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The Journal of Immunology, 1999, 162: 7049-7057.
Copyright © 1999 by The American Association of Immunologists

Diverse Fine Specificity and Receptor Repertoire of T Cells Reactive to the Major VP1 Epitope (VP1230–250) of Theiler’s Virus: Vß Restriction Correlates with T Cell Recognition of the C-Terminal Residue1

Byung S. Kim2,*, Young Y. Bahk*, Hee-Kap Kang*, Robert L. Yauch3,*, Jeong-Ah Kang*, Mi-Jung Park* and Nicholas M. Ponzio{dagger}

* Departments of Microbiology-Immunology and Pathology, Northwestern University Medical School, Chicago, IL 60611; and {dagger} Department of Laboratory Medicine and Pathology, University of Medicine and Dentistry–New Jersey Medical School, Newark, NJ 07103


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Theiler’s murine encephalomyelitis virus induces chronic demyelinating disease in genetically susceptible mice. The histopathological and immunological manifestation of the disease closely resembles human multiple sclerosis, and, thus, this system serves as a relevant infectious model for multiple sclerosis. The pathogenesis of demyelination appears to be mediated by the inflammatory Th1 response to viral epitopes. In this study, T cell repertoire reactive to the major pathogenic VP1 epitope region (VP1233–250) was analyzed. Diverse minimal T cell epitopes were found within this region, and yet close to 50% of the VP1-reactive T cell hybridomas used Vß16. The majority (8/11) of the Vß16+ T cells required the C-terminal amino acid residue on the epitope, valine at position 245, and every T cell hybridoma recognizing this C-terminal residue expressed Vß16. However, the complementarity-determining region 3 sequences of the Vß16+ T cell hybridomas were markedly heterogeneous. In contrast, such a restriction was not found in the V{alpha} usage. Only restricted residues at this C-terminal position allowed for T cell activation, suggesting that Vß16 may recognize this terminal residue. Further functional competition analysis for TCR and MHC class II-contacting residues indicate that many different residues can be involved in the class II and/or TCR binding depending on the T cell population, even if they recognize the identical minimal epitope region. Thus, recognition of the C-terminal residue of a minimal T cell epitope may associate with a particular Vß (but not V{alpha}) subfamily-specific sequence, resulting in a highly restricted Vß repertoire of the epitope-specific T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recently, the structural relationship among TCR, MHC, and the peptides involved in TCR recognition has been well demonstrated via x-ray crystallographic studies (1, 2, 3, 4). In addition, many studies have been previously performed to analyze the T cell repertoire involved in T cell responses, including the changes in the T cell repertoire following immunization with altered peptides (5). TCR Vß restrictions for Ag-specific T cells have also been identified in many systems, including experimental autoimmune encephalomyelitis (EAE)4 (6, 7, 8, 9). Such a restriction in the TCRs involved in autoimmune diseases may be useful for controlling unwanted responses by specifically eliminating the TCR-bearing pathogenic T cell populations (7, 8). However, this approach provided only limited success because of heterogeneous T cell responses to a given autoantigen or peptide. Most of the autoimmune models involve repeated immunizations of either peptides or autoantigens in potent adjuvants, and often such immunizations may not reflect true immune responses to native autoantigens during the development of autoimmune diseases.

We have initially examined the TCR Vß genes utilized by Theiler’s murine encephalomyelitis virus (TMEV)-specific T cells, because there is a restricted usage of particular TCR Vß genes by distinct neuroantigen epitope-specific T lymphocytes in other demyelinating diseases (i.e., multiple sclerosis and EAE) (6, 7, 8, 9). However, the T cell repertoire involved in the induction of autoimmune models is rather difficult to assess because these involve extensive immunizations resulting in many other incidental immune responses. In contrast, Theiler’s virus-induced demyelination system provides an important alternative model for studying the immune-mediated demyelination, since the TMEV system does not require repeated immunizations with any type of adjuvants. Thus, investigation of T cells involved in the initiation and progression of demyelination may provide insight into the immune-mediated demyelination induced following viral infection.

TMEV is a common enteric picornavirus in mice (10, 11). The BeAn strain induces a clinically undetectable early phase disease and a late severe white-matter disease (12, 13). In contrast to many other viruses, the persistent nature of TMEV infection leads to the development of a chronic, immune-mediated inflammation in the CNS (14). Recent studies indicate that Th1 responses to viral capsid proteins are involved in the pathogenesis of demyelination (15, 16, 17, 18). The major population of T cells during the course of disease recognizes three predominant (VP1233–250, VP274–86 and VP324–37) viral epitopes (17, 19, 20). The T cell populations specific for VP1 and VP2 epitopes are primarily the Th1 type and are found in the cellular infiltrate of demyelinating lesion (17), supporting the role of this type of T cells in the immune-mediated demyelinating disease. In addition, priming T cells specific for these VP1 and VP2 epitopes (but not VP3) result in acceleration of TMEV-induced demyelination (21), indicating that T cells reactive to these viral epitopes are likely involved in the pathogenesis of demyelination. Several recent studies also strongly suggest that the VP1 capsid protein, in particular, plays an important role in the pathogenesis of demyelination induced by TMEV. For example, many attenuated or nonpathogenic TMEV mutants selected for resistance to anti-viral Abs exhibit amino acid substitutions within the VP1 capsid protein (22, 23). In addition, a recent isolate of spontaneously occurring low-pathogenic TMEV variant displayed a single amino acid substitution in the entire capsid region at position 244 within the major VP1 T cell epitope (24).

In this study, the nature of T cell response to VP1233–250 region was thoroughly investigated. The recognition of minimal amino acid residues was extremely heterogeneous and distinct ranging from 237 to 241 for the N-terminus and from 242 to 250 for the C-terminus. The major T cell population recognized VP1237–245 and all the T cells utilized Vß16, demonstrating highly restricted Vß usage among these T cells, although their complementarity-determining region (CDR) 3 sequences, as well as TCR contact residues within the minimal epitope, were heterogeneous. However, such restriction in the V{alpha} usage among the T cells was not apparent, and even identical CDR3 sequences were shared by T cells with different fine epitope specificity. Thus, these results demonstrate for the first time the functional correlation between use of a Vß and the recognition of the C-terminal residue of a minimal T cell epitope. Interestingly, high concentrations of truncated viral peptides containing N-terminal 236 and C-terminal 246 residues abrogated the proliferative response of bulk T cells from virus-immunized mice, in contrast to the other major pathogenic viral epitope, VP274–86. Thus, this minimal epitope region (VP1237–245) may generate a quantitatively or qualitatively different stimulatory signal.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Four- to six-week-old SJL/J mice were purchased from either The Jackson Laboratory (Bar Harbor, ME) or Charles River Laboratories (Wilmington, MA) via the National Cancer Institute.

Viruses

A BeAn 8386 stock virus was propagated in BHK-21 cells in DMEM supplemented with 7.5% donor calf serum and purified by isopycnic centrifugation on Cs2SO4 gradients as previously described (25).

Synthetic peptides

The synthetic peptides representing the amino acid residues of TMEV were prepared using the RaMPS system (DuPont, Wilmington, DE) with 9-fluorenylmethyloxycarbonyl reagents. A major single peptide (>95%) was present in each of the peptide preparations according to reverse-phase HPLC analyses.

Infection and immunization of mice with TMEV

SJL/J mice were infected intracerebrally (1 x 106 PFU) with TMEV or injected s.c. in the base of the tail with 100 µl (50 µg) of a 1:1 emulsion of UV-inactivated TMEV in CFA. Nine days later, lymph node (LN) cells were pooled from two mice, and the level of T cell proliferation was subsequently assessed in vitro.

TMEV-specific T cell hybridomas

T cells used to produce hybridomas were derived from either the spleens of TMEV-infected or the LN of TMEV-immunized SJL/J mice. Single cell suspensions of spleens or LN were prepared and further stimulated in vitro for 4 days with UV-inactivated TMEV (25 µg/ml) or peptides (1–10 µM) in the presence of 5 x 106 irradiated (3000 rad) syngeneic splenocytes. Viable cells collected on a Histopaque gradient (Sigma, St. Louis, MO) were fused with the TCR {alpha}ß- variant of the AKR thymoma BW5147, as described previously (26).

Stimulation of T cells

Stimulation of T cell hybridomas was based on IL-2 production measured using the IL-2-dependent cell line CTLL-2. Briefly, T hybridoma cells (1 x 105 per well) were cultured for 24 h with Ag, peptide, or PBS in the presence of irradiated, syngeneic splenocytes (5 x 105) as APC. Culture supernatants (100 µl per well) were removed and added to 7.5 x 103 CTLL-2 cells in 100 µl of culture medium. After 24 h, cultures were pulsed with 1 µCi of [3H]TdR per well and were harvested for measurement of [3H]TdR uptake 14–18 h later. Results are expressed as {Delta}cpm ± the SEM for triplicate cultures. Levels of background response in the T hybridoma assays ranged from <1000 to approximately 2000 cpm.

T cell proliferation assay

Spleen or LN cells (5 x 105) were cultured in 96-well microculture plates in RPMI 1640 containing 0.5% syngeneic mouse serum and 5 x 10-5 M 2-ME. Triplicate cultures were stimulated with peptides for 72 h. Cultures were then pulsed with 1.0 µCi of [3H]TdR and harvested 18 h later. Measurements of [3H]TdR uptake by the cells were expressed as {Delta} cpm.

RT-PCR determination of Vß usage

Total cellular RNA of T cell hybridoma clones isolated with guanidine isothiocyanate (27) and mRNA was reverse transcribed into cDNA using oligo(dT)15–18. The concentrations of cDNA were determined based on the level of ß-actin amplification (35 cycles) by PCR. The Vß messages were assessed using the 5'-end sense sequence of the individual Vß and a common 3'-end antisense sequence of Cß as described previously (28).

Cloning and sequencing of TCR of ß- and {alpha}-chain CDR3 regions

The amplified PCR products of ß-chains were cloned into pGEM-T vector (Promega, Madison, WI) and then sequenced by the dideoxy nucleotide termination method using the Sequenase kit (Amersham Life Science, Arlington Heights, IL). To ensure the accuracy of sequencing, at least 3–4 clones per mRNA were sequenced. The first strand cDNAs for {alpha}-chains were synthesized using either oligo(dT) or a specific C{alpha}-2 (5'-GCTCCAGGCAATGGCCCCATT-3') antisense primer. An oligo(dG) tail sequence was introduced into the first strand cDNAs with terminal deoxynucleotidyl transferase for 2 h at 37°C. The second amplification was performed using C{alpha}-2 and oligo(dC) (~12–18 mer) primers and the PCR products were subcloned in the pGEM-T vector. The V{alpha} and J{alpha} sequences were designated according to the recent International Committee recommendations (29).

Assessment of MHC- and TCR-contacting residues

A functional competition assay was used to determine the relative I-As-binding ability of the various peptides (30). Hybridoma cells (1 x 105) were cultured with 5 x 105 APC in the presence of 1 µM of antigenic peptide (VP1236–245) plus 12.5 µM of competitor peptide. A1Bb was used as a negative control, non-MHC-binding peptide to assess the background level of nonspecific inhibition. T cell stimulation was based on IL-2 production, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Heterogeneity of the T cell repertoire recognizing the major VP1 epitope (VP1233–250) region

Many different T cell hybridomas reactive to the VP1 epitope were derived from either the spleens of TMEV-infected or the draining LNs of TMEV-immunized SJL/J mice following in vitro stimulation with either whole TMEV or T cell epitope-containing peptides (Table IGo). CD4+ T lymphocytes are able to recognize peptide Ags of approximately 8–14 amino acid residues in the context of the MHC class II molecule. The VP1233–250 peptide is 18 amino acids in length and thus may not reflect the minimal epitope. To define the minimal epitope within VP1233–250, we produced a panel of synthetic peptides with single amino acid truncations consecutively from the C-terminal (residue 250) or the N-terminal (residue 233) positions (Fig. 1Go). The fine epitope specificities of the hybridomas were assessed using these synthetic peptides. Fig. 1Go shows the results with representative hybridomas. For example, 11B4 requires arginine at position 237 and arginine at position 250 for an optimal stimulation. On the other hand, 4D1 requires arginine at position 237 and valine at position 245. Table IGo includes the summary of all the VP1233–250-reactive hybridomas tested. These results clearly indicate that the regions recognized by individual hybridomas are broad and largely overlapping and yet are not identical. The boundaries of the N-terminal residues ranged from 236 to 241 and those of the C-terminal residues from 242 to 250. In particular, the requirements of the C-terminal residues of the peptides to stimulate these hybridomas vary depending on the hybridoma clones. Therefore, fine epitopes of the VP1233–250 region recognized by T cells from either virus-infected or immunized mice appear to be extremely heterogeneous, reflecting a broad T cell repertoire for at least three nested fine epitopes within this VP1 epitope region.


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  		Table I. Analysis of the fine epitope specificity of SJL/J-derived, VP1233–250-specific T cell hybridomas

 


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  		FIGURE 1. Determination of the minimal N-terminal and C-terminal amino acid residues within the VP1233–250 epitope required for activation of representative VP1233–250-specific hybridoma clones. The graph represents IL-2 production upon stimulation of hybridoma clones with 10 µM of the respective overlapping synthetic peptides. A synthetic peptide representing residues 250–267 of the VP2 capsid protein was used as a negative control. Results are expressed as the net incorporation of [3H]TdR ({Delta}cpm ± SEM) by an IL-2-dependent cell line, CTLL-2, in the presence of Ag-stimulated hybridoma culture supernatants after subtracting the background cpm.

 
Skewed utilization of TCR Vß16 by VP1233–250-specific T lymphocytes

To analyze the diversity of TCR repertoire involved in the recognition of this VP1 epitope region, the TCR Vß genes utilized by the T cell hybridomas were determined by PCR and/or reactivity to the Vß16-specific superantigen (Fig. 2Go). As shown in Table IIGo, we examined the TCR Vß usage in 20 VP1233–250-specific T cell hybridomas. These T cell hybridoma clones expressed only one functionally rearranged Vß gene, suggesting that they are clonal. Analysis of the TCR Vß usage by individual epitope-specific T cell hybridomas demonstrated that 50% (10/20) of the VP1233–250-specific T cells utilized the Vß16 gene. The skewed Vß16 usage among the VP1-specific hybridomas was statistically significant (p = 0.04) as compared with other Vß usage. Thus, these data suggest that there is a preferential usage of the Vß16 gene segment by VP1233–250-specific T cells in TMEV-IDD.



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  		FIGURE 2. Assessment of Vß usage by the T cell hybridomas specific for TMEV. A, Vß usage was determined by RT-PCR using individual Vß sense primers and a Cß antisense primer. A typical PCR assay is shown here using a representative hybridoma clone 11B7. B, The expression of functional Vß16 was confirmed by IL-2 production upon stimulation with the NJ117NA lymphoma cell line, expressing the mouse mammary tumor virus LTR of superantigen for Vß16. Some examples of the reactivity are shown here. IL-2 production was measured after stimulation of various hybridoma clones (1 x 105/well) with 2 x 103 NJ117NA or NJ101 (negative control not expressing the LTR) lymphoma cells lines (10,000 rad). The result is expressed as the incorporation of [3H]TdR (cpm ± SEM) by an IL-2-dependent cell line, CTLL-2.

 

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  		Table II. Determination of Vß repertoire involved in the recognition of SJL/J-derived, VP1233–250-specific T cell hybridomas1

 
To confirm Vß16 expression as a functional receptor on these T cell hybrids, we utilized an SJL lymphoma cell line (NJ117NA) expressing a superantigen (RCS-Mtv) for Vß16 (31, 32, 33). As a control lymphoma line, NJ101 that does not express the RCS-Mtv (31, 34) was included. The data in Table IIGo and Fig. 2GoB demonstrate that all the T cell hybridomas expressing the Vß16 gene segment measured by RT-PCR were specifically stimulated by NJ117NA, but not T cells expressing other Vß genes. Effective stimulatory responses were detected at a responder to stimulator ratio of as low as a 1000:1. Thus, all the T cells positive for Vß16 by RT-PCR appear to express functional Vß16 TCR protein on the surface of the T cell hybridomas.

Vß usage among T cell hybridomas specific for another predominant viral epitope

To investigate whether such a predominance of Vß16 usage among T cells is specific for VP1233–250, Vß usage by similarly selected T cell hybridomas specific for another predominant T cell epitope, VP274–86, was analyzed (data not shown). The Vßs of the T cells specific for VP2 were very heterogeneous, and the use of any single Vß was not apparently favored, although the number of T cell hybridomas analyzed was limited. Only 12.5% (1/8) of the VP274–86-specific T cells used Vß16. Thus, T cells specific for the predominant VP1 region appear to preferentially utilize Vß16 TCR ß-chain subfamily as compared with the other major pathogenic viral epitope region, VP274–86.

Correlation of Vß usage with fine epitope specificity

To investigate the potential correlation between the fine epitope specificity and the Vß usage as well as the CDR3 sequences, the TCR ß-chains of VP1-specific T cell hybridomas were cloned, and the CDR3 regions including Jß usage were analyzed (Table IIGo). Interestingly, all the T cell hybridomas recognizing valine at the C-terminal epitope residue of VP1245 utilized Vß16. However, two Vß16-expressing T cell hybridomas recognized epitopes with a different C-terminal residue, indicating that some Vß-16-bearing T cells can recognize other minimal epitopes within the VP1 region. The N-terminal residue, arginine at VP1237, required as the minimal residue, was also very much restricted (Table IGo). In addition, all these hybridomas recognizing the minimal C-terminal residue (VP1245) have a similar CDR3 length (7–9 residues). However, neither the sequences of the CDR3 region nor the Jß usages were similar to each other. The heterogeneity in the CDR3, even among the Vß16+ T cell hybridomas, indicates that the T cell repertoire is extremely broad. Thus, the restriction of Vß16 usage for the T cells reactive to VP1237–245 may reflect the preferential interaction between the Vß region and the VP1 epitope peptide.

The lack of correlation between TCR {alpha}-chain repertoire and fine T cell specificity

To correlate the TCR {alpha}-chain repertoire and the fine VP1 reactivity, TCR {alpha}-chains of selective T cell hybridomas were analyzed (Table IIIGo). Many hybridomas expressed more than one functionally rearranged {alpha}-chain as described previously (35, 36, 37). Seven hybridomas recognizing VP1233–250 expressed V{alpha}13 and four V{alpha}4. It is interesting to note that 4 of 8 Vß16+ T cell hybridomas recognizing VP1237/245 utilized V{alpha}13. Thus, there is some preferential pairing between Vß16 and V{alpha}13 for this epitope specificity. However, V{alpha}13 usage was not restricted only to the hybridomas with this fine epitope specificity. For example, V{alpha}13 was also found in T cell hybridomas (e.g., 11B4 and 13B4) having a different fine epitope specificity (VP1238/249 and VP1237/243, respectively) within the VP1233–250 region. The CDR2 region of V{alpha}13, as well as V{alpha}4, contains a higher number of acidic residues as compared with other V{alpha}s. In addition, the CDR3 regions of the {alpha}-chains of T cell hybridomas recognizing the VP1 epitope region heavily utilize acidic residues. Therefore, this acidic V{alpha} subfamily may be preferred for the recognition of the positively charged epitope region (see Fig. 4Go). Taken together, the contribution of {alpha}-chains, in contrast to ß-chains, may not be crucial for identifying fine epitope differences, since identical {alpha}-chains can be shared for distinct epitopes within the VP1233–250 region.


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  		Table III. Analysis of TCR V{alpha} usage by SJL/J-derived, TMEV-specific T cell hybridomas

 


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  		FIGURE 4. The summary of MHC- and TCR-contacting residues recognized by four representative Vß16+, VP1237–245-specific T cell hybridomas. The methods of the experiments are described in the Fig. 3Go legend. Upward arrows indicate TCR-contacting residues, downward arrows indicate MHC class II-contacting residues, and bold dashes indicate spacer residues.

 
Involvement of diverse residues within VP1237–245 in the interaction with TCR and MHC

To further understand the heterogeneity of TCR repertoire of T cells reactive to the VP1 epitope region, the individual residues within VP1237–245 were analyzed using T cell hybridomas specifically recognizing this VP1 epitope region (Fig. 3Go). Individual amino acid residues within this epitope region were sequentially substituted with alanine at each position, and then the functional roles for TCR and/or MHC class II interactions were assessed as described previously (38). Results with four representative T cell hybridomas derived from virus-infected mice recognizing VP1237–245 are summarized in Fig. 4Go. The results indicate that minor class II binding residues can be different from each other depending on the T cell hybridomas even though they recognize the identical minimal epitope region of the VP1. However, lysine at position 242 was involved in MHC binding for all of these VP1-specific hybridomas. In contrast, arginine at position 237 is consistently involved in TCR contacting for all the VP1237–245-reactive hybridomas, although other residues are also involved in the interaction with TCR. Thus, the major TCR- and MHC-contacting residues are constant among these VP1237–245-specific hybridomas derived from virus-infected mice.



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  		FIGURE 3. Determination of TCR and MHC class II contacting residues within the VP1236–245 region. A typical assessment for TCR and class II contacting is shown here using a representative hybridoma, 11B7. A, Reactivity of the hybridoma with VP1236–245 peptides substituted with alanine at each position. A total of 1 µM of peptides were used to stimulate the hybridoma. B, Determination of TCR- and class II-contacting residues. The analogue peptides that failed to directly stimulate the T cell hybridoma were used as competitors for the stimulation with the native peptide, VP1236–245. The analogue peptides that failed to inhibit the T cell stimulation were considered to contain residues involved in the class II binding, and the peptides that significantly inhibited were considered as TCR-contacting residues. C, Ability of the analogue peptides to antagonize the IL-2 response of the T cell hybridoma. APC were prepulsed with a suboptimal dose of VP1236–245, then washed and further cultured with T hybridoma cells and various concentrations of the indicated analogue peptides, as described in Materials and Methods. Data represent the percentage inhibition of the IL-2 response levels generated in the absence of potential antagonist peptide.

 
Requirement of conserved residues at the C-terminus of VP1237–245 for Vß16+ T cells

Substitutions of the C terminus with various amino acid residues suggest a strong restriction in this position to maintain T cell reactivity specific for this minimal epitope region (Fig. 5Go). Charged residues substituted at position 245 of VP1236–245 peptide completely abrogated the reactivity with the Vß16+ hybridomas, and hydrophobicity as well as size differences in the substituted residues appear to be important factors for maintaining T cell reactivity. This is in sharp contrast to the reactivity of a T cell hybridoma whose core recognition (VP1237/243) does not require the presence of this C-terminal valine residue. Consequently, Vß16-bearing T cells may preferentially be activated in response to the minimal epitope region. Therefore, the overall predominance of the T cells bearing Vß16 may reflect the abundance of T cell populations reactive to this part of the VP1 epitope region.



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  		FIGURE 5. The stimulation levels of Vß16+, VP1237–245-specific T cell hybridoma by VP1236–245 peptides substituted with various amino acid residues at position 245. Five representative T cell hybridomas (11B7, A1.3, 5H9, 4D1, and 12H9) and a Vß17+, VP1237–243-specific control T cell hybridoma (13B4) were tested for the levels of stimulation (IL-2 production with 10 µM peptide concentration) by substituted peptides. A, Demonstrates examples of Vß16+ (A1.3) and non-Vß16+ (13B4) T cell hybridomas stimulated with various doses of substituted peptides at this position. B, Displays the comparison between the levels of T cell stimulation with peptide substituted with alanine at position 245 as compared with the levels with the native residue, valine, at the same position.

 
A broad T cell repertoire in bulk T cells from virus-immunized mice

The MHC class II-restricted T cells reactive to the major VP1 epitope region have been further investigated to understand the role of T cell repertoire specific to this region in the pathogenesis of demyelination induced after viral infection. This epitope is particularly interesting because it contains many positively charged amino acid residues (Fig. 1Go) and T cells specific for this region are apparently involved in the pathogenesis of demyelination (21). Initially, T cell proliferative responses of LN cells from TMEV-immunized mice were examined (Fig. 6Go). Significant levels of proliferative responses were detected with peptides from 239–250 to 233–243 at concentrations 1 and 10 µM. The range of proliferative responses expanded to 241–250/233–242 at the concentration of 50 µM. However, at this concentration, the proliferative responses to the peptides between 236–259 and 233–246, which induced high levels of proliferation at lower peptide concentrations, were completely abolished, although T cell proliferative response to the major VP274–86 was not reduced (Fig. 6Go). This reduction in the proliferative responses appears to be due to the apoptosis of the responding T cells (data not shown). Perhaps, this reflects the recognition of the peptides containing residues at position 236–246 with a relatively high affinity by the major T cell populations reactive to this epitope region. A similar pattern of splenic T cell proliferative responses was seen in virus-infected mice, although the levels of the responses were much lower (data not shown), suggesting that the quality of T cell responses to TMEV in virus-immunized mice is not significantly different from that from virus-infected mice. These results strongly suggest that T cell populations induced in response to TMEV include a diverse range of fine epitope reactivity within the predominant VP1 epitope region, VP1233–250. This high diversity in the T cell repertoire toward VP1233–250 is consistent with that seen with individual T cell hybridoma clones (Table IIGo).



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  		FIGURE 6. Representative proliferative responses of LN cells from TMEV-immunized SJL/J mice to synthetic peptides within VP1233–250. Eleven days after immunization with 50 µg of UV-BeAn, draining LN cells were pooled from three mice and cultured (6 x 105/well) for 4 days with 1, 10, or 50 µM of the respective peptide, before assessment of [3H]TdR uptake. Peptides containing single amino acid residue truncations from the N terminus or C terminus of VP1233–250 were used in this assay. Results are expressed as the {Delta}cpm ± SEM: mean cpm VP1 peptide-stimulated cultures - mean cpm HEL34–45-stimulated cultures. The positive control proliferative response to UV-BeAn = 109,880 cpm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have found the T cell repertoire toward a major capsid epitope of TMEV is very heterogeneous in susceptible SJL/J mice (Fig. 1Go and Table IGo). Based on the recognition of the C-terminal residues (i.e., >249, 248, 245, 243 and <242), the T cells can be separated into at least five different groups. This is consistent with the previous observation that T cell repertoire of high responder mice to a predominant T cell epitope is very heterogeneous with respect to the C-terminal residue recognition (38, 39). The heterogeneity in the T cell repertoire against the major Th1 epitopes responsible for the pathogenesis of demyelination (17, 21) may have a very important implication. For example, such a broad T cell repertoire to this region may be intended for protective immunity against virus and yet leading to the pathogenesis of demyelination.

The potential restriction in the TCR usage has been of keen interest to immunologists (6, 7, 8, 9, 40, 41) since such a restriction in the TCRs involved in autoimmune diseases may provide a powerful tool to control responses, by specifically eliminating the TCR-bearing T cell populations (7, 8). However, this approach provided only limited success because of heterogeneous T cell responses to a given autoantigen or peptide (28, 42). Most of the autoimmune models involve repeated immunizations of either peptides or autoantigens in potent adjuvants, and often such immunizations may not reflect the true immune responses to native autoantigens during the development of autoimmune diseases. In this paper, we demonstrate for the first time that a restricted usage of certain Vß subfamily is associated with the recognition of an identical C-terminal residue within a T cell epitope (Table IIGo). However, the T cell repertoire to the epitope region remains very heterogeneous overall. Thus, different T cell responses to individual minimal epitopes within an epitope region may have masked the potential restriction of the Vß usage in some of the previous studies (42). It is unlikely that the higher percentage of T cell hybridomas expressing Vß16 resulted from the fusion of expanded, sister T cell clones because these hybridomas do not share identical TCR CDR3 sequences of ß- and/or {alpha}-chains (Table IIIGo).

It is interesting to note that SJL/J mice develop a high incidence of B lymphoma-expressing mouse mammary tumor virus LTR that functions as a Vß16-specific superantigen (32). Thus, it is conceivable that the high levels of Vß16 usage among T cells specific for VP1233–250 may reflect the expanded pool of Vß16+ T cells in the selection for the epitope-specific T cells. However, such an expansion of Vß16+ T cells was not detected in the periphery by RT-PCR during the early stages of TMEV-IDD. This is consistent with the previous observation that expression of this SJL/J-specific superantigen arises only during the development of spontaneous reticulum cell sarcoma late in life (43). When injection of superantigens into mice in which the T cell response to the encephalitogenic autoantigen for EAE is dominated by the superantigen-reactive Vß-chain, demyelination can be modulated via apoptosis-mediated elimination of the autoreactive T cells (44, 45, 46). Therefore, such an effect may potentially be seen in this system if the superantigen is introduced to TMEV-infected mice. However, our preliminary results following transplantation of the superantigen-expressing tumor cells did not alter the course of the demyelinating disease (data not shown), suggesting that other T cell populations may also be important for the development of virally induced demyelination. This is consistent with our previous studies indicating that Th1 responses to at least another major epitope (VP274–86), in addition to the non-Vß16 response to the VP1233–250 region, are involved in the pathogenesis of TMEV-induced demyelination.

Recent x-ray crystallographic studies (4) have suggested that the Vß CDR1 and -2, as well as the ßHV4 regions interact with the C-terminal portion of the epitope peptides. In particular, ßHV4 is known to be involved in the interaction between Vß subfamilies and superantigens (47). Thus, the restriction of Vß16 usage for the T cells reactive to VP1237–245 containing the C-terminal valine residue may represent a favorable interaction between these elements. Relatively low levels of tolerance for substitution of the C-terminal residue with other nonconserved amino acids strongly support this possibility (Fig. 5Go). However, it is also conceivable that the lysine residue at position 244 may be involved in the interaction with Vß16. The C-terminal valine residue may strongly influence the interaction between the lysine at position 244 and the Vß by stabilizing the recognition. Neither the ß-chain CDR3 sequences nor certain {alpha}-chain subfamilies (CDR1, CDR2, and {alpha}HV4) and their CDR3 region influenced the C-terminal recognition. Again, this is consistent with the above structural study. The heterogeneity in the CDR3, even among the T cell hybridomas expressing the Vß16 subfamily (Table IIGo), indicates that T cell repertoire is extremely broad. This appears to be attributable to the differences in the recognition of individual residues within the minimal epitope by MHC class II and/or TCR molecules (Fig. 4Go). However, the heterogeneity in the CDR3 appears to be nonrandom and can be grouped into several subtypes expressing similar motifs depending on the recognition of individual residues. However, the biological significance of this Vß 16 restriction among the VP1-specific T cells is not yet clear. Since other major viral epitopes do not appear to be restricted, the overall TCR repertoire toward whole virus may be much more heterogeneous.

The distribution of V{alpha} subfamilies or {alpha}-chain CDR3 sequences among the T cell hybridomas is very distinct from that of the ß-chains. Many hybridomas expressed more than one functionally rearranged {alpha}-chain, and this is not surprising since the allelic exclusion of the {alpha}-chains is not as strict as that of ß-chains (35, 36, 37). However, the CDR3 regions of these TCR {alpha}-chains consistently contain negatively charged acidic residues. Since the predominant TCR contacting residues appear to be positively charged N-terminal residues (e.g. N-terminal arginine residue at position 237 for hybridomas recognizing VP1237–245), these {alpha}-chain CDR3 regions may be involved in the interaction with these epitope residues. Interestingly, about one third of the {alpha}-chains for the T cell hybridomas recognizing VP1233–250 express V{alpha}13 subfamily (Table IIIGo). However, the distribution of the V{alpha} subfamily is not dependent on the fine epitope specificity. It is noteworthy that V{alpha}13 is used for T cells specific for strongly basic, nucleosome autoantigens in mice with spontaneous lupus (48). The VP1 epitope is also very basic: six of the nine residues in the major minimal epitope within VP1233–250 are positively charged (Fig. 4Go). Therefore, this acidic V{alpha}-subfamily may be preferred for the recognition of the positively charged epitope. In addition, identical V{alpha} sequences (e.g. V{alpha}4S9) including the CDR3 region are also shared among certain hybridomas specific for different fine epitopes within this VP1233–250 region. Thus, the contribution of {alpha}-chains may not be crucial to the fine epitope specificity. Since the CDR2 and CDR3 regions of these V{alpha} subfamilies contain a higher level of negatively charged residues, the {alpha}-chains may be involved in the recognition of the overall charge of the epitope, while ß-chains are more responsible for determining fine epitope specificity.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants RO1 NS28752 and RO1 NS33008. Back

2 Address correspondence and reprint requests to Dr. Byung S. Kim, Department of Microbiology-Immunology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: Back

3 Present address: Dana-Farber Cancer Research Institute, Harvard Medical School, Boston, MA 02115. Back

4 Abbreviations used in this paper: EAE, experimental allergic (autoimmune) encephalomyelitis; TMEV, Theiler’s murine encephalomyelitis virus; CDR, complementarity-determining region; LN, lymph node; IDD, induced demyelinating disease. Back

Received for publication January 21, 1999. Accepted for publication March 30, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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