The role of CD8 suppressors versus destructors in autoimmune central nervous system inflammation

Human Immunology (2008) 69, 797-804

The role of CD8 suppressors versus destructors in autoimmune central nervous system inflammation Alla L. Zozulya, Heinz Wiendl* University of Würzburg, Department of Neurology, Würzburg, Germany Received 8 July 2008; received in revised form 22 July 2008; accepted 22 July 2008

KEYWORDS CD8 regulatory T cells; Effector T cells; Neuroinflammation; Autoimmunity

Summary Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) of putative autoimmune origin. Recent evidence indicates that MS autoimmunity is linked to defects in regulatory T-cell function, which normally regulates immune responses to self-antigens and prevents autoimmune diseases. MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have long been regarded as a CD4⫹ T-cell-mediated autoimmune disease. Studies addressing the role of CD8⫹ T cells, however, have only recently begun to emerge. Pathogenic function was attributed to CD8⫹ T cells because of their abundant presence or oligoclonal repertoire within MS lesions. However, CD8⫹ T cells appeared to have important regulatory functions, as demonstrated in EAE or human MS studies. We here review the contribution of CD8⫹ T cells to inflammation and immune regulation in CNS autoimmunity. The knowledge of distinct CD8⫹ T-cell populations exerting destructive versus beneficial functions is summarized. The long-term goal is to delineate the exact phenotypic and functional characteristics of regulatory CD8⫹ T-cell populations (natural as well as inducible) in humans. This knowledge may help to further develop concepts of reconstituting or enhancing endogenous mechanisms of immune tolerance in future therapeutic concepts for MS. © 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Introduction Multiple sclerosis (MS) is a central nervous system (CNS) disorder characterized by inflammation, demyelination, and axonal damage [1]. A complex interplay between genetic and environmental factors, as well as an impaired immune balance, contributes to disease susceptibility and course. Several studies document an elevated frequency of macrophages and autoreactive T lymphocytes in an activated state [2], as well as high * Corresponding author. Fax: 931-201-23488. E-mail address: [email protected] (H. Wiendl).

titers of serum and cerebrospinal fluid (CSF) antibodies against multiple viral and nonviral antigens [3]. Increased levels of general “immune reactivity” are accompanied by evidence for dysfunctions in several immunoregulatory immune subsets in MS patients (e.g., suppressor cells) [4,5]. MS has long been considered a prototypic CD4⫹ T helper-1 (Th-1)-mediated autoimmune disease [1,6]. Only recently has the importance of CD8⫹ T cells in the pathogenesis of CNS autoimmunity emerged. In some models of experimental autoimmune encephalomyelitis (EAE), CD8⫹ T cells convincingly served as beneficial regulators of pathology and clinical severity [7–10]. Other EAE models reported CD8⫹ T

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A.L. Zozulya and H. Wiendl

ABBREVIATIONS Ag APC CNS EAE GA IFN IL ILT mAb MBP MHC MOG MS ODC OVA PLP TCR TGF Th-1 TREG Ts

antigen antigen-presenting cell central nervous system experimental autoimmune encephalomyelitis glatiramer acetate interferon interleukin immunoglobulin-like transcripts monoclonal antibody myelin basic protein major histocompatibility complex myelin oligodendrocyte glycoprotein multiple sclerosis oligodendrocytes ovalbumin proteolipid protein T-cell receptor transforming growth factor T helper 1 regulatory T cells suppressor T cells

cells were destructive effector elements relevant for the onset, severity, and extent of CNS pathology [11–14]. An increasing number of human studies of MS patients report CD8⫹ T cells are key players involved in disease pathogenesis: CD8⫹ T cells outnumber CD4⫹ T cells in MS brain tissue [15,16] and the number of CD8⫹ T cells and macrophages correlates with the extent of axonal damage in MS lesions [17,18]. In contrast to CD4⫹ T cells, CD8⫹ T cells demonstrate oligoclonal expansions in the MS brain and CSF [19 – 23]. Expanded CD8⫹ T-cell clones exist in peripheral blood as, well as in the CNS, and persist over time [20]. Furthermore, distinct CD8⫹ T-cell clones in the CNS exist in different regions of MS brain specimens, further supporting their putative pathogenetic relevance [23,24]. The detrimental function of CD8⫹ T cells is further supported by their ability to kill oligodendrocytes as well as neuronal cells [18,25]. However, the antigen(s) or immunodominant epitopes recognized by these CD8⫹ T cells are yet to be determined. Over the past years, several myelin sheath components such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein (PLP) were identified as immunologically relevant antigens (Ag), possibly associated with pathogenic autoimmunity in MS patients. Autoreactive CD4⫹ and CD8⫹ T cells recognizing these Ag as targets are believed to be crucial in the initiation and maintenance of CNS autoinflammation, resulting in myelin destruction and neural damage. The existing peripheral regulation of immune responses dampens potential pathogenic autoimmunity. This natural regulatory mechanism of healthy individuals seems to be impaired in patients with MS. Although the role of autoreactive versus regulatory CD4⫹ T cells has been the subject of intense studies over the past 2 decades, autoreactive versus regulatory CD8⫹ T cells have received much less attention.

This review summarizes our current knowledge regarding the role of CD8⫹ T cells in CNS autoimmunity. Studies in animal models have elegantly demonstrated both effector and regulatory roles of CD8⫹ T cells. In the cascades of immunoregulatory and destructive inflammatory events during MS, evidence for both functions has been described. However, many open questions exist in relation to the phenotypic and functional characteristics of natural and inducible CD8⫹ regulatory T cells. Further studies are required to demonstrate the relevance of CD8⫹ regulatory T cells for the etiopathogenesis or treatment of MS.

Immunopathology through CD8ⴙ T cells CD8⫹ T cells emerging from the thymus are predestined to become cytotoxic lymphocytes. The relevance of CD8⫹ Tcell involvement during immune regulation is probably best understood for the control and clearance of viral infections, where these cells are mandatory. Because of their memory cell progeny, CD8⫹ T cells contribute to protection against subsequent encounters with the same foreign agent(s). The antiviral effects of CD8⫹ T cells can be mediated either by lysis of the infected target cell or by the secretion of potentially toxic cytokines such as interferon-(IFN)-␥ and tumor necrosis factor. Even after the infection has been cleared, dramatically expanded virus-specific primary CD8⫹ T cells remain in the blood for many years [26,27]. However, CD8⫹ T cells could induce immunopathology that has the potential to initiate autoimmune diseases. For example, self-antigens released from CD8⫹ T-cell lysis during the process of killing virus-infected cells can be presented by antigen-presenting cells to CD4⫹ or CD8⫹ T cells, rendering them autoreactive. Eventually, this might lead to damage at the site of inflammation or tissue containing the self-antigen (e.g., CNS) [28]. This concept implies also that without a direct infection of the target organ (CNS), viral antigens having similarities with self-CNS proteins can silently prime for autoimmunity by molecular mimicry, which can be triggered by totally different infections later in life [1]. If one considers the strong evidence of clonally expanded CD8⫹ T cells in MS CNS specimens [19 –23], together with the concept of how autoreactive T cells can be generated [29], it is tempting to consider CD8⫹ T cells as effector cells during CNS autoimmunity. The cells would most likely act as direct killers of target cells, contributing to immune-mediated demyelination and neural damage in MS.

CD8ⴙ T cells as putative effector cells in CNS inflammation Evidence from animal studies For many years, there was little interest in studying CD8⫹ T cells in EAE. This attitude of the community was aggravated by a clinical study demonstrating that CD4⫹ T-cell depletion in MS patients made only minor improvements in relapse rates or levels of magnetic resonance imaging activity [30], all fitting to the concept that CD8⫹ T cells play a minor role in the immunopathogenesis of MS. The relevance of myelinspecific CD8⫹ T cells and their function as effector cells during CNS autoimmunity have been challenged more

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CD8 suppressors versus destructors in autoimmune CNS inflammation recently. Classic, active EAE is a CD4 Th-1-mediated animal model. Furthermore, encephalitogenicity of adoptively transferred myelin-specific CD4⫹ T cells further manifested the relevance of CD4⫹ rather than CD8⫹ T cells in the pathogenicity of EAE. Therefore, many concepts and pathogenic hypotheses for MS have been deduced from this “CD4-biased” view (reviewed in [13]). Depletion of CD8⫹ T cells with monoclonal antibodies (mAbs) did not affect either the disease incidence or the severity of EAE [7]. Also, complete disruption of the CD8 gene led to more relapses but less mortality. Since then, several transgenic mouse models have been developed to study the pathogenetic role of CD8⫹ T cells in autoimmune destruction of the CNS. Some of the systems employed T-cell receptor (TCR)-transgenic T cells recognizing self- or neoself-antigens in a major histocompatibility complex (MHC) class I-restricted manner [12,31]. Indeed, two studies could demonstrate EAE development without any CD4⫹ T-cell help by adoptively transferring CD8⫹ T cells (Table 1) [12,14]. The relapsing/remitting EAE

Table 1

course was dominated by infiltrating CNS Ag-specific CD8⫹ T cells and demyelination, mainly in the brain rather than in the spinal cord. The studies also helped to later identify a MHC class I-(H-2Db)-restricted MOG37– 46 epitope and the use of MHC I-specific tetramers to identify MOG-specific CD8⫹ T cells in the course of the disease [11]. In some cases, self-tolerance mechanisms as a result of the synchronic expression of relevant antigens at the target organ and TCR specificity of transgenic mice usually precluded further analyses of EAE development. However, a pathologic role for endogenous CD8⫹ T cells in EAE could be demonstrated in mice overexpressing the costimulatory ligand CD86 on microglia cells [32]. Additionally, Cao et al. [33] demonstrated the encephalitogenic potential of CD8⫹ T cells in an “EAE model” using a mouse in which ovalbumin (OVA) was transgenically expressed and sequestered only on oligodendrocytes (ODC). In these OVA–ODC mice, the neoself-antigen remains invisible to CD4⫹ T cells, but is accessible to naive CD8⫹ T cells. Endogenously generated CD8⫹ T cells expressing the MHC class I-restricted, OVA-specific OT-I

Examples of studies demonstrating CD8⫹ T cells as putative effector cells in autoimmune CNS inflammation.

Experimental approach Mouse Immunization of C3H mice with MHC I-restricted MBP79–87 Adoptive transfer of MOG35–55/H-2Db reactive CD8⫹ T cells into C57Bl/6 and RAG-1-/- recipients

Key finding

Comments

Reference

Development of EAE mediated by MHC I-(H-2Kk)-restricted CD8⫹ T cells Severe and permanent EAE induction

First evidence of effector function of CD8⫹ T cells during EAE

[12]

Autoreactive CD8⫹ T cells are able to transfer the disease to wild-type and T-cell-deficient mice but not to ␤-2microglobulin gene-deleted (MHC class I-deficient) recipients Evidence of direct contribution of autoreactive CD8⫹ T cells to neuroinflammation under certain conditions CD8⫹ T cells can initiate autoimmunity in the CNS independently from CD4⫹ T cells

[14]

Mice overexpressing B7.2/CD86 on microglia

Spontaneous EAE triggered by clonally expanded CD8⫹ T cells in the CNS

Mice overexpressing neo-selfantigen OVA on oligodendrocytes, crossed with OTI Tg mice

Spontaneous EAE triggered by IFN-␥ expressing CD8⫹ T cells in the CNS

Human Analysis of brain postmortem from patients with progressive MS

Analysis of frozen sections of MS tissue at the level of single cell using single target amplification of TCR-␤ chain V-gene rearrangements Flow cytometric evaluation of autoreactive T-cell responses against various neuroantigenic targets in RRMS patients

Overrepresentation of CD8⫹ T cells compared with CD4⫹ T cells

First study identified clonally expanded CD8⫹ T cells in MS plaques

Identified a higher proportion of CNS-specific CD8⫹ responses in patients with RRMS compared with healthy donors; no differences existed for CD4⫹ T cells

First histological evidence of CD8⫹ T cells in MS lesion pathogenesis: number of CD8⫹ exceed CD4⫹ T cells in chronically inflamed MS plaques Reports of oligoclonal expansions in CD8⫹ T cells rather than CD4⫹ T cells in the CNS, CSF, and blood of MS patients First work demonstrating neuroantigen-specific CD8⫹ Tcell responses in patients with RRMS in peripheral blood

[32]

[33]

[16]

[19]

[34]

800 receptor resulting from a cross of ODC–OVA with OT-1 mice were highly encephalitogenic and developed spontaneous EAE (Table 1).

Evidence from human studies Significant numbers of CD8⫹ T cells exist in MS plaques. In most regions, CD8⫹ T cells outweigh CD4⫹ T cells in number (Table 1) [15,16]. To date, the function or Ag-specificity of CNSinfiltrating CD8⫹ T cells remains unknown (Table 1). Using single-cell TCR analysis, it could be demonstrated that infiltrating CD8⫹, but not CD4⫹ T cells are clonally expanded in the brain [19,23]. Furthermore, identical clones may exist in different brain regions of the same individual. Sequence analysis of the CDR3 region promotes the idea of an antigen-dependent affinity maturation and clonal selection. Interestingly, such clones persist in different compartments of the organism (CNS, cerebrospinal fluid (CSF), and peripheral blood) for many years, again supporting their putative pathogenic role [20]. Thus far, the antigens recognized by CNS-infiltrating CD8⫹ T-cell clones in MS remain elusive. Crawford et al. [34] reported a higher proportion of CD8⫹ T cells specific for different neuroantigens in peripheral mononuclear cells from patients with relapsing–remitting MS compared with healthy controls (Table 1). It is assumed that CNS-infiltrating CD8⫹ T cells in MS contribute to acute or chronic neural damage [18]. This assumption is supported by the principal ability of antigen-specific CD8⫹ T cells to kill MHC-class I-expressing neuronal cells [18,35]. An upregulation of MHC class I in MS lesions and the ability of virtually all cell types in the CNS to express MHC class I imply that various CNS cells might be direct targets of a cytotoxic CD8⫹ T-cell attack. Finally, an association of susceptibility to MS with certain MHC I molecules (e.g., HLA-A3) independent of MHC II gene linkage has been documented. Further, the combination of HLA-A3 and the HLA-DR2 alleles appeared to increase the risk of developing MS compared with individuals carrying only HLA-DR2-susceptible alleles [36,37].

CD8ⴙ T cells as putative regulators in CNS inflammation Lessons from animal models of MS As mentioned above, the majority of published data on the mechanism of neuroinflammation favored a CD4⫹ Th-1mediated type of disease. Clearly, experiments with ablation or blocking of CD4⫹ T cells, together with established models using MHC II-restricted myelin proteins such as MBP, PLP, or MOG as potent activators of encephalitogenic CD4⫹ T cells, supported the notion of these cells being main inducers of the disease. The dominantly accepted idea of CD4⫹ T cells controlling EAE pathogenesis was strengthened by two independent works demonstrating the regulatory or suppressive function of CD8⫹ T cells rather than the effector activity of these cells during EAE (Table 2) [7,8]. Later, CD8⫹ T cells derived from rodents fed MBP also suppressed immune responses to MBP in vitro and protected animals from induction of EAE via a transforming growth factor (TGF)-␤-mediated pathway [38]. Using a CD4/CD8 knockout approach, it was demonstrated that either deletion substantially reduced EAE sus-

A.L. Zozulya and H. Wiendl ceptibility. However, this protection was greater in CD8deficient mice. The depletion of CD4⫹ cells from CD8-/- mice protected them from MOG-induced EAE, whereas the frequency of EAE sick CD4-/- animals with depleted CD8⫹ cells was higher (Table 2) [39]. A later study in the MOG35–55induced EAE model confirmed that depletion of CD8⫹ T cells with monoclonal antibodies could enhance EAE when given before immunization [40]. Notably, the absence of the MHC class I-locus Qa-1 (equivalent to HLA-E in humans) also resulted in increased susceptibility of EAE. This suggested a regulatory role of Qa-1-recognizing CD8⫹ T suppressor cells [41,42]. Similarly, the lack of functional CD8⫹ T cells and MHC class I molecules in ␤-2 microglobulin knockout mice aggravated autoimmune tissue destruction in the CNS after induction of active EAE (Table 2) [9].

Role of regulatory CD8ⴙ T cells in MS Natural and inducible CD8ⴙ regulatory T cells Originally, immunoregulatory T cells were described as “suppressor” T cells and the majority of these suppressors were characterized by the coexpression of CD8 [43]. After pioneer studies by Sakaguchi and colleagues [44], CD4⫹CD25⫹ FoxP3-expressing T cells were discovered, now called regulatory T cells (TREG). Studies of TREG in CD4⫹ T cells have advanced tremendously, somehow outnumbering publications regarding CD8⫹ T regulatory cells. However, the CD8 regulatory field is also emerging. Still, many efforts to understand the cellular mechanisms of CD8⫹ T-cell-mediated suppression are hampered by difficulties in isolating and characterizing these cells, both in mice and in humans. Distinctive or indicative cellular markers are virtually lacking for CD8 suppressor cells so far. A summary of characteristics of CD8⫹ natural versus inducible TREG for both mice and humans is presented in Table 3. Among various CD8⫹ T cells associated with suppressive function, CD8⫹CD122⫹ T cells were identified as naturally occurring CD8 TREG [45]. These cells directly controlled IFN-␥ production and proliferation of CD8⫹ T cells without the intervention of antigen presentation. The mechanism is thought to act via an interleukin (IL)-10-dependent mechanism [46]. The latest study revealed a molecular mechanism of CD8⫹CD122⫹ TREG in the recognition of target cells. Thus, CD8⫹CD122⫹ TREG cells recognize previously activated T cells by interacting with cell surface molecules, including conventional MHC class I ␣␤TCR [47]. Another novel population of naturally occurring CD8⫹ TREG cells has been described recently in humans [48]. These cells are characterized by the constitutive expression of immune-tolerogenic HLA-G and suppress autologous CD4 or CD8 T cells via a non-cell-contact-dependent mechanism. HLA-G-expressing cells were also found in the thymus, suggesting that they belong to the physiologic immune repertoire [48]. Of note, HLA-G-expressing, naturally occurring TREG exist both in the CD4 and in the CD8 cell populations. Many CD8⫹ TREG populations or subsets with different markers have been identified and categorized as “inducedtype” CD8 TREG. The costimulatory molecule CD137 (4-1BB) was engaged in the generation of CD8⫹ TREG, which, in turn, could elevate TGF-␤ secretion suppressing antigen-specific

801

CD8 suppressors versus destructors in autoimmune CNS inflammation

Table 2 Examples of studies demonstrating the regulatory role of CD8⫹ T cells during CNS neuroinflammation using variants of the experimental autoimmune encephalomyelitis (EAE) model. Experimental approach MBP peptide-induced EAE in B10.PL mice followed by CD8⫹ T-cell depletion with mAbs against CD8 MBP protein-induced EAE in CD8-/- mice crossed to susceptible to EAE PL/JH-2u background

MOG-induced EAE in CD4 gene-deleted (CD4-/-) and CD8-/- DBA/1 mice

CD8⫹ T-cell depletion with mAb prior to active MOG35-55 immunization in C57Bl/6 mice PLP peptide-induced EAE in Qa-1-/- mice

MOG peptide-induced EAE in ␤2microglobulin-/- mice crossed to C57Bl/6 background

MOG peptide-induced EAE in CD8⫹CD28recipients

Key finding ⫹

Comments

CD8 T-cell depletion rendered mice normally resistant to EAE to become susceptible to EAE upon reimmunization Deletion of the complete CD8 gene converted EAE course from monophasic to chronic replasing and remitting, although with overall lower maximum severity Both deletions substantially reduced EAE susceptibility compared to wild-type animals. Only 20% of CD4-/mice depleted of CD8⫹ T cells developed EAE, although the majority of CD8-/- mice depleted of CD4⫹ were protected Enhanced EAE induction in recipients

Reference

First evidence of suppressive activity of CD8⫹ T cells during EAE

[7]

Confirms the regulatory (with few effector) function of CD8⫹ T cells in this EAE model

[8]

Follow-up study suggesting that CNS damage is partly mediated by CD8⫹ T cells; however, this work requires a cautious interpretation as cross-recognition of MHC II epitopes in addition to MHC I molecules as well as CD4 expression on activated CD 8⫹ T cells are now evident Verifies CD4⫹ T-cell importance in EAE initiation and implies the necessity of T cells expressing CD8 to downregulate the disease Increased susceptibility of QaExerts Qa-1-dependent and 1-/- mice to EAE -restricted CD8⫹ T cell inhibitory activity, which prevents pathogenic expansion of CD4⫹ T cells Significantly more severe EAE Evidence of regulatory role of CD8⫹ course in ␤2-microglobulin-/T cells in this EAE model; because of several factors such as the mice compared with wild type requirement of MHC I molecules for axonal regeneration and integrity, a lack of Qa-1 and CD1 expression, and “iron overload syndrome” in these mice, the interpretation of this study is complicated Although CD28-deficient mice Evidence of regulatory role of CD8⫹ are resistant to EAE, CD8⫹ T cells in the resistance of CD28deficient mice to disease T-cell depletion rendered them susceptible

[39]

[40]

[41]

[9]

[10]

MBP ⫽ myelin basic protein; mAbs ⫽ monoclonal antibodies; EAE ⫽ experimental autoimmune encephalomyelitis; MOG ⫽ myelin oligodendrocyte glycoprotein; PLP ⫽ proteolipid protein; MHC ⫽ major hictocompatibility complex.

proliferative responses of CD4⫹ T cells [49]. Najafian et al. [10] reported that a distinct population of antigen-specific CD8⫹CD28- cells have the ability to suppress immune responses by directly interacting with antigen-presenting cells and rendering them tolerogenic. A lack of CD28 resulted in significant exacerbation of EAE, a consequence that might not solely be explained by the function of CD8⫹CD28- TREG. Suciu-Foca and colleagues [50 –52] elucidated a highly relevant mechanism of how CD8⫹ TREG cells can be induced in human: the upregulation of immunoglobulin-like transcripts 3 (ILT-3) and ILT-4 on monocytes and dendritic cells trig-

gered by CD8⫹CD28- T cells seemed to be responsible for the propagation of antigen-specific CD4⫹ TREG-mediated suppression. Interestingly, these antigen-specific MHC I-restricted CD8⫹CD28- TREG obviously first induce tolerogenic dendritic cells to upregulate ILTs, which, in turn, generate CD4⫹TREG [53]. Gilliet and Liu [54] were the first to demonstrate that CD40L-activated plasmacytoid dendritic cells can induce IL-10-producing CD8⫹ TREG. Finally, CD103 (␣E␤7 integrin) was recently identified as a marker for alloreactive CD8⫹ TREG [55]. Alloantigen-induced and in vitro expanded CD8⫹CD103⫹ T cells had low proliferative and cytotoxic ca-

802 Table 3

A.L. Zozulya and H. Wiendl Known characteristics of natural (n) versus inducible (i) regulatory CD8⫹ T cells.

Origin MHC restriction Cellular proteins Cytokine secretion Mechanism of suppression In vivo generation

CD8 nTREG

CD8 iTREG

Thymus ?, classic and nonclassic MHC molecules CD122⫹⫹ [45]; HLA-G⫹⫹ [48]; CD25⫹ [64] FoxP3⫹ [65] (IL-10?) Non-cell-contact- and IL-10-dependent [46], Ag-nonspecific ?

Periphery Qa-1 (HLA-E), classic and nonclassic MHC molecules CD28- [10], CD103⫹ [55] IL-10 [66], TGF-␤ [67,68] Cell-to-cell contact-dependent, Ag-specific

In vitro expansion Not established

Through plasmacytoid dendritic cells [54]; TGF-␤ [69]; immature dendritic cells [50] TCR-signaling-dependent

nTREG ⫽ natural regulatory T cells; iTREG ⫽ inducible regulatory T cells; TCR ⫽ T-cell receptor; Ag ⫽ antigen.

pacity, but produced considerable amounts of IL-10 and suppressed T-cell proliferation in the mixed lymphocyte culture via a cell– cell dependent mechanism (Table 3) [55].

CD8ⴙ regulatory T cells in MS Numerous studies investigated the hypothesis that MS is linked to impaired function of certain subsets of TREG cells (reviewed in [4]). Within this concept, the role of CD8⫹ TREG (natural or inducible) has undergone a critical reappraisal. Recent studies demonstrated that CD8⫹ TREG can be considered relevant players in the immunopathogenesis of MS [56,57]. The possibility that the suppressor function of CD8⫹ T cells might be impaired in MS patients was originally demonstrated by Antel and colleagues [58,59]. Later, was reported that the majority of regulatory T cells isolated from CD4⫹ T-cell-vaccinated patients appeared to be CD8⫹ T cells. These cells inhibit the proliferation of vaccine T-cell clones upon Ag stimulation and specifically lyse vaccine CD4⫹ T cell clones in vitro [60,61]. Correale and Villa [56] studied the role of CD8⫹ TREG during MS exacerbations and remissions. They found CD8⫹ TREG specific clones recognizing and lysing activated myelinreactive CD4⫹ T cells, which were decreased in MS patients during exacerbations, especially in the CSF compartment. They could also characterize CD94/NKG2 receptors expressed on natural killer and CD8⫹ T cells. Their upregulation seemed to be responsible for the impairment of cytolytic activity during exacerbations. Blocking CD94 and NKG2 with specific antibodies could restore the cytolytic activity of CD8⫹ TREG from MS patients, suggesting an importance of HLA-E-restricted CD8⫹ TREG in the control of autoimmune disease [56]. The reconstitution of TREG function currently represents a viable therapeutic strategy to restore immune tolerance in MS. Immunotherapy with glatiramer acetate (GA), a polypeptide mixture with strong immunomodulatory potential, induced CD8⫹ T-cell responses in patients with MS [62]. Later, Tenakoon et al. [57] demonstrated that this immunomodulatory therapy exerts its beneficial action at least partly via CD8⫹ TREG cells. Using immunizations of MS patients with GA, an antigen-specific suppression of GA-loaded CD4⫹ T cells by GAinduced CD8⫹ TREG was reported, thereby demonstrating an upregulated suppressive ability of CD8⫹ T cells [57]. As mentioned above, a distinct subset of human thymusderived TREG has been identified based on expression of the

HLA-G molecule [48]. These cells were suppressive in vitro. This suppression depended on HLA-G, ILT-2, and IL-10 and could occur in a contact-independent manner ([48] and Y-H Huang, personal communication). The analysis of human clinical samples of other inflammatory diseases suggested a specific accumulation of HLA-G-expressing cells at the site of on-going inflammation. It was hypothesized that HLA-G belongs to endogenous tolerogenic mechanisms employed in the CNS to counterbalance inflammation. Interestingly, unlike naturally occurring FoxP3-expressing CD4⫹ nTREG, HLA-G TREG cells from MS patients were functionally unimpaired (Y.-H. Huang, C. Weidenfeller, A.L. Zozulya, I. Metz, D. Buck, K.V. Toyka, W. Brück and H. Wiendl, unpublished observation). A drop in numbers of CD4⫹ and CD8⫹ HLA-Gexpressing cells correlated with an increased risk of postpartum relapses in patients with MS [63]. The exact role of CD8 versus CD4 HLA-G-expressing TREG under physiologic and pathophysiologic conditions remains to be further elucidated.

Conclusions Accumulating evidence points to an increasing appreciation for the role of CD8⫹ T cells during CNS autoimmunity. A positive association of MS with certain MHC class I alleles, clonal expansion of CD8⫹ T cells in the CNS, and the presence of autoreactive myelin-specific CD8⫹ T cells in MS patients all argue for an important pathogenetic role of these cells in CNS autoinflammation. However, numerous studies also indicate an important protective function of CD8⫹ T cells. The latter might be impaired, thereby contributing to inflammation and neurodegeneration in MS. Further studies are clearly needed to address the role and function of regulatory CD8⫹ T cells in MS. Conceptually, regulatory CD8⫹ T cells might help to balance the disturbed systemic immune response against CNS antigens, counterbalance the destructive immune activity in the target organ, or help to repair CNS damage. Consequently, future therapeutic strategies might be directed to improving the expression of suppressor molecules on CD8⫹ TREG or promoting delivery to organs under immune attack. Eventually, this could help reconstitute the ability of CD8⫹ T cells to control autoimmunity. Therefore, the challenge ahead is to delineate specific markers that distinguish effector from regulatory CD8⫹ T cells in animals and humans. To date, only a few studies have identified

CD8 suppressors versus destructors in autoimmune CNS inflammation such markers or molecules associated with regulatory CD8 function (e.g., HLA-G, CD122, CD103). Further work is needed to evaluate these as valid biomarkers of CD8 suppressors.

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