Autoreactive cells in systemic Autoimmune disease

CD4+ Helper cells FACS sorted from spleens of 20 week old Male BXSB.Yaa and 20 week old Male BXSB.Yaa Nr4a2 cKO mice. Unbiased TCR repertoire analysis technology by Repertoire Genesis Inc., Osaka, Japan was used. Unbiased adaptor-ligation PCR was performed according to the previous reports (Yoshida, Yoshioka et al., 2000). After PCR amplification, index (barcode) sequences were added by amplification with Nextera XT index kit v2 setA (illumina, San Diego, CA). The indexed amplicon products were mixed in an equal molar concentration and quantified by a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA). Sequencing was carried out with the Illumina Miseq paired-end platform (2 x 300bp). All the paired-end reads were classified by index sequences. Assignment of sequences was performed by determining sequences with the highest identity in a data set of reference sequences from the international ImMunoGeneTics information system (IMGT) database (http://www.imgt.org). Data processing, assignment, and data aggregation were automatically performed using a repertoire analysis software originally developed by Repertoire Genesis Inc. Nucleotide sequences of CDR3 regions ranged from conserved Cysteine at position 104 (Cys104) of IMGT nomenclature to conserved Phenylalanine at position 118 (Phe118) and the following Glycine (Gly119) was translated to deduce amino acid sequences. A unique sequence read (USR) was defined as a sequence read having no identity in TRV, TRJ and deduced amino acid sequence of CDR3 with the other sequence reads.