Contribution of CD8 T lymphocytes to the immuno-pathogenesis of multiple sclerosis
and its animal models
Lennart T. Marsa,b, Philippe Saikalic,d, Roland S. Liblaua,b,⁎, Nathalie Arbourc,⁎
aINSERM, U563, Centre de Physiopathologie de Toulouse Purpan, Hôpital Purpan, Toulouse, F-31300, France
bUniversité Toulouse III, Paul-Sabatier, Toulouse, F-31400, France
cUniversité de Montréal, Department of Medicine, CRCHUM, 1560 Sherbrooke E Y-3609, Montreal, QC, Canada H2L 4M1
dMontreal Neurological Institute, McGill University, 3801 University Street, Montreal, QC, Canada H3A 2B4
a b s t r a c t a r t i c l ei n f o
Received 14 August 2009
Received in revised form 21 June 2010
Accepted 6 July 2010
Available online 15 July 2010
Cytotoxic T cell
Central nervous system
demyelination, axonal loss, and immune cell infiltration. Numerous immune mediators are detected within MS
lesions, including CD4+and CD8+T lymphocytes suggesting that they participate in the related pathogenesis.
recent literature pertaining to the potential roles of CD8+T lymphocytes both in MS and its animal models. The
CD8+T lymphocytes detected in MS lesions demonstrate characteristics of activated and clonally expanded cells
vivo models mediated by CD8+T lymphocytesrecapitulate important featuresof the human disease. Whether the
CD8+T cells can induce or aggravate tissue destruction in the CNS needs to be fully explored. Strengthening our
understanding of the pathogenic potential of CD8+T cells in MS should provide promising new avenues for the
treatment of this disabling inflammatory disease.
© 2010 Elsevier B.V. All rights reserved.
Multiple sclerosis (MS) is an immune-mediated disease of the
central nervous system (CNS) characterized by multi-focal demye-
lination, axonal loss, and activation of glial cells. Whereas components
of the immune system are detected within MS lesions, the
contribution of each of these immune mediators to injury remains
to be defined . A vast body of evidence gathered from the
experimental autoimmune encephalomyelitis (EAE) mouse models
points to the crucial role of CD4+T cells in the disease pathogenesis.
This considerable literature has led the scientific community to
transpose these observations to the human disease MS . EAE is an
autoimmune demyelinating disease induced by the active immuni-
zation of animals with myelin protein extracts or immunodominant
myelin peptides emulsified in complete Freund's adjuvant (CFA) .
The identification of CD4+T cells as main culprits in EAE pathogenesis
comes from the fact that immunization with major histocompatibility
complex (MHC) class II restricted peptides induces EAE in genetically
susceptible hosts . Alternatively, the adoptive transfer of activated
myelin-specific CD4+T cells is sufficient to induce the disease in
autologous hosts. Moreover, the implication of CD4+T cells in the
pathogenesis of MS is supported by the strongest genetic risk factor so
far described being conferred by specific MHC class II alleles .
However, MS patients treated with an anti-CD4 depleting antibody
did not gain any clinical benefits although the depletion was efficient
[5–8]. Thus, at least in the human disease, the picture is far more
complex and CD4+T lymphocytes are not the only perpetrators
involved in the pathogenesis. A growing body of evidence suggests
that CD8+T lymphocytes partake in MS related CNS damage raising
interest in the scientific community [9,10]. This review provides an
overview of the recent literature documenting the potential roles of
CD8+T lymphocytes both in MS and its animal models.
1. CD8+T lymphocytes as effector cells
1.1. CD8+T lymphocytes: crucial immune cells
CD8+T lymphocytes recognize, via their T cell receptor (TCR),
molecules [11,12]. The activation of a naïve CD8+T lymphocyte requires
at least two signals provided by a professional antigen presenting cell
(APC): the first one being the TCR engagement by a recognized peptide–
MHC class I complex and the second one provided by the interaction
between co-stimulatory molecules and co-activating receptors. Such
Biochimica et Biophysica Acta 1812 (2011) 151–161
⁎ Corresponding authors. N. Arbour is to be contacted at Department of Medicine,
Université de Montréal, CRCHUM-Notre-Dame Hospital, Pavilion JA DeSeve (Y-3609),
1560 Sherbrooke E, Montreal, QC, Canada H2L 4M1. Tel.: +1 514 890 8000x25112; fax:
+1 514 412 7602. R.S. Liblau, INSERM, U563, Centre de Physiopathologie de Toulouse
Purpan, Hôpital Purpan, Toulouse, F-31300, France. Tel.: +33 562 74 45 15; fax: +33
562 74 75 58.
E-mail addresses: firstname.lastname@example.org (R.S. Liblau),
email@example.com (N. Arbour).
0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
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efficient stimulation triggers a complex cascade of intracellular signaling
leading to the maturation (change in surface molecules), proliferation,
and production of mediators by cytotoxic T lymphocytes (CTL). Once
adequately activated, CD8+T cells survey the body and kill encountered
target cells expressing the appropriate peptide–MHC class I complex.
Since most nucleated cells express or can express MHC class I molecules,
they represent potential targets recognized by CD8+T lymphocytes.
Several mechanisms are deployed by CD8 T cells including the release of
lytic enzymes such as perforin and granzymes and the Fas–FasL
interaction [13,14]. Also, activated CD8+T cells secrete several pro-
inflammatory cytokines including interferon-γ (IFN-γ), tumor necrosis
factor (TNF), and interleukin-2 (IL-2). Through these combined effector
functions CD8+T cells play a crucial role in the control of intracellular
pathogens and neoplasic cells [15,16]. A fraction of the activated CD8+T
lymphocytes persists as memory cells, which provide protection with an
enhanced response upon secondary challenge. Memory CD8+T cells are
also subdivided into central and effector memory subsets, each group
bearing specific markers . Finally, subsets of CD8+T cells carrying
suppressor or regulatory properties have been described.
Numerous publications have documented that activated CD8+T
cells can utilize distinct mechanisms to recognize and attack CNS cells
[18,19]. Murine neurons are susceptible to CD8+T cell Fas/FasL
mediated cytotoxicity but are protected from a perforin-induced cell
death by the same effector cells  unless FasL is not expressed by
these neurons . In contrast, murine astrocytes quickly respond to
degranulation (release of lytic enzymes) by cytotoxic CD8+T cells
. Cell death of human neurons has been observed upon the
addition of granzyme B , another lytic enzyme than perforin.
Moreover, cytotoxic CD8+T cells can selectively attack neuronal
neurites supporting the notion that axonal damage observed in MS
lesions could be mediated by these cells . We have recently shown
that primary cultures of human adult oligodendrocytes express
ligands for NKG2D while neurons, microglia and adult astrocytes do
not. By disrupting the NKG2D–NKG2D ligand interaction, we could
specifically impede the CD8 T cell-mediated killing of oligodendro-
cytes, but not that of other CNS cells not expressing these ligands .
These observations illustrate the specific exchanges occurring
between cytotoxic CD8 T cells and distinct CNS cells and the
subsequent diversified cell death pathways induced.
1.2. Characteristics of peripheral effector CD8 T lymphocytes in MS
Although it is possible that in the highly inflamed CNS, APC on site
could activate/reactivate T cells, the first aggressive T cells infiltrating
this organ most likely would have been efficiently activated in the
periphery. Therefore, assessment of immune responses in MS patients
in the peripheral compartment is highly relevant and could identify
specific immune properties attributed to this autoimmune disease.
Moreover, the feasibility of obtaining repetitive peripheral blood
samples from patients makes this approach an attractive tool to assess
and monitor disease development. Several groups reported that the
peripheral T cell repertoire in MS patients varies according to disease
activity [25–27] supporting the notion that the brain inflammation is
associated with changes in the peripheral lymphocytes. Very early
during MS development, skewing of the TCR repertoire, more
importantly in the blood derived CD8+T cell compartment is
observed in these patients compared to controls. These observations
suggest that aberrant responses in the CD8+T cell subset alreadyexist
at the beginning of the disease . Moreover, in the peripheral blood
of MS patients increased levels of CD8+CCR7+CD45RA−(central
memory T cells) are detected compared to healthy controls ,
advocating that these patients carry an enhanced proportion of
previously activated CD8+T cells compared to controls.
Killestein and colleagues examined the cytokine profile of CD4+
and CD8+T cells upon a short in vitro stimulation in relationship to
magnetic resonance imaging (MRI) features of tissue destruction and
disability score in MS patients . The cytokine profile of CD8+T
cells rather than of their CD4+counterparts was more predictive of
specific chemokine receptors: CCR5 and CXCR3 on CD8+T lympho-
cytes was enhanced in MS patients compared to controls  and this
increase correlated with new lesion development as assessed by MRI.
In order to invade the CNS from the periphery, T cells need to adhere
to the CNS endothelium and then cross the blood brain barrier.
Interestingly, CD8+T cells but not CD4+T cells from relapsing-
remitting MS patients exhibited an augmented capacity to roll and
arrest on inflamed brain venules via a P-selectin glycoprotein ligand-1
dependent mechanism . These observations are compelling to
support the notion that CD8+T cells from MS patients have enhanced
capacityto cross from the periphery to the CNScompare to theirCD4+
counterparts. Taken together, there is considerable evidence impli-
cating distinct characteristics in peripheral CD8+T cells from MS
patients that could predict the development of CNS lesions.
1.3. MHC class I association with MS
As reviewed above, CD8+T cells recognize specific antigens that are
presented by autologous MHC class I molecules (HLA-A, B, and C in
humans or H-2D, H-2K, and H-2L in mice). The MHC alleles carried by
the host dictate which peptides (self or foreign) and the affinity with
which they are presented to autologous CD8+T cells. Therefore,
specific alleles could play a key role in the modulation of auto-
aggressiveimmuneresponses.Anincreasedprevalenceof specific HLA-
A alleles within the MS population compared to controls has been
reported more than 30 years ago . In the last decade, using modern
genetic tools different groups confirmed the association of specific
alleles with this disease. HLA-A*0301 has been shown to increase the
risk of developingMSinaddition toHLA-DR15 orindependentlyof
the HLA-DRB1*15, DQB1*06 [35,36]. In contrast, the HLA-A*0201 allele
decreases the risk of developing MS  and reduces the risk for
specific MHC class I alleles could impact on the presentation of self-
antigens to auto-aggressive CD8+T cells involved in MS.
1.4. Reactivity of CD8+T cells to CNS antigens
Most peptides loaded on MHC class I molecules originate from
intracellularly transcribed proteins; however, some phagocytosed
proteins can also gain access to the MHC class I loading machinery and
be presented by cross-presentation, especially by professional APC .
These mechanisms allow for CNS specific self-antigens to be efficiently
presented to CTL by professional APC, especially when the self-antigens
are derived from cells under attack in MS lesions such as oligoden-
drocytes and neurons. Proteins from the myelin sheath, which is
targeted antigens in the context of MS and include: myelin basic protein
(MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein
(MOG), and myelin associated glycoprotein (MAG). Identifying the
myelin-derived target epitopes for a given HLA class I allele permits the
patient. Early studies focused on HLA-A2, because it is the most frequent
epitopes derived from candidate target-antigens for MS were identified
and, after synthesis, their HLA-A2 binding properties were established.
HLA-A2-restricted CD8+T cell lines or clones could thus be generated
against MAG287–295, MAG509–517, MAG556–564, MBP87–95 MBP110–118,
PLP80–88[41–43], and Transaldolase168–176, (an enzyme of the pentose
phosphate pathway expressed at high levels by oligodendrocytes) .
Moreover, the frequency of CD8+T cells specific for HLA-A*0201:
MBP110–118or Transaldolase168–176,as well as other myelin and neural-
antigens, appears more elevated in the peripheral blood of MS patients
relative to healthy controls [41,43,45,46]. Importantly, human myelin-
L.T. Mars et al. / Biochimica et Biophysica Acta 1812 (2011) 151–161
specific CD8+T cells recognizing the HLA-A*0201:MBP110–118complex
could induce lysis of HLA-matched oligodendrocytes in vitro, in the
absence of exogenous antigen , indicating that this epitope is
CD8+T cells carriedeffector functions including productionof IFN-γand
TNF, proliferation and cytotoxicity when exposed to various epitopes of
CD8+T cells bore a naïve phenotype in control subjects whereas in MS
patients, they were activated and had an effector or memory phenotype
[43,45,48], suggesting that in patients these cells have already been
actively exposed to their cognate antigen. However, a recent analysis of
myelin-specific CD8+T cell responses reported that MS patients and
healthy controls had the same frequency of IFN-γ secreting CD8+T cells
using peripheral blood in response to a large array of myelin peptides
derived from MOG, MBP and PLP and presented by several HLA class I
alleles (A3, A2, B7, B27 and B44) . Other studies failed to detect
differences between MS and controls regarding the CD8+T cells
responses to neuronal or oligodendroglial antigens . Thus, an
increased peripheral frequency of autoreactive CD8+T cells targeting
myelin or neuronal epitopes has been occasionally observed in MS
patients compared to controls [41,43,45,46]. Additional studies will be
required to determine whether autoreactive CD8+T cells from MS
patients exhibit distinct properties, not only in frequency but also
regarding their effector properties, compared to controls.
1.5. Antigen presentation to CD8+T cells
efficient interaction with a professional APC [14,51]. APC located in
different areas in and around the CNS may present CNS-derived
antigens to CD8+T cells during MS or its animal models. Despite the
immune privileged status of the CNS, part of the cerebrospinal fluid
(CSF) drains to the cervical lymph nodes located in the neck. At this
location, antigens gathered from the CNS can be presented to the
peripheral immune cells. Interestingly, cervical lymph nodes obtained
from MSpatients and animals (marmosets and mice) affectedwith EAE
contain myelin-laden APC. These APC have engulfed myelin proteins
CD40, and MHC class I and class II molecules suggesting that they are
competent APC [52,53]. Indeed, a remarkable study indicated that after
and cross-present antigens to CD8+T cells . Upon peptide
presentation to antigen-specific CD8+T cells, these APC imprint a
particular integrin profile that directs these CD8+T cells to the tissue
from which the antigen was initially gathered. Accordingly, APC that
collected antigen from the CNS and presented them in the cervical
This implies that competent myelin-laden APC located in the cervical
to the CNS. Once in the CNS, these CD8+T cells can be re-activated
locally by the numerous myelin-laden APC (macrophages and micro-
glia) that are around lesions . Moreover, APC in the CNS have been
locally, activate peptide-specific CD8+T cells and trigger a cytotoxic
response . Finally, MHC class I is normally low but constitutively
expressed by microglia and endothelial cells in human CNS although
absent on other cell types. However, astrocytes, oligodendrocytes, and
neurons in acute MS lesions have been shown to express MHC class I
 supporting the notion that virtually all CNS cell types can be
targeted and potentially killed by CD8+T cells.
1.6. Antigen-driven retention of CD8 T lymphocytes in the CNS
CD8+T cells that have encountered their antigen in the presence of
appropriate co-stimulation and cytokines undergo a specific differenti-
the CNS of MS patients bear key features of an antigen-specific response.
Using CD8+T cells from the blood and CSF as well as by micromanip-
ulation of CD8+T cells in CNS tissue sections, these groups applied
complementarity-determining region (CDR) 3 spectratyping. This en-
abled them to determine the sequence of the β chain of the TCR, which
confers the specificity of the TCR by the select combination of V, D and J
gene segments. Babbe and colleagues studied two MS cases indetail
T cells in the lesion. Examining a larger study group consisting of 36 MS
patients, Jacobsenetal.  indicated thatCD8+T cells isolatedfrom the
CSF of MS patients contained also mainly clonally expanded cells. These
in a few patients studied longitudinally. Furthermore, a specific β chain
was enriched in CD8 T cells in the CSF compared to the blood
compartment (7.7% in the blood and 16.4% in the CSF) whereas the
frequency of a specific β chain was similar in both blood and CSF
compartments for the CD4+T cells. Skulina et al.  confirmed the
occurrence and enrichment of clonal populations of CD8+T cells in the
CSF samples taken 5 years apart from the same individual indicated the
persistence of the same CD8+T cell clone during that period. In contrast,
CD4+T cells has been detected in the CNS and blood of Rasmussen
encephalitispatients .Takentogether, there isconsiderableevidence
implicating antigen-driven CD8+T cell expansion in MS and potentially
CD8+T cells in the CNS of MS patients are not bystander cells, but rather
cells. Strong evidence confirming the oligodendroglial or neuronal
specificity of clonally expanded CD8 T cells in the CSF and CNS tissue of
1.7. CNS associated CD8+T lymphocytes bear effector functions
in post-mortem material from acute or relapsing-remitting MS patients
compared to more chronic cases or lesions . Moreover, reports from
several laboratories confirmed that specifically CD8+T lymphocytes are
present in MS lesions and that their number reaches or surpasses that of
CD4+T lymphocytes [58,63–68]. There is a vast body of evidence
scene’ of MS lesions. CD8+T lymphocytes are particularly detected
within the parenchyma and in close proximity to oligodendrocytes and
demyelinated axons with polarization of their cytolytic granules in MS
lesions [65,66,69]. Dendritic cells that have engulfed myelin (positive
either for oil-red O or myelin basic protein) are detected in MS lesions
. Moreover, these myelin-laden professional APC have interactions
cells, at the margin of the chronic and active lesions . Recently, over
IL-17, the prototypic cytokine of the new pathogenic T cell subset
Th/Tc17. Equal proportion of CD4+and CD8+T cells was detected
producing this cytokine in situ , demonstrating that both T cell
were enriched in both the CSF  and CNS tissue of MS patients
L.T. Mars et al. / Biochimica et Biophysica Acta 1812 (2011) 151–161
[48,69,72]. This memory subset usually resides in tissues and is able to
that enables lymphocytes to migrate back to lymph nodes (accordingly
with the effector memory profile) [74,75], indicating their possible
retention in the CNS. Moreover, the CSF of early diagnosed MS patients
has been shown to be enriched for CD8+T cells bearing a highly
differentiated effector memory phenotype: CCR7− CD45RA+/−, and
this enrichment was more important than in the CD4+compartment
, suggesting their role early on during the development to relapsing-
remitting MS. Finally, a significant correlation has been observed
between the number of CD8+T cells and the extent of axon damage in
MS lesions as measured by the accumulation of amyloid precursor
protein [76,77], advocating that CD8+T cells actively contribute to the
2. Effector CD8+T cells in animal models
2.1. HLA — humanized mouse models
Various mouse lines have been generated in which murine MHC
class I expression has been invalidated to impose the restriction of the
CD8+T cell repertoire to a transgenic human HLA class I allele. These
humanized mice have been used to study the pathogenic impact of
myelin-specific CD8+T cells with direct relevance to the human
pathology. To assess whether CD8+T cell responses targeting
HLA-A*0201 binding myelin epitopes could aggravate autoimmune
demyelination, we identified MOG epitopes that are conserved
between mouse and man. Using humanized HLA-A*0201 transgenic
mice, we could reveal in vivo that CD8+T cells targeting the
immunodominant, naturally processed MOG181–189 peptide can
potentiate the autoreactive CD4+T cell response by accelerating the
encephalitogenic process and worsening the disease evolution .
Other investigations have generated CD8+T cell lines against
PLP45–53in association with the HLA-A3 molecule [35,79,80]. To study
the functionalcontribution of HLA-A3 to MS pathogenesis, humanized
CD8+TCR transgenic mice were created on a C57Bl/6 background
. These humanized mice express HLA-A*0301 together with an
HLA-A*0301:PLP45–53specific TCR (2D1) isolated from an MS patient.
A small fraction of these mice (4%) developed spontaneous EAE
mediated by CD8+T cells and characterized by demyelination and
axonal damage. Immunization with PLP45–53peptide emulsified in
CFA led to a disease with a 25% incidence and a distinct biphasic
evolution. The first bout of disease was mediated by PLP45–53specific
CD8+T cells. The second phase, however, was mediated by an
encephalitogenic CD4+T cell response targeting the MOG35–55
peptide presented by mouse I-Ab. As such, these data prove that
CD8-mediated autoimmune demyelination can drive epitope spread-
ing , not only from one myelin-antigen to another, but also
between T cell compartments. In addition to proving the pathogenic-
ity of myelin-specific CD8+T cells in a humanized context, this study
provides an interesting hypothesis concerning the protective impact
of the HLA-A*0201 allele on MS. Indeed, to test the epistatic
interactions between the disease conferring HLA-A*0301 allele and
the protective HLA-A*0201 allele, humanized mice were created
expressing both HLA transgenes together with the 2D1 TCR. These
triple transgenic mice were fully protected from both spontaneous
and induced disease. This was the result of a strong negative selection
of 2D1 expressingCD8+T cells in the thymus induced by HLA-A*0201.
This suggests that HLA-A*0201 might protect from MS by purging
PLP45–53reactive CD8+T cells from the repertoire by presenting an, as
yet unidentified, self-antigen. Lastly, this and previous studies [81,83],
demonstrate the potential of introducing human disease associated
genes into rodents to reveal their functional roles in the pathogenesis
2.2. Pathogenicity of myelin and oligodendrocyte-specific CD8+T cells
To reassess the pathogenic potential of myelin-specific CD8+T
cells, several groups established experimental protocols to isolate
and expand myelin-specific CD8+T cells permitting their transfer
into syngeneic recipient mice. This approach revealed that CD8+T
cells targeting myelin antigens can be highly pathogenic. Joan
Goverman's laboratory generated CD8+T cell responses against
MBP79–87in C3H mice using a vaccination strategy, which is more
prone to induce CD8+T cell responses than protein/peptide
immunization in CFA . The adoptive transfer of these H-2Kk:
MBP79–87-specific CD8+T cells induced a severe autoimmune disease
that reproduced certain features of MS, not classically seen in CD4+-
mediated EAE. Clinically these mice exhibited upper motor neuron
impairment, ataxia, spasticity and only occasionally hind limb
paralysis. The disease evolution was rapid and severe, proving fatal
to all mice by day 14. The inflammatory lesions were located
exclusively in the brain, with focal involvement of grey and white
matter that was most pronounced in the white matter of the
cerebellum. CD8+T cell-mediated lesions associated vascular
damage and perivascular tissue insult reminiscent of ischemic injury.
Few inflammatory cells were found outside the perivascular cuffs.
Demyelination was severe in the adjacent nervous tissue. Tissue
destruction could be alleviated by injecting neutralizing anti-IFN-γ
mAbs, while tumor necrosis factor receptor (TNFR)-Fc fusion
proteins provided no clinical improvement. This is of interest as
IFN-γ plays a similar detrimental role in MS , by contrast, IFN-γ
has a beneficial role in the conventional model of EAE as it reduces
the severity of disease .
Other studies revealed that the transfer of MOG35–55-specific CD8+T
cells, into syngeneic recipients consistently induced chronic paralysis in
C57Bl/6 mice [88,89]. The disease was independent of a secondary CD4+
T cell response as both RAG−/−and SCID recipients developed disease.
Inflammatory lesions were observed in both the brain and spinal cord.
 and rich in neutrophils . Tissue damage was marked by
pronounced destruction of nerve fibers, rather than selective demyelin-
ation. The pathogenic CD8+T cells recognize the minimal epitope
MOG37–46in the context of H-2Db. This indicates that MOG35–55
contains at least 2 nested peptides that are encephalitogenic in C57Bl/6
mice. MOG40–48drives the encephalitogenic CD4+T cell response when
presented in the context of I-Ab, while MOG37–46activates H-2Db
restricted CD8+T cells.
Whether myelin-reactive CD8+T cells cause disease by killing
oligodendrocytes, or via bystander-mechanism driven by cross-presen-
tation of myelin antigens by CNS resident APC has not been addressed in
the models mentioned above. This issue was recently studied in two
distinct transgenic mouse models in which a ‘neo-self-antigen’ was
selectively expressed in oligodendrocytes [91,92]. In mice expressing
ovalbumin (OVA) under the proximal MBP promoter (ODC-OVA mice),
OVA protein was exclusively detected in the CNS and localized to the
cytosol of oligodendrocytes . Crossing the ODC-OVA mice with OT-I
mice expressing a transgenic TCR recognizing OVA257–264in the context
of H-2Kbcaused a lethal demyelinating disease. The disease was
mediated by CD8+T cells as both RAG−/−/OT-I/ODC-OVA triple
transgenic mice and the adoptive transfer of naïve OT-I CD8+T cells
into RAG−/−ODC-OVA mice caused disease in an IFN-γ dependent
manner. The ‘neo-self’ specific CD8+T cells got activated during the first
10 days after birth, so early in life the blood–brain barrier is not
completely developed therefore spontaneous release of CNS specific
antigen is most likely happening. During this period OVA was accessible
to CD8+T cells in the cervical and mesenteric lymph nodes, permitting
their priming and differentiation. In vitro studies indicated that
oligodendrocytes from ODC-OVA mice were direct targets for the
antigen-specific CD8+T cells. In addition to oligodendrocyte loss, the
L.T. Mars et al. / Biochimica et Biophysica Acta 1812 (2011) 151–161
slices of ODC-OVA mice . Interestingly, the spontaneous demyelin-
atingdiseaseobserved intheOT-I/ODC-OVAdouble-transgenic mice can
be blocked by the administration of a mAb specific for the H-2Kb:
OVA257–264complex, indicating the efficacy of blocking antigen-presen-
tation in preventing autoimmune CD8 T cell responses targeting
oligodendrocytes, which express low levels of MHC class I .
We expressed an alternative neo-self-antigen, Influenza virus
Hemagglutinin (HA), selectively in oligodendrocytes . This was
achieved using a double knock-in approach combining MOG-iCre
mice, expressing the Cre recombinase selectively in oligodendrocytes
and knock-in mice allowing the conditional (Cre-dependent) expres-
sion of HA. In the double knock-in ‘MOG-HA’ mice, Cre-mediated DNA
recombination and HA transcription were observed selectively in the
CNS, but HA protein expression was below detection levels of
immunohistochemistry. Crossing MOG-HA mice with Cl4 mice, in
which most CD8+T cells express a transgenic TCR specific for
HA512–520in the context of H-2Kd, resulted in a situation of immune
ignorance characterized by the indifference of antigen-specific CD8+
T cells to their cognate self-antigen in oligodendrocytes. This is in
contrast to the spontaneous EAE observed in the ODC-OVA transgenic
mice. The low level of HA expression, as well as the different genetic
background might have contributed to these differences. In addition,
the membrane embedded HA512–520 peptide is less soluble than
cytosolic OVA and might, therefore, be less efficiently drained to
secondary lymphoid organs . Strikingly, severe demyelinating
lesions could be induced in the MOG-HA mice upon adoptive transfer
of in vitro differentiated cytotoxic CD8+T cells specific for HA. Non-
irradiated MOG-HA recipients developed weight loss, and in more
severe cases tremors, reduced mobility, but no overt paralysis. The
inflammatory lesions were dominant in the optic nerve and spinal
cord, but also affected the brain. T cell infiltration initiated in the optic
nerve and caused microglia activation, oligodendrocyte apoptosis and
subsequent demyelination. Transducing the pathogenic CD8+T cells
with GFP prior to transfer permitted to trace these cells in the
demyelinating lesions. GFP+CD8+T cells were frequently found in
close apposition with oligodendrocytes and some had their Granzyme
B containing granules polarized towards the juxtaposed oligodendro-
cyte, suggesting directed degranulation. Oligodendrocytes displayed
nuclear condensation indicative of subsequent apoptosis. This loss of
oligodendrocytes preceded severe demyelination leading to severe
axonal damage and eventually their destruction. These studies
demonstrate the deleterious capacity of oligodendrocyte-specific
CD8+T cells in an inflammatory demyelinating pathology resembling
active MS lesions. Direct antigen-driven cytotoxicity of oligodendro-
cytes is implicated, although additional mechanisms may be at play. A
similar CD8+T cell-mediated autoimmune response targeting HA-
expressing astrocytes, caused their selective deletion without pro-
viding any evidence of secondary tissue damage . This study
indicates that CD8+T cells are less likely to induce bystander damage
than CD4+T cells.
2.3. Spontaneous demyelination driven by CD8+T cells
Modifications to the homeostasis of the CNS can breach immune
tolerance and cause secondary immune insult. Two transgenic mouse
models have recently illustrated initiation of secondary CD8+T cell
responses in the CNS. Proteolipid protein (PLP) transgenic mice
express a construct encoding for multiple copies of the wild-type Plp
gene. Homozygotes develop tremors and seizures due to dysmyelina-
tion causing rapid death. . Hemizygous mice only moderately
augment PLP expression and are clinically normal for long periods of
time . After 12–18 months, these mice develop progressive
neurological signs including ataxia, tremor and seizures. Affected
areas in the optic nerve and cerebral white matter exhibited
prominent demyelination, while the spinal cord revealed intense
axonal degeneration. Oligodendrocytes frequently persisted in de-
generative regions. Interestingly, activated CD8+T cells and macro-
phages accumulated in the CNS of these mice while only limited B and
CD4+T cells were present in the infiltrates . These infiltrating
CD8+T cells were shown to be pathogenic as RAG−/−PLP transgenic
mice reconstituted with bone marrow from wild-type but not
CD8-deficient mice developed disease . The target antigen
recognized by the pathogenic CD8+T cells has so far not been
identified. PLP and other myelin antigens might not be the strongest
candidates as no cytotoxicity was observed towards oligodendrocytes
that persist with the degenerating lesions. Taken together these
observations indicate that primary myelin damage might predispose
to secondary CD8-mediated tissue destruction. A similar set of
observations were made in mice with peroxisome-deficiency .
The second model was serendipitously generated when trans-
genic mice expressing the co-stimulatory molecule CD86 under the
H-2Kbpromoter were created . All lines were characterized by
strong constitutive CD86 expression on T cells, while B cells of
different sublines expressed either low, intermediate, or high levels
of CD86 . One subline, was found to express CD86 constitutively
in the CNS on CD45loCD11bhimicroglia . These mice developed
spontaneous neurological deficits, including ataxia and a difficulty to
right when overturned, ultimately leading to death. Inflammatory
lesions were observed in both grey and white matter of the spinal
cord and caused axonal damage and demyelination. The lesions
comprised CD4+and CD8+T cells, MHC-II expressing cells, but no B
cells. This spontaneous pathology is T cell dependent as CD86
transgenic Tcrβ−/−mice, which are deficient in αβ T cells, were fully
protected from disease . Importantly, the T cell-deficient CD86
transgenic mice retained the elevated CD86 expression on microglia
indicating that it is transgene driven and not inflammation induced.
Reconstituting these mice with TCR+/+bone marrow or the transfer
of purified T cells restored the T cell compartment, however, only
bone marrow or T cells from CD86 transgenic mice, and not wild-
type mice, caused CNS inflammatory demyelination, indicating that
the transgenic expression of CD86 on T cells also contributes to
disease pathogenesis. The CD8+T cell subset likely drives this
immune pathology as reconstituting CD86 transgenic Tcrβ−/−mice
with bone marrow from CD4 T cell-deficient (I-Ab−/−or Cd4−/−
CD86 transgenic mice) accelerated and aggravated the disease course
. In this model, activated oligoclonal CD8+T cells infiltrated the
CNS prior to disease initiation. The detrimental property of CD8+T
cells requires IFN-γ as invalidation of the IFN-γ receptor (Ifn-r−/−) in
the recipient CD86 transgenic mice prevented disease development.
Defining the target antigen(s) and the relative contribution of the
CD86 expression on T cells and microglia will provide further insight
in CD8-mediated demyelination and the tolerogenic mechanisms
protecting the CNS.
3. CD8+T cell immunity to CNS viral infections
3.1. Virus-induced demyelinating models
Viral infections of the CNS readily generate effector CD8+T cell
responses that infiltrate the CNS parenchyma aiming to control viral
spread via both lytic and non-cytolytic mechanisms . Delayed or
suboptimal anti-viral responses can exacerbate CNS infection causing
severe tissue damage and potentiating the risk of secondary
autoimmunity via determinant spreading or bystander immune
activation. In this context the model of Theiler's murine encephalo-
myelitis virus (TMEV) is noteworthy . TMEV is a picornavirus
that naturally infects mice. In experimental conditions, following
intracerebral inoculation of the DA strain of TMEV, the virus initially
replicates in neurons and all mice develop an acute grey matter
encephalomyelitis. In resistant mouse strains such as C57Bl/6, the
infection is cleared within 3 weeks by anti-viral CD8+T cells
L.T. Mars et al. / Biochimica et Biophysica Acta 1812 (2011) 151–161
recognizing the immunodominant VP2122–130epitope presented by
H-2Db. After viral clearing this intense cytotoxic CD8+T cell response
targeting infected neurons contracts and mice fully recover. In
contrast, in susceptible strains such as SJL, the inflammatory response
fails to clear the virus that persists in the spinal cord white matter
where it resides in microglia and oligodendrocytes. There, the virus
elicits chronic inflammatory and demyelinating lesions, which can
lead to flaccid paralysis. At this chronic phase the lesions are very
similar to those observed in MS. Both CD8+and CD4+T cells are
thought to contribute to demyelination: CD8+T cells by killing
infected oligodendrocytes and CD4+T cells via bystander mechan-
isms in response to either viral or myelin antigens [105–107].
However, CD8+T cells might play a unique role in causing the
neurological manifestations associated with TMEV infection. When
comparing TMEV infection in MHC-I-deficient (β2-microglobulin−/−),
MHC-II-deficient (I-Ab−/−) and wild-type SJL mice the intensity and
localization of demyelinating lesions were identical in the 3 groups,
yet only SJL and MHC-II-deficient mice developed severe neurological
deficits . In contrast, MHC-I-deficient mice exhibited preserved
hind limb motor-evoked conduction velocities, suggesting that in the
absence of CD8+T cells demyelinated axons retain functionality.
Histologically, demyelinated axons were indeed shown to resist in
MHC-I-deficient mice. Moreover, these axons exhibited increased
sodium channel densities augmenting the efficacy of impulse
conduction. It was, therefore, proposed that reactivation of CD8+T
cells in the vicinity of demyelinated axons may contribute to
functional neurological impairment . Cd8−/−mice similarly
revealed extensive demyelination without developing neurological
impairment, further supporting a role for CD8+T cells in neuronal
dysfunction after TMEV infection . These data substantiate the
previously mentioned correlation between the number of CD8+T
cells and the extent of axon damage in MS lesions [76,77].
CD8+T cells can cause demyelination by targeting oligodendro-
cytes. Using a model system in which lymphocytic choriomeningitis
virus (LCMV) proteins were selectively expressed in oligodendrocytes
by transgenesis, it was possible to test whether LCMV Armstrong
infection, which does not infect the CNS parenchyma, could initiate
CNS autoimmune demyelination due to the imposed mimicry. Indeed,
CD8+T cell-mediated demyelination was clearly illustrated in this
viral model but the effector mechanisms involved have remained
CD8+T cells can cause demyelination via bystander mechanisms.
This was shown using murine hepatitis virus (MHV), which infects
oligodendrocytes, astrocytes and neurons in the CNS . MHV
causes immune-mediated demyelination driven by T and B cells, as
indicated by the fact that infected RAG−/−mice fail to develop
demyelination despite harboring a large viral load in the CNS
[112,113]. Adoptive transfer of either CD4+or CD8+T cells restored
the inflammatory demyelinating phenotype in these mice .
Interestingly, even activated CD8+T cells of irrelevant specificity can
mediate bystander demyelination  involving IFN-γ .
Moreover, CNS viral infections have revealed the versatility of effector
CD8+T cells in exerting their immune functions. Intracerebral LCMV
infection leads to blood–brain barrier breakdown and convulsive
seizures caused by virus-specific CD8+T cells . Interestingly,
when the virus-specific CD8+T cells target the infected stromal cells
in the subarachnoid space the interaction was transient and
insufficient to cause apoptosis of the infected target cells. Rather,
the activated CD8+T cells released chemokines that stimulated the
permeability of the blood–brain barrier and triggered the influx of
pathogenic monocytes and neutrophils that contributed directly to
the lethal seizures . A recent study has illustrated that viral
infection could break tolerance of myelin-specific CD8 T cells not only
via a molecular mimicry mechanism but also by activating CD8 T cells
bearing dual TCRs that are able to simultaneously recognize both viral
and myelin antigens .
3.2. Epstein–Barr virus-specific CD8+T cells in the context of MS
Thirty years ago, Prineas reported the detection of organized
lymphoid tissue in the perivascular spaces of MS plaques, especially in
‘old’ plaques as stated by the author . More recently, Aloisi and
colleagues reported the detection of lymphoid follicle like structures
containing B cells, T cells and plasma cells  and showed that
these lymph node like structures contained B cells infected with
Epstein–Barr virus (EBV) and that some of the CD8+T cells in these
ectopic structures had their granules polarized toward these infected
B cells. Whether CD8+T cells in the CNS of MS patients while
targeting infected EBV-B cells actively contribute to the pathogenesis
of MS or represent a bystander population of infiltrating cells
attracted by the prolonged inflammatory state of the organ has not
been elucidated. In contrast, others detected CNS EBV infectionin only
rare cases of MS [121,122] suggesting that the presence of this virus in
the target organ of this autoimmune disease is not a common feature.
Moreover, whether EBV-specific CD8+T cell responses are distinct in
MS patients is still controversial. Increased or decreased frequency of
EBV-specific CD8+T cell responses was detected in MS patients
compared to controls [123,124]. When autologous EBV-infected
lymphoblastoid cells were used as APC, the frequency of EBV-specific
CD8+T cells was lower in MS patients than in controls .
However, using ELISPOT with peptides of 8–15 mers to stimulate
CD8+T cell responses, greater frequency of EBV-specific IFN-γ
producing CD8+T cells was found in clinically isolated syndrome
than in any other form of MS (RR-MS, SP-MS and PP-MS), other
neurological diseases or healthy controls . CD8+T cell responses
(studied in PBMC) to latent EBV proteins were higher in MS patients
than in controls as measured by intracellular cytokine staining
(IFN-γ) in response to EBV-infected and immortalized autologous B
cells, but no difference was observed between MS and controls for the
frequency of EBV-specific CD4+T cells . Overall, the potential
contribution of EBV-specific CD8+T cell responses both in the
periphery and locally in the CNS in the context of MS remains to be
4. Regulatory CD8+T cells in MS and EAE
The cellular immunity provided by both CD4+and CD8+T cells is
essential to fight infections and eliminate potentially neoplasic cells.
However, T cell responses have the potential to instigate immune-
mediated pathologies, such as autoimmune diseases, including MS.
Thus, a fine balance between protective and deleterious effects needs
to be sustained. Both CD4+and CD8+T cells bearing suppressor or
regulatory properties have been described. CD4+regulatory T cells
have been extensively studied; however, knowledge about their CD8
T cell counterparts is not as extensive. Subsets of CD8+T cells in both
humans and rodents have been shown to suppress immune responses
[127,128]. Whether suppressor CD8+T cells play a role in MS or are
deficient in MS patients remains a topic of considerable debate.
More than twenty years ago, MS patients were shown to have
defective CD8+T cell suppressor functions compared to healthy
controls [129,130] using a proliferation assay in which anti-CD3
stimulated and enriched CD8+T cell cultures used as suppressor cells
were added to fresh autologous peripheral lymphocytes in the
presence of concavalin A. Later on, a single clone of CD8+T cells
expanded from one healthy donor was reported to regulate
autologous MBP-specific CD4+T cells . More recently, treatment
of MS patients with glatiramer acetate, a synthetic copolymer of four
amino acids, increased the capacity of CD8+T cells to kill in a MHC
class I and HLA-E dependent manner CD4+T cells of any specificity as
long as these target cells were loaded with glatiramer acetate-derived
peptide in their MHC groove . The same group (Karandikar and
colleagues) showed that upon glatiramer acetate therapy, the TCR
repertoire of CD8+T cells responsive to this peptide mixture was
L.T. Mars et al. / Biochimica et Biophysica Acta 1812 (2011) 151–161
oligoclonal over time and more limited than that in the CD4
compartment . Furthermore, Correale and Villa advocated that
CD8+T cells can kill myelin-specific CD4+T cells in a HLA-E restricted
fashion . In vitro expanded myelin-specific CD4+T cell clones
from MS patients were used as antigen for the expansion of
autologous CD8+T cells. The amplified CD8+T cell clones killed
autologous myelin-specific CD4+T cell clones only when these target
cells were activated. It is not possible to rule out that the long-term in
vitro culture did trigger T cell functions that are not representative of
their in vivo properties. Although the authors implicated HLA-E
recognition in the cytotoxicity, they did not show whether their CD8+
T cell clones express either HLA-E receptor (NKG2A or NKG2C), nor
whether the target cells (CD4+T cell clones) expressed higher levels
of HLA-E upon activation since these cells at a resting state could not
trigger the CD8+T cell responses .
It has been suggested that within the CD8+T cell compartment,
CD28+expressing cells are cytotoxic whereas CD28−are suppressor
. Interestingly, CD8+CD28−T cells were detected in lower levels
in the peripheral blood of MS patients compared to controls .
Moreover, a recent study underlined the possibility that activation of
CD8+T cells with a single antigen, an immunodominant cytomega-
lovirus HLA-A2-restricted antigen, can lead to either cytotoxic CD8+T
cells (that were CD28+) or suppressor CD8+T cells (that were CD28
−) depending on the APC and environmental milieu . However,
the sole absence of CD28 is not a reliable marker of suppressor
capacity since CD8+CD28−T cells isolated from myeloma have been
shown to release inflammatory cytokines and to possess cytotoxic
capacity . Unfortunately, a specific marker distinguishing
suppressor or regulatory cells from other conventional CD8+T cells
has yet to be identified, although expression of Foxp3, the transcrip-
tion factor associated with CD4 regulatory T cells, may identify a
suppressive CD8 T cell subset . Thus, only suppressor capacity
confirmed by functional assays could substantiate that any CD8+T
cells possess regulator properties and whether these cells are
deficient in MS patients remains to be fully investigated.
4.1. CD8+T cell responses in active EAE
EAE is predominantly driven by distinct autoreactive CD4+T cell
subsets, with a varying importance of the humoral response . The
magnitude of CNS tissue damage correlates most strongly with the
frequency of activated macrophages/microglia . The active immu-
nization protocol with myelin proteins or peptides favors CD4+T cell
responses resulting in only low frequency CD8+T cell infiltration within
demyelinating lesions . During active EAE CD8+T cells are thought
to play a dual role. Indeed, CD8-deficient mice display reduced initial
sustain disease remission [142,143]. A similar aggravation of EAE was
observed in MOG35–55or MBP immunized β2m−/−mice, in which the
deficiency in β2-microglobulin prevents the expression of both classical
and non-classical MHC-I molecules . Further studies revealed that
CD8+T cells isolated from mice that had recovered from EAE could
eliminate MBP-specific CD4+T cells in vitro and in vivo . This
molecule Qa-1, the mouse equivalent to human HLA-E. These CD8+T
cells are thought to target a peptide from the Vβ8.2 chain of the
autoreactive TCR presented in the context of Qa-1 on the surface of
pathogenic CD4+T cells causing their apoptosis [146–149]. The
generation of Qa-1−/−mice, lacking these regulatory CD8+T cells, has
since permitted to assess their precise impact on EAE. These mice
developed a more severe disease due to the resistance of Qa-1-deficient
CD4+T cells to CD8+T cell-mediated regulation . Moreover,
them to express Qa-1 such that they become susceptible to NKG2A-
expressing suppressor CD8+T cells that can then block their capacity to
induce EAE .
Other reports of regulatory CD8+T cells in the context of EAE have
been published such as the identification of suppressor cells in the
polyclonal CD8+CD28−T cell subset that are thought to convey
protection against EAE . Also, CD8+T cells expressing CD122
(IL-2/IL-15 receptor β) have been shown to spontaneously arise
during EAE. While depletion of these cells exacerbated EAE symp-
toms, the adoptive transfer of these cells alleviated the disease from
naïve recipients . Overall, regulatory CD8+T cells have been
reported but their frequency and the mechanisms by which they
suppress other immune cells remain unexplored.
5. Concluding remarks
Major advances have recently been made in the understanding of
the pathophysiology of MS. Unraveling the complexity of the
inflammatory response has identified various immune mechanisms
involved in the disease pathogenesis. Notably, the humoral response
has been implicated in direct antibody-mediated demyelination while
new effector CD4+T cell subsets that contribute to CNS autoimmunity
have been identified. This review considers the detrimental traits of
the CD8+T cell lineage during inflammatory demyelinating responses
in the CNS. Experimental models strongly support the idea that CD8+
T cells can induceor aggravate tissue destructionin theCNS. However,
the interactions of CD8+T cells with the other immune components
implicated in the pathophysiology of MS, and how these interactions
can account for the heterogeneity in lesion formation and clinical
evolution remains to be fully explored. Experimental models will
therefore have to be employed to understand the migration, cytokine
secretion profile, and relative pathogenic impact of each of the
immune cell subsets individually and in synergy. Early indications
suggest that CD4+T subsets exhibit a distinct migratory behavior and
might differentially influence lesion localization in the CNS .
Such differential behavior might be extended to CD8+T cells, which
benefit from a unique antigen-driven mechanism to infiltrate the CNS
suggesting that the requirements for CNS entry might be distinct
between the CD4+and CD8+T cell subsets . Consequently, the
dominance of CD8+T cells might vary between CNS regions and over
time. Documenting the dynamics of the CD8+T cell response in MS
might point to distinct disease stages or alterations in disease severity
that correlate with CD8+T cell dominance.
A more detailed dissection of the functional variety of CD8+T cells in
cells characterized by distinct cytotoxic properties and cytokine profiles
have been observed in different immune settings [156–160] and might
the CNS . Moreover, existing observations regarding CD8-mediated
tissue damage during CNS viral infection might provide important leads
to the potential mechanisms employed by CD8+T cells during CNS
inflammation. For instance, the non-cytolytic function of CD8+T cells to
recruit immune components by releasing chemokines and promote
CD4+T cell compartment, CD8+T cell subsets portray regulatory
properties. Whether these CD8+T lymphocytes play a positive or
negative role in the inflamed CNS is still a matter of debate and
controversy [9,128]. Assessment of antigen specificity but also of specific
functions will be essential to unravel the contribution of CD8+T cells to
the pathogenesis of MS.
Lastly, current and future therapies for MS must be assessed for their
at selectively targeting the CD4+T cell response have produced little
cells by anti-CD52 mAb-mediated depletion (Alemtuzumab) , anti-
α4-integrin mAb-mediated inhibition of CNS infiltration (Natalizumab)
, and the immunomodulator FTY720 mediated inhibition of
lymphocyte egress from lymphoid organs reduce relapses and limit the
L.T. Mars et al. / Biochimica et Biophysica Acta 1812 (2011) 151–161
formation of new lesions. Strengthening our understanding of the
avenues for the treatment of this disabling inflammatory disease.
Research by RSL and LTM is supported by the European Union: FP6
Neuropromise, SUDOE Immunonet, the National Medical Research
Institute (INSERM), the Medical Research Foundation (FRM), the
ARSEP, and the Region Midi- Midi-Pyrénées. Research by NA and PS is
supported by the Multiple Sclerosis Society of Canada (MSSC). PS is
supported by a Canadian Graduate Scholarships doctoral research award
from the Canadian Institutes of Health Research. NA holds a Donald Paty
Career Development Award from the MSSC and a Chercheur-Boursier
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