Representative examples of MSG tissues that belong to the three SS subgroups, as these classified by the grade of inflammatory lesion (SS-I, mild; SS-II, intermediate; and SS-III, advanced MSG lesions). A, C, and E: Tissues stained by anti-CD3 antibody, which reveal the areas of T cells. B, D, and F: Detection of Foxp3⁺ cells (shown by arrowheads) in a serial section corresponding to the tissue area marked by square in A, C, and E, respectively. The figures that are incorporated in B, D, and F represent detail of the squared area, showing the typical nuclear staining of Foxp3⁺ cells. Differential incidence of Foxp3⁺-stained cells among the three SS subgroups is readily detected and cannot be attributed to the lack or decreased numbers of T cells, as shown in the respective panels presenting CD3⁺ T-cell distribution (A, C, E). Original magnifications: ×100 (A, C, E); ×200 (B, D, F); ×400 (insets B, D, F).

Immunohistochemical stainings of paraffin-embedded MSG biopsies that were obtained from one patient with SS (A, B), non-SS sialadenitis (related to infection by HCV, designated as HCV⁺) (C, D), and a sicca-complaining control (control) with negative biopsy (E, F). Foxp3⁺ cells displaying the typical nuclear staining (A, C, E) were solely detected among the infiltrating mononuclear cells and were located at the areas of CD3⁺ T cells (B, D, F). Double stainings revealed that Foxp3⁺ cells (brown, indicated by arrows) were also positive for the T-cell-specific CD3 (G) and the CD4 (H) antigens (red, arrowheads), but not the B-cell marker CD20 (I), the macrophage-specific CD68 molecule (J), or fascin (K) and S100 (L) proteins that characterize follicular and interdigitating DCs, respectively (red-stained cells, designated by arrowheads). Representative examples are shown. Original magnifications: ×100 (A–F); ×400 (G–L).

A: Plot indicating the distribution of infiltrating Foxp3⁺/CD3⁺ cells at the MSG inflammatory lesions of sicca-complaining controls with negative biopsy (designated as sicca-controls), non-SS sialadenitis controls (sialadenitis), SS patients (SS), and SS subgroups, as these classified by the grade of MSG autoimmune lesions (SS-I, SS-II, and SS-III: mild, intermediate, and advanced MSG lesions, respectively). Foxp3⁺/CD3⁺ cell percentage was not found to statistically differ between the SS cohort and sialadenitis-controls, whereas Foxp3⁺ cells were not detected in sicca-controls. Mann-Whitney test revealed that Foxp3⁺/CD3⁺ cell distribution varied significantly among the three SS subgroups, with the highest observed at the group with the intermediate grade of infiltration (SS-II) and the lowest at the group with mild lesions (SS-I). P values are designated by asterisks (*P < 0.05, ***P < 0.0001). Horizontal bars represent the mean value of the group. B: Plot showing the Spearman’s rank correlation analysis between the percentages of Foxp3⁺ cells to total infiltrating MNCs in the MSG inflammatory lesions and the serum C4 levels of SS patients (n = 26). C: Representative density plot presenting the flow cytometric analysis of the CD4⁺Foxp3⁺ cells in the peripheral blood of an SS patient, using a fluorescein isothiocyanate-conjugated anti-CD4 and a PE-conjugated anti-Foxp3 antibody. For analyses, CD4⁺Foxp3⁺ cells were expressed as a percentage of total CD4⁺ cells. D: Plot indicating the percentage of CD4⁺Foxp3⁺ cells to CD4⁺ T cells in the peripheral blood of healthy donors (HDs) and SS patients (SS), as well as SS subgroups (SS-I, SS-II, and SS-III), analyzed by flow cytometry. Mann-Whitney analyses revealed that similar distribution of CD4⁺Foxp3⁺ cells is observed in healthy donors and SS patients. The percentages of CD4⁺Foxp3⁺ cells were found to significantly vary among the three SS subgroups. The lowest percentages are observed in the subgroup with intermediate MSG lesions (SS-II), whereas the highest in patients with advanced MSG lesions (SS-III). P values are designated by asterisks (*P < 0.05, **P < 0.01). Horizontal bars represent the group mean. E: Plot displaying the significant negative correlation (r = −0.6679, P = 0.0065) between the incidence of Foxp3⁺ cells in the peripheral blood (expressed as percentage of CD4⁺ cells) and the autoimmune MSG lesions (expressed as a percentage of CD3⁺ cells). Analysis was performed by Spearman’s rank correlation test.