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

224 Myocarditis

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.

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-

Innate Lymphocyte Effectors

(Natural Killer, Natural Killer T and γδ T Cells) in Infection and Myocarditis 227

stimulation (see Figure 2), iNKT cells produce IL-13, a Th2 cytokine, which can induce TGFβ production from CD11b+Gr-1+ myeloid derived suppressor cells. TGFβ promotes FoxP3 expression and Treg cell differentiation (Mantel & Schmidt-Weber, 2010). Indirectly, NKT cells activate NK cells (Carnaud et al., 1999) and subpopulations of NK cells suppress adaptive immunity either through their effects on dendritic cells or T cells (Flodstrom-Tullberg et al., 2009; Lunemann et al., 2009). CD1d and NKT cells are crucial to Treg cell development (Sonoda et al., 1999), and treating mice with αGalCer increases Treg cell activation (La Cava et al. , 2006) and suppresses autoimmune diabetes in NOD mice (Cardell, 2006; Ly et al., 2006). NKT cells secret high levels of TGFβ and IL-10 (Sonoda et al., 2001; Stein-Streilein et al., 2000) which alter dendritic cell (DC) cytokine (IL-10) and accessory molecule (CD40, CD80 and/or CD86) expression (Kumanogoh et al., 2001; McGuirk & Mills, 2002; Salomon et al., 2000) that favors T regulatory cell responses. (Bach et al., 2004; Chen et al., 2009). NKT cells are protective in Chagas' disease (Duthie & Kahn, 2006; Olson et al., 2009) and IFNγ expression by the NKT cells appears to be crucial to their protective effect. Similarly, treating CVB3 infected mice with αGalCer is protective, again indicating a role for NKT cells in preventing myocarditis (Wu et al., 2010). However, whether these NKT cells also suppress immunopathogenicity by enhancing Treg cell responses is not clear. γδ T cells appear to have the opposite effect on Treg cell responses. IL-23 activated γδ cells prevent conversion of effector T cells to iTreg cells (Petermann et al., 2010). Similarly γδ cells reduce IL-10 producing Treg cells in the lung in an asthma model γδ cells (Hahn et al., 2008). Vγ2Vδ2 cells prevent IL-2 induced expansion of CD4+CD25+FoxP3+ T (Gong et al., 2009). γδ T cells promote dendritic cell maturation and enhance antigen presentation. Also, these innate effectors suppress IL-2 expression which is needed for Treg cell maintenance. Recently, it has been shown that CVB3-H3 infection of mice deficient in γδ cells results in significant increases in Treg cells and accumulation of a population of CD1d+ Treg cells (Huber, 2009, 2010). These CD1d+ Treg cells are substantially more immunosuppressive to myocarditis than the CD1d- Treg cells on a per cell basis. These CD1d+ Treg cells are absent in mice containing γδ cells, and adoptive transfer of activated γδ cells into CVB3-H3 infected γδKO mice both restores myocarditis susceptibility and eliminates the CD1d+ Treg cell population. Direct co-culture of the activated γδ cells on CD1d+ and CD1d- Treg cell populations shows that the γδ effector cells are lytic to the CD1d+ Treg in a CD1d- and caspase-dependent manner. (Huber, 2010).

Fig. 3. Cross-talk between innate effectors in control of adaptive immunity.

Santos et al., 2001; Soares et al., 2001). As with the CVB3 model, γδ cells are required for the pathogenic CD4 and CD8 responses in T. cruzi infections (Nomizo et al., 2006). However, unlike the CVB3 myocarditis model, the relevant γδ cell in T. cruzi infection expresses the Vγ1 T cell receptor. Why Vγ4 cells are operational in CVB3 disease while Vγ1 cells function in T. cruzi infection, is not currently known, but might reflect the difference between a virus and protozoa as the etiological agent. Nonetheless, the principle is the same: innate effectors control the activation of pathogenic antigen-specific adaptive immunity which subsequently causes cardiac damage.

The next question is how γδ cells control induction of adaptive immunity. Regulatory T cells (Tregs) are important negative immune modulators, constitute up to 10% of peripheral CD4+ T cells in naive mice and humans, and express CD25 (IL-2 receptor α chain) (Sakaguchi, 2005; Sakaguchi et al., 2008; Torgerson, 2006). There are several types of T regulatory cells which can basically be divided into natural (nTreg) and inducible (iTreg) populations. nTreg cells are generated in the thymus, and presumably arise from T cells with high affinity TCR for self antigens. nTreg cells are functionally mature when leaving the thymus and do not require antigen exposure peripherally to generate immunosuppressive activity. In contrast to nTreg cells, iTreg can be converted from effector T cell populations in the periphery subsequent to antigen challenge. The transcription factor, FoxP3, is usually associated with Treg cell development and transduction of exogenous FoxP3 into CD4+CD25- cells converts these cells into CD4+CD25+ Treg cells (Sakaguchi et al., 2008). Induction of FoxP3 expression and therefore, Treg cell activation depends upon the presence of TGFβ (Mantel & Schmidt-Weber, 2010). While FoxP3 is necessary for conversion of CD4+ cells to Treg cells, IL-2 is required for Treg cell maintenance/survival. Animals lacking either CD25 (IL-2R) or IL-2 develop lymphoproliferative and autoimmune diseases (Malek & Bayer, 2004) associated with a decrease in Treg cells. Three mechanisms have been proposed for Treg cell function (Sakaguchi et al., 2008). The first mechanism hypothesizes that Treg cells out-compete effector T cells for MHC-antigen complexes on antigen presenting cells which effectively blocks effector T cell activation. In the second mechanism, Treg cells directly interact with dendritic cells through CTLA4 which downregulates required accessory molecule expression needed for successful antigen stimulation of effector T cells. Finally, Treg cell secretion of TGFβ or IL-10 may inhibit T cell differentiation (Ozdemir et al. , 2009; Ray et al., 2010).

CVB3 infection up-regulates CD1d on infected cardiac myocytes and myeloid cells. CD1d activates NKT and Vγ4 T cells which can either recognize CD1d on myocytes leading to myocyte death or interact with dendritic cells (DC) through CD1d to alter antigen presentation function of the dendritic cells or to produce cytokines. NKT cells may also activate NK cells. NK and NKT cells promote activation of Treg cells by promoting myeloid derived suppressor cell differentiation, while Vγ4 T cells have the opposite effect and either directly kill Treg cells through CD1d expressed on the Treg cell population or induce dendritic cell maturation and enhanced antigen presentation to adaptive immune (CD4, CD8) effectors. Treg cells inhibit activation of CD4+IFNγ+ (Th1) cells which are required for generation of cytolytic, autoimmune CD8 effector cells. The CD8 effector cells are the primary immunopathogenic mediator to cardiac injury in CVB3 induced myocarditis.

Treg cells prevent autoimmunity in myocarditis (Frisancho-Kiss et al., 2006; Huber et al., 2006; Wang, 2010). Distinct populations of innate effector T cells can have dramatically different effects on Treg cell responses. iNKT cells can promote Treg cell activation (Nowak et al., 2006; Roelofs-Haarhuis et al., 2004; Roelofs-Haarhuis et al., 2003). Under appropriate

Santos et al., 2001; Soares et al., 2001). As with the CVB3 model, γδ cells are required for the pathogenic CD4 and CD8 responses in T. cruzi infections (Nomizo et al., 2006). However, unlike the CVB3 myocarditis model, the relevant γδ cell in T. cruzi infection expresses the Vγ1 T cell receptor. Why Vγ4 cells are operational in CVB3 disease while Vγ1 cells function in T. cruzi infection, is not currently known, but might reflect the difference between a virus and protozoa as the etiological agent. Nonetheless, the principle is the same: innate effectors control the activation of pathogenic antigen-specific adaptive immunity which subsequently

The next question is how γδ cells control induction of adaptive immunity. Regulatory T cells (Tregs) are important negative immune modulators, constitute up to 10% of peripheral CD4+ T cells in naive mice and humans, and express CD25 (IL-2 receptor α chain) (Sakaguchi, 2005; Sakaguchi et al., 2008; Torgerson, 2006). There are several types of T regulatory cells which can basically be divided into natural (nTreg) and inducible (iTreg) populations. nTreg cells are generated in the thymus, and presumably arise from T cells with high affinity TCR for self antigens. nTreg cells are functionally mature when leaving the thymus and do not require antigen exposure peripherally to generate immunosuppressive activity. In contrast to nTreg cells, iTreg can be converted from effector T cell populations in the periphery subsequent to antigen challenge. The transcription factor, FoxP3, is usually associated with Treg cell development and transduction of exogenous FoxP3 into CD4+CD25- cells converts these cells into CD4+CD25+ Treg cells (Sakaguchi et al., 2008). Induction of FoxP3 expression and therefore, Treg cell activation depends upon the presence of TGFβ (Mantel & Schmidt-Weber, 2010). While FoxP3 is necessary for conversion of CD4+ cells to Treg cells, IL-2 is required for Treg cell maintenance/survival. Animals lacking either CD25 (IL-2R) or IL-2 develop lymphoproliferative and autoimmune diseases (Malek & Bayer, 2004) associated with a decrease in Treg cells. Three mechanisms have been proposed for Treg cell function (Sakaguchi et al., 2008). The first mechanism hypothesizes that Treg cells out-compete effector T cells for MHC-antigen complexes on antigen presenting cells which effectively blocks effector T cell activation. In the second mechanism, Treg cells directly interact with dendritic cells through CTLA4 which downregulates required accessory molecule expression needed for successful antigen stimulation of effector T cells. Finally, Treg cell secretion of TGFβ or IL-10 may inhibit T cell

CVB3 infection up-regulates CD1d on infected cardiac myocytes and myeloid cells. CD1d activates NKT and Vγ4 T cells which can either recognize CD1d on myocytes leading to myocyte death or interact with dendritic cells (DC) through CD1d to alter antigen presentation function of the dendritic cells or to produce cytokines. NKT cells may also activate NK cells. NK and NKT cells promote activation of Treg cells by promoting myeloid derived suppressor cell differentiation, while Vγ4 T cells have the opposite effect and either directly kill Treg cells through CD1d expressed on the Treg cell population or induce dendritic cell maturation and enhanced antigen presentation to adaptive immune (CD4, CD8) effectors. Treg cells inhibit activation of CD4+IFNγ+ (Th1) cells which are required for generation of cytolytic, autoimmune CD8 effector cells. The CD8 effector cells are the primary immunopathogenic mediator to cardiac injury in CVB3 induced myocarditis. Treg cells prevent autoimmunity in myocarditis (Frisancho-Kiss et al., 2006; Huber et al., 2006; Wang, 2010). Distinct populations of innate effector T cells can have dramatically different effects on Treg cell responses. iNKT cells can promote Treg cell activation (Nowak et al., 2006; Roelofs-Haarhuis et al., 2004; Roelofs-Haarhuis et al., 2003). Under appropriate

causes cardiac damage.

differentiation (Ozdemir et al. , 2009; Ray et al., 2010).

stimulation (see Figure 2), iNKT cells produce IL-13, a Th2 cytokine, which can induce TGFβ production from CD11b+Gr-1+ myeloid derived suppressor cells. TGFβ promotes FoxP3 expression and Treg cell differentiation (Mantel & Schmidt-Weber, 2010). Indirectly, NKT cells activate NK cells (Carnaud et al., 1999) and subpopulations of NK cells suppress adaptive immunity either through their effects on dendritic cells or T cells (Flodstrom-Tullberg et al., 2009; Lunemann et al., 2009). CD1d and NKT cells are crucial to Treg cell development (Sonoda et al., 1999), and treating mice with αGalCer increases Treg cell activation (La Cava et al. , 2006) and suppresses autoimmune diabetes in NOD mice (Cardell, 2006; Ly et al., 2006). NKT cells secret high levels of TGFβ and IL-10 (Sonoda et al., 2001; Stein-Streilein et al., 2000) which alter dendritic cell (DC) cytokine (IL-10) and accessory molecule (CD40, CD80 and/or CD86) expression (Kumanogoh et al., 2001; McGuirk & Mills, 2002; Salomon et al., 2000) that favors T regulatory cell responses. (Bach et al., 2004; Chen et al., 2009). NKT cells are protective in Chagas' disease (Duthie & Kahn, 2006; Olson et al., 2009) and IFNγ expression by the NKT cells appears to be crucial to their protective effect. Similarly, treating CVB3 infected mice with αGalCer is protective, again indicating a role for NKT cells in preventing myocarditis (Wu et al., 2010). However, whether these NKT cells also suppress immunopathogenicity by enhancing Treg cell responses is not clear. γδ T cells appear to have the opposite effect on Treg cell responses. IL-23 activated γδ cells prevent conversion of effector T cells to iTreg cells (Petermann et al., 2010). Similarly γδ cells reduce IL-10 producing Treg cells in the lung in an asthma model γδ cells (Hahn et al., 2008). Vγ2Vδ2 cells prevent IL-2 induced expansion of CD4+CD25+FoxP3+ T (Gong et al., 2009). γδ T cells promote dendritic cell maturation and enhance antigen presentation. Also, these innate effectors suppress IL-2 expression which is needed for Treg cell maintenance. Recently, it has been shown that CVB3-H3 infection of mice deficient in γδ cells results in significant increases in Treg cells and accumulation of a population of CD1d+ Treg cells (Huber, 2009, 2010). These CD1d+ Treg cells are substantially more immunosuppressive to myocarditis than the CD1d- Treg cells on a per cell basis. These CD1d+ Treg cells are absent in mice containing γδ cells, and adoptive transfer of activated γδ cells into CVB3-H3 infected γδKO mice both restores myocarditis susceptibility and eliminates the CD1d+ Treg cell population. Direct co-culture of the activated γδ cells on CD1d+ and CD1d- Treg cell populations shows that the γδ effector cells are lytic to the CD1d+ Treg in a CD1d- and caspase-dependent manner. (Huber, 2010).

Fig. 3. Cross-talk between innate effectors in control of adaptive immunity.

Innate Lymphocyte Effectors

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