A total of 65 patients (51W/14 M) with a definite diagnosis of Hashimoto’s thyroiditis (HT) were enrolled in the study as previously described.21 Twenty-three of them (16 W/7 M) showed the presence of an isolated thyroiditis (IT). Fifteen patients (11W/4 M) had HT and non-segmental vitiligo (VIT), 16 patients (15 W/1 M) had HT and chronic atrophic gastritis (CAG), and 11 patients (9 W/2 M) had HT and celiac disease (CD). All patients were spontaneously or pharmacologically euthyroid, but in the IT group, patients were further divided into euthyroid (14 patients, 12 W/2 M) and hypothyroid (9 patients, 4 W/5 M). Twenty healthy donors (HD) (16W/4 M) were also enrolled and served as an internal control. The anthropometric and functional characteristics are reported in Table 1.

The diagnosis of Hashimoto’s thyroiditis has been made based on the presence at least of two out of the following three criteria: 1) characteristic US features (hypoechogenity, dysomogeneity, pseudonodules, echogenic septae, etc.); 2) the presence of high anti-thyroperoxidase auto-antibodies (anti-TPOAb) titers; and/or 3) hypothyroidism.22 The diagnosis of euthyroid Hashimoto thyroiditis has been based on the presence of both the remaining criteria (i.e., high anti-TPOAb titers and typical US pattern).

The diagnosis of each associated disease was defined based on diagnostic criteria approved by the specific Consensus Conference and confirmed by serologic and histological tests, where required.23–26

All patients and healthy donors enrolled were negative for infection and/or inflammatory disorders in the last 6 months; patients with different but relevant chronic disorders (cancer, diabetes, COPB, obesity, and renal failure) and those who were pregnant and nursing and/or treated with drugs interfering with immune response (NSAIDs, steroids, immunosuppressant drugs, and immunomodulatory drugs) were positively excluded. All CD patients were in gluten-free diet for almost 6 months. All patients were informed about the purpose of the study and gave a written informed consent. The study has been approved by the local ethical committee (ASL C, Roma-Latina, Italy) according to the local ethical rules and to the guidelines of the Declaration of Helsinki.

All patients gave fasting morning blood samples, collected in EDTA. CD20+ T lymphocytes were analyzed by flow cytometry with a staining no wash protocol. A volume of 100 μL of whole blood was incubated for 25 min on ice in the dark with the following directly conjugated antibodies: anti-CD4-APC-H7 (clone RPA-T4, 5 μL); anti-CD8-FITC (clone RPA-T8, 5 μL); anti-CD16-PE (clone 3G8, 2 μL); anti-CD19-PE (clone HIB19, 10 μL); anti-CD20-APC (clone 2H7, 10 μL); anti-CD45-PerCP (clone HI30, 5 μL); and anti-CD56-PE (clone B159, 10 μL). For the measurement of intrinsic cellular fluorescence (i.e., autofluorescence), 100 μL of whole blood was incubated in the same way without adding antibodies. At the end of the incubation, 1 mL of lysis buffer (BD Pharm Lyse, Becton Dickinson) was added to each sample and the samples were incubated for 15 min at room temperature, in the dark to allow the lysis of red blood cells which would interfere with the cytometric acquisition. At the end of this second incubation, the samples were directly acquired using a FACs ARIA II cell sorter (Becton Dickinson) equipped with a 488-nm solid-state laser and a 633-nm HeNe laser.

Total T lymphocytes were identified as CD45- and CD3-positive cells. CD16 and CD19 were used to exclude B lymphocytes, while CD56 was used to exclude natural killer cells. Total T lymphocytes were further characterized as CD4 positive, CD8 positive, and double negative cells (CD4−/CD8−). Finally, for each of these subsets, the percentage of CD20-positive cells was analyzed.

Results were presented as median values. The difference between two groups was calculated using the non-parametric Mann–Whitney U test. When comparing more than two groups, the non-parametric Kruskal–Wallis test followed by the Dunn post-test was used to compare all pairs of data. INSTAT GraphPad Prism 9.0 software for Windows was used for the statistical analysis.