**5. Cell types**

While inflammation and neurodegeneration are correlated in active lesions, research suggests that neurodegeneration may become independent from inflammation in progressive disease [64]. There are many MS therapeutics that suppress proinflammatory cytokines or their effector functions, but not all treatments show equal efficacy and can cause unintended effects. Currently, there is no cure. It is thus becoming clear that there is a need to elucidate the different populations important in initiating and progressing disease, and by studying their interactive networks, identify possible areas for targeted intervention.

### **5.1 T cells**

While there is overwhelming evidence of a role for T cells in the pathogenesis of MS, further studies in humans and in the mouse model of disease, experimental autoimmune encephalomyelitis (EAE), provides compelling evidence that other cell types

#### *Cellular Cytotoxicity and Multiple Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.105681*

play major roles. Linkage to the Human Leukocyte antigen (HLA) locus, including MHC I and II genes, was the first genetic locus identified, and still provides today the strongest linkage to MS. Further studies have identified an extended HLA haplotype, HLA-DRB1\*15:01, DQA1\*0102, DQB1\*0602, within the MHC class II region [65]. As MHC II molecules specifically present peptide antigens to activate CD4 T cells, this suggests that CD4 T cells are important in initiation and progression of MS.

Th1 cells are a lineage of CD4 effector T cells that promote cell mediated immune responses and are necessary for defence against intracellular viral and bacterial pathogens. They were originally believed to be the main pathogenic T cells in MS, not only because susceptibility genes were linked to MHC II molecules, but also because immune surveillance of a healthy brain to scan for infection, showed favouring towards infiltration by Th1 cells, and therapeutic strategies designed to induce a shift from Th1 to Th2 immune response resulted in beneficial outcomes in MS patients [66–68].

The development of Th1 cells is coupled to the involvement of cell-extrinsic and cell-intrinsic factors, including signal transducer activator 1 (STAT1), the transcription factor Tbx21, IL-21 and STAT4 [69]. The CD4-Th1 model for MS was further supported by a trial performed in 1987, which found that administering IFN-γ to RRMS patients exacerbated disease. An accompanying increase in circulating monocytes bearing class II (HLA-DR) surface antigens suggested that the attacks induced by the treatment were immunologically mediated [70].

Th1 cells are also known to drive EAE. However, it was found that transgenic mice that lacked Th1 cells developed more severe EAE, thereby contradicting the Th1 cell theory for MS [71]. This conundrum was partially resolved following further investigation involving IL-23, a heterodimer cytokine composed of a unique p19 subunit and a common p40 subunit shared with IL-12. IL-23 promotes development of Th17 cells as opposed to Th1 cells [72]. Early studies on Th17 cells therefore dismissed a role for the previously favoured Th1 cells, but more recent research suggests that both cell types may play distinct roles in pathology [73]. It was suggested that Th1 cells accessed the CNS initially and subsequently facilitated the recruitment of Th17 cells [73].

Analysis of CNS tissue revealed distinct histopathological features and immune profiles depending on cytokine modulated T cells. IL-12p70 driven disease was characterised by macrophage-rich infiltrates, however in IL-23 driven lesions it was found that neutrophils and the growth factor, granulocyte colony stimulating factor (CSF), were the most prominent [74]. Research has shown that while IL-23 is commonly associated with the expansion of Th17 cells or the stabilisation of the Th17 phenotype, a similar course of EAE has been reported following the transfer of MOG-specific T cells into either wild type or IL-23 knockout mice [75]. This suggests that once encephalitogenic cells have been generated, EAE can develop in the absence of IL-23. IL-23 may therefore only be necessary for disease induction and not the effector phase of disease.

While MHC II molecules were found to be the strongest associated with MS in genetic studies, the MHC I HLA-A\*0301 allele, independent of the HLA II haplotype DRB1\*15,DQB1\*06, was found to be increased in MS patients [65]. There was also a negative association with the MHC I HLA-A\*0201 and disease [76]. As MHC I molecules are recognised by CD8 T cells, this suggests that CD8 T cells play a role in MS.

In one of the first studies that shifted from a CD4 T cell focus, CD8 T cells outnumbered the CD4 T cell subset in all parenchyma samples from MS patients, regardless of the MS type, duration or speed of disease progression [77]. Research has also

shown that APCs, including dendritic cells (DCs), interact with T cells and proliferating lymphocytes, predominately CD8 T cells, at the margins of chronic active MS lesions [78]. CD8 T cells have also been found within active lesions of RRMS patients [77]. These T cells, and to a lesser extent, compartmentally differentially distributed B cells, have been shown to correlate with disease progression and damage.

CD8 T cells are an important subpopulation of MHC I restricted T cells, and are mediators of adaptive immunity. Cytotoxic T cells specialise in direct killing of cells that are infected, particularly with viruses, or are cancerous or damaged in other ways. Cytotoxic cells rely on two mechanisms for lytic activity: granule-dependent cytotoxicity (reviewed in [79]) and death receptor dependent cytotoxicity (reviewed in [80]). The principle mechanism used is granule-dependent cytotoxicity. In lesion prone areas of the CNS, T lymphocytes, including CD8 cytotoxic T lymphocytes (CTLs), are recruited to the affected tissue and brain cells are stimulated to present antigens to the T lymphocytes via de novo expression of MHC molecules. Although levels of MHC I and MHC II are very low in normal CNS parenchyma, neural injury leads to a massive increase in activated and phagocytotic microglial, which can serve as competent APCs [81]. To develop into functioning CD8 T cells, the TCR must recognise the MHC-peptide combination along with the costimulatory signal from APCs. While classical MHC I molecules necessary for CD8 T cell activation are not usually expressed on neural cells, they are induced in most inflammatory and degenerative CNS diseases [82].

Oligodendrocytes lack expression of costimulatory molecules and are thus unable to trigger the full effector of T cells, however they have been known to express MHC I in vitro [83]. Therefore, despite the lack of complete activation of the T cells, oligodendrocytes may still be targets of primed CTLs. MHC I expressing oligodendrocytes are susceptible to lysis by blood donor derived CD8 CTLs [83]. IFN-γ treated human oligodendrocytes also express Fas/CD95, and are therefore susceptible to death receptor dependent cytotoxicity [84]. Another component of the CNS, the neurons, were found to be capable of expressing MHC I when treated with IFN-γ [85, 86]. Medana and colleagues in 2000 discovered that hippocampal neurons were highly susceptible to direct application of cytotoxic granules, but showed no signs of perforin mediated lysis or membrane damage following attack by CTLs [87]. This effect was not observed in any other cell type.

Research to date indicates that all cellular elements of the CNS may act as targets to CTLs but that susceptibility and cytotoxic pathways involved vary dependent on the cell type and the immune activations during the course of the inflammatory process.

#### **5.2 B cells**

Historically, B cells have not been recognised as major players in regulatory function in the development of autoimmune diseases, although the identification of autoantibodies produced by autoreactive plasma cells and their pathogenic consequences are widely accepted [88]. B cells are considered effector cells as well as cells with immunoregulatory potential. B cells in MS patients express increased levels of costimulatory molecules, increasing the stimulation of antigen-reactive T cells [89]. It has been reported that MS patients have increased levels of IL-6 and GM-CSF, correlating with increased Th17 cells [90, 91]. B cell targeted therapies utilise B cell depleting monoclonal antibodies against the B cell marker CD20. These antibodies trigger B cell lysis through antibody dependent cellular cytotoxicity, complement dependent cytotoxicity or apoptosis induction [92].

## **5.3 NK cells**

Administration of daclizumab, an alpha subunit of IL-2 receptor blocking monoclonal antibody, to MS patients was found to strongly reduce brain inflammation. This therapy, while being associated with a decline in circulating CD4 and CD8 T cells, also correlated with a significant expansion of CD56bright NK cells in vivo. This provided supporting evidence of NK cell-mediated negative immunoregulation of T cells during daclizumab treatment [93], and the identification of NK cells in association with MS, where positive outcome was possibly due to the treatment's effect of increasing the NK cell numbers [94, 95].

For decades, NK cells have been classified as a component of the innate immune system. However, evidence suggests that, like B and T cells, NK cells are educated during development, possess antigen-specific receptors, undergo clonal expansion and generate memory cells (reviewed in [96]). Research originally suggested that NK cells developed and underwent differentiation within the bone marrow, however more recent extensive ex vivo characterisation of haematopoietic precursor cells (HPCs) and downstream NK cell development intermediates (NKDIs) reveals that they are enriched in secondary lymphoid tissues (STLs), including the tonsils, spleen and lymph nodes [97–100]. This suggests that NK cells in humans can differentiate in the SLTs, and may do so preferentially.

Human NK cells are phenotypically defined by expression of CD56 and the lack of CD3 expression [101]. CD56 is the 140-kDa isoform of neural cell adhesion molecule (NCAM) found on NK cells and a minority of T cells [102]. NK cells are categorised into two distinct populations depending on the cell surface density of CD56. The majority of human NK cells, approximately 90%, express low levels of CD56 (CD56dim) and high levels of FCyRIII (CD16), while the minority express higher levels of CD56 (CD56bright) [103]. CD56bright NK cells have long being associated with an immunoregulatory role, due to increased production of NK-derived immunoregulatory cytokines, including IFN-γ, TNF-β, IL-10, IL-13 and GM-CSF, and reduced cytotoxicity compared to CD56dim NK cells [104]. CD56bright NK cells express receptors for cytokines such as IL-12, IL-15 and IL-18, produced by APCs, which can trigger proliferation of CD56bright NK cells and their production of molecules, including IFN-γ, IL-10 and IL-13 [104]. It has been demonstrated that DCs are a key source of cytokines for the activation of CD56bright NK cells [105]. Modulation and proliferation of CD56bright NK cells can also occur due to DC-derived IL-27 [105]. Activated NK cells can modulate the function of APCs by stimulating monocytes to produce TNF-α and kill immature DCs by a perforin-dependent process referred to as DC editing [106, 107]. However, more recent research has challenged this commonly accepted concept of CD56bright as the primary source of immunoregulatory cytokines. Studies have shown that CD56dim NK cells are also a major source of proinflammatory cytokines and chemokines that are induced rapidly after target cell recognition [108, 109].

The absence of MHC class I molecules, as indicated by virally infected cells or cancerous cells with MHC I downregulated, is not always sufficient to induce NK cell mediated death, suggesting that there must be activating receptors on NK cells whose affinity for target cell ligands dominates over the inhibitory signals of the NK cell. Some activating receptors identified include NKG2D, the NCR, and NKp80 [110–112]. NKG2D is the best characterised of these activating NK cell receptors. It is a C-type lectin-like receptor expressed on the surface of all human NK cells and recognises at least six ligands, each with a MHC class I homology [113]. Following

receptor-ligand interaction, NKG2D phosphorylates an adaptor protein that recruits and activates phosphatidylinositol-3 (PI-3) kinase, which results in perforin-dependent cytotoxicity [114, 115]. Gunesh et al. found that the deletion of CD56 on the NK92 cell line lead to impaired cytotoxic function. The knockout CD56 cells failed to polarise during immunological synapse formation and had severely impaired exocytosis of lytic granules [116].

Treatment of MS patients with IFN-β caused an expansion of CD56bright NK cells, and resulted in the population of CD56dim cells being diminished [117]. The study also found that the proportion of CD56bright NK cells was significantly higher in the secondary lymphoid tissues compared to the peripheral blood for the control group [117]. This suggested that CD56bright NK cells may preferably locate within secondary lymphoid tissues, where they are able to interact with T cells and contribute to control of disease activity in MS [117].

There is an ongoing debate as to whether NK cells have a predominately beneficial or detrimental role in EAE, made even more complex by the lack of CD56 expression on murine NK cells. Studies have shown that enhancing the regulatory features of NK cells ameliorates the disease course of EAE. When the interaction between NKG2A and its ligand Qa-1 (the murine equivalent to the human HLA-E) expressed on target cells were blocked by antibodies specific for either antigen, it was found that NKG2Aexpressing NK cells in particular decreased CNS inflammation by killing microglial and T cells [118, 119].

Enrichment of NK cells through treatment with IL-2 coupled with a monoclonal antibody specific for IL-2 (IL-2 mAb) was also found to ameliorate EAE [120]. The IL-2 mAb supplements the proliferation of NK and CD8 T cells in mice by increasing the biological activity of the pre-existing IL-2 by formation of immune complexes [121]. Increased levels of IL-2 was also found to expand Tregs while preventing the induction of Th17 during EAE development [122]. However, NK cells have different effects during the early stages of EAE, and possibly MS, compared to the late stages. In the early stages NK cells were found to protect the CNS whereas NK cells were found to kill neural stem cells (NSCs) during the late stages of EAE, as a result of reduced expression of Qa-1 on NSCs [120, 123].

## **5.4 NKT cells**

NKT cells are unique T lymphocytes that express NK cell lineage markers, and act as a bridge between the innate and adaptive immune system. NKT cells account for a small percentage of lymphocytes, but have profound immunomodulatory roles in a variety of diseases [124]. There are two categories of NKT cells, type I and type II. Type I NKT cells, also known as invariant NKT cells (iNKT cells), express a semiinvariant Vα24-Jα18 (Vα14-Jα18 in mice), paired with a restricted range of β chains, that recognises α-galactosylceramide (α-GalCer) presented by CD1d [125, 126]. Type II NKT cells use TCRα and β chains that are reactive to a broad range of antigens, but do not recognise α-Galcer [127].

Nonobese diabetic (NOD) mice are susceptible to MOG-induced EAE. However, if NKT cells are increased either by transgenesis or adoptive transfer, the mice show protection from disease [128]. EAE protection has been correlated with inhibition of Ag-specific IFN-γ production in the spleen, modulating the encephalitogenic Th1 response [128]. There is conflicting evidence as to the effects of deletion of NKT cells on EAE. Some studies resulted in no effect on disease [129], with other studies

showing disease exacerbation in CD1d-deficient and Jα18-deficient mice [130, 131]. Activation of type I NKT cells by α-GalCer has been shown to improve EAE outcome. These improvements arise by indirectly enhancing Th2 response and reducing the Th1 response, or potentiating the differential of immunosuppressive myeloid cells [131–134]. However conflicting studies showed that high doses of α-GalCer could worsen EAE by directly enhancing Th17 and Th1 differentiation through phosphorylation of STAT3 and activation of NK-κB [135].

NKT cells from MS patients have been reported to have an increased production of cytokines. IL-4 production was increased by CD4 NKT cell clones in RRMS compared to other MS progression types, causing significant Th2 bias [136]. However, NKT cells in progressive MS patients displayed proinflammatory profiles [137]. It has also been suggested that the current available drugs for MS treatment may function through NKT cell targeting. A large reduction of type I NKT cells in peripheral blood was associated with remission of MS [136]. Type 1 interferon-β (T1IFN-β), a popular disease modifying therapy (DMT) for RRMS treatment, has been noted to promote expansion and functionality of type I NKT cells in vitro and to prevent disease in in vivo models of MS [138]. Research indicates a diverse role for NKT cells in MS pathology due to cytokine production.
