Eur. J. Immunol. 2012. 42: 195–205 Leukocyte signaling 199 Figure 3. Ligand-binding interactions are required for CD6 accumulation at the immunological synapse but not for downmodulation of Ca21 signaling. (A) Conjugates formed between sAg-pulsed Raji cells and rCD6-YFP-expressing T cells. Left panels: differential interference contrast (DIC) images. Upper right panel: localization of rCD6 (visualized in red) when rCD166 is not expressed on the surface of Raji cells; lower right panel: localization of rCD6 (red) and rCD166 (green) when rCD166 is expressed on the surface of Raji cells. Fluorescence overlays (yellow) of rCD6-YFP and rCD166-CFP localization are shown. Magniﬁcation is 40 , scale bar 5 10 mm. (B) Intracellular calcium was measured in rCD61 T cells establishing contacts with rCD1661 Raji cells (thin line, n 5 16), rCD61 T cells establishing contacts with rCD166 Raji cells (dashed line, n 5 11), or rCD6 T cells establishing contacts with rCD1661 Raji cells (thick line, n 5 15). Raji cells were previously pulsed with sAg. Averaged responses shown are from the calcium proﬁles of individual cells from each set, from two independent experiments. (C) Expression of human CD166 in untransfected (left) or hCD166-cDNA-transfected cells (middle), and of rat CD166 in rCD166-cDNA-transfected cells (right). Cells were gated based on the expression of hCD166 or rCD166, respectively, and these gates were used for the analysis in (D). The negative control (Neg) is also shown. (D) Binding of hCD6 tetramers (hCD6-tet, gray lines) to untransfected K562 cells (left), K562-hCD166 cells (middle), and K562-rCD166 cells (right). Binding of hCD58 tetramers (hCD58-tet, thick lines) to the cells was used as negative control, as K562 cells do not express the hCD58 ligand, hCD2. Proﬁles of hCD58- tet overlapped with the proﬁles of unlabeled cells (data not shown). (E) Interactions between one T cell expressing rCD6-YFP (T in red) and three sAg-loaded Raji cells, one of which expresses rCD166-CFP (B in green) and two that do not express rCD166 (B in white). Fluorescence overlays (yellow) of rCD6 and rCD166 localization are shown. Magniﬁcation is 40 , scale bar 5 10 mm. (F) Raji cells transfected with a rCD166-CFP vector were pulsed with a sAg mix before incubation with ex vivo T cells expressing rCD6Dd3, a naturally occurring isoform devoid of the rCD166 binding domain. Cells were ﬁxed after 45 min of interaction. Localization of rCD166-CFP (green) and rCD6Dd3-YFP (red) as well as ﬂuorescence overlays (yellow) are shown with a magniﬁcation of 40 . Scale bar 5 10 mm. (G) Intracellular calcium was measured sequentially every 10 s in T cells interacting with sAg-pulsed Raji cells. Calcium mobilization was measured for T cells lacking rCD6 (rCD6 , solid line) or T cells expressing rCD6Dd3 (dashed line), interacting with rCD166-expressing Raji cells. Averaged responses were determined from 12 rCD6 T cells and 16 rCD6Dd3- expressing T cells interacting with sAg-loaded Raji cells from two independent experiments. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu