Eur. J. Immunol. 2012. 42: 195–205 DOI 10.1002/eji.201040528 Leukocyte signaling 195 CD6 attenuates early and late signaling events, setting thresholds for T-cell activation Marta I. Oliveira1,2, Carine M. Gonc-alves1,2, Mafalda Pinto1, St´ephanie Fabre3,4, Ana Mafalda Santos1,2,5, Simon F. Lee1, Mo´nica A. A. Castro1, Raquel J. Nunes1,2, Rita R. Barbosa1, Jane R. Parnes6, Chao Yu5, Simon J. Davis5, Alexandra Moreira1, Georges Bismuth3,4 and Alexandre M. Carmo1,2, 1 Group of Cell Activation and Gene Expression, IBMC-Instituto de Biologia Molecular e Celular, Porto, Portugal 2 ICBAS-Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, Porto, Portugal 3 Institut National de la Sante´ et de la Recherche Me´dicale, Paris, France 4 Institut Cochin, Universite´ Paris Descartes, Centre National de la Recherche Scientiﬁque (UMR 8104), Equipe labellise´e par la Ligue Nationale contre le Cancer, Paris, France 5 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, Nufﬁeld Department of Clinical Medicine, University of Oxford, Oxford, UK 6 Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA, USA The T lineage glycoprotein CD6 is generally considered to be a costimulator of T-cell activation. Here, we demonstrate that CD6 signiﬁcantly reduces early and late T-cell responses upon superantigen stimulation or TCR triggering by Abs. Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react signiﬁcantly less well compared with T cells not expressing the exogenous receptor. When the cytoplasmic domain of rat CD6 was removed, calcium responses were recovered, indicating that the inhibitory properties of CD6 are attributable to its cytoplasmic domain. Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deﬁcient cells or cells expressing a cytoplasmic deletion mutant of human CD6. Similarly, calcium signals triggered by anti-CD3 were enhanced in human T lymphocytes following morpholino-mediated suppression of CD6 expression. Finally, the proliferation of T lymphocytes was increased when the CD6–CD166 interaction was blocked with anti-CD166 Abs, but inhibited when anti-CD6 Abs were used. Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufﬁcient to restrain signaling in T cells. Key words: Cellular activation . Cell-surface molecules . Inhibitory molecules . Signal transduction . T cells Supporting Information available online Correspondence: Dr. Alexandre M. Carmo e-mail: acarmo@ibmc.up.pt & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu