**3.2 Natural killer T cells and CD1-restricted γδ T cells**

Many T cells respond to CD1 molecules (Barral & Brenner, 2007; Kaufmann, 1996) and express either T cell receptors (TCR) consisting of α/β or γ/δ polypeptide chains. Group 1 CD1-restricted αβ T cells are clonally diverse with fine antigen specificity, recognition of both self and foreign lipid antigens and either double negative (CD4-CD8-) or single positive (CD4+ or CD8+) (Barral & Brenner, 2007; Kaufmann, 1996; Vincent et al., 2005). The αβ T cell response is slow, similar to classical MHC αβ T cell responses indicating that these CD1-restricted effectors probably do not belong to the innate immune system. There are two major populations of γδ T cells in humans (Vδ1 and Vδ2) with Vδ2 cells primarily present in the circulation and Vδ1 cells primarily found in tissues and intestine (Das et al., 2004). Subsets of both γδ populations recognize antigens in context of non-classical MHC class Ilike molecules including group 1 CD1(Rincon-Orozco et al., 2005; Russano et al., 2007). Activation of the group 1 CD1 restricted effectors requires IL-12, NKG2D activation on the effector and adhesion molecule interactions (LFA3/CD2, LFA1/ICAM1) in addition to TCR engagement. Since mice lack Group 1 CD1 molecules, this species does not have Group 1 CD1-restricted immunity. However, these effectors may function in humans.

T cells reacting to CD1d (Group 2 CD1) are also diverse. CD1d-restricted natural killer T (NKT) cells are designated as either invariant NKT (iNKT, also known as Type 1) or diverse NKT (also known as Type 2) cells (Barral & Brenner, 2007; Kronenberg, 2005; Ronchi & Falcone, 2008; Taniguchi et al., 2010). Type 1 iNKT cells have a TCR comprised of a single type of TCRα chain (Vα14Jα18 for mice and Vα24Jα18 for humans) and one of a limited number of distinct TCRβ chains resulting in limited clonal diversity. In contrast, Type 2 NKT cells use TCR comprised of diverse α and β chains. iNKT cells comprise between 2-40% of CD3+ cells in various tissues (Bendelac et al., 2007; Terabe & Berzofsky, 2008), have a constitutively activated phenotype, and rapidly secret large amounts of cytokines (IFN-γ, IL-4, IL-17, IL-5, and IL-13) upon activation due to the presence of pre-formed cytokine mRNA in the cells (Kronenberg, 2005; Michel, 2007; Olson et al., 2009; Stetson et al., 2003). Three mechanisms of iNKT cell activation have been described (Figure 2). Direct activation involves recognition of microbial antigens presented by CD1d on antigen presenting cells (TCR-mediated). In contrast, indirect activation either involves microbial stimulation of antigen presenting cells to release cytokines (IL-12 and Type 1 IFN) and presentation of

Innate Lymphocyte Effectors

**3.3 NKT and γδ T Cells in myocarditis** 

(Natural Killer, Natural Killer T and γδ T Cells) in Infection and Myocarditis 225

There are three major mechanisms for activating NKT cells. Direct activation involves phagocytosis of microbes and binding of microbial lipids into the CD1d groove in the late endosome with transport of the CD1d-lipid complex to the antigen presenting cell surface. NKT cells activated through this pathway produce a broader range of Th1 and Th2 cytokines. A second pathway involves microbial stimulation of TLR on the antigen presenting cell which can both affect self-lipid expression/availability and stimulate cytokine expression from the antigen presenting cells. NKT cells stimulated by the recognition of self-lipid/CD1d and the cytokine milieu secrete primarily Th1 cytokines. The third mechanism is either not or substantial less dependent upon CD1d recognition by the

Several cases of clinical cardiomyopathy have been reported where substantial numbers of γδ cells are in the inflammatory infiltrate (Eck et al., 1997; Takeda et al., 2008; Takeda et al., 2005). However, there is little direct evidence for a pathogenic role for these innate effectors in humans. As indicated above for NK cells, the lack of direct evidence for innate effectors in clinical myocarditis may simply reflect the fact that innate immunity should function early after infection and may disappear from the heart by the time that clinical symptoms are evident. In contrast to myocarditis in humans, substantial evidence implicates innate lymphocyte effectors in mouse models of coxsackievirus B3 (CVB3) and Borrelia burgdorferi (Lyme disease) myocarditis (Figure 3). As described above, mice lacking CD1d fail to develop myocarditis subsequent to CVB3-H3 (highly myocarditic variant of CVB3 (Knowlton et al., 1996)) infection despite high levels of virus replication in the heart (Huber et al., 2003). Infecting iNKT deficient mice with CVB3-H3 had no effect indicating that iNKT cells do not contribute to pathogenesis with this CVB3 variant. Surprisingly, CD1d deficient mice had significantly reduced numbers of activated γδ T cells belonging to the Vγ4 subset, and further analysis demonstrated that these Vγ4 cells are CD1d restricted as they killed CVB3-H3 infected CD1d+ but not infected CD1d- cardiac myocytes and cytotoxicity of the CD1d+ myocytes was blocked by anti-CD1d antibodies but not by antibodies to the classical MHC I and MHC II antigens (Huber, 2000; Huber et al., 2003). More importantly, activation of Vγ4 cells correlated to induction of CD4+IFNγ+ (Th1) virus-specific cells, which indicates that γδ cells might impact myocarditis through their effects on the antigen-specific, adaptive immune response (Huber & Sartini, 2005a; Huber et al., 2002). Previous studies had shown that heart-specific, autoimmune CD8+ cytolytic T lymphocytes are the primary immunopathogenic effector in CVB3 induced myocarditis (Guthrie et al., 1984; Henke et al., 1995; Huber & Lodge, 1984; Huber et al., 1988; Huber et al., 2002). These autoimmune CD8 cells kill uninfected cardiocytes through recognition of cardiac myosin epitopes (Huber & Gauntt, 2000) and can adoptively transfer myocarditis into uninfected recipients (Huber et al., 1987). However, the autoimmune CD8 T cell response is absolutely dependent on CD4+IFNγ+ cells (Huber et al., 2002). This is not surprising as many studies have shown that CD4+ Th1 cells promote CD8 T cell activation (Krawczyk et al., 2007; Serre et al., 2006). Although Vγ4 cells are required for generation of CD4+IFNγ+ cells, once the CD4+IFNγ+ cells exist, Vγ4+ cells are no longer necessary for autoimmune CD8 cell induction or myocarditis (Huber et al., 2002). The CVB3 model is not the only one showing that γδ T cells are required for immunopathogenic CD4 and CD8 T cell responses. Trypanosoma cruzi, the etiological agent in Chagas' disease, causes myocarditis with cardiac injury at least partially mediated by T cells and IFNγ (dos Santos et al., 2001; Marin-Neto et al., 2007; Ribeiro-Dos-

NKT cells but NKT cell activation is primarily induced through cytokines alone.

self/altered self lipid antigens on CD1d; or cytokines (IL-12 and IL-18) in the absence of CD1d antigen presentation (Brigl et al., 2003). Both inflammation and TLR activation can affect expression of enzymes involved in lipid metabolism (Khovidhunkit et al., 2004; Salio et al., 2007) and this may either increase total self lipid in endosomes or alter self lipids making them appear more foreign to the immune system. The mechanism of iNKT cell activation can impact the types of cytokines released with direct CD1d activation resulting in both Th1 (IFNγ) and Th2 (IL-4/IL-13) release while indirect activation causes predominantly Th1 (IFNγ) expression (Brigl et al., 2003). iNKT cells producing Th2 cytokines modulate NK cells to express TGFβ and TGFβ promotes T regulatory cell activation(Chen et al., 2009; Monteiro et al., 2010). Thus, depending upon the mode of iNKT cell activation, these effectors can be either pro- or anti-inflammatory. Type 2 NKT cells also can have a Th1 or Th2 phenotype with corresponding cytokine profiles, and therefore may have either potentiating or protective roles in infections and autoimmune diseases (Arrenberg et al., 2009). A number of reports indicate that Type 1 and Type 2 NKT cells are antagonistic to each other and form a regulatory network to control adaptive immunity. Most reports suggest an anti-inflammatory role for Type 2 NKT cells which can be protective in autoimmune diabetes in NOD mice (Duarte et al., 2004), experimental allergic encephalomyelitis (Jahng et al., 2004) and Con-A induced hepatitis (Halder et al., 2007). Furthermore, while type 1 NKT cells may increase tumor immunosurveillance, Type 2 NKT cells may suppress anti-tumor immunity (Ambrosino et al., 2007; Terabe & Berzofsky, 2008). Activation pathways for γδ T cells can also be diverse with both direct antigen presentation in MHC or MHC-like molecules or indirect with minimal or no antigen presentation (Kaufmann, 1996). Unlike NKT cells, γδ T cells may either react to CD1 itself in the absence of any antigen or to antigen without presentation by MHC/MHC-like molecule involvement. Although both human and mouse γδ T cells have been found to recognize antigens presented by Group 1 CD1 and other non-classical MHC antigens (Chien & Konigshofer, 2007; Cui et al., 2009; Spada et al., 2000; Van Kaer et al., 1991), only this laboratory has reported a subpopulations of γδ cells (Vγ4 TCR) recognizing CD1d (Huber et al., 2003).

Fig. 2. Mechanisms for NKT cell activation.

There are three major mechanisms for activating NKT cells. Direct activation involves phagocytosis of microbes and binding of microbial lipids into the CD1d groove in the late endosome with transport of the CD1d-lipid complex to the antigen presenting cell surface. NKT cells activated through this pathway produce a broader range of Th1 and Th2 cytokines. A second pathway involves microbial stimulation of TLR on the antigen presenting cell which can both affect self-lipid expression/availability and stimulate cytokine expression from the antigen presenting cells. NKT cells stimulated by the recognition of self-lipid/CD1d and the cytokine milieu secrete primarily Th1 cytokines. The third mechanism is either not or substantial less dependent upon CD1d recognition by the NKT cells but NKT cell activation is primarily induced through cytokines alone.
