Eur. J. Immunol. 2012. 42: 195–205 Leukocyte signaling 197 Figure 1. CD6 decreases the amplitude of Ca21 responses. (A) Cytometric analysis of ex vivo human T cells transfected with an rCD6-YFP vector. Live cells were gated based on the Forward Scatter (FSC) and Side Scatter (SSC) proﬁles. The expression of rCD6 is indicated by a thin line (upper panel) and the negative control is shown (in all panels) as a thick line. Expression of hCD6, (middle panel), and human CD3 (hCD3, lower panel) is shown before (thin line) and after transfection (dashed line) of T cells with the vector. (B) Raji cells preincubated with sAg interacted with Fura-2 loaded T lymphocytes transfected with a cDNA construct encoding rCD6-YFP. Time-lapse video imaging with 20 magniﬁcation showing superimposed differential interference contrast (DIC) and calcium signals (upper panel) and rCD6 expression (lower panel) on one rCD6 T cells (black arrow) and one rCD61 T cell (white arrow), interacting with APCs in the same ﬁeld. Scale bar 5 10 mm. (C) Percentage of rCD6 or rCD61 T cells that increase (ratio 41.5) or not their calcium after conjugate formation with sAg-pulsed Raji cells. Bars represent the mean (7SD) values of three separate experiments with at least 30 individual cells analyzed in each. (D) Intracellular calcium was measured sequentially every 10 s in T cells interacting with sAg-pulsed Raji cells. Calcium was measured for T cells expressing (dashed line) or not (solid line) rCD6-YFP. Calcium signals were averaged from individual responses of all the cells in each set. Results are from one of the three experiments showing similar results. (E) Dot plots showing maximal calcium signals obtained in individual cells following stimulation by sAg-loaded Raji cells. Each dot represents one cell. Horizontal lines indicate mean values [rCD6 (n 5 12); rCD61 (n 5 10)]. The probability that the Ca21 signals of rCD61 T cells in response to sAg stimulation were similar to those of rCD6 T cells was assessed using Students t-test (p 5 0.038). the calcium responses were signiﬁcantly lower for the rCD61 cells than those of the rCD6 cells (po0.05; Fig. 1E). To determine whether endogenous hCD6 would exhibit comparable inhibitory behavior in these cells, we suppressed its expression using hCD6-speciﬁc morpholino oligomers (MOs); we were successful in reducing expression by over 95% as assessed by western blotting (Fig. 2A). Using CD3 mAb (OKT3), we acti- vated the untransfected T cells as well as T cells treated with the MOs, and measured calcium mobilization. At 1 mg/mL to activate 3 106 cells/mL, OKT3 induced a very modest increase in calcium in hCD6-expressing T cells, whereas a pronounced increase in calcium signaling was observed in the hCD6-depleted T cells (Fig. 2B), conﬁrming the observations made for the rCD6 transfections. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Localization of CD6 at the immune synapse is not required for the inhibition of calcium signaling In order to promote rCD6 localization to the immune synapse (IS), we transfected Raji cells with a cDNA-encoding rat CD166 (rCD166) fused to CFP. As expected, when Raji cells lacked expression of rCD166, rCD6 in T cells was distributed homo- geneously over the cell surface and there was no particular enrichment at the IS (Fig. 3A, upper panels). In contrast, for Raji cells expressing rCD166, both the rCD6 expressed by the T cells and the rCD166 expressed by the Raji cells accumulated in the contact area (Fig. 3A, bottom panels). Measurements of calcium mobilization in rCD61 cells establishing contacts with Raji cells expressing rCD166 (rCD1661; n 5 16) or not expressing rCD166 www.eji-journal.eu