Detection of skewed T-cell receptor V-β gene usage in the peripheral blood of patients with multiple sclerosis

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Abstract

The ex vivo analysis of the T-cell receptor V-beta (TCRBV) gene usage by circulating T lymphocytes in Multiple Sclerosis (MS) patients may contribute to understanding disease pathogenesis. In the present study, TCRBV gene usage was analyzed in freshly collected unstimulated peripheral blood mononuclear cells (PBMC) isolated from 40 MS patients and 20 healthy controls. Nine patients presented abnormal repertoires, with expansion of one or more TCRBV segments. Among these patients, six presented expansion of TCRBV9 chain expression, three also having an expansion of TCRBV1, TCRBV11 and TCRBV22 segments. The most frequently observed TCRBV chain expansion, TCRBV9, was further analyzed and identified as polyclonal. Evaluation of clinical variables showed that median disease duration was shorter in patients with TCRBV gene expression abnormalities. Longitudinal evaluation of five patients with a skewed repertoire showed regression of expanded TCRBV chains expression to normal values. These data indicate that certain MS patients have abnormal TCRBV gene expression. Such abnormalities are caused by polyclonal expansions of T lymphocyte subpopulations that use the same TCRBV gene families, are unstable and preferentially observed early in the course of the disease.

Introduction

Several lines of evidence indicate that multiple sclerosis (MS) is a disease mediated by an autoimmune response specific to myelin components of the central nervous system (CNS) (Martin and McFarland, 1995). Perivenular and white matter mononuclear cell infiltrates characterize MS lesions. CD4+ and CD8+ T lymphocytes expressing the α/β T cell receptor (TCR), as well as CD4–CD8-T cells expressing the γ/δ TCR are observed in both acute and chronic MS lesions (Prineas, 1985; Wucherpfennig et al., 1992). In susceptible animals, passive transfer of myelin antigen-specific T lymphocytes (but not of immune serum or B cells) induces experimental autoimmune encephalomyelitis (EAE), a disease that clinically and pathologically resembles MS (Martin and McFarland, 1995). In addition, transgenic mice expressing a myelin basic protein (MBP)-specific TCR virtually in the whole T-lymphocyte repertoire have been shown to develop spontaneous (Goverman et al., 1993). The observation of a restricted fine specificity and TCR gene usage by encephalitogenic T cells allowed the development of specific immunotherapies of (Acha-Orbea et al., 1988; Vandenbark et al., 1989). For the same purpose—to identify and possibly inactivate the potentially encephalitogenic antigen(s)-specific T cells responsible for initiating the autoimmune damage—the T-cell response has been thoroughly investigated in MS patients. Several studies have previously characterized the TCRBV usage of myelin antigen-specific T-cell clones (TCC) generated in vitro, but failed to demonstrate consistent restrictions of TCRBV gene usage. Although preferential TCRBV17, TCRBV12 (Wucherpfennig et al., 1990), TCRBV5S2, and TCRBV6S1 usage (Kotzin et al., 1991) by myelin basic protein (MBP)-specific T-cell lines obtained from MS patients have been reported, these observations have not been confirmed by other studies (Meinl et al., 1993). It has become clear that the T-cell response to MBP and other myelin antigens and their immunodominant epitopes is more complex and heterogeneous in MS than in the experimental model of the disease, both in terms of TCR usage and of antigen specificity (Meinl et al., 1993; Martin and McFarland, 1995).

It should also be noted that myelin antigen-specific TCC can be easily isolated from the peripheral blood of patients as well as healthy controls, and that their frequency and phenotype may not differ (Martin and McFarland, 1995). It is likely that in vitro culture and expansion select clones that are not representative of the whole circulating T cell repertoire.

In the present study TCRBV usage was analyzed in freshly collected, unstimulated PBMC obtained from MS patients and healthy controls. The advantage of this approach is that it provides a description of the whole repertoire of circulating T cells without making assumptions on antigen specificities. The T-cell repertoire was analyzed by using a method that combines the sensitivity of polymerase chain reaction (PCR) and the simplicity of a conventional immunoassay with remarkable reproducibility (Bettinardi et al., 1992). Taking advantage of this technique, the expression of TCRBV genes was studied for the first time in a large number of MS patients and controls. A skewing of TCRBV gene usage due to polyclonal T cell-expansion was observed in MS patients compared with normal controls. This skewing was more frequently observed in patients with short disease duration.

Section snippets

Patient inclusion criteria

Inclusion criteria were: clinically definite or laboratory-supported definite MS according to Poser et al. (1983), age from 18 to 55 years, relapsing–remitting (RR) or secondary chronic progressive (CP) clinical course, defined according to Lublin and Reingold (1996), and informed consent. Forty patients from the Inpatient and Outpatient Clinics of the University of Florence Department of Neurology were studied: 26 F and 14 M; mean age 36.7±9.3 years, range 21–55; mean disease duration 9.3±7.9

TCRBV repertoire in normal controls

The results of TCRBV repertoire analysis in healthy controls (N=20) are reported in Fig. 1 (panel A). The overall profile is consistent with published reports in which the expression of a sufficiently high number of TCRBV chains was studied (Moss and Bell, 1995).

TCRBV repertoire in MS patients

Expanded expression of a total of 7 TCRBV segments (TCRBV1, TCRBV4, TCRBV9, TCRBV11, TCRBV15, TCRBV19, and TCRBV22) was observed in nine patients. A high expression of the TCRBV9 chain was observed in six RR MS patients and was the most

Discussion

In this study TCRBV gene usage was analyzed in freshly collected, unstimulated PBMC obtained from MS patients free of clinically detectable infections or systemic inflammation. The aim of the study was (a) to investigate TCRBV gene expression in a large number of patients and establish possible differences from normal controls and (b) to analyze the repertoire ex vivo, thus avoiding biases of T-cell selection due to in vitro T cell culture and expansion (Jason and Inge, 1996). In order to

Acknowledgements

This article is dedicated to the memory of Professor Luigi Amaducci. Bruno Gran was supported by a Fellowship of the Fondazione per le Biotecnologie, Torino, Italy. This study was partially funded by a grant of the Associazione Italiana Sclerosi Multipla (AISM). We thank Drs. M. Londei, A. Uccelli, R. Martin and H.F. McFarland for critical reading of the manuscript and helpful discussions. Presented in part at the 120th Annual Meeting of the American Neurological Association, Washington DC,

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    Present address: Neuroimmunology Branch, NINDS, National Institutes of Health, Building 10, Room 5B-16, 10 Center DR, MSC 1400, Bethesda, MD 20892-1400, USA. Tel.: +1 301 4024488; fax: +1 301 4020373; e-mail: [email protected]

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