A Sensitive Method for Detecting Peptide-specific CD4+ T Cell Responses in Peripheral Blood from Patients with Myasthenia Gravis.

Sapna Sharma,Martina Verolin,Christopher Lindberg,Nils Yngve Lycke,Karin Schön,Clas Malmeström,Sarah Meisel

doi:10.3389/fimmu.2017.01370

Abstract

Myasthenia gravis (MG) is an autoimmune neurological disorder typified by skeletal muscle fatigue and most often production of autoantibodies against the nicotinic acetylcholine receptor (AChR). The present study was undertaken to assess the extent of AChR-peptide recognition in MG patients using co-culturing (DC:TC) of autologous monocyte-derived dendritic cells (moDCs) and highly enriched CD4+ T cells from the blood as compared to the traditional whole peripheral blood mononuclear cell (PBMC) cultures. We found that the DC:TC cultures were highly superior to the PBMC cultures for detection of reactivity toward HLA-DQ/DR-restricted AChR-peptides. In fact, whereas DC:TC cultures identified recognition in all MG patients the PBMC cultures failed to detect responsiveness in around 40% of the patients. Furthermore, reactivity to multiple peptides was evident in DC:TC cultures, while PBMC cultures mostly exhibited reactivity to a single peptide. No healthy control (HC) CD4+ T cells responded to the peptides in either culture system. Interestingly, whereas spontaneous production of IFNγ and IL-17 was observed in the DC:TC cultures from MG patients, recall responses to peptides enhanced IL-10 production in 9/13 MG patients, while little increase in IFNγ and IL-17 was seen. HCs did not produce cytokines to peptide stimulations. We conclude that the DC: TC culture system is significantly more sensitive and better identifies the extent of responsiveness in MG patients to AChR-peptides than traditional PBMC cultures.

Highlights

Myasthenia gravis (MG) is an organ-specific autoimmune disease with a reported prevalence of 14–17 cases/100,000 in a Swedish/European or US population [1, 2]
Most MG patients carried serum antibodies against acetylcholine receptor (AChR), but none had anti-MUSK antibodies and all healthy controls (HC) were negative for both types of antibodies (Table 1)
We demonstrate that the dendritic cells (DC):TC co-culture technique is significantly more sensitive and reliable than conventional peripheral blood mononuclear cell (PBMC) culture techniques for detecting CD4+ T cell peptide recognition in MG patients

Summary

Introduction

Myasthenia gravis (MG) is an organ-specific autoimmune disease with a reported prevalence of 14–17 cases/100,000 in a Swedish/European or US population [1, 2] It is an antibody-mediated, CD4+ T cell-dependent neuro-inflammatory disorder, which results in loss of function in the acetylcholine receptor (AChR) at the neuromuscular junction [3, 4]. A third group is MG patients presenting with thymoma (TOMG) [7] Beside these three groups, there is a small group of patients who lack AChR antibodies but have antibodies against the muscle-specific receptor tyrosine kinase (MuSK MG). There is a small group of patients who lack AChR antibodies but have antibodies against the muscle-specific receptor tyrosine kinase (MuSK MG) This group is likely to be MG with a different etiology [8]

Methods

Results

Conclusion