Proc. Natl. Acad. Sci. USA Vol. 94, pp. 1925–1930, March 1997 Immunology The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes CONRAD C. BLEUL*, LIJUN WU†,JAMES A. HOXIE‡,TIMOTHY A. SPRINGER*, AND CHARLES R. MACKAY†§ *The Center for Blood Research and Harvard Medical School, Department of Pathology, 200 Longwood Avenue, Boston, MA 02115; †LeukoSite, Inc., 215 First Street, Cambridge, MA 02142; ‡Hematology–Oncology Division, Clinical Research Building, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104 Contributed by Timothy A. Springer, December 13, 1996 ABSTRACT The chemokine receptors CXCR4 and CCR5 ing the heart, and in B lymphocyte development, and its function as coreceptors for HIV-1 entry into CD41 cells. knockout is lethal (12). SDF-1 signals through a seven- During the early stages of HIV infection, viral isolates tend to transmembrane-domain receptor previously known as use CCR5 for viral entry, while later isolates tend to use LESTRyfusin, now designated CXC-chemokine receptor 4 CXCR4. The pattern of expression of these chemokine recep- (CXCR4) (13, 14). tors on T cell subsets and their regulation has important The importance of chemokine receptors for HIV entry and implications for AIDS pathogenesis and lymphocyte recircu- AIDS pathogenesis has been illustrated by numerous publi- lation. A mAb to CXCR4, 12G5, showed partial inhibition of cations over the last year. Before its identity as a chemokine chemotaxis and calcium influx induced by SDF-1, the natural receptor was known, CXCR4 was shown to mediate entry of ligand of CXCR4. 12G5 stained predominantly the naive, T cell line-tropic (T-tropic) HIV-1 strains (15), a process that unactivated CD26low CD45RA1 CD45R02 T lymphocyte sub- subsequently was shown to be inhibited by SDF-1 (13, 14). The set of peripheral blood lymphocytes. In contrast, a mAb CC-chemokine receptors CCR5, and to a lesser extent CCR3 specific for CCR5, 5C7, stained CD26high CD45RAlow and CCR2b, mediate entry of macrophage-tropic (M-tropic) CD45R01 T lymphocytes, a subset thought to represent pre- viral strains (16–20). The importance of chemokine receptors viously activatedymemory cells. CXCR4 expression was rap- in HIV-1 pathogenesis is underscored by the observation that idly up-regulated on peripheral blood mononuclear cells dur- individuals deficient in CCR5 and peripheral blood mononu- ing phytohemagglutinin stimulation and interleukin 2 prim- clear cells (PBMC) from these individuals are resistant to ing, and responsiveness to SDF-1 increased simultaneously. infection by HIV-1 (19, 21–24). CCR5 expression, however, showed only a gradual increase Thus, the expression of chemokine receptors and their over 12 days of culture with interleukin 2, while T cell regulation is thought to influence lymphocyte migration, as activation with phytohemagglutinin was ineffective. Taken well as HIV infection and AIDS pathogenesis. Here, using a together, the data suggest distinct functions for the two specific mAb, we report on the expression of CXCR4 on receptors and their ligands in the migration of lymphocyte leukocytes and compare this expression with that of CCR5. subsets through lymphoid and nonlymphoid tissues. Further- Our findings show distinct expression patterns as well as more, the largely reciprocal expression of CXCR4 and CCR5 differential regulation during phytohemagglutinin (PHA) among peripheral blood T cells implies distinct susceptibility stimulation and interleukin 2 (IL-2) priming of the two core- of T cell subsets to viral entry by T cell line-tropic versus ceptors on T lymphocytes. macrophage-tropic strains during the course of HIV infection. MATERIALS AND METHODS Chemokines and their receptors are postulated to direct migration of distinct leukocyte subsets to sites of inflammation Cells, Cell Lines, and Cell Culture. Human PBMC, neutro- and to their specific niches in lymphoid organs (1, 2). Lym- phils, and eosinophils were obtained from healthy donors as phocyte migration experiments in vivo have revealed prefer- described (10, 25). For lymphocyte stimulation experiments, freshly prepared PBMC were seeded into 24 well-plates (Corn- ential pathways of recirculation for certain subsets of T cells 6 (3). Among T lymphocytes, the naive CD45RA1 subset is the ing) at 2 3 10 cells per ml in RPMI 1640 medium with 10% predominant lymph node homing subset, whereas CD45R01 fetal calf serum containing either 150 unitsyml recombinant memory type T cells are the predominant population migrating IL-2 (kindly provided by Antonio Lanzavecchia, Basel, Swit- through peripheral tissues (1, 4, 5). Virtually all T cell che- zerland) or 2.5 mgyml PHA (Sigma). Half the volume was replaced with fresh medium supplemented with IL-2 and moattractants described to date selectively attract memoryy activated T cells (6–9). We recently have described a CXC- PHA, respectively, on days 6 and 9. The stably CXCR4- chemokine, SDF-1, that attracts unactivated, freshly isolated transfected Chinese hamster ovary cell line 1C2 was grown as peripheral blood lymphocytes with high efficacy (10). SDF-1 described (26). mAbs. attracts lymphocytes and monocytes, but not neutrophils, in A mAb to CXCR4, termed 12G5, was generated by vitro and in vivo (10). SDF-1 is unusually well conserved immunizing BALByc mice with Sup-T1 cells that were chron- ically infected with the SIVmac variant CP-MAC (27). mAbs between mouse and man (11), and its gene is located on to CCR5 were generated in C57 BL6 mice using CCR5- chromosome 10, while the other CXC- and CC-chemokines y transfected L1.2 cells (L.W. and C.R.M., unpublished results), cluster on chromosomes 4 and 17, respectively. SDF-1 func- two of which (5C7 and biotinylated 3D8) were used in this tions in the development of nonhematopoietic organs, includ- study. These mAbs specifically stain L1.2 cells transfected with CCR5 but not other CC-chemokine receptors. In addition, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: CXCR4, CXC-chemokine receptor 4; CCR5, CC- chemokine receptor 5; T-tropic, T cell line-tropic; M-tropic, macro- Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA phage-tropic; PBMC, peripheral blood mononuclear cells; PHA, 0027-8424y97y941925-6$2.00y0 phytohemagglutinin; IL-2, interleukin 2; PE, phycoerythrin. PNAS is available online at http:yywww.pnas.org. §To whom correspondence should be addressed. 1925 Downloaded by guest on September 25, 2021 1926 Immunology: Bleul et al. Proc. Natl. Acad. Sci. USA 94 (1997) these mAbs are unreactive against leukocytes from CCR5- deficient individuals. Phycoerythrin (PE)-conjugated mAbs to CD20 (L27), CD26 (L272), CD56 (MY31), CD69 (L78), CD45R0 (UCHL-1), and a peridinin-chlorophyll-protein (PerCP)-conjugated mAb to CD3 (SK7) as well as PE- and PerCP-labeled control antibodies were obtained from Becton Dickinson. A PE-conjugated mAb to CD45RA (MEM56) was purchased from Caltag Laboratories (Burlingame, CA). Three-color flow cytometry was carried out by incubating 2 3 105 cells in PBSy1% BSAy5 mM EDTA for 30 min with supernatant or purified antibody at a final concentration of 5 mgyml. Cells were washed, incubated with a 1:50 dilution of a fluorescein isothiocyanate-conjugated goat anti-mouse Ig F(ab9)2 (Caltag Laboratories) for 20 min and washed again before incubation with 10% normal mouse serum in PBS to block unoccupied sites on the goat anti-mouse Ig F(ab9)2. Finally, the appropriate directly conjugated mAb(s) were added. Cells were washed and analyzed using a FACScan (Becton Dickinson). Fluorescence intensity of chemokine receptor-expressing cells was quantitated in terms of number of molecules of equivalent soluble fluorochrome (see Table 1) using Quantum 26 beads (Flow Cytometry Standards, San Juan, PR) according to the manufacturer’s recommendations. Chemokines and Chemotaxis Assay. Synthetic full-length human SDF-1a-(1–67) and its N-terminal truncation form SDF-1a-(6–67) (10, 13) were a kind gift of Ian Clark-Lewis (Vancouver, Canada). MIP-1b was purchased from Genzyme. Chemotaxis assays were carried out as described (10, 13) with the exception that PBMC were migrated for 2 h. Briefly, 5 3 FIG. 1. The CXCR4-specific mAb 12G5 partially blocks SDF-1- 105 PBMC or T cell blasts in 100 ml of RPMI 1640 medium induced cellular responses. (A) mAb 12G5 blocks the majority of containing 0.25% human serum albumin were transmigrated SDF-1-induced lymphocyte chemotaxis. Freshly isolated PBMC were through 5-mm pore-size bare filter Transwell inserts (Costar). preincubated with the indicated concentrations of mAbs to CXCR4 Migrated cells were counted by FACS analysis scatter-gating (12G5) and CCR3 (7B11) for 15 min and subsequently added to the top chamber of bare filter Transwell inserts. SDF-1 at the optimal on lymphocytes. For antibody inhibition, cells were incubated concentration of 1.5 mgyml was added to the bottom chamber. for 15 min with differing concentrations of mAb to CXCR4 Transmigrated cells were counted by flow cytometry scatter-gating on (12G5) or CCR3 (7B11) before addition to the top chamber of the lymphocytes. Results are shown as percentage of input. The the chemotaxis assay. Chemotaxis then was carried out in the experiment shown was representative of four independent experi- presence of mAb to an optimal concentration of 1.5 mgyml ments. (B) 12G5 partially blocks SDF-1-induced increases in intra- SDF-1a-(1–67). cellular calcium. CXCR4 stably transfected CHO cells (1C2) loaded Calcium Fluorimetry. Measurement of intracellular calcium with fura-2 AM were exposed to 1 mgyml of full-length SDF-1a-(1–67) was carried out as described (13). Briefly, CXCR4 stably or truncated inactive SDF-1a-(6–67) (negative control) in the pres- transfected CHO cells, termed 1C2, were loaded with fura-2 ence (lower reading) or absence (upper reading) of 10 mgyml 12G5 mAb.