198 Marta I. Oliveira et al. Eur. J. Immunol. 2012. 42: 195–205 Figure 2. Expression of hCD6 downregulates calcium signals. (A) Puriﬁed human primary T lymphocytes were treated with speciﬁc hCD6 MOs to suppress endogenous hCD6 expression (hCD6 kd), or with control scramble morpholinos (Control). Knockdown of hCD6 was evaluated by Western blot of cell lysates, using b-actin as a loading control. Numbers on the left indicate molecular mass markers (MM) in kilodaltons (kDa). (B) Cells were incubated with calcium indicators and after stimulation with CD3 mAb (OKT3), cells expressing hCD61 (grey line) and cells where hCD6 was knocked down (hCD6kd, black line) were evaluated for calcium mobilization by cytometry. Data are shown from one out of the three experiments with similar results. (rCD166 ; n 5 11) revealed no differences in signaling for T cells interacting with the two sets of target cells (Fig. 3B and Supporting Information Fig. 1A). This clearly indicates that the localization of rCD6 at the IS per se is not required in order for it to suppress calcium signaling. Human CD6 does not cross-react with rat CD166 A potential complication in the interpretation of these experiments is that hCD6, whose movement is not tracked, could interact with the rCD166 transfected into the Raji cells. Since mouse CD166 is reported to bind human as well as mouse CD6 [23], we investigated whether cross-species binding between human and rat forms could be occurring in our heterologous system. For this purpose, we expressed the extracellular region of hCD6 and assembled phycoerythrin-labeled CD6 tetramers that were then used to screen for CD6 binding to rat or human CD166-expressing cells. K562 cells, of human origin, do not express CD166, and were transfected with cDNAs encoding rat or human CD166 (Fig. 3C). Although, as expected, the CD166 K562 cells did not bind the hCD6 tetramer (Fig. 3D, left panel), hCD166-expressing K562 cells were clearly recognized (Fig. 3D, middle panel). In contrast, rCD166-expressing K562 cells were essentially negative for binding the hCD6 tetramers (Fig. 3D, right panel). Additional evidence that cross-species binding does not mediate CD6 translocation in our system is obtained from the observation that when rCD6-expressing cells contacted Raji- expressing rCD166 versus rCD166 Raji, there was a clear preference for the accumulation of rCD6 in synapses with Raji- expressing rCD166 (Fig. 3E). A ligand nonbinding form of CD6 suppresses calcium responses The experiments described above suggest that the interaction of CD6 with its ligand does not enhance its signaling suppressing & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim effects. Further conﬁrmation was obtained by expressing, on ex vivo human T cells, ratCD6Dd3 (rCD6Dd3), a natural occurring isoform lacking domain 3, the CD166-binding domain of rCD6 [21]. T cells expressing rCD6Dd3 fused to YFP were allowed to form conjugates with Raji cells expressing rCD166 and we observed that rCD6Dd3 failed to accumulate at the immune synapse according to ﬂuorescence microscopy-based analysis (Fig. 3F). Measurements of the calcium signals arising in T cells that express rCD6Dd3 (n 5 16) and interact with Raji cells expressing rCD166, versus cells lacking the construct (rCD6 ; n 5 12) indicate once again that the expression of rCD6Dd3 downmodulates calcium signals (Fig. 3G and Supporting Information Fig. 1B). This supports our preceding observations, i.e. that it is the expression of CD6 per se, rather than its interactions with its ligand or its synapse localization, that is responsible for the inhibition of calcium signaling. The inhibitory effects of CD6 depend on its cytoplasmic domain We addressed the role of the cytoplasmic domain of CD6 using an engineered isoform of rCD6 lacking its cytoplasmic domain, rCD6CY5 fused to YFP, which was expressed in ex vivo human T cells. rCD6CY5 moved to the synapse when the T cells were allowed to form conjugates with rCD166-expressing Raji cells (data not shown). Calcium signals were measured in individual cells conjugated with rCD1661 Raji cells, and we found that the fraction of T cells expressing rCD6CY5 that increased their calcium levels following conjugate formation (i.e. 66%; Fig. 4A) closely matched that of rCD6 cells (Fig. 1C). For the responding T cells expressing rCD6CY5 (n 5 10), we measured calcium signals and compared these with signals from responding T cells expressing full-length rCD6 (n 5 15; Fig. 4B and Supporting Information Fig. 2). The results indicate that rCD6-expressing cells displayed lower and more transient calcium signals than cells expressing rCD6CY5, and thus the inhibitory effects of CD6 can be assigned to its cytoplasmic tail. We have also addressed the role of hCD6, and of its cyto- plasmic domain, by the analysis of calcium ﬂuxes in whole-cell populations. E6.1 Jurkat cells, which already express very low levels of hCD6, were FACS-sorted and gated on the most-negative hCD6 cells, E6.1-hCD6 , which were used for our assays. These E6.1-hCD6 cells were stably reconstituted with either full-length hCD6, to give E6.1-hCD6-FL, or the human cytoplasmic deletion mutant, to yield E6.1-hCD6CY5 (Fig. 4C). Before each experi- ment, E6.1-hCD6-FL and E6.1-hCD6CY5 were FACS sorted so that the levels of hCD6-FL and hCD6CY5, respectively, were equivalent. We conﬁrmed by immunoprecipitation of hCD6 from surface biotinylated cells and Western blotting, that E6.1-hCD6- FL cells expressed only the full-length hCD6 protein ( 130 kDa) and that E6.1-hCD6CY5 cells expressed only the cytoplasmic domain-deleted form of hCD6 ( 90 kDa; Fig. 4D). Calcium www.eji-journal.eu