Cytotoxic T lymphocyte-mediated cell death in paraneoplastic sensory neuronopathy with anti-Hu antibody

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Abstract

Paraneoplastic sensory neuronopathy (PSN) has been shown to harbor characteristic anti-neuronal autoantibody ‘anti-Hu’ in their sera and cerebrospinal fluid. Creation of animal models exhibiting clinical or pathological features seen in PSN by means of passive transfer of anti-Hu positive IgG has not been achieved. Although, anti-Hu antibody was shown to induce neuronal cell lysis in vitro, this result has not been reproduced so far. Since prominent T cell infiltration are seen in the central nervous system and posterior spinal ganglion of the patients with anti-Hu syndrome, we studied cytotoxic T cell (CTL) activity in peripheral mononuclear cells from a patient with PSN harboring anti-Hu antibody. The activated CD8+ T cells from the patient’s venous blood were shown to lyse her own fibroblasts which were incubated with interferon-γ to induce HLA class I molecules on their surface and the recombinant HuD protein was injected into the cells by microinjector. This is the first report showing the existence of CTL in a patient with PSN.

Introduction

Paraneoplastic sensory neuronopathy (PSN) is a rare syndrome starting with dysesthetic pain and numbness in the distal extremities, then spreading through all four limbs and trunk causing severe sensory ataxia. More than 60% of PSN patients have small-cell lung cancer (SCLC) which usually is discovered after neurological symptoms appear. Many patients with PSN associated with SCLC harbor an antibody in their sera called anti-Hu, or ANNA-1, which mainly labels neuronal nuclei immunohistochemically and binds to several bands at about 35–42 kDa on Western blots of neuronal tissue homogenates [3]. This antibody is a good marker for diagnosing PSN and underlying lung cancer, but little evidence have been presented that this antibody directly induces neuronal cell loss. In contrast, patients with PSN show pleocytosis in their cerebrospinal fluid (CSF), and the hallmarks of their pathological features are inflammatory lymphocyte infiltration and loss of neurons in the dorsal root ganglia and other areas of the central nervous system, indicative of T cell-mediated neuronal damage. As there have been no reports verifying the role of T cells in PSN, we studied cytotoxic T cell (CTL) activity in patients with PSN associated with SCLC.

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Materials and methods

Recombinant HuD protein was constructed following the DNA sequence reported by Szabo et al. [12]. Briefly, a primer pair for the PCR was designed that corresponded to nucleotide positions 148–169 for the 5′ site and 448–467 for the 3′ site. The PCR product was ligated to the pET-16b plasmid vector carrying a His Tag sequence, transformed to E. coli BL21(DE3) cells and grown in rich broth media. Recombinant HuD protein production was induced with isopropyl-β-d-thiogalactopyranoside (IPTG). Cells

Results

Recombinant HuD protein production was recognized on SDS-PAGE at the expected molecular size, and anti-Hu antibody reacted specifically with that band. Immunohistochemistry clearly showed that the injected HuD protein was present in the cytoplasms of the fibroblasts (Fig. 2). Fibroblast viability was favorable (more than 90% of the cells were alive) for up to 4 h after the injection of bovine serum albumin or plasmid vector or HuD protein. The disappearance rate (results of quadruple

Discussion

Specific autoantibodies such an ‘anti Yo, Hu, Ri’, react with both tumor and nervous tissue, have been detected in some patients with paraneoplastic neurological syndromes. Patients who have antibodies tend to have a better cancer prognosis, the tumor being small, less metastatic and more slowly growing [2]. These patients tend to have rapidly progressive neurological symptoms and high titer autoantibodies in their sera and CSF. This suggests that these autoantibodies have an important role in

Acknowledgements

This work was supported by Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan.

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