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VLA-4 Integrin Concentrates at the Peripheral Supramolecular Activation Complex of the Immune Synapse and Drives T Helper 1 Responses

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VLA-4 Integrin Concentrates at the Peripheral Supramolecular Activation Complex of the Immune Synapse and Drives T Helper 1 Responses VLA-4 integrin concentrates at the peripheral supramolecular activation complex of the immune synapse and drives T helper 1 responses Marı´aMittelbrunn*†, Ana Molina†‡, Marı´aM. Escribese‡, Marı´aYa´ n˜ ez-Mo´ *, Ester Escudero‡, Angeles´ Ursa*, Reyes Tejedor*, Francisco Mampaso‡, and Francisco Sa´ nchez-Madrid*§ *Servicio de Inmunologı´a,Hospital de la Princesa, Universidad Auto´noma de Madrid, Madrid 28006, Spain; and ‡Departamento de Patologı´a,Hospital Ramo´n y Cajal, Universidad de Alcala´, Madrid 28001, Spain Edited by Ralph M. Steinman, The Rockefeller University, New York, NY, and approved June 10, 2004 (received for review November 28, 2003) ␣ ␤ ␣ ␤ The integrin 4 1 (VLA-4) not only mediates the adhesion and whereas the involvement of L 2 during Ag presentation is well ␣ ␤ ␣ ␤ transendothelial migration of leukocytes, but also provides co- known, the role of 4 1 has not been addressed. The L 2 stimulatory signals that contribute to the activation of T lympho- integrin mediates T cell adhesion to APCs, facilitating the cytes. However, the behavior of ␣4␤1 during the formation of the formation of the immunological synapse (IS) (6). The pair ␣ ␤ ␣ ␤ ͞ immune synapse is currently unknown. Here, we show that 4 1 L 2 intracellular adhesion molecule-1 (ICAM-1) forms an is recruited to both human and murine antigen-dependent immune adhesion ring that is called the peripheral supramolecular acti- synapses, when the antigen-presenting cell is a B lymphocyte or a vation complex (pSMAC), that surrounds the T cell receptor– dendritic cell, colocalizing with LFA-1 at the peripheral supramo- peptide–MHC complexes localized at the central SMAC of the ␣ ␤ ͞ lecular activation complex. However, when conjugates are formed IS (7, 8). Several studies in mouse models revealed that L 2 in the presence of anti-␣4 antibodies, VLA-4 colocalizes with the ICAM-1 interaction could be important for driving Th1 polar- CD3-␨ chain at the center of the synapse. In addition, antibody ization (9, 10). engagement of ␣4 integrin promotes polarization toward a T The ␣4␤1 integrin is predominantly expressed on hematopoi- helper 1 (Th1) response in human in vitro models of CD4؉ T cell etic cells and serves as a receptor for fibronectin and vascular differentiation and naı¨ve T cell priming by dendritic cells. The in cell adhesion molecule 1 (VCAM-1). In addition to mediating vivo administration of anti-␣4 integrin antibodies also induces an leukocyte adhesion to endothelium and extracellular matrix, immune deviation to Th1 response that dampens a Th2-driven ␣4␤1 has been implicated in T cell costimulation (11–13). The autoimmune nephritis in Brown Norway rats. These data reveal a dual role of ␣4␤1 as an adhesion and costimulatory molecule regulatory role of ␣4 integrins on T lymphocyte-antigen presenting suggests that this integrin could be involved in the modulation of cell cognate immune interactions. the T cell response during Ag presentation. However, the behavior of ␣4␤1 and its possible function during the establish- fter the recognition of antigens (Ag) presented by dendritic ment of an IS has not been examined. Here, we show that ␣4␤1 Acells (DCs), naı¨ve T lymphocytes proliferate and differen- is recruited to the pSMAC of IS colocalizing with LFA-1 tiate into T helper (Th) 1 or 2 effector cells. These effector integrin. We also demonstrate the functional involvement of this lymphocytes are characterized by distinct patterns of cytokine integrin in the priming of T lymphocytes toward a Th1 response production and homing behavior. Th1 cells mainly produce in vitro and in vivo. IFN-␥ and IL-2 and have a key role in the cellular immune Materials and Methods responses. Conversely, Th2 cells produce IL-4, IL-5, IL-6, and Cells, Antibodies, and Reactives. The Jurkat-derived human T cell IL-10 and promote the humoral immune responses (1). DCs are ϩ the only Ag-presenting cells (APCs) involved in the priming of line V␤8 J77cl20, the lymphoblastoid B cell line Raji, the naı¨ve Th cells and their polarization toward Th1 or Th2 differ- murine Th cell clone SR.D10, the I-Ak transgenic B lymphoma entiation. To acquire this capacity, DCs must undergo a matu- cell line TAK, and the S3085B (S3) human T cell clone have been ration process characterized by the loss of their Ag-capturing described (14). Human monocyte-derived DCs were generated capacity and the increase of their expression of costimulatory as described (14) and matured with lipopolysaccharide (LPS) (10 and adhesion molecules, including ␣4␤1 integrin (2). However, ng͞ml) (Sigma) or calcium ionophore A23187 (150 ng͞ml) other APCs (e.g., B lymphocytes) are also involved in regulating (Sigma) for 48 h or with tumor necrosis factor (TNF)-␣ (50 the cytokine profiles of Th cell responses, indicating the impor- ng͞ml) plus IL-1␤ (10 ng͞ml) for 4 days. The phenotype of tance of postpriming events (3). mature DCs (CD83 high CD86 high CD14low) was confirmed by The interaction between T cells and APCs plays an important flow cytometry. The murine anti-human ␣4 integrin mAbs role in directing Th cell polarization. The strength of antigenic HP2͞1 (epitope B1) and HP2͞4 (epitope B2) are mouse IgG1␬, stimulation, the duration of T cell receptor engagement, the bind with comparable affinity to ␣4 integrin, and cross-react with presence of different cytokines, and the participation of distinct rat ␣4 integrins (15); and anti-␣4 integrin mAb HP1͞7 (epitope costimulatory molecules are critical in determining the pheno- A) have been described (16). Anti-human ␤1 integrin TS2͞16, type of differentiated T cells. The cytokine IL-12, high doses of Ag, and CD28͞B7–1 interaction promote Th1 differentiation, whereas an environment enriched in IL-4, low doses of Ag, and This paper was submitted directly (Track II) to the PNAS office. CD28͞B7–2 or inducible costimulator (ICOS)͞ICOS ligand Abbreviations: Ag, antigen; DC, dendritic cell; Th, T helper; APC, Ag-presenting cell; SMAC, supramolecular activation complex; pSMAC, peripheral SMAC; VCAM-1; vascular cellular participation promote Th2 responses (4). adhesion molecule 1; ICAM-1, intracellular adhesion molecule 1; IS, immunological syn- Integrins are a large family of ␣␤ heterodimeric transmem- apse; LPS, lipopolysaccharide; TNF, tumor necrosis factor; CMAC, chloromethyl derivative of brane proteins that mediate cell–cell and cell–extracellular aminocoumarin; SEB, staphylococcal enterotoxin B; SEE, staphylococcal enterotoxin E; BN, matrix adhesion. Several integrins, lymphocyte function- Brown Norway; PMA, phorbol 12-myristate 13-acetate. ␣ ␤ †M.M. and A.M. contributed equally to this work. associated (LFA-1; L 2), very late activation antigen-4 (VLA-4; ␣4␤1), and VLA-1 (␣1␤1) have been involved also in the §To whom correspondence should be addressed. E-mail: [email protected]. transduction of costimulatory signals in T cells (5). However, © 2004 by The National Academy of Sciences of the USA 11058–11063 ͉ PNAS ͉ July 27, 2004 ͉ vol. 101 ͉ no. 30 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307927101 Downloaded by guest on September 29, 2021 ␣ ͞ ͞ ␮ ͞ anti-CD3 T3b, anti- L integrin TP1 40, anti-CD45 D3 9, anti- in 96-well culture plates (Costar) in the presence of 2 g ml human CD9 VJ1͞10, and anti-CD28 (CD28.2) mAbs have been HP2͞1, HP2͞4, or control Ab anti-␣3 integrin VJ1͞6. On day 5, described (14). Rat anti-mouse ␣4PS͞2 mAb was provided by T. IL-2 (10 units͞ml) was added. On day 14, Th cells were stimu- Issekutz (Dalhousie University, Halifax, Canada), rat anti- lated with PMA͞A23187, and IL-4 and IFN-␥ levels were mouse ICAM-1 was provided by D. Vestweber (University of measured as described above. Mu¨nster, Mu¨nster, Germany), anti-JAM-A antibody was pro- vided by E. Dejana (Instituto FIRC di Oncologia, Milan), and Flow Cytometry Analysis. Mature DCs were incubated with human rabbit anti-human CD3-␨ chain 448 was provided by B. Alarco´n ␥-globulin (100 ␮g͞ml) in PBS for 20 min and then with anti-␣4 (Centro de Biologı´a Molecular, Madrid). integrin HP2͞1 or anti-CD3 T3b mAb (negative control) for 30 Mouse anti-rat mAbs specific for CD45RC (FITC-OX22), min. After washing, cells were incubated with an FITC- CD4 (PE-W6͞25), and CD45 (OX1) were purchased from conjugated secondary Ab and analyzed in a FACScan flow Serotec (Oxford). Secondary Alexa 488- and Rhodamine red cytometer (Becton Dickinson). X-labeled Abs, streptavidin, and the fluorescent tracker CMAC (chloromethyl derivative of aminocoumarin) were from Molec- In Vivo Treatment Protocols. Brown Norway (BN) rats were sep- ular Probes. Poly(L -lysine) was from Sigma, and the superan- arated into two main groups. Group 1 served as a normal tigen staphylococcal enterotoxin E (SEE) was from Toxin control, whereas group 2 animals received five s.c. injections of ͞ Technology (Sarasota, FL). The human recombinant IL-2 was HgCl2 (1 mg kg body weight) over a period of 2 weeks (15). obtained through the National Institutes of Health AIDS Re- Within each group, rats were i.p. injected with vehicle (phos- search and Reference Reagent Program, Division of AIDS, phate buffer, pH 7.5) or 0.5 mg of HP2͞1, HP2͞4, or OX1 National Institute of Allergy and Infectious Diseases, contrib- (anti-rat CD45) mAbs on days 0, 8, and 13. At days 0, 6, 9, 13, uted by M. Gately (Hoffmann-LaRoche, Nutley, NJ). 15, and 23, rats (n ϭ 6) were bled and killed, and their spleens were removed. Blood was either heparinized for flow cytometry Cell Conjugate Assays and Immunofluorescence Microscopy. APCs studies or allowed to clot to obtain serum. were loaded for 20 min at 37°C with the fluorescent tracker Serum samples were tested for IFN-␥ levels by ELISA (R & CMAC (10 ␮M) (Molecular Probes) and incubated for 20 min ␮ ͞ ␮ ͞ D Systems).
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