Cells by Virus-Induced Molecular Mimicry Functional Activation Of

Functional Activation of Myelin-Specific T Cells by Virus-Induced Molecular Mimicry Julie K. Olson, Todd N. Eagar and Stephen D. Miller This information is current as J Immunol 2002; 169:2719-2726; ; of September 26, 2021. doi: 10.4049/jimmunol.169.5.2719 http://www.jimmunol.org/content/169/5/2719 Downloaded from References This article cites 34 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/169/5/2719.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Functional Activation of Myelin-Speciﬁc T Cells by Virus-Induced Molecular Mimicry1

Julie K. Olson, Todd N. Eagar, and Stephen D. Miller2

Molecular mimicry is the process by which T cells activated in response to determinants on an infecting microorganism cross-react with self epitopes, leading to an autoimmune disease. Normally, infection of SJL/J mice with the BeAn strain of Theiler’s murine encephalomyelitis virus (TMEV) results in a persistent CNS infection, leading to a chronic progressive, CD4؉ T cell-mediated demyelinating disease. Myelin damage is initiated by T cell responses to virus persisting in CNS APCs, and progressive demyelinating disease (50 days postinfection) is perpetuated by myelin epitope-specific CD4؉ T cells activated by epitope spreading. We developed an infectious model of molecular mimicry by inserting a sequence encompassing the immunodominant myelin epitope, proteolipid protein (PLP) 139Ð151, into the coding region of a nonpathogenic TMEV variant. PLP139-TMEV-infected mice ؉ developed a rapid onset paralytic inflammatory, demyelinating disease paralleled by the activation of PLP139–151-specific CD4 Downloaded from Th1 responses within 10Ð14 days postinfection. The current studies demonstrate that the early onset demyelinating disease induced by PLP139-TMEV is the direct result of autoreactive PLP139–151-specific CD4؉ T cell responses. PLP139–151-specific CD4؉ T cells from PLP139-TMEV-infected mice transferred demyelinating disease to naive recipients and PLP139–151-specific tolerance before infection prevented clinical disease. Finally, infection with the mimic virus at sites peripheral to the CNS induced early demyelinating disease, suggesting that the PLP139–151-specific CD4؉ T cells could be activated in the periphery and traffic to the CNS. Collectively, infection with PLP139Ð151 mimic encoding TMEV serves as an excellent model for molecular mimicry http://www.jimmunol.org/ :by inducing pathologic myelin-specific CD4؉ T cells via a natural virus infection. The Journal of Immunology, 2002, 169 2719Ð2726.

utoimmune diseases have been suggested to be initiated ular mimicry in autoimmune disease, the evidence was indirect in and perpetuated as a result of virus infections. Three that autoreactivity was not induced as a direct consequence of A possible mechanisms have been suggested for initiating infection. Recently, several reports have provided more direct ev- autoimmune disease following virus infection: bystander activa- idence of infection-induced molecular mimicry by showing that tion, molecular mimicry, and epitope spreading (reviewed in Ref. infection with HSV type I leads to the development of herpes 1). Molecular mimicry is the process by which cross-reactive T stromal keratitis, an inflammatory eye disease (10), and that a by guest on September 26, 2021 cells are generated during a virus infection that recognizes both Theiler’s virus variant engineered to encode a molecular mimic of viral and self epitopes (2). Over the past decade, evidence for a an encephalitogenic proteolipid protein epitope (PLP139Ð151)3 role of molecular mimicry in the initiation of autoimmune diseases could induce demyelinating disease upon infection (11). has resulted from a number of approaches. Initial molecular mim- Multiple sclerosis (MS) is a CD4ϩ T cell-mediated autoimmune icry studies showed that transgenic mice expressing virus proteins demyelinating disease affecting humans that has a suspected virus in the context of self tissues developed autoimmune disease fol- etiology (12). Theiler’s murine encephalomyelitis virus (TMEV)- lowing infection with the corresponding virus (3, 4). Previous induced demyelinating disease serves as a relevant mouse model studies have also demonstrated that viral peptides sharing limited of MS due to numerous pathological and immunological similar- sequence homology with self myelin peptides could stimulate au- ities (13). Infection of susceptible SJL/J mice with the BeAn strain toreactive T cell clones isolated from patients with multiple scle- of TMEV results in a chronic progressive CD4ϩ T cell-mediated rosis (5, 6). Mimic epitopes have been identified in Borrelia burg- CNS demyelinating disease onsetting 30Ð35 days postinfection dorferi that may be involved in Lyme arthritis and neuroborreliosis and characterized by persistent infection of CNS microglia (14). using patient T cell clones (7, 8), and heart disease was shown to Myelin destruction is initiated by virus-specific CD4ϩ T cells that be induced by immunizing mice with a Chlamydia pneumoniae eventually lead to the activation of myelin-specific, autoreactive peptide that shared sequence homology with heart-muscle protein CD4ϩ T cells via epitope spreading, with responses to PLP139Ð (9). Although these studies provided support for a role of molec- 151 first detected at 50 days postinfection (15, 16). Therefore, TMEV-induced demyelinating disease is a highly relevant model for studying virus-initiated autoimmune disease. Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Northwestern University Medical School, Chicago, IL 60611 Recently, we developed a molecular mimicry model for initiation of autoimmune demyelination following virus infection with Received for publication March 22, 2002. Accepted for publication June 20, 2002. TMEV containing a self myelin epitope (11). Native or mimic The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance sequences of the immunodominant PLP139Ð151 epitope were in- with 18 U.S.C. Section 1734 solely to indicate this fact. serted into a nonpathogenic TMEV variant. Infection of SJL mice 1 This work was supported by U.S. Public Health Service National Institutes of Health Research Grants NS34819, NS30871, and AI/DK-51973, and National Multiple Scle- rosis Society Grant RG2275. 3 Abbreviations used in this paper: PLP, proteolipid protein; DTH, delayed-type hy- 2 Address correspondence and reprint requests to Dr. Stephen D. Miller, Department persensitivity; EAE, experimental autoimmune encephalomyelitis; i.c., intracerebral; of Microbiology-Immunology, Northwestern University Medical School, 303 East MBP, myelin basic protein; MS, multiple sclerosis; SI, stimulation index; SP, spleno- Chicago Avenue, Chicago, IL 60611. E-mail address: [email protected] cyte; TMEV, Theiler’s murine encephalomyelitis virus; WT, wild type.

with TMEV encoding native mouse PLP139Ð151, an altered pep- was determined with triplicate wells for each peptide concentration and tide ligand of PLP139Ð151, or a PLP139Ð151 mimic present in then expressed as cpm Ϯ SEM. The stimulation index (SI) was calculated the protease IV protein of Hemophilus influenzae induced a rapid by dividing cpm in experimental wells by the cpm in PBS-containing wells. For cytokine analysis, a duplicate set of proliferation wells was used to onset demyelinating disease associated with the induction of ϩ collect supernatants at 48 and 72 h. Supernatants were analyzed for IL-2, PLP139Ð151-specific CD4 T cell responses. The current studies IFN-␥, or IL-5 secretion by ELISA (Endogen, Cambridge, MA). were conducted to determine whether the early onset demyelinating disease occurred directly through early activation of PLP139Ð Coupled cell tolerance 151-specific CD4ϩ T cells. The results show that the PLP139Ð 151-specific CD4ϩ T cells induced in PLP139-TMEV-infected Spleens were removed from naive mice and mechanically disrupted, and mice were encephalitogenic, as demonstrated by serial transfer of the RBCs were lysed. The splenocytes were incubated with ethylcarbodiimide (150 mg/ 3.2 ϫ 108 cells) and peptide (1 mg/ml PLP139Ð151 or these T cells to naive mice, and that infection of mice with OVA 323Ð339) on ice, shaking periodically for 2 h. The coupled cells were PLP139-TMEV at sites peripheral to the CNS resulted in activa- washed and centrifuged to remove cell clumps. The single cell suspension tion of PLP139Ð151-specific CD4ϩ T cells leading to CNS de- was resuspended in BSS, and 5 ϫ 107 coupled cells were injected i.v. into myelination. Most compellingly, PLP139Ð151-specific tolerance each recipient mouse. Recipient mice were given peptide-coupled splenocytes at day 7 before infection and again at day 3 following infection with induction before PLP139-TMEV infection resulted in reduced PLP139-TMEV virus. clinical demyelinating disease corresponding to a reduced PLP139Ð151-specific CD4ϩ T cell response. Collectively, these results demonstrate that infection with a virus expressing a self Serial disease transfer epitope mimic can directly induce autoreactive T cells with patho- Female SJL mice 5Ð6 wk old were infected with PLP139-TMEV virus, as Downloaded from logic potential in the absence of CFA. described above. At 21 days postinfection, spleens were removed from the mice and mechanically disrupted, and the RBCs were lysed. The resulting Materials and Methods splenocytes were cultured with 20 ␮g/ml peptide (PLP139Ð151 or VP2 70Ð86) for 4 days in DMEM supplemented with 10% FCS, 0.1 mM non- Construction of mimic BeAn viruses essential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, 100 ␮g/ml ␮ The cDNA encoding the BeAn strain of TMEV was modified to contain a streptomycin, and 5.5 M 2-ME. After the incubation, some of the cells ClaI restriction site at bp 1137, resulting in the deletion of 23 aa from the were incubated with anti-CD4-coated MACS beads (Miltenyi Biotec, Au- http://www.jimmunol.org/ ⌬ burn, CA), and then sorted on the AutoMacs (Miltenyi Biotec) column to virus leader (designated Cla-BeAn), as previously described (11). Briefly, ϩ PCR methods were used to insert ClaI sites into PLP DNA at both ends of separate out the CD4 T cells. Both the whole splenocytes and the sorted ϫ 6 a 30-aa piece, PLP130Ð159, which encompassed the encephalitogenic T cells were washed and resuspended in BSS. Then 3 10 blast cells PLP139Ð151 epitope. This piece was then inserted into the ClaI site in the were transferred i.v. to each mouse. ⌬ClaI BeAn virus cDNA. Viral RNA was produced from the cDNA for each recombinant using a T7 promoter transcription kit (Roche Molecular Biochemicals, Indianapolis, IN) and then transfected using Lipofectin (Life Viral plaque assay Technologies, Rockville, MD) into BHK-21 cells to produce infectious The brains and spinal cords were removed from the infected mice at the virus. BHK-21 cells were repeatedly infected with virus in serum-free indicated days postinfection. The organs were homogenized and then di- DMEM medium (Sigma-Aldrich, St. Louis, MO) until a viral titer of 1 ϫ luted in serum-free DMEM for the assay. BHK-21 cells were seeded in by guest on September 26, 2021 9 10 PFU/ml was obtained for the viral construct. tissue culture dishes for 2 days and grown to confluency. The cells were Infection and clinical evaluation of mice washed with serum-free DMEM before the addition of the homogenized tissue dilutions. The cells were then incubated at room temperature for 1 h Five- to 6-wk-old female SJL mice were purchased from Harlan Labs with occasional swirling. A 2% agar solution was added in equal volume (Bethesda, MD) and housed in the Northwestern University animal facility. to 2ϫ DMEM supplemented with 2% serum, plus 5 mM L-glutamine, 200 Mice were infected by intracerebral (i.c.) injection with 3 ϫ 107 PFU virus. ␮g/ml penicillin, and 200 ␮g/ml streptomycin. Following a 1-h incubation, Mice were scored at daily intervals on a clinical scale of 0Ð5: score 0, mice the DMEM/agar mixture was added to the cells, and the cells were incu- show no signs of disease; score 1, mice show mild gait abnormalities; score bated at 34¡C for 6 days in a humidified environment, at which time the 2, mice show more severe gait abnormalities; score 3, mice had paralysis agar layer was removed from the cells. The cells were fixed with methanol, in one limb; score 4, mice had more than one paralyzed limb; score 5, mice and then stained with crystal violet solution (0.12% crystal violet in 20% were moribund. ethanol). The plaques were counted on each plate and multiplied by the dilution and the amount of homogenate added to the plate to determine the Peptides PFU/ml. The weight of the tissue (mg)/ml homogenate was then used to calculate PFU/mg. PLP139Ð151 (HSLGKWLGHPDKF), PLP178Ð191 (NTWTTCQSIAFPSK), VP2 70Ð86 (WTTSQEAFSHIRIPLPH), and OVA323Ð339 (ISQAVHAA HAEINEAGR) were purchased from Peptides International (Louisville, KY). The amino acid composition was verified by mass spectrometry, and purity Results was assessed by HPLC. Infection with PLP139-TMEV results in a rapid onset severe demyelinating disease Delayed-type hypersensitivity response Intracerebral injection of mice with the wild-type (WT) BeAn Delayed-type hypersensitivity (DTH) assays were performed by injecting mice s.c. with 5 ␮g challenge peptide, PLP139Ð151 or VP2 70Ð86, into strain of TMEV leads to the development of a chronic progressive the ears. The ear thickness was determined before injection using a Mitu- demyelinating disease with clinical signs appearing ϳ30Ð40 days toyo model 7326 engineers micrometer (Schlesinger’s Tools, Brooklyn, postinfection (Fig. 1). A nonpathogenic variant of the BeAn virus, NY). After 24 h, the ears were again measured, and differences in ear ⌬Cla BeAn, was constructed by removing a 23-aa coding region swelling over prechallenge thickness were expressed in units of 10Ϫ4 inches Ϯ SEM. from the leader sequence and inserting a ClaI restriction digest site into which the immunodominant myelin epitope PLP139Ð151 was T cell proliferation and cytokine assays then inserted to produce PLP139-TMEV (11). Mice infected with Spleens were removed from infected mice at various times following in- PLP139-TMEV developed an earlier onset severe demyelinating fection. Splenocytes were cultured at 1 ϫ 106 cells/well (in 96-well mi- disease compared with WT TMEV-infected mice (Fig. 1). As a con- crotiter plates) in HL-1 medium (BioWhittaker, Walkersville, MD) sup- trol, a virus was constructed with an irrelevant epitope OVA323Ð339 plemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml ⌬ streptomycin. Peptides were added to the wells at increasing concentrations inserted into the Cla BeAn virus, and mice infected with this from 1 to 100 ␮M. Plates were incubated at 37¡C for 72 h and then pulsed virus developed a late onset demyelinating disease similar to the with 1 ␮Ci [3H]TdR for 24 h before harvesting and counting. Proliferation WT BeAn virus. The Journal of Immunology 2721 Downloaded from

FIGURE 2. PLP139Ð151-speciﬁc T cells from PLP139-TMEV-in-

fected SJL mice transfer EAE to naive recipients. Splenic T cells from mice http://www.jimmunol.org/ infected 21 days with PLP139-TMEV were cultured with PLP139Ð151 FIGURE 1. PLP139-TMEV infection results in an early onset demyeli- peptide or VP2 70Ð86 peptide for 3 days. A total of 5 ϫ 106 viable blasts ϭ nating disease. SJL mice (n 10/group) were infected with PLP139- was then transferred to naive SJL mice (n ϭ 10) by i.v. injection. One ⌬ ϩ BeAn, WT BeAn, Cla BeAn, or OVA323-BeAn virus and then followed group of the PLP139Ð151-reactivated cells was sorted for CD4 T cells for clinical signs of disease for 90 days postinfection. The mean clinical before transfer. The mice were followed for signs of clinical disease se- severity of disease (A) and the percentage of mice affected (B) were de- verity (A) and incidence (B) for 40 days following transfer. Results shown termined for each infected group. are from one representative experiment of ﬁve total experiments.

PLP139-TMEV virus infection results in encephalitogenic by guest on September 26, 2021 ϩ rectly mediate demyelinating disease. These experiments demon- PLP139Ð151-specific CD4 T cells strated that infection with a virus that encodes a self epitope di- ϩ Serial transfer experiments were conducted to determine whether rectly activates self-reactive CD4 T cells with the potential to infection with PLP139-TMEV virus in the absence of CFA in- initiate an autoimmune disease. duces the differentiation of encephalitogenic PLP139Ð151-specific CD4ϩ T cells. Previous studies have demonstrated that PLP139Ð PLP139-TMEV virus infection at peripheral routes results in the 151-specific CD4ϩ T cells isolated from spleens of mice immu- development of CNS demyelinating disease nized with PLP139Ð151/CFA can transfer experimental autoim- PLP139-TMEV virus administered directly to the CNS, via i.c. mune encephalomyelitis (EAE) to naive recipient mice (17). injection, results in demyelinating disease in the CNS. In an at- Splenocytes from SJL mice 21 days postinfection with PLP139- tempt to determine whether local CNS inflammation at disease TMEV were reactivated in vitro with either PLP139Ð151 peptide or viral peptide, VP2 70Ð86, as a control. One group of PLP139Ð 151-reactivated splenocytes was magnetically sorted to isolate CD4ϩ T cells. Peptide-activated splenocytes or sorted CD4ϩ T cells were transferred i.v. to naive mice. Mice that received the PLP139Ð151-reactivated splenocytes developed clinical signs of demyelinating disease onsetting ϳ10 days posttransfer (Fig. 2, A and B). However, mice that received VP2 70Ð86-activated splenocytes did not develop demyelinating disease, despite the fact VP2 70Ð86-reactive cells are found in the spleens of PLP139-TMEV- infected mice (11). Furthermore, purified PLP139Ð151-reactivated splenic CD4ϩ T cells also were able to transfer disease to naive mice similar to the PLP139Ð151-reactivated splenocytes. Mean- while, the reactivated splenocytes that were not CD4ϩ (those re- maining after the CD4ϩ purification) were unable to transfer disease to naive recipients (data not shown). Next, the CD4ϩ T cell responses were examined in the transfer recipients (Fig. 3). Mice FIGURE 3. Recipients of PLP139Ð151-specific CD4ϩ T cells from that received the PLP139Ð151-reactivated splenocytes or CD4ϩ T ϩ PLP139-TMEV-infected donors display DTH responses to PLP139Ð151, cells displayed specific CD4 T cell responses to PLP139Ð151, but not TMEV VP2 70Ð86. At day 14 following adoptive transfer, DTH ϩ but not to virus peptide. Therefore, PLP139Ð151-specific CD4 T responses to challenge with 5 ␮g PLP139Ð151 or VP2 70Ð86 were de- .Indicates significant DTH response over naive mice, p Ͻ 0.05 ,ء .cells that arise during infection with PLP139-TMEV virus can di- termined 2722 MYELIN-SPECIFIC AUTOIMMUNITY INDUCED BY VIRUS INFECTION initiation was a prerequisite for the development of virus-induced molecular mimicry, we asked whether PLP139Ð151-specific CD4ϩ T cells and clinical disease could be activated by PLP139- TMEV infection at sites peripheral to the CNS. SJL mice were infected with PLP139-TMEV virus via i.v., i.p., and s.c. routes and followed for development of clinical disease signs. As shown in Fig. 4, SJL mice infected at peripheral sites developed early onset demyelinating disease with similar incidence of disease and kinet- ics as by the i.c. route, but with slightly reduced severity. There- fore, infection with epitope mimic PLP139-TMEV virus at sites peripheral to the CNS target organ leads to development of organ- specific autoimmune disease. To determine the relative efficiency of activation of PLP139Ð 151-specific CD4ϩ T cell responses in mice infected by the different routes, splenic proliferative responses of PLP139-TMEV- infected mice were analyzed at varying times postinfection (Fig. 5). Mice infected by all the different routes of infection developed significant CD4ϩ T cell responses to the major viral epitope, VP2 70Ð86, and to PLP139Ð151 by 14 days postinfection. Interest- Downloaded from ingly, proliferative responses in the mice infected by the peripheral routes, particularly i.v., were somewhat higher than those in mice infected by the i.c. route (Fig. 5A). This same pattern was observed when CD4ϩ T cells were analyzed for secretion of the Th1 cyto- kines, IFN-␥ and IL-2, in response to viral and myelin epitopes at

14 days postinfection (Fig. 6). Splenic proliferative and cytokine http://www.jimmunol.org/ responses to both VP2 70Ð86 and PLP139Ð151 persisted in all groups at 28 and 56 days postinfection. Responses in mice infected by the i.v. route remained greater than the other routes at later times postinfection. Mice infected with PLP139-TMEV virus by the various routes did not result in induction of CD4ϩ T cells that secreted IL-4 or IL-5 in response to either viral or myelin peptides (data not shown). These results suggest that infection with the

ϩ FIGURE 5. CD4 T cell responses in mice infected by peripheral by guest on September 26, 2021 routes with PLP139-TMEV virus. SJL mice infected with PLP139-TMEV virus by the various routes of infection were analyzed using splenic T cell proliferation assays to determine CD4ϩ T cell responses to viral peptide, VP2 70Ð86, and myelin peptides, PLP139Ð151 and PLP178Ð191. Prolif- eration assays were conducted at days 14 (A), 28 (B), and 56 (C) postin- Indicates signiﬁcant increase over PBS controls for each group ,ء .fection (SI Ն 3). Results shown are from a representative experiment of four replicates.

mimic virus at sites peripheral to the CNS can activate CD4ϩ Th1-type T cells in the periphery, resulting in the development of autoimmune demyelinating disease in the CNS. TMEV is a CNS-tropic virus that is naturally transmitted to the CNS secondary to infection via the gut mucosa. The infection eventually spreads to the CNS, where the virus can persist, leading to the development of autoimmune demyelinating disease. There- fore, mice infected at the sites peripheral to the CNS were analyzed by plaque assays on brains and spinal cord tissue to determine the relative ability of the virus to spread to the CNS. At 21 days postinfection, mice infected by i.v., i.p., or s.c. routes have similar virus loads in both the brain and spinal cord (Table I). However, the virus loads in the mice infected by the peripheral routes were significantly lower than those in the brains and spinal cords from mice infected i.c. with PLP139-TMEV, which had similar virus load as FIGURE 4. Induction of rapid onset demyelinating disease in mice in- WT BeAn-infected mice. Therefore, PLP139-TMEV mimic virus fected at sites peripheral to the CNS with PLP139-TMEV virus. SJL mice (n ϭ 10) were infected with PLP139-TMEV virus by i.c., i.v., i.p., or s.c. can spread to the CNS in mice infected at sites peripheral to the routes. Mice were monitored for signs of clinical disease for 60 days, CNS, but at significantly reduced levels compared with mice in- which is expressed both as disease severity (A) and percentage of mice fected in the CNS, even though the severity of the disease course affected (B). Results shown are from one representative experiment of five in mice infected by the peripheral and CNS routes is similar. This total experiments. suggests that the rapid onset CNS disease is primarily due to the The Journal of Immunology 2723

Table I. CNS virus titers in SJL mice infected with PLP139-TMEV by various routes

Virus Load Virus Load a Ϯ Route of Infection Tissue (PFU/mg SD) (log10) PLP139-TMEV i.c. Brain 744 Ϯ 35 2.87 Spinal cord 52.8 Ϯ 4.3 1.72 PLP139-TMEV i.v. Brain 17 Ϯ 2.1 1.23 Spinal cord 3.0 Ϯ 0.7 0.48 PLP139-TMEV i.p. Brain 15.7 Ϯ 1.3 1.2 Spinal cord 4.3 Ϯ 0.8 0.63 PLP139-TMEV s.c. Brain 12.5 Ϯ 2.1 1.1 Spinal cord 5.6 Ϯ 1.3 0.75 WT TMEV i.c. Brain 849 Ϯ 45 2.93 Spinal cord 58.9 Ϯ 3.0 1.77 Naive Brain 0 0 Spinal cord 0 0

a Brains and spinal cords from groups of three to four naive SJL mice or SJL mice infected 21 days previously by the i.c., i.v., i.p., or s.c. injections with PLP139-TMEV were pooled, and virus titers were determined by a standard plaque assay on BHK-21

cells. These data are representative of three separate experiments. Downloaded from

The various treatment groups were next examined for CD4ϩ T cell responses. Mice infected with PLP139-TMEV virus develop CD4ϩ T cell responses, as measured by both DTH and T cell proliferation to the dominant virus epitope, VP2 70Ð86, and to the myelin mimic epitope, PLP139Ð151, at early times postinfection, http://www.jimmunol.org/ and the responses continue through later times postinfection (Fig. 8). PLP139Ð151-tolerized mice exhibited normal CD4ϩ T cell-

FIGURE 6. Infection by peripheral routes with PLP139-TMEV leads to early CD4ϩ Th1-type T cell responses. SJL mice infected with PLP139- TMEV virus at sites peripheral to the CNS were analyzed at day 14 postinfection for CD4ϩ Th1-type T cell responses to virus peptide, VP2 70Ð86, and myelin peptides, PLP139Ð151 and PLP178Ð191. Supernatants from the T cell proliferation cultures were collected at 48 h and analyzed for by guest on September 26, 2021 Cytokine responses were ,ء .IFN-␥ (A) and IL-2 (B) secretion by ELISA signiﬁcantly greater than those from PBS controls, p Ͻ 0.01. ability of infection with PLP139-TMEV by the various routes to activate a peripheral PLP139Ð151-speciﬁc CD4ϩ T cell response that then mediates CNS myelin destruction.

Tolerance to PLP139Ð151 before infection with PLP139-TMEV reduces demyelinating disease in PLP139-TMEV-infected mice The data to this point show that PLP139-TMEV infection induces the peripheral activation of PLP139Ð151-specific T cells with pathologic potential following in vitro reactivation. To directly determine whether the early onset demyelinating disease was a direct result of the activation of PLP139Ð151-specific CD4ϩ T cells aris- ing via molecular mimicry, the effects of PLP139Ð151-specific tolerance on disease induction in mice infected with the PLP139- TMEV virus were determined. We have previously reported that tolerance induced with splenocytes pulsed with PLP139Ð151 and fixed with ethylcarbodiimide (PLP139Ð151-splenocyte (SP)) is an effective means to prevent development of PLP139Ð151/CFA-induced R-EAE in SJL mice (18). Previous studies also showed that coupled cell tolerance developed independent of CD8ϩ T cells; thus, PLP139Ð151-specific tolerance was CD4ϩ T cell dependent (19). Groups of 10 SJL mice were tolerized by the i.v. injection of FIGURE 7. Tolerance induction to PLP139Ð151 peptide before i.c. infection with PLP139-TMEV virus results in decreased clinical disease. SJL 5 ϫ 107 PLP139Ð151-SP or OVA -SP (as a control) 7 days 323Ð339 mice (n ϭ 10) were tolerized with PLP139Ð151-SP or control treated with before and again 3 days after infection with PLP139-TMEV virus. OVA323Ð339-SP 7 days before and 3 days after i.c. infection with PLP139Ð151 tolerant mice displayed a significantly reduced dis- PLP139-TMEV virus on day 0. Mice were monitored for signs of clinical ease severity (Fig. 7A) and a lower incidence of disease (Fig. 7B) disease for 60 days, which is expressed both as disease severity (A) and compared with control OVA323Ð339-tolerized mice and with mice percentage of mice affected (B). Data are representative of four separate that received no tolerance treatment. experiments. 2724 MYELIN-SPECIFIC AUTOIMMUNITY INDUCED BY VIRUS INFECTION Downloaded from http://www.jimmunol.org/ FIGURE 8. PLP139Ð151-specific CD4ϩ T cell responses are specifically inhibited in mice tolerized with PLP139Ð151-SP before infection with PLP139-TMEV virus. DTH and splenic T cell proliferation were used to assess T cell responses in mice tolerized with PLP139Ð151-SP or control treated with OVA323Ð339-SP 7 days before and 3 days after i.c. infection with PLP139-TMEV virus on day 0. DTH was determined upon ear challenge at days Indicates significant DTH response ,ء .A) and 28 (B) postinfection with 5 ␮g PLP139Ð151 or with the immunodominant TMEV peptide, VP2 70Ð86) 14 over naive mice, p Ͻ 0.05. Splenic CD4ϩ T cell proliferative responses were performed in cultures stimulated with the myelin peptides, PLP139Ð151 and Indicates significant increase over PBS controls for each group ,ء .PLP178Ð191, or viral peptide VP2 70Ð86, and at days 14 (C) and 35 (D) postinfection (SI Ն 3). Results shown are from a representative experiment of four replicates.

specific responses to VP2 70Ð86, but significantly reduced re- Organ-specific autoimmune diseases have been strongly sug- by guest on September 26, 2021 sponses to PLP139Ð151 at early as well as at later times post- gested to be initiated and/or exacerbated by virus infection (1). The PLP139-TMEV infection. In contrast, mice tolerized to control possible mechanisms by which virus infection can trigger an au- ϩ OVA323Ð339 peptide developed CD4 T cell-specific responses to toimmune response include molecular mimicry, bystander activa- both VP2 70Ð86 and PLP139Ð151 similar to untreated mice. Sim- tion, and epitope spreading. Molecular mimicry involves the de ilarly, PLP139Ð151-, but not VP2 70Ð86-induced Th1 (IL-2 and novo activation of autoreactive T cells due to the cross-reactivity IFN-␥) cytokine responses were inhibited in PLP139Ð151-SP- between self epitopes and viral epitopes during a virus infection tolerized mice (Fig. 9). These results directly demonstrate that the (2). Bystander activation is the nonspecific activation of autoreac- early demyelinating disease in mice infected with PLP139-TMEV tive T cells resulting from the direct inflammatory and/or necrotic virus is dependent on the PLP139Ð151-specific CD4ϩ Th1 re- effects of virus infection on tissue in the target organ (20). Epitope sponses, as demonstrated by specifically reduced PLP139Ð151- spreading is the activation of autoreactive T cells due to the tissue specific CD4ϩ T cell responses accompanied by reduced demy- damage following virus infection releasing self epitopes as a result elinating disease in PLP139Ð151-tolerized mice. of the immune response to an organ-localized persistent virus infection (15, 21). Discussion Molecular mimicry theoretically results from infection with a These studies demonstrate that a nonpathogenic strain of TMEV virus expressing a peptide determinant(s) that shares homology encoding a self myelin epitope (PLP139-TMEV) can induce an with a self peptide, resulting in activation of T cells that can cross- early onset autoimmune demyelinating disease via activation of react with the self epitope. TCR degeneracy has been ascribed as autoreactive PLP139Ð151-specific CD4ϩ T cells upon virus infec- the reason for recognition of multiple peptides by the same TCR. tion of SJL mice in the absence of CFA. PLP139Ð151-specific Recent studies have shown degeneracy in the TCR specific for the CD4ϩ T cells activated upon infection with PLP139-TMEV were human myelin basic protein MBP85Ð99 peptide, with the TCR encephalitogenic, as demonstrated by their ability to transfer au- requiring only a few critical residues for recognition (22). T cell toimmune disease to naive mice. Second, infection with the clones specific for MBP85Ð99 established from MS patients were PLP139-TMEV by routes peripheral to the CNS resulted in acti- shown to cross-react with viral peptides expressed by a number of vation of PLP139Ð151-specific CD4ϩ T cells in the periphery, viruses, including HSV, adenovirus, reovirus, and human papillo- which could initiate autoimmune demyelinating disease in the mavirus (5). Likewise, a few critical residues were shown to be CNS. Finally, PLP139Ð151 epitope-specific tolerance before in- necessary for recognition of PLP139Ð151 by its TCR (23, 24). fection with PLP139-TMEV resulted in the specific reduction of PLP139Ð151-specific T cell hybridomas derived from SJL mice PLP139Ð151-specific CD4ϩ Th1 responses that directly correlated were also shown to cross-react with peptides expressed by various with a significant reduction in the incidence and severity of the mouse pathogens, demonstrating degeneracy in the PLP139Ð151 early onset demyelinating disease. TCR (25). Therefore, myelin-specific T cells have been shown by The Journal of Immunology 2725

glial cells (29). Initially following infection with the WT BeAn strain of TMEV, mice display CD4ϩ T cell responses only to virus Ags; however, beginning ϳ50 days postinfection, CD4ϩ T cell responses to immunodominant PLP139Ð151 myelin epitope develop. Subsequently, as the disease progresses, epitope spreading leads to development of CD4ϩ T cell responses to a variety of additional myelin epitopes (15). Furthermore, the demyelinating disease develops independent of MHC class I presentation to CD8ϩ T cells (30). Tolerance to intact TMEV virions coupled to syngeneic splenocytes before TMEV infection anergizes virus- specific CD4ϩ T cell responses, resulting in a reduction of incidence and severity of demyelinating disease (31, 32). In contrast, induction of peripheral tolerance to mouse spinal cord homogenate before TMEV infection did not affect the initiation of TMEV- induced clinical disease and had no effect on the development of virus-specific CD4ϩ T cell responses (33). Together, these studies indicate that the initial virus-specific T cell response and continued response to persistent virus infection are critical for the activation of the myelin-specific CD4ϩ T cell response and the development Downloaded from of the chronic progressive autoimmune demyelinating disease. Recently, our laboratory developed a virus-induced model of molecular mimicry for the induction of autoimmune demyelinating disease (11). A nonpathogenic TMEV variant (⌬ClaI-BeAn) was used to construct a virus that contained a self myelin sequence,

PLP130Ð159, within the virus coding region. Mice infected with http://www.jimmunol.org/ this virus developed an early onset autoimmune demyelinating dis- FIGURE 9. PLP139Ð151-specific IFN-␥ and IL-2 production are spe- ease as well as displaying early CD4ϩ T cell responses to both cifically inhibited in mice tolerized with PLP139Ð151-SP before infection TMEV epitopes and PLP139Ð151, unlike infection with WT with PLP139-TMEV virus. Cytokine-specific ELISAs were used to deter- TMEV, in which early T cell responses are only seen in response ␥ mine the concentration of IFN- (A) and IL-2 (B) from 48-h supernatants to virus epitopes. In addition, mimic PLP139Ð151 sequences were of splenic proliferative cultures prepared from mice at 14 days postinfec- constructed in which amino acid substitutions were made at the -Cytokine responses were significantly greater than those from PBS ,ء .tion primary (aa 144) or secondary (aa 147) TCR contact residues. stimulated controls, p Ͻ 0.01. Infection with the virus carrying a substitution in the secondary TCR contact residue induced early onset demyelinating disease by guest on September 26, 2021 in vitro studies to have the potential to cross-react with viral and activated cross-reactive PLP139Ð151-specific CD4ϩ T cells. epitopes, supporting the molecular mimicry model described in In contrast, infection with the virus substituted at the primary TCR these studies. contact residue (position 144) failed to induce early demyelinating EAE is a well-established experimental model for studying self disease or activation of cross-reactive PLP139Ð151-specific CD4ϩ myelin peptide-induced autoimmune demyelinating disease. PLP139Ð T cells. An additional mimic virus was constructed by inserting a 151/CFA immunization of SJL mice results in a relapsing-remit- sequence from H. influenzae that shared only 6 of 13 aa with the ting demyelinating disease mediated in the initial phase by core PLP139Ð151 epitope (11). More significant to a role for mo- PLP139Ð151-specific CD4ϩ Th1-type T cells with the following lecular mimicry in induction of autoimmune disease, infection relapses mediated by myelin-specific Th1 cells specific for endog- with this mimic virus resulted in early onset demyelinating disease enous myelin epitopes such as PLP178Ð191 and MBP84Ð104 ac- and activation of Th1 cells cross-reactive with the native PLP139Ð tivated via epitope spreading (18, 21, 26). The pathologic signif- 151 determinant. This model is the first to directly demonstrate that icance of epitope spreading is illustrated by the finding that a virus encoding a mimic of an encephalitogenic self myelin tolerance to PLP178Ð191, but not to PLP139Ð151, is required to epitope could induce an autoreactive CD4ϩ T cell response leading prevent disease relapses in mice treated following recovery from to a CNS demyelinating disease. acute disease (18). Additionally, PLP139Ð151-specific CD4ϩ T As illustrated by the ability of PLP139Ð151-specific CD4ϩ T cells from the immunized mice are encephalitogenic and can trans- cells recovered from PLP139-TMEV-infected mice to transfer dis- fer disease to naive recipient mice (18, 27). However, induction of ease to naive recipients and by the ability of peptide-specific tol- EAE initiated by either the native mouse myelin peptides or mimic erance to inhibit induction of PLP139-TMEV-induced disease, the peptides requires the presence of CFA to induce this hierarchical current results clearly demonstrate that the early onset demyeli- cascade of peptide-specific autoimmune responses. CFA creates an nating disease is due to the activity of the autoreactive PLP139Ð artificial inflammatory milieu that does not reflect the natural en- 151-specific Th1 cells and not due to any direct or indirect effects vironment in which self or mimic peptides would be normally of the virus infection. The failure of splenic T cells reactivated in encountered; thus, an infectious model as described in the current vitro with VP2 70Ð86 to transfer disease (Fig. 2) also indicates studies is more appropriate for the study of molecular mimicry. there is no cross-reactivity between the immunodominant TMEV TMEV is a natural mouse pathogen that upon infection can lead epitope and any myelin epitopes. Our previous report (11) showed to the development of an autoimmune demyelinating disease in that insertion of even a nonself epitope (OVA317Ð346) into the which the chronic phase of disease is mediated by myelin-specific nonpathogenic TMEV parental virus led to a late onset (50 days CD4ϩ Th1 cells (21, 28). Previous studies have shown that the postinfection) disease, indicating that reintroduction of nonspecific development of autoimmune demyelinating disease is dependent 30 mer into the leader of the ⌬ClaI-BeAn parental strain restored on persistent virus infection of the CNS, predominantly in micro- the ability of the virus to persist in vivo. Mice infected with 2726 MYELIN-SPECIFIC AUTOIMMUNITY INDUCED BY VIRUS INFECTION

ϩ OVA323-TMEV displayed CD4 T cell responses to VP2 70Ð86 13. Miller, S. D., and S. J. Gerety. 1990. Immunologic aspects of Theiler’s murine within 14 days postinfection, but developed demonstrable encephalomyelitis virus (TMEV)-induced demyelinating disease. Semin. Virol. ϩ 1:263. PLP139Ð151-specific CD4 T cell responses only at later times 14. Olson, J. K., A. M. Girvin, and S. D. Miller. 2001. Direct activation of innate and (Ն90 days postinfection). Nevertheless, the current studies were antigen presenting functions of microglia following infection with Theiler’s vi- critical in definitively ascribing the early onset disease to the ac- rus. J. Virol. 75:9780. 15. Miller, S. D., C. L. Vanderlugt, W. S. Begolka, W. Pao, R. L. Yauch, tivity of the autoreactive PLP139Ð151-specific T cell population K. L. Neville, Y. Katz-Levy, A. Carrizosa, and B. S. Kim. 1997. 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