Separation and analysis of mononuclear cells infiltrating the thyroid of patients with Graves' disease

Abstract

A simple method was established for separating lymphocytes infiltraing the thyroid from thyroid epithelial cells. Namely, suspensions of minced thyroid from patients with Graves' disease were layered on a Percoll two-step density gradient (p = 1.050 and 1.077 g/ml) and centrifuged (400g, 30 min, 4°C). In this way 0.1–18 × 10 5 lymphocytes/g of thyroid tissue with a purity of 65–95% were obtained. Thyroid lymphocytes were analyzed quantitatively with monoclonal antibodies by laser flow cytometry and compared with peripheral lymphocytes. The proportion of OKT3 + cells was decreased with increase in OKIa + cells. The percentage of OKIa + cells was significantly correlated with that of Leu12 + cells. The percentages of OKT4 + cells and OKIa + cells were higher when analyzed with an extended gate window, which was arranged for detection of activated, large-sized lymphocytes. The percentages of OKT8 + and Leu7 + cells were not significantly different from those in peripheral blood. From these results it was concluded that (1) the proportion of B lymphocytes is increased and that of T lymphocytes is decreased, (2) the proportion of activated B lymphocytes is increased, (3) some helper/inducer T cells are activated in the thyroid gland in Graves' disease, and (4) these activated lymphocytes may be important in local production of antithyroid autoantibodies.