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In vivo analysis of intracellular signaling pathways during lymphocyte trafficking and activation

Applicant Stein Jens Volker
Number 120640
Funding scheme Project funding (Div. I-III)
Research institution Theodor Kocher Institut Medizinische Fakultät Universität Bern
Institution of higher education University of Berne - BE
Main discipline Immunology, Immunopathology
Start/End 01.04.2008 - 31.03.2011
Approved amount 476'915.00
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All Disciplines (2)

Discipline
Immunology, Immunopathology
Cellular Biology, Cytology

Keywords (4)

Lymphocyte trafficking; Chemokines; G-protein-coupled receptors; Two-photon microscopy

Lay Summary (English)

Lead
Lay summary
Lymphocytes are constantly moving through the cell body in search of foreign Antigen. This is an important step in the immune surveillance of our body and our ability to rapidly respond to pathogens. Our group is interested in the molecular mechanisms which govern lymphocyte trafficking and activation, in particular within lymphoid organs. To this end. we are studying chemokines, small secreted polypeptides, and other signaling molecules which bind to so-called G-protein coupled receptors on expressed on lymphocytes. These molecules play a central role during lymphocyte trafficking and activation. A second interest of our research group is the visualization of these processes in vivo, using so-called two-photon microscopy. Our interest is therefore to obtain a better understanding of the immune system, in order to provide the basis for improved treatment of diseases.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Sensory innervation of the dorsal longitudinal ligament and the meninges in the lumbar spine of the dog.
Waber-Wenger Barbara, Forterre Franck, Kuehni-Boghenbor Kathrin, Danuser Renzo, Stein Jens Volker, Stoffel Michael Hubert (2014), Sensory innervation of the dorsal longitudinal ligament and the meninges in the lumbar spine of the dog., in Histochemistry and cell biology, 142(4), 433-47.
The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density.
Lee Kit M, Danuser Renzo, Stein Jens V, Graham Delyth, Nibbs Robert J B, Graham Gerard J (2014), The chemokine receptors ACKR2 and CCR2 reciprocally regulate lymphatic vessel density., in The EMBO journal, 33(21), 2564-80.
Maturation of lymph node fibroblastic reticular cells from myofibroblastic precursors is critical for antiviral immunity.
Chai Qian, Onder Lucas, Scandella Elke, Gil-Cruz Cristina, Perez-Shibayama Christian, Cupovic Jovana, Danuser Renzo, Sparwasser Tim, Luther Sanjiv A, Thiel Volker, Rülicke Thomas, Stein Jens V, Hehlgans Thomas, Ludewig Burkhard (2013), Maturation of lymph node fibroblastic reticular cells from myofibroblastic precursors is critical for antiviral immunity., in Immunity, 38(5), 1013-24.
OPTiSPIM: integrating optical projection tomography in light sheet microscopy extends specimen characterization to nonfluorescent contrasts.
Mayer Jürgen, Robert-Moreno Alexandre, Danuser Renzo, Stein Jens V, Sharpe James, Swoger Jim (2013), OPTiSPIM: integrating optical projection tomography in light sheet microscopy extends specimen characterization to nonfluorescent contrasts., in Optics letters, 39(4), 1053-6.
Endothelial cell-specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation.
Onder Lucas, Danuser Renzo, Scandella Elke, Firner Sonja, Chai Qian, Hehlgans Thomas, Stein Jens V, Ludewig Burkhard (2012), Endothelial cell-specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation., in The Journal of experimental medicine, 210(3), 465-73.
HIV-1 Nef interferes with T-lymphocyte circulation through confined environments in vivo.
Stolp Bettina, Imle Andrea, Coelho Fernanda Matos, Hons Miroslav, Gorina Roser, Lyck Ruth, Stein Jens V, Fackler Oliver T (2012), HIV-1 Nef interferes with T-lymphocyte circulation through confined environments in vivo., in Proceedings of the National Academy of Sciences of the United States of America, 109(45), 18541-6.
CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells.
Lamana Amalia, Martin Pilar, de la Fuente Hortensia, Martinez-Muñoz Laura, Cruz-Adalia Aranzazu, Ramirez-Huesca Marta, Escribano Cristina, Gollmer Kathrin, Mellado Mario, Stein Jens V, Rodriguez-Fernandez Jose Luis, Sanchez-Madrid Francisco, del Hoyo Gloria Martinez (2011), CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells., in The Journal of investigative dermatology, 131(7), 1503-12.
In vivo analysis of uropod function during physiological T cell trafficking.
Soriano Silvia F, Hons Miroslav, Schumann Kathrin, Kumar Varsha, Dennier Timo J, Lyck Ruth, Sixt Michael, Stein Jens V (2011), In vivo analysis of uropod function during physiological T cell trafficking., in Journal of immunology (Baltimore, Md. : 1950), 187(5), 2356-64.
Critical roles for Rac GTPases in T-cell migration to and within lymph nodes.
Faroudi Mustapha, Hons Miroslav, Zachacz Agnieszka, Dumont Celine, Lyck Ruth, Stein Jens V, Tybulewicz Victor L J (2010), Critical roles for Rac GTPases in T-cell migration to and within lymph nodes., in Blood, 116(25), 5536-47.
Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors.
Sreeramkumar Vinatha, Hons Miroslav, Punzón Carmen, Stein Jens V, Sancho David, Fresno Manuel, Cuesta Natalia (2010), Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors., in Immunology and cell biology, 94(1), 39-51.
Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions.
Coelho Fernanda M, Natale Daniela, Soriano Silvia F, Hons Miroslav, Swoger Jim, Mayer Jürgen, Danuser Renzo, Scandella Elke, Pieczyk Markus, Zerwes Hans-Günter, Junt Tobias, Sailer Andreas W, Ludewig Burkhard, Sharpe James, Figge Marc Thilo, Stein Jens V (2010), Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions., in Blood, 121(20), 4101-9.
CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.
Gollmer Kathrin, Asperti-Boursin François, Tanaka Yoshihiko, Okkenhaug Klaus, Vanhaesebroeck Bart, Peterson Jeffrey R, Fukui Yoshinori, Donnadieu Emmanuel, Stein Jens V (2009), CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway., in Blood, 114(3), 580-8.
Comprehensive analysis of lymph node stroma-expressed Ig superfamily members reveals redundant and nonredundant roles for ICAM-1, ICAM-2, and VCAM-1 in lymphocyte homing.
Boscacci Rémy T, Pfeiffer Friederike, Gollmer Kathrin, Sevilla Ana Isabel Checa, Martin Ana Maria, Soriano Silvia Fernandez, Natale Daniela, Henrickson Sarah, von Andrian Ulrich H, Fukui Yoshinori, Mellado Mario, Deutsch Urban, Engelhardt Britta, Stein Jens V (2009), Comprehensive analysis of lymph node stroma-expressed Ig superfamily members reveals redundant and nonredundant roles for ICAM-1, ICAM-2, and VCAM-1 in lymphocyte homing., in Blood, 116(6), 915-25.
Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway.
Kumar Varsha, Scandella Elke, Danuser Renzo, Onder Lucas, Nitschké Maximilian, Fukui Yoshinori, Halin Cornelia, Ludewig Burkhard, Stein Jens V (2009), Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway., in Blood, 115(23), 4725-33.

Associated projects

Number Title Start Funding scheme
135649 Investigating the molecular factors controlling lymphocyte motility and activation by in vivo imaging 01.04.2011 Project funding (Div. I-III)
107510 Intracellular signaling pathways during lymphocyte trafficking 01.04.2005 Project funding (Div. I-III)
125447 Examining the function of lymphoid organ structure during antiviral immune responses using microscopic and mesoscopic imaging 01.06.2009 Sinergia

Abstract

Continuous lymphocyte trafficking through secondary lymphoid organs (SLO), such as spleen and peripheral lymph nodes (PLN), forms the basis for efficient immune surveillance by the adaptive immune system. Inside PLN, lymphocytes disseminate into T cell zones and B cell follicles where they screen Antigen-presenting cells (APC) before departure via efferent lymphatic vessels. Together with adhesion receptors, lymphocyte recruitment from blood, interstitial migration and egress is regulated by G-protein coupled receptors (GPCR), such as chemokine receptors (CKR) and sphingosine-1-phosphate receptor 1 (S1P1). Although GPCR and their ligands responsible for homeostatic trafficking have been largely identified, relatively little is known about intracellular signaling molecules, which transmit signals for adhesion, transmigration, interstitial motility and dynamic lymphocyte-APC interactions. In previous studies by our group, we have described two largely independent pathways leading to T cell migration mediated by the Rac Guanine exchange factor DOCK2 and phosphoinositide-3-kinase (PI3K) g, respectively. In this proposal, we will continue this line of research, also including multiphoton intravital microscopy for in situ observation of cellular dynamics within lymphoid tissue. In the main subproject presented here, we propose to further examine the signaling networks formed by DOCK2 and PI3K isoforms, as well as their function during lymphocyte trafficking and cell-cell interactions. Using pharmacological inhibitors and genetically modified primary murine B cells, we will perform a thorough in vitro functional analysis of migration, static adhesion and biochemical analysis of DOCK2 and PI3Kd downstream CKR and S1P1. Furthermore, we will examine in detail the role of novel DOCK2-interacting proteins identified in a yeast-two-hybrid screen. Using multiphoton microscopy, we will study in situ APC-T cell interactions in absence of DOCK2 or PI3Kg/d activity. We also propose to analyze the role of DOCK2 during dynamic lymphocyte transmigration through PLN microvessels in vivo, again based on multiphoton microscopy. Finally, the function of the Tec kinase family member Itk during PI3K-mediated T cell migration will be critically examined in vitro and in vivo.In a second subproject, we will carry out a thorough analysis of phospholipase C (PLC) isoforms during lymphocyte migration in vitro and in vivo, as pharmacological PLC inhibitors block lymphocyte trafficking. We will carry out a thorough characterization of PLC isoform expression in T and B cells. Using a combination of PLCb2/b3-deficient T and B cells and a siRNA approach to suppress additional isoforms, we will perform adhesion/migration assays, as well as intravital microscopy of PLN microcirculation and multiphoton microscopy to assess interstitial motility.The impact of the T cell-expressed GPCR receptor TP during interstitial migration is the subject of a third subproject. Its ligand, the lipid mediator thromboxane A2 (TXA2), is expressed by activated DC and increases random T cell motility in vitro, thereby eficiently reducing T cell-DC interactions. Using control and TP-deficient TCR transgenic CD4 cells, we will employ multiphoton microscopy to investigate the influence of TXA2 on migration and T cell-APC contacts under steady state and proinflammatory conditions.In a final fourth subproject, we will further develop and exploit a highly promising imaging technique called Optical Projection Tomography (OPT). OPT is ideally suited to quantitatively analyze large-scale structures, such as vascular networks or B cell follicles, of entire mouse PLN and other SLO. Based on the recent acquisition of a dedicated OPT scanner, we propose to explore changes in internal PLN architecture during various inflammation models, as well as genetically modified or pharmacologically treated mice. We also aim to increase image quality through collaboration with leading OPT laboratories.In summary, this proposal links cell biology and in vivo imaging to analyze molecular mechanisms of lymphocyte trafficking, with a special emphasis on the characterization of DOCK2/PI3K/PLC mediated pathways and appliance of cutting-edge imaging technology.
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