**2. Biology of natural killer T cells**

Natural killer T cells were first recognised in 1990 as CD4-CD8- double negative (DN) thymocytes bearing the murine natural killer (NK) cell marker NK1.1 or the orthologous CD161 in humans (Ballas & Rasmussen, 1990). Since their discovery, the unexpected ontological complexity, development, function and pathophysiological roles of NKT cells have begun to unravel. Here, we briefly review the biology of NKT cells and focus on a subset known as invariant NKT (iNKT) cells by virtue of their CD1d-restricted, semiinvariant T cell receptor in order to better understand their significance in autoimmune diseases such as rheumatoid arthritis.

#### **2.1 CD1 molecules & the ontogeny of the NKT cell**

The first mouse anti-human monoclonal antibody recognised an antigen found on human thymocytes and certain B cell lymphoma lines subsequently termed the first cluster of differentiation or CD1. Five CD1 protein isoforms encoded on chromosome 1 bear resemblance to the α chains of MHC class I encoded on chromosome 6. CD1 molecules associate non-covalently with ϐ2-microglobulin, but unlike MHC class I molecules they

Invariant Natural Killer T Cells in Rheumatoid Arthritis and Other Inflammatory Arthritides 21

activated to release a diverse range of cytokines, proliferate and influence the subsequent

Unlike conventional T cells, iNKT cells appear to be selected in the thymus for their ability to recognise both microbial lipid antigen and self-antigen. The direct pathway of activation involves recognition of microbial lipid antigen presented on CD1d. In the case of the lipoglycan α-GalCer, CD1d-restricted antigen presentation is sufficient for iNKT cell activation although co-stimulatory signalling mediated by constitutive CD28 expression can further augment the iNKT response. Alternatively, the indirect pathway activates iNKT cells in response to microbial organisms lacking cognate glycolipid antigens by recognition of self-antigen, together with co-stimulatory cytokine signals (IL-12, IL-18) from toll-like receptor (TLR) ligand-activated APCs. Finally, cytokine-mediated iNKT cell activation independently of CD1d involvement has also been observed in response to

By virtue of their semi-invariant TCR, all iNKT cells can recognise glycolipids consisting of a galactose or glucose moiety α-linked to the polar head of a lipid. The prototypical extrinsic iNKT antigen is the α-linked galactosylceramide α-GalCer, a glycosphingolipid extracted from the non-sterile marine sponge *Agelas mauritianus.* α-GalCer and related glycosphingolipids are not present in mammalian cells but can be found in the cell walls of *Novosphingobium*, previously known as *Sphingomonas* bacteria that colonise the marine sponge. Glycosylated diacylglycerol lipids in *Borrelia burgdorferi* are also recognised by a sub-population of murine iNKT cells and iNKT cell deficiency is associated with reduced spirochete clearance and chronic inflammation. Other microbial CD1d-restricted lipids are thought to be present in *Plasmodium falciparum*, *Trypanosoma* spp, *Leishmania* spp, *Ehrlichia* spp, *Streptococcus pneumoniae*, *Helicobacter pylori* and *M bovis*. The importance of these other microbial lipid antigens is however unclear as they are not strong TCR agonists (Brigl & Brenner, 2010). The nature of relevant lipid self-antigen(s) has remained a matter of considerable debate. Phospholipids such as phosphatidylinositol, phosphatidylethanolamine, and phosphatidylglycerol can be eluted from CD1d but most however are not stimulatory or stimulate only a small fraction of iNKT cells. In contrast, sphingolipids such as the tumour-derived ganglioside GD3 or the lysosomal ϐ-linked glycosphingolipid isoglobotrihexosylceramide (iGb3) have been shown to be recognised by iNKT cells (Gapin, 2010). Lysophosphatidylcholine (LPC) is produced from membrane phosphatidylcholine by phospholipase-A2, an enzyme produced by myeloid APC and activated during the inflammatory response. Thus increased presentation of LPC to iNKT cells during inflammation has been proposed to lead to their enhanced activation and cytokine production (Fox et al., 2010). In addition to constitutive stimulatory self-antigens, neo-selfantigens have been proposed to arise following exposure of APC to microbial or viral danger signals resulting in increased iNKT cell sensitivity to existing self-lipids, generation of novel lipid entities that are not constitutively expressed or increased antigen presentation

Stimulatory antigens such as α-GalCer rapidly activate iNKT cells within hours, upregulating surface markers such as CD25 and CD69 and producing cytokines through

adaptive immune response (Fox et al., 2010).

and co-stimulation (Reilly et al., 2010).

**2.4 iNKT cell activation and effector functions** 

**2.3 Antigen and antigen-independent activation pathways** 

lipopolysaccharide (LPS) or viral CpG-activated APCs (Reilly et al., 2010).

show very limited polymorphism. Human CD1a, -1b and -1c (CD1 group 1) are widely expressed on dendritic cells and other professional antigen presenting cells (APC). CD1d (CD1 group 2) expression appears to be independent of group 1 isoforms and can be found on lymph node mantle zone B cells, cortical thymocytes, activated T cells, gut and liver tissues. CD1b was first shown to present microbial antigen in 1992 which was later identified as lipid antigen. Since then it has become clear that CD1 molecules bind diverse hydrophobic ligands, with the exception of CD1e which remains intracellular and is thought to be involved in lipid antigen processing and loading (Brigl & Brenner, 2004; Strominger, 2010).

After the discovery of NK1.1+ CD4-CD8 double negative (DN) thymocytes, further work led to iNKT cells bearing a TCR consisting of an invariant Vα14-Jα18 rearrangement and a limited repertoire of Vϐ8.2, -7 or -2 chains in mice or the homologous Vα24-Vα18 and Vϐ11 chains in humans. Later, the TCR of iNKT cells was shown to be CD1d-restricted and these cells were coined the type I NKT cells (Godfrey et al., 2004). Like iNKT cells, type II NKT cells are CD1d-restricted but possess a more diverse TCR repertoire that also displays bias. They have been shown to possess regulatory and pathogenic functions but have been less well studied than iNKT cells. Non-CD1d restricted T cells that possess NK cell markers also exist and are referred to as NKT-like or type III NKT cells. In mice, 20-80% of CD3+NK1.1+ T cells stain with α-GalCer/CD1d-tetramer. In humans, while 20-25% of T cells are CD161+, less than 1% are α-GalCer/CD1d-tetramer+ and many staining cells are CD161- . These cells comprise CD1 group 1-restricted NKT cells that can be αϐ+, γδ+, CD4+, CD8+, or CD4-CD8- T cells, and may also include mucosa associated invariant T cells (MAIT) that express an invariant TCRα chain restricted to the MHC class I-like molecule MR1 (Godfrey et al., 2010).

#### **2.2 iNKT cell development and homeostasis**

iNKT cells develop in the thymus when invariant TCRα rearrangement and CD1d recognition initiate positive and negative selection, but unlike conventional T cells positive selection of iNKT cells involves double-positive CD4+CD8+ cortical thymocytes rather than cortical epithelial cells. TCR, SLAM and other co-stimulatory signals (CD28, ICOS, TGF-ϐ) are required for maturation, expression of activation (CD44, CD69, CD122) and NK (KLRG1 and NK1.1) markers, and acquisition of innate effector functions dependent on the transcriptional regulator promyelocytic leukaemia zinc finger protein (PZLF). Maturation to NK1.1+ or CD161+ occurs in the periphery in mice and humans, or in the thymus in mice where a long-lived population of thymic mature iNKT cells may contribute to fine-tuning the negative selection of conventional T cells (D'Cruz et al., 2010).

Similar to naïve conventional T cells, peripheral iNKT cell are not expanded in unchallenged mice, with an average clonal size of 5-10 cells that do not require interaction with CD1d for homeostasis. Nevertheless, iNKT cell frequency is orders of magnitude higher than that of naïve MHC class I and II restricted T cells. Humans have fewer iNKT cells than mice although there is wide variation amongst individuals, varying between undetectable and 3% of peripheral lymphocytes. It is not known whether this is related to thymic development and migration, or peripheral proliferation and maintenance differences but the phenomenon appears to be a stable, genetically determined phenotype in both mice and humans (Van Kaer et al., 2011).

iNKT cells are most abundant in the liver and spleen in mice but exceed the number of antigen-specific T cells in the lymph nodes by 500-5000 fold. In keeping with their innate function, NKT cells also accumulate in inflammatory lesions where they can rapidly become activated to release a diverse range of cytokines, proliferate and influence the subsequent adaptive immune response (Fox et al., 2010).

#### **2.3 Antigen and antigen-independent activation pathways**

20 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

show very limited polymorphism. Human CD1a, -1b and -1c (CD1 group 1) are widely expressed on dendritic cells and other professional antigen presenting cells (APC). CD1d (CD1 group 2) expression appears to be independent of group 1 isoforms and can be found on lymph node mantle zone B cells, cortical thymocytes, activated T cells, gut and liver tissues. CD1b was first shown to present microbial antigen in 1992 which was later identified as lipid antigen. Since then it has become clear that CD1 molecules bind diverse hydrophobic ligands, with the exception of CD1e which remains intracellular and is thought to be involved in lipid antigen processing and loading (Brigl & Brenner, 2004; Strominger,

After the discovery of NK1.1+ CD4-CD8- double negative (DN) thymocytes, further work led to iNKT cells bearing a TCR consisting of an invariant Vα14-Jα18 rearrangement and a limited repertoire of Vϐ8.2, -7 or -2 chains in mice or the homologous Vα24-Vα18 and Vϐ11 chains in humans. Later, the TCR of iNKT cells was shown to be CD1d-restricted and these cells were coined the type I NKT cells (Godfrey et al., 2004). Like iNKT cells, type II NKT cells are CD1d-restricted but possess a more diverse TCR repertoire that also displays bias. They have been shown to possess regulatory and pathogenic functions but have been less well studied than iNKT cells. Non-CD1d restricted T cells that possess NK cell markers also exist and are referred to as NKT-like or type III NKT cells. In mice, 20-80% of CD3+NK1.1+ T cells stain with α-GalCer/CD1d-tetramer. In humans, while 20-25% of T cells are CD161+,

comprise CD1 group 1-restricted NKT cells that can be αϐ+, γδ+, CD4+, CD8+, or CD4-CD8- T cells, and may also include mucosa associated invariant T cells (MAIT) that express an invariant TCRα chain restricted to the MHC class I-like molecule MR1 (Godfrey et al., 2010).

iNKT cells develop in the thymus when invariant TCRα rearrangement and CD1d recognition initiate positive and negative selection, but unlike conventional T cells positive selection of iNKT cells involves double-positive CD4+CD8+ cortical thymocytes rather than cortical epithelial cells. TCR, SLAM and other co-stimulatory signals (CD28, ICOS, TGF-ϐ) are required for maturation, expression of activation (CD44, CD69, CD122) and NK (KLRG1 and NK1.1) markers, and acquisition of innate effector functions dependent on the transcriptional regulator promyelocytic leukaemia zinc finger protein (PZLF). Maturation to NK1.1+ or CD161+ occurs in the periphery in mice and humans, or in the thymus in mice where a long-lived population of thymic mature iNKT cells may contribute to fine-tuning

Similar to naïve conventional T cells, peripheral iNKT cell are not expanded in unchallenged mice, with an average clonal size of 5-10 cells that do not require interaction with CD1d for homeostasis. Nevertheless, iNKT cell frequency is orders of magnitude higher than that of naïve MHC class I and II restricted T cells. Humans have fewer iNKT cells than mice although there is wide variation amongst individuals, varying between undetectable and 3% of peripheral lymphocytes. It is not known whether this is related to thymic development and migration, or peripheral proliferation and maintenance differences but the phenomenon appears to be a stable, genetically determined phenotype in both mice and humans (Van

iNKT cells are most abundant in the liver and spleen in mice but exceed the number of antigen-specific T cells in the lymph nodes by 500-5000 fold. In keeping with their innate function, NKT cells also accumulate in inflammatory lesions where they can rapidly become

. These cells

less than 1% are α-GalCer/CD1d-tetramer+ and many staining cells are CD161-

**2.2 iNKT cell development and homeostasis** 

the negative selection of conventional T cells (D'Cruz et al., 2010).

2010).

Kaer et al., 2011).

Unlike conventional T cells, iNKT cells appear to be selected in the thymus for their ability to recognise both microbial lipid antigen and self-antigen. The direct pathway of activation involves recognition of microbial lipid antigen presented on CD1d. In the case of the lipoglycan α-GalCer, CD1d-restricted antigen presentation is sufficient for iNKT cell activation although co-stimulatory signalling mediated by constitutive CD28 expression can further augment the iNKT response. Alternatively, the indirect pathway activates iNKT cells in response to microbial organisms lacking cognate glycolipid antigens by recognition of self-antigen, together with co-stimulatory cytokine signals (IL-12, IL-18) from toll-like receptor (TLR) ligand-activated APCs. Finally, cytokine-mediated iNKT cell activation independently of CD1d involvement has also been observed in response to lipopolysaccharide (LPS) or viral CpG-activated APCs (Reilly et al., 2010).

By virtue of their semi-invariant TCR, all iNKT cells can recognise glycolipids consisting of a galactose or glucose moiety α-linked to the polar head of a lipid. The prototypical extrinsic iNKT antigen is the α-linked galactosylceramide α-GalCer, a glycosphingolipid extracted from the non-sterile marine sponge *Agelas mauritianus.* α-GalCer and related glycosphingolipids are not present in mammalian cells but can be found in the cell walls of *Novosphingobium*, previously known as *Sphingomonas* bacteria that colonise the marine sponge. Glycosylated diacylglycerol lipids in *Borrelia burgdorferi* are also recognised by a sub-population of murine iNKT cells and iNKT cell deficiency is associated with reduced spirochete clearance and chronic inflammation. Other microbial CD1d-restricted lipids are thought to be present in *Plasmodium falciparum*, *Trypanosoma* spp, *Leishmania* spp, *Ehrlichia* spp, *Streptococcus pneumoniae*, *Helicobacter pylori* and *M bovis*. The importance of these other microbial lipid antigens is however unclear as they are not strong TCR agonists (Brigl & Brenner, 2010).

The nature of relevant lipid self-antigen(s) has remained a matter of considerable debate. Phospholipids such as phosphatidylinositol, phosphatidylethanolamine, and phosphatidylglycerol can be eluted from CD1d but most however are not stimulatory or stimulate only a small fraction of iNKT cells. In contrast, sphingolipids such as the tumour-derived ganglioside GD3 or the lysosomal ϐ-linked glycosphingolipid isoglobotrihexosylceramide (iGb3) have been shown to be recognised by iNKT cells (Gapin, 2010). Lysophosphatidylcholine (LPC) is produced from membrane phosphatidylcholine by phospholipase-A2, an enzyme produced by myeloid APC and activated during the inflammatory response. Thus increased presentation of LPC to iNKT cells during inflammation has been proposed to lead to their enhanced activation and cytokine production (Fox et al., 2010). In addition to constitutive stimulatory self-antigens, neo-selfantigens have been proposed to arise following exposure of APC to microbial or viral danger signals resulting in increased iNKT cell sensitivity to existing self-lipids, generation of novel lipid entities that are not constitutively expressed or increased antigen presentation and co-stimulation (Reilly et al., 2010).

#### **2.4 iNKT cell activation and effector functions**

Stimulatory antigens such as α-GalCer rapidly activate iNKT cells within hours, upregulating surface markers such as CD25 and CD69 and producing cytokines through

Invariant Natural Killer T Cells in Rheumatoid Arthritis and Other Inflammatory Arthritides 23

(Scott et al., 2010). Given the dual pro-inflammatory and regulatory potential of iNKT cells, the study of their frequency and phenotype in RA, and their role in animal models of

Until the advent of α-GalCer/CD1d-tetramers and invariant TCR chain-specific monoclonal antibodies, much of the earlier work on iNKT cells had been muddied by the lack of specific reagents for reliable iNKT cell identification. However, despite the limitations posed by older identification methods, most studies have consistently shown that NKT or iNKT cell absolute and relative frequencies are reduced in RA. Here we review studies examining the peripheral and synovial compartments, individual iNKT cell subsets and the relationship

In the earliest published study on NKT cells in RA, Yanagihara et al. (1999) looked at CD3+NKR-P1A+(CD161+) NKT cells in 60 patients with established RA compared with 36 healthy controls. They found a 5.8 fold difference in NKT cells but no difference in NK cells. Although patients and controls were mismatched for age, no correlation with age was found in either group. There was no apparent correlation with disease duration, clinical disease

Recent studies of iNKT cell frequency using more specific detection reagents have confirmed results from earlier studies. Parietti et al. (2010) detected iNKT cells with a monoclonal antibody (mAb) against the canonical Vα24Jα18 invariant TCR chain in 36 RA, 43 SLE and 31 healthy subjects. The investigators confirmed the lower frequencies and percentages of iNKT in RA and SLE vs controls (0.09% and 0.01% vs 0.26%, respectively).

Our own group analysed the frequency of Vα24+Vϐ11+ NKT cells among 46 RA and 22 healthy controls, taking care to use a statistically robust minimum number of lymphocytegated events set at 500,000 in order to reliably measure the infrequent iNKT cells. Our results showed that RA patients have a 15-fold lower iNKT cell relative frequency compared to healthy controls (0.001% vs 0.21%, respectively), either before or after commencing

In a comparative analysis of NKT cell frequency in different compartments, Spadaro et al. (2004) studied 29 patients with psoriasis and psoriatic arthritis (PsA), 27 patients with RA and 27 healthy controls. Blood and synovial fluid (SF) lymphocyte subsets, including CD3+CD16+CD56+ NKT cells, were measured and compared. In peripheral blood, there was no statistically significant difference in NKT cell absolute or relative numbers between PsA, RA and healthy control subjects (61 cells/μL or 3.6%, 93 cells/μL or 5% and 89 cells/μL or 3.9%, respectively). SF NKT cells however were significantly reduced in both absolute and relative numbers as compared to peripheral blood in PsA and RA (2% vs 3.2% and 1.6% vs

Linsen et al. (2005) studied 23 RA and 22 healthy control patients using peripheral blood and, when available, synovial fluid and tissue specimens. They found that Vα24+Vϐ11+CD3+ NKT cells were significantly reduced in relative frequency in RA as compared to control

inflammatory arthritis, have been of significant interest to researchers in the field.

between iNKT cell frequency, disease activity and treatment response.

They found no effect of age, gender or treatment on iNKT cell frequency.

activity, inflammatory markers, RF status or drug treatment.

immunosuppressive treatment (Tudhope et al., 2010).

**3.1.2 Synovial compartment** 

4.1%, respectively) (Spadaro et al., 2004).

**3.1 iNKT cell frequency** 

**3.1.1 Peripheral blood compartment** 

constitutive expression of mRNA for IL-4 and IFN-γ. iNKT cells proliferate and expand up to 10-fold in the spleen, 5-fold in blood, bone marrow and lymph nodes, and 2- to 3-fold in liver with a peak at 3-4 days after antigen exposure. Unlike conventional T cells, iNKT cells show not only a lack of secondary memory response but a hyporesponsive state of immunological anergy lasting up to 2 months (Van Kaer et al., 2011).

Upon activation, iNKT cells have been reported to be capable of producing a wide variety of both Th1 and Th2 cytokines (IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17, IL-21, IFN-γ, TNF-α, GM-CSF) (Coquet, 2008). The precise pattern of cytokines produced may depend on factors such as tissue distribution, iNKT cell subset, antigen processing, activation pathway, TCR signal strength and cytokine milieu. In addition, chemokines and chemokine receptors allow homing to inflammatory sites where iNKT cells form a bridge between the innate and adaptive immune systems by jump starting antigen-specific responses (Salio et al., 2010).

Activated iNKT cells can stimulate inflammatory myeloid APC, NK and B cell function by the production of CD40L and pro-inflammatory cytokines such as IFN-γ and TNF-α which can induce maturation with up-regulation of co-stimulatory molecules, perpetuation of cytokine and chemokine production, thus enhancing MHC-restricted T cell stimulation and ensuing adaptive immune responses. In contrast, iNKT cells have also been shown to potentiate antigen-specific immune tolerance in a number of animal models of autoimmunity, organ transplantation and therapeutic mucosal immune tolerance induction. The mechanisms by which iNKT cells induce or maintain tolerance may be mediated by a shift in secretion toward regulatory cytokines such as IL-10 and IL-4 although experimental data have not been consistent and cytokine-independent mechanisms such as generation of regulatory DCs may play a role (Hegde et al., 2010).

#### **2.5 Pathophysiological roles of iNKT cells**

In summary, iNKT cells are a unique subset of T cells that can help orchestrate both proinflammatory and regulatory immune responses. Despite their small population size, they can simultaneously promote resistance against microbial infection, participate in tumour immunosurveillance, maintain peripheral tolerance and prevent autoimmunity.

iNKT cells have long been known to react to self. Such autoreactivity is TCR and CD1d dependent in both mice and human iNKT cells. It has been suggested that the antigens responsible for autoreactivity are the same as those involved in thymic selection so that iNKT cells are autoreactive by design (Gapin, 2010). An immunosuppressive role for iNKT cells has now been shown in a number of animal models of autoimmunity including type I diabetes in non-obese diabetic (NOD) mice, experimental autoimmune encephalomyelitis as a model of multiple sclerosis in C57BL/6 and NOD mice, models of systemic lupus erythematosus (SLE) and graft-versus-host disease (GvHD). In some cases, iNKT cells have been shown to play a pathogenic rather than protective role. Despite such apparently conflicting results, iNKT function can be harnessed for tolerance induction, as demonstrated most notably in the prevention of GvHD. In this chapter, we will focus our discussion on the possible roles of iNKT cells in rheumatoid arthritis (RA) and other inflammatory arthritis.

#### **3. iNKT cells in rheumatoid arthritis**

RA is an inflammatory arthritis affecting small and large synovial joints, mediated by a destructive interplay between T cells, B cells, macrophage-like synoviocytes and fibroblastlike synoviocytes, ending with synovial cartilage invasion and ultimately joint destruction (Scott et al., 2010). Given the dual pro-inflammatory and regulatory potential of iNKT cells, the study of their frequency and phenotype in RA, and their role in animal models of inflammatory arthritis, have been of significant interest to researchers in the field.

#### **3.1 iNKT cell frequency**

22 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

constitutive expression of mRNA for IL-4 and IFN-γ. iNKT cells proliferate and expand up to 10-fold in the spleen, 5-fold in blood, bone marrow and lymph nodes, and 2- to 3-fold in liver with a peak at 3-4 days after antigen exposure. Unlike conventional T cells, iNKT cells show not only a lack of secondary memory response but a hyporesponsive state of

Upon activation, iNKT cells have been reported to be capable of producing a wide variety of both Th1 and Th2 cytokines (IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17, IL-21, IFN-γ, TNF-α, GM-CSF) (Coquet, 2008). The precise pattern of cytokines produced may depend on factors such as tissue distribution, iNKT cell subset, antigen processing, activation pathway, TCR signal strength and cytokine milieu. In addition, chemokines and chemokine receptors allow homing to inflammatory sites where iNKT cells form a bridge between the innate and adaptive immune systems by jump starting antigen-specific responses (Salio et al., 2010). Activated iNKT cells can stimulate inflammatory myeloid APC, NK and B cell function by the production of CD40L and pro-inflammatory cytokines such as IFN-γ and TNF-α which can induce maturation with up-regulation of co-stimulatory molecules, perpetuation of cytokine and chemokine production, thus enhancing MHC-restricted T cell stimulation and ensuing adaptive immune responses. In contrast, iNKT cells have also been shown to potentiate antigen-specific immune tolerance in a number of animal models of autoimmunity, organ transplantation and therapeutic mucosal immune tolerance induction. The mechanisms by which iNKT cells induce or maintain tolerance may be mediated by a shift in secretion toward regulatory cytokines such as IL-10 and IL-4 although experimental data have not been consistent and cytokine-independent mechanisms such as generation of

In summary, iNKT cells are a unique subset of T cells that can help orchestrate both proinflammatory and regulatory immune responses. Despite their small population size, they can simultaneously promote resistance against microbial infection, participate in tumour

iNKT cells have long been known to react to self. Such autoreactivity is TCR and CD1d dependent in both mice and human iNKT cells. It has been suggested that the antigens responsible for autoreactivity are the same as those involved in thymic selection so that iNKT cells are autoreactive by design (Gapin, 2010). An immunosuppressive role for iNKT cells has now been shown in a number of animal models of autoimmunity including type I diabetes in non-obese diabetic (NOD) mice, experimental autoimmune encephalomyelitis as a model of multiple sclerosis in C57BL/6 and NOD mice, models of systemic lupus erythematosus (SLE) and graft-versus-host disease (GvHD). In some cases, iNKT cells have been shown to play a pathogenic rather than protective role. Despite such apparently conflicting results, iNKT function can be harnessed for tolerance induction, as demonstrated most notably in the prevention of GvHD. In this chapter, we will focus our discussion on the possible roles of iNKT cells in rheumatoid arthritis (RA) and other inflammatory arthritis.

RA is an inflammatory arthritis affecting small and large synovial joints, mediated by a destructive interplay between T cells, B cells, macrophage-like synoviocytes and fibroblastlike synoviocytes, ending with synovial cartilage invasion and ultimately joint destruction

immunosurveillance, maintain peripheral tolerance and prevent autoimmunity.

immunological anergy lasting up to 2 months (Van Kaer et al., 2011).

regulatory DCs may play a role (Hegde et al., 2010).

**2.5 Pathophysiological roles of iNKT cells** 

**3. iNKT cells in rheumatoid arthritis** 

Until the advent of α-GalCer/CD1d-tetramers and invariant TCR chain-specific monoclonal antibodies, much of the earlier work on iNKT cells had been muddied by the lack of specific reagents for reliable iNKT cell identification. However, despite the limitations posed by older identification methods, most studies have consistently shown that NKT or iNKT cell absolute and relative frequencies are reduced in RA. Here we review studies examining the peripheral and synovial compartments, individual iNKT cell subsets and the relationship between iNKT cell frequency, disease activity and treatment response.
