Analysis of retroorbital T cell antigen receptor variable region gene usage 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 disease 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 from patients with early and late stages of clinically severe GO and from patients with non-GO orbital conditions. Ribonucleic acid extracted from orbital tissue and peripheral blood lymphocytes (PBL) was reverse transcribed and amplified using the polymerase chain reaction and 22 V alpha and 24 V beta gene-specific oligonucleotide primers. The resulting TcR V alpha and V beta transcripts were verified by Southern hybridization analysis using TcR C region-specific, digoxigenin-labeled oligonucleotide probes. Compared with matched PBL, the retroorbital TcR V alpha and V beta gene repertoire expressed was heterogeneous, but revealed marked restriction of V gene usage in samples derived from retroorbital connective tissue and extraocular muscle of all eight patients with severe GO of short duration studied. In contrast, greater diversity of the TcR V beta gene repertoire and loss of TcR V alpha gene restriction was noted in four patients with late GO undergoing reconstructive eye muscle surgery. Unrestricted TcR V gene usage was demonstrated in orbital tissue and PBL samples obtained from control subjects. These results suggest that retroorbital TcR V gene usage is variable but markedly restricted during the earlier stages of GO. With increasing disease duration, greater diversity of the TcR V gene repertoire appears 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-infiltrating T lymphocytes in GO.