Impaired T Cell Function in RANTES-Deficient Mice Yasuhiko Makino,* , † Donald N. Cook,‡ Oliver Smithies,§ Olivia Y. Hwang,* Eric G. Neilson,* Laurence A. Turka,* Hiroshi Sato, ¶,1 Andrew D. Wells,* and Theodore M. Danoff* *Penn Center for Molecular Studies of Kidney Disease, Renal-Electrolyte and Hypertension Division, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; †Department of Molecular Diagnosis and ¶ Department of Molecular Immunology, Chiba University Graduate School of Medicine, Chiba, Japan; ‡Schering-Plough Research Institute, Kenilworth, New Jersey 07033; and §Department of Pathology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 The chemokine RANTES is a chemoattractant for monocytes and T cells and is postulated to participate in many aspects of the immune response. To evaluate the biological roles of RANTES in vivo, we generated RANTES-deficient (/) mice and characterized their T cell function. In cutaneous delayed-type hypersensi- tivity assays, a 50% reduction in ear and footpad swell- ing was seen in / mice compared to / mice. In vitro, polyclonal and antigen-specific T cell prolifera- tion was decreased. Quantitative analysis using the fluorescent dye carboxy-fluorescein succinimidyl es- ter revealed that this proliferative defect was due both to fewer antigen-reactive T cells and to a reduction in the capacity of these cells to proliferate. In addition, IFN- and IL-2 production by the / T cells was dra- matically decreased. Together, these data suggest that RANTES is required for normal T cell functions as well as for recruiting monocytes and T cells to sites of in- flammation. © 2002 Elsevier Science (USA) Key Words: chemokines; knockout; delayed-type hy- persensitivity; cellular proliferation; Th1/Th2. INTRODUCTION The chemokines are involved in many aspects of the immune response including lymphocyte chemoattrac- tion, adhesion, and activation (1–3). The family con- tains more than 40 members, which have been divided into subfamilies based on a conserved pattern of cys- teines (2). Members of the CC subfamily have overlap- ping in vitro biological activities including chemoat- traction for monocytes and T cells (4). However, recent in vivo studies using receptor antagonists, neutralizing antibodies, and gene-targeted mice suggest that there is a relatively high degree of specificity to their in vivo function (5, 6). The CC chemokine RANTES is expressed in vitro by many cell types including T cells (7), macrophages (8, 9), fibroblasts (10), mesangial cells (11), renal tubular cells (12), and endothelial cells (13), when appropri- ately stimulated. RANTES is also stored in platelet granules and released with thrombin stimulation (14). Like many of the other CC chemokines, RANTES acts as a chemoattractant for monocytes, T cells (15), eosinophils (16), NK cells (17), and dendritic cells (18). It also promotes T cell adherence to activated endothe- lial cells (19) and stimulates T cell proliferation (20). Given this diverse spectrum of in vitro activities and the observation that many chemokines with overlap- ping in vitro activities are expressed at sites of inflam- mation, it has been difficult to accurately define the biological role of RANTES. To better define its in vivo activity, we have used gene targeting to produce RANTES-deficient mice. Analysis of these mice reveals that RANTES plays a role in the delayed-type hypersensitivity (DTH) 2 re- sponse, T cell proliferation, and the production of IL-2 and IFN-. MATERIALS AND METHODS Generation of RANTES-Deficient Mice A replacement vector was derived from a 6.6-kb Apa- I–XbaI fragment isolated from a Lambda Fix II library prepared from mouse strain 129/Sv genomic DNA (Stratagene). A 334-bp BglII fragment spanning the transcription and translation start site in exon 1 (-267 to 67) was replaced with a hygromycin-resistance cas- sette containing a Pgk-1 promoter (Hygro, generously supplied by Beverly Jones, Princeton, NJ). The Hygro cassette is flanked by an XhoI and SalI site and, after a partial fill-in reaction, was ligated into the BglII 1 To whom correspondence and reprint requests should be ad- dressed at Department of Molecular Immunology, Chiba Univer- sity Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. Fax: +81-43-227-1498. E-mail: hirosato@chiba. email.ne.jp. 2 Abbreviations used: DTH, delayed-type hypersensitivity; MCP-1, monocyte chemoattractant protein 1; MIP-1, macrophage inflam- matory protein 1 alpha; KLH, keyhole limpet hemocyanin; APC, antigen-presenting cell; CFSE, carboxy-fluorescein succinimidyl es- ter; PPD, purified protein derivative. Clinical Immunology Vol. 102, No. 3, March, pp. 302–309, 2002 doi:10.1006/clim.2001.5178, available online at http://www.idealibrary.com on 1521-6616/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. 302