Norbert Goebels

Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection, single-cell PCR, and CDR3-spectratype analysis

Clonal expansions of CD8+ T cells have been identified in muscle and blood of polymyositis patients by PCR techniques, including T cell receptor (TCR) complementarity-determining region (CDR)3 length analysis (spectratyping). To examine a possible pathogenic role of these clonally expanded T cells, we combined CDR3 spectratyping with laser microdissection and single-cell PCR of individual myocytotoxic T cells that contact, invade, and destroy a skeletal muscle fiber. First, we screened cDNA from muscle biopsy specimens by CDR3 spectratyping for expanded TCR β chain variable region (BV) sequences. To pinpoint the corresponding T cells in tissue, we stained cryostat sections with appropriate anti-TCR BV mAbs, isolated single BV+ T cells that directly contacted or invaded a muscle fiber by laser-assisted microdissection, and amplified their TCR BV chain sequences from rearranged genomic DNA. In this way, we could relate the oligoclonal peaks identified by CDR3-spectratype screening to morphologically characterized microdissected T cells. In one patient, a large fraction of the microdissected T cells carried a common TCR-BV amino acid CDR3 motif and conservative nucleotide exchanges in the CDR3 region, suggesting an antigen-driven response. In several cases, we tracked these T cell clones for several years in CD8+ (but not CD4+) blood lymphocytes and in two patients also in consecutive muscle biopsy specimens. During immunosuppressive therapy, oligoclonal CDR3-spectratype patterns tended to revert to more polyclonal Gaussian distribution-like patterns. Our findings demonstrate that CDR3 spectratyping and single-cell analysis can be combined to identify and track autoaggressive T cell clones in blood and target tissue. This approach should be applicable to other inflammatory and autoimmune disorders.

The role of autoimmune T lymphocytes in the pathogenesis of multiple sclerosis

Autoimmune T cells play a key role as regulators and effectors of autoimmune disease.In multiple sclerosis (MS), activated T cells specific for myelin components or other locally expressed autoantigens enter the CNS and recognize their antigen(s) on local antigen-presenting cells. After local stimulation, the T cells produce a plethora of cytokines and inflammatory mediators that have profound effects on the local cellular environment, induce and recruit additional inflammatory cells, and contribute to myelin damage. An increasingly detailed knowledge of these processes will greatly facilitate the development of new immunotherapies. This article focuses on the role of T cells in MS. We provide a brief overview of the principles of T-cell immunology, discuss the experimental techniques available for studying T cells, address the role of T cells in the pathogenesis of MS, and highlight modern concepts for immunotherapy. NEUROLOGY 1995;45(Suppl 6): S33-S38

T-Cell-Mediated Autoimmunity: Novel Techniques to Characterize Autoreactive T-Cell Receptors

Histological samples of autopsy or biopsy tissue provide the best available evidence that autoreactive T cells are involved in the immunopathogenesis of many autoimmune diseases. However, morphology alone does not provide information on the antigen-specific T-cell receptor (TCR) of these cells, let alone on their antigen specificity. In this review article we discuss a number of emerging possibilities for identifying TCR sequences directly from biopsy tissue. We also review the methods for expressing presumably autoreactive TCR molecules and speculate on how the expressed TCR might be used to identify target antigens. Such information should eventually provide new insights into disease pathogenesis which lead to better therapies.

Cross-reactive T-Cell Receptors in Tumor and Paraneoplastic Target Tissue

BACKGROUND According to established criteria, paraneoplastic encephalomyelitis with adrenal neuroblastoma comprises a definite paraneoplastic neurologic syndrome. OBJECTIVE To detect T-cell clones that cross-react against antigens shared between tumor and nervous system. DESIGN Case study. SETTING Academic research. Patient A 22-year-old woman having paraneoplastic encephalomyelitis with adrenal neuroblastoma. MAIN OUTCOME MEASURES We compared the T-cell receptor repertoires expressed in blood, cerebrospinal fluid, and neuroblastoma tumor tissue using complementary determining region 3 (CDR3) spectratyping and clone-specific polymerase chain reaction. RESULTS The T-cell receptor repertoire in cerebrospinal fluid was narrow compared with that in tumor and blood. Four T-cell clones from different tissues had identical T-cell receptor beta chains. Remarkably, the chains showed identical amino acid sequences but different nucleotide sequences. CONCLUSIONS These T cells represent ontogenetically distinct clones but share functionally identical receptors. They recognize the same antigen in nervous system and tumor tissue and represent an attractive target for selective therapy.

Myositis Diagnosis and Management

INTRODUCTION The inflammatory myopathies comprise a group of acquired myopathies in which muscle weakness and inflammatory infiltrates are the principal clinical and histological findings. Traditionally, a distinction is made between polymyositis, dermatomyositis and inclusion body myositis. This brief review will focus on polymyositis. CLINICAL FEATURES Diagnostic criteria Polymyositis is characterized by symmetrical proximal muscle weakness. Respiratory, pharyngeal and neck muscles may also be involved during later stages of the disease (Dalakas & Sivakumar 1996; Mantegazza et al. 1997; Dalakas 1998a). Up to half the patients suffer from muscle pain or arthralgia. The history, clinical symptoms and signs, elevated serum levels of muscle enzymes, electrophysiological changes and histological findings together provide the basis for the diagnosis. The main diagnostic criteria and features are summarized in Table 1 for polymyositis and for the two other forms of idiopathic inflammatory myopathies, dermatomyositis, and inclusion body myositis. Clinically, poylmyositis and dermatomyositis are distinguished by