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Brain, Vol. 127, No. 5, 981-995, 2004
© 2004 Guarantors of Brain
doi: 10.1093/brain/awh119

Blood T-cell receptor ß chain transcriptome in multiple sclerosis. Characterization of the T cells with altered CDR3 length distribution

David-Axel Laplaud1,2, Catherine Ruiz1, Sandrine Wiertlewski2, Sophie Brouard1, Laureline Berthelot1, Marina Guillet3, Benoît Melchior1, Nicolas Degauque1, Gilles Edan4, Philippe Brachet1, Philippe Damier1,2 and Jean-Paul Soulillou1

1 Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 437: ‘Immunointervention dans les Allo- et Xénotransplantations’, Institut de Transplantation et de Recherche en Transplantation (ITERT), CHU Hôtel Dieu, 30 Bd Jean Monnet, 2 Clinique Neurologique and Centre d’Investigation Clinique, Hôpital G. et R. Laennec, Bd J. Monod, 3 TcLand S.A., 30 Bd Jean Monnet, 44093 Nantes Cedex and 4 Service de Neurologie, Hôpital Pontchaillou, 35000, Rennes, France

Correspondence to: Jean-Paul Soulillou, INSERM U437, 30 Bd Jean-Monnet, 44093 Nantes Cedex 01, France E-mail: jps{at}nantes.inserm.fr


   Summary
Top
 Summary
Introduction
 Material and methods
 Results
 Discussion
 References
 
Multiple sclerosis is an inflammatory demyelinating disease of the CNS associated with T cells autoreactive for myelin components. In this study, we analysed the T-cell receptor (TCR) usage of the variable ß (Vß) chain transcriptome in the blood of multiple sclerosis patients at various stages of the disease using a global and quantitative comparison of the complementarity-determining region 3 length distribution (CDR3-LD) of transcripts of the 26 Vß genes. We investigated 35 patients: 12 with a high risk of multiple sclerosis, 10 with clinically definite multiple sclerosis, 13 with a relapsing–remitting worsening and active multiple sclerosis and 13 healthy individuals. Cells bearing the TCR transcripts with altered CDR3-LD were sorted and studied for CD4 or CD8 phenotype, cytokine transcript accumulation and response to human myelin basic protein (MBP). We show that patients from all the groups have a significantly skewed blood T-cell repertoire. Vß transcriptome patterns were more altered in patients from the clinically definite multiple sclerosis group and the worsening and active multiple sclerosis group than in the high risk group. The T cells sorted from Vß families with altered CDR3-LD concerned both CD4 and CD8 T cells, with a more pronounced skewing in the CD8 compartment. These cells displayed a significantly increased level of interferon-{gamma}, interleukin-2 and tumour necrosis factor-{alpha} transcripts compared with their counterparts from the healthy individual group. Furthermore, using interferon-{gamma} enzyme-linked immunospot (ELISPOT) assays, T cells from four out of seven altered Vß families tested from multiple sclerosis patients responded to human MBP, whereas no response was observed with human albumin or with altered Vß families from healthy individuals. Our data support the concept of an early autoimmune component in the disease and emphasize the possible involvement of CD8-positive T cells in multiple sclerosis.

Key Words: CD4/CD8; CDR3; cytokines; multiple sclerosis; T cells; Vß genes

Abbreviations: APC= antigen-presenting cell; CDMS = clinically definite multiple sclerosis; CDR3 = complementarity-determining region 3; CDR3-LD = complementarity-determining region 3 length distribution; ELISPOT = enzyme-linked immunospot; HI = healthy individuals; HLA = histocompatibility leukocyte antigen; HPRT = hypoxanthine phosphorylribosyl transferase; HRMS = high risk of multiple sclerosis; IFN-{gamma} = interferon-{gamma}; IL = interleukin; MBP = myelin basic protein; PBL = peripheral blood lymphocyte; PBMC = peripheral blood mononuclear cell;TCR = T-cell receptor; TNF-{alpha} = tumour necrosis factor-{alpha}; Vß = variable ß; WMS = worsening multiple sclerosis

Received October 1, 2003. Revised December 10, 2003. Accepted December 14, 2003.


   Introduction
Top
 Summary
 Introduction
Material and methods
 Results
 Discussion
 References
 
Multiple sclerosis is a chronic inflammatory and demyelinating disease of the CNS whose aetiology remains unknown. Autoimmunity seems to play a key role in multiple sclerosis pathogenesis, as suggested by the presence of autoreactive T cells for myelin components in the peripheral blood and CNS of patients (Noseworthy et al., 2000Go). A better characterization of the peripheral blood T lymphocytes in multiple sclerosis would probably serve to evaluate the disease severity, to forecast its evolution and to adopt new therapeutic strategies. One way to characterize the peripheral lymphocytes possibly implicated in multiple sclerosis is to analyse the skewing of the complementarity-determining region 3 length distribution (CDR3-LD) of the T-cell receptor (TCR) variable ß chain (Vß). Such an analysis was developed initially on the basis of a semi-quantitative assessment of CDR3-LD by reverse transcription–polymerase chain reaction (RT–PCR) technology (spectratype/Immunoscope® methods) (Pannetier et al., 1995Go). Several studies have addressed the question of CDR3-LD in unmanipulated T cells collected from the blood of multiple sclerosis patients and detected skewed ß gene usage, a fact that supports the possibility of oligoclonal T-cell expansions in the blood (Musette et al., 1996Go; Gran et al., 1998Go; Lozeron et al., 1998Go; Muraro et al., 2002Go; Matsumoto et al., 2003Go). In addition, some studies have focused on myelin basic protein (MBP)-specific T lymphocytes, showing a preferential use of certain Vß genes (Wucherpfennig et al., 1990Go; Kotzin et al., 1991Go; Oksenberg et al., 1993Go). However, these latter studies were performed following in vitro T-cell clone stimulation with myelin peptides, a procedure that may select and promote the growth of low frequency committed T cells and therefore may not reflect the actual autoreactive T-cell pool size involved.

Recently, we developed an approach which integrates both the analysis of CDR3-LD alterations (Immunoscope®/spectratype) and quantitative real-time PCR-based measurements of Vß/hypoxanthine phosphoribosyl transferase (HPRT) transcript ratios, for all the possible CDR3 lengths. The data are displayed as a global ‘T-cell landscape’ of the whole ß chain transcriptome (Sebille et al., 2001Go; Guillet et al., 2002Go). In this study, we used this approach to investigate the Vß CDR3-LD and, more specifically, to estimate the magnitude of the skewed T-cell repertoire at various stages of the disease, including at the appearance of the first clinical symptoms. Thirty-five patients suffering from different stages of multiple sclerosis [patients with a high risk of multiple sclerosis (HRMS), patients with clinically definite multiple sclerosis (CDMS) or patients with a relapsing–remitting worsening and active multiple sclerosis (WMS)] and 13 healthy individuals (HI) were compared. Our data show that there is a detectable T-cell immune component in the blood of multiple sclerosis patients at all stages of the disease. The blood T-cell CDR3-LD alterations concerned the CD4- and CD8-positive cells but were prominent in the CD8+ fraction, involving numerous ß chain families in CDMS and WMS compared with patients with HRMS. In addition, the T cells from multiple sclerosis patients with a selected TCR accumulated more proinflammatory cytokine transcripts than their normal HI counterparts. Furthermore, using an interferon-{gamma} (IFN-{gamma}) enzyme-linked immunospot (ELISPOT) assay, we show that most of the T cells from altered Vß families of the three groups of patients respond to human MBP, suggesting their possible role in the disease process.


   Material and methods
Top
 Summary
 Introduction
 Material and methods
Results
 Discussion
 References
 
Patients
Thirty-five patients divided into two groups were studied (their clinical characteristics are summarized in Table 1). Group I was composed of 22 patients at the onset of the disease. This group was divided into two subgroups. The HRMS group (n = 12) was composed of patients presenting a first demyelinating event clinically well defined and confirmed by neurological or ophthalmological examination and the presence of at least three Barkhof’s criteria on a spinal or cerebral MRI (Barkhof et al., 1997Go). Patients in this group were referred to as HRMS patients (CHAMPS Study Group, 2002Go). The CDMS group (n = 10) was composed of patients undergoing a second or third relapse, reaching a clinically definite multiple sclerosis according to Poser’s criteria (Poser et al., 1983Go). Blood from group I patients was collected at the time of a relapse. None of the patients were under immunosuppressive or immunoregulatory drug treatment at the time of or before the study. All patients were interviewed to confirm the absence of infectious illness or other autoimmune diseases, and all had a blood cell count within the normal range. Group II or the WMS group (n = 13) was composed of patients with a clinically definite relapsing–remitting multiple sclerosis (according to Poser’s criteria) considered as requiring an immunosuppressive treatment with mitoxantrone (Edan et al., 1997Go). Patients from the WMS group were referred to as patients with a worsening and active multiple sclerosis. The criteria for mitoxantrone treatment were the loss of at least 1 point on the Expanded Disability Status Score during the previous 6 months and/or the occurrence of several relapses despite treatment compliance and the presence of at least one gadolinium-enhanced T1 lesion according to MRI. None of the patients suffered from other detectable autoimmune, inflammatory or infectious diseases. Results from laboratory tests performed at the time of blood sampling were within the normal ranges. All WMS group patients had undergone an immunomodulatory treatment that had been stopped at least 1 month before testing. Histocompatibility leukocyte antigen (HLA)-DR typing was performed for 32 patients. HLA class I typing was available for 14 patients.


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  		Table 1 Main clinical characteristics of the patients
 
HI group
Blood from 13 HI (mean age: 31.8, eight females, five males), who had been interviewed previously to rule out autoimmune or inflammatory disease, was taken for comparison. All multiple sclerosis patients and HI gave their informed consent for this study according to French legislation.

Blood harvesting, RNA extraction and cDNA synthesis
A 100 ml aliquot of blood was collected by venopuncture. Peripheral blood lymphocytes (PBLs) were recovered after a Ficoll gradient (Eurobio, Les Ulis, France). After washing, 2 x 107 cells were frozen in Trizol® reagent (InvitrogenTM, Life Technologies, CA) for RNA extraction according to the manufacturer’s instructions. The rest of the cells were frozen at –80°C in AB serum containing 7.5% dimethylsulfoxide. One day later, cells were transferred to a liquid nitrogen tank. The RNA concentration for each sample was determined by optical density measurement, and a quality control on a 1% agarose gel was performed. A 2 µg aliquot of RNA was reverse transcribed using an Invitrogen cDNA synthesis kit (Boeringher Mannheim, Indianapolis, IN) and diluted to a final volume of 100 µl.

TCR repertoire analysis
cDNA was amplified by PCR using a Cß primer and one of the 26 Vß-specific primers. The amplifications were performed in a 9600 Perkin-Elmer thermocycler (Applied Biosystems, Foster City, CA) as previously described (Gagne et al., 2000Go). Analysis of CDR3-LD was performed using Immunoscope® software (Pannetier et al., 1995Go; Douillard et al., 1996Go; Brouard et al., 1999Go). The percentage of CDR3-LD alteration for each Vß family and a global percentage of CDR3-LD alteration for each individual or each group was obtained as described in Gorochov et al. (1998Go). Briefly, the percentage of alteration was defined as the difference between the frequency of each CDR3 length in the distribution profile of the Vß family studied and the control distribution, calculated from the 13 age- and sex-matched HI. The global CDR3-LD alteration is represented as a Topview TcLandscape® (see below) enabling an easy appraisal of the ‘qualitative’ measurement of the CDR3-LD bias. Only CDR3 lengths with an alteration >25% were taken into account. The level of Vß RNA was measured by real-time quantitative PCR and expressed as a ratio of a non- or minimally regulated gene, the HPRT gene, in order to normalize the values, and the primers used were especially designed for quantitative PCR as previously described (Gagne et al., 2000Go). The data were displayed as a three-dimensional TcLandscape® (Guillet et al., 2001Go; Sebille et al., 2001Go). Percentages of CDR3-LD alterations are represented as a colour code, from deep blue (–30%) to dark red (+30%). The x-axis displays the 26 human Vß families, the y-axis gives the Vß/HPRT ratios, and the z-axis gives the CDR3 lengths. The colour code is the same for the three-dimensional TcLandscape® and the corresponding Topview.

Cytokine transcript quantification
Cytokine transcript measurement was performed on RNA from sorted Vß families. The cells were sorted using phycoerythrin-coupled Vß monoclonal antibodies (Immunotech, Marseille, France) on a FACSvantage (Becton-Dickinson, Mountain View, CA). The purity of sorted cells was >95%. RNA was extracted as described above and cDNA was obtained using a Boehringer SMART kit (Boehringer, IN) according to the manufacturer’s recommendations. Briefly, RNA samples were mixed with cDNA synthesis primer and SMART II oligonucleotide, incubated at 72°C for 2 min and chilled on ice. First-strand buffer, dithiothreitol, deoxynucleotide triphosphate (dNTP) and PowerScript Reverse Transcriptase were then added and the tubes incubated at 42°C for 1 h, before being put on ice. A non-specific PCR was then performed, allowing a linear amplification. Pure water, Advantage 2 PCR buffer, dNTP, PCR primers and Advantage Polymerase Mix were added to the first strand cDNA obtained previously. The tubes were then placed in a Perkin-Elmer 9600 automater, and thermal cycling was performed using the following program: 1 min at 95°C, n cycles with 5 s at 95°C, 5 s at 65°C, and 6 min at 68°C (the number of cycles was determined by the quantity of RNA as assessed by optical density measurement). Real-time quantitative PCR was performed subsequently using interleukin (IL)-2, IFN-{gamma}, tumour necrosis factor-{alpha} (TNF-{alpha}), IL-10, IL-13 and IL-2-R{alpha} (CD25) chain primers (IL-2, 5'-AAACACAGCTACAACTGGAGCA-3' and 3'-GCTGATTAAGT CCCTGGGTCTT-5'; IFN-{gamma}, 5'-TGTCCAACGCAAAGCAATA CA-3' and 3'-TTCGCTTCCCTGTTTTAGCTG-5'; TNF-{alpha}, 5'-T TAAGCAACAAGACCACCACT-3' and 3'-TCAAGGAAGTCTG GAAACATCT-5'; IL-10, 5'-CTGCCTAACATGCTTCGAGATC-3' and 3'-AACCCTTAAAGTCCTCCAGCAA-5'; IL-13, 5'-GGCA GCATGG TATGGAGCA-3' and 3'-TTCAGTTGAACCGTCCC TCG-5'; IL-2-R{alpha}, 5'-CAAGGGTCAGGAAGATGGATTC-3' and 3'-CCAGGACGAGTGGCTAGAGTTT-5') and normalized against HPRT transcript levels (Guillet et al., 2001Go).

CD4/CD8 T cell characterization
CD4+ or CD8+ T-cell selection was performed using MACS microbeads according to the manufacturer’s recommendations (Miltenyi Biotec, Germany). The kit consists of an indirect magnetic labelling system composed of a hapten–monoclonal antibody cocktail (anti-CD8/anti-CD4, CD16, CD56, CD11b, CD19 and CD36) and iron microbeads coupled with an anti-hapten antibody which enables the magnetic depletion of non-T cells. The magnetic bead-labelled cells are depleted by passing the cells through a MACS column in the magnetic field of an autoMACS. Purity was >90%. The cells were mixed with Trizol® and the RNA extracted and retrotranscribed using the SMART procedure as explained above. The cDNA was then amplified and the CDR3-LD analysis performed as described above.

In order to assign a selected CDR3 length to a population of CD4+ or CD8+ cells, the Immunoscope profiles of unselected PBL, CD4+ and CD8+ fractions were compared. The percentage corresponding to the frequency of a given selected CDR3 length in a Vß family was calculated and compared in the PBL, CD4+ and CD8+ fractions. The skewing was assigned to the CD8+ cells when the calculated percentage was higher for CD8 than for CD4+ cells.

ELISPOT assay protocol
T cells from seven Vß families with an altered CDR3 length were sorted from six patients (HRMS4, Vß 17; HRMS7, Vß 3; HRMS12, Vß 8 and Vß 14; CDMS1, Vß 23; WMS7, Vß 3; and WMS8, Vß 4), and three Vß families with an altered CDR3 length were sorted from two HI (HI9, Vß 21 and Vß 22; and HI10, Vß 2) and studied for human MBP reactivity. Additionnally, three other Vß families with a Gaussian-like CDR3-LD were sorted from three multiple sclerosis patients (WMS2, Vß 8, WMS12, Vß 3; and HRMS10, Vß 23) and also studied for MBP reactivity, in the same conditions. Peripheral blood mononuclear cells (PBMCs) were thawed and washed in phosphate-buffered saline. T cells with a Vß family with an altered CDR3 length were sorted using the corresponding phycoerythrin-labelled anti-Vß antibody and anti-phycoerythrin microbeads in an autoMACS (Miltenyi Biotech, Germany). The purity of the sorted Vß families was measured by flow cytometry and was >75%. The remaining fraction of the PBMCs was irradiated for 315 s at 15 Gy and they were used as autologous antigen-presenting cells (APCs). First, human MBP (Sigma, France) at a concentration of 20 µg/ml, or human albumin (Sigma, France) at the same concentration was mixed with irradiated PBMCs. Next, 4 x 104 PBMCs were added to a 96-well anti-IFN-{gamma}-coated ELISPOT plate (AID, Germany) and 4 x 104 of the T cells from sorted Vß families were added to the plate. A control with irradiated PBMCs alone at the same concentration was performed systematically. The cells were incubated for 24 h at 37°C in 5% CO2 and washed three times with washing buffer according to the manufacturer’s instructions. A secondary biotinylated anti-IFN-{gamma} antibody was then added at 100 µl/well for 2.5 h. Plates were washed three times with buffer, and streptavidin–horseradish peroxidase was added for 2 h at room temperature. Plates were washed in buffer and spot colour was developed for a maximum of 1 h by adding 3-amino-9-ethylcarbazol substrate diluted in acetate buffer containing H2O2. Plates were then washed with distilled water to stop the reaction. After drying, images of the wells were acquired using the AID ELISPOT software. All the experiments were run at least in duplicate or triplicate depending on the number of cells available. The results were expressed as means of the du(tri)plicates.

Statistical analysis
A {chi}2 test, a Kruskal–Wallis test and a Dunn’s multiple comparison test were performed between each group of multiple sclerosis patients (HRMS, CDMS and WMS) and HI for comparison of the Vß/HPRT transcript ratios and the global CDR3-LD profiles. A Kruskal–Wallis test was performed on cytokine transcript values. A Mann–Whitney test was performed for the different ELISPOT assays. Differences were defined as statistically significant when P < 0.05.


   Results
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 Summary
 Introduction
 Material and methods
 Results
Discussion
 References
 
HI display few Vß families with significant CDR3-LD alterations with low Vß/HPRT transcript ratios
Figure 1 shows one representative example of the CDR3- LD profiles from the HI group. The TcLandscape pattern of every normal individual tested is available online (www.nantes.inserm.fr/u437/sitesconnexes.html). Only two of the 13 normal individuals expressed Vß families with a significantly altered CDR3 length (>25%). All details concerning the alteration of Vß families in the HI group are given in Fig. 5. Furthermore, the mean global percentage of CDR3-LD alteration in this group was 10.5 ± 1.9% (Fig. 6A). The mean Vß/HPRT transcript ratio in the HI group was 2.3 ± 0.9. In addition, 67% of the Vß families of HI had a Vß/HPRT transcript ratio between 0 and 2, while 23% of the Vß families had a ratio between 2 and 5, and 10% a ratio >5 (Fig. 6C).



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  		Fig. 1 TcLandscape® of blood T cells from one representative HI. The data are displayed for each individual as a three-dimensional TcLandscape® and as a Topview for easier assessment of global CDR3-LD alterations. The percentages of CDR3-LD alterations are represented as a colour code. The x-axis displays the 26 human Vß families, the y-axis gives the Vß/HPRT ratios, and the z-axis gives the 13 possible CDR3 lengths.

 


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  		Fig. 5 CDR3 length alterations for all the patients and HI. CDR3 length alterations >25% appear in black.

 


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  		Fig. 6 (A) Values of global CDR3-LD for each patient and HI. The mean value for each group is indicated by a bar. A Kruskal–Wallis test and a Dunn’s multiple comparison test were performed, **P < 0.01. (B) Distribution of the mean Vß/HPRT transcript ratios in the patient groups and HI (each dot represents the mean Vß/HPRT transcript ratio for each patient and HI). A Kruskal–Wallis test and a Dunn’s multiple comparison test were performed to compare values. A bar indicates the mean value for each group. **P < 0.01. (C) Distribution of the Vß/HPRT transcript ratios between the groups according to a grading scale of 0–2, 2–5 and >5. A {chi}2 test was performed to compare the distribution between the groups.

 
HRMS patients express a significantly higher mean CDR3-LD alteration than HI
Figure 2 displays the CDR3-LD of the 12 HRMS patients. Four of the 12 patients displayed a CDR3 length with an alteration >25%. Details of the CDR3 length alterations are given in Fig. 5. Neither the number of patients with significantly altered Vß (presence of a CDR3 length alteration >25%) nor the number of Vß families with a significantly altered CDR3 length were different from the HI group. However, the mean percentage of CDR3-LD alterations (16.5 ± 2.9%, Fig. 6A) was significantly higher than that of the HI group (10.58 ± 1.9%, P < 0.01).



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  		Fig. 2 TcLandscape® and Topview representation of blood T cells from the HRMS group. CDR3 length alterations >25% appear in red.

 
The blood T cells from CDMS patients display more extensive CDR3-LD alterations than those from HRMS patients and HI
Nine of the 10 CDMS patients displayed a CDR3 length with an alteration >25% (TcLandscapes® and Topviews, Fig. 3). Details of the Vß families concerned are given in Fig. 5. The number of significant CDR3 length alterations in the CDMS group was significantly different from that of the HI and HRMS groups (P < 0.01). The number of patients displaying at least one altered CDR3 length in the CDMS group was also significantly different from the HI and HRMS groups (P < 0.01). In addition, the mean percentage of CDR3-LD alterations (17.8 ± 1.6%, Fig. 6A) was significantly different from that observed in HI (10.58 ± 1.98%, P < 0.001) and in the HRMS group (16.5 ± 2.9%, P < 0.03).



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  		Fig. 3 TcLandscape® and Topview representation of blood T cells from the CDMS group. CDR3 length alterations >25% appear in red.

 
The blood T cells from WMS patients display significantly more CDR3-LD alterations than those from HI and HRMS patients
Ten of the 13 WMS patients displayed CDR3 lengths with alterations >25% (TcLandscapes® and Topviews, Fig. 4). Details concerning the Vß families with significant CDR3 length alterations are summarized in Fig. 5. Patients with at least one significantly altered CDR3 length (alteration >25%) were significantly more numerous than in the HI and HRMS groups (P < 0.01 and P < 0.05, respectively). Furthermore, the number of alterations >25% in the WMS group was significantly different from the HI group (P < 0.05). Additionally, the mean percentage of CDR3-LD alterations in WMS patients (15.5 ± 1.5%, Fig. 6A) was significantly higher than that of the HI group (10.58 ± 1.9%, P < 0.01) but not that of HRMS patients (16.5 ± 2.9%).



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  		Fig. 4 TcLandscape® and Topview representation of blood T cells from the WMS group. CDR3 length alterations >25% appear in red.

 
Vß/HPRT transcript ratios
Vß/HPRT transcript ratio data are an indirect reflection of the pool size of T-cell populations with different CDR3-LD. The mean Vß/HPRT transcript ratios in the HRMS (0.94 ± 0.5), CDMS (0.6 ±  0.4) and WMS groups (0.7 ± 0.3) were significantly lower than those in the HI group (2.3 ± 0.9, P < 0.001, Fig. 6B). Furthermore, when Vß/HPRT transcript ratios were compared according to a grading scale of 0–2, 2–5 and >5, their distribution was significantly different compared with the HI group (Fig. 6C, P < 0.01).

Vß transcript CDR3-LD alterations according to HLA class I and class II typing and MRI activity
Distribution of CDR3-LD alterations according to HLA-DR typing was analysed in 32 patients, 13 of them being DR2 positive. Ten of these 13 patients displayed significant alterations versus 12 of the 18 negative for DR2 (NS). No public skewed CDR3-LD was observed in HLA-DR2 patients. No correlation between Vß family alterations and HLA class I typing was found. Correlation of CDR3-LD alterations with T1 gadolinium-enhanced lesions was also studied. For the patients of group I, 14 exhibited at least one gadolinium-enhanced lesion. Eight of them exhibited at least an altered CDR3 length >25% versus four out of eight with no gadolinium-enhanced lesion (NS).

T cells from multiple sclerosis patients with altered CDR3-LD Vß accumulate proinflammatory cytokine transcripts
In order to better understand the role played by the altered T cells identified by TcLandscape®, five Vß families with highly altered CDR3-LD were sorted from three different patients (one patient per group: HRMS8, Vß 3; CDMS4, Vß7.1, Vß7.2 and Vß17; WMS1, Vß17). These families were compared with sorted Vß families with Gaussian-like CDR3-LD (HI10, Vß 3 and Vß 7; HI11, Vß 17; HI12, Vß 4 and Vß 17; HI13, Vß 1 and Vß 16) and three Vß families with altered CDR3-LD from HI (HI9 and HI12, Vß 21, Vß 22 and Vß 13.1, respectively) for accumulation of different cytokine transcripts. The data are summarized in Fig. 7. The mean IFN-{gamma} and IL-2 mRNA levels in sorted Vß families with altered CDR3-LD from multiple sclerosis patients was significantly higher than for families with a Gaussian-like pattern or altered CDR3-LD in the HI group (P < 0.01 and P < 0.05, respectively). TNF-{alpha} mRNA was also significantly accumulated in the multiple sclerosis group, compared with the HI group (P < 0.05). IL-10, IL-13 and IL-2-R{alpha} chain mRNA transcripts were not significantly different between the three groups. Despite the fact that the number of patients analysed was low, these data suggest the presence of T cells with highly altered CDR3-LD able to produce proinflammatory cytokines in the blood of multiple sclerosis patients.



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  		Fig. 7 Cytokine/HPRT transcript ratios from sorted T cells. Cytokine mRNA was studied for T cells sorted from families with CDR3 length alteration >25% in multiple sclerosis patients compared with T cells sorted from Vß families with a Gaussian-like pattern (HI-G) or an altered CDR3 length (HI-A) from healthy individuals. The median range is indicated for each group. Kruskal–Wallis test, *P < 0.05, **P < 0.01.

 
The alterations of CDR3-LD are more prominent in CD8+ T cells
Sorted CD4+ and CD8+ T cells from nine patients (16 Vß families) and two HI (six Vß families) were studied and their immunoscope profile compared with that of unselected PBLs (Fig. 8A and B). In some cases, the CD4 or CD8 nature of a selected CDR3 length was obvious (Fig. 8A, examples CDMS1 and CDMS9). In other cases (Fig. 8B: HRMS12, Vß 14; CDMS4, Vß 15, Vß 17 and Vß 21; WMS7, Vß 7; WMS9, WMS10 and HI9, Vß 6.4), the correspondence of the immunoscope profiles of total PBLs with that of the fraction studied (CD4 or CD8) suggested that the selected CDR3 length in question belonged to this T-cell subpopulation, which was corroborated by the comparison of the frequency of the prominent CDR3 length in the three cellular fractions. Finally, in other Vß families (Fig 8B: CDMS4, Vß 7; CDMS7; CDMS10; WMS7, Vß 4 and Vß 15; HI9, Vß 21, Vß 22 and Vß 24; HI7, Vß8 and Vß 24), no clear assignment to CD4+ or CD8+ cells was possible. Using this approach, 11 of the 16 Vß families from multiple sclerosis patients and one of the six Vß families from HI with an altered CDR3 length were found to have a more prominent skewing in the CD8+ T cells, suggesting a more pronounced bias in the CD8+ repertoire in multiple sclerosis than in HI.



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  		Fig. 8 CD4/CD8 phenotype of blood T cells from Vß families with an altered CDR3 length. (A) Examples from Vß families with an altered CDR3 length. The phenotype was obtained by comparison of the immunoscope profile of each population (CD4, CD8 and unselected PBLs). (B) Summary of the CD4/CD8 pattern of the Vß families studied. CD8>CD4 means that the CD8+ fraction of T cells is more represented in the selected CDR3 length than the CD4+ fraction (see Material and methods). The percentage indicated represents the frequency of the more prominent CDR3 length in the Vß family studied. The comparison of the percentages for the same CDR3 length in the different cell fractions (PBL, CD4 and CD8) indicates the fraction that is responsible for the skewing.

 
Sorted T cells from Vß families with an altered CDR3-LD from multiple sclerosis patients produce IFN-{gamma} in the presence of human MBP
IFN-{gamma} ELISPOT assays were performed with T cells from Vß families with altered CDR3–LD from six multiple sclerosis patients (three from the HRMS group, one from the CDMS group and two from the WMS group) and two HI. In addition, because the analysis of Vß families with Gaussian-like CDR3-LD from patients already studied for their skewed Vß family was technically impossible (due to an insufficient quantity of cells), three Vß families with Gaussian-like CDR3-LD were studied from three other patients. Only background ELISPOT reactivity (<10 spots) was detected when irradiated PBMCs (used as APCs) were tested (data not shown). Figure 9A shows the ELISPOT score obtained when human MBP was added to a culture of purified sorted T cells and irradiated PBMCs used as APCs (black boxes). For comparison, the same cultures were tested with human albumin (white boxes). In addition, the irradiated PBMCs alone were also stimulated by human MBP (grey boxes). The figure shows that purified T cells from multiple sclerosis patients were highly reactive in response to human MBP (P < 0.01 versus human albumin). Some reactivity was also observed in the irradiated PBMC fraction when human MBP was present in the culture. However, the sorted T-cell response was much higher than that of the PBMCs alone in four out of seven cases. Importantly, Fig. 9B shows that sorted T cells from the HI counterparts cultured in the same conditions with syngenic PBMCs were not reactive to human MBP compared with altered Vß families from multiple sclerosis patients (P < 0.05). Furthermore, the number of IFN-{gamma} spots obtained with the Gaussian Vß families from the three additional patients was as low as for the altered Vß families from HI (mean number of spots: 2.3 ± 1), suggesting a specific response of the skewed Vß families from multiple sclerosis patients.



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  		Fig. 9 IFN-{gamma} ELISPOT in T cells sorted from Vß families with an altered CDR3 length. (A) ELISPOT score of irradiated PBMCs (APCs) in multiple sclerosis patients (grey boxes) and irradiated PBMCs with T cells from sorted CDR3-LD-altered Vß families in the same multiple sclerosis patients (black boxes) in the presence of human MBP. White boxes represent the control score obtained with PBMCs and sorted T cells from the same multiple sclerosis patients in the presence of human albumin. (B) Comparison of reactivity for human MBP in irradiated PBMCs with T cells sorted from CDR3-LD-altered Vß families (black squares) or HI (white squares). Mann–Whitney test, P < 0.01.

 

   Discussion
Top
 Summary
 Introduction
 Material and methods
 Results
 Discussion
References
 
In this study, we analysed the TCR Vß chain transcriptome both qualitatively (alteration of CDR3-LD) and quantitatively (amount of transcripts concerned) in three groups of patients with relapsing–remitting multiple sclerosis at various stages of their disease compared with age-matched HI. Despite the fact that our study only concerned Vß biases (analysis of V{alpha} chain patterns may provide further information), significant T-cell repertoire alterations, which were more prominent in CD8+ T cells and mostly concerned Vß families reactive to human MBP, can be detected in the blood of these patients from the onset of the disease [occurrence of first clinical symptoms (HRMS)]. Furthermore, we show that sorted, circulating T cells from families with altered CDR3-LD are characterized by a transcriptional pattern with significant accumulations of IFN-{gamma}, IL-2 and TNF-{alpha} mRNA, without any further in vitro stimulation. Our data provide new information that supports the concept of early peripheral T-cell activation in multiple sclerosis. Our results also suggest that multiple sclerosis patients could benefit from immunoregulatory treatment early in the course of their disease.

TCR biases in patients with multiple sclerosis have been reported by several groups in the past (Kotzin et al., 1991Go; Oksenberg et al., 1993Go; Musette et al., 1996Go; Gran et al., 1998Go; Lozeron et al., 1998Go; Muraro et al., 2002Go; Matsumoto et al., 2003Go). However, this study is the first to analyse additionally a cohort of patients identified as HRMS patients. Although healthy age-matched individuals also exhibited some level of CDR3-LD alterations (~10%), HRMS patients displayed highly significant global CDR3-LD alterations (~16%). However, the Vß/HPRT transcript ratios did not suggest blood accumulation of the T-cell populations using the altered CDR3 Vß transcript species. This apparent lack of peripheral accumulation may be related to a continuous flux of these selected T cells toward other compartments including the CNS. Such a flux has been suggested recently by the effect of natalizumab, a monoclonal antibody inhibiting T-cell homing into the CNS compartment through endothelial cells (Miller et al., 2003Go).

Significant global CDR3-LD alteration at a stage where the disease is in fact only suspected suggests that strategies aimed at inhibiting activated T-cell transfer to the CNS or at regulating activation of selected T cells could be useful in the very early stage of the disease. One study has analysed patients at the onset of a confirmed disease (CDMS) (Musette et al., 1996Go). However, only a partial exploration of the T-cell repertoire was performed, with only a few Vß families analysed. This latter study of patients with confirmed disease (i.e. at a later stage than HRMS) was performed on Vß 5 and Vß 17 and also reported CDR3-LD alterations. Our data confirm and extend these initial observations by showing that most of the patients at the CDMS stage have both significantly more families with altered CDR3-LD and a more altered global transcriptome (allowing a statistical comparison) than patients at the HRMS stage (see Figs 5 and 6A). The fact that 11 of the 26 Vß families exhibited selected CDR3-LD at this stage of the disease suggests early epitope spreading. Spreading of T-cell responses against different epitopes has been well characterized in experimental models of multiple sclerosis (Vanderlugt and Miller, 2002Go). In humans, Tuohy et al. (1999Go) have also described a serial analysis showing the decreasing frequency of clones directed against a single epitope during the progression of the disease from a monosymptomatic demyelinating syndrome (a group of patients similar to our HRMS group) to the stage of CDMS and the appearance of new clones directed against other epitopes of the same molecule or of other myelin proteins.

It is known that the CD8 T-cell repertoire in healthy subjects is more altered than the CD4 repertoire (Gorochov et al., 1998Go). In this study, only six altered Vß families from two HI were observed and only one alteration clearly belonged to the CD8+ cell fraction. To our knowledge, the CD4/CD8 distribution of selected T cells in the blood of multiple sclerosis patients has rarely been investigated (Monteiro et al., 1996Go). The reason for a prominent bias in CD8+ T cells in multiple sclerosis may be due to a response against viruses since it has been shown that the relapses of the disease are favoured by infections (Edwards et al., 1998Go; Buljevac et al., 2002Go). However, this explanation is not likely because patients with a recent or ongoing clinical infection were not enrolled in the study. Furthermore, the patients from the WMS group were not included at the moment of a relapse. Five patients of the CDMS group were able to be analysed more closely for the phenotype of the T cells of Vß families with altered CDR3-LD, and six out of the nine Vß families analysed turned out to correspond in the majority to CD8+ cells (see Fig. 8A and B). The fact that the altered CDR3 length species represent a large majority of the T cells of these families (as assessed from the area under the curve of the immunoscope pattern of the amplified CDR3 segments) strongly suggests that these selected clones belong more to the CD8 than the CD4 phenotype. These observations are in agreement with the findings of Battistini et al. (2003Go) who have reported that circulating CD8+ T cells from multiple sclerosis patients express adhesion molecules allowing them to cross the blood–brain barrier and may explain the significant CD8+ T-cell infiltrate seen in CNS lesions of multiple sclerosis (Booss et al., 1983Go; Gay et al., 1997Go). Moreover, these data suggest that, even if HLA class II-restricted CD4+ cells play a role in disease susceptibility (Haines et al., 1998Go), spreading involving different class I restricted effectors occurs rapidly throughout the course of the disease, possibly by a phenomenon of cross-presentation (for a review see Carbone et al., 1998Go). In this respect, these data also suggest that besides class II tetramers (Reddy et al., 2003Go), class I tetramers may be a pertinent tool to test the blood T cells of multiple sclerosis patients. Interestingly, the only patient analysed at the HRMS stage also displayed alterations of CD8+ T cells (Fig. 8A and B). The concept that CD8+ T cells may be important in multiple sclerosis has been suggested by the possibility of inducing autoreactive CD8+ T cells for myelin peptides (Tsuchida et al., 1994Go). Furthermore, the role of CD8 T cells has been highlighted recently by the findings that the majority of cells with clonal expansions and memory phenotype in the CSF and brain lesions of patients with multiple sclerosis were also CD8+ T cells (Babbe et al., 2000Go; Jacobsen et al., 2002Go). These data and our own showing circulating CD8+ CDR3-LD selected T cells in the blood of multiple sclerosis patients support the possibility that CD8+ cytotoxic T lymphocytes could damage class I-expressing brain cells including oligodendrocytes and neurons (Medana et al., 2001Go; Liblau et al., 2002Go; Neumann et al., 2002Go). Our observations also suggest a potential use of selected anti-Vß antibodies in patients exhibiting expansions of circulating T cells with altered CDR3-LD as an alternative therapy.

This trend of CD8+ selection was also found in two out of five Vß families with altered CDR3-LD in the WMS group. However, these patients, who had been defined as presenting an exacerbated disease (see Material and methods; all of them were included in a mitoxantrone regimen and complied with the usual criteria for worsening disease), exhibited fewer alterations of their Vß transcriptome than CDMS patients, despite still being significantly different from age-matched HI. Furthermore, these patients did not have more Vß/HPRT transcript ratios with altered CDR3-LD in their blood than normal individuals. Whether this lack of more severe global Vß transcriptome alterations in this group of patients with active disease is related to the fact that the blood sampling was not concomitant with disease exacerbation, that most of these patients (as opposed to those of other groups) had previously received immunologically oriented treatments or that an exacerbated flux of activated circulating T cells into the CNS occurred is unknown.

We were able to analyse three patients further (one from each of the clinical groups) for their transcriptional profile (real-time PCR) of the major Th1/Th2-related cytokines as well as TNF-{alpha}. This study was performed immediately following cell sorting using anti-Vß antibodies, without in vitro stimulation to avoid artefactual selection. Interestingly, despite the low number of Vß families studied, the transcriptional pattern of T cells from altered families from multiple sclerosis patients differed significantly from those observed in families with either biased or Gaussian CDR3-LD from normal individuals, reinforcing the idea that these expanding peripheral T cells may be involved in the disease process. Furthermore, IFN-{gamma} ELISPOT assays were performed to test T cells from Vß families with altered CDR3 lengths in each group of patients and HI. There was a striking difference in response to human MBP between T cells from Vß families with altered CDR3-LD in multiple sclerosis patients and those from the controls. The T cells from more than half of such Vß families from multiple sclerosis patients produced IFN-{gamma} when stimulated with MBP, whereas none of the Vß families tested from HI did so. The reactivity of these cells to human MBP is probably due to the CD4 fraction of these cells, since it is known that peripheral APCs may not be optimal for cross-presentation. The lack of response of three Vß families from multiple sclerosis patients could be explained by a reactivity to other myelin or non-myelin antigen(s) or a production of cytokines other than the one tested.

These observations fit with other studies which also reported IFN-{gamma} and/or TNF-{alpha} mobilization in this disease (Huang et al., 1999Go; Pelfrey et al., 2000Go; Tejada-Simon et al., 2001Go) despite the fact that in two of these three studies, the patterns were analysed following in vitro stimulation with a putative multiple sclerosis antigen and that TNF-{alpha}/IFN-{gamma}-producing CD8+ T-cell clones directed against myelin peptides were found (Tsuchida et al., 1994Go).

Taken together, our data support the concept of circulating TCR-selected T cells in multiple sclerosis and that CD8+ T cells may be important in the pathophysiological processes of the disease. Our data also suggest that surveying the blood T-cell abnormalities in multiple sclerosis may be helpful for drug response studies, particularly when global Vß transcriptome analysis is used.


   Acknowledgements
 
We wish to thank Nelly Robillard and Yannick Oudinet for excellent technical help (cell sorting), Joanna Ashton for manuscript editing, and Mrs M. J. Mérienne, Dr T. Ronzière and Dr E. Lepage for clinical data harvesting (Rennes, France). This work was supported in part by grants from the ARSEP (Association pour la Recherche sur la Sclerose En Plaques), the Collège des Enseignants de Neurologie, the Académie Nationale de Médecine and the Direction of Clinical Research at Nantes University Hospital.


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