Identification of Novel Myelin-Associated CD4+ T cell Autoantigens Targeted in MS Using a High-Throughput Gene Synthesis Technology
Abstract
Myelin-specific CD4+ T cells are implicated in the pathogenesis of MS. Attention has focused on the autoantigens MBP, MOG, PLP, and MOBP however, it is unclear whether these are the major targets of the CD4+ T cell response in MS. Over 300 myelin-associated proteins have been identified in the CNS, most of which have not been evaluated as antigens in MS. The purpose of this project was to test a high throughput gene synthesis and T cell screening approach to identify new MS autoantigens. We built a synthetic minigene library encoding the peptide compliment of 342 myelin-associated proteins fused to the LC3 targeting sequence that delivers peptides into the class II antigen presentation pathway. Library accuracy was determined by sequencing and specific amplification of accurate minigenes for a subset of 50 myelin proteins. Minigenes were pooled in a 96 well plate format, transcribed in vitro, and mRNA pools were transfected into activated B cells as antigen presenting cells in T cell stimulation assays. The library was validated using an influenza hemagglutinin-specific CD4+ T cell clone. To identify novel class II myelin epitopes, we screened the library using peripheral CD4+ T cells from a flaring MS patient. T cells that responded to library encoded peptides by upregulating CD154 expression were enriched and expanded as T cell lines for further screening to identify the specific peptide targets. We conclude that CD4+ T cell antigens can be detected using minigene encoded peptides but that an enrichment step is required to increase sensitivity.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 2013
- Accession Number
- ADA598720
Entities
People
- Karen Cerosaletti
Organizations
- Benaroya Research Institute