1290 The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 9 November 2004
A viral epitope that mimics a self antigen
can accelerate but not initiate
Urs Christen,1 Kurt H. Edelmann,2 Dorian B. McGavern,2 Tom Wolfe,1 Bryan Coon,1
Meghann K. Teague,3 Stephen D. Miller,3 Michael B.A. Oldstone,2 and Matthias G. von Herrath1
1Immune Regulation Lab, Department of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California, USA.
2Division of Virology, Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California, USA. 3Department of Microbiology-Immunology,
Northwestern University Medical School, Chicago, Illinois, USA.
We document here that infection of prediabetic mice with a virus expressing an H-2Kb–restricted mimic
ligand to a self epitope present on β cells accelerates the development of autoimmune diabetes. Immunization
with the mimic ligand expanded autoreactive T cell populations, which was followed by their trafficking to
the islets, as visualized in situ by tetramer staining. In contrast, the mimic ligand did not generate sufficient
autoreactive T cells in naive mice to initiate disease. Diabetes acceleration did not occur in H-2Kb–deficient
mice or in mice tolerized to the mimic ligand. Thus, arenavirus-expressed mimics of self antigens accelerate a
previously established autoimmune process. Sequential heterologous viral infections might therefore act in
concert to precipitate clinical autoimmune disease, even if single exposure to a viral mimic does not always
cause sufficient tissue destruction.
Evidence obtained by study of genetically identical twin pairs
indicates that environmental or epigenetic factors play a role
during the pathogenesis of human autoimmune diseases (1).
Furthermore, epidemiological studies have associated infections
with many different viruses with autoimmune diseases like type
1 diabetes (T1D) (2–5) and MS (6–9). However, it is still unclear
how these infectious agents would cause autoimmunity. Molec-
ular mimicry is one hypothesis to explain such a link mecha-
nistically, and it postulates that cross-reactions with “foreign”
(infectious) ligands during host defense activate autoreactive
lymphocytes or, conversely, that lymphocytes activated during
antiviral responses recognize autoantigens, thus initiating an
autoimmune process. Evidence for this concept (reviewed in refs.
10, 11) has involved the demonstration of T lymphocytes or anti-
bodies cross-reactive with host proteins (12, 13). For example, T
lymphocyte clones recovered from patients with MS recognized
both viral epitopes as well as self (myelin) ligands (14). In human
T1D, Honeyman and colleagues have found a significant link
between the occurrence of rotavirus infections in young children
and the emergence of islet antibodies, suggesting cross-reactivity
between rotavirus and islet antigens (15). However, the associa-
tion of T1D and rotavirus infections is still controversial, as a
similar study from Finland (16) could not confirm the results
obtained in the Australian series (15).
Although molecular mimicry has been studied extensively as an
initiating event for autoimmunity, little is known about its effect
on an already established, ongoing autoimmune process. This was
the focus of our study here. Murine animal models of autoimmu-
nity have defined three separate mechanisms that may act alone
or together in the causation of autoimmune disease: molecular
mimicry (reviewed in refs. 17–20), epitope spreading (19, 21), and
bystander activation (22–26). We focused here on molecular mim-
icry using a murine T1D model of heterologous, sequential viral
infections. We elucidated a novel mechanistic link between infec-
tious events and autoimmune disease by showing that mimicry
between viral and self antigens can accelerate but not easily initiate
Heterologous virus infections have typically been considered
positive enhancers of overall immune health through the repeated
activation of cross-reactive memory T lymphocytes (27). Via this
mechanism, immunity to one organism can provide an advan-
tage to the host in combating new infections without severely
diminishing immune memory to the first organism. The benefits
of heterologous infections to the immune system, however, carry
within them a potential downside: a second or tertiary infection
may alter T cell hierarchy in such a way that self-reactive, initially
subdominant T cell populations generated during previous infec-
tions are expanded, leading to enhanced immunopathology and
autoimmune disease. We investigated here whether activation and
expansion of autoreactive lymphocyte populations can occur after
interaction with lower-avidity viral ligands that mimic self ligands
and whether this might lead to clinical disease after repeated stim-
ulation with heterologous viral infections and/or within an already
established local inflammatory environment (28) within the target
organ. The rat insulin promoter–lymphocytic choriomeningitis
virus–nucleoprotein (RIP-LCMV-NP) transgenic mouse model of
autoimmune diabetes, in which the NP from LCMV is expressed
transgenically as a self antigen in pancreatic β cells as well as the
thymus, coupled with LCMV infection leads to LCMV-specific
Nonstandard abbreviations used: EDCI, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; GP, glycoprotein; HSK, herpes stromal keratitis; HSV-1, murine
herpesvirus 1; Kb(–), H-2Kb–deficient; Kb(+), H-2Kb–sufficient; LCMV, lymphocytic
choriomeningitis virus; LCMV-Arm, LCMV strain Armstrong; LCMV-Arm-Var,
LCMV-Arm escape variant; LCMV-Past, LCMV strain Pasteur; NGS, normal goat
serum; NP, nucleoprotein; PDLN, pancreatic draining lymph node; poly(I:C),
polyinosinic-polycytidylic acid; PV, Pichinde virus; RIP, rat insulin promoter; T1D,
type 1 diabetes; TMEV, Theiler murine encephalomyelitis virus.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J. Clin. Invest. 114:1290–1298 (2004).
The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 9 November 2004
autoreactive CD8 and CD4 lymphocytes (29) that initiate the
destruction of β cells (30). Islet destruction requires both perforin
and IFN-γ (31). Importantly, in these mice, the full autoimmune
process in the islets develops after the systemic antiviral T cell
response to the triggering virus (LCMV) has receded and virus
has been cleared. At that time, antibodies against other islet anti-
gens like insulin and glutamic acid decarboxylase appear. Thymic
expression of the viral protein leads to elimination of the majority
of the LCMV-NP–specific autoreactive CD8 T cells, resulting in a
slower disease development that requires both CD4 and CD8 T
cells (29). Thus, the viral NP, which is genetically passed to prog-
eny mice, acts like a true self antigen in RIP-LCMV mice. One can
envision a similar scenario in humans affected by T1D: we know
that many islet antigens are expressed in the thymus; consequent-
ly, the numbers and/or avidity of autoreactive lymphocytes are low
and viral infections that affect the pancreas, among other organs,
are not infrequent.
We found here that heterologous sequential viral infections can
augment autoreactive cross-reactive T lymphocytes in the target
organ above the disease-initiating threshold (32), so that major
tissue injury and diabetes develop much more rapidly. The out-
come of our study here clearly demonstrates that cross-reactivity
between viral and self epitopes can augment but not initiate auto-
immune disease in this model. In addition, our studies show that
the infectious agent essential for initiating the autoimmune pro-
cess can be cleared by the host immune response prior to a second-
ary virus infection, which is responsible for the disease accelera-
tion. The resulting more rapid diabetes development occurs only
if the secondary infection takes place during the prediabetic phase,
but not in healthy, unprimed animals. Thus, in humans, disease
acceleration could be the result of the combined effect of a few to
several immunological cross-reactive viruses.
Challenge of unprimed, naive animals with viruses that express lower-avid-
ity mimic ligands of β-cell epitopes fails to induce autoimmune diabetes.
Infection of RIP-LCMV-NP H-2b transgenic mice, which express
the LCMV strain Armstrong (LCMV-Arm) NP protein (LCMV-NP)
in β cells, with LCMV-Arm leads to T1D 2–4 months after infec-
tion in more than 95% of mice. By 10–14 days after LCMV infec-
tion, virus is cleared from infected mice (29). In order to identify
the T cell epitopes of the viral NP molecule responsible for induc-
tion of diabetes, we infected transgenic mice with LCMV-Arm that
contains LCMV-NP396 and LCMV-NP205 cytotoxic T cell epitopes
or with an LCMV-NP396 cytotoxic T lymphocyte (CTL) escape vari-
ant (LCMV-Arm escape variant; LCMV-Arm-Var) that contains a
single amino acid substitution of phenylalanine to leucine at posi-
tion 403 (substitution underlined: FQPQNGQFI to FQPQNGQ-
LI; Table 1) (33). While infection with LCMV-Arm caused diabetes
with the expected kinetics, infection with LCMV-Arm-Var, which
lacked the LCMV-NP396 epitope but still expressed the normal
LCMV-NP205 epitope (33), did not (Figure 1A). Thus, the LCMV-
NP396 epitope that is recognized with comparatively high avidity
(34) was essential for initiation of T1D in RIP-NP H-2b mice.
To assess the contribution of the LCMV-NP205 CD8 T cell epitope
for induction of diabetes, we utilized Pichinde virus (PV), another
member of the arenavirus family, which contains the cross-reactive
H-2Kb–restricted epitope PV-NP205, YTVKFPNM, that shares six
of eight amino acids with the LCMV-NP205 epitope, YTVKYPNL
(substitutions underlined; Table 1) (27). Although binding of the
LCMV-NP205 and PV-NP205 epitopes to H-2Kb was similar (despite
differences in the MHC anchoring residues at positions 209 and
212), they exhibited differential antigenic properties. PV infection
elicited more CD8 T cells to the PV-NP205 than to the LCMV-NP205
epitope in earlier studies (27), indicating that true cross-reactivity
of the interaction of the NP205-peptide presented by MHC class I in
conjunction with TCR was operational rather than reduced bind-
ing affinity of the mimic peptide to MHC. Despite the presence
of this cross-reactivity with the LCMV-NP transgene, infection of
transgenic mice with PV failed to induce diabetes (Figure 1A). This
lack of disease induction was likely associated with the approxi-
mately 100-fold lower avidity of PV-NP205 compared with LCMV-
NP396 in cytotoxicity assays (27), resulting in a failure to induce a
robust primary CD8 T cells response to the lower-avidity PV-NP205
after LCMV infection. Collectively, these data indicate that upon
infection of RIP-LCMV-NP mice with LCMV-Arm, induction of
diabetes is dependent on the higher-avidity H-2Db–restricted
LCMV-NP396 epitope and not the lower-avidity H-2Kb–restricted
LCMV-NP205 epitope. Based on this study (Figure 1A) and previ-
ous studies (35), we conclude that naturally occurring viral mimics
recognized with comparatively lower avidity are unable to activate
a sufficient number of naive autoreactive lymphocytes to cause
clinical diabetes, even if the infecting virus is tropic to the pancreas
and induces local inflammation of the target organ (36, 37).
A lower-avidity viral mimic ligand of a β-cell CD8 T cell epitope can sig-
nificantly accelerate an ongoing autoimmune process and the development
of clinical disease. We next asked whether the lower-avidity LCMV-
NP205 epitope could influence the diabetic outcome of mice in
the prediabetic stage in which autoreactive processes were already
established. We addressed this question through the use of sequen-
tial heterologous infections of transgenic mice with LCMV-Arm
and PV separated by a 4-week interval. As described earlier (27),
immunity to PV exhibits clear cross-reactivity of the PV epitope
PV-NP205 to the LCMV-NP epitope LCMV-NP205. Indeed, as shown
in Figure 1B, PV infection administered 1 month after the initial
autoimmunity-initiating LCMV infection considerably accelerated
T1D in RIP-LCMV-NP mice. Importantly, the PV infection had to
occur at a time when islet destruction was already ongoing (initi-
ated by LCMV infection 4 weeks earlier), as the reverse scenario
(when PV was given first followed by secondary PV or LCMV infec-
tion) did not accelerate T1D (Figure 1B). This observation shows
that PV can enhance LCMV-induced T1D in RIP-LCMV-NP mice
CD8 T cell epitopes of LCMV-NP and PV-NP
Immunodominant NP epitopes
Subdominant NP epitopes
Immunodominant and subdominant CD8 T cell epitopes of LCMV-NP
and PV-NP are shown with their individual MHC restrictions. Amino
acids shared between LCMV-NP205 and PV-NP205 are underlined.
1292 The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 9 November 2004
when given after but not before the initiation of islet destruction
by LCMV-NP396–specific CTLs.
To define the precise role of cross-reactive CD8 T cells that spe-
cifically react to the NP205 epitope of LCMV and PV (LCMV/PV-
NP205–specific cross-reactive CD8 T cells) in these different infec-
tion scenarios, we quantified their numbers and functional activity
after primary and secondary PV and LCMV infection, respectively.
Figure 2 shows the frequencies of IFN-γ–producing CD8 T cells in
response to the LCMV H-2Db–restricted LCMV-NP396 and domi-
nant LCMV-glycoprotein 33 (LCMV-GP33) peptides, the LCMV
cross-reactive subdominant H-2Kb–restricted LCMV-NP205 peptide,
and the PV dominant PV-NP38 peptide (internal PV control) after
primary LCMV or PV infection and after secondary infection with
LCMV or PV. As expected, primary and secondary PV infection
expanded the dominant PV-NP38–specific population (Figure 2B).
Furthermore, as described earlier (29), infection of RIP-LCMV-NP
mice with LCMV alone resulted in high numbers of LCMV-GP33–
specific CD8 T cells but much lower numbers of LCMV-NP396–spe-
cific CD8 T cells, as this mouse line expresses LCMV-NP in the thy-
mus as well as in the pancreas, resulting in thymic negative selection
of a significant proportion of NP-specific CD8 T cells. The slower
development of disease in RIP-LCMV-NP mice than in RIP-LCMV-
GP mice (38) can be attributed to this fact. Important for our
investigation here is that secondary PV infection in LCMV-infected
RIP-LCMV-NP mice strongly and selectively expanded the LCMV-
NP205–specific CD8 T cell population but none of the other LCMV-
specific populations (LCMV-GP33 and LCMV-NP396). This finding
indicates that LCMV/PV-NP205–specific cross-reactive CD8 T cells
are alone not sufficient to cause T1D in RIP-LCMV-NP mice but
are important for the acceleration of disease observed after second-
ary PV infection. Most likely they must be present in islets together
with LCMV-NP396–specific CD8 T cells in order to cause disease, as
diabetes never developed after single PV infection (Figure 1A). The
selective expansion of LCMV/PV-NP205–specific cross-reactive CD8
T cell populations is in concordance with observations of Brehm
et al. (27) and strengthens the hypothesis that PV-NP205–specific
CD8 T cells play a role in the acceleration of diabetes in mice with
preclinical diabetes. Challenge of mice immune to LCMV (LCMV-
immune mice) with a secondary LCMV infection did not result in
a long-lasting expansion of either LCMV-NP396–specific or LCMV-
NP205–specific cross-reactive CD8 T cell populations (Figure 2B).
This stands well in agreement with our earlier findings that sequen-
tial infection with LCMV-Arm has no influence on the incidence
and kinetics of diabetes in RIP-LCMV-NP mice (39).
We further investigated whether the expansion/activation of
previously primed LCMV/PV-NP205–specific CD8 T cell popula-
tions by the mimic epitope expressed by PV enhanced the effec-
tor functions of these cells. Primary infection of C57BL/6 wild
type or RIP-LCMV-NP mice with PV induced no detectable CD8
T cells response to whole LCMV-NP protein (Table 2). However,
PV-challenged, LCMV-primed mice made clearly detectable recall
responses to whole LCMV-NP, as evidenced by killing assays (Table
2), and to LCMV-NP205 as shown by intracellular cytokine stain-
ing for IFN-γ by flow cytometry (Figure 2C). In agreement with
the results presented in Figure 2C is the substantially increased
number of PV-NP205 CTL precursors in mice that received LCMV
and then PV sequentially (Figure 2D). Thus, expansion of LCMV/
PV-NP205 cross-reactive CD8 T cell populations with lytic activ-
ity (31) and IFN-γ production occurs after PV infection only in
Sequential infection with LCMV and PV results in accumulation of
PV-NP205–specific CD8 T cells in islets. Histological examination of the
pancreas at week 3 after secondary infection of LCMV-primed RIP-
LCMV-NP mice with PV revealed increased lymphocyte infiltration
and destruction of the islets of Langerhans (Figure 3A, lower left
panel). There was profound difference in islet infiltration by CD8
T cells in these mice versus mice that had only been administered
a single LCMV infection (Figure 3A, upper left panel).
A critical component of the hypothesis that LCMV/PV-NP205–
specific cross-reactive CD8 T cells participate in the acceleration
of diabetes is the identification of these cells at the right time
and place after secondary PV infection in relation to the pancreas
Molecular mimicry can accelerate but not easily initiate autoim-
mune diabetes. (A) Molecular mimicry is insufficient to prime naive
autoreactive CD8 T cells and cause autoimmune diabetes. RIP-
LCMV-NP mice were infected with 105 PFU LCMV-Arm (open circles),
LCMV-Arm-Var (filled triangles), or PV alone (filled circles). (B) Primed
autoreactive cells can become activated via molecular mimicry and
accelerate disease. RIP-LCMV-NP mice were infected with either 105
PFU LCMV-Arm (open circles, filled circles) or 105 PFU PV (open tri-
angles, filled triangles) on day 0 and, as indicated, received a second-
ary inoculation (2nd inf.) with PV (open triangles, filled circles) 28 days
after the priming LCMV infection. As a comparison, the incidence data
to RIP-LCMV-NP mice infected with LCMV alone are displayed (open
circles). For both studies, blood glucose values were determined at
weekly intervals. Mice with blood glucose levels above 300 mg/dl were
considered diabetic. It is evident from these studies that secondary
infection but not primary infection with PV can accelerate T1D develop-
ment. Statistical analysis was done using the log rank test. Note that
the diabetes onset curves for the groups LCMV alone versus LCMV-
PV are significantly different (P = 0.0066).
The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 9 November 2004
and, more specifically, the target of the autoimmune response
(the islets of Langerhans). Thus, we generated H-2Kb–PV-NP205
tetramers in attempt to perform in situ tetramer staining for
PV-NP205–specific CD8 T cells in quick-frozen pancreas sections,
similar to what we previously reported for LCMV-GP33–specific
CD8 T cells in the CNS (40). Histological examination using spe-
cific immunohistochemical staining for CD8 T cells and selective
H-2Kb–PV-NP205 tetramer staining on tissue sections for NP205-
specific CD8 T cells revealed that PV-NP205 CD8 T cells were pres-
ent exclusively in islets of mice initially primed with LCMV and
secondarily infected with PV. Between 1 and 3 PV-NP205–specific
CD8 T cells were found in about 50% of all islet sections analyzed.
In contrast, none of the islet sections examined in mice infected
with LCMV alone contained any PV-NP205–specific CD8 T cells
(Figure 3A, upper right panel). Quantification of our data indi-
cated that a frequency of 2–4% of all infiltrating CD8 T cells were
PV-NP205 specific in sequentially infected mice, which reflected the
frequencies displayed in Figure 3B, assessed by flow cytometry in
peripheral lymphoid organs, and corroborated frequencies of islet-
infiltrating GP33-specific CD8 T cells observed both in the RIP-
LCMV-GP fast-onset diabetes model (GP not expressed in thymus)
using H-2Db–LCMV-GP33 tetramers (D.B. McGavern, unpublished
results) and in LCMV-mediated leptomeningitis (40).
The expansion of LCMV/PV-NP205–specific cross-reactive CD8
T cell populations was also demonstrated by flow cytometry
after staining with H-2Kb–PV-NP205 tetramers (Figure 3B) and
intracellular cytokine staining for IFN-γ (Figure 3B, lower panels) in
the blood as well as in the pancreatic draining lymph nodes (PDLNs)
after sequential infection of transgenic mice with LCMV and PV.
In particular, in the PDLNs, H-2Kb–PV-NP205 tetramer+ CD8 T cell
populations expanded to a frequency of 4% after secondary PV infec-
tion, compared with only 0.4% after a single LCMV infection (Figure
3B). Thus, if PV infection occurs after LCMV infection, a selective
and considerable expansion of initially subdominant, lower-avidity
LCMV/PV-NP205–specific cross-reactive CD8 T cell populations is
seen in the target organ and PDLNs.
Activation of LCMV/PV-NP205–specific cross-reactive CD8 T cells through
molecular mimicry is absolutely essential for acceleration of diabetes and
does not occur in mice genetically deficient in H-2Kb and in mice that were
tolerized to PV-NP205. Further evidence that LCMV/PV-NP205–specific
cross-reactive CD8 T cells were responsible for the acceleration of
T1D was obtained from experiments using RIP-LCMV-NP mice
bred onto a H-2Kb–deficient [Kb(–)] background (41). Because these
mice still present the LCMV-NP396 epitope, which is H-2Db restrict-
ed, diabetes still occurred with the expected slower kinetics after
a single LCMV infection (data not shown). However, sequential
infection of these mice with LCMV followed by PV did not result
in accelerated disease, which occurred as expected in RIP-LCMV-
NP × H-2Kb–sufficient [Kb(+)] littermates (Figure 4A). Overall, the
incidence of T1D in RIP-LCMV-NP × Kb(+) littermates mice was
lower and its kinetics slower than in the original RIP-LCMV-NP
line, because background genes introduced by the SV129 embry-
CD8 T cell populations specific for the mimicking epitope PV-NP205 are significantly expanded after sequential infection with PV. (A and B) RIP-
LCMV-NP and RIP-LCMV-NP × Kb(–) mice were infected with 105 PFU LCMV or PV. After 4 weeks, the mice received a secondary infection of
either LCMV or PV. (A) Intracellular cytokine staining (ICCS) after stimulation with PV-NP205 is displayed for 1 representative mouse infected first
with LCMV (LCMV alone) and then with PV (LCMV-PV) (mean frequencies are indicated in boxed areas). (B) The frequency of epitope-specific
CD8 T cells in the blood was determined by ICCS for IFN-γ after stimulation with the indicated peptides (key) immediately before (upper panel)
and 7 days after (lower panel) secondary infection. (C) Numbers of H-2Kb–restricted PV-NP205–specific lymphocytes after LCMV or PV infec-
tion, assessed by ICCS for IFN-γ. Splenocytes were harvested on day 35 from mice that received LCMV at day 0 (d0) and, for the PV group,
PV at day 28 (d28). Means (± SEM) are displayed. (D) Lytic precursors after LCMV-NP396 versus PV-NP205 antigenic stimulation for 10 days.
In addition to lytic activity, IFN-γ production was assessed in the supernatant of each well; wells with IFN-γ levels of more than 0.05 ng/ml by
ELISA were counted as positive. IFN-γ production was on average 13 (± 3.5) ng/ml in LCMV-NP396–stimulated cultures and 7.1 (± 3.1) ng/ml in
PV-NP205–stimulated cultures. This experiment was repeated three times and mean values (± SEM) are displayed. *P < 0.05.
1294 The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 9 November 2004
onic stem cells from knockout lines are known to protect from
T1D in RIP-LCMV mice (42). In contrast to RIP-LCMV-NP × Kb(+)
littermates, RIP-LCMV-NP × Kb(–) mice did not have accelerated
T1D after secondary PV infection. In parallel with the lack of accel-
eration of diabetes in the RIP-LCMV-NP × Kb(–) mice, secondary
infection with PV did not result in expansion of LCMV/PV-NP205–
specific CD8 T cell populations (Figure 2B, right column) or in
enhanced islet infiltration (Figure 4B).
Furthermore, RIP-LCMV-NP mice that were tolerized to the
PV-NP205 epitope showed no acceleration of T1D after second-
ary infection with PV (Figure 4C). Tolerization to PV-NP205 was
achieved by i.v. injection of PV-NP205–coated splenocytes that
were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)-car-
bodiimide (EDCI) (43, 44). Mice that were treated with 2 × 107
syngeneic EDCI–PV-NP205 cross-linked splenocytes 5 days before
primary LCMV infection did not display accelerated T1D after
subsequent infection with PV 1 month after infection with LCMV
(Figure 4C). In contrast, mice that received splenocytes that were
cross-linked with EDCI in absence of PV-NP205 displayed acceler-
ated T1D after secondary infection as usual (Figure 4C). These
experiments are consistent with the necessity of H-2Kb–restricted
NP205-specific T cells in the acceleration of T1D.
LCMV/PV-NP205–specific cross-reactive CD8 T cell populations expand
and accelerate disease in LCMV-immune mice challenged with PV-NP205
peptide and polyinosinic-polycytidylic acid. Final evidence that LCMV/
PV-NP205–specific cross-reactive CD8 T cells are responsible for the
acceleration of T1D was obtained from experiments using RIP-
LCMV-NP mice that were administered PV-NP205 peptide together
with synthetic polyinosinic-polycytidylic acid [poly(I:C)], a “mimic”
of double-stranded viral RNA (Figure 4, D and E). LCMV/PV-NP205–
specific CD8 T cell populations were significantly expanded in
LCMV-immune RIP-LCMV-NP mice that received 100 μg PV-NP205
peptide (i.p.) together with a single injection of 7.5 μg poly(I:C) per
gram body mass (i.p.) at week 4 after LCMV infection (Figure 4D).
The frequency of LCMV/PV-NP205–specific CD8 T cells was much
higher than after administration of the irrelevant H-2Kb–binding
OVA peptide SIINFEKL followed by poly(I:C) or PV-NP205 alone and
even exceeded the frequency observed when LCMV-immune mice
received a secondary infection with PV (Figure 4D). As expected,
T1D was accelerated in mice treated with
PV-NP205 plus poly(I:C) in a way similar
to that in mice that received a secondary
PV infection (Figure 4E). Mice that were
administered OVA peptide (SIINFEKL)
and poly(I:C) had hyperglycemia (mean
blood glucose value of 225 mg/dl) but
no diabetes (Figure 4E). Furthermore,
treatment with PV-NP205 alone did not
accelerate diabetes (Figure 4E). These
data demonstrate that in the proper
inflammatory environment, the NP205
epitope that confers molecular mimicry
between LCMV and PV is sufficient to
accelerate autoimmune diabetes and
requires no further assistance by other
LCMV and/or PV epitopes.
In this study we have provided evidence
that the expansion of previously primed
autoreactive T cell populations via heterologous virus infections
and molecular mimicry can lead to the acceleration of autoimmune
diabetes in prediabetic hosts but not to de novo induction of diabe-
tes in naive mice. We have identified the cellular factors responsible
for disease acceleration in the H-2b RIP-LCMV-NP diabetes model
and have shown that the mechanism involves the expansion/acti-
vation and participation in islet destruction of previously primed
CD8 T cell populations specific for the subdominant NP205 epitope.
Important for understanding the pathogenesis of human autoim-
mune diabetes are (a) our findings that molecular mimicry alone,
in the context of arenavirus infection, is not likely to lead to auto-
immune diabetes, unless islet inflammation is already present (pre-
diabetic, ongoing disease state), and (b) the fact that heterologous
viral infections can expand T cell populations with lower-avidity
autoreactive specificities that are unable to initiate disease them-
selves after a single infection. With regards to the association of
sequential viral infections with the pathogenesis of human T1D,
our conclusions indicate the following possible scenario: a viral
infection necessary for the initiation of T1D can be cleared prior to
a second virus infection mimicking an autoantigen. Such a sequen-
tial infection(s) can occur after a relative long latency period. It is
important to note that the clinical manifestation of T1D requires
more than 90% of all β cells to be destroyed, whereas the clinical
manifestation of CNS disease (manuscript submitted for publica-
tion) occurs at comparably much lower degrees of destruction. This
might explain why virally expressed mimics cannot precipitate dia-
betes in naive mice, whereas they can cause significant damage in
a model of virally induced CNS inflammatory disease (manuscript
submitted for publication).
What are the implications of our observations for the role of
molecular mimicry in autoimmune disease? Previously published
findings in our model systems indicate that LCMV-GP33–specific
CD8 T cells that are of comparable avidity to LCMV/PV-NP205–
specific cross-reactive CD8 T cells can induce disease; thus, avid-
ity alone does not determine the ability of an epitope to initi-
ate autoimmune diabetes. One must additionally consider that
there is a critical threshold number of autoreactive lymphocytes
required (32), short of which clinical disease cannot develop.
Therefore, the cross-reactive immune response to an autoanti-
Cytotoxic T cells specific for LCMV-NP are found in LCMV-immune but not naive mice after
LCMV (day 0) + PV (day 42)
C57BL/6J (day 7 after PV)
RIP-LCMV-NP (day 7 after PV)
RIP-LCMV-NP (day 28 after PV)
2 wk stimulation on LCMV APCs
RIP-LCMV-NP (day 28 after PV)
2 wk stimulation on PV APCs
C57BL/6J (day 7 after LCMV)
C57BL/6J (day 7 after PV)
Specific 51Cr release from H-2b targets infected with:
E:T LCMV PV
50:1 14 ± 2 30 ± 4
50:1 15 ± 4 32 ± 4
5:1 35 ± 8 0
5:1 12 ± 4 20 ± 6
50:1 45 ± 8 0
50:1 0 20 ± 7
25 ± 6
10 ± 3
23 ± 7
15 ± 4
11 ± 4
34 ± 8
Primary ex vivo cytotoxic T lymphocyte activities were determined 7 days after LCMV or PV infection as
described in Methods. Secondary in vitro stimulation was performed where indicated using LCMV- or
PV-infected irradiated APCs. E:T, effector/target; T1D dev., development of T1D; ND, not determined.
The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 9 November 2004
gen elicited by an infectious agent conferring molecular mim-
icry would have to be of sufficient magnitude, avidity, and/or
persistence in order to become clinically relevant and precipitate
disease in a healthy individual not predisposed to autoimmune
disease. The failure of the NP205 mimic epitope to initiate auto-
immune disease is not a property restricted to subdominant
epitopes, because the numbers of LCMV/PV-NP205–specific
cross-reactive CD8 T cells were about equal to the numbers of
LCMV-NP396–specific CD8 T cells in RIP-LCMV-NP mice (Fig-
ure 2). Thus, the usually subdominant LCMV-NP205 epitope (in
C57BL/6 H-2b mice) is not subdominant in RIP-LCMV-NP mice
that express NP in the thymus in addition to the pancreas. This
can be explained by the fact that thymic selection affects selec-
tively epitopes with higher functional avidity, as demonstrated
by us previously in the RIP-LCMV thymic expresser mice (45).
In addition, a mimic of a dominant epitope showed similar
results. Infection of H-2d RIP-LCMV-NP mice with LCMV strain
Pasteur (LCMV-Past), which carries a mutation of the dominant
Ld-restricted LCMV-Arm–NP118 CD8 epitope (35), also failed to
develop diabetes, in contrast to mice infected with LCMV-Arm
(32). The LCMV-Past mimic epitope, like PV-NP205, was found
to be of low avidity compared with the wild-type epitope. Taken
together, those previous results and our observations here indi-
cate that the important factors in determining the ability to initi-
ate autoimmune pathology are epitope avidity and numbers of
CD8 T cells induced. Molecular mimicry appears therefore less
likely to precipitate autoimmune diabetes de novo in healthy
individuals with no preexisting islet damage (Figure 1).
Alternatively, as shown here, CD8 T cells specific for mimic
foreign or self ligand that are incapable of initiating disease by
themselves may become dangerous if they encounter a “fertile
field” of ongoing inflammatory responses (28). Such a scenario
can be envisioned in the presence of a persistent infection in which
a chronic inflammatory milieu drives the activation and expan-
sion of cross-reactive T cell populations, as in animal models of
murine herpesvirus 1–induced (HSV-1–induced) herpes stromal
keratitis (HSK) (25) and in recombinant Theiler murine encepha-
lomyelitis virus–induced (TMEV-induced) demyelination (20).
Indeed, infection of mice with a HSV-1 mutant virus with a single
amino acid change in the UL6 protein of HSV-1 affecting the puta-
tive mimicry epitope failed to induce HSK. However, the mutant
HSV-1 was able to induce disease in predisposed mice that received
CD4 cells from donors infected with wild-type virus (25). In the
TMEV model, recent data suggest that a molecular mimic of an
encephalitogenic myelin proteolipid epitope, when expressed by
an engineered TMEV, can both initiate CNS autoimmune disease
and exacerbate a previously established disease (manuscript sub-
mitted for publication). However, our model differs significantly
from those two other experimental systems in that diabetes mani-
festation requires destruction of around 90% of β cells. In contrast
to MS, we have demonstrated for diabetes that the enhancement
of disease requires at least two consecutive acute viral infections
that can be nonpersistent. In our case, LCMV-NP396–specific CD8
T cells (Figure 2) were essential for the precipitation of disease
(Figure 1) and contributed to providing an inflammatory milieu in
Sequential infection with LCMV and PV results in accumulation of PV-
NP205–specific CD8 T cells in the islets of Langerhans. (A) RIP-NP
mice were infected with 105 PFU LCMV. After 4 weeks, one group
of mice received a secondary infection of PV (105 PFU, i.p.). Left
panels, pancreata were harvested at week 3 after secondary infec-
tion and 6-μm tissue sections were stained for cellular infiltration with
a monoclonal antibody against CD8. Sections were counterstained
with hematoxylin. Right panels, pancreata were harvested at day 5
after secondary infection and 6-μm tissue sections were cut and were
stained for CD8 T cells with rhodamine X–conjugated anti-CD8 (red)
and for PV-NP205–specific CD8 T cells with allophycocyanin-conjugat-
ed H-2Kb–PV-NP205 tetramers (green). Note that only after sequential
infection with LCMV followed by PV are PV-NP205–specific CD8 T lym-
phocytes (yellow) found in the islets of Langerhans. Original magnifica-
tion, ×20. (B) Expansion of PV-NP205–specific CD8 T cell populations
in blood and pancreatic lymph nodes after secondary PV infection.
Upper panels, flow cytometry of PV-NP205–specific CD8 T cells in the
blood of LCMV-immune mice that did or did not receive secondary
infection with PV, as detected by H-2Kb–PV-NP205 tetramers; mean
frequencies are indicated in boxed areas. Lower panel, frequencies
of PV-NP205–specific CD8 T cells were determined by flow cytometry
using H-2Kb–PV-NP205 tetramer staining and by ICCS for IFN-γ expres-
sion after 5 hours of in vitro stimulation with PV-NP205 peptide.
1296 The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 9 November 2004
the islets for subsequently arising PV-activated LCMV/PV-NP205–
specific cross-reactive CD8 T cells during the prediabetic phase.
Indeed, in support of this argument, the reverse scenario in which
PV was either given twice (Figure 3) or given as a first infection
followed by infection with LCMV (data not shown), resulting in
significant numbers of activated LCMV-PV-NP205– but not LCMV-
NP396–specific CD8 T cells, respectively, did not result in acceler-
ated diabetes. Earlier studies with the RIP-LCMV model suggest
that transient infection of the pancreas with LCMV is likely to be
important for the subsequent accumulation of autoreactive CD8
T cells and β-cell damage. In agreement with this is the observation
that LCMV-NP–specific lymphocyte lines that have been grown
in vitro and transferred into RIP-LCMV-NP recipient mice were
able to traffic to the target organ are unable to cause T1D unless
the co-stimulator molecule B7.1 is coexpressed on β cells, mimick-
ing APC activation (38). Similarly, a recent study by Darabi et al.
demonstrated that myelin oligodendrocyte glycoprotein–specific
Th1 cells can elicit autoimmune pathology in the CNS only when
the APCs in the target organ are activated by local inflammation
caused by intracerebral deposition of toll-like receptor 9–activat-
ing CpG oligonucleotides (46).
We observed that the sequence in which the heterologous
virus infections occurred affected the autoimmune outcome.
This finding is important for understanding viral associations
with autoimmune diseases, as not only the heterologous viral
strains but also the order in which they infect the host appear
to be important. These data contribute to a growing literature
describing how the history of heterologous infections can skew
T cell hierarchies, thereby altering antiviral immunity and sub-
sequent immunopathologies (27, 47, 48). A major implication is
that the infectious history of a patient is of growing importance
in defining agents that potentially can induce autoimmunity
or push a preexisting autoimmune condition toward clinical
disease. The primary triggering event, which might by itself be
insufficient to cause clinically evident disease, would be followed
by one or multiple secondary antigenic encounters that change
the established T cell hierarchy. As a consequence, autoreactive T
cells accumulate and become activated until they reach a critical
mass and rapidly destroy enough cells or tissue, leading to clini-
cal disease. Molecular mimicry could be involved in some or all
of the above steps. Coupled with a certain genetic predisposition
and possibly other unrelated inflammatory events of the target
organ, molecular mimicry can thus be a highly adverse and dis-
advantageous event, even if no disease and only subclinical auto-
immunity would result in otherwise healthy, nonpredisposed
individuals. Future studies should investigate whether there is a
significant time-wise association between critical infections and
the development of clinical signs of autoimmune disease. Fac-
tors such as time and pattern of (heterologous) infections might
account for the controversial association of T1D and rotavirus
infections (15, 16). Additionally, because autoreactive lympho-
cytes frequently exhibit an activated phenotype in patients but
H-2Kb–restricted, autoreactive, LCMV/PV-NP205–specific cross-
reactive CD8 T cells mediate the acceleration of diabetes. (A and
B) RIP-LCMV-NP or RIP-LCMV-NP × Kb(–) mice were infected
with LCMV or PV. After 4 weeks, the mice received a secondary
infection of PV. (A) Blood glucose of RIP-LCMV-NP, RIP-LCMV-
NP × Kb(–), and RIP-LCMV-NP × Kb(+) littermates was measured
in weekly intervals. The diabetes onset curves (blood glucose
values > 300 mg/dl) for the groups [RIP-LCMV-NP × Kb(–) vs.
RIP-LCMV-NP × Kb(+)] are significantly different (log rank test;
P = 0.0167). (B) Pancreas sections from 3–4 mice per group at
week 3 after secondary infection with PV were stained for cellular
infiltration of CD8 T cells. Sections of 1 representative RIP-LCMV-
NP × Kb(–) and RIP-LCMV-NP × Kb(+) mouse are shown. Original
magnification, ×20. (C) Mice were tolerized to PV-NP205 by injec-
tion of 2 × 107 ECDI–PV-NP205–coupled autologous splenocytes
(ECDI + NP205) or with 2 × 107 splenocytes treated with EDCI
alone, 5 days before infection with 105 PFU LCMV. After 4 weeks,
mice were infected with PV. Diabetes incidence (blood glucose
values > 300 mg/dl) at week 4 after PV infection is displayed;
numbers of mice analyzed per group are indicated in parenthe-
ses. (D and E) Groups of 3–4 mice were infected with LCMV.
After 4 weeks, the mice received 100 μg of PV-NP205 peptide or
an H-2Kb–restricted control peptide (OVA; SIINFEKL). In addi-
tion, mice received three injections of poly(I:C) (7.5 μg/g body
mass) at the time of peptide injection and then at days 2 and 4
thereafter. Controls received PV-NP205 only or PV infection. (D)
The frequency of blood LCMV/PV-NP205–specific cross-reactive
CD8 T cells was assessed by flow cytometry using H-2Kb–PV-
NP205 tetramers (day 7 after peptide injection). (E) Mean blood
glucose values (± SEM) measured at week 2 after peptide and/or
poly(I:C) injection is displayed.
The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 9 November 2004
not healthy controls, these cells should be evaluated selectively
for their ability to cross-react with microbial ligand candidates.
Ideally, appropriate searches will take conformational as well as
sequential homologies into account. Possibly the prevention of
certain infections found to present self-antigenic mimics or ther-
apies aimed at skewing the T cell hierarchy away from a known
autoimmune predisposition could in this way decrease the inci-
dence of autoimmunity.
Transgenic mouse lines. The mouse line expressing the LCMV-NP (transgen-
ic founder line RIP-NP 25-3) was generated in H-2b C57BL/6-J and H-2d
BALB/c mice using the RIP vector and cDNA clones for LCMV-NP and
-GP. RIP-NP 25-3 mice expressed the transgene in both their pancreatic β
cells and their thymus but not in other tissues (29). Screening was done by
PCR as described previously (29). Kb(–) mice were obtained from B. Perar-
nau and F.A. Lemonnier (Departement SIDA-Retrovirus, Institut Pasteur,
Paris, France) (49). RIP-LCMV-NP × Kb(–) mice were screened for H-2Kb
deficiency by flow cytometry using phycoerythrin-conjugated anti–H-2Kb
(BD Biosciences — Pharmingen). The study was approved by the La Jolla
Institute for Allergy and Immunology Animal Care Committee.
Viruses. LCMV-Arm clone 53b and LCMV-Arm-Var (33) were plaque-puri-
fied three times on Vero cells and stocks were prepared by a single pas-
sage on BHK-21 cells. Six- to ten-week-old mice were infected i.p. with a
single dose of 105 PFU (29). PV was kindly provided by M.J. Buchmeier (The
Scripps Research Institute, La Jolla, California, USA) (50) and was injected
at a dose of 105 PFU per mouse.
Blood glucose values. Blood glucose was monitored at weekly intervals with
the blood glucose monitoring system OneTouch Ultra (LifeScan Inc.). Dia-
betes was defined as a blood glucose values of more than 300 mg/dl (38).
Immunohistochemistry. Tissues were immersed in Tissue-Tek OCT
(Bayer) and were quick-frozen on dry ice. Using cryomicrotome and
sialin-coated Superfrost Plus slides (Fisher Scientific), 6- to 10-μm tissue
sections were cut. Sections were then fixed with 90% ethanol at –20°C
and, after sections were washed in PBS, incubated with an avidin/biotin
blocking kit (Vector Laboratories). Primary and biotinylated secondary
antibodies (Vector Laboratories) were incubated for 60 minutes each,
and color reaction was obtained by sequential incubation with avidin-
peroxidase conjugate (Vector Laboratories) and diaminobenzidine–
hydrogen peroxide. Primary antibodies used was rat anti–mouse CD8a
(Ly2; BD Biosciences — Pharmingen).
CTL assays. Lytic activity of LCMV-specific CTLs was measured in a
standard 5-hour in vitro 51Cr-release assay using syngeneic (MC57, H-2b)
target cells infected with LCMV (MOI = 0.1). Secondary CTL assays were
performed after splenocyte restimulation for 5–6 days with irradiated,
syngeneic, LCMV-infected peritoneal exudate cells (PECs) (51).
Flow cytometry. For intracellular stains, single-cell suspensions were
restimulated for 5 hours with 1 μg/ml MHC class I–restricted viral peptides
in the presence of brefeldin A. Cells were stained for surface expression of
CD4 and CD8, fixed, permeabilized, and stained for intracellular IFN-γ
(antibodies were obtained from BD Biosciences — Pharmingen). Samples
were acquired using a FACSCalibur (BD).
In vitro restimulation. For LCMV-specific responses, memory splenocytes
were cultured for 6–10 days on LCMV-infected, irradiated APCs (peritoneal
exudate macrophages from H-2b mice). After culture, viable cells were col-
lected and cells were incubated for 5 hours with peptide in the presence of
brefeldin A before being stained for intracellular IFN-γ (38).
Precursor frequency analysis. For precursor frequency analysis, spleen
cells were harvested on day 60 after primary LCMV infection. These cells
were serially diluted and were cultivated in 96-well flat-bottomed plates
in the presence of T cell growth factor and syngeneic, irradiated, LCMV-
infected (103 PFU/ml) spleen cells (105 cells/well). After 5–9 days, each well
was assayed for CTL lysis (described above) on target cells that were left
uninfected or were infected with LCMV. The fraction of positive cultures
(lysis > 11%) was determined for each dilution (38).
In situ tetramer stains. Briefly, fresh frozen sections 6 μm in thick-
ness were cut from the organs of interest and were stained with a
phycoerythrin-labeled MHC class I tetramer (1.0 μg/ml) with 2% normal
goat serum (NGS) and rat anti-CD8α (0.5 μg/ml). Staining with tetramer
containing an irrelevant peptide was used as a negative control. After
an overnight incubation at 4°C, sections were washed in PBS and then
fixed for 30 minutes at room temperature with PBS-buffered 2% form-
aldehyde. Sections were washed again in PBS and were incubated for 3
hours at 4°C with polyclonal rabbit anti-phycoerythrin diluted 1:2,500 in
PBS with 2% NGS. Afterward, sections were washed and were incubated
for 3 hours at 4°C with rhodamine red X–conjugated donkey anti-rabbit
and FITC-conjugated goat anti-rat diluted 1:1,000 in PBS with 2% NGS.
Rhodamine red X–positive and FITC-positive T lymphocytes were ana-
lyzed with a confocal microscope (40).
EDCI tolerization. Splenocytes were isolated from C57BL/6 mice and were
incubated for 1 hour on ice at a concentration of 3.2 × 108 cells/ml with 1
mg NP205 peptide in presence of 30 mg/ml EDCI (Sigma-Aldrich) (43, 44).
Cross-linked splenocytes were washed and 2 × 107 cells were injected retro-
orbitally into recipient mice. Primary infection with 105 PFU LCMV-Arm
was at day 5 after adoptive transfer. At week 4 after LCMV infection, mice
received a secondary infection with 105 PFU PV.
Poly(I:C) treatment. Mice were infected with 105 PFU LCMV. At week 4
after infection, mice received a single dose (i.p.) of 100 μg NP205 peptide or
the H-2Kb–binding control OVA peptide (SIINFEKL). At the same time,
mice received 7.5 μg/g body mass of the synthetic double-stranded viral
RNA mimic poly(I:C).
This is Publication 625 of the Department of Developmen-
tal Immunology, La Jolla Institute for Allergy and Immunol-
ogy. M.G. von Herrath was supported by NIH grants AI44451,
DK51091, and AI51973. U. Christen was a recipient of a Juvenile
Diabetes Research Foundation fellowship and a career develop-
ment award by the American Liver Foundation. K.H. Edelmann
was supported by NIH training grant NS041219. M.B.A. Old-
stone was supported by NIH grants DK58541 and AI09484. We
thank Michael J. Buchmeier (The Scripps Research Institute, La
Jolla, California, USA) for providing us with PV. We are grate-
ful to J. Lindsey Whitton (The Scripps Research Institute, La
Jolla, California, USA) and Howard M. Grey (La Jolla Institute
for Allergy and Immunology, San Diego, California, USA) for
critically reviewing our manuscript, and we thank Diana Frye for
assistance with the manuscript.
Received for publication June 28, 2004, and accepted in revised
form September 10, 2004.
Address correspondence to: Matthias G. von Herrath, Department
of Developmental Immunology, La Jolla Institute for Allergy and
Immunology, 10355 Science Center Drive, San Diego, California
92121, USA. Phone: (858) 558-3671; Fax: (858) 558-3579; E-mail:
Urs Christen and Kurt H. Edelmann contributed equally to
1298 The Journal of Clinical Investigation http://www.jci.org Volume 114 Number 9 November 2004
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