Abstract
OBJECTIVE—To use a panel of monoclonal antibodies (Mab) which recognise HLA class II alleles associated with rheumatoid arthritis for fluorescence activated cell sorter (FACS) analysis of peripheral blood mononuclear cells (PBMNC) from patients with early and established rheumatoid arthritis and to compare these results against DNA oligotyping of HLA class II molecules in the same patients. METHODS—27 patients (18 from an early arthritis clinic, nine with established rheumatoid arthritis) were studied using both techniques. PBMNC were stained with Mab which recognise the shared epitope, the HLA-DRB1*04 molecule and its *0401, *0404 subtypes in the presence of bound peptide. Mab stained cells were analysed by FACS. Genomic DNA was prepared from PBMNC and used for DNA oligotyping and sequencing by standard methods. RESULTS—FACS analysis of Mab stained PBMNC gave identical results to those obtained by DNA oligotyping in 26/27 patients. The antibodies identified the shared epitope in 14/14 cases and the presence of an HLA-DRB1*04 molecule in 12/12 cases. HLA-DRB1*0404 was identified in 4/4 cases. HLA-DRB1*0401 was identified in 5/6 cases. One patient oligotyped as HLA-DRB1*0401, but consistently failed to react with the *0401 Mab. DNA sequencing of the second exon of the HLA-DRB1*0401 allele in this patient confirmed a normal HLA-DRB1*0401 genotype. CONCLUSIONS—FACS analysis of PBMNC stained with Mab recognising the shared epitope and rheumatoid arthritis associated HLA susceptibility molecules provides a rapid, reliable, and more accessible alternative to DNA oligotyping. The apparent discordance between phenotypic and genetic analysis of HLA-DRB1*0401 in one patient, may reflect variability in HLA-DRB1*0401 gene expression or in class II peptide presentation.
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Two-colour FACS profiles of gated PBMNC stained with FITC conjugated murine anti-CD3 (X axis) and murine anti-HLA-DRB1*04, shared epitope, *0401, *0404, or negative control antibodies (Y axis). Cells were then washed and stained with phycoerythrin conjugated goat anti-mouse antibody. Genotypes (*0401, *0101, *0404) are listed above the figure. Anti-CD3 coated T cells also stain with the goat anti-mouse secondary antibody and are then clearly demarcated as a "double positive" population in the upper right quadrant. Positive HLA class II staining on B cells (in the lower left quadrant) is noted as a shift in fluorescence in the phycoerythrin channel.