ANALYSIS OF RETROORBITAL T-CELL ANTIGEN RECEPTOR VARIABLE REGION GENEUSAGE IN PATIENTS WITH GRAVES OPHTHALMOPATHY

To date, it has remained unclear whether orbit-infiltrating T cells in patients with Graves' ophthalmopathy (GO) represent a primary immune response in which a limited number of T cell clones driving the diseas e are activated against specific antigens, or whether they participate in a non-specific inflammatory process. To characterize these T cells at the molecular level, we examined the T cell antigen receptor (TcR) V gene repertoire in situ in retroorbital tissue specimens obtained f rom patients with early and late stages of clinically severe GO and fr om patients with non-GO orbital conditions. Ribonucleic acid extracted from orbital tissue and peripheral blood lymphocytes (PBL) was revers e transcribed and amplified using the polymerase chain reaction and 22 V alpha and 24 V beta gene-specific oligonucleotide primers. The resu lting TcR V alpha and V beta transcripts were verified by Southern hyb ridization analysis using TcR C region-specific, digoxigenin-labeled o ligonucleotide probes. Compared with matched PBL, the retroorbital TcR V alpha and V beta gene repertoire expressed was heterogeneous, but r evealed marked restriction of V gene usage in samples derived from ret roorbital connective tissue and extraocular muscle of all eight patien ts with severe GO of short duration studied. In contrast, greater dive rsity of the TcR V beta gene repertoire and loss of TcR V alpha gene r estriction was noted in four patients with late GO undergoing reconstr uctive eye muscle surgery. Unrestricted TcR V gene usage was demonstra ted in orbital tissue and PBL samples obtained from control subjects. These results suggest that retroorbital TcR V gene usage is variable b ut markedly restricted during the earlier stages of GO. With increasin g disease duration, greater diversity of the TcR V gene repertoire app ears to develop, and oligoclonality of the T cell response may be lost . Selection of patients with early stages of GO will be important when further dissecting TcR usage and antigen specificity of orbit-infiltr ating T lymphocytes in GO.