Circulating Treg subpopulation
We first analyzed the frequencies of total Tregs and activated Tregs in peripheral blood using FOXP3 intracellular staining in patients with AIN (Figure 1a ). The frequencies of both total Tregs and activated Tregs in patients with AIN were significantly lower than those observed in age-matched control subjects, as shown in Figure 1b (4.49 ± 0.99% vs. 5.87 ± 1.44% and 0.78 ± 0.17% vs. 1.00 ± 0.33%; p = 0.0123 and p = 0.0123, respectively). This result was partly consistent with those reported in our previous study (9). The low frequency of total Tregs may be caused by the decrease in activated Tregs that play an important role in suppressive function to avoid autoantibody production in autoimmune disease (including AIN).
For the analysis of the TCR repertoire, Tregs were defined as CD4+CD25+CD127lowT cells substituted for FOXP3 intracellular staining using a flow cytometer (Figure 1c ). When the frequency of CD4+CD25+CD127lowTregs was compared between patients with AIN and control subjects, Tregs in the former group showed a lower frequency than that noted in control subjects (6.32 ± 1.59% vs. 7.69 ± 1.36%, respectively; p = 0.0113) (Figure 1d ). There was no difference in the frequency of CD4+CD25 conventional T cells between patients with AIN and control subjects.