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Leukosite Inc., Cambridge, Massachusetts 02142; and 
Millennium Biotherapeutics Inc., Cambridge, Massachusetts 02139
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–inducible protein 10 (IP-10) and monokine induced by IFN- (Mig). Although CCR5 was expressed on both Th1 and Th2 lines, it was absent in several Th2 clones and its expression was markedly influenced by interleukin 2. Chemokine receptor expression and association with Th1 and Th2 phenotypes was affected by other cytokines present during polarization. Transforming growth factor β inhibited CCR3, but enhanced CCR4 and CCR7 expression, whereas interferon 
inhibited CCR3 but upregulated CXCR3 and CCR1. These results demonstrate that chemokine receptors are markers of naive and polarized T cell subsets and suggest that flexible programs of chemokine receptor gene expression may control tissue-specific migration of effector T cells.
Abbreviations used: CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; IP-10, IFN-–inducible protein 10; Mig, monokine induced by IFN-; MIP, macrophage inflammatory protein; mRNA, messenger RNA; TARC, thymus and activation regulated chemokine.
It is well established that after activation, T lymphocytes acquire effector functions that are best exemplified by the pattern of cytokines produced (1, 2). Th1 effector cells produce IL-2 and IFN-
In addition to effector function, activated T lymphocytes acquire different migratory capacities, a fact that is the key to an efficient regulation of the immune response (3). The regulation of leukocyte migration is an intricate process involving the participation of adhesion molecules such as selectins and integrins (4, 5), as well as chemokines and chemokine receptors (6). The combined action of adhesion molecules and chemokines is thought to provide an address code for leukocyte migration to different sites (7, 8). Indeed, early work has identified markers for T cell subsets that preferentially migrate to distinct anatomical sites. Thus, T cells that home to skin express the cutaneous lymphocyte–associated antigen (9), whereas those homing to the gut express the β7 integrins (10). In addition to adhesion molecules, chemokine receptor expression has been shown to be important for selective leukocyte migration. For instance, expression of the CC chemokine receptor (CCR)31 on eosinophils and the IL-8 receptors on neutrophils allows these cells to migrate to sites where agonists such as eotaxin or IL-8 are produced (11, 12).
Presumably, chemokines and chemokine receptors are also important for the selective migration of T cells, especially subsets of effector T cells. Indeed, a strong relationship between migratory capacity and cytokine production is emerging. It was shown recently that Th1s, but not Th2s express a functional ligand for E- and P-selectin that allows these cells to migrate to sites of inflammatory reactions (13, 14). In addition, we have shown recently that the eotaxin receptor CCR3 is expressed on Th2s and not Th1s (15). These results suggest that migration and effector function are coordinately acquired as part of a common developmental program.
In this study, we demonstrate that distinct profiles of chemokine receptors are acquired by T cells after polarization and are modulated by cytokines. CCR3 and CCR4 are expressed exclusively on Th2s, whereas CXC chemokine receptor (CXCR)3 and CCR5 are preferentially found on Th0s and Th1s. In addition, TGF-β inhibits CCR3, but enhances CCR4 and CCR7 expression, whereas IFN-
Cells, T Cell Lines, and Clones.
FACS® Analysis.
Cytokine Detection at the Single Cell Level.
Ca2+ Flux Measurement.
RNase Protection Assay.
Northern Blot Analysis. and activate mononuclear phagocytes thus protecting against intracellular pathogens. In contrast, Th2 effector cells produce IL-4 and -5 and are involved in responses dominated by IgE, eosinophils and basophils. inhibits CCR3 and CCR4 but upregulates CXCR3 and CCR1. These results demonstrate flexible programs of chemokine receptor gene expression that best serve the complex requirements for tissue and microenvironment selective migration.
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Materials and Methods
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Media and Reagents.
The medium used throughout was RPMI 1640 supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% pyruvate, 50 µg/ml kanamycin (GIBCO BRL, Gaithersburg, MD), 5 x 10–5 M 2-mercaptoethanol (Merk, Darmstadt, Germany), and 5% human serum (Swiss Red Cross, Bern, Switzerland) or 10% FCS (Hyclone Laboratories, Inc., Logan, UT). Human recombinant IL-2 and -4 were produced in our laboratory by PCR cloning and expression in the myeloma expression system (16). Eotaxin, IFN-–inducible protein 10 (IP-10), monokine induced by IFN- (Mig), macrophage inflammatory protein (MIP)-1β, and thymus and activation regulation chemokine (TARC) were purchased from Peprotech (London, UK).
Blood samples were obtained from healthy volunteers and PBMCs were separated by the standard Ficoll-paque method. Cord blood T cells were stimulated with PHA (1 µg/ml; Murex Ltd., Dartford, UK) and rIL-2 (40 U/ml) under Th1-polarizing conditions (human rIL-12 [2 ng/ml; Hoffmann–La Roche, Nutley, NJ] plus neutralizing mAb to human IL-4 [200 ng/ml; PharMingen, San Diego, CA]) or Th2-polarizing conditions (human rIL-4 [200 U/ml] plus neutralizing mAb to human IL-12 [2 µg/ml; R&D Sys., Inc., Minneapolis, MN]). After 1 and 2 wk, the cultures were restimulated with PHA and irradiated allogeneic PBMCs (3,000 rad from a 137Cs source) in the same polarizing conditions and analyzed after a further 10 d. Cell lines were also generated from sorted CD4+ or CD8+ T cells. In some experiments, IFN- (1,000 U/ml; Hoffmann–La Roche) or TGF-β1 (2 ng/ml; R&D Sys., Inc.) were added to Th1- or Th2-polarizing cytokines. T cell blasts were cloned by limiting dilution and maintained by periodic restimulation with PHA, irradiated allogeneic PBMCs, and rIL-2 as previously described (17).
Cell staining was performed using mAbs followed by FITC- or PE-conjugated, affinity-purified, isotype-specific goat anti–mouse antibodies (Southern Biotechnology Associates, Inc., Birmingham, AL). For three-color analysis biotin-labeled goat anti–mouse antibodies were used followed by APC-conjugated streptavidin (Molecular Probes, Eugene, OR). The following mouse antibodies were used: anti-CXCR3 (1C6, IgG1; reference 18), anti-CCR3 (7B11, IgG2a; reference 19), anti-CCR5 (5C7, IgG2a; reference 20), anti-CXCR4 (12G5, IgG2a; R&D Sys., Inc.), anti-CD4 (OKT4, IgG2b and 6D10, IgG1; American Type Tissue Collection, Rockville, MD), anti– TCR-/β (BM 031, IgG2b), anti-CD45RA (ALB11, IgG1), and anti-CD45RO (UCHL1, IgG2a) (all from Immunotech, Marseille, France). Because of the need for three antibodies of different isotypes in the indirect three-color staining, we used anti-CD45RO or -CD45RA mAbs to distinguish between naive and memory T cells. The samples were analyzed on a FACSCalibur® (Becton Dickinson, Mountain View, CA) using propidium iodide to exclude dead cells.
T cells were stimulated with 10–7 M PMA plus 1 µg/ml ionomycin (Sigma Chemical Co., St. Louis, MO) for 4 h. Brefeldin A (10 µg/ml, Sigma Chemical Co.) was added during the last 2 h. Cells were fixed with 2% paraformaldehyde, permeabilized with phosphate buffered saline containing FCS (1%) and saponin (0.5%) and stained with FITC-labeled anti–IFN- (IgG1) and PE-labeled anti–IL-4 (IgG2b) mAbs (Becton Dickinson).
T cells were loaded with Indo-1 (Sigma Chemical Co.) and their response to various concentrations of chemokines was analyzed on a FACS Vantage® flow cytometer (Becton Dickinson) as described (21). In some experiments, fluxing and nonfluxing cells were isolated by cell sorting over a 1-min period and further analyzed for cytokine production.
Total RNA was extracted from T cells using RNAzol B (Tel-Test, Inc., Friendswood, TX). Multiprobe template sets hCR5 (containing DNA templates for CCR1, CCR3, CCR4, CCR5, CCR8, CCR2a+b, CCR2a, CCR2b, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and hCR6 (containing DNA template for CXCR1, CXCR2, CXCR3, CXCR4, Burkitt's lymphoma receptor (BLR)-1, BLR-2/CCR7, V28, and GAPDH) were purchased from PharMingen. The DNA templates were used to synthesize the -[32P]UTP (3,000 Ci/mmol, 10 mCi/ml; Amersham Life Science, Buckinghamshire, UK) –labeled probes in the presence of a GACU pool using a T7 RNA polymerase (Promega, Madison, WI). Hybridization with 5–15 µg of each target RNA was performed overnight followed by digestion with RNase A (Boehringer Mannheim, Rotkreuz, Switzerland) and T1 (Calbiochem, La Jolla, CA) according to the PharMingen standard protocol. The samples were treated by proteinase K (Boehringer Mannheim) –SDS mixture and then extracted with chloropan and precipitated in the presence of ammonium acetate. The samples were loaded on an acrylamide-urea sequencing gel next to labeled DNA molecular weight markers and to the labeled probes, and run at 50 watts with 0.5x Tris-borate/EDTA electrophoresis buffer (TBE). The gel was adsorbed to filter paper, dried under vacuum, and exposed on film (X-AR; Kodak, Rochester, NY) with intensifying screens at –70°C.
10–15 µg of total RNA were loaded on a RNA formaldehyde denaturing gel and run with 3-[N-morpholino]propane sulfonic acid (MOPS)/EDTA buffer for 3 h, followed by an overnight transfer to Genescreen membrane (New England Nuclear, Boston, MA) with 10x standard saline citrate (SSC). RNA was covalently fixed to the membrane by UV cross-linking using Stratalinker (Stratagene, La Jolla, CA). The membranes were hybridized with either full-length IL-12Rβ2 or with β-actin. The probes were labeled with -[32P]dCTP (Amersham) by random labeling (random primed DNA labeling kit; Boehringer Mannheim), purified on Biospin 30 spun column (BioRad Labs., Hercules, CA), heat inactivated, and quenched on ice before adding to the hybridization mixture. The hybridization mix was composed by 50% of deionized formamide, 5x Denhardt's, 5x SSC, 10 mM EDTA, pH 8.0, 1% SDS buffered with 25 mM NaH2PO4, and 25 mM Na2HPO4, and supplemented with 200 µg/ml heat denaturated salmon sperm DNA (Sigma Chemical Co.).
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Chemokine Receptor Expression on Naive and Memory T Cells in Peripheral Blood.
Using specific monoclonal antibodies, we analyzed the expression of chemokine receptors in T cells expressing different CD45 isoforms that identify naive or memory T cells (22; Fig. 1). CXCR4 was present at high levels on all cord blood T cells and on CD45RA+ adult naive T cells, whereas it was present at lower levels and only on a fraction (15–40%) of CD45RA– memory T cells. In contrast, CCR3, CCR5, and CXCR3 were absent in cord blood T cells and were expressed preferentially in adult peripheral blood T cells with memory phenotype. CCR3 was expressed on a small fraction (1–10%) of CD45RA– T cells that has been previously identified to comprise Th2s (15). CCR5 was expressed on 18–32% of CD45RA– T cells, whereas CXCR3 was expressed at high levels on a much larger fraction (58–73%) of CD45R0+ memory T cells. Interestingly, CXCR3 was also present on a population of CD3+ CD45R0– cells present in both cord blood and adult blood. In conclusion, CXCR4 is a major receptor in naive T cells, whereas CCR3, CCR5, and CXCR3 are selectively expressed on memory CD45R0+ RA– T cells.
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and TGF-β. and TGF-β, which have been shown to affect T lymphocyte polarization (23, 24), may modulate chemokine receptor expression when added to Th1- or Th2-polarizing conditions (Fig. 3).
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did not show any effect on Th1 polarization. However, when added to Th2-polarizing conditions, IFN- induced a shift from Th2 to Th1 as shown by IFN- production and by upregulation of the IL-12Rβ2 subunit, which is a marker of Th1s (23). Interestingly, this treatment also resulted in an upregulation of CXCR3 and inhibition of CCR3 expression. These results are compatible with the notion that IFN- is dominant over IL-4 and induces a complete switch to a Th1 phenotype characterized by expression of higher levels of CXCR3 and lower levels of CCR3.
T cells polarized under Th2 conditions in the presence of TGF-β completely lacked both IL-4 production and CCR3 expression in keeping with the described effect of this cytokine on Th2 development (24). However, this was not accompanied by a switch to Th1, since the cells generated did not produce IFN-, expressed low levels of CXCR3, and lacked the IL-12Rβ2 subunit.
Differential Response to Chemokines by Th1 and Th2 Lines.
Polarized T cell lines were tested for their capacity to respond to chemokines that selectively engage distinct receptors. Fig. 4 shows the percentage of T cells that undergo [Ca2+]i increase in response to various concentrations of chemokines. Low concentrations of the CXCR3 agonist IP-10 (25) triggered [Ca2+]i increase in Th1 lines, whereas they were ineffective on Th2 lines. Indeed, Th2 lines responded only to 10-fold higher concentrations of IP-10, with a lower percentage of fluxing cells and with a lower [Ca2+]i increase. These results indicate that Th1s have a higher capacity to respond to IP-10 (and Mig, data not shown) than Th2s, demonstrating that the level of receptor expression determines the sensitivity to the agonist. On the other hand, the CCR5-specific agonist MIP-1β (26, 27) showed the same effect on Th1s and Th2s, in keeping with the expression of comparable levels of this receptor on both cell lines. Finally, the CCR3 agonist eotaxin (28) triggered [Ca2+]i increase in Th2, but not Th1 lines. Interestingly, TARC, which has been reported to be a selective agonist for CCR4 (29), efficiently induced a [Ca2+]i increase in Th2, but not Th1 lines, suggesting that the cognate receptor might be selectively expressed on Th2s.
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and TGF-β, Respectively. (Fig. 6).
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showed changes in CCR3 and CXCR3 mRNAs that were consistent with those observed using mAbs.
Remarkably, CCR4 mRNA was expressed at high levels in Th2 lines and was virtually absent on Th1 lines. In addition, CCR4 was upregulated by TGF-β and downregulated by IFN-. Taken together with the selective response of Th2s to TARC, these data indicate that CCR4, like CCR3, is selectively expressed on Th2s. However, CCR4 is regulated differently from CCR3 in response to TGF-β.
Two receptors were expressed similarly on Th1s and Th2s, but were differentially modulated by IFN- and TGF-β. CCR1 was expressed at low levels in Th1s and Th2s polarized under standard conditions. Addition of IFN- resulted in a dramatic increase in CCR1 message, whereas addition of TGF-β resulted in an almost complete inhibition. In contrast, CCR7 was absent in Th1 and Th2 lines, but its expression was strongly upregulated by TGF-β.
Taken together, the above results indicate that IFN- and TGF-β can add further functionality to polarized T cells by modulating the expression of chemokine receptors.
Chemokine Receptor Expression on T Cell Clones.
A large panel of T cell clones were analyzed for IL-4 and IFN- production and for chemokine receptor expression. Three examples are shown in Fig. 7, A–C. In general, clonal populations showed a considerable heterogeneity in the pattern of cytokine production, since they comprised cells that did not make any cytokine as well as cells that made all the possible combinations of IFN- and IL-4. Indeed, even typical Th1 or Th2 clones contained a few cells that produced the reciprocal cytokine combination. The expression of CXCR3 and CCR3 on individual clones reflected their expression on polyclonal lines. CCR3 was expressed only by Th2 clones, whereas CXCR3 was expressed at higher levels on Th1 than on Th2 clones, with few exceptions (Fig. 7 D). In contrast, CCR5 expression on T cell clones was variable since the receptor was absent on several (but not all) Th2 clones (Fig. 7 E). In addition, CCR5 expression was influenced by the activation state and by the presence of IL-2. Indeed, IL-2 withdrawal for 4 d resulted in a complete loss of CCR5 expression, whereas CXCR3 or CCR3 were retained (data not shown).
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and TGF-β. CXCR4 is expressed at high levels on cord blood T cells and on adult peripheral blood CD45RA+ naive T cells, but is expressed at lower levels on adult CD45RA– memory T cells, and is even absent on some of them. In addition, while CXCR4 is expressed on short-term T cell lines and is even upregulated by stimulation with IL-2 and PHA (20), it was absent on most long-term T cell clones (data not shown). Taken together, these results suggest that CXCR4 is progressively lost after repeated antigenic stimulations.
We have previously shown that CCR3 is selectively expressed on Th2s (15), and in this work, we identify CCR4 as an additional Th2-specific marker. Th2 lines express high levels of CCR4 message and undergo [Ca2+]i increase in response to TARC, a selective ligand for CCR4 (29). In addition, T cell responsiveness to TARC can be used to directly isolate an almost pure population of Th2s from cell lines that contain mixed populations of Th1s, Th0s, and Th2s. Interestingly, CCR4 is also expressed on basophils, while CCR3 is expressed on both eosinophils and basophils (19, 30, 31). The sharing of chemokine receptors between Th2 and the effector cells of allergy is likely to represent a key mechanism for the generation of allergic reactions. CCR3 and CCR4 ligands may selectively attract not only eosinophils and basophils, but also Th2s that, upon activation by antigen, could provide a source of IL-4 and IL-5 required for activation and survival of the effector cells (15).
CXCR3 is selectively expressed on lymphocytes and binds the IFN-–induced chemokines IP-10 and Mig (25). We have shown that CXCR3 is present on most peripheral blood CD45R0+ T cells and is expressed at higher levels on Th0s and Th1s and at low levels on Th2s. The responsiveness to IP-10 and Mig, together with the expression of ligands for E- and P-selectin (13), may favor the migration of Th1 and Th0 cells to sites of inflammatory reaction dominated by IFN- production (32).
CCR5 is a receptor for regulated on activation normal T cell expressed and secreted (RANTES), MIP-1 and MIP-1β (27) and was expressed at comparable levels on Th1 and Th2 lines. However, when clones were analyzed, CCR5 appeared to be expressed at higher levels on Th1, whereas many Th2 clones were negative. One should note that CCR5 expression is influenced by the activation state of the T cells and, as other receptors (33), is upregulated by IL-2 (20). Thus, while CXCR3 is expressed as a stable marker of memory Th1s and Th0s, CCR5 reflects the activation state of the cells. This may be the reason why CCR5 is present on fewer peripheral blood memory T cells than CXCR3.
IFN- and TGF-β have a major influence on Th1/Th2 polarization and also affect the expression of chemokine receptors. It has been shown that in humans, IFN-, and similarly IL-12, induce signal transducer and activator of transcription 4 (STAT4) phosphorylation (34) and consequently dominantly induce Th1 differentiation (23). We have shown that when added to Th2-polarizing conditions (IL-4 plus anti–IL-12 antibodies) IFN- not only reverts the cytokine profile from Th2 to Th1, but also the chemokine receptor profile, since it prevents the acquisition of CCR3 and CCR4 and allows expression of high levels of CXCR3. In addition, IFN- upregulates CCR1 expression in all polarizing conditions, thus conferring additional properties to Th1s besides those induced by IL-12.
TGF-β has been reported to inhibit Th2 development and maintain T cells in a state close to naivety (24). We have shown that TGF-β indeed prevents the development of the IL-4–producing phenotype, as well as CCR3 expression. However, the expression of CCR4 is not inhibited, but rather enhanced by TGF-β, indicating that CCR3 and CCR4, although both expressed on Th2s, are differentially regulated. Thus, only CCR3 represents a reliable marker for Th2s, since it correlates precisely with the acquisition of the IL-4–producing phenotype. On the other hand, CCR4, although expressed on Th2s, does not appear to be a selective Th2 marker since it is also expressed on cells that are not capable of producing IL-4.
In addition to upregulating CCR4, TGF-β also upregulates CCR7, a receptor specific for EBV-induced gene 1 (EBI1) ligand chemokine (ELC)/MIP-3β (35). Furthermore, CCR7 was also found on CD45RA+ naive T cells by RNase protection (F. Sallusto, unpublished data). It is interesting to speculate that T cells activated under the aegis of TGF-β may be in a particular "seminaive" state characterized by low effector function combined with the capacity to migrate to secondary lymphoid organs in response to the cognate chemokines ELC/MIP-3β to complete their education.
Our studies suggest that chemokine expression will be a central factor controlling the type of T cell immune response in a tissue, for instance, Th1 or Th2. Expression of the CXCR3 ligands IP-10 or Mig would lead to a preferential Th1 recruitment, whereas expression of CCR3 or CCR4 ligands such as eotaxin and TARC would lead to preferential Th2 recruitment. The regulation of chemokine expression is still poorly understood. IP-10 and Mig are induced by IFN- (36) and IP-10 is found at sites of Th1 type immune responses and delayed-type hypersensitivity reactions (32). Eotaxin is induced in Th2 type immune responses, such as in allergic airway disease, but is largely absent from arthritic lesions (12; and Mackay, C.R., unpublished data).
Besides their role in leukocyte migration, some chemokine receptors such as CXCR4, CCR5, and CCR3 behave as coreceptors for HIV entry into target cells (37–40). The differential expression of these receptors on naive and polarized T cells will determine their susceptibility to be infected by viruses carrying different surface glycoproteins. It is interesting that the CC chemokine macrophage-derived chemokine (MDC) has been recently shown to be a powerful and broad spectrum inhibitor of HIV infection, raising the possibility that the cognate receptor, CCR4, plays a major role as a coreceptor for viral entry (41). We have shown that both CCR3 and CCR4 are expressed on Th2s and that CCR4 is upregulated on all cells by TGF-β. In addition, CCR4 is expressed also on peripheral blood memory T cells at high levels (F. Sallusto, unpublished data).
In conclusion, our data show that chemokine receptor expression on T cells is controlled by activation and cytokines present in the milieu and correlates with distinct effector function. The expression of chemokine receptors is not only a useful marker of polarized T cells and a tool to study T cell differentiation, but also a possible target to interfere with an immune response by inhibiting the recruitment of distinct functional subsets.
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Submitted: 6 November 1997
Revised: 13 January 1998
The present address of Charles R. Mackay is Millenium Biotherapeutics Inc., Cambridge, MA 02139.
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