Objective: In this study we performed single-cell analysis of the intracellular cytokine expression in peripheral CD4+ and CD8+ lymphocytes from patients with Hashimoto's thyroiditis (HT) to investigate the type-1 response separately for the two lymphocyte sub-populations. Design and methods: Twenty-nine patients affected by HT and 20 healthy subjects, matched for sex and age, were enrolled. After the analysis of the lymphocyte sub-populations, the intracellular content of interferon-γ (IFN-γ) in phorbolmyristate acetate (PMA)-stimulated CD4+ and CD8+ lymphocytes was assayed. Moreover, the CD4+ lymphocytes were also evaluated for the intracellular expression of IL-4. Results: No significant differences in CD3+, CD4+ and CD8+ lymphocytes were found between HT patients and control subjects. However, the HT patients showed higher numbers of CD4+IFN-γ+, CD4+IL-4+ and CD8+IFN-γ+ (t-test, P ≤ 0.001) cells than the control subjects. Analysing the intracellular expression of IFN-γ and IL-4 in relation to thyroid function, we found that the euthyroid patients (18 cases) showed more expression of IL-4 in CD4+ lymphocytes than the control subjects, without any significant modification of IFN-γ expression in CD4+ and CD8+ lymphocytes. However, the hypothyroid patients (11 cases) showed an increase of IFN-γ expression in both CD4+ and CD8+ lymphocytes with respect to the control subjects and the euthyroid patients. Moreover, the expression of IL-4 in CD4+ cells from hypothyroid patients was significantly lower than that seen in the euthyroid cases and comparable to that found in the control subjects. Conclusions: Our study has demonstrated that the peripheral CD4+ and CD8+ T lymphocytes from the HT patients show a type-1 activation strictly correlated to the occurrence of hypothyroidism. Further studies will be needed to clarify the exact role of peripheral lymphocytes in HT and whether they could provide a reliable marker of thyroid immune involvement.
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