200 Marta I. Oliveira et al. Eur. J. Immunol. 2012. 42: 195–205 Figure 4. The inhibitory role of CD6 is dependent on its cytoplasmic domain. (A) Percentage of calcium responsive (ratio 41.5) or un- responsive (ratio o1.5) T cells expressing a rCD6 cytoplasmic deletion mutant (rCD6CY5-YFP), 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. (B) Calcium signals were acquired sequentially every 10 s from rCD6CY5-YFP T cells (solid line) versus rCD61 T cells (expressing full- length rCD6, dashed line), interacting with sAg-pulsed Raji cells. Averaged responses were determined from 15 rCD61 T cells and 10 rCD6CY5-expressing T cells interacting with sAg-loaded Raji cells. Results are from one of two experiments, both gave similar results. (C) Surface expression of hCD6 in untreated E6.1 Jurkat cells (top), in Jurkat cells following transfection with a cDNA construct encoding full-length hCD6 (hCD6-FL) (middle), and in Jurkat cells following transfection with a cDNA construct encoding a cytoplasmatic deletion mutant of hCD6 (hCD6CY5) (bottom). Live cells were gated based on the FSC and SSC proﬁles. (D) E6.1-hCD6-FL and E6.1-hCD6CY5 cells were cell-surface biotinylated, lysed in detergent, and CD6 species were immunoprecipitated using MEM-98 mAb and run on SDS-PAGE. (E) E6.1 Jurkat cells expressing hCD6-FL (black lines) or hCD6CY5 (gray lines), and CD6 cells (dashed lines) were preincubated with the calcium indicators Fluo-3 and Fura-red and, upon anti-CD3 stimulation, total intracellular calcium levels were monitored by cytometry during 5 min. Results are from one of three experiments, all gave similar results. responses upon CD3 stimulation (OKT3 at 1 mg/mL using 3 106 cells/mL) were similarly high in both hCD6 cells and cells expressing hCD6CY5, whereas cells expressing full-length hCD6 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim exhibited much poorer signaling capabilities (Fig. 4E), conﬁrm- ing the cytoplasmic domain dependence of calcium signaling by both human and rCD6. CD6 modulates late signaling responses We next evaluated whether CD6 was also able to exert an inhibitory effect on downstream functional responses, such as IL-2 production. To address this, E6.1-hCD6 or E6.1-hCD6-FL cells were stimulated with OKT3, or with a negative control Ab. Following stimulation, cells expressing hCD6 produced three-fold less IL-2 than the parental cells (Fig. 5A). We also assessed the role of CD6 in untransfected ex vivo T cells interacting with APCs, by testing whether blocking the CD6–CD166 interaction altered the proliferative behavior of the T cells. Ex vivo lymphocytes were allowed to interact with sAg-loaded Raji cells, which express endogenous human CD166, in the presence or absence of blocking anti-human CD166 and CD6 Abs [21]. As shown in Fig. 5B and C, preincubating the Raji cells with the CD166 Ab resulted in enhanced cell proliferation such that the fraction of CD6–CD166 blocked T cells completing two rounds of prolifera- tion after 6 days of activation was double that of control cells. In contrast, when we blocked the interaction using a CD6 Ab, cellular proliferation was inhibited, suggesting that binding of the mAb to CD6 induces inhibitory signaling in the T cells (Fig. 5C). We concluded that CD6 constrains both short-term and long-term signaling events in T cells. Discussion In this report, we present evidence that, like CD5, CD6 is capable of generating inhibitory signals in T lymphocytes. Our main ﬁndings are (i) that transfected rCD6 and hCD6 reduce Ca21 responses in human T cells and a T-cell line, respectively, and that the suppression of hCD6 expression increases these responses; (ii) that the ligand-dependent localization of CD6 to the synapse is neither required for these effects nor, surprisingly, further enhances them; (iii) that the cytoplasmic domains of human and rCD6 are responsible for these inhibitory effects; and (iv) that, in addition to receptor proximal signaling, CD6 expression suppresses longer term events such as cytokine secretion and T-cell proliferation. We conclude from these experiments that CD6 has an important role in establishing thresholds for T-cell activation. CD6 was previously regarded to be a costimulatory receptor however, this assumption was largely built on reports by ourselves and others in which CD6 was cross-linked together with the TCR using Abs [6, 7, 13]. An explanation for these observa- tions is that CD6 associates with protein tyrosine kinases such as Lck, Fyn, and ZAP-70 [13], and it could be expected that mAb- induced coaggregation of CD6 with the TCR might promote the Lck/Fyn-mediated phosphorylation of CD3 ITAMs, leading to signaling. CD5 was originally proposed to be a costimulatory www.eji-journal.eu