Long-term expression of an HLA-DQ molecule in the EBV-transformed bare lymphocyte cell line, BLS-1, using a plasmid vector.
The HLA class II molecule, DQ6, confers strong natural protection against the development of type 1 diabetes. The mechanism of disease protection is unknown, but is likely to be related to the function of the molecule in antigen presentation. In order to investigate this function, we have created an in vitro model which expresses DQ6 in isolation by introducing the relevant DQ alleles into an Epstein-Barr virus (EBV)-transformed, human leucocyte antigen (HLA) class II-deficient B cell line, bare lymphocyte syndrome (BLS)-1. A recent report suggested that the expression of transferred genes in human EBV-transformed B cells might be limited in duration. We present a plasmid-based transfection method that allows long-term stable expression of the DQ molecule. The DQA1*0102 and DQB1*0602 alleles were cloned into the pCIneo expression vector and the constructs were introduced into BLS-1 by electroporation. Stable transfectants were selected using magnetic sorting and cloned by limiting dilution. Two clones were shown to express functionally active DQ6 molecules even after 14 months of continuous culture. These clones will be used in functional studies to investigate the antigen binding and T-cell activation properties of the DQ6 molecule.
Kelly
M A
MA
Department of Medicine, Division of Medical Sciences, University of Birmingham and Birmingham Heartlands Hospital, Birmingham, UK. m.a.kelly@bham.ac.uk
Rayner
M L
ML
Mijovic
C H
CH
Barnett
A H
AH
D017951:Antigen Presentation / Q000276:immunology; D001324:Autoantigens; D002461:Cell Line, Transformed; D004247:DNA / Q000276:immunology; D018274:Electroporation; D005434:Flow Cytometry; D006683:HLA-DQ Antigens / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D018189:Immunomagnetic Separation; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000276:immunology / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000235:genetics; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016511:Severe Combined Immunodeficiency / Q000276:immunology* / Q000473:pathology; D013601:T-Lymphocytes / Q000276:immunology
D001324:Autoantigens; D006683:HLA-DQ Antigens; C063831:HLA-DQ6 antigen; D008565:Membrane Proteins; D010446:Peptide Fragments; D012313:RNA; D004247:DNA; C516862:PTPRN protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8