Patients with SLE (n = 14), SSc (n = 5), pSS (n = 7) and sSS (n = 5) were recruited. These patients were, with few exceptions (one SLE and one SSc), female; median ages ranged from 48 to 63 years in different groups. Clinically, patients had mild to moderate disease activity and were stable on current therapy (Supporting information, Table S1). CD4 and CD8 (CD4–) T cells were gated in HD PBMC after exclusion of doublets and gating on intact lymphocytes by light scatter. The strategy is shown in Fig. 1 for a representative sample, analysed ex vivo. Baseline CD146 expression was detected on small percentages

of CD4+ and CD4− lymphocytes, but clearly above isotype controls. The cells were stimulated in vitro with anti-CD3 and anti-CD28 antibodies, and subsequent up-regulation of CD146 and activation markers on T cells was measured. Activation was confirmed by up-regulation of CD69 (Fig. 2a) and CD25 (Fig. 2b). CD146 was induced Target Selective Inhibitor Library datasheet on activated CD4 and CD8 T cells, starting at 24 h and persisting until at least 96 h, similar to CD25. From day 2 onwards, CD146 expression continued to increase, even as activated cells began to lose CD69. T cells underwent blast transformation (not shown), although cell division was not tracked. Thus, both CD4 and CD8

T cells were Trichostatin A purchase capable of up-regulating CD146 expression in response to stimulation via CD3 and CD28 in vitro. Representative ex-vivo analyses of CD4 versus CD146 staining, gated on CD3+ lymphocytes from HDs, are shown in Fig. 3a. The frequencies of CD146+ cells ranged from 0·3 to 3·6% of CD4+ T cells (Fig. 3b, left; median: 1·70%, IQR: 1·00–2·60%), as reported previously [7]. Within the CD8 (CD4–CD3+) subset, CD146+ T cells were less frequent (Fig. 3b; P < 0·0001 for HD, paired analysis by Wilcoxon test). Isotype control staining did not differ between the T cell subsets (not shown). As described further below, most CTD patients had normal frequencies

of CD146+ T cells, but a minority showed 4��8C increased frequencies. First, we examined whether or not HD T cells expressing CD146 were enriched or depleted of activation markers. Ex vivo, a minority of total CD4+ T cells in HDs expressed CD25 (Fig. 4a, left). The small subset of CD146+ cells appeared to be shifted towards raised CD25 fluorescence intensity compared to the double-negative population, even though most of these cells did not exceed the threshold for positivity. This suggested that most CD146+ T cells express low levels of CD25. If the two markers were expressed independently of each other, the frequency of CD25+ cells in the CD146+ subset should be the same as in bulk CD4 T cells. However, CD25+ cells comprised a greater proportion of CD146+ cells than of total CD4 cells, indicating a mutual association, which was highly reproducible between donors (Fig. 4b, left; numerical P-values indicate a significant association, as assessed by a paired, non-parametric analysis).