202 Marta I. Oliveira et al. Eur. J. Immunol. 2012. 42: 195–205 these kinases away from the TCR apparatus or it could function analogously to the IgSF glycoprotein CD2, which transduces mitogenic signals via its association with lipid rafts and the tyrosine kinases Lck and Fyn [30, 31], and inhibitory signals via its association with CD5 [32–34]. Since CD5 and CD6 also associate at the surface of T cells and one major effect of CD6 stimulation is the phosphorylation of CD5 [13], it is feasible that CD5 can integrate or transform the signals generated by the triggering of CD6. A striking ﬁnding of the present study is that CD6 appears to be capable of strong inhibitory signaling in the absence of ligand. Our ﬁndings suggest that CD6 is a general attenuator of T-cell activation, whose level of expression at the plasma membrane, rather than its interactions with ligand or localization at the immunological synapse, determines the amplitude of the signal. At present, we are unable to offer an explanation for why the localization of CD6 to the synapse does not induce stronger inhibitory signaling. A correlation between the levels of CD6 expression and the heightened inhibition of cell responsiveness has been proposed previously [9]. However, given that very few T cells do not express CD6, ﬁner-grain regulation may be achieved by the generation of CD6 intracellular isoforms produced at distinct stages of activation or differentiation [12, 35, 36]. Since, as we show, the cytoplasmic domain is crucial for the inhibitory role of CD6, variation in its composition may translate into different associations with effector molecules that could very conceivably ﬁne-tune activation thresholds in T cells. Nevertheless, the full-length isoform seems to be the most abundant, and hence at least at some stages of immune respon- ses, CD6 will display some capacity for downmodulating activation signals as was shown for the full-length constructs used in the present study. As the cytoplasmic part of CD6 is highly tyrosine phosphorylated after T-cell activation, the example of CD5 could be followed [16, 17], with a systematic and careful analysis of the various tyrosine residues present within the domain of CD6 undertaken. The observation that, like CD6, CD5 also attenuates signaling in T and B cells stimulated via their physiological receptors in a ligand independent manner, suggests that CD6 is not alone in its capacity to establish activation thresholds via its expression per se, and that this could be a general feature of inhibitory signaling proteins. Materials and methods Plasmid constructs rCD6FL/pEYFP-N1, rCD6Dd3/pEYFP-N1 lacking the ligand-bind- ing domain of CD6, and a cytoplasmic deletion mutant, rCD6CY5/pEYFP-N1, as well as rCD166/pECFP-N2 have been described previously [21]. To produce a truncated form of hCD6, the expression vector encoding full-length hCD6, CD6/pBJ-neo [12], was used as a template for the ampliﬁcation of a PCR fragment, with a forward & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim primer at the beginning of the transmembrane domain (50-CAA GGAATCTCGGGAGCTAATGCTCC-30) and a reverse primer includ- ing the ﬁrst tyrosine residue codon of the cytoplasmic tail, followed by a stop codon and a XbaI restriction site (50-ACTATCTAGAC TACTACTCGAGCGGCCGCCACTGTGCTGGATATCTGCAGAATTAA TATT-30). The PCR product was then subcloned into CD6C Y5/pcDNA3.1/CT-GFP-TOPO [21], in the EcoRI-XbaI restriction sites. Human CD166 cDNA was obtained from activated PBMC, using forward (50-TAGTAGAAGCTTATGGAATCCAAGGGGG CCAG-30) and reverse primers (50-ACAATCACAAAACTGAA GCCCGCGGTAGTAG-30) in a PCR reaction, and cloned into the pECFP-N2 vector in HindIII-SacII restriction sites. Cells and transfections Cells lines Raji [37], Jurkat E6.1 [38], K562 [39], and CHO-K1 [40] were maintained in RPMI 1640 supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, penicillin G (50 U/mL), and streptomycin (50 mg/mL). Human peripheral blood lympho- cytes were obtained from peripheral blood of healthy donors after centrifugation over Lymphoprep (Nycomed Pharma). Human primary T cells were isolated from peripheral blood by centrifuga- tion in a Ficoll gradient followed by negative depletion on magnetic beads (T-cell negative isolation kit, Dynal Biotech). Transient transfections of primary T lymphocytes and of Raji cells were performed as described previously [21]. Jurkat E6.1 cells were stably transfected as described previously [31]. K562 cells (5 106) were transfected with 50 mg of hCD166/ pECFP-N2 or rCD166/pECFP-N2 and by electroporation at 500 mF and 875 V and cultured in complete RPMI supplemented with 5 mg/mL of G418 (Invitrogen) to select for positive cells. Flow cytometry and cell sorting Flow cytometry was performed as described previously [33]. Monoclonal Abs used were OX52 recognizing rCD6 [41], MEM- 98 recognizing hCD6 [42], and UCHT1, recognizing human CD3 [43]; polyclonal Abs were rabbit anti-mouse (RAM) FITC-labeled (DakoCytomation) and rabbit F(ab0)2 anti-mouse RPE (Dako). Fluorescence-activated cell sorting was performed using a FACSAria (Becton Dickinson). Conjugate formation and ﬂuorescence analysis Conjugate formation and ﬂuorescence analysis were performed as described previously [21]. Calcium measurements Single-cell calcium measurements were performed as described previously [22]. To measure total calcium release in human www.eji-journal.eu