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The Journal of Immunology, 2007, 179, 6604 -6612
Copyright © 2007 by The American Association of Immunologists, Inc.
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*Autoimmune Diseases

Molecular Mimics Can Induce Novel Self Peptide-Reactive CD4+ T Cell Clonotypes in Autoimmune Disease1

Anne M. Ercolini and Stephen D. Miller2

Department of Microbiology-Immunology, Interdepartmental Immunobiology Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It has been postulated that infectious agents may precipitate autoimmune disease when T cell responses raised against the pathogen cross-react with self-peptides, a phenomenon known as molecular mimicry. However, there are very little data available characterizing the similarity between the repertoire of the cross-reactive self-specific T cell population compared with the pathogen-specific T cell repertoire. In this study, we use immunoscope analysis to identify the T cell populations induced upon priming SJL/J mice with a pathogen-derived mimic of the immunodominant encephalitogenic myelin peptide PLP139–151, which is contained within the protease IV protein of Haemophilus influenzae (HAE574–586). We describe an IFN-{gamma}-producing Vβ19+ T cell population in HAE574–586-primed mice that appears to be the "public clonotype" as it expanded in response to peptide in all mice tested. Critically this Vβ19+ T cell population is not expanded in mice primed with the self-peptide PLP139–151, indicating that mimics can induce the expansion of new self-reactive populations not initially present in the periphery of a host. This is the first description of the use of immunoscope analysis to characterize the cross-reactive anti-self T cell response induced by a molecular mimic.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Molecular mimicry is defined as immunologic cross-reactivity between one protein or peptide and another. Mimicry between self-Ags and Ags found in infectious agents is postulated to either precipitate autoimmune disease or exacerbate already established disease symptoms (1, 2, 3, 4). For example, there is compelling evidence indicating that rheumatic heart disease is caused by molecular mimicry between T cell epitopes derived from streptococcal M proteins and epitopes from cardiac myosin (5, 6). In the autoimmune disease Multiple Sclerosis (MS),3 disease relapses or flares have long been associated with upper respiratory and other infections (7, 8, 9). Several studies have shown that myelin basic protein-specific T cells from MS patients can also recognize peptide epitopes derived from pathogens such as EBV and human herpesvirus-6, indicating that mimicry may play a role in the etiology of this disease (10, 11, 12, 13).

An ongoing area of interest in infection-induced mimicry is the characterization of the pathogen-specific immune response in comparison to the cross-reactive immune response to the self autoepitope. In T cell-mediated autoimmunity, is the T cell response raised against the foreign peptide exactly the same T cells which are cross-reactive to self epitopes, or are there differences? The cross-reactive T cell population may be of different avidity or have a different cytokine profile. Questions remain on the single T cell level about whether the foreign Ag activates already established self-specific populations in the periphery or can create new self-specific peripheral populations, and whether the self-specific population induced by cross-reactivity is a distinct subset of T cells generated against the foreign peptide.

Immunoscope analysis is a powerful tool to identify T cell populations based on TCR CDR3 length (14, 15). Using primers specific for TCR Vβ and Cβ or Jβ regions, PCR amplification of T cell cDNA is performed and the products run on a sequencing gel along with size standards. Within each Vβ population, eight or more CDR3 lengths will resolve (often termed "peaks") whose magnitude can be quantified. T cells undergoing clonal expansion will share identical CDR3 regions, which will be reflected in the immunoscope profile. In this paper, we use immunoscope analysis in a mimicry model of MS. Carrizosa et al. (16) previously identified HAE574–586 (a peptide found in the bacteria Haemophilus influenzae) as a CD4+ T cell mimic of the SJL/J mouse immunodominant encephalitogenic myelin peptide PLP139–151. We show in mice primed with HAE574–586 that T cell populations specific for the mimic are identical in CDR3 length and sequence to the populations cross-reactive to the self peptide. We also identify a particular Vβ19+ T cell population in HAE574–586-primed mice that appears to be the "public clonotype" as it expanded in response to peptide in all mice tested. Lastly, we show that this same Vβ19+ T cell population is not expanded in mice primed with the self-peptide PLP139–151, indicating that mimics can induce the expansion of new self-reactive populations not initially present in the periphery of a host.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Five- to 6-wk old female SJL/J mice were purchased from Harlan Laboratories and housed in the Northwestern University Center for Comparative Medicine. All protocols were approved by the Northwestern University Animal Care and Use Committee. Mice were maintained on standard laboratory food and water ad libitum. Mice were primed by s.c. injection of 100 µl of peptide emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco), distributed over three sites on the flanks.

Peptides

PLP139–151 (HSLGKWLGHPDKF), OVA323–339 (ISQAVHAAHAEINEAGR), and HAE574–586 (EQLVKWLGLPAPI) were purchased from Peptides International. The amino acid composition was verified by mass spectrometry, and purity (96–99%) was assessed by HPLC.

T cell preparation and culture

Spleens were removed from mice, dissociated over wire mesh, and red cells lysed by 5 min room temperature incubation in hypotonic buffer (17 mM Tris, 140 mM NH4Cl (pH 7.2)). Splenocytes were cultured at 5 x 106 cells/well in a 24-well plate in HL-1 medium (BioWhittaker) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 55 µM 2-ME (Invitrogen Life Technologies). Plates were incubated for indicated amounts of time in a humidified chamber (37°C, 7.5% CO2). CD4+ T cells were purified using the autoMACS cell separation system (Miltenyi Biotec) as per kit instructions; purity of sorted samples was assessed to be ≥93% via flow cytometry analysis using Abs against CD90.2 and CD4 (eBiosciences). IFN-{gamma}-secreting cells were enriched using the autoMACS cell separation system as per kit instructions. In brief, cells are removed from culture and incubated with Cytokine Catch Reagent, which are cytokine-specific Abs that attach to the surface of leukocytes via binding to CD45. Cells are then allowed to secrete cytokines for a period of 45 min at 37°C. The secreted IFN-{gamma} is "captured" on the cell surface. The cells are then labeled with magnetic microbeads conjugated to a second IFN-{gamma}-specific Ab. The cells can then be magnetically enriched.

Tolerance induction

Peripheral tolerance induction using peptide-coupled splenocytes was performed as previously described (17). In brief, tolerance was induced by the i.v. injection of 5 x 107 peptide-coupled splenocytes into syngeneic recipient mice one week before and 3 days after priming with peptide in CFA.

Primers

Primer sequences are shown in Table I.


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  		Table I. Primer sequences employed in the immunoscope analyses

 
Immunoscope

RNA was extracted from purified T cell samples using RNeasy columns (Qiagen) and treated with DNase I (Sigma-Aldrich). Treated RNA was checked for contamination with genomic DNA by PCR with actin-specific primers. Up to 1 µg DNase-treated RNA was used in synthesizing cDNA with the Advantage RT-for-PCR kit (Clontech). cDNA was diluted with water for a final volume of 20–40 µl in proportion to the amount of RNA used to start the reaction. For Vβ-Cβ immunoscopes, 1 µl cDNA, 0.25 µM final concentration of each Vβ primer and FAM-labeled (fluorescent) Cβ primer, and Amplitaq Gold PCR Mastermix (Applied Biosystems) were mixed to a final volume of 10 µl. Cycling conditions were as follows: 94°C for 10 min, then 30 cycles of 94°C for 40 s, 60°C for 20 s, and 72°C for 40 s. Before final elongation at 72°C for 10 min, 1 µl 25 mM MgCl2 was added to reduce the formation of "doublets" (18). Two microliters of PCR product was loaded along with ROX-labeled (fluorescent) size standard (Applied Biosystems) onto ABI PRISM 3100 Automated Capillary DNA Sequencer and data analyzed using the Genemapper 3.5 software (Applied Biosystems). Each PCR product represented T cell populations of different CDR3 lengths (in base pairs) for each Vβ-Cβ pairing. Using the Genemapper software, each product resolved as "peaks". The area underneath each "peak" for each product length was normalized to the total peak area for each Vβ-Cβ product and expressed as percentage of total area. TCR Jβ usage was determined using "nested" PCR as follows: Vβ-Cβ PCR was performed similar to above conditions, except the Cβ primer was unlabeled and 35 cycles were used. This PCR product was purified from primers and enzyme with the HighPure PCR Product Purification Kit (Roche), divided in 12 tubes, and amplified as above for five cycles using the 5' Vβ19 primer and FAM-labeled 3' Jβ primers. Immunoscope profiles shown are representative of single experiments.

TA cloning

Cloning of Vβ19-Jβ2.1 PCR products was done using the TOPO TA Cloning Kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. In brief, PCR was performed for 30 cycles as above with unlabeled 5' Vβ19 primer and 3' Jβ2.1 primer, and 4 µl product was used to anneal this PCR product to the cloning vector before bacterial transformation. Plasmid DNA from transformed colonies was extracted using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced using the T7 primer.

Real-time PCR

Real time PCR on cDNA samples was performed using the Light Cycler Fast Start DNA Master Sybr Green I Kit (Roche) according to the manufacturer’s instructions. In brief, 2 µl of cDNA and 0.5 µM final concentration each primer was used with an annealing temperature of 60°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Immunoscope characterization of CD4+ T cells in HAE574–586/CFA primed mice

We have previously shown that SJL mice infected with a nonpathogenic strain of Theiler’s murine encephalomyelitis virus (TMEV) engineered to encode a molecular mimic (HAE574–586) of the immunodominant encephalitogenic myelin PLP139–151 epitope develop an early onset demyelinating disease mediated by PLP139–151-specific CD4+ T cells (17, 19, 20, 21). To characterize the HAE574–586-specific and PLP139–151 cross-reactive populations in HAE574–586/CFA primed mice, spleens from individual peptide-primed mice were cultured in vitro with HAE574–586, PLP139–151, or left unstimulated. After 3 days, CD4+ T cells were purified from these cultures and cDNA synthesized from extracted RNA (Fig. 1A). Vβ-Cβ PCR was performed on a total of 10 mice, and products run on an automated DNA sequencer along with size standards to obtain the immunoscope for each individual mouse. Fig. 1, B and C, show representative immunoscopes from an individual mouse stimulated in vitro with 50 µM or 10 µM peptide, respectively. SJL mice express 14 Vβ products; the various CDR3 lengths ("peaks") for each Vβ-Cβ pairing are identified by their size in base pairs. The area underneath each peak was determined, normalized to the total peak area of each Vβ-Cβ product, and expressed as the percentage of the total area. To determine which T cell populations expanded following peptide stimulation, percentage peak area from the unstimulated samples was subtracted from percentage peak area of the peptide-stimulated samples. As shown in Fig. 1, B and C, several T cell populations expanded in response to both PLP139–151 and HAE574–586. Notably, the T cell populations that expanded in response to HAE574–586 overall also expanded in response to PLP139–151. Expansions seen in response to one peptide, but not the other tended to be limited. Fig. 2A summarizes the Vβ-Cβ immunoscopes of all 10 mice analyzed; displayed for each peak is the number of mice showing expansion of at least 1% in response to PLP139–151 alone, HAE574–586 alone, or both peptides. Again, the majority of population expansions occurred in response to both peptides. Most importantly, there were no populations that consistently expanded in response to one peptide, but not the other. Thus, in HAE574–586/CFA-primed mice, the HAE574–586-specific and PLP139–151 cross-reactive T cells are identical populations as determined by TCR CDR3 length.


Figure 1
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  		FIGURE 1. Immunoscope analysis of HAE574–586/CFA-primed SJL mice shows overlap in the HAE574–586-specific and PLP139–151-specific CD4+ T cell populations. A, Schematic of immunoscope priming, culture, and purification conditions. Eight days after priming with 100 µg HAE574–586 in CFA, splenocytes were extracted from individual mice and cultured for 3 days with peptide or with medium alone (unstimulated). CD4+ T cells were purified from cell cultures and PCR performed on extracted cDNA using primers specific for TCR Vβ regions (variable) and the TCR Cβ (constant) region. Products

 

Figure 2
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  		FIGURE 2. Analysis of Vβ-Cβ immunoscope from ten mice primed with HAE574–586/CFA shows that Vβ19/p188 is a "public clonotype". A, Number of HAE574–586/CFA-primed mice (out of ten) showing expansions of at least 1% in response to PLP139–151 alone ({square}), HAE574–586 alone (Figure 2), or both peptides ({blacksquare}). B, Vβ19-Cβ immunoscope of HAE574–586/CFA-primed mice stimulated in vitro with PLP139–151 (white columns), HAE574–586 (gray columns), or (C) OVA323–339. D, Mice primed with CFA alone and stimulated in vitro with PLP139–151 (white columns) or HAE574–586 (gray columns).

 
One particular population, Vβ19 peak size 188 (Vβ19/p188) was shown to expand in response to PLP139–151 and HAE574–586 in all 10 mice analyzed (Fig. 2A). As shown in Fig. 2B, expansions in this population ranged from 3 to 18%. In contrast, mice primed with HAE574–586/CFA and stimulated in vitro with the control peptide OVA323–339 showed only small expansions of <3% in Vβ19/p188 in two of three mice (Fig. 2C), and mice primed with CFA alone and stimulated with PLP139–151 or HAE574–586 likewise showed small, inconsistent expansions in Vβ19/p188 (Fig. 2D). Therefore, the Vβ19/p188 population is a public clonotype important in the response to PLP139–151 and HAE574–586 in HAE574–586/CFA-primed mice.

Further characterization of the Vβ19/p188 response in HAE574–586 /CFA-primed mice

We next wanted to ascertain whether the overlap in the PLP139–151 and HAE574–586-specific populations could be seen at the level of the CDR3 region amino acid sequence. Because the Vβ19/p188 response is the most consistent, Vβ19-Jβ immunoscopes were performed to determine which Jβ regions were utilized. Fig. 3A summarizes the Vβ19-Jβ immunoscope data from all 10 mice. As was the case with the Vβ-Cβ immunoscopes, these data showed that the HAE574–586- and PLP139–151-specific populations overlap (indicated by • for each Jβ region). Specifically, Vβ19-Jβ2.1/p141 was found to expand in response to both HAE574–586 and PLP139–151 in the vast majority (9/10) of mice. Therefore, Vβ19-Jβ2.1 PCR was performed on primed and in vitro restimulated samples and these products cloned to identify the CDR3 region amino acid sequences. Clones were obtained from five individual mice and the sequences corresponding to the peak of interest analyzed (Fig. 3B). CDR3 region amino acid sequences from all mice followed a common twelve-residue motif (Q/H-S/R/L/G-D/G/W-W/L/G-G-G/N/V-Y/L-A-E-Q-F-F), and did not differ between PLP139–151 and HAE574–586-specific T cells.


Figure 3
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  		FIGURE 3. Further analysis of the Vβ19/p188 public clonotype. A, Nested PCR was performed using Vβ19 and Jβ primers on CD4+ splenocytes from the ten individual HAE574–586/CFA-primed mice shown in Fig. 2B. • indicates those Vβ19-Jβ products that showed expansion in identical CDR3 length "peaks" after in vitro stimulation with either PLP139–151or HAE574–586. B, Samples from primed and in vitro peptide-stimulated samples were amplified using Vβ19 and Jβ2.1 primers and cloned to obtain CDR3 region sequences (underlined, as defined previously (26 )). (Primers used in Fig. 4 were designed based on the highlighted sequences). Shown for each sample is the Vβ19-Jβ2.1 immunoscope (product size in b.p. along x-axis and product height along y-axis). Note that CDR3 "peaks" from unstimulated samples exhibit a Gaussian distribution that becomes skewed after in vitro stimulation with peptide.

 
Next, we wanted to determine whether the Vβ19+ population identified through immunoscope analysis after in vitro stimulation could be found directly ex vivo in HAE574–586-primed mice. Primers were designed to recognize two specific CDR3 regions identified by TA cloning in Fig. 3B (QSGLGGL and QSDWGN) and used for real-time quantitative PCR. CD4+ T cells purified ex vivo from the spleens of HAE574–586-primed mice showed higher levels of Vβ19+ T cells with the above mentioned CDR3 regions as compared with mice primed with CFA alone (Fig. 4, A and B). Interestingly, there was no increase in overall Vβ19 expression in the two groups (Fig. 4C).


Figure 4
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  		FIGURE 4. Real-time quantitative PCR shows higher levels of Vβ19+ cells expressing identified CDR3 sequences in CD4+ T cells taken ex vivo from mice primed with HAE574–586/CFA as compared with mice primed with CFA alone. CD4+ T cells were purified from the spleens of individual mice 13 days after priming with 100 µg HAE574–586 in CFA or CFA alone. Real-time PCR was performed on extracted cDNA using primers specific for Vβ19 and the identified CDR3 sequence QSGLGGL (A), Vβ19 and the identified CDR3 sequence QSDWGN (B), or Vβ19 and Cβ (C). The copy number of each product was normalized to the CD4 copy number for each sample. Data shown is representative of replicate experiment. Differences between groups are significant using the two-tailed Student t test.

 
Functionality of the Vβ19/p188 response in HAE574–586/CFA-primed mice

We next wanted to determine whether the identified Vβ19/p188 population has the potential to drive disease in this model. Presently, there is no commercially available Ab to Vβ19, which precludes us from sorting these T cells for analysis. Both MS and the murine model of this disease, experimental autoimmune encephalomyelitis, are driven by CD4+ T cells that secrete Th1 or Th17-type cytokines (2, 22). Thus, we decided to see whether Vβ19/p188 T cells are enriched in the population of T cells that secrete IFN-{gamma} in response to peptide. Spleens from HAE574–586-primed mice were stimulated overnight with 50 µM peptide and cells secreting IFN-{gamma} were enriched from the population (Fig. 5A). Cells in the IFN-{gamma}– fraction were further purified for CD4+ T cells and the immunoscope of this population compared with the immunoscope of IFN-{gamma}+ cells. The Vβ19/p188 population was enriched in the IFN-{gamma}-secreting population as compared with the population that did not secrete IFN-{gamma}, indicating that the Vβ19/p188 population can potentially drive disease (Fig. 5B). This was seen when the cells were stimulated in vitro with HAE574–586 or with PLP139–151, further demonstrating the similarity between the mimic-specific and self-specific CD4+ T cells.


Figure 5
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  		FIGURE 5. The Vβ19/p188 population is enriched in IFN-{gamma}-secreting splenocytes from HAE574–586-primed mice. A, Spleens from primed mice were harvested on day 8 and stimulated overnight with 50 µM peptide. IFN-{gamma}-secreting cells were enriched, and the IFN-{gamma}– fraction was further purified to obtain IFN-{gamma}– CD4+ T cells. B, Vβ19-Cβ immunoscope of mice stimulated in vitro with PLP139–151 (white columns) or HAE574–586 (gray columns). Shown for each product is the percentage of normalized area of the IFN-{gamma}+ fraction – percentage of normalized area of the IFN-{gamma}– CD4+ fraction.

 
We next determined the fate of the Vβ19/p188 population after induction of tolerance to the HAE574–586 peptide. Mice were tolerized to HAE574–586 using peptide-coupled splenocytes or sham-tolerized with PBS-incubated splenocytes (Fig. 6). Spleens were taken from HAE574–586-primed mice at day 8 and cultured for 3 days with varying concentrations of PLP139–151 or HAE574–586. Immunoscope analysis shows little to no expansion of the Vβ19/p188 population in mice tolerized to HAE574–586 (Fig. 6, D–H) as compared with sham-tolerized mice (Fig. 6, A–C). Interestingly, other populations in the HAE574–586-tolerized mice were still present (Fig. 6, I–M). These data are consistent with other reports showing that tolerance mainly affects the "public" clonotype that drives the response in an animal while "private" clonotypes (those not part of the common response to an Ag, but rather found only in individual animals) remain unaffected (15, 23).


Figure 6
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  		FIGURE 6. Tolerizing mice to HAE574–586 eliminates the "public" Vβ19/p188 clonotype from the spleens of HAE574–586-primed mice while leaving other "private" clonotypes intact. Tolerance was induced by the i.v. injection of 5 x 107 HAE574–586-coupled splenocytes into syngeneic recipient SJL mice 1 wk before and 3 days after priming with HAE574–586 in CFA. Sham-tolerized mice were injected with PBS-incubated splenocytes. Eight days after priming spleens were cultured with the indicated concentrations of PLP139–151 or HAE574–586 for 3 days and a Vβ-Cβ immunoscope performed as in Fig. 1. Shown for each product is the percentage of normalized area after peptide stimulation – percentage of normalized area of the unstimulated sample.

 
Analysis of the Vβ19/p188 response in PLP139–151/CFA-primed mice

Lastly, we asked whether the Vβ19/p188 population was part of the self-specific repertoire already present in SJL mice. CD4+ splenocytes from mice primed with PLP139–151/CFA showed little to no expansion in Vβ19/p188 after in vitro stimulation with PLP139–151 (Fig. 7), indicating that priming with the HAE574–586 mimic can induce the expansion of a PLP139–151 cross-reactive population separate from the endogenous population in SJL mice. Thus, interestingly mimic epitopes can expand dominant clonotypes that are not expanded by priming with the dominant self epitope.


Figure 7
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  		FIGURE 7. PLP139–151-primed mice do not show significant Vβ19/p188 expansion in response to PLP139–151. Vβ19-Cβ immunoscope was performed as in Fig. 1 on mice primed with PLP139–151/CFA and in vitro stimulated with 50 µM PLP139–151. were run on an automated DNA sequencer along with size standards to obtain the immunoscope for each individual mouse. A total of ten mice were analyzed. B, Representative Vβ-Cβ immunoscope of splenocytes from a mouse stimulated in vitro with 50 µM PLP139–151 (white columns) or 50 µM HAE574–586 (gray columns). Shown for each Vβ are the different product lengths (in base pairs) corresponding to CDR3 regions of different amino acid lengths. Shown for each product is the (% normalized area after each peptide stimulation) – (% normalized area of the unstimulated sample). Numbers >0 indicate expansion of that population in response to in vitro stimulation with peptide. C, Representative Vβ-Cβ immunoscope of splenocytes from a mouse stimulated in vitro with 10 µM peptide. Expansions of at least 1% are shown in bold.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this paper, we identify the "public clonotype" CD4+ T cell response in SJL/J mice primed with the myelin PLP139–151 peptide mimic HAE574–586. The public T cell repertoire is defined as Ag-specific T cells that can be found in all individuals of a particular MHC haplotype after identical antigenic challenge (15). This "shared" T cell repertoire may quantitatively comprise only a small fraction of the Ag-specific response in any individual, but appears to be the most important in driving the overall immune reaction (14). It was found in BALB/c mice primed with hen-egg-lysozyme that the Vβ8.2+ public clone is more sensitive to toler-ance induction than other T cell clones recognizing the same region of the protein (23). Another study showed preferential expansion of Vβ5.2+ T cells in the PBL of MS patients as compared with healthy controls and the presence of this subset in the cerebrospinal fluid of all MS patients analyzed (24). Similarly, a single T cell clonotype was found in several patients infected with human CMV following reactivation of the latent virus or chronic inflammation (25). In our model, the Vβ19/p188 population was present in all HAE574–586-primed mice analyzed, could secrete IFN-{gamma} in response to restimulation with either the mimic or the self PLP139–151 peptide, and was deleted after peptide-specific tolerance, all indications that this population is important for driving the cross-reactive anti-self response in HAE574–586-primed mice. One goal in identifying the public repertoire is to develop specific therapies based on the identified clonotype, whether it is to block the response (in the case of autoimmune diseases) or to enhance it (in the case of anti-tumor therapy). In our model, we plan to determine whether specifically inactivating the Vβ19/p188 T cells will limit the overall immune response to HAE574–586 and/or block the cross-reactivity to PLP139–151.

We also compared the cross-reactive response to PLP139–151 to the HAE574–586-specific response. To our knowledge, this is the first report using immunoscope analysis to characterize and define the cross-reactive anti-self response induced by a peptide mimic at the TCR CDR3 level. We found that the cross-reactive cells were not a particular subset of the HAE574–586-specific response, but rather included most if not all of the TCR Vβ clonotypes generated in the mouse in response to HAE574–586. This is not to say, however, that the mimic-and self-specific T cell response in HAE574–586-primed mice is identical in every way. It is possible for the two populations to have the same TCR CDR3 usage but be qualitatively different. In HAE574–586/CFA-primed mice, the proliferative response to HAE574–586 is much more robust than the response to PLP139–151, and the level of cytokine release in response to PLP139–151 is very minimal (16, 19). Similarly, T cell clones from patients with rheumatic heart disease were shown to have weaker responses to self-cardiac myosin than to the streptococcal M protein (5). This may indicate that the PLP139–151-specific T cells are of lower frequency and/or of lower avidity. Although we showed a common CDR3 motif for TCR Vβ19+ cells, it is possible that HAE574–586- and PLP139–151-specific cells utilize different TCR V{alpha}-chains; this would likely affect the avidity of the T cells. The generation of an Ab to murine TCR Vβ19 as well as HAE574–586- and PLP139–151-specific tetramers will hopefully provide answers to these questions by allowing us to purify the specific T cells of interest.

An enduring question in this model is why these HAE574–586/CFA-primed mice have been shown not to develop experimental autoimmune encephalomyelitis (16, 19) if myelin-reactive T cells are generated. As stated above, it may be a question of T cell frequency or avidity, but may also be due to an absence of certain T cell clonotypes critical for disease induction. We plan to further analyze the immunoscopes of PLP139–151/CFA-primed mice to ascertain whether there are significant differences from what is seen in HAE574–586/CFA-primed mice. We have found that although mice primed with the mimic peptide in CFA do not develop clinical symptoms, mice infected with TMEV encoding the same HAE574–586 mimic peptide display gait abnormalities and T cell immune infiltrates into the CNS and, importantly, that this disease can be inhibited by prior induction of tolerance to the self PLP139–151 epitope (17, 19, 20, 21). Immunoscope analysis of self-reactive T cells in these mice along with intracellular cytokine analysis will help elucidate whether delivery of the mimic peptide in the context of the potent innate immune response induced by TMEV infection provides critical signals necessary to initiate CNS disease in this murine model of MS.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by United States Public Health Service, National Institutes of Health Grants NS-040460 and NS-023349, and National Multiple Sclerosis Society (NMSS) Research Grant RG-3166-A-4. A.M.E. was supported by National Multiple Sclerosis Society (NMSS) Postdoctoral Fellowship Grant FG-1596-A-1. Back

2 Address correspondence and reprint requests to Dr. Stephen D. Miller, Northwestern University Medical School, 303 East Chicago Avenue, Chicago IL. E-mail address: s-d-miller{at}northwestern.edu Back

3 Abbreviations used in this paper: MS, Multiple Sclerosis; TMEV, Theiler’s murine encephalomyelitis virus. Back

Received for publication May 18, 2007. Accepted for publication September 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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