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Originally published online as doi:10.1189/jlb.0906584 on November 16, 2006

Published online before print November 16, 2006
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(Journal of Leukocyte Biology. 2007;81:696-710.)
© 2007 by Society for Leukocyte Biology

Characterization of CD8+ T cell repertoire in identical twins discordant and concordant for multiple sclerosis

Paolo Somma*, Giovanni Ristori{dagger}, Luca Battistini{ddagger}, Stefania Cannoni{dagger}, Giovanna Borsellino{ddagger}, Adamo Diamantini{ddagger}, Marco Salvetti{dagger}, Rosa Sorrentino* and Maria Teresa Fiorillo*,1

* Department of Cell Biology and Development and
{dagger} Neurology and Center for Experimental Neurological Therapy, S. Andrea Hospital, University of Rome "La Sapienza," Rome, Italy; and
{ddagger} Neuroimmunology Unit, European Centre for Brain Research, Santa Lucia Foundation, Rome, Italy

1 Correspondence: Department of Cell Biology and Development, University of Rome "La Sapienza," Via dei Sardi 70, 00185 Rome, Italy. E-mail: mariateresa.fiorillo{at}uniroma1.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Autoreactive CD4+ and CD8+ T cells directed against CNS autoantigens may play a role in the development of multiple sclerosis (MS). Identical twins share the same genetic background but not the TCR repertoire that is shaped by the encounter with self or foreign antigens. To gain insights into the interplay between MS and T cell repertoire, peripheral blood CD4+ and CD8+ T lymphocytes and their CCR7+/CCR7– subsets from five pairs of identical twins (four discordant and one concordant for MS; none of which had taken disease-modifying therapy) were compared by TCR ß-chain (TCRB) complementary-determining region 3 (CDR3) spectratyping. CD4+ T cells generally showed a Gaussian distribution, whereas CD8+ T cells exhibited subject-specific, widely skewed TCR spectratypes. There was no correlation between CD8+ T cell oligoclonality and disease. Sequencing of predominant spectratype expansions revealed shared TCRB-CDR3 motifs when comparing inter- and/or intrapair twin members. In many cases, these sequences were homologous to published TCRs, specific for viruses implicated in MS pathogenesis, CNS autoantigens, or copaxone [glatiramer acetate (GA)], implying the occurrence of naturally GA-responding CD8+ T cells. It is notable that these expanded T cell clones with putative pathogenic or regulatory properties were present in the affected as well as in the healthy subject, thus suggesting the existence of a "MS predisposing trait" shared by co-twins discordant for MS.

Key Words: autoimmunity • monozygotic twins • T cell receptor • CDR3 spectratyping


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS, in which genetic and environmental factors, especially viral infections, are involved [1 2 3 ]. Autoimmune T cell responses against myelin proteins, occurring in MS patients and in experimental autoimmune encephalomyelitis (EAE) animal models, are suspected to play a key role in disease pathogenesis [4 5 6 7 8 9 10 11 ]. The association of certain HLA Class II alleles with MS and the ability to induce EAE have focused the attention on autoreactive CD4+ T cells as major effectors [2 , 12 , 13 ]. Recently, the interest toward CD8+ T lymphocytes has been strengthened by several reports showing their contribution to CNS tissue damage and the correlation with progression and severity of disease [10 , 14 , 15 ]. In demyelinating plaques of patients with MS, CD8+ usually outnumber CD4+ T cells and undergo local clonal expansions more frequently than CD4+ T cells, as assessed by the analysis of TCR ß-chain (TCRB) rearrangements at a single-cell level [16 ]. Moreover, specific enrichment and oligoclonal expansions of memory CD8+ T cells were found in the cerebrospinal fluid of MS patients [17 ]. One important point is whether the expanded CD8+ T populations found at different sites in the damaged tissues of MS patients are relevant to the disease. A strong support for a pathogenic hypothesis comes from a recent study showing that CD8+ T cell clones infiltrating the CNS persist in the cerebrospinal fluid (CSF) and in the blood for many years as clonal expansions [18 ]. However, the possibility that these expanded CD8+ T cell clones are also implicated in a regulatory network cannot be ruled out [15 ].

Further insights into the relative role of the two major T cell subsets may come from the analysis of the skewing of the complementary-determining region 3 (CDR3)-length distribution of the TCRB in monozygotic twins, discordant or concordant for MS. This approach has the advantage of not introducing any bias and allows the comparison of T cell repertoires in individuals with the same genetic background and presumably, under similar environmental pressure.

Many studies investigating TCR repertoires have shown a skewed ß gene use in ex vivo T cells of patients with MS, suggesting the presence of oligoclonal T cell expansions in the blood [19 20 21 22 23 24 ]. Other reports have revealed a preferential use of certain Vß genes by myelin basic protein (MBP)-specific T lymphocytes [25 , 26 ]. These results are controversial and although some TCRBV appeared to be preferred by MBP-responsive T cell clones in single MS patients, there was a high variability [27 ]. This suggests that the genetic background is crucial in shaping the subject-specific TCR repertoire. In this respect, MS patients differ from EAE animal models, as a highly restricted V-gene use in MBP recognition has been observed in some rodent strains [28 , 29 ].

In this report, we examined the TCRBV repertoire on peripheral CD8+ T cells and CCR7+ and CCR7– subsets in monozygotic twin pairs, discordant or concordant for MS, using the CDR3 spectratyping. Moreover, a large number of TCR sequences corresponding to CD8+ T cell expansions have been determined in MS-affected or in healthy co-twin members. The comparison of these sequences with TCR, available in the public databases, provided a clue regarding the putative, functional role of these expanded clones within the CD8+ T cell repertoire.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Identical twin pairs
Five pairs of monozygotic twins were studied; four of these are discordant, and one is concordant for clinically definite MS. They were subjected to leukapheresis, and a gadolinium-enhanced magnetic resonance imaging was obtained within 24 h from the procedure. At the time of sampling, affected subjects were free from immune modulatory therapies for at least 3 months. For this study, each subject had only one blood-draw. Informed consent has been obtained from each patient and the healthy twin. Characteristics of the twins enrolled for this study are described in Table 1 .


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  		Table 1. Characteristics of Monozygotic Twins Selected for This Study

 
T cell preparation
PBMCs from monozygotic, MS-affected and normal twin pairs were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia, Uppsala, Sweden). Highly purified CD4+, CD8+ T cells and their CCR7+ or CCR7– subsets were sorted by high-speed cell sorting on a Dako Cytomation MoFlo cell sorter (Dako, Denmark) using the following antibodies: anti-CD3 Alexa-488 (Becton Dickinson, San Jose, CA, USA; PharMingen, San Diego, CA, USA), anti-CD4 energy-coupled dye, anti-CD8 PE-Cy7 (Coulter, Fullerton, CA, USA), and anti-CCR7 PE (R&D Systems, Minneapolis, MN, USA). T cell fractions were resuspended in 1 ml Trizol/1 x 106 cells (Invitrogen, Carlsbad, CA, USA) and stored at –80°C.

cDNA synthesis, PCR amplification, and CDR3 spectratyping
Total RNA was extracted from each T cell subset (from 5x105 to 2x106 cells) using TRIzol, according to the manufacturer’s instructions (Invitrogen) and 1 µg total RNA used for cDNA synthesis using oligo(dT)12–18 as primer and Superscript III as RT (Invitrogen). To normalize the conditions of TCRB-CDR3 spectratype analysis, TCRB constant regions were amplified using ß-chain-specific primers and cDNA samples at three different concentrations. The amounts of PCR product obtained after 22 and 26 cycles were estimated by densitometry on 1.5% agarose gel. For PCR amplifications of the different BV families, we used a panel of 26 TCRBV-specific forward primers together with a reverse primer specific for the TCRB constant region labeled with [{gamma}-32P]ATP as described elsewhere [30 ]. PCR reaction mix was prepared as follows: 1.5 U AmpliTaq Gold (Applied Biosystems, Foster City, CA, USA), 1x PCR buffer, 200 µM dNTPs, 1.5 mM MgCl2, 0.5 µM TCRBV-specific primer, 0.5 µM radiolabeled TCRB constant region primer, and from 0.2 to 2 µl cDNA. After 8 min at 95°C, PCR amplifications were performed for 32 cycles (30 s at 95°C, 30 s at 58°C, and 30 s at 72°C) with a final 7-min extension at 72°C. PCR samples in formamide/dye-loading buffer were heated at 94°C for 2 min and run on a prewarmed 6% acrylamide/7 M urea sequencing gel. Radiolabeled bands, which constitute the TCRBV-CDR3 spectratypes, were visualized by autoradiography.

Cloning and sequencing of specific TCRBV segments
cDNA of the selected samples was reamplified using a specific primer for the TCRBV segment of interest and the unlabeled TCRB constant region primer and the PCR product cloned into pGEM-T vector according to the TA cloning procedure (Promega, Madison, WI, USA). When the TCRB-CDR3 spectratype was oligoclonal, positive clones containing the expected TCR were identified by PCR amplification of the insert using 3'-radiolabeled primer. Afterwards, the corresponding TCRB-CDR3 spectratype and the radiolabeled PCR products from single clones were compared by acrylamide gel electrophoresis. Nucleotide sequences were determined by automated DNA sequencing (www.mwg-biotech.com) using the forward pUC/M13 primer. Deduced TCR amino acid sequences determined in this study were compared with those reported in the public protein databases (http://www.ncbi.nlm.nih.gov) using the BLAST program.

Statistical analysis
The correlation coefficient (r) was calculated using the parametric correlation test (Pearson) comparing the number of altered TCRB-CDR3 spectratypes in patients with MS versus healthy twins.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CDR3 spectratype analysis in the CD4+ T cell populations
The CDR3 length distribution of the different TCRBV segments was analyzed on CD4+ T cells derived from identical twin pairs (four discordant for MS: RP/RS, UD/US, TP/TE, PS/PM; and one concordant: CN/CI; Table 1 ). The spectratypes revealed a Gaussian distribution in the majority of the 26 Vß families analyzed, with minor exceptions in twin pairs UD/US (data not show) and CN/CI. A representative example of regular CDR3 distribution is shown in Figure 1 , where the spectratype of different TCRBV families expressed by a discordant co-twin pair is reported. The specific TCRBV products appeared as a ladder of bands spaced by 3 bp, and the highest density is in the middle (the optimal length for that specific Vß family). An exception is observed in Vß10 and Vß19 spectra, where only few discrete bands were visible on a smeared background as already observed by others [19 , 30 ]. Figure 2 A shows the TCRB-CDR3 spectratypes from CN/CI, the only identical twin pair concordant for MS. The comparison of their CD4+ TCR repertoire highlighted some relevant differences: the CDR3 spectratypes in CN, with few exceptions, showed a Gaussian-like distribution of bands, whereas in the co-twin Cl, which became ill in 2002, 8 years later than CN, there were oligoclonal expansions in some TCRBV families (Vß1, Vß6.1, Vß6.2, Vß16). This is in agreement with other data supporting the finding that the most pronounced skewing of the TCRBV gene use can be detected during the first period (1–2 years) after the MS onset [21 ]. This could be attributed to early T cell activation events occurring in the periphery. It is notable that when the CCR7+/CD4+ (central memory; Fig. 2B ) andCCR7–/CD4+ (effector memory; Fig. 2C ) T cell subsets of patient CI were analyzed separately, the former showed a normal CDR3 spectratype distribution, whereas the latter contained all the oligoclonal expansions. TCR sequences of some oligoclonal bands from CI, present in bulk CD4+ T cells and in the corresponding CCR7–/CD4+ subfraction (Vß6.1 and Vß6.2 in Fig. 2A and 2C ), were determined and found to be the same (Fig. 2D) .


Figure 1
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  		Figure 1. Spectratypes in the CD4+ T cell subset of the twin pair discordant for MS PM/PS, showing a regular CDR3 length profile (Gaussian distribution). For each Vß family, the spectratypes of intrapair twins have been run in adjacent lanes. Vß spectra not reported here were under the threshold of detection. PM, diseased twin; PS, healthy twin.

 

Figure 2
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  		Figure 2. Spectratype patterns from the twin pair concordant for MS CN/CI in the bulk CD4+ T cell population (A), in the CCR7+/CD4+ T cell subset (B), and in the CCR7–/CD4+ T cell subset (C). In the twin CI, the Vß6.1 and Vß6.2 expansions found in the bulk CD4+ T cells and in the CCR7–/CD4+ T cell subset are circled in black and correspond to the same TCRB whose CDR3 sequence is reported in D.

 
Increased T cell oligoclonality in the CD8+ T cell repertoire
CD8+ TCRB-CDR3 spectratypes in MS concordant and discordant twins were notably different from those of CD4+ T cells as a result of the frequent skewing found in all subjects under study (Fig. 3A 3B 3C 3D 3E ). Deviations from Gaussian distribution in some cases appeared as restricted spectra without prevalent expansions (RP Vß13, RS Vß22, TP Vß15, TE Vß20, PM Vß17, CI Vß6.1), and in other cases, one or few bands dominated the spectra indicating monoclonal (RP Vß18, UD Vß23, TP Vß11, TE Vß23) or oligoclonal (RS Vß11, US Vß3, US Vß15, UD Vß4, TE Vß12, CN Vß1, CI Vß12) T cell expansions. For a semiquantitative comparison of altered TCRB-CDR3 spectratypes within the CD8+ T cell population in diseased versus healthy twin members, we arbitrarily divided the spectra in two distinct categories: the first including spectra with perfect Gaussian distributions or with minor distortions and the second, comprehensive of spectra with an evidently irregular profile or dominated by oligo- or monoclonal expansions. By this criterion, we estimated that the frequency of altered TCRB-CDR3 length distributions was 50.4% (67 out of 133 total spectra evaluated) in the group of twins with MS and 49.4% (44 out of 89) in the group of healthy twins. A nonsignificant correlation between altered CD8+ TCRB-CDR3 spectratypes and the disease was evident (r=0.037; P=ns). Moreover, among all individuals analyzed, the highest frequency of altered CDR3 spectratype distributions was found in the healthy twin UD, who also showed a biased TCRB-CDR3 length distribution in the CD4+ T cell compartment (data not shown). These data indicated that an increased skewing of the CD8+ T cell repertoire was not associated with the clinical conditions. It is notable that within each pair, the majority of oligoclonal expansions was subject-specific, except in a few cases in which the expanded bands had equal size (Vß11 RP/RS, Vß4 UD/US, Vß22 TP/TE, Vß14 PM/PS in Fig. 3A 3B 3C 3D , respectively). This is true for the discordant twin pairs as well as for the concordant pair in which there is no apparent increase of dominant bands with the same size that could correspond to shared clonal expansions (Fig. 3E) . A general criterion to establish a possible relevance of T cell expansions in disease pathogenesis is that the expansion should be present in the patients but not in the healthy subjects. In our study, we found that Vß23 expansions were more frequent in affected (US, TE, PM, and CN) than in healthy twins (UD; Fig. 3B 3C 3D 3E ). This is in agreement with previous data showing increased skewing of the Vß23 family in the CD8+ T cell repertoire of MS patients compared with healthy controls [19 ].


Figure 3
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  		Figure 3. TCRB-CDR3 size spectratypes in the entire CD8+ T cell population show distortions from Gaussian distribution and frequent clonal expansions. Results from discordant twin pairs are reported in A for RP/RS, in B for UD/US, in C for TP/TE, and in D for PS/PM, healthy/MS-affected, respectively. The concordant twin pair CN/CI is reported in E.

 
Analysis of CDR3 spectratypes in the CCR7+/CD8+ and CCR7–/CD8+ T cell subsets
The expression of the chemokine receptor CCR7 divides human memory T cells into two functionally different subsets [31 ]. CD8+ T cells from identical twins were separated in the two CCR7+ and CCR7– phenotypes and analyzed further. Principally, we addressed our attention to CCR7–/CD8+ memory T cells (see below), which may be relevant for the disease, as they possess immediate effector functions (effector memory) and the ability to migrate toward inflamed tissues. However, we also studied the pool of CCR7+/CD8+ T cells, which in the peripheral blood, includes central memory T cells devoid of immediate effector functions, and naive T cells, thus outnumbering the CCR7– T cell fraction by far. TCRB-CDR3 spectratypes in CCR7+/CD8+ (Fig. 4 ) and in CCR7–/CD8+ (Fig. 5 ) T cell subsets were analyzed from three disease-discordant twin pairs (RP/RS, TP/PE, and UD/US) and from the concordant twin pair (CN/CI; Fig. 6 ). In members of the three discordant twin pairs, the CDR3 spectratypes were skewed in CCR7+/CD8+ (Fig. 4A 4B 4C) as well as in CCR7–/CD8+ (Fig. 5A 5B 5C) T cell subfractions. This suggested that effector memory (CCR7–) and central memory (CCR7+) T cells contributed to oligoclonality found in the bulk CD8+ T cell population. Sometimes Vß expansions detected in the total fraction of CD8+ T cells (Fig. 3A 3B 3C) were tracked out in CCR7+/CD8+ (RP Vß11, UD Vß7 in Fig. 4A and 4B ), in CCR7–/CD8+ T cells (US Vß3, UD Vß9, TE Vß4, TE Vß12, TE Vß23 in Fig. 5B and 5C ), or in both subsets (UD Vß4, UD Vß16 in Figs. 4B and 5B ). In contrast, the only twin pair concordant for MS generally showed a regular CDR3-length distribution in the CCR7+ fraction (Fig. 6A) . Thus, the majority of Vß expansions noticed in the entire CD8+ T cell fraction was found in the CCR7– effectormemory T cell pool (Fig. 6B) . To verify that such Vß expansions corresponded to the same clones, we characterized some expansions further. As an example, we focused on the Vß5.3 chain expansion from the concordant twin CN, which was detectable in the bulky CD8+ T cell population and even more evident in the CCR7– subset (see lower bands of Vß5.3 spectra in Figs. 3E and 6B ). Consistent with our expectation, the TCR sequencing confirmed the identity of these two expanded bands possessing CASS-GDSR-TQYFG (BJ2.3 segment) as a CDR3 motif.


Figure 4
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  		Figure 4. CDR3 spectratype patterns in the CCR7+/CD8+ T cell subsets of twins discordant for MS. RP/RS (A); UD/US (B); TP/TE (C).

 

Figure 5
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  		Figure 5. CDR3 spectratype patterns in the CCR7–/CD8+ T cell subsets of twins discordant for MS. RP/RS (A); UD/US (B); TP/TE (C).

 

Figure 6
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  		Figure 6. TCRB-CDR3 size spectratypes in the CCR7+/CD8+ (A) and in the CCR7–/CD8+ (B) T cell subsets derived from the concordant twins CN/CI. This is the only case in which spectra of CCR7+/CD8+ T cells show a Gaussian distribution of bands.

 
CDR3 sequence analysis of predominant TCRBV segments within CD8+ T cells of MS-affected and healthy monozygotic twins
To gain insight into the shaping of CD8+ T cell repertoires in MS-affected twins versus the healthy counterpart, we determined the sequence of more than 130 TCRB-CDR3 spectratype bands. Two-thirds of these TCRB sequences derived from the diseased members of the discordant pair (RS, TE, PM, and US) or of the concordant pair (CN and CI) and one-third from the unaffected twins (RP, TP, PS, and UD). We focused on spectratype bands corresponding to monoclonal or oligoclonal T cell expansions detected predominantly in the CCR7–/CD8+ T cell subset (~80 TCRB-CDR3 segments sequenced) but also in the CCR7+ fraction or in the bulk CD8+ T cell population. A main concern was to check whether predominant bands were actually composed of single expanded T cell clones. Therefore, PCR products from TCRB-CDR3 monoclonal spectra were subjected to direct DNA sequencing, and those corresponding to oligoclonal spectra were cloned into the pGEM-T vector, and more clones having the DNA insert of expected size were sequenced. In the majority of cases, one single, readable sequence was obtained by direct sequencing, and identical TCRB-CDR3 sequences were yielded from the positive clones. Only a negligible number of TCRs, reported here, did not match with expanded T cell clones.

Several important points could be addressed by the analysis of TCRB-CDR3 sequences obtained from discordant and concordant twin pairs. First, we asked whether affected twins displayed a preferential use of specific BJ segments compared with healthy counterparts. No clear-cut differences could be highlighted. We found instead that the BJ2.1 segment was most frequently represented in TCRB derived from patients (26%) as well as from normal twins (32%). Other segments used quite frequently by both cohorts were BJ2.7 and BJ1.1, which were present, respectively, in 17% and 15% of diseased twins and in 20% and 11% of healthy twins. Another major issue was to establish the occurrence of public TCR sequences shared by MS-affected twins and presumably implicated into the disease process [32 ]. For this, each TCRB-CDR3 amino acid motif was compared with all other determined TCR sequences. No identical TCRB shared by more than one subject was disclosed. However, the comparison of intrapair twin members or even affected versus affected, affected versus normal, and normal versus normal twins of the different pairs revealed the existence of a number of TCRB chains, which although not identical, were highly homologous. Table 2 shows several TCRs having common BV segments besides highly homologous N-D-N regions and identical BJ segments. It may be speculated that highly homologous TCRs shared between nonintrapair members had been selected by the same peptide/HLA Class I complex, as the individuals from whom they derive have at least one HLA Class I gene in common (HLA-B18 for TE and CN; HLA-A2 for RP/RS and UD; HLA-B7 for PM and RS). In addition, many other TCRB-CDR3 sequences, shown in Table 3 , could be grouped on the basis of common CDR3 motifs such as GTX, TSG, VAG, GRX, LR, GGS, DR, and DS.


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  		Table 2. Sequence Homologies among TCRB-CDR3 Regions of Clonally Expanded CD8+ T Cells Derived from MS-Affected and Healthy Twins

 

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  		Table 3. Recurrent TCRB-CDR3 Motifs Found in CD8+ T Cells of MS-Affected and Healthy Co-Twins

 
TCRs from predominant CD8+ T cell expansions show homology with published sequences
An extensive database search was performed by using the BLAST program and the CDR3 motifs shown in Tables 2 and 3 as query sequences. It is interesting that such comparative analyses revealed a significant degree of similarities between the TCRB-CDR3 sequences in the CD8+ T cell populations of identical twins reported here and others belonging to T cells directly or indirectly implicated in MS pathogenesis or with immune regulatory functions. As shown in Table 4 , a certain number of CDR3 motifs partially matched with those reported by Babbe et al. [16 ] and used by clonally expanded CD8+ T cells infiltrating actively demyelinating brain lesions in MS patients. It is notable that some of these TCRs belong to those T cell clones persistent in the CSF and the blood for many years [18 ]. Moreover, there were several other CDR3 sequence homologies with TCRs used by myelin-specific T cell clones from patients with MS (Table 4) . About one-third of these TCRs derive from the healthy members of the pairs. This finding is in agreement with other data showing the presence of neuroantigen-specific T cells in MS patients as well as in healthy individuals [42 ].


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  		Table 4. Sequence Homologies among TCRs of Clonally Expanded CD8+ T Cells and Published TCRs Found in Patients with MS

 
Transaldolase (TAL), an enzyme expressed selectively at high levels by oligodendrocytes, has been described as a novel autoantigen in MS. TAL Peptide 168-176 evokes a cytotoxic T cell response in HLA-A2-positive MS patients but not in HLA-matched controls [43 ]. Stimulation with the entire protein or even with peptide 168-176 elicits a skewed T cell response, as all TAL-specific CD8+ T cells expressed TCRs with Vß14 chains. It is interesting that the alignment of some of the Vß14 TCR sequences reported here, from affected or normal twins, with those adopted by TAL-responsive CTL lines revealed a surprisingly high homology in the CDR3 motif besides the use of the same BJ segment (Table 5 ). It is noteworthy that CD8+ T cells expressing these TCRs possessed the CCR7– phenotype and derived exclusively from HLA-A2-positive individuals (RP/RS and US/UD twin pairs). This evidence further supports the possibility that these expanded T cell clones are actually targeting the TAL protein.


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  		Table 5. Sequence Homologies with TCRs Recognizing 168–176 TAL Peptide

 
Viruses leading to latent and/or persistent infections, such as EBV and other herpes viruses, have been long associated with MS [3 ]. Therefore, we searched in the public databanks for TCRB-CDR3 sequence homologies between ours and published TCRs, expressed by T cells, driven by viral antigens. Table 6 shows the best matches between TCR sequences derived from MS-affected twins or from healthy co-twins under study and those published as specific for EBV.


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  		Table 6. Sequence Homologies among TCRs from Expanded CD8+ T Cells of Twins and TCRs of EBV-Specific T Cells

 
Glatiramer acetate [(GA) or copaxone] is a synthetic, random polymer whose efficacy in the clinical treatment of MS has been demonstrated amply. Evolution of the GA-induced CD8+ T cells and their TCR repertoire has been analyzed recently in patients with MS during the course of GA therapy [50 ]. Twins enrolled in our study have never been treated with GA. Nevertheless, in their CD8+ repertoire, there were TCRB-CDR3 sequences with significant homologies to those used by GA-specific CD8+ or CD4+ T cells obtained by GA-treated patients and controls (Table 7 ).


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  		Table 7. TCR Sequences Matching with Published TCRB-CDR3 Regions of Copaxone-Specific T Cells

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This paper reports a snapshot of CD8+ and CD4+ T cell repertoires in monozygotic twin pairs, four discordant and one concordant for MS, with the assumption that in such stringent setting, the differences in the TCRBV spectratyping could be influenced, at least in part, by the disease. Another major goal was to gain information about the TCRBV-CDR3 sequence motifs used by the predominant T cell expansions. The study focused on CD8+ T cells, toward which there is growing attention as pathogenic and regulatory cells [15 ]. This dual role makes a therapeutic approach aimed at modulating the balance in favor of the regulatory functions conceivable and therefore, encourages a better comprehension of their biology.

It is evident from the results shown here that a skewed and subject-specific TCRB repertoire in the peripheral CD8+ T cell population characterizes not only the twins with MS but also their healthy siblings. A global estimation of the frequency of oligoclonal or monoclonal TCR expansions in diseased compared with healthy co-twins did not reveal significant differences. Therefore, in such conditions of shared genetic background and perhaps also of environmental conditions, CD8+ T cell repertoires are not apparently marked by the pathologic state.

Many reports have described a more biased TCR repertoire in the blood of patients with MS compared with control individuals, and when a differential study was carried out on CD4+ and CD8+ subsets, a more prominent skewing was detected in the CD8+ T cells [20 21 22 23 24 ]. Accordingly, in our study, only occasional alterations have been detected in the CD4+ TCRB spectratypes of twins analyzed. However, as for CD8+ T cells, we failed to correlate the occurrence of such CD4+ T cell expansions with a clinically overt disease. Actually, the highest number of CD4+ TCRB expansions has been found in the healthy subject UD (data not shown), which also showed the most skewed CD8+ T cell repertoire (Fig. 3B) .

Different assumptions can be made to explain this lack of disease-distinctive features: first, CD8+ T cells oligoclonality occurs frequently in normal individuals as a result of infections, immune regulation, and senescence [51 , 52 ] and may increase in autoimmune diseases such as MS [19 ]. In our case, the contribution of the aforementioned factors could be so strong as to mask the expansions related to the disease. Accordingly, only in the case of the Vß23 family were we able to disclose a prevalence of CD8+ T cell expansions in the cohort of affected twins. Alternatively, the comparable degree of oligoclonality detected in both members of discordant twin pairs could be explained by the presence of a MS-associated trait, also carried by the unaffected sibling [53 ]. In this regard, consistent with our data, a shift in the CDR3 repertoires of naive CD4+ T cells in identical discordant twins compared with healthy controls has been reported [54 ]. Such a shift of CDR3 repertoires could represent a predisposing factor for MS, which, however, is not sufficient by itself to cause the disease. Sequencing data, which we have obtained from TCRB chains used by CD8+ T cell-expanded clones of affected and healthy co-twins, also support the possibility of a "common trait" shared by pairs of identical twins discordant for MS.

However, the real possibility to highlight differences in the expression of the TCRBV chains in peripheral blood T cells of MS patients has been questioned by Jacobsen and co-workers [17 ]. They noticed, in MS patients, an overexpression of single TCRBV chains in CSF-derived CD8+ T cells compared with those from peripheral blood and argued that the T cell repertoire analysis on peripheral blood does not always give a clue about what is happening in the disease-targeted tissues. A more informative analysis may possibly be obtained by subdividing the CD8+ T cell fraction in CCR7+ and CCR7– subsets. By this approach, we enriched our samples of ready effector memory T cells (CCR7– phenotype) able to reach the inflamed organs [31 ] and therefore, potentially important for the disease. In the three pairs of MS discordant twins (RP/RS, TP/PE, and UD/US), the separate analysis of CCR7+/CD8+ and CCR7–/CD8+ cells revealed a highly skewed repertoire distributed in both subsets (Figs. 4 and 5) . On the contrary, for MS concordant twins CN and CI, we were able to verify that all T cell expansions detected in the bulky CD8+ T cells were contributed by the CCR7– subset (Fig. 6) . The same observation was made by analyzing, separately, the CCR7+CD4+ and CCR7–CD4+ subsets in this concordant twin pair (Fig. 2) , suggesting that at least in the concordant twin pairs, T cell expansions are in charge of the population of effector memory T cells.

Comparison of a high number of TCRB-CDR3 sequences from the CD8+ T cells (from the bulk or from CCR7– and CCR7+ subsets) expanded in the repertoire of affected and healthy twins never showed 100% identity. Little is known about the sharing of TCR gene use in healthy identical twins or in pairs where MS is present [55 , 56 ]. However, our data are in favor of a low concordance rate in the TCR repertoire, also within twins concordant for MS. Nevertheless, we have found several TCRB sequences from siblings of the same or different pairs exhibiting a close match (Table 2) . Furthermore, there was a frequent sharing of several CDR3 motifs (DS, GTG, LGG, TSG; see Table 3 ), recurrent in MBP-reactive T cell clones from humans and rats [21 , 29 ].

A BLAST search for published sequences, matching the TCRB-CDR3 regions of these prominent CD8+ T cell expansions, has given a hint about the potential self and foreign antigens driving the repertoire of the twins under study. This analysis revealed a high number of TCRs sharing homology with sequences reported to be specific for CNS antigens, such as myelin components (Table 4) [36 37 38 39 40 41 ] or TAL (Table 5) , a key enzyme of the pentose phosphate pathway, abundant in oligodendrocytes, which elicits CD8+ T cell reactivity exclusively in HLA-A2-positive patients with MS [43 ]. In some cases, TCR matched sequences derived from clones of undefined specificity reported to dominate T cell infiltrates of brain lesions in MS patients [16 ].

The role of EBV as a possible infectious agent causing MS is supported by several findings. First, more than 90% of patients with MS show high concentrations of oligoclonal IgG antibodies in the brain and CSF, and a portion of this humoral immune response is directed against specific, latent EBV proteins [3 , 57 ]. Second, increased CD8+ T cell responses directed against EBV latent antigens are present in MS patients versus normal controls. Accordingly, our data suggest that in twins, part of the expanded CD8+ T cell repertoire could be driven by EBV-derived antigens (Table 6) .

In the clinical treatment of MS, GA (or copaxone) is used for its immune modulatory effects. GA administration induces not only CD4+ but also CD8+ specific T cell responses [58 ], which are regulatory/suppressive in nature and able to kill CD4+ T cells in a GA-specific and HLA Class I-restricted manner [59 ]. Very recently, in a longitudinal study, the clonal and functional dynamics of CD8+ T cell responses in patients with MS, during the period of GA therapy, have been evaluated [50 ]. TCRBV analysis revealed that GA treatment induces the development of a focused and oligoclonal CD8+ T cell repertoire with dominant clones that often persist over time. It is interesting that several TCRB-CDR3 sequences within the pool of expanded CD8+ T cells from our twins shared significant homologies with those used by GA-responsive CD8+ or CD4+ T cells (Table 7) [50 ]. It is noteworthy that the twins recruited for this study have never been under a GA regimen. Therefore, this finding gives a clue to the presence of naturally occurring CD8+ T cells, with bona fide regulatory properties in the healthy or in MS-affected twins, which are likely to be expanded further by GA administration.

In conclusion, the results reported here disclose a large overlapping of CD8+ T cell repertoires in affected versus healthy co-twins. It is interesting that these sequences share homology with published TCRs specific for viruses, self-antigens, and synthetic compounds with regulatory functions, all related to MS pathogenesis and disease-modulation, suggesting that 1) these T cells are not per se pathogenic, being found in healthy and diseased twins; 2) immunoregulatory T cells are likely to be present in both twins and therefore, may represent a therapeutical target; and 3) epigenetic-environmental factors, which impair the balance between autoaggressive and regulatory T cells, rather than the expansion of a disease-specific T cell repertoire, may be responsible for the disease.


   ACKNOWLEDGEMENTS
 
This project has been supported by the Associazione Italiana Sclerosi Multipla (FISM), project numbers 2001/R54 and 2003/R59, by the Ministero, Istruzione, Università e Ricerca (MIUR), fondo per gli investimenti della ricerca di base (FIRB) to R. S., and by the Ministero della Salute (Finalizzato) to L. B. We express our gratitude to all twins for their cooperation. We thank Dr. M. P. Perrone for HLA typing of twins and F. Lucantoni for technical assistance.

Received September 22, 2006; accepted October 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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