REVIEWS

EPITOPE SPREADING IN IMMUNE-

MEDIATED DISEASES: IMPLICATIONS
FOR IMMUNOTHERAPY
Carol L. Vanderlugt and Stephen D. Miller
Evidence continues to accumulate supporting the hypothesis that tissue damage during an
immune response can lead to the priming of self-reactive T and/or B lymphocytes, regardless
of the specificity of the initial insult. This review will focus primarily on epitope spreading at the
T-cell level. Understanding the cellular and molecular basis of epitope spreading in various
chronic immune-mediated human diseases and their animal models is crucial to
understanding the pathogenesis of these diseases and to the ultimate goal of designing
antigen-specific treatments.

CRYPTIC EPITOPE
A cryptic epitope is defined as a
hidden or sequestered epitope
that is processed and presented
more efficiently as a result of an
inflammatory immune response
initiated by either a dominant
epitope, as in a response to an
infectious agent, or revealed as a
result of the diversification of
the response secondary to self
tissue damage, as in an
autoimmune response.
Department of
Microbiology-Immunology
and Interdepartmental
Immunobiology Center,
Northwestern University
Medical School,
303 E. Chicago Avenue,
Chicago, IL 60611, USA
Correspondence to S.D.M.
e-mail: s-d-miller@
northwestern.edu
DOI: 10.1038/nri724
A typical immune response against a self or foreign pro-
tein is usually focused on one or two epitopes within
that protein, which are termed dominant. Epitope
spreading was initially defined as the diversification of
epitope specificity from the initial focused, dominant
epitope-specific immune response to subdominant
epitopes on that protein1,2. The hierarchy of dominant
and CRYPTIC EPITOPES in this scenario is thought to be due
to a combination of differential protein processing and
presentation by various antigen-presenting cell popula-
tions, and also to the availability of epitope-specific
T cells, taking into consideration central and peripheral
tolerance mechanisms3.
Our laboratory and others, extending this view,
have studied epitope spreading specifically in the con-
text of chronic tissue damage, which can lead to
autoimmunity. In these studies, regardless of the initial
antigenic stimulus — be it viral, graft rejection or
autoimmune — the specificity of the immune
response spreads to include self epitopes other than
that which initiated the inflammatory process (FIG. 1).
In this scenario, infection, organ transplantation or
autoimmunity (organ-specific or systemic) leads to tis-
sue damage. The resulting inflammation and tissue
damage then primes a hierarchical cascade of autoreac-
tive T-cell specificities, even allowing cryptic or
sequestered epitopes to be processed and presented.
In most of the models to be discussed, epitope
spreading initiated as a result of tissue damage is
thought to play an active role in ongoing disease
pathology. However, there is some evidence that
epitope spreading is an important component of
protective immune responses, acting to enhance the
efficiency of the immune response, as seen in tumour
clearance, and as a mechanism to downregulate
immune responses, such as those occurring in
autoimmunity.
At present, most treatment regimens for autoim-
mune diseases like multiple sclerosis, type-1 diabetes
and myasthenia gravis rely on generalized suppression
of the immune system and/or neutralization of
inflammatory mediators. It is, however, desirable to
design antigen-specific forms of immunotherapy
that leave the immune system intact and able to neu-
tralize opportunistic pathogens. Mounting evidence
for a pathological role for epitope spreading in
chronic autoimmune disease makes the development
of epitope-specific therapies problematic4. Therefore,
a more complete understanding of the cellular and
molecular basis of the epitope spreading phenomenon
is required to promote the development of antigen-
specific treatments for human autoimmune diseases,
more efficient vaccines and more effective methods to
control transplant rejection.

NATURE REVIEWS | IMMUNOLOGY VOLUME 2 | FEBRUARY 2002 | 8 5

© 2002 Macmillan Magazines Ltd
REVIEWS

a Primary epitope Secondary epitope

MHC II TCR MHC II TCR

APC TH 1 APC TH 1

CD28
Peripheral CD80/86
lymphoid tissue b Proliferation and differentiation j

Blood
vessel
Mono TH 1 TH 1

c Adhesion and penetration

Target
d i
tissue Chemokines
IFN-γ
MIP-1α Tissue
TH 1 APC debris

h
e
LT/TNF-β
Cytokines Tissue
IFN-γ g destruction
Mono
IFN-γ
MIP-1α Phagocytosis
O2 radicals, NO
Proteolytic enzymes
TNF-α
f Activation
Macrophage

Figure 1 | Epitope spreading in autoimmune and virus-induced tissue immunopathology. Presentation of the primary
epitope (the immunodominant self or viral epitope) occurs in peripheral lymphoid tissue (a), resulting in activation and differentiation
of autoreactive TH1 cells (b). The activated TH1 cells migrate (c) into the target tissue, where they encounter antigen presented by
resident APCs. (d). After antigen restimulation, the pathologic TH1 cells release a cascade of chemokines and cytokines (e), leading
to recruitment of additional mononuclear phagocytes from the peripheral blood, which are activated along with resident APCs (f).
Activated mononuclear cells then lead to bystander tissue destruction (g) via phagocytic mechanisms and release of TNF-α,
proteolytic enzymes, NO and O2 radicals. The tissue debris (h) is processed and presented on resident and peripheral APCs (i),
leading to the activation and differentiation of a second wave of TH1 cells (j), which can re-enter the tissue and cause additional
tissue destruction. APC, antigen presenting cell; IFN-γ, interferon-γ; LT/TNF-β, lymphotoxin/tumour necrosis factor β; MIP-1α,
macrophage inflammatory protein 1α; MHC II, major histocompatibility complex class II; Mono, monocyte; TCR, T-cell receptor;
TH1, T helper cells type 1 (see glossary).

Epitope spreading in autoimmune disease
Characterization of the cellular and molecular basis of
epitope spreading in various chronic immune-mediated
diseases and disease models is extremely important both
to understanding the pathogenesis of those diseases and
to achieving the ultimate goal of designing antigen-
specific treatments for human autoimmune diseases.
Although immune responses are complex, involving
both humoral and cellular immune components,
some autoimmune diseases are predominately CD4+
T-cell mediated, whereas others seem to be primarily
antibody mediated. We will focus on the role of epi-
tope spreading in T-cell-mediated autoimmune dis-
eases. Although we present some intriguing human
studies, a pathological role for epitope spreading is
very difficult to verify in human disease because the
initiating antigenic specificity is usually impossible to
define. Animal models have therefore been useful
because the peptide specificity of the initial immune
response can be manipulated, genetically identical ani-
mals can be used and the immune response can be
serially assessed in different peripheral lymphoid
organs, as well as in the target tissue of the disease.
Autoimmune models of multiple sclerosis. Multiple scle-
rosis is a human autoimmune demyelinating disease
thought to be CD4+ T-cell mediated and to be charac-
terized by mononuclear cell infiltration into the central
nervous system (CNS). Autoimmune and virus-induced
animal models of multiple sclerosis have yielded many
insights into the possible mechanisms involved in the
human disease. The study of experimental autoimmune
encephalomyelitis (EAE), which is induced by priming
the animal with myelin antigens — including myelin

86 | FEBRUARY 2002 | VOLUME 2 www.nature.com/reviews/immunol

© 2002 Macmillan Magazines Ltd

T HELPER TYPE 1 (TH1)
CD4+ T cells have been divided
into at least two distinct types.
TH1 cells produce IFN-γ,
lymphotoxin and TNF-α, and
mediate macrophage
inflammatory responses such as
delayed-type hypersensitivity
(DTH). Demyelination in
multiple sclerosis models is
thought to be due to TH1 cells.
TH2 cells produce IL-4, IL-10
and/or TGF-β, and can
downregulate TH1 responses.
INTRAMOLECULAR EPITOPE
SPREADING
Spreading from one epitope to
another on the same molecule,
for example, from PLP139–151 to
PLP178–191.
INTERMOLECULAR EPITOPE
SPREADING
Spreading of the specificity of an
immune response from an
epitope on one molecule to one
on a different molecule is termed
intermolecular epitope
spreading. An example would be
the spread in EAE induced with
PLP139–151, an epitope on
proteolipid protein, to an
epitope on myelin basic protein,
such as MBP84–104.
Table 1 | Clinical disease in induced mouse models of multiple sclerosis
Mouse strain
SJL
(SJLxSWR)F1
B10.PL
C57BL/B6
*Relapsing–remitting disease is characterized by an acute paralytic episode, followed by a significant
clinical recovery (remission) period and, later, a significant worsening of clinical symptoms. There can
be multiple relapses.
‡
Monophasic disease is characterized by an acute paralytic episode followed by complete recovery
from clinical symptoms.
§
Chronic disease is characterized by an acute paralytic episode followed by stabilization or, often, a
gradual worsening of disease symptoms.
MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; PLP, proteolipid protein.
NATURE REVIEWS | IMMUNOLOGY
basic protein (MBP), proteolipid protein (PLP), myelin
oligodendrocyte glycoprotein (MOG) and myelin pep-
tides — led to the initial description of epitope spread-
ing1,2. EAE has been an excellent model in which to
study this process because in this model disease is easily
induced by a defined peptide, and the changing speci-
ficity of the immune response to endogenous myelin
epitopes after disease initiation and subsequent tissue
damage can be followed over time. In addition, EAE
presents as either an acute, relapsing–remitting or
chronic disease (as does multiple sclerosis), depending
on the mouse strain used and the priming myelin epi-
topes (TABLE 1). Multiple investigators have characterized
a hierarchical order of epitope spreading in various EAE
systems and have shown a pathological role for this
process in chronic disease progression3,5–9.
Our laboratory has extensively characterized the role
of epitope spreading in relapsing EAE initiated by prim-
ing SJL mice with the immunodominant PLP epitope,
PLP139–151. In this model, PLP139–151-specific CD4+ T-cell
reactivity is induced within 3 days of priming and is
maintained throughout the disease course. Immediately
before, and continuing during, the first relapse,
PLP178–191 reactivity (INTRAMOLECULAR EPITOPE SPREADING) is
detected by T-cell proliferation and delayed-type hyper-
sensitivity (DTH) assays, and during the second relapse,
MBP84–104 responses (INTERMOLECULAR EPITOPE SPREADING)
are identified (FIG. 2a). The development of these
responses has been shown to correlate with the extent
of myelin destruction during the acute disease phase,
and T cells isolated from the CNS of sick mice prolifer-
ate to the spread epitopes PLP178–191 and MBP84–104.
These T cells can also transfer disease to naive recipi-
ents9. More convincingly, peptide-specific tolerance to
the relapse-associated epitopes blocks disease progres-
sion, even though peripheral T-cell responses against
the disease-inducing peptide PLP139–151 remain intact10.
For example, PLP178–191-specific tolerance induction
during remission from PLP139–151-induced EAE reduced
the relapse incidence from 80% in controls to 20% in
tolerant mice.
Inducing epitope Disease pattern
PLP139–151 Relapsing–remitting*
PLP178–191 Relapsing–remitting*
MBP84–104 Relapsing–remitting*
PLP139–151 Relapsing–remitting*
MBP1–11 Monophasic‡
PLP178–191 Relapsing-remitting
MOG35–55 Monophasic/chronic§
PLP178–191 Monophasic‡
© 2002 Macmillan Magazines Ltd
REVIEWS
It is also interesting that the hierarchical order of
epitope spreading during relapsing EAE in the SJL
mouse correlates with the size of the T-cell repertoire,
with the pattern of spreading proceeding from the
most to the least immunodominant epitope (that is,
PLP139–151>PLP178–191>MBP84–104)10. In the SJL mouse, it
has long been known that the response to the PLP139–151
epitope is dominant in EAE induced by mouse spinal
cord homogenate11. This is in agreement with recent
findings of a very high precursor frequency of
PLP139–151-reactive T cells in the periphery of naive SJL
mice. This high precursor frequency is partly due to
lack of thymic deletion to PLP139–151, because an alter-
native isoform of PLP (DM20, which lacks residues
116–150) is more abundantly expressed in the thymus
than is full-length PLP12. Even though other encephali-
togenic epitopes such as PLP178–191 and MBP84–104 bind
the SJL major histocompatibility complex (MHC) class
II molecule with efficiency close to that of PLP139–151,
induction of EAE with any myelin epitope other than
PLP139–151 invariably leads to spreading first to
PLP139–151-specific T cells9,10.
Some circumstantial evidence that epitope spreading
occurs in human autoimmune disease is presented later
in this review. If epitope spreading proves to be an inte-
gral part of chronic human autoimmune disease, pep-
tide-specific therapies may be problematic, as the speci-
ficity of the pathogenic response would change over
time. However, full T-cell activation, and therefore func-
tional epitope spreading, requires co-stimulatory signals
such as those provided by CD28–CD80/86 and
CD154–CD40 interactions. In this regard, short-term
blockade of CD28–CD80 co-stimulation using anti-
CD80 F(ab) fragments administered after recovery
from acute PLP139–151-induced relapsing EAE resulted in
a striking reduction in relapse incidence13. This protec-
tion was long lasting, and worked efficiently even when
treatment began after the first relapse. Importantly,
CD28 blockade seemed to lead to specific tolerance of
T-cell responses to the relapse-associated PLP178–191 epi-
tope10. For example, when administered before the first
relapse, anti-CD80 F(ab) therapy resulted in PLP178–191-
specific tolerance and protection from disease relapse,
even though peripheral T HELPER TYPE 1 (T 1) responses to
H
the initiating PLP139–151 epitope were unaffected.
Similarly, we have shown that short-term blockade of
the CD40–CD154 interaction using a monoclonal anti-
CD154 antibody administered either at the peak of
acute disease, or during remission from acute disease,
significantly inhibited disease relapse concomitant with
suppression of T-cell reactivity to the relapse-associated
myelin epitopes14. These results indicate that epitope
spreading plays a major role in EAE progression in SJL
mice, and short term co-stimulatory blockade can
specifically inhibit initiation of T-cell responses to the
spread epitope and expression of clinical relapses.
Interestingly, treatment of SJL mice during ongoing
relapsing EAE using intact antibodies or F(ab) frag-
ments specific for the downregulatory co-stimulatory
molecule cytotoxic T-lymphocyte-associated protein 4
(CTLA-4) led to exacerbated disease progression and
VOLUME 2 | FEBRUARY 2002 | 8 7
REVIEWS

a Acute phase Primary relapse Secondary relapse b Disease onset Early chronic Late chronic

Anti-PLP139–151 Anti-PLP178–191 Anti-MBP84–104 Anti-TMEV Anti-TMEV Anti-TMEV

Anti-PLP139–151 Anti-PLP139–151
Anti-PLP178–191
Anti-PLP56–70

Clinical severity

Clinical severity
Anti-MOG92–106
Anti-MBP84–104

10 20 30 40 50 60 70 20 40 60 80 100 120 140

Days post-induction Days post-infection
PLP139–151 TMEV
Primed

Figure 2 | Hierarchical pattern of intramolecular and intermolecular epitope spreading in PLP139–151-induced relapsing
EAE and Theiler’s virus-induced demyelinating disease (TMEV-IDD). a | The relapsing–remitting clinical course and pattern
of epitope spreading in relapsing-EAE in the SJL mouse induced by priming with the immunodominant PLP139–151 epitope. Acute
disease is mediated by PLP139–151-specific TH1 cells, which are then downregulated, resulting in disease remission. Myelin
destruction during the acute clinical episode leads to the priming of T cells to a secondary epitope, PLP178–191 (intramolecular
epitope spreading), which mediates the primary relapse. After regulation of these cells, a third wave of TH1 cells is induced to the
lesser immunodominant MBP84–104 epitope (intermolecular epitope spreading), which mediates the secondary relapse. b | The
clinical course and pattern of epitope spreading in TMEV-IDD in the SJL mouse. Disease onset is initiated by TMEV-specific CD4+
T cells, which arise several days after intracerebral infection and release proinflammatory cytokines upon encountering virus
epitopes presented by persistently infected antigen-presenting cells in the CNS. These virus-specific responses persist throughout
the chronic disease course. Myelin debris released as a result of the initial tissue damage leads to the induction of CD4+ TH1
responses against myelin epitopes, which occurs in a hierarchical order, beginning with responses to the immunodominant
PLP139–151 epitope and then progressing to less-dominant myelin epitopes. MBP, myelin basic protein; MOG, myelin
oligodendrocyte glycoprotein; PLP, proteolipid protein; TMEV, Theiler’s murine encephalomyelitis virus.

ISOLATED MONOSYMPTOMATIC
DEMYELINATING SYNDROME
(IMDS). IMDS is a group of
distinct clinical disorders often
associated with eventual
progression toward clinically
definite multiple sclerosis.
TCR Vβ CDR3 SPECTRATYPING
Polymerase chain reaction-
based method of identifying
pseudoclonal TCR usage by
analyzing Vβ family gene usage.
In independent reactions,
Vβ–Cβ products across the
CDR3 region are amplified from
cDNA, tagged with a
fluorochrome, and resolved on a
polyacrylamide gel
electrophoresis gel. Expanded
pseudoclonal Vβ–Cβ products
of a single length are
distinguished from other
Vβ–Cβ products by size
differences introduced at the
coding junction.
enhanced T-cell reactivity to both inducing and relapse-
associated epitopes15,16. This indicates that normally,
CTLA-4-mediated signalling negatively regulates the
dynamic spread of autoreactive T-cell responses during
the course of autoimmune disease.
Relapsing EAE induced in (SWR×SJL)F1 mice has
also been extensively characterized. The immune
response to the inducing peptide PLP139–151 declines with
time, whereas responses to new myelin epitopes
emerge5,17. Interestingly, interferon-β (IFN-β) treat-
ment, which reduces disease relapses in these mice and
in multiple sclerosis patients18,19, inhibits epitope spread-
ing in the mouse model20. Responses to PLP139–151 after
IFN-β treatment are essentially unchanged, with one
important exception. Splenocytes from mice treated
with IFN-β and activated ex vivo with PLP139–151 pro-
duced significantly more interleukin-10 (IL-10) and less
IL-12 than control mice, indicating that IFN-β may
reduce the relapse rate by creating a TH2-like environ-
ment in which epitope spreading is blocked20. IL-10
inhibits expression of proinflammatory cytokines21,22,
MHC class II molecules23 and co-stimulatory24 mole-
cules by antigen-presenting cells (APCs). These proper-
ties would probably limit tissue destruction and the abil-
ity of APCs to activate T cells that are specific to new
antigens; that is, epitope spreading.
Epitope spreading has also been described by
McFarland et al. in the nonhuman primate (marmoset)
MP4-induced EAE model. MP4 is a fusion protein con-
sisting of PLP and MBP. In this model, demyelination
was found to be associated with a humoral response to
MOG, a myelin protein not included in the inducing
MP4 fusion protein25.
Although more difficult to prove, there is evidence
that epitope spreading occurs in human multiple sclero-
sis. Recently, Tuohy et al. followed T-cell responses over
several years in three IMDS (ISOLATED MONOSYMPTOMATIC
DEMYELINATING SYNDROME) patients, showing that T-cell
autoreactivity to particular PLP epitopes decreased with
time after diagnosis. Interestingly, when two of these
patients progressed to clinically definite multiple sclero-
sis, autoreactivity to PLP peptides other than those first
observed appeared17,26,27. Goebeles et al. studied five
multiple sclerosis patients by isolating MBP-specific
T-cell lines at five time points. Specificity was deter-
mined using a panel of overlapping peptides and rep-
ertoire analysis was done by TCR Vβ CDR3 SPECTRATYPING.
It was concluded that a pattern of a focused, stable epi-
tope response is the exception rather than the rule in
progressive multiple sclerosis28.
Virally induced models of multiple sclerosis. Viruses
and other infectious insults are implicated in the aeti-
ology of many human autoimmune diseases, includ-
ing multiple sclerosis 29. There are various mecha-
nisms by which infection can lead to the initiation of
an autoimmune response. These include molecular
mimicry, bystander activation, release of cryptic epi-
topes and epitope spreading (FIG. 3). Spreading from
viral to self epitopes has been shown or suggested to

88 | FEBRUARY 2002 | VOLUME 2 www.nature.com/reviews/immunol

© 2002 Macmillan Magazines Ltd

play a pathological role in several virus-induced
autoimmune disease models, whereas bystander acti-
vation and molecular mimicry are thought to be
involved in other diseases (reviewed in REF. 30) (TABLE 2).
We have examined the role of epitope spreading in
initiating anti-myelin autoimmune responses in the
chronic stages of Theiler’s murine encephalitogenic
virus-induced demyelinating disease (TMEV-IDD).
TMEV, a picornavirus and natural mouse pathogen,
induces a life-long persistent infection of CNS-resi-
dent APCs, leading to a chronic–progressive CD4+
T-cell-mediated demyelinating disease in susceptible
mouse strains such as SJL/J. Myelin destruction is ini-
tiated by virus-specific CD4 + T cells, which target
CNS-persistent virus31. Peripheral T-cell responses to
myelin epitopes develop during progressive disease in
a hierarchical order, beginning with the dominant
PLP139–151 peptide (FIG. 2b), but only after the onset of
demyelination32,33. As disease progresses, responses to
various other less-dominant encephalitogenic myelin
epitopes (such as PLP178–191, PLP56–70 and MOG92–106)
develop. Autoreactivity is initiated in part by the
in vivo processing of myelin debris and subsequent
presentation of myelin epitopes by CNS-resident
APCs34. CNS APCs harvested before any signs of
demyelination and disease (<40 days after infection)
present viral peptides, but not myelin peptides34.
However, by 90 days post-infection, microglia and
macrophages isolated from the spinal cords of diseased
mice endogenously present both viral and myelin epi-
topes to T cell lines and hybridomas34. Borrow et al. also
showed that DTH responses to various myelin epitopes
were activated during ongoing TMEV-IDD35.
Significantly, induction of tolerance to multiple
encephalitogenic myelin epitopes, using the MP-4
fusion protein in SJL mice with ongoing TMEV-IDD,
attenuated disease progression and resulted in signifi-
cantly less demyelination and decreased inflammatory
cell infiltration in the CNS36. These results support the
hypothesis that tissue damage initiated by virus-specific
T cells results in presentation of self antigens and acti-
vation of autoreactive T cells in the inflammatory CNS
environment, indicating a pathological role for epitope
spreading in virus-induced autoimmune disease.
Table 2 | Proposed mechanisms for virus-induced autoimmunity
Animal virus model Human autoimmune
disease
Theiler’s murine encephalomyelitis Multiple sclerosis
virus (TMEV)
Recombinant TMEV* Multiple sclerosis
Semliki forest virus (SFV) Multiple sclerosis
Coxsackie B3 virus Myocarditis
INSULITIS Coxsackie B4 Virus Diabetes
Inflammation surrounding the Mouse cytomegalovirus (MCMV) Myocarditis
insulin-producing β-cells in the
pancreas. Diabetes occurs when
Herpes simplex virus type 1 (HSV-1) Herpes stromal keratitis
β-cells can no longer produce
adequate amounts of insulin. *Induced by infection with a TMEV variant engineered to express an epitope mimic of proteolipid protein residues 139–151.
NATURE REVIEWS | IMMUNOLOGY
© 2002 Macmillan Magazines Ltd
REVIEWS
NOD mouse model of type 1 diabetes. Type 1 diabetes is
characterized by a chronic autoimmune response
against islet β-cells, which begins early in life. Clinical
diabetes, which is the result of the subsequent loss of
these cells and their ability to produce insulin, occurs
several years later37. INSULITIS — and subsequently dia-
betes — develops spontaneously in the non-obese dia-
betic (NOD) mouse and is thought to be primarily
dependent on CD4+ TH1 responses. Much work has
been done to characterize the specificity of T-cell
responses in NOD mice as diabetes develops38–44. The
45-kd isoform of glutamic acid decarboxylase (GAD45)
is considered by many (although not all45) investigators
to be the initial target of CD4+ T-cell autoimmunity in
this model of diabetes and early tolerance to this
response has been shown to block epitope spreading
and disease39.
Recent work in human diabetes has focused mainly
on humoral responses. Bonifacio et al. examined serum
antibodies to insulin, GAD and protein tyrosine phos-
phatase (islet antigen 2, IA-2) in offspring of diabetic
parents at various times, starting at birth. Two distinct
autoimmune profiles were observed: a rapid disease
onset at the first appearance of an autoantibody
response and a slower epitope spreading stage with a
later onset of disease. The early response in both sub-
sets (at ~ 2–3 yr of age) was typically immunoglobulin
G1 (IgG1) and most commonly to insulin. IgG1 anti-
bodies to GAD and IA-2 often appeared simultane-
ously or soon after 2–3 yr of age. In the group with
slower disease onset, these initial responses declined
(due to antigen-specific regulation) and responses to
other antigens appeared46. These investigators also
examined epitope spreading, specifically in the anti-
GAD antibody response, from birth to disease onset in
offspring of diabetic parents. The initial response usu-
ally involved anti-GAD64 residues 96–444 and epitope
spreading was evident in most offspring47. GAD-spe-
cific epitope spreading was also seen in another large
study group of diabetic patients48.
Interestingly, pancreas graft rejection in diabetic
recipients (under generalized immune suppression)
was associated with increased or sustained high levels
of humoral islet autoimmunity and with epitope
Proposed mechanism for References
virus-induced autoimmunity
Epitope spreading 32
Molecular mimicry 84
Bystander activation, 85,86
molecular mimicry
Bystander activation, 87,88
molecular mimicry
Bystander activation 89
Bystander activation 87,88
Bystander activation, 90,91
molecular mimicry
VOLUME 2 | FEBRUARY 2002 | 8 9
REVIEWS

A Molecular mimicry B Epitope spreading

a Persistent
microbial infection

c Self-tissue d Self-tissue b Microbial

destruction e Self-tissue peptide
d Self-tissue destruction peptides
peptide mimic
Inflammatory
cytokines
Macrophage Macrophage sTH1
b Inflammatory TCR MHC II
MHC II TCR cytokines

APC mTH1 mTH1 APC

c
a

f Epitope spread from

Infectious microbial to self-peptides sTH1
agent
Peptide
MHC II TCR

mTH1

Macrophage
a Microbial
C Bystander activation D Cryptic antigens infection of
tissue

d IFN-γ

Increased Self-tissue f a Microbial

self-antigen destruction infection e Activation
presentation
f APC engulfs b Microbial
of APC self peptides peptides i Self-tissue
destruction
b Microbial
APC peptide
mTH1 APC
c Inflammatory c
cytokines
and chemokines
d Activation
of APC

h Inflammatory
mTH1 APC sTH1 cytokines

g Cryptic antigen
processed and
sTH1 e Recruitment presented
of self-reactive
T cells

Figure 3 | Mechanisms of infection-induced autoimmunity. After a microbial infection, activated microbe-specific TH1 (mTH1)
cells migrate to the infected organ. A | Molecular mimicry describes the activation of crossreactive TH1 cells that recognize both the
microbial epitope (mTH1) and the self epitope (sTH1) (a). Activation of the crossreactive T cells results in the release of cytokines and
chemokines (b) that recruit and activate monocytes and macrophages, which mediate self-tissue damage (c). The subsequent
release of self-tissue antigens and their uptake by APCs perpetuates the autoimmune disease (d). B | Epitope spreading involves a
persistent microbial infection (a) that causes the activation of microorganism-specific TH1 cells (b,c), which mediate self-tissue
damage (d). This results in the release of self peptides (e), which are engulfed by APCs and presented to self-reactive TH1 cells (f).
Continual damage and release of self peptides results in the spread of the self-reactive immune response to multiple self-epitopes
(f). C | Bystander activation is the nonspecific activation of self-reactive TH1 cells. Activation of microorganism-specific TH1 cells
(a,b) leads to inflammation (c,d) and results in the increased infiltration of T cells at the site of infection and the activation of
self-reactive TH1 cells by TCR-dependent and -independent mechanisms (e) Self-reactive T cells activated in this manner mediate
self-tissue damage and perpetuate the autoimmune response (f). D | Cryptic antigen model describing the initiation of
autoimmunity by differential processing of self peptides. Following microbial infection (a) IFN-γ is secreted by both activated
microbe-specific TH1 cells (b,c) and microbe-infected tissue cells (d). This activates APCs (e) and can lead to APC engulfing
self-antigens (f). Cytokine activation of APCs can induce increased protease production and different processing of captured
self-antigens, resulting in presentation of cryptic epitopes. The presentation of these cryptic epitopes can activate self-reactive
TH1 cells (g), leading to self-tissue destruction (h,i). APC, antigen-presenting cell; MHC II, major histocompatibility complex class II;
TCR, T-cell receptor. Modified with permission from REF. 30.

90 | FEBRUARY 2002 | VOLUME 2 www.nature.com/reviews/immunol

© 2002 Macmillan Magazines Ltd

HEAT-SHOCK PROTEIN
Heat-shock proteins are
expressed in all cells, including
microbes, when they are
stressed; for example, when they
experience high temperatures.
These proteins can then become
targeted by an immune
response.
COMPLETE FREUND’S ADJUVANT
Used to trigger an immune
response to proteins or peptides
emulsified in the adjuvant; it
consists of freeze-dried
Mycobacterium, emulsifying
agents and mineral oil.
NATURE REVIEWS | IMMUNOLOGY
spreading, but not with the levels of GAD- or IA-2-spe-
cific serum antibody seen before the allotransplanta-
tion49. This implies that recurrent autoimmunity, along
with epitope spreading, leads to transplant rejection in
diabetic patients.
Models of myasthenia gravis. Myasthenia gravis is char-
acterized by the presence of anti-acetylcholine receptor
(AChR) antibodies, which leads to a decreased number
of AChRs at the neuromuscular junction, impaired
neuromuscular transmission and muscle weakness.
Experimental autoimmune myasthenia gravis (EAMG)
can be induced in animals by immunization with
AChR, leading to AChR-specific T-cell responses and
antibody production50. In rabbits, after induction of
disease by immunization with three human extracel-
lular peptides, serum antibodies to native AChR are
present and bind strongly to rabbit, rather than to
human, AChR50,51. This suggests that endogenous pre-
sentation of rabbit AChR — which has recently been
reported to be mediated by myoblasts52 — during the
initial anti-human AChR immune response leads to
epitope spreading.
In another model of EAMG, in which C57BL/6
mice are immunized with AChR, anti-CTLA-4 anti-
body treatment resulted in severe EAMG — which had
a rapid onset — enhanced T-cell responses to AChR
and increased anti-AChR-antibody production. Anti-
CTLA-4 antibody is thought to block a downregulatory
signal and therefore increase any ongoing T-cell
response. Interestingly, mice immunized with the
immunodominant peptide α(146–162) (an extracellu-
lar sequence of the AchR) and treated with anti-CTLA-4
showed symptoms of clinical EAMG. Diversification of
the autoantibody repertoire and enhanced T-cell prolif-
eration against not only the immunizing α(146–162)
peptide, but also other subdominant epitopes, was also
evident. Control mice did not show any signs of clinical
disease, indicating that treatment with anti-CTLA-4
antibody augmented the initial α(146–162)-specific
immune response and led to epitope spreading in both
the T- and B-cell components of this pathological
autoimmune response53.
Human myasthenia gravis — both early and late
onset — is a complex disease that is often characterized
by thymic pathologies such as hyperplasia, atrophy or
thymoma. Several studies have suggested heterogeneity
of T-cell responses against epitopes on the α-, γ- and
δ-subunits of AChR; however, much work remains to
be done50. In addition to T-cell and antibody epitopes
on AChR, antibodies against striated muscle antigens
such as titin are characteristic of generalized myasthe-
nia gravis54. This suggests that the immune response
in myasthenia gravis is not focused on a dominant
epitope. However, studies in patients with the disease
in which responses are monitored over time will be
necessary to determine whether there is evidence of
epitope spreading.
Models of arthritis. Arthritis is an inflammatory disease
of the joint synovium. T-cell reactivity towards various
© 2002 Macmillan Magazines Ltd
REVIEWS
synovium- or cartilage-associated antigens has been
reported, along with reactivity against microbial and self
HEAT-SHOCK PROTEINS (HSPs), which are upregulated by
cells under stress and released by necrotic cell death.
Adjuvant arthritis, a model of rheumatoid arthritis55,56,
is induced in the Lewis rat by injection of COMPLETE
FREUND’S ADJUVANT, which contains heat-killed
Mycobacterium tuberculosis. There is evidence to suggest
that epitope spreading has a role in the pathogenesis of
adjuvant arthritis. Moudgil et al. found that the initial
T-cell response towards the 65-kDa HSP of M. tubercu-
losis was focused on epitopes in the middle of this pro-
tein, with some reactivity near the N-terminus. By 8–10
weeks after disease onset, reactivity to peptides towards
the C-terminus of Mycobacterial hsp65 was evident,
and tolerance induced by pretreatment with these
C-terminal peptides in synthetic adjuvant blocked
arthritis induction57,58. Epitope spreading in this sce-
nario may be secondary to tissue damage, as the spread
epitopes represent conserved sequences shared with rat
Hsp60. Therefore, epitope spreading might have a pro-
tective outcome in this setting. Similarly, synovial T cells
taken just before disease remission from patients with
juvenile chronic arthritis or rheumatoid arthritis show
enhanced responses to self Hsp65 (REF. 59) and are a
good indicator of a favourable prognosis60,61. Further
evidence of epitope spreading in rheumatoid arthritis
comes from a study by Alam et al., who sequenced more
than 650 T-cell receptor (TCR) Vβ D–J junctional
regions from synovial and peripheral blood mononu-
clear cells of two patients with rheumatoid arthritis.
Their results indicate that a dynamic TCR selection
process takes place during disease progression62.
Epitope spreading in organ allograft rejection
Acute transplant rejection is thought to be the result of
direct cytotoxic T lymphocyte (CTL) recognition of
MHC class I molecules expressed on donor APCs,
because elimination of passenger dendritic cells from
the organ substantially reduces transplant rejection. By
contrast, indirect helper CD4+ T-cell recognition of
recipient MHC class II molecules plus alloantigenic
peptide (transplanted tissue processed and presented by
host APCs) is most likely to be responsible for chronic
rejection63. In human studies, onset of acute rejection in
heart transplant recipients was shown to correlate with
T-cell responses to a single dominant epitope on one
alloantigen64,65. However, in allograft recipients with
recurring episodes of rejection or in patients in which
chronic rejection was at its onset, recipient T-cell reac-
tivity could spread to other epitopes within the allo-
geneic MHC molecule, as well as to other alloantigens
expressed by graft tissue. In a recent study, recipients of
heart allografts were monitored for reactivity against
donor human leukocyte antigen (HLA)-DR peptides
before and up to 36 months after transplantation64.
In patients who received a doubly mismatched trans-
plant (HLA-DR disparity at both loci), chronic rejection
was significantly more likely when intermolecular epi-
tope spreading occurred than if reactivity was directed
only to the initially targeted HLA-DR molecule65.
VOLUME 2 | FEBRUARY 2002 | 9 1
REVIEWS
Implications for immunotherapy
Substantial evidence from studies of numerous animal
models and of various human diseases supports the
hypothesis that tissue damage, regardless of the initiat-
ing event, can lead to epitope spreading, which can then
contribute to ongoing disease. Chronic or persistent
infections may trigger, sustain or exacerbate autoim-
mune disease through epitope spreading via antigen
presentation of epitopes released within injured tis-
sue30,66,67. The initial focused immune attack on organ
allografts also spreads to include new T- and B-cell reac-
tivity, leading to chronic graft rejection. However,
tumour vaccination studies suggest that epitope spread-
ing may increase the efficacy of vaccination protocols.
The immune response therefore appears to be
extremely dynamic, with a constantly changing focus.
Although the hierarchy and functional significance of
responses to spread epitopes can be characterized in
defined animal models of immune-mediated disease,
gathering this knowledge in human disease is difficult.
Treatment of autoimmunity and transplant rejection.
What implications do the phenomena of epitope
spreading have for the future of antigen-specific
immunotherapies? Currently, it would be difficult to
define the pattern of epitope spreading in a chronic
human autoimmune disease because the initiating epi-
tope specificity is not known and the epitope domi-
nance and hierarchy of immune responses to multiple
tissue antigens would vary depending on the individ-
ual’s HLA genotype. Therefore, the use of peptide-
specific tolerance to treat chronic autoimmune diseases
in humans may not be practical at this time. However, as
shown in the relapsing EAE models discussed previ-
ously, it is theoretically possible to achieve long-term
amelioration of autoimmune tissue destruction by
inducing peptide-specific tolerance to endogenous tis-
sue epitopes in mice with ongoing disease10. In addition,
using an animal model of myasthenia gravis, Xu et al.
developed a monoclonal antibody that recognizes the
T-cell receptor (TCR) of α(100–116) AChR-specific
T cells. Treatment with this mAb reversed ongoing
EAMG (when given early after disease onset) and
reduced serum antibodies, including antibodies to the
main immunogenic region, α(61–76), of the AChR in
this rat model68. Although these examples show that, in
some disease models, peptide-specific treatments can
ameliorate the ongoing disease, direct translation to
treatment of human disease is currently difficult.
Another mechanism by which peptide-specific toler-
ance may result in long-lasting disease inhibition,
despite epitope spreading, is via bystander suppression.
Treatment of mice to induce TH2-directed immune
deviation before or shortly after priming for disease,
using therapies induced by self peptide administration
by intravenous, oral or nasal routes69–71 or by altered
peptide ligands (APLs)72, has been shown to inhibit
induction of EAE by other epitopes. However, therapy
in all of these studies was started before the onset of
clinical disease and therefore induced a pre-established
anti-inflammatory TH2/regulatory environment. There
92 | FEBRUARY 2002 | VOLUME 2
© 2002 Macmillan Magazines Ltd
is a surprising lack of reports in which bystander sup-
pression is used to treat established disease in animal
models of autoimmunity, so the efficacy of this
approach in the light of ongoing chronic inflammation
is not known. As a cautionary note, a recent clinical trial
in multiple sclerosis that employed an APL of the HLA-
DR2-restricted MBP83–99 epitope led to disease exacerba-
tions in a significant number of patients, in spite of evi-
dence that TH2 responses had been induced73. This may
indicate that TH2 cells can contribute to organ-specific
autoimmunity in a pre-existing inflammatory setting in
which the blood–tissue barrier is compromised.
Autoreactive TH2 cells have been shown to be capable of
mediating diabetes74 and CNS demyelination75 when
transferred to immunodeficient recipients.
As discussed above, our group and others have shown
that short-term blockade of either the CD28–CD80/86
or CD40–CD154 co-stimulatory pathways in animals
with established relapsing EAE specifically inhibits the
initiation of responses to spread epitopes, resulting in
long-term protection from disease progression. Short-
term co-stimulation blockade, therefore, has the net
effect of inducing antigen-specific tolerance, in that
these reagents specifically target ongoing T-cell activa-
tion (epitope spreading), but only short-term treatment
is required.
We have focused primarily on epitope spreading as a
mechanism involved in magnification of pathological
immune responses. It is likely, however, that epitope
spreading is an intrinsic part of an immune reaction,
both in magnifying the initial attack and in its subse-
quent downregulation. Therefore, initiation of TH2
responses via epitope spreading may be an important
intrinsic immunoregulatory mechanism in TH1-
initiated diseases, geared to limit tissue destruction and
promote re-establishment of tissue-specific immune
tolerance. Interestingly, in the NOD model of type 1 dia-
betes, early induction of a TH2 (anti-inflammatory)
response to one specific β-cell autoantigen (βCAA), by
neonatal priming in incomplete freunds adjuvant, accel-
erated epitope spreading of TH2 responses to other
βCAAs. This spreading of TH2 autoimmunity resulted
in active tolerance and reduced disease incidence42,44.
Early treatment with any one of several different
βCAAs prevents the development of diabetes in the
NOD mice, presumably through TH2-mediated epitope
spreading leading to the production of anti-inflamma-
tory cytokines. Although the efficiency of this treatment
in NOD mice declines when initiated at a later stage in
the disease progression, the hope remains that early
treatment of human diabetes might lead to therapeutic
epitope spreading and disease reduction.
Alloantigen-specific tolerance studies also provide
evidence that epitope spreading may be important for
immunoregulation. A study examining skin-graft toler-
ance after donor-specific transfusions found that toler-
ance induced by transfusions mismatched at one MHC
class-I locus actually spread to skin transplants that were
mismatched at two MHC-class-I loci or at one MHC-
class-I locus plus multiple minor histocompatibility
antigens76. This data was interpreted to be the result of
www.nature.com/reviews/immunol

INFECTIOUS TOLERANCE
Production of anti-inflammatory
cytokines (e.g. IL-4, IL-10,
TGF-β) by an antigen-specific
regulatory T cell, which suppress
immune responses to additional
epitopes in a non-specific
manner.
1. Lehmann, P. V., Forsthuber, T., Miller, A. & Sercarz, E. E.
Spreading of T-cell autoimmunity to cryptic determinants of
an autoantigen. Nature 358, 155–157 (1992).
The first description of epitope spreading in an
autoimmune disease.
2. Lehmann, P. V., Sercarz, E. E., Forsthuber, T., Dayan, C. M.
& Gammon, G. Determinant spreading and the dynamics of
NATURE REVIEWS | IMMUNOLOGY
epitope spreading of tolerance, but it might involve INFEC-
+
TIOUS TOLERANCE mediated by CD4 regulatory T cells77.
Implications for tumour and microbial vaccination.
Epitope spreading has also been shown to be involved in
the magnification of beneficial (infection and cancer)
immune responses. For example, El-Shami et al. have
described epitope spreading after ovalbumin
(OVA)257–264 peptide immunization plus EG.70VA (thy-
moma cell line of C57BL/6 transfected with the cDNA
of chicken OVA) challenge in C57BL/6 mice78. Injection
of EG.7OVA alone results in tumour growth and a CTL
response restricted to the immunodominant OVA257–264
epitope. Mice immunized with peptide-pulsed irradi-
ated cells to induce an OVA257–264-specific CTL response
and later challenged with a lethal dose of live EG.7OVA
tumour cells were completely protected. In addition to
OVA257–264, CTL responses in these mice targeted addi-
tional OVA peptides (intramolecular epitope spreading)
and EL4 cells (intermolecular epitope spreading). Mice
that underwent epitope spreading as a result of
OVA257–264 vaccination and EG.7OVA-tumour challenge
were protected when challenged with live EL4 tumour
cells (no OVA antigen present), indicating that the inter-
molecular epitope spreading induced during the vacci-
nation process was protective. Similar results have been
reported in immunity to P815 tumour cells79.
Two recent studies in human cancer research vali-
date research into peptide-based vaccines and indicate
that epitope spreading may play an important role in
their effectiveness. ERBB2 (HER-2/neu) is a self antigen
that is overexpressed in 15–30% of human adenocarci-
nomas. Patients with ERBB2-overexpressing breast or
ovarian tumours received ERBB2 peptides inoculated
together with granulocyte–macrophage colony-stimu-
lating factor (GM-CSF), which has been shown in rats
to elicit CD4+ T-cell responses to the intact protein.
Before vaccination, these patients had no ERBB2-
specific responses80. After vaccination with specific pep-
tides, patients acquired immune responses not only to
ERBB2 peptide but also to peptides not included in the
vaccine (epitope spreading). In another study of
breast/ovarian cancer, patients were injected with autol-
ogous dendritic cells pulsed with ERBB2- or mucin-1-
derived peptides. Data from several patients in this
study indicate that immunization with a single tumour
antigen may induce CTL reactivity towards several
tumour antigens (that is, epitope spreading) in vivo81.
These results indicate that epitope spreading may
increase the efficiency of protein vaccination. Although
the data described here were obtained from tumour
immune responses, the concept may be applicable to
microbial vaccination.
the autoimmune T-cell repertoire. Immunol. Today 14,
203–208 (1993).
3. Lehmann, P. V., Targoni, O. S. & Forsthuber, T. G. Shifting
T-cell activation thresholds in autoimmunity and determinant
spreading. Immunol. Rev. 164, 53–61 (1998).
4. Steinman, L. Despite epitope spreading in the pathogenesis
of autoimmune disease, highly restricted approaches to
© 2002 Macmillan Magazines Ltd
REVIEWS
Concluding remarks
The immune response is remarkably complex and
strives to maintain a balance between pro- and anti-
inflammatory responses. Understanding the tempo-
ral dynamics of sequential immune responses to
newly arising specificities and how these affect dis-
ease pathology and regulation remains an important
area of study in autoimmunity, transplantation and
vaccination. The immune response consists of both
the initial magnification phase — which can be either
deleterious, as in autoimmune disease, or beneficial,
as in vaccinations or infectious disease — and a later
downregulatory phase to return the immune system
to homeostasis. Epitope spreading may be an impor-
tant component of both phases. Knowledge of the
pattern of epitope spreading in human autoimmune
disease or transplant rejection could be used to
design antigen-specific therapies that block ongoing
tissue destruction or organ rejection, respectively.
Alternatively, therapies could be designed to enhance
the natural downregulatory mechanisms. For exam-
ple, magnification of TH2 responses via epitope
spreading could be used to suppress pathogenic TH1
responses for protection against the development of
diabetes, as discussed above.
As discussed, identifying the hierarchy and func-
tional significance of epitopes involved in human
autoimmune diseases is difficult at present. Technical
limitations aside, short-term co-stimulatory blockade
(CD28 or CD154) in animal models has been shown
to affect long-term antigen-specific regulation of
responses to spread epitopes. It is also important to
note that epitope spreading during magnification of
immune responses is not always pathological, but can
be beneficial. For example, the use of peptide vaccines
for tumour or microbial vaccination may be more effi-
cient than once thought, given that responses to the ini-
tial peptide may be followed by responses to additional
tumour or microbial peptides via epitope spreading,
enhancing the efficacy of protection or therapy.
Reagents like anti-CTLA-4, which lower the threshold
of T-cell activation and enhance the magnification of
T-cell responses, thereby accelerating epitope spread-
ing, may improve the efficacy of tumour or microbial
peptide vaccines15,82,83.
As described in this review, attempts to manipulate
the pro-inflammatory/anti-inflammatory balance of
the immune system can lead to unexpected conse-
quences. Although the studies discussed here indicate
that there are multiple possible therapeutic avenues to
explore, a much greater understanding of the regulation
of the immune system is required before effective and
safe therapies can be applied to human disease.
immune therapy may still succeed [with a hedge on this bet].
J. Autoimmun. 14, 278–282 (2000).
5. Yu, M., Johnson, J. M. & Tuohy, V. K. A predictable sequential
determinant spreading cascade invariably accompanies
progression of experimental autoimmune encephalomyelitis:
a basis for peptide-specific therapy after onset of clinical
disease. J. Exp. Med. 183, 1777–1788 (1996).
VOLUME 2 | FEBRUARY 2002 | 9 3
REVIEWS
6. Vanderlugt, C. L. et al. The functional significance of epitope
spreading and its regulation by co-stimulatory molecules.
Immunol. Rev. 164, 63–72 (1998).
7. Kumar, V. Determinant spreading during experimental
autoimmune encephalomyelitis: is it potentiating, protecting or
participating in the disease? Immunol. Rev. 164, 73–80 (1998).
8. Tuohy, V. K. et al. The epitope spreading cascade during
progression of experimental autoimmune encephalomyelitis
and multiple sclerosis. Immunol. Rev. 164, 93–100 (1998).
9. McRae, B. L., Vanderlugt, C. L., Dal Canto, M. C. & Miller, S. D.
Functional evidence for epitope spreading in the relapsing
pathology of experimental autoimmune encephalomyelitis.
J. Exp. Med. 182, 75–85 (1995).
The first demonstration that epitope spreading has
pathological significance in ongoing autoimmunity.
10. Vanderlugt, C. L. et al. Pathologic role and temporal
appearance of newly emerging autoepitopes in relapsing
experimental autoimmune encephalomyelitis. J. Immunol.
164, 670–678 (2000).
A demonstration that ongoing autoimmunity and
epitope spreading can be specifically inhibited by
peptide-specific tolerance or blockade of
CD80/86–CD28 co-stimulation.
11. Kennedy, M. K. et al. Inhibition of murine relapsing
experimental autoimmune encephalomyelitis by immune
tolerance to proteolipid protein and its encephalitogenic
peptides. J. Immunol. 144, 909–915 (1990).
12. Anderson, A. C. et al. High frequency of autoreactive myelin
proteolipid protein-specific T cells in the periphery of naive
mice: mechanisms of selection of the self-reactive
repertoire. J. Exp. Med. 191, 761–770 (2000).
13. Miller, S. D. et al. Blockade of CD28/B7-1 interaction
prevents epitope spreading and clinical relapses of murine
EAE. Immunity 3, 739–745 (1995).
14. Howard, L. M. et al. Mechanisms of immunotherapeutic
intervention by anti-CD40L (CD154) antibody in an animal
model of multiple sclerosis. J. Clin. Invest. 103, 281–290
(1999).
Evidence that blockade of CD40/CD154 co-
stimulation can ameliorate ongoing autoimmunity and
epitope spreading.
15. Karandikar, N. J., Eagar, T. A., Vanderlugt, C. L., Bluestone,
J. A. & Miller, S. D. CTLA-4 downregulates epitope
spreading and mediates remission in autoimmune disease.
J. Neuroimmunol. 109, 173–180 (2000).
16. Karandikar, N. J., Vanderlugt, C. L., Bluestone, J. A. & Miller,
S. D. Targeting the B7/CD28:CTLA-4 costimulatory system
in CNS autoimmune disease. J. Neuroimmunol. 89, 10–18
(1998).
17. Tuohy, V. K., Yu, M., Yin, L., Kawczak, J. A. & Kinkel, P. R.
Regression and spreading of self-recognition during the
development of autoimmune demyelinating disease.
J. Autoimmun. 13, 11–20 (1999).
18. Rudick, R. A. Disease-modifying drugs for relapsing-
remitting multiple sclerosis and future directions for multiple
sclerosis therapeutics. Arch. Neurol. 56, 1079–1084 (1999).
19. Arnason, B. G. Immunologic therapy of multiple sclerosis.
Annu. Rev. Med. 50, 291–302 (1999).
20. Tuohy, V. K. et al. Modulation of the IL-10/IL-12 cytokine
circuit by interferon-β inhibits the development of epitope
spreading and disease progression in murine autoimmune
encephalomyelitis. J. Neuroimmunol. 111, 55–63 (2000).
21. Fiorentino, D. F., Zlotnik, A., Mosman, T. R., Howard, M. H.
& O’Garra, A. IL-10 inhibits cytokine production by activated
macrophages. J. Immunol. 147, 3815–3822 (1991).
22. Fiorentino, D. F. et al. IL-10 acts on the antigen-presenting
cell to inhibit cytokine production by TH1 cells. J. Immunol.
146, 3444–3451 (1991).
23. De Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C. G. &
De Vries, J. E. Interleukin 10 (IL-10) inhibits cytokine
synthesis by human monocytes: an autoregulatory role of
IL-10 produced by monocytes. J. Exp. Med. 174,
1209–1220 (1991).
24. Ding, L. & Shevach, E. M. IL-10 inhibits mitogen-induced
T cell proliferation by selectively inhibiting macrophage
costimulatory function. J. Immunol. 148, 3133–3139 (1992).
25. McFarland, H. I. et al. Determinant spreading associated
with demyelination in a nonhuman primate model of multiple
sclerosis. J. Immunol. 162, 2384–2390 (1999).
26. Tuohy, V. K., Yu, M., Weinstock-Guttman, B. & Kinkel, R. P.
Diversity and plasticity of self recognition during the
development of multiple sclerosis. J. Clin. Invest. 99,
1682–1690 (1997).
An initial demonstration of spreading and focusing of
responses to PLP epitopes during the progression of
multiple sclerosis.
94 | FEBRUARY 2002 | VOLUME 2
27. Tuohy, V. K., Yu, M., Yin, L., Kawczak, J. A. & Kinkel, R. P.
Spontaneous regression of primary autoreactivity during
chronic progression of experimental autoimmune
encephalomyelitis and multiple sclerosis. J. Exp. Med. 189,
1033–1042 (1999).
28. Goebels, N. et al. Repertoire dynamics of autoreactive T
cells in multiple sclerosis patients and healthy subjects:
epitope spreading versus clonal persistence. Brain 123,
508–518 (2000).
29. Kurtzke, J. F. Epidemiologic evidence for multiple sclerosis
as an infection. Clin. Microbiol. Rev. 6, 382–427 (1993).
30. Olson, J. K., Croxford, J. L. & Miller, S. D. Virus-induced
autoimmunity: potential role of viruses in initiation,
perpetuation, and progression of T cell-mediated
autoimmune diseases. Viral Immunol. 14, 227–250 (2001).
31. Karpus, W. J., Pope, J. G., Peterson, J. D., Dal Canto, M. C.
& Miller, S. D. Inhibition of Theiler’s virus-mediated
demyelination by peripheral immune tolerance induction.
J. Immunol. 155, 947–957 (1995).
32. Miller, S. D. et al. Persistent infection with Theiler’s virus
leads to CNS autoimmunity via epitope spreading. Nature
Med. 3, 1133–1136 (1997).
The first description that a persistent virus infection
can lead to autoimmunity via epitope spreading.
33. Katz-Levy, Y. et al. Temporal development of autoreactive
TH1 responses and endogenous antigen presentation of self
myelin epitopes by CNS-resident APCs in Theiler’s virus-
infected mice. J. Immunol. 165, 5304–5314 (2000).
34. Katz-Levy, Y. et al. Endogenous presentation of self myelin
epitopes by CNS-resident APCs in Theiler’s virus-infected
mice. J. Clin. Invest. 104, 599–610 (1999).
Evidence of endogenous presentation of self epitopes
by resident APCs in the target tissue of the disease.
35. Borrow, P. et al. Investigation of the role of delayed-type-
hypersensitivity responses to myelin in the pathogenesis of
Theiler’s virus-induced demyelinating disease. Immunology
93, 478–484 (1998).
36. Neville, K. L., Padilla, J. & Miller, S. D. Myelin-specific
tolerance attenuates the progression of a virus-induced
demyelinating disease: implications for the treatment of MS.
J. Neuroimmunol. (In the press).
37. Eisenbarth, G. S. Type I diabetes mellitus. A chronic
autoimmune disease. N. Engl. J. Med. 314, 1360–1368
(1986).
38. Delovitch, T. L. & Singh, B. The nonobese diabetic mouse as
a model of autoimmune diabetes: immune dysregulation
gets the NOD. Immunity 7, 727–738 (1997).
39. Kaufman, D. L. et al. Spontaneous loss of T cell tolerance to
glutamic acid decarboxylase in murine insulin-dependent
diabetes. Nature 366, 69–72 (1993).
40. Zechel, M. A., Elliott, J. F., Atkinson, M. A. & Singh, B.
Characterization of novel T-cell epitopes on 65 kDa and
67 kDa glutamic acid decarboxylase relevant in autoimmune
responses in NOD mice. J. Autoimmun. 11, 83–95 (1998).
41. Zechel, M. A., Chaturvedi, P. & Singh, B. Characterization of
immunodominant peptide determinants of IDDM-associated
autoantigens in the NOD mouse. Res. Immunol. 148,
338–348 (1997).
42. Tian, J., Lehmann, P. V. & Kaufman, D. L. Determinant
spreading of T helper cell 2 (TH2) responses to pancreatic
islet autoantigens. J. Exp. Med. 186, 2039–2043 (1997).
Evidence of protective epitope spreading.
43. Zechel, M. A., Krawetz, M. D. & Singh, B. Epitope
dominance: evidence for reciprocal determinant spreading
to glutamic acid decarboxylase in non-obese diabetic mice.
Immunol. Rev. 164, 111–118 (1998).
44. Tian, J. et al. Infectious TH1 and TH2 autoimmunity in
diabetes-prone mice. Immunol. Rev. 164, 119–127 (1998).
45. Durinovic-Bello, I. Autoimmune diabetes: the role of T cells,
MHC molecules and autoantigens. Autoimmunity 27,
159–177 (1998).
46. Bonifacio, E., Scirpoli, M., Kredel, K., Fuchtenbusch, M. &
Ziegler, A. G. Early autoantibody responses in prediabetes
are IgG1 dominated and suggest antigen-specific
regulation. J. Immunol. 163, 525–532 (1999).
47. Bonifacio, E., Lampasona, V., Bernasconi, L. & Ziegler, A. G.
Maturation of the humoral autoimmune response to
epitopes of GAD in preclinical childhood type 1 diabetes.
Diabetes 49, 202–208 (2000).
48. Sohnlein, P. et al. Epitope spreading and a varying but not
disease-specific GAD65 antibody response in type I
diabetes. The Childhood Diabetes in Finland Study Group.
Diabetologia 43, 210–217 (2000).
49. Braghi, S. et al. Modulation of humoral islet autoimmunity by
pancreas allotransplantation influences allograft outcome in
patients with type 1 diabetes. Diabetes 49, 218–224 (2000).
© 2002 Macmillan Magazines Ltd
50. Vincent, A. et al. Determinant spreading and immune
responses to acetylcholine receptors in myasthenia gravis.
Immunol. Rev. 164, 157–168 (1998).
51. Vincent, A., Jacobson, L. & Shillito, P. Response to human
acetylcholine receptor α 138–199: determinant spreading
initiates autoimmunity to self-antigen in rabbits. Immunol.
Lett. 39, 269–275 (1994).
52. Curnow, J., Corlett, L., Willcox, N. & Vincent, A.
Presentation by myoblasts of an epitope from endogenous
acetylcholine receptor indicates a potential role in the
spreading of the immune response. J. Neuroimmunol. 115,
127–134 (2001).
53. Wang, H. B. et al. Anti-CTLA-4 antibody treatment triggers
determinant spreading and enhances murine myasthenia
gravis. J. Immunol. 166, 6430–6436 (2001).
54. Yamamoto, A. M. et al. Anti-titin antibodies in myasthenia
gravis: tight association with thymoma and heterogeneity of
nonthymoma patients. Arch. Neurol. 58, 885–890 (2001).
55. Van Eden, W. et al. Heat-shock protein T-cell epitopes
trigger a spreading regulatory control in a diversified
arthritogenic T-cell response. Immunol. Rev. 164, 169–174
(1998).
56. Sonderstrup, G. & McDevitt, H. Identification of autoantigen
epitopes in MHC class II transgenic mice. Immunol. Rev.
164, 129–138 (1998).
57. Moudgil, K. D. et al. Diversification of T cell responses to
carboxy-terminal determinants within the 65-kD heat-shock
protein is involved in regulation of autoimmune arthritis.
J. Exp. Med. 185, 1307–1316 (1997).
58. Moudgil, K. D. Diversification of response to hsp65 during
the course of autoimmune arthritis is regulatory rather than
pathogenic. Immunol. Rev. 164, 175–184 (1998).
59. Prakken, A. B. et al. Autoreactivity to human heat-shock
protein 60 predicts disease remission in oligoarticular
juvenile rheumatoid arthritis. Arthritis Rheum. 39,
1826–1832 (1996).
60. Prakken, A. B. et al. T-cell reactivity to human HSP60 in
oligo-articular juvenile chronic arthritis is associated with a
favorable prognosis and the generation of regulatory
cytokines in the inflamed joint. Immunol. Lett. 57, 139–142
(1997).
61. deGraeff-Meeder, E. R. et al. Juvenile chronic arthritis: T cell
reactivity to human HSP60 in patients with a favorable
course of arthritis. J. Clin. Invest. 95, 934–940 (1995).
62. Alam, A. et al. Persistence of dominant T cell clones in
synovial tissues during rheumatoid arthritis. J. Immunol.
156, 3480–3485 (1996).
63. Bradley, J. A. Indirect T cell recognition in allograft rejection.
Int. Rev. Immunol. 13, 245–255 (1996).
64. Suciu-Foca, N., Harris, P. E. & Cortesini, R. Intramolecular
and intermolecular spreading during the course of organ
allograft rejection. Immunol. Rev. 164, 241–246 (1998).
65. Ciubotariu, R. et al. Persistent allopeptide reactivity and
epitope spreading in chronic rejection of organ allografts.
J. Clin. Invest 101, 398–405 (1998).
Evidence of epitope spreading in allograft rejection.
66. Di Rosa, F. & Barnaba, V. Persisting viruses and chronic
inflammation: understanding their relation to autoimmunity.
Immunol. Rev. 164, 17–27 (1998).
67. Dorries, R. The role of T-cell-mediated mechanisms in virus
infections of the nervous system. Curr. Top. Microbiol.
Immunol. 253, 219–245 (2001).
68. Xu, L., Villain, M., Galin, F. S., Araga, S. & Blalock, J. E.
Prevention and reversal of experimental autoimmune
myasthenia gravis by a monoclonal antibody against
acetylcholine receptor-specific T cells. Cell Immunol. 208,
107–114 (2001).
69. Leadbetter, E. A. et al. Experimental autoimmune
encephalomyelitis induced with a combination of myelin
basic protein and myelin oligodendrocyte glycoprotein is
ameliorated by administration of a single myelin basic
protein peptide. J. Immunol. 161, 504–512 (1998).
70. Al-Sabbagh, A., Nelson, P. A., Akselband, Y., Sobel, R. A. &
Weiner, H. L. Antigen-driven peripheral immune tolerance:
suppression of experimental autoimmmune
encephalomyelitis and collagen-induced arthritis by aerosol
administration of myelin basic protein or type II collagen.
Cell. Immunol. 171, 111–119 (1996).
71. Anderton, S. M. & Wraith, D. C. Hierarchy in the ability of
T cell epitopes to induce peripheral tolerance to antigens
from myelin. Eur. J. Immunol. 28, 1251–1261 (1998).
72. Nicholson, L. B., Murtaza, A., Hafler, B. P., Sette, A. &
Kuchroo, V. K. A T cell receptor antagonist peptide induces
T cells that mediate bystander suppression and prevent
autoimmune encephalomyelitis induced with multiple myelin
antigens. Proc. Natl Acad. Sci. USA 94, 9279–9284 (1997).
www.nature.com/reviews/immunol

73. Bielekova, B. et al. Encephalitogenic potential of the myelin
basic protein peptide (amino acids 83–99) in multiple
sclerosis: results of a phase II clinical trial with an altered
peptide ligand. Nature Med. 6, 1167–1175 (2000).
74. Pakala, S. V., Kurrer, M. O. & Katz, J. D. T helper 2 (TH2)
T cells induce acute pancreatitis and diabetes in immune-
compromised nonobese diabetic (NOD) mice. J. Exp. Med.
186, 299–306 (1997).
75. Lafaille, J. J. et al. Myelin basic protein-specific T helper 2
(TH2) cells cause experimental autoimmune
encephalomyelitis in immunodeficient hosts rather than
protect them from the disease. J. Exp. Med. 186, 307–312
(1997).
76. Yang, L., DuTemple, B., Gorczynski, R. M., Levy, G. &
Zhang, L. Evidence for epitope spreading and active
suppression in skin graft tolerance after donor-specific
transfusion. Transplantation 67, 1404–1410 (1999).
77. Waldmann, H. & Cobbold, S. Regulating the immune
response to transplants: a role for CD4+ regulatory cells?
Immunity 14, 399–406 (2001).
78. el Shami, K. et al. MHC class I-restricted epitope spreading
in the context of tumor rejection following vaccination with a
single immunodominant CTL epitope. Eur. J. Immunol. 29,
3295–3301 (1999).
Evidence of MHC-class-I–restricted epitope
spreading in tumour immunity.
79. Markiewicz, M. A., Fallarino, F., Ashikari, A. & Gajewski, T. F.
Epitope spreading upon P815 tumor rejection triggered by
vaccination with the single class I MHC-restricted peptide
P1A. Int. Immunol. 13, 625–632 (2001).
NATURE REVIEWS | IMMUNOLOGY
80. Disis, M. L., Grabstein, K. H., Sleath, P. R. & Cheever, M. A.
Generation of immunity to the HER-2/neu oncogenic
protein in patients with breast and ovarian cancer using a
peptide-based vaccine. Clin. Cancer Res. 5, 1289–1297
(1999).
81. Brossart, P. et al. Induction of cytotoxic T-lymphocyte
responses in vivo after vaccinations with peptide-pulsed
dendritic cells. Blood 96, 3102–3108 (2000).
82. Leach, D. R., Krummel, M. F. & Allison, J. P. Enhancement of
antitumor immunity by CTLA-4 blockade. Science 271,
1734–1736 (1996).
83. Hurwitz, A. A., Yu, T. F., Leach, D. R. & Allison, J. P. CTLA-4
blockade synergizes with tumor-derived
granulocyte–macrophage colony-stimulating factor for
treatment of an experimental mammary carcinoma. Proc.
Natl Acad. Sci. USA 95, 10067–10071 (1998).
84. Olson, J. K., Croxford, J. L., Calenoff, M., Dal Canto, M. C. &
Miller, S. D. A virus-induced molecular mimicry model of
multiple sclerosis. J. Clin. Invest. 108, 311–318 (2001).
Evidence that epitope spreading can be initiated after
induction of autoimmunity via molecular mimicry.
85. Mokhtarian, F., Shi, Y., Zhu, P. F. & Grob, D. Immune
responses, and autoimmune outcome, during virus infection
of the central nervous system. Cell. Immunol. 157, 195–210
(1994).
86. Mokhtarian, F., Zhang, Z., Shi, Y., Gonzales, E. & Sobel, R. A.
Molecular mimicry between a viral peptide and a myelin
oligodendrocyte glycoprotein peptide induces autoimmune
demyelinating disease in mice. J. Neuroimmunol. 95, 43–54
(1999).
© 2002 Macmillan Magazines Ltd
REVIEWS
87. Lawson, C. M. Evidence for mimicry by viral antigens in
animal models of autoimmune disease including
myocarditis. Cell Mol. Life Sci. 57, 552–560 (2000).
88. Fairweather, D., Kaya, Z., Shellam, G. R., Lawson, C. M. &
Rose, N. R. From infection to autoimmunity. J. Autoimmun.
16, 175–186 (2001).
89. Horwitz, M. S. et al. Diabetes induced by Coxsackie virus:
initiation by bystander damage and not molecular mimicry.
Nature Med. 4, 781–786 (1998).
90. Zhao, Z.-S., Granucci, F., Yeh, L., Schaffer, P. A. & Cantor, H.
Molecular mimicry by herpes simplex virus-type 1:
Autoimmune disease after viral infection. Science 279,
1344–1347 (1998).
91. Deshpande, S. P. et al. Herpes simplex virus-induced
keratitis: evaluation of the role of molecular mimicry in lesion
pathogenesis. J. Virol. 75, 3077–3088 (2001).
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink
CD28 | CD40 | CD154 | CD80 | CD86 | CTLA-4 | GAD | GM-CSF |
ERBB2 | HLA-DR | HLA-DR2 | IA-2 | IgG1 | IL-10 | IL-12 | IFN-β |
IFN-γ | MBP | MOG | PLP | TNF-α
OMIM: http://www.ncbi.nlm.nih.gov/Omim
type-1 diabetes | multiple sclerosis | myasthenia gravis |
rheumatoid arthritis
Access to this interactive links box is free online.
VOLUME 2 | FEBRUARY 2002 | 9 5