Analysis of extraocular muscle‐infiltrating T cells in thyroid‐associated ophthalmopathy (TAO)

TAO is characterized by an autoimmune process affecting the orbital contents. T cells have been suggested to have a major role in pathogenesis, but so far only limited data are available to clarify the extraocular muscle (EOM)‐infiltrating T cell phenotype, antigenic reactivity and cytokine profile in TAO patients. In the present study, biopsies of affected EOM were taken and the infiltrating T cells isolated and expanded in vitro with mitogen. Their phenotype was determined by flow cytometric (FACS) analysis and compared with peripheral blood‐derived T cell lines, treated in the same way from the same patient. Cytokines present in the supernatant after mitogen stimulation of the T cell lines were assayed by ELISA. In addition, cytokine mRNA present at the time of biopsy was determined by rapid RNA extraction from EOM and reverse transcription‐amplification with specific cytokine oligonucleotide probes (IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12, IL‐13, IL‐15, interferon‐gamma (IFN‐γ), tumour necrosis factor‐alpha (TNF‐α)). In the T cell lines from two patients, proliferation assays were carried out with antigens derived from thyroid gland, EOM and a thyrotropin (TSH) receptor preparation. Most T cell lines were CD4 + , CD45RO + , and TCR α/β + , both from the EOM and the peripheral blood. A wide variety of cytokines was detected by analysis of supernatants or mRNA, but the profiles were not identical comparing the two approaches. However, IL‐4 was detected by both. Dose‐dependent proliferation was observed in response to thyroid extract in a biopsy‐derived T cell line. In conclusion, EOM‐infiltrating T cells from patients with TAO, expanded in vitro , were chiefly CD4 + and produced a mixture of cytokines, including IL‐4. The proliferation data suggest that there are thyroid‐reactive T cells in EOM.