http://immunol.nature.com • march 2002 • volume 3 no 3 • nature immunology Eric Sebzda 1 , Madelon Bracke 2 ,Tamara Tugal 1 , N ancy H ogg 2 and D oreen Ann Cantrell 1 T cell receptor (TCR) stimulation activates the small GTPase Rap1A, which is reported to antagonize Ras signaling and induces T cell anergy.To address its role in vivo, we generated transgenic mice that constitutively expressed active Rap1A within the T cell lineage. We found that active Rap1A did not interfere with the Ras signaling pathway or antagonize T cell activation. Instead of anergy, the T lymphocytes that constitutively expressed active Rap1A showed enhanced TCR-mediated responses, both in thymocytes and mature T cells. In addition, Rap1A activation was sufficient to induce strong activation of the β 1 and β 2 integrins via an avidity-modulation mechanism. This shows that, far from playing an inhibitory role during T cell activation, Rap1A positively influences T cells by augmenting lymphocyte responses and directing integrin activation. 1 Lymphocyte Activation Laboratory, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London W C2A 3PX , UK. 2 Leukocyte Adhesion Laboratory, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London W C2A 3PX , UK. Correspondence should be addressed to D. A. C. (doreen.cantrell@ cancer.org.uk). Rap1A positively regulatesT cells via integrin activation rather than inhibiting lymphocyte signaling T cell activation, which is initiated by antigen receptors and costimulatory molecules, produces coordinated signaling pathways that lead to antigen- specific clonal expansion and effector functions. T cell receptor (TCR)- mediated signals regulate p21 ras activity, which is critically involved in cytokine gene induction and T cell development. Similarly, a second Ras family member, Rap1, is activated by antigen receptors 1,2 , although the con- sequences of this activity remain controversial. Rap1 was first identified as a protein capable of reverting the mor- phological phenotype of Ras-transformed fibroblasts 3 . Rap1 has an effector-binding domain homologous to that of Ras, and its ability to compete for target proteins such as Raf-1 was thought to explain its abil- ity to reverse Ras-mediated transformation 4,5 . Thus, it is proposed that Rap1 antagonizes the actions of p21 ras specifically by preventing Ras activation of the Raf-1→MAPK kinase (MEK)→extracellular signal–regulated kinase 1 (Erk1) and Erk2 pathway. This model became immunologically relevant when it was shown that anergic T cells express high amounts of activated Rap1A 6 . Ras signaling and Erk acti- vation are defective in these cells, which supports the concept of Rap1A as a negative regulator. Similarly, a correlation between weak Erk acti- vation and strong Rap1A activation has been noted in studies that exam- ined Ras induction in T cell lines 7 . In addition, stimulation of T cells with anti-CD28 partially inhibits TCR-mediated Rap1 activation 1,8 , which suggests that optimal T cell responses require subversion of Rap1 activation. A RTICLES 251 Published online: 11 February 2002, D O I: 10.1038/ni765 KpnI NotI Prim1a Prim2a Prim1b Prim2b hCD2 promoter HA tag V12Rap1A LCR 11.9 kb PDBu Ionomycin Rap1 α-CD3 IL-2 0 1 5 10 0 1 5 10 0 1 5 10 0 1 5 10 Time (min) a b Figure 1. Generation of constitutively active Rap1A- transgenic mice. (a) Immunoblot detection of active Rap1A in human T cells. After T cell stimulation, active Rap1A was isolat- ed using a GST-fusion protein that contained the RalGD S-bind- ing domain specific for active Rap1 (RalGD SRBD -GST). Protein extract was analyzed by immunoblotting with anti-Rap1. (b) Scheme showing the V12Rap1A transgene. A 0.75-kb fragment containing H A-tagged human V12Rap1A cD N A was subcloned using a SmaI site within the human CD 2 vector.The transgene is shown relative to the human CD 2 promoter and LCR. Arrows indicate the position of the transgene-specific primer pairs used in PCR-based screening for transgenic mice. (c) Immunoblot detection of V12Rap1A transgene. Cell extracts that corresponded to an equal number of thymocytes from normal littermate control (N LC) and transgenic lines 1 and 2 (Tg1 and Tg2) were loaded and analyzed by immunoblotting with Rap1- or H A-specific antibodies. (d) Transgenic V12Rap1A is constitutively active.Active Rap1 was extracted from control or V12Rap1A-transgenic thymocytes with a RalGD SRBD -GST fusion protein and ana- lyzed by immunoblotting with anti-Rap1. Upper band, H A-tagged transgenic V12Rap1A. Lower band, endogenous Rap1. NLC Tg1 Tg2 HA-tag Rap1A - + - + NLC Tg1 PDBu Rap1A d c © 2002 Nature Publishing Group http://immunol.nature.com