196 Marta I. Oliveira et al. Eur. J. Immunol. 2012. 42: 195–205 Introduction CD6 is an integral membrane protein expressed on peripheral T lymphocytes and medullary thymocytes [1]. CD6 is likely to be involved in the regulation of T-cell development and activation [2], and to have an important role in the context of human inﬂammatory responses and cellular expansions, given that the CD6 gene is strongly associated with susceptibility to multiple sclerosis (MS) according to genome-wide association studies and meta analyses [3, 4]. Anti-CD6 IgM Abs have been used in clinical trials with MS patients, although they are yet to have a clinical impact [5]. Given this level of clinical interest, however, it is of considerable importance that the signaling pathways regulated by CD6 are better characterized. CD6 has long been regarded as a costimulatory molecule largely because Ab-mediated CD6 cross-linking potentiates proliferative T-cell responses [6, 7], and also because soluble, monomeric CD6 present in antigen (Ag)-primed mixed lymphocyte reactions inhibited Ag-speciﬁc T-cell responses [8–10]. Upon TCR engagement, CD6 is phosphorylated on tyro- sine residues suggesting that interactions with SH2 domain- containing intracellular mediators very likely occur [11]. The cytoplasmic domain of CD6 additionally contains multiple potential binding sites for SH3 domain-containing signaling effectors [12]. Accordingly, the rat homologue of CD6 has the capacity to associate with protein tyrosine kinases of different families, such as the Src-family kinases Lck and Fyn, the Syk family kinase ZAP-70, and the Tec-family kinase Itk [13]. Beyond this neither the signaling pathways directly triggered by CD6 stimulation nor even the character of the resulting signals is well understood. Recent studies have shown that CD6 associates with SLP-76, a positive regulator of T-cell activation [9], and with the scaffold- ing protein syntenin-1, possibly coupling CD6 to the cytoskeleton and other signaling effectors [14]. CD6 has also been shown to associate at the surface of T cells with the structurally related receptor CD5 [13, 15], an inhibitor of T-cell responses [16–18]. CD5 and CD6 exhibit a shared pattern of expression during ontogeny, and the possibility of overlapping effects of these two proteins on repertoire selection has been previously suggested [19]. Moreover, stimulation of rat T cells with anti- CD6 Abs enhanced the tyrosine phosphorylation of CD5 [13], suggesting that CD6 triggering may increase the inhibitory potential of CD5. The molecular basis of the interaction between CD6 and its physiological ligand, CD166, has been comprehensively studied [2]. CD166 is expressed on cortical and medullary thymic epithelial cells, monocytes and transiently on activated leuko- cytes, indicating that the contact between CD6 and CD166 may be important during thymic selection and during and after the activation of mature T cells [9, 20]. CD6 is also reported to accumulate at the immunological synapse upon T cell–APC conjugate formation [8], with the interaction depending on the binding of APC-expressed CD166 to the third scavenger receptor cysteine-rich extracellular domain of CD6 [21]. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim We have investigated the signaling role of CD6 during Ag presentation and in vitro stimulation. Surprisingly, contrary to expectation based on the effects of CD6 cross-linking, which activates T cells [6, 7, 13], we consistently observe that the expression of CD6 at the surface of T cells abrogates both early and late signaling responses triggered by Ag or by direct stimu- lation of the TCR/CD3 complex, even in the absence of ligand binding. Our results strongly suggest that, as in the case of the evolutionarily related molecule CD5, CD6 is a negative modulator of T-cell activation whose expression alone at the T-cell surface is sufﬁcient to constrain T-cell activation. Results CD6 expression inhibits calcium responses in T cells To investigate the signaling role of CD6, we used ex vivo human T lymphocytes and induced the expression of exogenous rat CD6 (rCD6) at their cell surface. We then measured calcium responses of individual T cells, either expressing or not rCD6, interacting with superantigen (sAg)-loaded APCs. Transient manipulations of T-cell signaling molecules in this system have been useful for identifying critical parameters for T-cell activation previously [22]. Following transfection with a cDNA encoding rCD6 fused to YFP, the human T cells expressed variable levels of the chimeric protein (Fig. 1A, top panel). Endogenous human CD6 (hCD6) as well as human CD3 (hCD3) were highly expressed and their expression was unaffected by the coexpres- sion of rCD6 (Fig. 1A, middle and bottom panels, respectively). Similarly, Raji cells (used as APCs) express human CD166 (hCD166), and so presumably in T cell–APC conjugates, T-cell surface hCD6 interacts with hCD166 expressed in Raji cells. We have considered untransfected T cells interacting with untransfected Raji the standard experimental setting, and chan- ges that may occur following the expression of rCD6 and rCD166 should be due to speciﬁc functions of these heterologous rat molecules. T cells were allowed to interact with sAg-loaded Raji cells, and intracellular calcium levels in individual T-cell/Raji cell conju- gates were followed by ﬂuorescence imaging. Strikingly, T cells expressing high levels of exogenous rCD6 appeared to be much less responsive to APCs than T cells that were low or negative for rCD6; a typical example is shown in Fig. 1B (see also Supporting Information movie 1). To quantitate this effect, we selected T cells expressing either high levels of rat CD6 (rCD61), or cells for which rCD6 expression was not detectable in the ﬂuorescence microscope (rCD6 ). Sixty percent of rCD61 cells failed to respond to the APCs, compared with only 36% of the rCD6 cells (Fig. 1C). For the responding cells of both the rCD61 and the rCD6 groups, we measured the calcium signals in individual cells. On average, rCD61 cells (n 5 10) displayed lower calcium signals than rCD6 cells (n 5 12), and the response returned to baseline more quickly (Fig. 1D). 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