Annals of the Rheumatic Diseases 1997;56:135–139 135 Use of monoclonal antibodies to detect disease Ann Rheum Dis: first published as 10.1136/ard.56.2.135 on 1 February 1997. Downloaded from associated HLA-DRB1 alleles and the shared epitope in rheumatoid arthritis Ian Wicks, GeoV McColl, Angela D’Amico, Loretta Dougherty, Brian Tait Abstract earlier and more aggressive therapeutic Objective—To use a panel of monoclonal intervention. Early stratification of patients antibodies (Mab) which recognise HLA who are at risk for severe disease is an class II alleles associated with rheumatoid important clinical issue for these reasons. arthritis for fluorescence activated cell Many studies have confirmed an association sorter (FACS) analysis of peripheral blood between rheumatoid arthritis and a restricted mononuclear cells (PBMNC) from pa- number of alleles at the highly polymorphic tients with early and established rheuma- HLA-DRB1 locus (primarily DRB1*0401, toid arthritis and to compare these results *0404, and *0101).23 Each of these alleles against DNA oligotyping of HLA class II shares sequence homology in the third molecules in the same patients. hypervariable region of the â chain, which is Methods—27 patients (18 from an early referred to as the shared epitope. In a study of arthritis clinic, nine with established 201 patients with rheumatoid arthritis, who rheumatoid arthritis) were studied using were unselected for disease severity, the both techniques. PBMNC were stained presence of a single copy of *0401, *0404, or with Mab which recognise the shared *0101 was associated with an odds ratio for epitope, the HLA-DRB1*04 molecule and rheumatoid arthritis of 3.5.4 Two identical its *0401, *0404 subtypes in the presence of copies of these alleles gave an odds ratio of 7.8 bound peptide. Mab stained cells were and this increased to 25.6 with *0401/*0404 analysed by FACS. Genomic DNA was compound heterozygosity. Similar results were prepared from PBMNC and used for DNA obtained from an analysis of 120 patients pre- oligotyping and sequencing by standard senting with early arthritis.5 In this study, 10/11 methods. compound heterozygotes (*0401/*0404) dev- Results—FACS analysis of Mab stained eloped erosions at one year. Conversely, PBMNC gave identical results to those patients without the HLA-DRB1 susceptibility obtained by DNA oligotyping in 26/27 alleles are at low (10-20%) risk for progressive patients. The antibodies identified the rheumatoid disease.23 These data raise the http://ard.bmj.com/ shared epitope in 14/14 cases and the pres- possibility of using HLA typing, together with ence of an HLA-DRB1*04 molecule in other clinical and laboratory indices, to 12/12 cases. HLA-DRB1*0404 was identi- compile a profile of the patient with early fied in 4/4 cases. HLA-DRB1*0401 was inflammatory arthritis who is at risk for more identified in 5/6 cases. One patient severe rheumatoid disease. oligotyped as HLA-DRB1*0401, but HLA analysis has traditionally been Reid Rheumatology consistently failed to react with the *0401 performed in the tissue typing laboratory, on September 29, 2021 by guest. Protected copyright. Laboratory, Burnet Mab. DNA sequencing of the second exon initially using serological and cellular typing Clinical Research of the HLA-DRB1*0401 allele in this reagents and more recently with molecular Unit, Walter and Eliza Hall Institute of patient confirmed a normal HLA- techniques. The standard method for Medical Research, DRB1*0401 genotype. determining HLA-DR and its subtypes now Parkville, Victoria, Conclusions—FACS analysis of PBMNC involves isolation of genomic DNA from Australia I Wicks stained with Mab recognising the shared peripheral blood cells, the polymerase chain G McColl epitope and rheumatoid arthritis associated reaction (PCR) with sequence specific A D’Amico HLA susceptibility molecules provides a oligonucleotide primers and dot blotting, L Dougherty rapid, reliable, and more accessible followed by hybridisation with probes directed Tissue Typing alternative to DNA oligotyping. The appar- at hypervariable region sequence diVerences. Laboratory, Royal ent discordance between phenotypic and These molecular techniques, which are Melbourne Hospital, genetic analysis of HLA-DRB1*0401 in one generally carried out in the context of HLA Parkville, Victoria, Australia patient, may reflect variability in HLA- matching for organ transplantation, require B Tait DRB1*0401 gene expression or in class II specialised expertise and equipment and strin- peptide presentation. gent quality control. While HLA typing has Correspondence to: Dr Ian Wicks, Reid been used as a research tool in the study of Rheumatology Laboratory, (Ann Rheum Dis 1997;56:135–139) rheumatoid arthritis, it has not been generally Burnet Clinical Research Unit, Walter and Eliza Hall accessible as an aid to clinical management. Institute of Medical Rheumatoid arthritis is a common but Recently, monoclonal antibodies (Mab) Research, PO Royal Melbourne Hospital, Victoria clinically heterogeneous disease. Recent work which recognise HLA-DR4, its subtypes *0401 3050, Australia. suggests that much of the joint damage in more and *0404, and the shared epitope severe cases occurs in the first few years of the (QKAA/QRAA) have been generated.67 These Accepted for publication 1 4 September 1996 disease. Accordingly, there is a trend towards antibodies are conformationally dependent 136 Wicks, McColl, D’Amico, Dougherty, Tait and require peptide loading of the MHC mol- by double staining (figure). Increased B cell Ann Rheum Dis: first published as 10.1136/ard.56.2.135 on 1 February 1997. Downloaded from ecule.6 Fluorescence activated cell sorter fluorescence in the phycoerythrin channel was (FACS) analysis of peripheral blood cells usually obvious from inspection, but quadrants potentially oVers a faster and more accessible were set on all samples, using goat anti-mouse method for identifying patients carrying (GAM) conjugated phycoerythrin alone rheumatoid arthritis associated HLA-DRB1 (negative control) and NFLD.M6 (positive alleles early in the course of the disease. In this control) to estimate staining in the phycoeryth- study, we compared the results of FACS analy- rin channel. The following formula was used to sis and genotyping in 27 patients, 18 of whom quantify fluorescence in the phycoerythrin came from an early inflammatory arthritis channel: clinic and nine had established rheumatoid arthritis. (%Test) - (%Negative control) × 100 (%Pos control) - (%Neg control) Methods SUBJECTS A figure of greater than 20% was recorded as Eighteen consecutive patients, all of whom ful- positive staining. FACS results were analysed filled the 1987 revised American Rheumatism without knowledge of the genotype. Association (ARA) criteria for rheumatoid arthritis, were enrolled from a study of early HLA DNA OLIGOTYPING inflammatory polyarthritis conducted at the DNA was isolated using the method of Miller Royal Melbourne Hospital (RMH). Patients et al 8 from the buVy coat of anticoagulated came from the community of suburban blood samples. Genomic DNA was amplified Melbourne. Nine patients with established by the PCR, dot blotted, and probed with dig- rheumatoid arthritis—drawn from the RMH oxygenin labelled (Boehringer Mannheim, outpatient rheumatology clinic—were also Germany) oligonucleotides specific for HLA- studied. DRB1 variable sequences. A two step procedure was used, consisting of PCR ampli- ANTIBODY STAINING fication of the DRB1 second exon using Peripheral blood was collected into generic primers and hybridisation with probes heparinised tubes and the mononuclear cells providing a serologically equivalent DR assign- isolated on a LeucoSep Ficoll density gradient ment (including DR4 and DR0101/0102). (Oropharma AG, Zurich). Peripheral blood Secondly, primers amplifying only the DR4 mononuclear cells (PBMNC, 2 × 106) were group of alleles were used in the PCR with stained with 30 µl of irrelevant mouse genomic DNA and the product probed with a immunoglobulin, Mab NFLD.D1 (anti-DR4; series of oligonucleotides which defined B1*04), Mab NFLD.D2 (anti-shared epitope *0401-*0422. A similar procedure was used to QKRAA/QRRAA), Mab NFLD.D11 (anti- define the DR1 alleles. Following hybridisation DR4Dw4; B1*0401), Mab NFLD.D13 and stringent washing, bound probes were (anti-DR4Dw14; B1*0404), or NFLD.M6 detected using an anti-digoxygenin-alkaline http://ard.bmj.com/ (Mab to monomorphic determinant on phosphatase conjugate and chemiluminescent HLA-DR; positive control) (supplied as super- substrate (Boehringer Mannheim, Germany). natants by Terra Nova Biotech, Newfound- Hybridisation signals were visualised by land, Canada). In cellular enzyme linked exposure to x ray film (Kodak XAR-5). immunosorbent assays (ELISA), the NFLD.D2 also showed limited binding to QARAA 7 HLA-DR4 1 SEQUENCING containing alleles. Fluorescein isothiocyanate Primers flanking the second exon of the HLA- on September 29, 2021 by guest. Protected copyright. (FITC) conjugated Mab anti-CD3 (1:40) DR4 1 gene were used to PCR amplify a 300 (Dako, Denmark) was added to all tubes in base pair fragment from the genomic DNA of order to demarcate clearly the HLA class II patient 15. The PCR product was purified with expressing B cells. Cells were held for 20 min- a High Pure PCR product purification kit utes on ice and washed once through fetal calf (Boehringer Mannheim, Germany) and serum. Phycoerythrin conjugated goat anti- sequenced in both directions using a Taq Dye mouse Mab was added (Terra Nova Biotech, deoxy terminator