells in humans are limited.

Similarly, in mice, CD1d-restricted NKT cells that recognize α-GalCer but do not use the canonical Vα14-Jα18 TCR α-chain have also been described, and predominantly express a semi-invariant Vα10-Jα50 TCR α-chain paired with a Vβ8+ TCR β-chain (Vβ8.1/0.2 or Vβ8.3). Vα10+ NKT cells have been identified in thymus, spleen, and liver of Jα18−/− mice by CD1d-α-GalCer tetramer staining, and displayed a CD44high CD69int pre-activated phenotype, similar to type I NKT cells. Like other NKT cell populations, their development required expression of CD1d. In addition to α-GalCer, Vα10+ NKT cells preferentially recognize other glucose-based glycolipids such as α-GlcCer, GSL-1 from *Sphingomonas* or α-GlcA-DAG from *Mycobacterium smegmatis*, and in response to stimulation with these glycolipids produce large amounts of cytokines including IFN-γ, IL-4, IL-13, and IL-17 (46). Thus, recognition of CD1d/α-GalCer complexes by NKT cells in humans and mice is not uniformly restricted to the use of Vα24-Jα18/Vβ11 or Vα14-Jα18 TCRs, respectively, but can be mediated by a range of Vα and Vβ domains, highlighting the variation in antigen recognition among CD1d-restricted α-GalCer-reactive TCRs. Furthermore, the different antigen specificities of some of these non-canonical α-GalCer-reactive NKT cells may correlate with distinct functional capabilities, and it remains to be explored if this subset harbors NKT cells that can be expanded *in vivo* and form memory responses.

#### Type II NKT Cells

CD1d-restricted T cells that do not express the Vα14-Jα18 rearrangement and do not recognize α-GalCer were first described in MHC II-deficient mice among the remaining CD4+ T cells (47). From then called diverse NKT (dNKT), type II NKT, or variant NKT (vNKT) cells, this NKT cell population, found in both mice and humans, exhibits a more heterogeneous TCR repertoire (**Table 1**). For example in mice, the type II NKT cells that have been described use Vα1, Vα3, Vα8, or Vα11 TCR α-chains paired with Vβ8 or Vβ3 TCR β-chains, or Vα4 paired with Vβ5 or Vβ11, and appear to contain oligoclonal Vα3.2-Jα9/Vβ8 and Vα8/Vβ8 TCR families (48–50). Currently, no direct and specific tools exist to identify the entire type II NKT cell population *in vivo*, but different approaches have been developed to study these cells in mice. The first is to compare the immune responses between Jα18−/− mice (lacking only type I NKT cells) and CD1d−/− mice (lacking both type I and type II NKT cells). It should be noted that Jα18−/− mice exhibit lower TCR repertoire diversity due to deficits in rearrangements of several Jα segments (51). This raises the possibility that defects described in Jα18−/− mice are not solely due to type I NKT cell deficiency and a more specific type I NKT cell-deficient mouse model is needed. The second tool used to study type II NKT cell function is 24αβ TCR transgenic mice that were generated by overexpressing the Vα3.2/Vβ9 TCR from the type II NKT cell hybridoma VIII24 (52). A third approach is the use of Jα18 deficient IL-4 GFP (Jα18−/− 4get) reporter mice. This model is based on the finding that some type II NKT cells spontaneously express IL-4 mRNA transcripts, similar to type I NKT cells (53, 54). However, this approach does not identify all type II NKT cells since, for example, GFP+ type II NKT cells from Jα18−/− 4get mice do not respond to sulfatide, an antigen that is recognized by a significant number of type II NKT cells. The fourth approach to identify type II NKT cells is the use of lipid antigen-loaded CD1d tetramer reagents, similar to the use of α-GalCer/CD1d tetramers that are used to detect type I NKT cells. The discovery of sulfatide as a potent type II NKT cell ligand led to the generation of sulfatide-loaded CD1d tetramers and revealed an oligoclonal TCR repertoire among sulfatide-specific type II NKT cells with predominant use of Vα3/Vα1-Jα7/Jα9 and Vβ8.1/Vβ3.1-Jβ2.7 genes (50, 55, 56). However, sulfatide/CD1d tetramers appear to be more difficult to use compared to α-GalCer/CD1d tetramers, likely due to difficulties in loading the antigen and/or greater instability of sulfatide/CD1d complexes and, moreover, not all type II NKT cells recognize sulfatide (50, 54). Similar to sulfatide, β-glucosylceramide (β-GlcCer)- or glucosylsphingosine β-GlcSph-loaded CD1d tetramers stained a subset of human and mouse type II NKT cells (57). Furthermore, the recent discovery of several microbial antigens recognized by different type II NKT hybridomas enables the design of antigen-loaded CD1d tetramers that are likely to be useful to characterize type II NKT cells *in vivo* (58, 59). Another approach to study type II NKT cells is the use of dNKT hybridomas that were initially identified by their recognition of CD1d-expressing APC and their use of TCR α-chains different from Vα14-Jα18 (47–49, 60, 61). These dNKT hybridomas were used to characterize the TCRs expressed by type II NKT cells and continue to be used to identify self- and microbial lipid antigens that are recognized by type II NKT cells.

Using the approaches described above, many type II dNKT cells appear to share phenotypic and functional features with type I NKT cells such as high autoreactivity (62), PLZF- and SAPdependent thymic development (54, 63), constitutive expression of IL-4 mRNA (54), and the ability to secrete a wide range of cytokines rapidly after stimulation, including IFN-γ, IL-2, IL-4, IL-10, IL-17, GM-CSF, and cytolytic mediators such as perforin (54, 63). Furthermore, many type II NKT cells have a CD44high CD69+ CD122+ activated/memory phenotype, whereas CD62L is more or less expressed dependent on which transgenic mouse model is used, and can be divided into different subsets depending on CD4 and NK1.1 expressions (54, 63–65). However, several studies suggest that type II NKT cells exist that are phenotypically and functionally distinct from type I NKT cells. For example, most of the T cells stained with sulfatide/CD1d tetramers in C57BL/6 mice did not express the early activation marker CD69 (50). Moreover, in 24αβ TCR transgenic mice on the non-obese diabetic (NOD) background, the majority of DN 24αβ NKT cells were CD44int, CD45RBhigh, CD62Lhigh, CD69−/low, similar to conventional T cells, whereas the majority of CD4+ 24αβ NKT cells exhibited the typical type I NKT CD44high, CD45RBlow, CD62Llow, CD69high activated/memory phenotype (66). In addition, in both humans and mice, type II NKT-TFH populations have recently been described that recognized β-GlcCer or β-GlcSph (57). The human type II NKT-TFH population utilized Vα24−/Vβ11− TCRs with diverse Vβ chains and displayed a naïve CD45RA+, CD45RO−, CD62high, CD69−/low phenotype. The majority of these cells expressed a TFH-like phenotype in mice and humans (CXCR5+, PD-1high, ICOShigh, Bcl6high, FoxP3−, IL-21+) at steady state and mainly secreted IL-5, IL-6, IL-10, and IL-17 following activation. Their TFH properties were associated with the induction of GC B cells and lipid-specific antibodies *in vivo* in a CD1d-dependent manner.

In humans, CD1d-restricted type II NKT cells appear to be much more frequent than type I NKT cells. In human bone marrow, approximately 25% of CD3+ T cells expressed CD161 and half of the CD161+CD3+ cells recognized CD1d. Interestingly, the majority of these CD1d-restricted T cells used Vα24−/Vβ11− TCRs (67). In PBMC of healthy individuals, approximately 0.5% of CD3+ lymphocytes stained with β-GlcCer/CD1d tetramers, similar to numbers in Gaucher's disease patients, whereas 1–2% of CD3+ lymphocytes in these patients stained positive with β-GlcSph/CD1d tetramers, compared to 0.2% in healthy individuals (57). In myeloma patients, lysophosphatidylcholine (LPC)-loaded CD1d dimers stained on average 0.6% of T cells in PBMC, several fold higher than type I NKT cell numbers determined with α-GalCer-loaded CD1d dimers (68), whereas in healthy controls, both LPC- and α-GalCer-loaded CD1d dimers stained approximately 0.05% of PBMC.

Thus, type II NKT cells can be distinguished from type I NKT cells by their use of more diverse TCRs and their distinct antigen specificities. Many type II NKT cells are phenotypically and functionally similar to type I NKT cells, however, some type II NKT cells appear to have a naïve T cell phenotype and further studies are required to test if these cells are capable to form antigen-specific memory responses. In addition, similar to type I NKT cells, type II NKT cells may also harbor functionally distinct subsets, and it will be important to determine to what extent functional differentiation correlates with antigen specificities.

### CD1d-Restricted **γδ** and **δ**/**αβ** T Cells

In addition to the use of αβ TCRs, CD1d-restricted T cells expressing γδ TCRs have recently been described in both mice and humans (**Table 1**). According to their Vδ-chain expression, human γδ T cells can be divided into two major populations: Vδ2+ and "non-Vδ2" subsets, the latter comprise Vδ1+ γδ T cells and the less prevalent Vδ3+ γδ T cells (69, 70). Vδ1+ γδ T cells are mainly tissue-resident and are found in the skin and at mucosal surfaces, whereas Vδ2+ γδ T cells are predominant in human blood. Compared with αβ T cells, the types of antigens recognized by γδ T cells and the role and function of antigen presentation in γδ TCR recognition are much less clear. Interestingly, some γδ T cells have recently been found to directly recognize CD1dpresented lipid antigens (71). Indeed, Spinazzo and colleagues showed that only γδ T cells, but not αβ T cells, from peripheral blood and nasal mucosa of cypress pollen-sensitive subjects were activated in a CD1d-dependent manner by phospholipids extracted from pollen grains (72–74). Up to now thought of as type I NKT cell-specific ligands, α-GalCer and some of its derivatives including OCH were recognized by a subset of circulating human Vδ1+ γδ T cells (75). Similarly, sulfatide-reactive CD1drestricted Vδ1+ γδ T cells were found in human blood and among gut T cells (76, 77). In addition, Vδ3+ γδ T cells found in the liver of patients with leukemia or chronic viral infection recognized CD1d molecules and killed CD1d+ cells (78). In mice, development of myocarditis following infection with coxsackievirus B3 (CVB3) relied on CD1d up-regulation and CD1d-dependent activation of Vγ4+ γδ T cells (79). Furthermore, α-GalCer reactive, CD1d-restricted T cells using TCRs in which the Vδ1 gene is fused to Jα and Cα domains that are paired with an array of TCR β-chains to form a δ/αβ TCR have recently been described (80). Thus, lipid-specific γδ and δ/αβ TCRs expand the TCR diversity among CD1d-restricted T cells. Understanding the full range of antigens that are recognized by CD1d-restricted T cells that use TCR γ/δ genes, the extent of permissive TCR diversity and the numbers of these cells under physiologic conditions and during infection and other pathologies constitute important areas for future exploration.

### Mechanisms of CD1d-Restricted T Cell Activation

In contrast to the high degree of TCR diversity and antigen specificity of adaptive MHC-restricted T cells, CD1d-restricted T cells utilize a more restricted TCR repertoire and recognize antigens presented by a monomorphic antigen-presenting molecule. Yet, these cells are able to respond to highly diverse infectious agents and become activated in a variety of non-infectious pathological conditions. Recent progress has started to shed light on how the self- and exogenous lipid antigens that are presented by CD1d, the molecular underpinnings of TCR-mediated recognition of lipid/CD1d complexes, the antigen-presenting pathways that result in formation of stimulatory lipid/CD1d complexes, and the non-TCR signals all contribute to the activation of type II NKT cells and other non-invariant CD1d-restricted T cell populations.

#### CD1d-Presented Ligands

α-galactosylceramide and numerous related derivatives have helped to define the range of antigen specificity among type I NKT cells and provide powerful agents for their pharmacologic stimulation with various functional outcomes (**Figure 1**). Furthermore, several α-linked GSLs and diacylglycerols that stimulate type I NKT cells have been isolated from various microbes, confirming that α-linked GSLs are indeed important natural antigens for type I NKT cells. In addition, an intensive search for the self-antigens that are critical for type I NKT cell development has revealed several candidates, including iGb3, β- and α-linked GSLs, and various phospholipids. All of these antigens have been the topic of excellent recent reviews and will not be discussed in detail here (81–84). Similarly, a number of endogenous and exogenous glycerol- or ceramide-based lipid antigens that stimulate non-invariant CD1d-restricted T cells have been identified, but significant differences in antigen specificity exist between the various CD1d-restricted T cell subsets as a result of their varied TCR usage. For example, type II NKT cells display specificities for antigens that are not typically considered to be agonists for type I NKT cells, but some overlap in antigen specificity between these two NKT cell subsets is becoming apparent. In contrast, only subtle differences in antigen specificity have been observed between type I NKT cells and the non-canonical Vα24− NKT cells and Vα10+ in humans and mice, respectively. Interestingly, CD1d-restricted T cells with γ/δ TCRs have been identified that recognize α-GalCer or sulfatide, antigens that were thought to be specific for type I and type II NKT cells, respectively. Thus, antigen specificity alone is not a reliable criterion to define CD1d-restricted T cell subsets (**Table 1**; **Figure 2**).

#### Self-Ligands

Type II NKT cells were originally identified based on their autoreactivity to CD1d-expressing cells and subsequently individual stimulatory self-lipid antigens were identified. In addition, numerous studies have now demonstrated the activation of non-invariant CD1d-restricted T cells in diseases where foreign lipids are not present, such as autoimmunity, cancer, viral infection, or in response to toll-like receptor agonists, suggesting that the recognition of self-lipids is critical for their peripheral activation.

Sulfatide (SO3-3Galβ1Cer) is a self-GSL recognized by a subset of type II NKT cells that predominantly express Vα3/Vα1 and Vβ8.1/Vβ3.1 TCRs (**Figure 1**) (50, 55). Native sulfatide is a mixture of multiple sulfatide isoforms with different lengths and saturation of their fatty acid chains, and is found in many organs (brain, kidney, pancreatic β-cells) and tumor cells. A major component of the myelin sheets of the central nervous system, the mono-unsaturated C24:1 sulfatide isoform activates XV19 type II NKT cells in a CD1d-dependant manner, unlike its saturated (C24:0) or shortened (C18:1) isoforms. Although found in the nervous system in only minor quantities, the lysosulfatide (LSF) that lacks the entire fatty acid chain, is the most potent sulfatide isoform described (85). However, the XV19 type II NKT hybridoma exhibits high autoreactivity toward splenocytes from CST−/− mice that lack cerebroside sulfotransferase and therefore cannot generate sulfatide, and sulfatide-reactive NKT cells were still detected in CST−/− mice (55), indicating that sulfatide is not required for type II NKT cell development. Administration of sulfatide to mice has widely been used to study the function of sulfatide-specific type II NKT cells *in vivo*. In humans, the majority of T cells in PBMC that stained with sulfatide-loaded CD1d tetramers were γδ T cells that use a semi-invariant Vδ1+ γδ TCR (76).

Another β-linked GSL, β-glucosylceramide (β-GlcCer), has recently been shown to activate type I and type II NKT cells (**Figure 1**). Stimulation of APC by diverse TLR agonists can modify the GSL biosynthesis pathway by enhancing the expression of several glycosyltransferases and, as a consequence, the accumulation of endogenous GSL, including β-GlcCer, that are then presented by CD1d (86, 87). However, two recent studies have questioned the role of β-GlcCer in the activation of type I NKT cells. The studies attributed the stimulatory activity of synthetic or mammalian β-GlcCer to co-purified α-GlcCer and suggest that mammalian immune cells produce constitutively small quantities of stimulatory α-glycosylceramides under control of catabolic enzymes of the ceramide and glycolipid pathway (88, 89). For type II NKT cells, the β-GlcCer-22:0 isoform and its deacylated product, glucosylsphingosine or β-GlcSph, have been shown to activate type II NKT subsets in humans and mice and CD1d tetramers loaded with these antigens can be used to stain these cells (57). In another example of induced self-lipidmediated activation, Zhao and co-workers showed that type II NKT cells in the Jα18-deficient IL-4 reporter mouse were activated in response to CpG ODN, a potent synthetic agonist of TLR9, which mimics a hallmark of microbial DNA (54). Similar to what has been observed for type I NKT cells (86), type II NKT cell activation by CpG ODN resulted in production of IFN-γ but not IL-4 or IL-13, and was partially CD1d-dependent. Type II NKT cells isolated from Jα18-deficient IL-4 reporter mice recognized β-GlcCer but not sulfatide or phospholipids. However, the lipids responsible for the activation of type II NKT cells following CpG ODN stimulation were not identified. Several type II NKT hybridomas (i.e., XV19, VIII24, VII57) were unresponsive to α-glycosylceramides such as α-GclCer (90) and autoreactivity of the XV19 type II NKT cell hybridoma persisted with use of β-GlcCer- or GSL-deficient cell lines, suggesting that glycolipids are not the only self-lipids responsible for the autoreactivity of type II NKT cells (91).

Mammalian phospholipids are a major component of cell membranes and have been identified as natural antigens that are recognized by CD1d-restricted NKT cells. Mouse CD1drestricted type I and type II NKT cell hybridomas exhibit distinct but overlapping antigen recognition specificities, particularly in response to phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) (**Figure 1**) (62). Mammalian phospholipids differ from their

microbial counterparts in the fatty acyl chain composition or the position of the fatty acyl molecules at the *sn*-1 and *sn*-2 positions of the glycerol backbone. Such modifications of the lipid tails can considerably impact type I NKT cell activation (92). Surprisingly, C16:0/18:1 PG isolated from murine skin, bovine DPG mainly composed of (C18:2/C:18:2)2 species and their respective synthetic version have high potency to activate the 14S.10 type II NKT cell hybridoma in a CD1d-dependent manner (58), suggesting that both self- and microbial phospholipids

Lysophospholipids are produced after phospholipid hydrolysis by a phospholipase and act as lipid messengers in many physiological processes. These lipids are found in high concentration at inflammatory sites, suggesting a role in the etiology of disorders such as autoimmune diseases, obesity, atherosclerosis, and cancer. Markedly up-regulated in myeloma patients, LPC species were recognized by human type I NKT cell clones (93) and Vα24−/Vβ11− type II NKT cells (**Figure 1**) (68). Chang et al. also reported that human LPC-reactive type II NKT cells predominantly secreted IL-13 in response to LPC, suggesting an immunosuppressive function of these cells (68). LPC isoforms C18:0 and C16:0 were the most potent to activate sulfatide-reactive type II NKT cells compared to the C24:0 isoform (94). Notably, LPC is not recognized by murine type II NKT cells. Similar to LPC, lysosphingomyelin (LSM), which displays the same head group, choline, activated both type I and XV19 type II NKT cells (93, 94). Recently, Zeissig and co-workers showed that lysophosphatidylethanolamine (LPE), which accumulated during hepatitis B virus (HBV) infection, activated type II NKT cells from Jα18-deficient IL-4 reporter mice but not type I NKT cells (53). This activation could be induced by different LPE isoforms, including C16:0, C18:0, and C18:1. Thus, activation of CD1d-restricted T cells in inflammatory contexts where exogenous microbial lipid antigens are not available can result from recognition of induc-

Thus, several self-lipids are recognized by subsets of type II NKT cells and recognition of these lipids is important for type II NKT cell function. Indeed, during microbial infection and non-infectious inflammatory conditions, up-regulation and presentation of self-lipids or stimulatory lyso forms converts innate

danger signals into activation of type II NKT cells.

can be recognized by these T cells.

ible self-lipids.

cells), and phenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonate (PPBF, type II NKT cells).

microbial counterparts in the fatty acyl chain composition or the position of the fatty acyl molecules at the *sn*-1 and *sn*-2 positions of the glycerol backbone. Such modifications of the lipid tails can considerably impact type I NKT cell activation (92). Surprisingly, C16:0/18:1 PG isolated from murine skin, bovine DPG mainly composed of (C18:2/C:18:2)2 species and their respective synthetic version have high potency to activate the 14S.10 type II NKT cell hybridoma in a CD1d-dependent manner (58), suggesting that both self- and microbial phospholipids can be recognized by these T cells.

Lysophospholipids are produced after phospholipid hydrolysis by a phospholipase and act as lipid messengers in many physiological processes. These lipids are found in high concentration at inflammatory sites, suggesting a role in the etiology of disorders such as autoimmune diseases, obesity, atherosclerosis, and cancer. Markedly up-regulated in myeloma patients, LPC species were recognized by human type I NKT cell clones (93) and Vα24−/Vβ11− type II NKT cells (**Figure 1**) (68). Chang et al. also reported that human LPC-reactive type II NKT cells predominantly secreted IL-13 in response to LPC, suggesting an immunosuppressive function of these cells (68). LPC isoforms C18:0 and C16:0 were the most potent to activate sulfatide-reactive type II NKT cells compared to the C24:0 isoform (94). Notably, LPC is not recognized by murine type II NKT cells. Similar to LPC, lysosphingomyelin (LSM), which displays the same head group, choline, activated both type I and XV19 type II NKT cells (93, 94). Recently, Zeissig and co-workers showed that lysophosphatidylethanolamine (LPE), which accumulated during hepatitis B virus (HBV) infection, activated type II NKT cells from Jα18-deficient IL-4 reporter mice but not type I NKT cells (53). This activation could be induced by different LPE isoforms, including C16:0, C18:0, and C18:1. Thus, activation of CD1d-restricted T cells in inflammatory contexts where exogenous microbial lipid antigens are not available can result from recognition of inducible self-lipids.

Thus, several self-lipids are recognized by subsets of type II NKT cells and recognition of these lipids is important for type II NKT cell function. Indeed, during microbial infection and non-infectious inflammatory conditions, up-regulation and presentation of self-lipids or stimulatory lyso forms converts innate danger signals into activation of type II NKT cells.

### Microbial and Other Exogenous Ligands

Until recently, no microbial antigen had been identified that could be directly recognized by non-iNKT cells. Based on the ability of human CD1b molecules to present lipid antigens from *Mycobacterium tuberculosis* (*Mtb*) to diverse CD1b-restricted T cells (7) and due to the similarities between CD1b and CD1d intracellular trafficking and localization in humans (95), we investigated *Mtb* lipids for antigens that may be recognized by type II NKT cells. Purification, isolation and structural analysis of *Mtb* polar lipids revealed PG species with different acyl chain combinations (including C19:0/C16:0, C19:0/C16:1, C18:1/ C16:0, and C18:1/C16:1) to stimulate several type II NKT cell hybridomas (14S.6, 14S.10, TBA7, VII68, and XV19) in a CD1ddependent manner (**Figure 1**) (93). Moreover, PG, diphosphatidylglycerol (DPG or cardiolipin) and PI from *Corynebacterium glutamicum* (*Cg*), with C18:1/C16:0, (C18:1/C16:1)2, and C18:1/ C16:0 fatty acid composition, respectively, also activated type II NKT cells, in particular 14S.6, 14S.10, and TBA7 hybridoma cells. In addition, *Mtb* and *Cg* apolar lipids activated type II NKT cell clones in a CD1d-dependent manner, but specific stimulatory lipid species have not yet been identified from this lipid fraction. Similarly, Wolf and colleagues recently demonstrated that PG and DPG isolated from *Listeria monocytogenes* (*Lm*) were also potent ligands for type II NKT clones (TBA7 and 14S.10 especially) (59). Interestingly, whereas in both studies neither PG nor DPG from *Mtb*, *Cg*, and *Lm* succeeded in activating type I NKT cell hybridomas, DPG can be recognized by a subset of CD1d-restricted γδ T cells. Indeed, hepatic and splenic γδ T cells secreted IFN-γ and RANTES (CCL5) in response to DPG in a CD1d-dependent manner (74).

Phospholipids are also found in plant, especially in pollen, which are an important source of environmental allergens. Extracted from cypress grains, phospholipids, as PC in particular, stimulated non-Vα24 NKT and γδ T cells from cypress pollen-sensitive subjects in a CD1d-restricted fashion (72, 73, 96). However, the structure of the acyl chain combination of the stimulatory PC from pollen has not been defined. Interestingly, non-lipidic small molecules can also be associated with CD1d proteins with low affinity. These sulfur-containing molecules, including phenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonate (PPBF) and structurally related compounds (**Figure 1**), activated human non-Vα24− CD1d-restricted T cells that expressed a Vα2/

FIGURE 1 | Antigens recognized by CD1d-restricted T cells. Shown are the structures of lipid antigens for type I and/or type II NKT cells: the prototypical type I NKT cell glycolipid α-galactosylceramide (α-GalCer); the

*tuberculosis* (*Mtb*) (type II NKT cells); the mammalian diphosphatidylglycerol (DPG or cardiolipin) whose *Mtb* counterpart shares the same alkyl chains as *Mtb* PG (Type II NKT cells); lysophosphotidylethanolamine (lysoPE; type I and type II NKT cells), lysophasphatidylcholine (LysoPC; type I and type II NKT cells), and phenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonate (PPBF,

type II NKT cells).

lipid self-antigen sulfatide (type II NKT cells); the mammalian phosphatidylglycerol (PG; type I and type II NKT cells) and its microbial counterparts from *Corynebacterium glutamicum* (*Cg*) or *Mycobacterium*  Vβ21 TCR (97). Thus, in addition to the prominent recognition of self-lipid antigens, non-invariant CD1d-restricted T cells also recognize a range of microbial and exogenous lipids presented by CD1d.

#### CD1d/Antigen Recognition

Considering the differences in TCR usage and antigen specificities among the different CD1d-restricted T cell populations, unraveling the molecular mechanisms by which their TCRs recognize CD1d/antigen complexes is central to understanding their functions. Indeed, recent structural analyses have brought to light key differences in antigen/CD1d recognition by the TCRs of type I NKT cells, Vα24−/Vα10+ NKT cells, type II NKT cells, and γδ T cells.

The invariant type I NKT cell TCR docks over the F′ pocket of CD1d in an orthogonal conformation (**Figures 3A,B**) (98, 99). Only the α-chain, via the complementary-determining region 1α (CDR1α) and CDR3α, is in contact with the head group of α-linked glycolipids such as α-GalCer, and thus has an important role in specificity for, and recognition of, glycolipids. CDR3β and CDR2β loops stabilize the complex by interacting with the CD1d molecule. Variations in TCR β-chain usage cause subtle structural modifications in the conformation of the TCR α-chain, which indirectly contribute to the preferential recognition of some antigens or modulate the affinity for antigen/CD1d complexes (99–101). For the recognition of β-linked ligands, type I NKT TCRs operate through induced-fit molecular mimicry by pushing the β-linked headgroup into a flattened position that is similar to that of the headgroup of stimulatory α-linked lipids (102–104). Therefore, the mode of antigen/CD1d recognition by type I NKT TCRs has been compared to the function of pattern-recognition receptors that interact in an innate-like and conserved manner with ligands that represent microbe-associated molecular patterns or endogenous stress-induced ligands (105, 106).

The Vα24− and Vα10+ NKT TCRs from humans and mice, respectively, display very similar conformation compared with type I NKT TCRs in complex with α-GalCer/CD1d, except that the Vα10+ CDR2α loop is also in contact with the antigen (46) and that the Vα24− CDR1α loop interacts with the galactose headgroup highly contributing to the TCR fine specificity for the antigen (44).

In contrast, structural analysis of the tri-molecular sulfatide/ CD1d/XV19 TCR complex showed that this type II NKT TCR docks above the extreme end of the A′ pocket and adopts a parallel mode vis-a-vis of the CD1d molecule, similar to the interaction of conventional T cell with MHC molecules (**Figures 3A,C**). Both TCR α- and β-chains interact with CD1d and CDR1β and CDR3β loops confer recognition and discrimination for the antigen (107, 108). Unlike the recognition of β-linked glycolipids by the type I NKT TCR, the type II NKT TCR does not flatten the sulfatide headgroup during ligation. Thus, differences at the TCR-CD1d/antigen interfaces between type I and type II NKT cells provide insights into the molecular basis for the different ligand specificities and highlight how altered TCR use results in a differing docking solution on a monomorphic antigen-presenting molecule. The extent to which the TCR diversity of type II NKT cells results in different docking solutions for distinct TCRs remains to be determined.

Similar to type II NKT TCRs, the TCRs of CD1d-restricted γδ T cells dock above the A′ pocket and all CDRδ loops (CDR1δ, CDR2δ, CDR3δ) are in contact with CD1d (**Figure 3D**). Notably, the CDR3δ loop also contacts the antigen. Whereas sulfatide recognition requires a central position of the δ-chain above the lipid portal with no contribution of the TCR γ-chain (77), the interaction with α-GalCer involves γ-chain/CD1d contacts and a docking mode closer to the extreme end of the A′ pocket (75). Thus, γδ T cells can utilize a variety of interactions with their TCR γ- and δ-chains to recognize lipid/CD1d complexes.

#### CD1d Antigen Presentation

In humans, the CD1 family is composed of five isoforms divided into two groups: CD1a, CD1b, CD1c, and CD1e forming group 1 and CD1d forming group 2. In mice, only the CD1d isoform is expressed. CD1 molecules are transmembrane glycoproteins that display structural similarities to MHC class-I: one heavy chain composed of three domains (α1, α2, and α3) non-covalently linked to β2-microglobulin via the α3 domain. The CD1d ligandbinding site is composed of α1 and α2 helices, which form two deep hydrophobic pockets, called A′ and F′. The pockets accommodate hydrocarbon chains of glycolipid antigens leading to the protrusion of the lipid head group on the surface and thus its accessibility to the TCR of CD1d-restricted T cells (109, 110). The α3 domain connects the ligand-binding region to a transmembrane domain, followed by a short cytoplasmic tail. The cytoplasmic tail of human and mouse CD1d is critical for its intracellular localization as deletion of a tyrosine-based motif encoded in it interrupts CD1d recycling between the plasma membrane and endolysosomal compartments (**Figure 4**) (111, 112). Moreover, the internalization of murine CD1d and its subsequent lysosomal localization require the binding of the adaptor protein complex AP-3 to the tyrosine-based motif (113, 114). Interestingly, human CD1d fails to bind the AP-3 complex, unlike human CD1b (113). In humans, CD1d trafficking from ER to cell surface and lysosomes is orchestrated by its physical interaction with MHC II and invariant chain (115, 116). By extension from its role in CD1d trafficking, the tyrosine-based motif in the cytoplasmic tail of CD1d is also critical for type I NKT cell function. Indeed, its deletion impaired type I NKT cell development and activation by endogenous and exogenous antigens such as α-GalCer (60, 112, 117). In contrast, CD1d with a truncated cytoplasmic tail is still capable of activating type II NKT cells and does not impair their development, suggesting that loading of type II NKT cell self-antigens onto CD1d occurs within the ER (60, 111, 112). Recently, Shin and co-workers have shown that three consecutive arginine residues between the transmembrane region and the cytoplasmic tail are involved in the intracellular trafficking of CD1d and the presentation of endogenous glycolipids to both type I and type II NKT cells (118). Selection and editing of CD1dbound lipids is also influenced by accessory lipid-binding and -loading molecules such as the microsomal triglyceride transfer protein (MTP) and saposins that facilitate the formation of lipid/CD1d complexes in the ER and throughout the endocytic pathway, respectively (**Figure 4**). The ability of CD1d molecules to differentially stimulate type I versus type II NKT cells based on their distinct intracellular trafficking routes is associated with

loading of distinct self-lipids onto CD1d. Indeed, using lipid elution and mass spectrometry, Yuan et al. demonstrated that ER-retained CD1d molecules associated with phosphatidylcholine (PC), whereas CD1d molecules that trafficked through the secretory pathway were loaded with sphingomyelin for which terminal synthesis occurs within the Golgi apparatus (119). Interestingly, recycling CD1d molecules carried a combination of both sphingomyelin and PC but also lysophospholipids that result from lipid degradation by phospholipase in lysosomes. In contrast to the presentation of endogenous lipid antigens, activation of type II NKT cells by exogenous self- or microbial lipids requires cellular lipid uptake and intersection of lipid loading and intracellular CD1d trafficking in secretory and/or lysosomal compartments (93, 120). Thus, different intracellular antigen presentation pathways result in formation of stimulatory lipid/ CD1d complexes for type I and type II NKT cells.

#### Cytokine- and NK Receptor-Mediated Activation

Activation of naïve MHC-restricted T cells is controlled by TCR signals that result from highly specific recognition of peptide antigen/MHC complexes in combination with receptor-mediated co-stimulatory signals, whereas the immediate cytokine milieu is critical to guide the differentiation of T cell effector functions. In contrast, innate T cells such as type I NKT cells have adopted a strategy for their activation that integrates TCR-mediated signaling and stimulation by pro-inflammatory cytokines to result in rapid activation (16). Dependent on the context and on the affinity of the CD1d-presented lipid antigens, one signal prevails over the other. For example, a high-affinity lipid antigen such as a potent induced self-lipid or microbial lipid can result in strong and predominantly TCR-mediated activation with no or only limited need for cytokine stimulation. In contrast, presentation of low-affinity microbial or self-antigens requires stimulation with antigen-presenting cell-derived inflammatory cytokines to result in overt NKT cell activation. This cytokine-mediated stimulation can be so strong that it alone results in overt activation, obviating the need for TCR-mediated stimulation altogether. This mechanism ensures activation of type I NKT cells early in an ensuing inflammatory response during infection even when no microbial CD1d-presented lipids are expressed, as is the case, for example, during viral infections, and similar mechanisms may explain type I NKT cell activation in tumor immunity and autoimmune diseases (121, 122). At steady state, type I NKT cells highly express a wide range of cytokine receptors and have therefore the ability to respond to multiple cytokines like IL-12 (123, 124), IL-18 (125), and IL-23 alone or in combination with IL-1β (126, 127), IL-25 (30), and IFN type 1 (86). Depending on the cytokines present in the inflammatory environment, type I NKT cells secrete different cytokines such as IFN-γ in response to IL-12, IL-18 or IFN type 1, IL-17 with IL-23, and IL-22 with IL-23/IL-1β in combination.

Rolf and co-workers have compared by gene expression profiling the expression of cytokine receptors between type I NKT cells and type II NKT cells from 24αβ TCR transgenic mice (128). Interestingly, whereas *IL-18r1*, *IL-18rap* (IL-18 receptor associated protein), and *IL2ra* (CD122) were expressed at high and similar levels in both subsets, type I NKT cells present a higher expression of *IL12rb1* (up to 3-fold) and *IL2ra* (CD25) (up to 11-fold). Compared to conventional naïve CD4+

T cells, both type I and type II NKT cells displayed low levels of *IL1r2*, *IL6ra*, and *Ifngr2*. Recently, IL-25 has been shown to activate type II NKT cells *in vivo* and promote their production of IL-13 through IL17RB (129, 130). This suggests that, similar to type I NKT cells, type II NKT cells can respond to stimulation with inflammatory cytokines. Interestingly, during HBV infection activation of type II NKT cells was primarily driven by recognition of CD1d-presented lysophospholipids and largely independent of IL-12 signaling, whereas activation of type I NKT cells was significantly reduced in the absence of IL-12 (53). Whether indirect mechanisms also contribute to the activation of CD1d-restricted γδ T cells remains to be determined.

Inflammation and viral infection lead to alterations of the endogenous lipid

Initially named owing to their expression of NK1.1, NKT cells express a multitude of both activating (for example, NKG2D, NK1.1, CD160, NKR-P1A, NKp46) and inhibitory (Ly49c, Ly49G2, NKG2A, 2B4) NK receptors that play an important role in their activation and regulation. For example, stimulation of NK1.1 and NKG2D was sufficient to directly activate type I NKT cells (131, 132), and NKG2D–ligand interactions were critical for type II NKT cell-mediated disease induction in a mouse model of HBV infection (133). At steady state, both NKT cell subsets highly express *Klrk1* (NKG2D), *Klrb1c* (NK1.1), and *CD160* but only type II NKT cells display high levels of *Klra3* (Ly49c), *Klra7* (Ly49G2), and in a lesser manner *Ncr1* (NKp46). For their part, type I NKT cells express higher levels of *Klrb1a* (NKR-P1A) and *Klrc1* (NKG2A) (128).

Thus, in order to overcome the limitations of restricted antigen specificity that result from limited TCR diversity and the recognition of a monomorphic antigen-presenting molecule, both type I and type II NKT cells appear able to integrate stimulatory signals provided by TCR-mediated stimulation and inflammatory cytokines with modulation by NK receptor-signaling, to ensure their rapid activation in various infectious and inflammatory contexts.

### Functions of Type II NKT Cells

MTP, microsomal triglyceride transfer protein.

In contrast to the large body of literature previously mentioned that has documented a role for type I NKT cells in various pathological states, progress in understanding the role of other CD1d-restricted T cell populations has been hampered by the limited ability to track these cells and the lack of models to assess their function *in vivo*, in particular for Vα24−/Vα14− NKT cells and CD1d-restricted γδ or δ/αβ T cells. However, several lines of evidence suggest that type II NKT cells can contribute to and modulate a range of immune responses, occasionally in opposing roles to type I NKT cells.

#### Microbial Infections Viral Infections

The first indication that type II NKT cells can contribute to protective immunity during viral infection came from studies in mice using diabetogenic encephalomyocarditis virus-induced pathology (EMCV-D) that is characterized by hind-limb paralysis and impaired glucose-tolerance resulting from virus cytopatic effects on neuronal cells and islet cells, respectively. Whereas EMCV-D infection resulted in similar disease severity in WT and Jα18−/− mice, CD1d−/− mice exhibited a higher incidence and exacerbated severity of the disease, suggesting a protective role of type II NKT cells during this infection (**Figure 5**) (134). However, this protective function was not unique to type II NKT cells since activation of type I NKT cells by α-GalCer provided protection of WT mice during EMCV-D infection. An additional example of the protective potential of type II NKT cells during viral infection comes from studies in humanized SCID mice that were infected with HIV-1, in which administration of sulfatide resulted in reduced viral replication (135). Thus, natural or sulfatide-induced activation of type II NKT cells during viral infections can enhance protective innate and adaptive immune responses (**Figure 5**).

Infection with human HBV is a common cause of acute and chronic liver injury, including cirrhosis and hepatocellular carcinoma. The immune response during HBV infection plays a dual role, by eliciting tissue damage in response to viral antigens and by controlling viral replication. Mice engineered to express HBV envelope proteins in hepatocytes, or a terminally redundant HBV DNA construct that results in intrahepatic HBV replication has been used to study HBV infection in mice, and showed that type II NKT cell responses contributed to acute hepatitis and tissue injury in a CD1d- and NKG2D-dependent manner (133, 136). More recently, Zeissig and colleagues used HBV-expressing adenoviral particles to reproduce HBV infection in mice and uncovered a protective role for type II NKT cells (53). Type II NKT cell activation in response to HBV was dependent on the expression of CD1d and MTP (53) which transfers endogenous phospholipids onto CD1d (137, 138), suggesting an essential role for endogenous lipid presentation. Furthermore, as observed both in patients with viral hepatitis and HBV-infected mice, HBV infection increased the transcription of the secretory phospholipase A2 (sPLA2) (139) and expression of one of its substrates, PE. Interestingly, the analysis of microsomal lipids revealed an increase of LPE isoforms able to activate type II NKT cells. Type II NKT cell activation during HBV infection was predominantly based on LPE recognition, as IL-12 neutralization had little effect. By contrast, type I NKT cells did not recognize LPE but required IL-12 and MTP, suggesting that their activation occurred after type II NKT activation and APC maturation. In addition to their critical role in initiating an innate immune response against HBV in this model, type II NKT cells appear to also be involved in modulating the generation of protective adaptive immune responses. Indeed, HBV-infected CD1d−/− mice (lacking both type I and type II NKT cells) displayed significantly reduced activation of CD4 and CD8 T cells, whereas Jα18−/− mice (lacking only type I NKT cells) showed only reduced activation of CD4 T cells. This type II NKT cell-dependent reduction of adaptive CD8+ T cell responses during HBV infection led to a defect in viral control and resulted in chronic inflammation (53). Thus, in mouse models of HBV infection, type II NKT cells can both contribute to immune-mediated tissue damage and to the innate and adaptive immune responses that control viral replication (**Figure 5**). In humans, type II NKT cells that produced large amounts of IFN-γ, but little IL-13 or IL-4, accumulated in the liver during hepatitis C (HCV) infection, but their role during infection remains to be determined (140, 141).

#### Bacterial Infections

As described above, type II NKT cells are activated in response to diverse bacterial antigens, suggesting a role for these cells in host defense against these bacterial pathogens. During sepsis, the immune response consists of a primary phase characterized by a potentially lethal cytokine burst induced by activated leukocytes, and a secondary latent phase where host defense is reduced. Despite the rapid activation of type I NKT cells (increased CD69 expression, proliferation) after *Staphylococcus aureus* inoculation, Jα18−/−, CD1d−/−, and WT mice presented comparable rates of mortality, suggesting that neither type I nor type II NKT cells play a significant protective or deleterious role in *S. aureus* sepsis (142). However, administration of sulfatide concomitant with *S. aureus* inoculation improved the survival rate of mice (**Figure 5**). Interestingly, the protective effect of sulfatide was CD1d-dependent and type I NKT cell-independent, suggesting that activation of sulfatide-reactive type II NKT cells is essential and sufficient for this protective effect. Sulfatide treatment was accompanied by a decrease of the inflammatory cytokines TNFα and IL-6. Thus, activated sulfatide-reactive type II NKT cells reduced the cytokine storm of the primary hyper-reactive phase during *S. aureus* sepsis while maintaining an adequate immune response to limit bacterial growth and clearance.

During *Mtb* infection, type I NKT cells produced IFN-γ through stimulation with IL-12/IL-18 and inhibited intracellular bacterial replication by their CD1d-restricted secretion of GM-CSF (143, 144). As described above, several type II NKT hybridomas were activated by *Mtb*-infected APC through the specific recognition of *Mtb* phospholipids (93), suggesting that, similar to type I NKT cells, type II NKT cells may also contribute to protective immunity during *Mtb* infection. Similarly, given the recognition of *Lm* antigens by type II NKT cells, the increased bacterial burden in CD1d−/− during *Lm* infection as well as the ameliorated *Lm* infection following treatment with blocking anti-CD1d antibodies suggests that type II NKT cells may contribute to protective immunity or have a regulatory function during *Lm* infection (145, 146).

#### Parasitic Infections

Schistosomiasis is a chronic parasitic disease caused by the extracellular parasite *Schistosoma*, and a strong Th2 response, triggered by parasite eggs, is essential for host survival.

CD1d-deficient mice developed a markedly reduced Th2 response during schistosomiasis (147) indicating an important role of CD1d-restricted T cells in the development of a protective immune response. Whereas type I and type II NKT cells were not essential during the very early phase (1 and 4 weeks) of infection, the two subsets played distinct but complementary roles during the acute phase (7 and 12 weeks) of infection (148). Indeed, type II NKT cells promoted the Th2 response as their lack induced a decrease of Th2 cytokines (IL-4, IL-5, and IL-10) and specific IgG1 production (**Figure 5**), a feature also described in aluminduced humoral immune response (149). On the other hand, secretion of IFN-γ by type I NKT cells contributed to the production of specific IgG2b, a marker of Th1 responses. Interestingly, schistosome egg-sensitized DC activated type I but not type II NKT cells and this activation required the presentation of selfantigen rather than parasite-derived antigens or TLR2/TLR3 engagement (148). Nonetheless, Magalhaes and co-workers have shown that administration of schistosoma-derived LPC or cercaria induced secretion of IL-5 and IL-13 (150) dependent on TLR2, and lead to the recruitment of eosinophils at the site of infection. The cellular source(s) of Th2 cytokines have not been investigated in this model, but considering that LPC is a potent type II NKT cell antigen, it is likely that these cells are activated during infection.

Natural killer T cell subsets also displayed opposing roles during infection with *Trypanosoma cruzi* (151). *T. cruzi* infection causes a chronic inflammatory disease in which the anti-*T. cruzi* immune response that controls the persistent parasites can also contribute to the inflammatory tissue damage of the myocardium and gastro-intestinal tract that causes Chagas disease. Indeed, Jα18−/− mice were more sensitive to infection with *T. cruzi* compared to WT or CD1d−/− mice as indicated by greater morbidity and mortality. The increased susceptibility to infection in the absence of type I NKT cells was accompanied by an enhanced inflammatory response with infiltrates of activated cells (NK and T cells), B cells and DC in lymphoid organs and greater muscle inflammation and pro-inflammatory cytokine secretion (IFNγ, TNF-α, nitric oxide). Moreover, the humoral response was impaired as shown by a decreased anti-*T. cruzi* IgG2a antibody titer, compared to WT and CD1d−/− mice. This suggests that during *T. cruzi* infection, type II NKT cells augment the inflammatory anti-parasite response, whereas type I NKT cells limit this response (**Figure 5**).

Together, these examples show that in a range of viral, bacterial, and parasitic infections type II NKT cells can either promote protective innate and adaptive cell-mediated immune responses, or contribute to infection-induced pathology. Understanding the mechanisms that determine these opposing functions of type II NKT cells during infection will be important areas of future research.

#### Anti-Tumor Immunity

Type I NKT cells critically contribute to natural anti-tumor responses, as demonstrated by the prompt growth of spontaneous tumors in type I NKT cell-deficient Jα18−/− mice compared to WT mice (152–154). Furthermore, the activation of type I NKT cells by α-GalCer provides potent effects against hematologic malignancies and solid tumors through their IFN-γ-production and the subsequent activation of DC and NK cells (155, 156). By contrast, sulfatide-activated type II NKT cells repress antitumor immunity (**Figure 5**) (157–159) by abrogating type I NKT activation in response to α-GalCer, in terms of cytokine secretion and expansion (160). Moreover, their IL-13 production, in combination with TNF-α, led to up-regulation of TGF-β secretion by myeloid-derived suppressor cells (MDSC), and resulted in decreased cytotoxic T cell activity (161). Interestingly, in contrast to their notable immunoregulatory role in anti-tumor responses, two recent studies have highlighted the ability of type II NKT cells to contribute to anti-tumor immunity in response to CpG in a B16 melanoma model (54), and by their ability to directly kill lymphoma cells *in vitro* (63). Thus, type II NKT cells can suppress anti-tumor immunity, counteracting the anti-tumor activity of type I NKT cells, but can also contribute to defense against tumor growth. Understanding the factors that determine the role of type II NKT cells in tumor immunity will be critical to harness their potential in novel anti-tumor strategies.

#### Autoimmunity and Tolerance

Type II NKT cells help maintain tolerance to self-antigens and thereby prevent autoimmune disease. On the other hand, they also can mediate tissue damage and play a pathogenic role in autoimmunity.

#### Experimental Autoimmune Encephalomyelitis

An important role for type II NKT cells has been shown in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. During EAE, sulfatide-mediated activation of type II NKT cells induced tolerogenic DC polarization, abrogated activation of both type I and type II NKT cells, class-II MHC effector myelin protein reactive T cells, and microglial cells and suppressed autoimmunity (**Figure 5**) (55, 162).

#### Type 1 Diabetes

NOD mice, which spontaneously develop type 1 diabetes, were protected from disease following sulfatide administration that led to type II NKT cell activation and secretion of IL-10 by DC, resulting in inhibition of type I NKT cells and activation and expansion of diabetogenic T cells (**Figure 5**) (163). In contrast, in another study using a different experimental approach, sulfatide failed to protect NOD mice from diabetes (164). A non-sulfatidereactive type II NKT cell subset was also able to protect NOD mice from diabetes by regulating diabetogenic T cells through PD-1/PD-L1 and ICOS/ICOSL pathways (66, 165).

#### Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by antinuclear autoantibodies (IgG2a particularly) and multiorgan injury such as proteinuria and immune complex glomerulonephritis, which is dependent on IFN-γ (166, 167). Whereas α-GalCer administration exacerbated the disease by inducing a strong Th1 response, CD1d-blocking slowed down the development of the disease mediated by a decrease of IgG2a production (167). Interestingly, transfer of splenic CD4+, CD8+, or DN Vα4.4/Vβ9 type II NKT cells, purified from transgenic mice generated with CD1d-reactive T cells initially derived from irradiated mice, promoted the disease by producing large amount of IFN-γ. By contrast, NKT cells derived from bone marrow and displaying a DN phenotype prevented SLE by secreting large amounts of IL-4 (166).

#### Colitis

Type II NKT cells exposed *in vivo* to high levels of CD1d expression could directly contribute to the spontaneous development of colitis in mice (168). Their harmful role was mediated through their IL-13 secretion induced by IL-25 and their cytotoxic activity against epithelial cells (**Figure 5**) (56, 129, 169).

#### Liver Diseases, GVHD, and Obesity

Halder and co-workers have shown that presentation of sulfatide by hepatic plasmacytoid DC (pDC) to type II NKT cells led to the recruitment of anergic type I NKT cells, in a IL-12 and MIP-2-dependent manner and prevented concanavalin A-induced hepatitis (170). The same group has also shown that type II NKT cells neutralized the pathogenic role of type I NKT cell during hepatic ischemic reperfusion disease (50) and alcoholic liver disease (94). Indeed, activation of type II NKT cells after sulfatide administration suppressed IFN-γ production by hepatic type I NKT cells and inhibited the recruitment of myeloid cells in the liver that have been shown to enhance injury (**Figure 5**) (171).

In bone-marrow (BM) transfer, both host-residual and donorderived NKT cells exert protective functions that are particularly well described in graft-versus-host disease (GVHD). Whereas protection by host-residual T cells was provided by the type I NKT subset, type II NKT cells played critical roles in donorderived protective effects (172–175). Indeed, on the one hand, adoptive transfer of type I NKT cells or the administration of α-GalCer attenuated GVHD in recipient mice due to the vigorous secretion of IL-4 by type I NKT cells and the subsequent Th2 polarization of the immune response (172–175). On the other hand, donor BM type II NKT cells produced IL-4, like type I NKT cells, but also IFN-γ which induced apoptosis of donor CD4+ and CD8+ T cells in a Fas-dependent manner (174, 176). Moreover, Exley and co-workers have shown that human CD161+ CD1dreactive BM-derived non-invariant T cells specifically suppress mixed-lymphocytes reaction (MLR) and could induce tolerance to allografts (67).

In obesity, activation of type II NKT cells by lipid excess initiated adipose tissue and liver inflammation leading to obesity (177). By contrast, Hams and colleagues found that type II NKT cells activated by IL-25 regulated weight and glucose homeostasis by recruiting to visceral adipose tissue eosinophils and activated macrophages that are found in lean individuals (130).

Thus, in a context-dependent manner, type II NKT cells provide protective or deleterious effects in a number of diverse diseases including microbial infection, anti-tumor immunity, and autoimmunity (**Figure 5**), suggesting that these cells may constitute promising therapeutic targets for a broad range of diseases.

#### Summary and Future Directions

Significant progress has been made in defining the diversity among CD1d-restricted T cells that, in addition to the widely studied type I NKT cells, include α-GalCer-reactive NKT cells that use non-canonical TCRs, type II NKT cells that use a more diverse repertoire of αβ TCRs, as well as T cells that use γδ or δ/αβ TCRs. Critical differences distinguish the members of the CD1d-restricted T cell family beyond their use of different TCRs, including their antigen specificities and modes of antigen/CD1d recognition. A major hurdle in unraveling the biology of the CD1d-restricted T cell subsets that do not recognize α-GalCer is the difficulty to reliably detect and analyze these cells in humans and mice *ex vivo* and *in vivo*. Discovery of new self- and foreign antigens in combination with strategies and tools to directly detect these cells such as antigen-loaded CD1d tetramers, and availability of suitable small animal models will be necessary to characterize the entire repertoire and to determine the degree of diversity versus oligoclonality among all CD1d-restricted T cell subsets. Furthermore, it is currently unknown to what extent the differences in TCR usage and antigen specificity of these subsets correlate with phenotypic attributes or immunologic functions that are distinct from those of type I NKT cells. For example, in anti-tumor immunity and in response to microbial infection, type II NKT cells have been shown to have opposing functions to those of type I NKT cells. In addition, while many non-invariant CD1d-restricted T cells appear to display a type I NKT cell-like activated/memory phenotype, several studies have indicated that CD1d-restricted T cells exist that are more similar to conventional MHC-restricted T cells. Importantly, it remains to be determined if truly naïve CD1d-restricted T cells exist that can mount antigen-specific memory T cell responses following primary stimulation and expansion. Given their ability to recognize bacterial antigens including ones expressed by *Mtb*, this would provide a rationale to target these cells in

#### References

vaccine strategies. In analogy to the functionally distinct type I NKT cell subsets that comprise NKT1, NKT2, NKT17, and NKTFH cells, the Th2-bias observed among some human type II NKT cells and following sulfatide administration, and the discovery of TFH-like type II NKT cells in humans and mice suggest that functionally distinct subsets may also exist among non-invariant CD1d-restricted T cells. Correlation of such functional differences with antigen specificity would allow therapeutic targeting of these cells with activating or inhibitory ligands.

Several examples illustrate that non-invariant NKT cells, in particular type II NKT cells, critically contribute to the immune responses in a wide range of diseases, including microbial infection, anti-tumor immunity, and autoimmunity. Central to our understanding of the varied roles of CD1d-restricted T cells is unraveling the mechanisms that regulate their activation in these diverse conditions. During infection, recognition of inducible self-lipid antigens in combination with stimulation by the inflammatory milieu through cytokines, or direct recognition of microbial antigens results in the rapid activation of CD1d-restricted T cells. How these T cells distinguish between self- and microbial lipids, whether activation by antigens with different affinities results in distinct functional outcomes, and whether recognition of inducible self-antigens extends to non-infectious conditions such as tumor immunity or autoimmunity are currently not known. Defining the context-dependent mechanisms of activation, decrypting the disease-specific balance of protective and potentially harmful functions of the different CD1d-restricted T cell populations and a better understanding of their effector functions and interactions with other immune cells present significant ongoing challenge and will ultimately determine the success of targeting CD1d-restricted T cells for preventive or therapeutic interventions.

### Acknowledgments

The authors would like to thank R. Tatituri for providing the lipid structures shown in **Figure 2**, and Patrick Brennan, Laurent Gapin, Francois Trottein, Christelle Faveeuw, and Mayra Cruz Tleugabulova for insightful discussions and comments. This work was supported by grants from the US National Institutes of Health (5K08AI077795, 1R21AI103616) and from the William F. Milton Fund.

receptor-related orphan receptor (gamma)t+ and respond preferentially under inflammatory conditions. *J Immunol* (2009) **183**:2142–9. doi:10.4049/ jimmunol.0901059

producing IFN-gamma and IL-4. *J Immunol* (2007) **179**:6579–87. doi:10.4049/ jimmunol.179.10.6579

**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.

*Copyright © 2015 Macho-Fernandez and Brigl. 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.*

# **MR1-restricted mucosal-associated invariant T cells and their activation during infectious diseases**

*Lauren J. Howson, Mariolina Salio and Vincenzo Cerundolo\**

*MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK*

MR1-restricted mucosal-associated invariant T (MAIT) cells recognize vitamin B metabolites, which are generated by a broad range of bacteria, from *Escherichia coli* to *Mycobacterium tuberculosis* and BCG. MAIT cells have been described as innate sensors of infection as they accumulate early in infected tissues. MAIT cells maintain an activated phenotype throughout the course of infections, secrete inflammatory cytokines, and have the potential to directly kill infected cells, playing an important role in shaping the host response. In this review, we will discuss the current knowledge regarding the molecular mechanisms that underline MAIT cells activation in sterile and non-sterile inflammatory conditions.

#### *Edited by:*

*S. M. Mansour Haeryfar, Western University, Canada*

#### *Reviewed by:*

*Marcelo B. Sztein, University of Maryland School of Medicine, USA Olivier Lantz, Institut Curie, France*

#### *\*Correspondence:*

*Vincenzo Cerundolo, MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headley Way, Oxford, OX3 9DS, UK<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

> *Received: 27 February 2015 Accepted: 27 May 2015 Published: 16 June 2015*

#### *Citation:*

*Howson LJ, Salio M and Cerundolo V (2015) MR1-restricted mucosal-associated invariant T cells and their activation during infectious diseases. Front. Immunol. 6:303. doi: 10.3389/fimmu.2015.00303* **Keywords: MAIT cells, infection, MR1, bacteria, vitamins**

### **Introduction**

Innate-like lymphocytes, such as invariant natural killer T (iNKT) cells, have gained great interest since their discovery, as these cells lie at the interface between innate and adaptive immune responses. iNKT cells are memory cells bearing a semi-invariant T cell receptor (TCR) through which they recognize self- and bacterial-derived lipid antigens presented by CD1d molecules. During pathogen infection, iNKT cells facilitate adaptive immune responses by inducing dendritic cell maturation (1). Over the last few years, in addition to iNKT cells, another innate-like lymphocyte cell subset has been the focus of much research: mucosal-associated invariant T (MAIT) cells, recognizing a unique family of bacterial-derived metabolites in the context of the major histocompatibility complex (MHC) class I-like molecule MR1.

### **MR1 Structure and Function**

The MR1 gene was first described in 1995 in a genome screen aimed at identifying novel MHCrelated genes (2). The human MR1 gene is located on chromosome 1, like CD1 genes, and despite a similar intron/exon organization to classical MHC molecules, it is non-polymorphic. MR1 is highly conserved among mammalian species, with 90% sequence homology between human and mouse MR1-ligand binding domains and a high level of functional cross-reactivity, a feature reminiscent of CD1 molecules (3, 4). Similar to MHC class I molecules, MR1 associates with β2-microglobulin and interacts with the peptide-loading complex during biosynthesis in the endoplasmic reticulum (5, 6). In 2003, the connection between MR1 and MAIT cells was made by demonstrating the central role of MR1, along with commensal flora and B cells, for MAIT cell development (7). Although MR1 is ubiquitously transcribed, its physiological expression at the cell surface has been difficult to demonstrate, with the exception of B cell subsets in the intestinal mucosa (8). A study of mouse MR1 reported that only the folded form of MR1 was able to activate MAIT cells and certain mutations in the putative binding groove disrupted MAIT cell activation, through presentation of yet unknown antigens (9). These results led to great interest in determining the identity of the antigens presented.

The observation that MAIT cells have anti-microbial activity provided important insights into the identification of the MAIT agonists presented by MR1. Kjer-Nielsen et al. (10) showed that, similar to other MHC class I and MHC class I-like molecules, MR1 was unable to fold correctly in refolding buffer alone. This suggested that it required the presence of its ligand in order to stabilize its structure. Refolding was attempted in a number of conditions using RPMI-1640 as a control. Surprisingly, it was found that MR1 molecules were able to fold in this cell culture media, which was known to contain a number of vitamins produced by microbes, but not present in mammals. This observation-based approach led Kjer-Nielsen and colleagues to test the ability of vitamins to bind and stabilize MR1, and ultimately led to the discovery of a new class of antigens: vitamin B metabolites (10). The first crystal structure of the MR1-ligand complex was with the folic acid (vitamin B9) metabolite 6-formylpterin (6-FP), which was shown to be unable to activate MAIT cells (10). To determine the identity of MAIT stimulatory ligands, MR1 molecules were refolded in the presence of the supernatant from *Salmonella typhimurium* bacterial cultures, as it was known that *Salmonella*-infected cells strongly activate MAIT cells (11, 12). By high-resolution mass spectrometry (MS) [ESI–TOF-MS], MR1 was shown to associate with a compound at *m/z* 329.1100, with an atomic composition matching that of several riboflavin (vitamin B2) derivatives: 6,7-dimethyl-8--ribityllumazine (RL-6,7-diMe), 7-hydroxy-6-methyl-8--ribityllumazine (RL-6- Me-7-OH), and reduced 6-hydroxymethyl-8--ribityllumazine (rRL-6-CH2OH). In functional assays, these compounds were stimulatory and induced CD69 expression as well as interferongamma (IFNγ) and tissue necrosis factor-alpha (TNFα) production by MAIT cells (10).

In order to understand the origin of the above MAIT cell ligands, *Lactocossus lactis* mutants of the four-gene operon, which controls riboflavin biosynthesis, were studied (13). These results confirmed that riboflavin is necessary and sufficient to generate natural MAIT cell ligands. Furthermore, these results pointed to the compound 5-amino-6--ribitylaminouracil (5- A-RU) as an intermediate necessary for MAIT cell activation. This intermediate does not bind to MR1, but forms MAITstimulating ligands through a non-enzymatic condensation with glyoxal or methylglyoxal species, which can be of bacterial or host cell origin (such as byproducts of glycolysis). The yields of these condensation reactions are the unstable, yet potent intermediates 5-(2-oxoethylideneamino)-6--ribitylaminouracil (5- OE-RU) and 5-(2-oxopropylideneamino)-6--ribitylaminouracil (5-OP-RU). These molecules can be captured and stabilized through Schiff bases with Lys43 in the MR1 groove. Furthermore, these compounds are the substrates for conversion to RL-6,7 diMe. Mass spectrometry analysis of MR1 refolded in the presence of culture supernatants from *S. typhimurium, E. coli* (DH5α), or rRL-6-CH2OH also revealed species with matching properties to the synthetic 5-OP-RU, raising the possibility that the compound initially identified as MR1 ligand was indeed 5-OP-RU (10, 13). These results provide evidence that MAIT cells are able to sense a wide range of bacteria through detection of vitamin metabolites, including transitory intermediates, presented by MR1 molecules.

### **MAIT Cell Phenotype**

Mucosal-associated invariant T cells were identified based on their surface phenotype and mucosal tissue localization. The MAIT cell invariant TCR Vα7.2–Jα33/12/20 in humans was first described in 1993 as one of the few preferentially used TCRs in the double-negative T cell compartment (14). This finding was the first evidence of a new subset of T cells possibly recognizing a limited set of antigens in the context of non-polymorphic antigen presenting molecules. It was not until 1999 that the MAIT cell subset was defined as a conserved subpopulation distinct from MHC class I- and CD1-restricted cells with an activated/memory phenotype (15). They also reported that the human MAIT cell TCR β chain usage was primarily TRBV6 or TRBV20. Initial research into MAIT cells has been hampered by the lack of specific reagents; however, the generation of a monoclonal antibody specific for the Vα7.2 TCR chain (16) and MR1 tetramers (17) have recently enabled the functional and phenotypic analysis of MAIT cells and brought them to the forefront of innate-like lymphocyte research. MAIT cells are defined as CD3+ Vα7.2+ CD161++ and either CD8+ or doublenegative T cells (12, 16). MR1-loaded tetramer experiments have also identified a small subset of MAIT cells that are CD4+ (17). All human MAIT cell subsets express the transcription factor PLZF, known to direct the effector program of the iNKT cell lineage (18). However, murine MAIT cells do not express PLZF (16), thus the functional relevance of this observation is currently unclear. MAIT cells can also be defined based on co-expression of interleukin (IL)-18R (12) and CD26 (19). Furthermore, in adults, peripheral blood MAIT cells have an effector memory phenotype defined as CD45RO+, CD62Llo, CD95hi CD122int , CD127int, and they express tissue-homing chemokine receptors: CCR5, CCR6, CXCR6, and CCR9 (20). By contrast, MAIT cells do not express CCR7 that is a marker for homing to lymph nodes. The distinct memory phenotype and peripheral location of these cells are linked to their unique developmental pathway.

### **MAIT Cell Development**

Mucosal-associated invariant T cells develop and undergo selection in the thymus. Like iNKT cells, MAIT cells are selected by CD4/CD8 double-positive (DP) thymocytes (21). MR1 expression on DP thymocytes is essential, as MR1-deficient mice do not develop T cells expressing the MAIT cell TCR at detectable levels (7). Although thymic selection occurs independently of B cell and commensal flora (16), B cells are essential for MAIT cell peripheral expansion and memory phenotype acquisition. However, the endogenous antigen(s) capable of selecting MAIT cells in the thymus to date remain unclear. Furthermore, unlike iNKT cells, human MAIT cells egress the thymus as naïve cells, that in the periphery acquire a memory phenotype prior to birth (22).

In mice, the interaction of MAIT cells with B cells in the periphery drives the acquisition of the MAIT cell memory phenotype (16). In humans, a recent study analyzed MAIT cell development in second trimester fetal tissues, and reported acquisition of innate-like anti-microbial activity already *in utero* (22). This study suggested that MAIT cell maturation begins in the secondary lymphoid organs, where they reported Vα7.2+ CD161+ cells acquire expression of the transcription factor PLZF, CD62L, and CD45RO. In fetal liver and mucosal tissues, Vα7.2+ CD161+ cells expressed high levels of PLZF, IL-18Rα, CD62L, and CD45RO and were capable of responding to bacterial antigen stimulation both through proliferation and production of IFNγ and IL-22. While murine studies have revealed the importance of the microbiota in shaping MAIT cell differentiation, as they are absent in germ-free mice (7), this study suggests that human MAIT cells develop prenatally, before establishment of the commensal microflora, although the factors driving this maturation remain to be identified (22). However, it remains unclear whether Vα7.2+ CD161+ T cells described by Leeansyah and colleagues are MR1 restricted MAIT cells, as MR1-tetramer staining or MR1-blocking antibody experiments were not performed (22).

The interaction of MR1 expressing B cells and MAIT cells in the periphery is necessary for MAIT cell expansion in mice (16). Likewise in humans, individuals with mutated Bruton's tyrosine kinase, and thus with disrupted B cell development, have reduced MAIT cell TCR transcripts in the blood compared to healthy controls (7). These results are consistent with the observation that both primary B cells and B cell lines can efficiently present bacterial antigens to MAIT cells in an MR1-dependent manner (23).

Together, the results from these studies suggest that MAIT cells are selected and undergo maturation prior to exposure to commensal bacteria. It can be speculated that peripheral expansion of MAIT cells may occur via presentation of an endogenous antigen present at mucosal sites or through maternally derived commensal flora antigen, possibly carried in the amniotic fluid.

### **MAIT Tissue Distribution**

Mucosal-associated invariant T cells are primarily located in the mucosal compartments and blood. The expression of chemokine receptors CXCR6 and CCR9 at the surface of MAIT cells suggested that they traffic to the tissues, particularly the intestine, lung, and liver (20). Indeed, these tissues have a higher proportion of MAIT cells, composing 20–40% of liver T cells and 4–10% intestinal T cells (20, 24). MAIT cells compose 1–8% of T cells in the blood. Analysis of the Vα7.2–J33 and Vα7.2–Jα12 transcripts in different tissues revealed that MAIT cells are also present in the kidney and at a lower frequency in the tonsils and lymph nodes (25). They also reported variable expression of these transcripts in the prostate and ovary (25). Analysis of the α chains revealed a predominance of Vα7.2–Jα33 in the blood and Vα7.2–Jα12 in tissues indicating that these two MAIT subsets might differ in their tissue-homing properties (25). Further investigation is needed to determine whether these two subtypes are functionally distinct in terms of their antigen/pathogen specificity or are related to the infection history of the individual.

In humans, circulating MAIT cells are more abundant than iNKT cells, while in mice they are far fewer (15, 16, 20). Studies assessing the influence of age and gender of MAIT cells found that MAIT cells decrease with age, but are not significantly different between males and females (26–28). The MAIT cell phenotype also changes according to the tissue compartment. For example, liver MAIT cells have a more activated phenotype compared to blood MAIT cells and express higher levels of CD69, CD38 and HLA-DR, possibly reflecting continuous antigen exposure (24).

### **MR1-Dependent and -Independent MAIT Cell Activation**

In 2010, two publications described a wide range of phylogenetically diverse bacteria and yeast that were able to activate MAIT cells. These microbes included: *E. coli*, *Pseudomonas aeruginosa*, *Klebsiella pneumoniae*, *Lactobacillus acidophilus*, *Staphylococcus aureus, Staphylococcus epidermidis, Saccharomyces cerevisiae, Candida glabrata*, and *Candida albicans* (11, 12). Conversely, viruses and few bacterial species such as *Enterococcus faecalis* and *Streptococcus* group A did not elicit MR1-dependent MAIT cell activation. The later discovery of the MAIT cell ligand offered the explanation for the lack of response as these bacteria lack the vitamin B metabolites that bind MR1 molecules and stimulate MAIT cells (10).

#### **Cytokine Production**

Mucosal-associated invariant T cells can produce proinflammatory cytokines upon activation (**Figure 1**). Stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, as well as bacteria, showed that human MAIT cells are capable of producing IFNγ, TNFα, IL-2, and IL-17 but not IL-10 (20, 24). *Ex vivo* multiparametric mass cytometry in three donors recently confirmed that there are distinct subsets of circulating MAIT cells releasing different cytokine combinations, most frequently MIP1β, IFNγ, TNFα, and IL-2, and to a lesser extent IL-4 and IL-10 (25). Furthermore, MAIT cells from the small intestine are capable of producing IL-22 in response to *E. coli*, but this was not observed in MAIT cells from the liver or lung (22). Thus, MAIT cells have a mixed Th1/Th17 cytokine profile with differences according to their peripheral location, possibly reflecting local environmental imprinting.

 Cytotoxicity occurs primarily via granule exocytosis and relies on the effector cell containing components that enable destruction of a target cell. These include perforin and granzymes that, in humans, consist of five isoforms with different substrate specificities: granzyme (Gr) A, GrB, GrH, GrK, and GrM (29). GrB and perforin have been directly linked to the cytotoxic activity of CD8+ T cells (30). It has been shown that MAIT cells are able to lyse bacteria-infected epithelial cells (31) and *E. coli-*infected THP1 cells (32) in an MR1-dependent manner. Interestingly, human MAIT cells have a unique cytotoxic profile, characterized by low perforin expression, but high GrA and GrK, contained in CD107+ lytic granules (32). Upon antigen recognition, an MR1-dependent GrB upregulation and an MR1-independent perforin upregulation was observed, suggesting that antigen pre-exposure licenses MAIT to acquire a stronger cytotoxic phenotype (32). The ability to kill infected target cells suggests that MAIT cells could have an important role

directly kill infected cells.

in controlling infections by intracellular bacteria, such as *Shigella* and *Salmonella,* although the latter may escape MAIT cell detection by preventing vacuole–lysosomal fusion (33). Indeed, it has been shown that, while HeLa cells infected with *Shigella flexneri* can be readily killed by MAIT cells, HeLa cells infected with *S. typhimurium* failed to be detected or lysed by MAIT cells (31).

hepatocytes can act synergistically to enhance MAIT cell activation. Viruses (2)

#### **MR1-Independent Activation**

Like other innate-like cells, MAIT cells can also respond to infection in an MR1-independent manner. For example, the *in vitro* MAIT cell response to cells infected with *Mycobacterium bovis* Bacillus Calmette–Guérin (BCG) is largely MR1-independent (34). The high expression of IL-18R on MAIT cells suggested that IL-18 could be one of the cytokines able to activate MAIT cells (20, 35). Therefore, cytokine-dependent activation of MAIT cells was explored in more detail and it was reported that MAIT cells could produce IFNγ when their TCR is blocked and they are cultured with IL-18 and IL-12 (36). Furthermore, cytokines can synergize with TCR stimulation to enhance MAIT cell activation. For example, liver-resident MAIT cells express high levels of IL-7R and IL-7 potentiates TCR-dependent secretion of Th1 cytokine and of IL-17A (24). These results suggest that multiple factors could shape MAIT cells responsiveness during bacterial infection, and their ability to respond to cytokine stimulation, independent of TCR cross-linking, may account for their response to microbes not containing MR1 ligands, such as viruses and some bacterial species (**Figure 1**).

### **MAIT Cells in Bacterial Infections**

Murine infection models and natural human infections have been researched in order to further our understanding of MAIT cells and their activation during pathological conditions.

In a mouse model of lung infection with live *Francisella tularensis* (LVS), it was demonstrated that MAIT cells colonize the lung in both early and intermediate phases of the infection, reach their peak expansion in the late clearance phase of infection, and persist following clearance of the bacteria (37). From day 8 of infection onward, MAIT cells in the lung produced anti-bacterial cytokines (IFNγ, TNFα, and IL-17A). The evidence for the importance of MAIT cells in controlling infection came from MR1 knockout mice, which showed a delay in clearance of the bacteria in the lungs (but not in the spleen or liver) and delayed appearance of adaptive immune responses in the lung (37). A similar increase in bacterial load in comparison with wild type mice was found when mice lacking MAIT cells were infected with *K. pneumonia* or *M. bovis* BCG (34, 38). Together, these studies indicate that the role of MAIT cells in controlling infection is not redundant, and is vital for timely mounting of effective adaptive immune responses.

#### **Tuberculosis**

In humans, the contribution of MAIT cells to anti-bacterial immunity has been studied mostly during *Mycobacterium tuberculosis* infection. MAIT cells have been shown to be able to respond *in vitro* to *M. tuberculosis* infected primary lung epithelium and dendritic cells (11, 39). In individuals with active *M. tuberculosis* infection, it was found that circulating MAIT cells were in lower absolute numbers and proportions compared to healthy individuals (11, 12). This suggested that MAIT cells may migrate to inflamed tissues, where they can have a localized response to infection. There is also evidence that MAIT cells are enriched in the lung of healthy donors and therefore could respond early during the course of infection (11).

To assess whether the reduced number of MAIT cells is accompanied by an altered functionality, a study analyzed the cytokine production by MAIT cells from individuals with tuberculosis (TB) in response to different stimuli. Despite lower frequencies, a higher proportion of MAIT cells in individuals with active TB secreted IFNγ and TNFα in response to BCG stimulation as compared to healthy controls (although the overall response was very low) (40). More convincingly, a lower proportion of MAIT cells responded to *E. coli* in the same cohort of infected individuals compared to healthy controls. These results suggest that there may be an overall enrichment of MAIT cells specific for BCG compared to *E. coli* upon *in vivo* antigenic exposure.

This concept of expansion of pathogen-specific MAIT T cell clones is suggested by a recent paper reporting that heterogeneity in the TCR β chain usage by MAIT cell clones was directly related to their ligand specificity, and thus, to responses to different microbes (41). While this is a very attractive possibility, this observation requires further confirmation as the MAIT cells assessed in the above study were defined as Vα7.2+ CD8+ cells that produced TNFα in response to bacterial stimulation. Without using MAIT specific markers, such as CD161, IL-18R, or CD26, this strategy could have included a proportion of conventional Vα7.2+ T cells. Furthermore, no MR1 blocking experiments were included to demonstrate differential microbe recognition. Nevertheless, a contribution of the TCRβ chain sequence to antigen binding has been also suggested by a second study (42), where six MAIT TCRs with differing β chains were refolded and their biophysical parameters were measured. This study showed that the CDR3β loop usage directly impacts MAIT cell recognition of ligands through altering the TCR flexibility and contact with MR1 and the ligand (42); thus, suggesting a role of the TCR CDR3β loop in fine-tuning MAIT cells' specificity.

These findings open up an exciting area of research, suggesting that this innate-like lymphocyte subset may be capable of forming memory-like responses upon selective expansion of MAIT cell clones following lifetime microbe exposure. The ability to form memory responses could have implications for the therapeutic use of MAIT cells.

#### **Other Bacterial Infections**

Decreased numbers of circulating MAIT cells have also been reported in individuals with cystic fibrosis during infection with the opportunistic pathogen *P. aeruginosa* (43). In these individuals, the lower MAIT cell numbers also correlated with lung disease severity, systemic inflammation, and clinical status. However, whether the reduction in numbers was a consequence of their localization to the airway mucosa, or by itself influenced colonization and disease progression, was not addressed.

The relevance of MAIT cells in a model of invasive enteric infection was addressed in a study investigating the efficacy of an attenuated strain of *Shigella dysenteriae* (31)*.* A specific reduction in circulating MAIT cells was observed at day 11 in subjects who received bacteria compared to controls. Individuals who responded to the vaccine, producing a specific IgA response, also had a trend toward a higher baseline proportion of MAIT cells and at day 11 had an increased proportion of activated MAIT cells as determined by HLA-DR upregulation.

Mucosal-associated invariant T cells have also been investigated in cases of severe infection with *Vibrio cholerae*. It was reported that MAIT cells activation, as measured by CD38 expression, peaks 7 days after the onset of infection (44). In adults, the proportion of MAIT cells did not change upon infection, but interestingly, in children a significant and persistent decrease in MAIT cell proportion was observed. The reason for the adult/child discrepancy is not clear and requires further investigation. Although *V. cholerae* is known to have a riboflavin biosynthetic pathway, *in vitro* experiments aimed at demonstrating the role of vitamin B2 metabolites in this recognition were not performed.

A specific decrease in MAIT cell counts (but not iNKT or γδ T cells) was also observed in severe infections. Critically ill patients admitted to the intensive care unit (ICU) with sepsis had lower circulating levels of MAIT cells compared to healthy controls and non-septic critically ill individuals (45). Patients with lower MAIT numbers were also more susceptible to hospital-acquired infections. Of interest, it was reported that streptococcal infections (which do not stimulate MAIT cells *in vitro*) induced a less pronounced decrease in MAIT cell numbers than non-streptococcal infections, although there was a large overlap between groups that prevented statistical significance.

### **MAIT Cells in Viral Infection**

Although it has been established that virally infected cells do not directly activate MAIT cells (12), several publications have investigated the numbers and activation of MAIT cells in human immunodeficiency virus (HIV) infection. The rationale behind investigating MAIT cells in the context of HIV is due to the effects this virus has on increasing the permeability of intestinal epithelia. This alteration causes translocation of microbial products from the gastrointestinal tract, resulting in systemic immune activation (46, 47).

#### **Human Immunodeficiency Virus**

Two groups reported that circulating MAIT cells are reduced early in HIV infection compared to healthy controls (48, 49). This trend was also observed in individuals with chronic HIV, and numbers could not be restored following anti-retroviral therapy (49). Consistent with these findings, staining with MR1 tetramers loaded with 5-OP-RU confirmed that the tetramer-positive MAIT population is reduced during HIV infection (50).

The activation and function of circulating MAIT cells in HIV were also assessed, and it was found that they express lower levels of CD69 and produce less IFNγ and TNFα in response to *ex vivo* bacterial stimulation compared to healthy controls (49). Interestingly, after anti-retroviral therapy, a partial restoration of cytokine production was observed. However, this impaired activation and function of MAIT cells were not confirmed in a separate study (50).

The authors also investigated MAIT cells in the rectal and colon mucosa of HIV infected individuals, which were reduced, like circulating MAIT cells. However, mucosal numbers were better preserved and could be restored after anti-retroviral therapy (49, 51).

The decline and possible impaired activation of MAIT cells in patients infected with HIV are likely to affect the MAIT cells' ability to control bacteria and yeast infections and, as a result, leave the host extremely vulnerable and contributing to acquired immune deficiency syndrome (AIDS) pathogenesis.

#### **Hepatitis B and C**

As previously discussed, a large proportion of liver-resident T cells are MAIT cells. These liver-resident immune cells are of great interest as viruses, such as hepatitis B virus (HBV) and hepatitis C virus (HCV), can establish persistent infections in the liver. To determine whether liver MAIT cells could respond to innate signals upon viral infection, Toll-like receptor (TLR) agonists were used to stimulate liver intra-sinusoidal cells. It was found that TLR8 agonist ssRNA40 was able to activate liver MAIT and NK cells to produce large amounts of IFNγ (52). The response was MR1-independent and driven by IL-12 and IL-18 released by TLR8-stimulated monocytes. A similar activation of hepatic MAIT cells was observed in individuals infected with HBV and HCV. This preservation of anti-viral activity of MAIT cells in diseased livers could provide a possible therapeutic target for the treatment of viral infection.

### **MAIT Cells in Sterile Disease**

The capacity of MAIT cells to respond to cytokine stimulation, as described above, raises the possibility that these cells may also play a role in sterile inflammation such as autoimmune disorders and cancer.

#### **Multiple Sclerosis (MS)**

The role of MAIT cells in MS has been extensively researched. MAIT cells' TCR was identified in central nervous system MS lesions using single-strand conformation polymorphism analysis in autoptic specimens (53). Normal frequency of MAIT cells was found in the blood of MS patients and MAIT cells were also detected in the cerebrospinal fluid of relapsing patients. This suggested a role for MAIT cells in the progression of this disease, which was functionally explored by a study using a mouse model for MS, experimental autoimmune encephalitis (EAE). In transgenic mice overexpressing the MAIT cell TCR, MAIT cells protected against EAE through suppression of Th1 cytokine production and increased production of IL-10 primarily by B cells (54). It was also observed that, in MR1 knockout mice, EAE was exacerbated, suggesting a new regulatory role for MAIT cells.

Conflicting results have emerged from studies assessing the distribution of circulating MAIT cells in MS patients. A study of an Italian cohort assessing MAIT cells of monozygotic twins with discordant disease states showed a greater number of circulating CD8+ CD161++ cells in MS patients compared to their healthy twin (55). By contrast, a study of a Japanese cohort showed decreased circulating MAIT cells in MS patients compared to healthy controls (56). This discrepancy between the two studies findings underlines the difficulties of studies with human subjects and suggests the variation in the commensal flora or lifetime infection burden may have affected MAIT cell frequencies in the two populations.

#### **Psoriasis**

It has previously been reported that IL-17A is a vital component in the etiology of psoriasis (57). As MAIT cells are able to produce this cytokine, it was thought that they might be the CD8+ cells previously observed in psoriatic lesions. It was found that MAIT cells are indeed present in the psoriatic lesions along with Th17 cells (58). This provided evidence that MAIT cells may contribute to psoriasis, however, their activation in this setting is yet to be explored.

#### **Rheumatic Diseases**

Mucosal-associated invariant T cells have also been studied in the context of rheumatic diseases. It was found that individuals with systemic lupus erythromatosis (SLE) and rheumatoid arthritis (RA) had lower circulating levels of MAIT cells (59). Furthermore, MAIT cells have been detected in the synovial fluid of RA patients in higher proportions than in the blood. Individuals with SLE had MAIT cells with impaired activation, as they had a lower production of IFNγ when challenged with bacteria or stimulated with PMA and ionomycin. This impaired function was more prominent in individuals with lower MAIT cell numbers, suggesting a correlation between MAIT cell numbers and function. Conversely, in RA patients, the IFNγ secretion was preserved. To assess whether the inhibitory molecule PD-1 correlated with MAIT cell dysfunction, its expression on MAIT cells was assessed and found to be higher on MAIT cells in individuals with SLE compared to individuals with RA or healthy controls. However, after PD-1 blockade, MAIT cell activation was only partially restored, indicating other negative regulation mechanisms may be contributing to MAIT cell dysfunction in SLE.

#### **Inflammatory Bowel Disease and Celiac Disease**

The location of MAIT cells in the small intestine mucosa makes them of great relevance in the pathogenesis of intestinal inflammatory disorders, such as inflammatory bowel diseases (IBD): Crohn's disease and ulcerative colitis. A study reported that the number of circulating MAIT cells was reduced in individuals with Crohn's disease and ulcerative colitis compared to healthy controls (60). A similar observation was reported with circulating MAIT cell numbers in individuals with celiac disease (61). It was also reported that MAIT cells were higher in number in inflamed compared to healthy tissues (60). Functional assessment of MAIT cells from individuals with IBD showed increased IL-17 production upon stimulation along with increased IL-22 in ulcerative colitis but decreased IFNγ secretion in individuals with Crohn's disease. This first demonstration of activated MAIT cells in inflamed gut tissues warrants further investigation as to their potential pathogenic role.

#### **Cancer**

A less explored area is the potential role of MAIT cells in cancer. A publication has suggested MAIT cells may be present in renal and brain cancers through detection of their TCR in these tissues (62). This presence correlated with the presence of pro-inflammatory cytokines in the tumor tissue, suggesting they could have antineoplastic functions. However, this has not been explored any further.

## **MAIT Cell as a Potential Therapeutic Target**

With their ability to alter the cytokine microenvironment to proinflammatory, direct killing of infected cells, potential to enhance adaptive immune responses, and the recent discovery of their activating ligands, MAIT cells may be an ideal target to manipulate for enhancing immune responses. An understanding of the potential of these cells could aid the designing of more targeted vaccines and effective immunotherapies.

The use of bacteria as adjuvants to enhance immune responses was first explored in 1891 when William Coley conducted intratumoral injections of *S. pyogenes* and *Serratia marcescens* to cause inflammation and subsequent destruction of the tumor cells (63). This form of immunotherapy, in which bacteria are used to stimulate anti-tumor immune responses, has not been highly successful with the notable exception of the intra-vesical instillation of BCG, which is currently the gold standard treatment of non-invasive bladder cancer. Although successful, it is still not well understood what the immune principles are that underlie its success.

### **References**

It has previously been discussed that MAIT cells are able to respond to BCG-infected cells *in vitro* (34, 40). Understanding whether MAIT cells are key players in mediating the success of BCG immunotherapy may help in designing more targeted therapies and also opens up the potential for MAIT cell-targeted immunotherapies for other cancers.

## **Outstanding Questions**

A number of questions still need to be addressed in the field of MAIT cell research. First, the full spectrum of MAIT-activating ligands remains to be explored. Whether other possible vitamin metabolites or small organic compounds could bind MR1 and stimulate MAIT cell responses is still an open question. Also, a priority will be identification of endogenous ligand(s) involved in shaping MAIT cell selection and maturation during development.

Second, although MAIT cells can respond to yeast species *in vitro*, the role of MAIT cells during yeast infections *in vivo* remains to be addressed.

Third, it is still not clear what factors determine the relative contribution of antigen-dependent versus cytokine-dependent MAIT cell response during an infection. Early findings suggest that the presence of the ligand, mode of infection, and subcellular localization of the bacteria may all impact the way MAIT cells respond during infection.

Finally, it is still not known whether the MAIT cell subset could be an effective therapeutic target. The identification of potent antigens for MAIT cells could provide a possible adjuvant in vaccine and immunotherapies targeting both infections and also non-sterile diseases.

## **Concluding Remarks**

The role of MAIT cells during infection is only beginning to be established, with their early infiltration into infected tissues, production of pro-inflammatory cytokines, and role in assisting an effective adaptive immune response. The effector functions of MAIT cells in sterile disease conditions are also being explored. Such an integral role in the progression of infection, and possibly sterile diseases, makes these cells an ideal target for enhancing immune responses.

## **Acknowledgments**

This work was supported by Cancer Research UK (Programme Grant C399/A17722), the Medical Research Council, and the Radcliffe Department of Medicine, University of Oxford.

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**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.

*Copyright © 2015 Howson, Salio and Cerundolo. 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.*

# **The role of mucosal associated invariant T cells in antimicrobial immunity**

#### *Ruth J. Napier 1 , Erin J. Adams 2 , Marielle C. Gold1,3,4 and David M. Lewinsohn1,3,4 \**

*1 Pulmonary and Critical Care Medicine, Oregon Health & Science University, Portland, OR, USA, 2 The University of Chicago, Chicago, IL, USA, 3 VA Portland Health Care System (VAPORHCS), Portland, OR, USA, 4 Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, USA*