Cutting Edge: Hierarchy of Chemokine Receptor and TCR Signals Regulating T Cell Migration and Proliferation 1 Shannon K. Bromley,* Daniel A. Peterson,* Michael D. Gunn, ² and Michael L. Dustin 2 * Chemokines play an important role in establishing the distri- bution of lymphocyte subpopulations in primary and second- ary lymphoid tissues and in the recruitment of leukocytes to sites of inflammation. However, the potential of chemokines to down-regulate immune responses has not been demonstrated. We now show that certain chemokine gradients have the po- tential to suppress T cell activation by preventing formation of the immunological synapse, the specialized cell-cell junction that forms before a T cell can be fully activated. Our data reveals an immunosuppressive potential of chemokines engag- ing the CXCR3 and CCR7 receptors, but not the CXCR4, CCR2, CCR4, or CCR5 receptors. These results suggest a novel mechanism for T cell ignorance of agonist MHC-peptide complexes based on dominant chemokine gradients. The Journal of Immunology, 2000, 165: 15–19. T cell activation is an essential process for elimination of many microbial infections and for chronic inflammatory processes. T cell activation is initiated by formation of a specialized junction between T cells and APCs, aptly described as an immunological synapse (IS) 3 (1). The first manifestation of IS formation in response to agonist MHC-peptide complexes is that the T cell stops migrating (2– 4). The ability of agonist MHC- peptide complexes to deliver a stop signal through the TCR may play an important role in selective retention of Ag-specific T cells in tissues and in T cell proliferation. Chemokines have also been shown to arrest T cell rolling under flow (5). However, in tissues, chemokine gradients are thought to motivate and direct T cell mi- gration (6). For example, chemokine gradients attract activated T cells to APCs (7). In contrast, a gradient leading past the APC would have the potential to suppress the T cell response by pre- venting T cell stopping and IS formation. The ability of chemokine receptor signaling to compete with TCR signaling for control of T cell migration has not been systematically examined. Therefore, to understand the basic issues in the competition between TCR and chemokine signals, we tested the ability of a panel of chemokines to compete with TCR signals for T cell migration and proliferation. Our results demonstrate that chemokines can be divided into two groups with respect to TCR signals, dominant and subordinate. Dominant chemokine gradients override the TCR-mediated stop signal, while subordinate chemokine gradients do not prevent or reverse the TCR-mediated stop signal. Dominant chemokines have the potential to suppress T cell responses as demonstrated here by inhibition of T cell proliferation. The cells that have bypassed MHC-peptide complexes under the influence of a dominant che- mokine gradient still proliferate to subsequent MHC-peptide ex- posure. This suggests that dominant chemokine gradients render T cells ignorant of agonist MHC-peptide complexes, as opposed to anergic. Materials and Methods T cells Splenocytes from 3A9 TCR transgenic mice (8), provided by E. R. Unanue (St. Louis, MO), were stimulated for 3 days with 1 M hen eggwhite lysozyme (HEL). Cells were then expanded with EL-4 supernatant con- taining 50 U/ml IL-2 activity and used on day 7. T cells from 3A9 TCR transgenic mice interact specifically with IA k HEL 48–62 . The 3A9 T cells prepared in this manner make IFN- and no IL-4 and are therefore Th1 like. Transmigration assays A total of 10 5 T cells in 100 l were added to 5-m pore-size, polycar- bonate 24-well tissue culture inserts (Costar, Cambridge, MA), with 600 l media (or chemokine dilution) in the lower well. After the indicated time the cells in the lower well were counted with the aid of a hemocytometer. All points were determined in triplicate. Murine EBI1 ligand chemokine was a kind gift from J. Cyster (San Francisco, CA), and secondary lym- phoid tissue chemokine (SLC) was provided by M. D. Gunn. All other chemokines were obtained from R&D Systems (Minneapolis, MN). IA k HEL 48–62 was provided by E. R. Unanue. ICAM-1 expressed in Chinese hamster ovary cells was solubilized with buffered Triton X-100 and cap- tured on YN1/1 Sepharose. ICAM-1 was eluted at low pH in 1% octyl- glucoside. Molecular densities were determined by immunoradiometric as- say using iodinated AW3.18.152 and YN1/1 for IA k and ICAM-1, respectively. Inserts were coated with 50 l of 25 g/ml fibronectin for 1 h at 37°C. Then, liquid was aspirated from the inserts, and the inserts were dried at 37°C for 2 h. Alternatively, filters were coated with ICAM-1, IA k , *Center for Immunology and the Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110; and ² Department of Medicine, Duke University Medical Center, Durham, NC 27710 Received for publication February 29, 2000. Accepted for publication May 5, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant AI43542 and by an Arthritis Research Grant. 2 Address correspondence and reprint requests to Dr. Michael L. Dustin, Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8118, St. Louis, MO 63110. E-mail address: dustin@immunology. wustl.edu 3 Abbreviations used in this paper: IS, immunological synapse; HEL, hen eggwhite lysozyme; SLC, secondary lymphoid tissue chemokine; SDF, stromal cell-derived factor; MIP, macrophage inflammatory protein; IP-10, IFN-inducible protein-10; MDC, macrophage-derived chemokine; MCP, macrophage chemoattractant protein. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 ● ●