Fig. 2

Reductions in invariant natural killer T (iNKT) cells and increases in T follicular helper (Tfh) and T regulatory (Treg) cells in anti-nuclear antibodies (ANA)⁺ individuals regardless of diagnosis. Representative gating, together with scatterplots show frequencies in each subject group for peripheral iNKT (a, b), Treg (c, d), and Tfh (e, f) cells. For the iNKT cell plot, the cells were first gated on lymphocytes, whereas for the Tfh and Treg cell plots, they were first gated on CD3⁺CD4⁺ cells within the lymphocyte pool with the scatterplots showing the percentage of these cells as a proportion of the CD3⁺ T cell population. g Representative gating for interferon (IFN)-γ-, IL-17A-, and IL-21-producing CD4 T cells following phorbol-12-myristate-13-acetate (PMA) and ionomycin stimulation of peripheral blood mononuclear cells (plots shown have been gated on CD3⁺CD4⁺ lymphocytes). h–j Proportions of cytokine-producing cells within the CD3⁺CD4⁺ T cell population for each subject group. Statistical comparisons on the left side of each figure are between healthy controls (HC) and asymptomatic ANA⁺ individuals (ANA+), patients with undifferentiated connective tissue disease (UCTD), or pooled patients with systemic autoimmune rheumatic disease (SARD), whereas those on the right side of the figure are between the individual SARDs and HC. Bars represent the mean with SEM. Every data point represents an individual patient. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s post-hoc test for multiple comparisons, as compared to HC; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. SLE, systemic lupus erythematosus; SjD, Sjogren’s disease; SSc, systemic sclerosis