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*
Antigen Presentation Laboratory, Division of Immunology and Cell Biology and
Human Genetics Group, Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra, Australia; and
Cellular Immunology Laboratory and
§
Institut National de la Santé et de la Recherche Médicale, CJF 9608, Pitie-Salpetriere Hospital, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MBP1–11 is a dominant epitope in MBP that induces EAE in susceptible mouse strains expressing I-Au MHC class II molecules (5, 6, 15). Previously, we and others have studied this peptide extensively to determine how it binds to I-Au class II molecules, stimulates MBP-reactive T cells clones, and induces EAE. We also determined just how much of this peptide sequence was in fact required for stimulation of T cells and for the induction of EAE (15). We showed that an 11-amino acid polyalanine peptide with just five native MBP residues could induce EAE (16).
Wucherpfennig and Strominger have provided strong evidence that peptides derived from certain viruses and bacteria could stimulate MBP-specific T cell clones generated from MS patients (17). We have utilized a similar approach of data base search based on MHC and TCR contact residues and identified a herpesvirus Saimiri (HVS) peptide with homology to the disease causing MBP1–11 peptide (15). This peptide stimulates MBP1–11-specific T cell hybridomas and induces clear clinical signs of EAE in some (PL/J x SJL/J)F1 mice. We show that this is due to cross-recognition between MBP1–11:I-Au and HVS peptides:I-Au complexes by CD4+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PL/J and (PL/J x SJL/J)F1 mice (8–12 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred at the animal facility at the John Curtin School of Medical Research (Canberra, Australia) in specific pathogen-free conditions.
Peptides
Peptides were synthesized by standard F-moc chemistry using the Applied Biosystems (Foster City, CA) model 431A peptide synthesizer. Peptides were analyzed by HPLC and purified if necessary; structure was confirmed by amino acid analysis and mass spectrometry.
Cell incubations, medium, and conditions
RPMI 1640 supplemented with either 10% FCS or 2% normal mouse serum, 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM glutamine, and 0.05 mM 2-ME was used in most cultures. Cells were incubated at 37°C in a 5% CO2 atmosphere.
Lymphoproliferation assay
Popliteal lymph nodes were removed 10 days after initial immunization of mice with MBP1–11 emulsified in CFA with 400 µg of heat-killed Mycobacterium tuberculosis H37 Ra (Difco, Detroit, MI). Single-cell suspensions were prepared, and cells (2 x 105) were cultured in flat-bottom 96-well microtiter plates in 2% normal mouse serum with or without peptides for 72 h, pulsed with [3H]thymidine, harvested, and counted as described (7). Data are presented as stimulation index on triplicate wells. The SDs were 1–15% of the mean.
T cell hybridoma assays
The T cell hybridomas were established from clones PJR-25, PJPR7.5, PJB20, and BR4, as described previously (6). Activation of T cell hybridoma (2 x 104) was assessed by incubating cells with an I-Au-expressing B cell line (2 x 104) as APC and various concentrations of peptides. After 24 h, 50 µl of supernatant were harvested from each well and tested for IL-2 production by using an IL-2-dependent cell line HT-2, as described previously (16).
Computer modeling
A structural model of I-Au has previously been
described (18) and was obtained from the authors. Initial models for
MBP and HVS peptides were constructed testing all four nongapped
alignments of the 11-residue peptide sequences with the 15-residue
class II-associated invariant chain peptide (CLIP). These preliminary
models were then scanned for general complementarity between residues
of the peptide and the class II molecules as well as their potential to
explain observed experimental data. For both the MBP and HVS peptides,
the third binding mode was selected, and these models were subjected to
further refinement as follows. First, the models were minimized, then
shaken with a short burst of molecular dynamics, and then further
minimized, making use of the DISCOVER program in the Biosym package
(Biosym, San Diego, CA). Structures were initially minimized with
respect to the consistent valence force field (CVFF) energy potential
in vacuum for 100 steps, using steepest descent minimization, with no
cross or morse terms but including charges, a distance-dependent
dielectric constant, and a nonbound interaction cutoff of 12 Å. Final
minimization was performed until the maximum derivative converged to
0.05 Kcal/mol Å. Molecular dynamics was used to shake the system and
was performed for 100 cycles at 303 °K, allowing 1000 steps for
equilibration, with all other conditions as for minimization. The final
structures are shown in Figure 3
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EAE was induced as described (6, 15, 16) by injecting 200 µg MBP or HVS peptides emulsified in CFA s.c. at the base of the tail in a total volume of 0.1 ml. Two hundred nanograms of pertussis toxin (JRH Biosciences, Woodland, CA) was injected i.v. at the time of immunization and again 48 h later. Mice were examined daily for 30 to 40 days and were scored as follows: 1, limp tail; 2, partial hind limb paralysis; 3, complete hind limb paralysis; 4, hind and fore limb paralysis; 5, moribund. The data are presented as cumulative incidence, calculated as total number of mice that showed signs of EAE at any point during the experiment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown that a polyalanine peptide with a Gln-Lys-Arg-Pro
(QKRP) motif at positions 3, 4, 5, and 6 can bind
I-Au, stimulate MBP-reactive T cells, and, most
importantly, induce EAE in mice (16). Wraith and coworkers had
previously identified Gln at position 3 and Pro at position 6 to be the
main TCR contact residues of MBP1–11 peptide, while Lys at position 4
to be an MHC contact residue (6). The finding that a specific in vivo
response can be generated by a peptide containing only five native MBP
residues provided evidence that TCR from disease-inducing T cells, at
least in this case, recognized only a few residues of the MHC-bound
antigenic peptides. Since only five native residues in a peptide are
sufficient to induce EAE, it is conceivable that a pathogen with
homology to self proteins at only a few residues may trigger autoimmune
disease. We therefore identified a few closely matched peptides of
viral origin (Ref. 15 and Table I) and
used one of them as a model unrelated viral peptide to test the
hypothesis that a viral peptide with little homology with MBP1–11 can
stimulate self-reactive T cells and cause EAE.
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). A peptide from HVS,
identified in a computer database search as a close match with
MBP1–11, was acetylated and used to examined the ability of this
peptide to stimulate MBP-reactive T cells. Figure 1B shows
that the HVS.1 peptide was able to stimulate all four T cell hybridomas
but not as well as the wild-type MBP1–11 peptide. Even a short HVS
peptide of 8 amino acids stimulated MBP-reactive T cell hybridoma (Fig. 1C). These results indicate that a cross-reactive
viral peptide can stimulate self-reactive T cells.
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If molecular mimicry is one of the mechanisms implicated in
autoimmune diseases, one would predict that immunization with a
homologous nonself peptide can generate a sufficient number of T cells
that can be stimulated by the self peptide. We examined this by
immunizing two sets of (PL/J x SJL/J)F1 mice
with either HVS.1 or MBP1–11 peptides. T cells from both groups were
then stimulated in vitro by either HVS or MBP1–11 peptides (Fig. 2). These results show that lymph node
cells from MBP1–11-immunized mice can be stimulated by the HVS.1
peptide. However, three- to fourfold higher concentrations of HVS.1
peptide were required for the maximum proliferation. Similarly, lymph
node cells from mice immunized with HVS peptide responded to MBP1–11,
but overall these responses were lower than the stimulation by HVS
peptide (Fig. 2B). These results show clearly that a
large number of T cells can be generated in vivo that can cross-react
with unrelated peptides that have some structural similarity with the
immunizing peptide. Indeed, the computer modeling studies have
suggested that MBP and HVS peptides are held in the groove by a similar
network of hydrogen bonds, including both main chain and side chain
atoms of the peptide and I-Au molecule (Fig. 3
). The major differences between MBP
(Fig. 3A) and HVS (Fig. 3B)
peptides are confined to the C-terminal region of the peptides.
Strikingly, the backbone orientation of ArgP8 is such that
the side chain is projected into the ridge made by the irregularity of
the ß-domain helix. Moreover, ProP9 is constrained to the
main chain with an angle of -60 degrees and
PheP10 must be oriented in such a way as to
occupy Asp
73, Leu77,
Tyrß30, and Tyrß37 pocket. These
differences may explain slightly different patterns of immune responses
initiated by the HVS peptides compare to MBP1–11 peptide (See Figs. 1, 2, and 4).
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The most challenging test to address cross-reactivity and
molecular mimicry is to assess whether HVS peptides can induce EAE. To
test this, we immunized (PL/J x SJL/J)F1 mice
with either HVS or MBP1–11 peptides to induce EAE. Figure 4 shows that
up to 40% of HVS.1 peptide-immunized mice developed EAE albeit with a
slightly reduced severity. While MBP-immunized mice had a mean clinical
score of 3.5, the HVS.1 peptide-immunized mice had a mean clinical
score of 2.5. Even a short 8-amino acid HVS.2 peptide can induce EAE in
20 to 30% of mice. EAE in these mice was associated with typical
perivascular cellular infiltrations in the spinal cord (Fig. 5b). The mice that did
not develop clear clinical signs of EAE showed some cellular
infiltration in the CNS (data not shown). Immunization of control mice
with peptides with no sequence homology with MBP1–11 (e.g.,
ova323–339 or MBP89–101) have never resulted in any clinical or
histologic signs of EAE (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have shown previously that a polyalanine peptide with only a few native MBP amino acids, Gln-Lys-Arg-Pro (QKRP), could induce EAE with a severity identical to that of MBP1–11 (16). This led us to propose that a microbial peptide with some similarity with MBP1–11 could stimulate MBP-reactive T cells and cause EAE (15). The HVS peptide (AAQRRPSRPFR) selected in this study is radically different from MBP1–11, especially on the C-terminal end. In HVS peptide, even the core sequence differs by 1 amino acid (Lys to Arg at position 4). McDevitt and colleagues have shown previously that there is a dramatic change in the binding properties of MBP1–11 peptide to I-Au if position 4 is substituted with certain amino acids (6, 16). It is also important to note that His at position 10 in MBP1–11 improves binding of this peptide to I-Au and enhances induction of EAE (16). Another major difference between MBP1–11 and HVS peptides is the addition of a Proline residue at position 9. This may lead to a "kink" in the peptide. As shown here, the incidence of EAE by HVS peptides is much reduced compared with MBP1–11. This could be due to their ability to stimulate only a subset of MBP1–11-specific T cells, and/or the HVS peptides may act as weak or partial agonist for MBP-reactive TCRs
Four mechanisms have been proposed to explain the activation of an autoimmune process by infections. The first is molecular mimicry, implying some level of homology between a self Ag and an infectious agent (8, 19, 21, 22, 23). This mechanism can operate for both Ab- and T cell-mediated autoimmune diseases (8, 21, 22, 23, 24). The second is activation of a subset of T cells containing self-reactive lymphocytes by a bacterial or viral superantigen. Experimental evidence for such a mechanism exists (9), and indication that such a mechanism could be at play in a human autoimmune disease has recently been published (11). These two mechanisms do not require that the infection take place in the tissue that would be a target of the subsequent autoimmune disease. Thirdly, infection in a tissue may favor the release of self Ags that can be processed and presented to self-reactive T cells leading to the tissue damage. Evidence for such a mechanism has recently been provided in the Theiler’s virus encephalomyelitis (25). Finally, viral-specific T cells in a tissue may activate bystander self-reactive T cells (26). This mechanism has not yet been worked out at the molecular level but appears attractive in view of the fact that cytokine combinations can activate both naive and memory T cells.
How would microbial pathogens initiate an autoimmune disease? One possibility is that self-reactive T cells that have escaped the thymic deletion encounter cross-reactive microbial peptides bound to MHC molecules. This may result in the low level stimulation of self-reactive T cells, migration into a site, and the causing of some tissue damage. Once the tissue destruction has begun, the release of self Ag from the target tissue may perpetuate the immune response against its own Ags even after the microbial pathogen has been cleared. It is important to note that, at least in our model of EAE, MBP1–11 peptide binds I-Au poorly (6, 16). This poor binding of MBP1–11 could result in an inefficient negative T cell selection for this peptide in the thymus. Once in periphery, these cells could be potentially autoreactive, waiting to be stimulated by a trigger such as a virus or bacteria. As has been shown by others, TCR is capable of being stimulated by a variety of peptides presented by the same MHC class II molecule (16, 17, 20, 27).
A question also arises whether a similar viral peptide could contribute to MS pathology. Molecular mimicry has been described between MBP and several viral peptides (Refs. 12, 21, 22; for a brief review see also Refs. 8, 23). Moreover, MS-like symptoms have been observed in a series of patients following hepatitis B surface Ag vaccination (28). There is also convincing evidence for molecular mimicry between Campylobacter jenjuni and Guillain-Barre syndrome (a peripheral nerve inflammatory demyelinating disease) (24). Clearly we need to investigate whether cross-reactive peptides can 1) be generated from microbial pathogens and 2) be loaded on to MHC molecules. There are several examples in the literature that demonstrate clearly that MHC class II molecules can present peptides derived from intracellular proteins (29, 30, 31, 32). This would suggest that a cross-reactive peptide from an intracellular virus could in theory be loaded onto class II MHC molecules and presented to CD4+ encephalitogenic T cells. Peptides derived from extracellular pathogens have not yet been well studied for stimulating self-reactive CD4+ T cells. However, it is plausible that cross-reactive peptides generated from extracellular pathogens via a well-defined class II Ag-processing pathway could result in cross-recognition by autoaggressive T cells (for review on class II see 2 .
In conclusion, we have presented experiments that show that a cross-reactive nonself peptide from a viral protein can generate immune response such that it could lead to a clinical autoimmune disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anand M. Gautam, M & E Biotech A/S, Kogle Alle 6, DK-2970 Horsholm, Denmark. E-mail address:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CNS, central nervous system; MBP, myelin basic protein; MS, multiple sclerosis; HVS, herpesvirus Saimiri.
Received for publication February 3, 1998. Accepted for publication February 26, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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