Cells were cultured for 5 days with a T cell expander capable of preserving the original T cell function (CD3/CD28 Dynabeads, Dynal Biotech). rIL-2 (Chiron) was added at 30 U/mL on day 1. On the last day of culture, cell proliferation was tested by the addition of 0.25 μCi of [3H]thymidine/well, and this was followed by harvesting 18 hours later. Incorporated [3H]thymidine see more was assessed with a β-counter (Canberra Packard, Ltd., Pangbourne, United

Kingdom). The inhibition percentage was calculated as follows: The normality of the variable distribution was assessed by the Kolmogorov-Smirnov goodness-of-fit test; once the hypothesis of normality was accepted (P > 0.05), a comparison was performed with the Student t test. If the values were not normally distributed, the analysis was performed with the Mann-Whitney test. Categorical variables were compared

with Fisher’s exact test. Correlations were assessed with Pearson’s or Spearman’s correlation coefficient. A P value < 0.05 was considered significant. The percentage of CD4+CD25hi T cells in patients with AIH was lower than that in HCs; this difference reached statistical significance not only when undivided AIH patients were considered but also when the subgroups of [A] patients and [R] patients were analyzed separately ([A] patients versus HCs, P = 0.05; [R] patients versus HCs, P = 0.02). CD45RO and CD62L expression did not differ between patients and controls; it was present in about 70% of CD4+CD25hi T cells in both groups. CD8+CD28− T cell numbers were similar in patients and controls. The number of CD3+CD56+ (NKT) cells mirrored the pattern of CD4+CD25hi buy Aloxistatin cells: they were significantly lower in undivided AIH patients versus controls and lower in [A]

patients versus [R] patients (AIH [A] patients versus HCs, P = 0.001; AIH [R] patients versus HCs, P = 0.005). The numbers of γδTCR-expressing cells were comparable between AIH and controls, but the physiological ratio of circulating Vδ1 and Vδ2 cells, preserved in [R] patients, MCE公司 was inverted in [A] patients (AIH [A] patients versus HCs, P = 0.001). The expression of FOXP3 and CTLA-4 was evaluated in magnetically purified CD4+CD25+ lymphocytes and recorded both as the percentage of positive cells and as the mean fluorescence intensity (MFI; Table 3). FOXP3 expression was significantly reduced in AIH patients versus controls (P = 0.038 for the percentage and P = 0.012 for the MFI), regardless of disease activity, whereas CTLA-4 was similarly expressed in AIH patients and controls. CD8+CD28− lymphocytes from both HCs and AIH patients did not produce significant amounts of IL-10 in our ex vivo culture setting (data not shown). Granzyme B expression by γδTCR-positive cells was higher in AIH patients versus controls in terms of both the percentage of positive cells and the fluorescence intensity (Table 3), the latter mirroring disease activity and being significantly higher in [A] patients versus [R] patients (44.05 ± 7.83 and 22.39 ± 4.04, P = 0.