Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) characterized by an attack of the myelin sheath that surrounds and protects CNS axons by autoreactive T cells. Its murine model, experimental autoimmune encephalomyelitis (EAE), is triggered by active immunization with myelin antigens or transfer of activated autoreactive myelin-specific T cells to naïve recipients. Until recently, defects in regulatory mechanisms had only been described in MS in cells of the adaptive compartment (28).

Conflicting data on a beneficial vs. detrimental role of NK cells in EAE have been published (29–31), but studies on regulatory NK cells in mice are difficult to translate into humans, because murine NK cells do not express CD56 and the murine counterparts of CD56^bright and CD56^dim subsets have not been identified with certainty.

Enhancing regulatory features of NK cells ameliorates the disease course of EAE. In particular, NK cells expressing NKG2A (which is expressed by all CD56^bright NK cells in humans) were shown to decrease CNS inflammation by killing T cells and microglial cells, when the interaction between NKG2A and its ligand Qa-1 (the murine equivalent to the human HLA-E) expressed on the target cells was blocked by antibodies specific for either antigen (32, 33). Decreased expression of Qa-1 on microglial cells upon CNS inflammation rendered them more sensitive to NKG2A⁺ NK cell-mediated lysis. Importantly, enrichment of NK cells through treatment with IL-2 coupled with IL-2 mAb (“IL-2 complexes”) (34) ameliorated EAE (35). A recent work from the same group has shed light on the differential effects of NK cells in early vs. late stages of EAE and possibly MS (36), which may depend on their interactions with neural stem cells (NSCs). Indeed, Liu et al. found that, in MS and EAE brains, NK cells are in contact with NSCs and that, in EAE NSCs released IL-15 upon contact with NK cells, thereby supporting NK cell proliferation and survival; in turn, NK cells killed NSCs, particularly during the late stages of EAE, as a result of reduced expression of Qa-1 on NSCs. Accordingly, removal of NK cells during the late phase of EAE reduced disease severity (36). These observations suggest that cytotoxicity of NK cells may be a double-edged sword in EAE, with NK cells attenuating inflammation in the acute phase of disease by killing immune cells (T cells, microglial cells), but impairing potential repair during the late stage by killing NSCs.

While Liu et al. did not ascertain whether NK cells in contact with NSCs belonged to the CD56^bright or CD56^dim NK subset (36), others analyzed the phenotype of NK cells in the cerebrospinal fluid (CSF). The majority of intrathecal NK cells in healthy individuals, MS patients and patients with other neurological diseases are CD56^bright NK cells (21, 37, 38), suggesting that CSF enrichment in CD56^bright NK cells is not MS specific, but rather CNS specific (37). This may reflect organ-specific, rather than blood-specific, tropism of CD56^bright NK cells (39). The recently discovered lymphatic vessels in the brain (40, 41) may be the route of entry for CD56^bright NK cells, which were shown to circulate in the lymph (42). Of note, a higher migratory capacity of CD56^bright compared to CD56^dim NK cells was observed in a model of the human blood–brain barrier (BBB) (21).

Given the evidence that CD56^bright NK cells are a regulatory population of the IS in healthy individuals, we have explored their function in untreated MS patients or patients with clinically isolated syndrome suggestive of MS (25). The number of CD56^bright NK cells was similar in MS patients and healthy subjects (HS). However, upon stimulus with pro-inflammatory cytokines, CD56^bright NK cells from MS patients suppressed much less efficiently the proliferation of autologous CD4⁺ T cells compared to those from HS. This was associated with an increased expression of HLA-E on CD4⁺ T cells in MS and was reverted by blocking HLA class I on T cells, suggesting that the cytotoxic function of CD56^bright NK cells on their targets is inhibited through binding of HLA-E on T cells to the NK cell inhibitory ligand NKG2A. HLA-E is a non-classic major HLA class I molecule expressed by immune cells and, outside the immune compartment, by endothelial cells, which release its soluble form upon inflammation (43). HLA-E upregulation was found in MS CNS within white matter lesions, in endothelial cells and astrocytes (44, 45). Similarly, Morandi et al. detected increased levels of soluble HLA-E in the CSF and expression of HLA-E within immune cells and neural cells in MS plaques, which correlated with decreased NK cell cytotoxicity (46). The causes of such upregulation are, as yet, unknown. Thus, an impairment of CD56^bright NK cell immunoregulatory function in MS may occur not only in the periphery but also within the CNS. Recently, we also described decreased cytolytic activity of NK cells in MS as a consequence of reduced upregulation of CD155 on T cells after activation, concomitantly with a reduced NK cell surface expression of DNAM-1 (21). These studies point toward a resistance of T cells to NK cell suppressive functions rather than an intrinsic defect in the NK cells in MS.

The relevance of the immunoregulatory function of NK cells in MS is emphasized by studies from the group of Takashi Yamamura, who described a particular peripheral NK phenotype to be characteristic of MS patients in remission, with increased production of the anti-inflammatory cytokine, IL-5 (“NK2” cells), and high expression of CD95 (47), which inhibited the production of IFN-γ by Th1 clones (48). Among these patients, a further division of NK2 cells as CD11c-high (not producing IL-5) and CD11c-low (producing IL-5) subsets identified CD11c-high patients at risk of relapse (49), suggesting that CD11c + NK cells are pro-inflammatory. In another study, a subpopulation of NK cells characterized by low expression of CD8, a phenotype associated with CD56^bright NK cells, was observed to be reduced in untreated patients with relapsing-remitting MS (50).

Previous infections may influence the development of MS (51). Interestingly, infections not only activate NK cells but also shape NK cell functions (52–54). Thus, cytomegalovirus (CMV) induces expansion of NK cells that produce IL-10 in mice, to prevent excess of activation of CD8 T cells (55, 56). In humans, CMV infection is associated with expansion of terminally differentiated NK cells bearing the NKG2C receptor and has been implicated both in MS etiology and/or “protection” (57). In this context, Martinez-Rodriguez et al. explored the expression of NKG2C on NK cells from MS patients and controls, in relation to their CMV⁺ serostatus and to the NKG2C genotype, finding that the expansion of NKG2C⁺ NK cells in CMV⁺ patients was associated to lower risk of disease progression, suggesting that CMV may exert a beneficial influence on MS, either through expansion of NKG2C⁺ NK cells or through other mechanisms (58). Differently to CMV, infection with Epstein–Barr virus, which has also been associated with an increased risk of MS, expands early differentiated NKG2A + CD56^dimNK cells (59, 60), but whether such cells have any role in the pathogenesis of MS is unknown.

In addition to first-line MS therapies, interferon beta (IFN-β) and glatiramer acetate, novel immune-modulating therapies such as the anti-inflammatory dimethyl fumarate, the T cell proliferation inhibitor teriflunomide, the migration inhibitors natalizumab and fingolimod (FTY720), the IL-2 receptor-modulating daclizumab, and the immune cell-depleting alemtuzumab are now available for treatment of MS (61, 62). Many of these immune-modulating biologicals alter the NK cell compartment (Figures 2A,B) by increasing NK cell frequencies (Figure 2A) and/or NK-mediated immune regulatory functions (Figure 2B) (61, 63), which points to an immune-protective role of NK cells in MS. Furthermore, antibody-dependent cell-mediated cytotoxicity (ADCC) by CD56^dim NK cells has been proposed as an essential therapeutic mechanism in alemtuzumab-mediated T and B cell depletion as well as rituximab-mediated B cell depletion (Figure 2C) (64, 65).

One therapeutic approach in MS is the reduction of inflammatory lesions by inhibiting infiltration of autoreactive lymphocytes into the CNS. While the humanized monoclonal antibody (hMA) anti-CD49d (alpha 4 integrin) natalizumab prevents transmigration of circulating lymphocytes across the BBB (67), the sphingosine 1-phosphate receptor (S1P) modulator FTY720 reduces CNS inflammation in MS (68) indirectly by preventing lymphocyte egress from LNs (69). A natalizumab-induced increase of total NK cells and CD56^bright NK cells in blood concomitantly with reduced NK cell numbers in CSF (Figure 2A) (21, 70, 71) suggests CD49d-dependent transmigration of NK cells into the CNS. Trafficking of NK cells in steady state and inflammatory conditions requires S1P (72) and decreased numbers of circulating CD56^bright NK cells have been observed 6 h after FTY720 treatment (73) (Figure 2A). Long-term treatment also resulted in reduced numbers of both NK cell subsets in the CSF (own observations). Despite reduced NK cell numbers, a relative increase in peripheral and intrathecal NK cell subsets within the lymphocyte compartment (74) indicates that FTY720 inhibits NK cell emigration less than that of other lymphocytes (Figure 2A). This might be due to the fact that egress of NK cells from LNs is regulated through both S1P₁ and S1P₅ (72, 75), whereas other lymphocytes use only S1P₁ (76). Since S1P₁ and S1P₅ seem to trigger the activation of distinct intracellular signal transduction pathways, it has been suggested that S1P₅ might be less susceptible to FTY720 than S1P₁ (72). Along this line, a higher expression of S1P₅ on CD56^dim NK cells than on CD56^bright ones (72) might explain the relative increase of the CD56^dim NK cell subset (77). While treatment of MS patients with FTY720 alters NK cell trafficking, it has no impact on cytokine secretion or cytolytic function of NK cells (77).

In contrast, treatment with daclizumab affects both peripheral (20, 78) and intrathecal CD56^bright NK cell numbers (79), as well as their immunoregulatory function (19, 21) (Figures 1 and 2B). Daclizumab is a recently approved hMA directed against IL-2Rα, which showed enhanced efficacy in MS compared to IFN-β [DECIDE trial (80)]. Daclizumab enhances endogenous mechanisms of immune tolerance by reducing early T cell activation (81), expanding CD56^bright NK cells (20, 78), while reducing lymphoid tissue-inducer cells (82), and restoring defective NK cell-mediated control of T cell activity in MS (19, 21). The mechanism of the effect of daclizumab on CD56^bright NK cells has been discussed in chapter 1.3 (Figure 2C). Daclizumab both boosts NK cell cytolytic function in a DC-dependent manner and renders antigen-activated T cells more sensitive toward NK-mediated lysis, thus restoring defective NK cell-mediated control of T cell activity in MS (19, 21) (Figure 2B).

Alemtuzumab is a hMA directed against the cell surface molecule CD52, which demonstrated a high clinical efficacy in MS (83, 84). CD52 is highly expressed on T and B cells and to a lower degree on NK cell subsets (85, 86). Accordingly, a relative increase of circulating NK cells with increased numbers of CD56^bright NK cells was observed 6 months after alemtuzumab therapy, whereas the CD56^dim subset remained unaltered. However, there was no change in NK cell cytolytic function (87). ADCC is mediated via the FcγRIII (CD16)-expressing CD56^dim NK cell subset (88). Since intrathecal CD56^dim NK cells are sparse (21, 89), the therapeutic efficacy of alemtuzumab within the CNS might be limited (Figure 2C). Along this line, insufficient disease inhibition in progressive MS by intrathecal application of rituximab was proposed to be due to low numbers of CD56^dim NK cells and reduced levels of complement proteins within the CNS (66). Further studies are required to shed more light on NK cell-mediated ADCC as a mechanism of action of human monoclonal antibody-mediated depleting therapies in MS.

NK cells are important players in controlling T cell activation in CNS autoimmunity, and impaired immune regulatory function of NK cells might be one of the driving forces in the pathogenesis of MS. Thus, a better understanding of the underlying mechanisms of NK cell-mediated regulation of T cell activation might help to improve treatment strategies in MS.

CG wrote chapter 3, conceptualized Figure 2, revised the manuscript, and approved the final version. AS-M drew Figure 2, revised the manuscript, and approved the final version. HW wrote chapter 3, revised the manuscript, and approved the final version. EM critically reviewed the manuscript for important intellectual content and approved the final version. NKdeR conceptualized Figure 1, critically reviewed the manuscript for important intellectual content, and approved the final version. AU critically reviewed the manuscript for important intellectual content and approved the final version. AL organized the sections of the manuscript, wrote chapters 1 and 2, conceptualized and drew Figure 1, revised the manuscript, and approved the final version.

CG received speaker honoraria and travel expenses for attending meetings from Genzyme, Novartis Pharma GmbH, and Bayer Health Care. AS-M has no financial disclosures. HW received compensation for serving on Scientific Advisory Boards/Steering Committees for Bayer Healthcare, Biogen, Merck Serono, Novartis, and Sanofi-Genzyme. He also received speaker honoraria and travel support from Bayer vital GmbH, Bayer Schering AG, Biogen, CSL Behring, Fresenius Medical Care, Glaxo Smith Kline, GW Pharmaceuticals, Lundbeck, Merck Serono, Omniamed, Novartis, and Sanofi-Genzyme. He received compensation as a consultant from Biogen, Merck Serono, Novartis, and Sanofi-Aventis. He received research support from Bayer Vital, Biogen, Genzyme, Merck Serono, Novartis, Sanofi-Genzyme Germany, and Sanofi US. EM has no financial disclosures. NKdeR has no financial disclosures. AU received grants and contracts from Fondazione Italiana Sclerosi Multipla (FISM), Novartis, Fondazione Cariplo, and Italian Ministry of Health; received honoraria or consultation fees from Biogen, Roche, Teva, Merck Serono, Genzyme, and Novartis. He is a member of a company advisory board, board of directors, or other similar groups for Roche and Genzyme. AL received honoraria for speaking by Biogen, Novartis, and Teva, consulting fees by Merck Serono, Sanofi-Genzyme, and Novartis, and funding for travel from Teva, Merck Serono, Biogen, and Novartis.