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Patterns of Chemokine Receptor Expression on Peripheral Blood T Lymphocytes: Strong Expression of CCR5 Is a Selective Feature of V2/V9 T Cells¹

Andrea Glatzel^2,*, Daniela Wesch^*, Florian Schiemann, Ernst Brandt, Ottmar Janssen^* and Dieter Kabelitz^3,*

^* Institute of Immunology, University of Kiel, Kiel, Germany; and Forschungszentrum Borstel, Laborgruppe Biologische Chemie, Borstel, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
T lymphocytes play an important role in the immune defense against infection, based on the unique reactivity of human V2V9 T cells toward bacterial phosphoantigens. Chemokines and their corresponding receptors orchestrate numerous cellular reactions, including leukocyte migration, activation, and degranulation. In this study we investigated the expression of various receptors for inflammatory and homeostatic chemokines on peripheral blood T cells and compared their expression patterns with those on T cells. Although several of the analyzed receptors (including CCR6, CCR7, CXCR4, and CXCR5) were not differentially expressed on vs T cells, T cells expressed strongly increased levels of the RANTES/macrophage inflammatory protein-1/-1 receptor CCR5 and also enhanced levels of CCR1–3 and CXCR1–3. CCR5 expression was restricted to V2 T cells, while the minor subset of V1 T cells preferentially expressed CXCR1. Stimulation with heat-killed extracts of Mycobacterium tuberculosis down-modulated cell surface expression of CCR5 on T cells in a macrophage-dependent manner, while synthetic phosphoantigen isopentenyl pyrophosphate and CCR5 ligands directly triggered CCR5 down-modulation on T cells. The functionality of chemokine receptors CCR5 and CXCR3 on T cells was demonstrated by Ca²⁺ mobilization and chemotactic response to the respective chemokines. Our results identify high level expression of CCR5 as a characteristic and selective feature of circulating V2 T cells, which is in line with their suspected function as Th1 effector T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemokines are a large family of low m.w. proteins that play important roles in leukocyte migration, activation, and degranulation. They are classified on the basis of structural features into major subclasses of CXC chemokines, where two of four conserved cysteines are separated by an amino acid X, and CC chemokines, where these two cysteines are located side by side. Minor subgroups of chemokines are characterized by the absence of two cysteines (C chemokine lymphotactin) or the presence of three amino acids between two cysteines (CX3C chemokines) (see Ref. 1 for review). Based on such structural features, a new systematic nomenclature for human chemokines has been proposed (2). The corresponding chemokine receptors are seven-transmembrane G protein-coupled receptors that share structural features and can be grouped according to the corresponding ligand into CXCR, CCR, CR, and CX3CR families. An alternative classification of chemokines and receptors is based on functional and physiological features and distinguishes between inflammatory (or inducible) and homeostatic (or constitutive) chemokines (3). Inflammatory chemokines are frequently up-regulated in nonlymphoid tissue under inflammatory conditions and are instrumental in the recruitment of effector T lymphocytes.

With regard to T lymphocyte biology, chemokines and their corresponding cellular receptors are involved in intrathymic T cell development (4, 5), in the orchestration of T-B cell interactions, as well as in the differentiation of effector T cells and the development of memory T cells (6, 7). Although naive T lymphocytes express CXCR4 and CCR7, various CCR and CXCR are differentially expressed on effector and memory T cells (6, 8). Coordinated expression of chemokine receptors has been associated with functionally distinct T lymphocyte subsets. In this respect, CCR5, CXCR3, and CCR1 have been found preferentially on Th1 cells producing IFN-, while polarized Th2 cells producing IL-4 frequently express the eotaxin (CCL11) receptor CCR3 (9, 10, 11, 12). Furthermore, subsets of memory T lymphocytes can be distinguished on the basis of their CCR7 expression. It appears that CCR7 is gradually lost as T cells differentiate from CCR7⁺ naive cells via CCR7⁺ lymph node-homing noneffector memory cells toward CCR7^- tissue-homing effector memory cells (7, 13). Importantly, the cell surface expression of some chemokine receptors is modulated by cytokines and Ag recognition via the TCR, suggesting that T cells might change their migration pattern after antigenic stimulation (14, 15). Moreover, it is quite clear that the correlation of a committed functional phenotype with a particular pattern of cell surface chemokine receptors is not absolute. CCR3 can thus be induced on polarized Th1 cells, and CCR5 can be induced on Th2 cells in the presence of an appropriate cytokine milieu (16).

Although the vast majority of mature T lymphocytes expresses a heterodimeric TCR, a small subset (1–5%) of peripheral blood T cells carries the alternative TCR (17). Major differences between and T cells concern the diversity of the TCR germline repertoire and the Ags recognized by the respective TCR molecules (see Ref. 18 for review). The majority (50–95%) of human peripheral blood T cells express a V2V9-encoded TCR, whereas T cells using other V/V elements are usually rare in peripheral blood, but constitute major T cell populations in other anatomical localizations such as the small intestine (17, 19, 20). V2V9 T cells recognize small phosphorylated molecules derived from bacterial metabolic pathways (phosphoantigens) that cannot be seen by T cells (17, 21, 22, 23, 24). These features together with results from in vivo studies in animal models suggest that T cells play an important and nonredundant role in the immune defense against infectious micro-organisms (17, 25). It is likely that T cells have additional functions, such as the immune surveillance of stressed cells and of certain tumor cells (17, 26, 27).

Although the expression and significance of chemokine receptors on T lymphocytes has been the subject of extensive studies, little information is available on the chemokine receptor expression of T cells. Functional studies with purified T cells and T cell clones indicate that human T cells migrate in response to CC chemokines such as monocyte chemoattractant protein 1 (MCP-1)⁴ (6) (or CCL2), RANTES (CCL5), macrophage inflammatory protein 1 (MIP-1 or CCL3), and MIP-1 (CCL4), but not in response to CXC chemokines IL-8 (CXCL8) or IFN-inducible protein 10 (IP-10 or CXCL10) (28). The expression of the corresponding chemokine receptors was not investigated in this study (28). More recently, it was shown by RNase protection assay that phosphoantigen-activated V2 T cell lines rapidly down-regulated their expression of CC chemokine receptors, most notably CCR5, following re-exposure to the synthetic phosphoantigen isopentenyl pyrophosphate (IPP) (29). In addition, CXCR3 expression was found on TCR-expressing thymocytes that migrated in response to the corresponding ligands IP-10 (CXCL10), monokine-induced by IFN- (or CXCL9), and IFN-inducible T cell -chemoattractant (or CXCL11) (30).

In this study, we present the first comparative analysis of chemokine receptor expression on peripheral blood and T lymphocytes. Our results reveal significant differences between circulating and T cells in their surface expression of certain chemokine receptors, most notably CCR5. We discuss these findings with respect to the migration properties and effector phenotype of peripheral blood T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lymphocyte populations

PBMC were isolated from buffy coats or from heparinized peripheral blood obtained from healthy adult donors by Ficoll-Hypaque density gradient centrifugation. and T cell clones were established from E-rosette-purified T cells (for clones) or from MACS-purified T cells by limiting dilution as previously described (19, 31). Briefly, T cells were seeded at 0.3 cells/well in 96-well microtiter plates in the presence of 2 x 10⁵ irradiated PBMC feeder cells, PHA (0.5 µg/ml), and IL-2 (10 U/ml). T cell clones were expanded in RPMI 1640 medium (Life Technologies, Karlsruhe, Germany) supplemented with L-glutamine (2 mM), antibiotics, 10% heat-inactivated FCS, and IL-2 and were restimulated every 2 wk with irradiated feeder cells and PHA as previously described (31). To investigate activation-induced modulation of chemokine receptor expression, PBMC or E-rosette-purified T cells (1 x 10⁶/ml) were stimulated for 24 h in 24-well culture plates with 1 µg/ml PHA, a 1/10.000 dilution of heat-killed Mycobacterium tuberculosis (M. tb.) H37Ra extract (32), 1 µg/ml LPS (L 2654 from Sigma-Aldrich, Deisenhofen, Germany), or 2 µg/ml IPP (Sigma-Aldrich) (33).

Flow cytometry

PBMC were stained with FITC-conjugated pan-TCR mAb (Endogen, Woburn, MA) or pan-TCR mAb (BD PharMingen, Heidelberg, Germany) and anti-human chemokine receptor mAb. All chemokine receptor Ab were used as PE conjugates with the exception of anti-CCR7 and anti-CXCR4, which were not directly fluorochrome labeled. PE-conjugated goat F(ab')₂ anti-mouse Ab (Caltag Laboratories, Burlingame, CA) was used as a second-step reagent to detect these primary Abs. We used the following anti-chemokine receptor mAb: CCR1, CCR2, CCR3, CCR6, CCR7, CXCR1, CXCR2, CXCR3, CXCR4, and CXCR5 (all from R&D Systems, Wiesbaden, Germany) and CCR5 (BD PharMingen). In addition, we used Tricolor-conjugated mAb against CD45RO and CD45RA (Caltag Laboratories). Appropriate fluorochrome-labeled isotype controls were included. The TCR V and V usage of established T cell clones was analyzed with appropriate mAb as previously described (19). All analyses were measured on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). Results are presented as histograms or dot plots of mean fluorescence intensity. In the latter case the background of isotype controls has been subtracted. Statistical analysis was performed using paired Student’s t test.

Ca²⁺ mobilization

Changes in the cytosolic free Ca²⁺ concentration in response to chemokine receptor ligands was visualized in Fluo-3/AM-loaded T cell clones by flow cytometry. Briefly, T cell clones (5 x 10⁶/ml) were incubated (25 min, 37°C) in HBSS containing 4 µm Fluo-3/AM (Molecular Probes, Leiden, The Netherlands). Afterward, T cell suspensions were diluted 1/5 in RPMI 1640 and incubated for an additional 30 min at 37°C. Cells were washed three times and resuspended (5 x 10⁶ cells/ml) in assay buffer (137 mM NaCl, 5 mM KCl, 1 mM Na₂HPO₄, 5 mM glucose, 1 mM CaCl, 0.5 mM MgCl₂, 10 mM HEPES, and 1 g/L BSA (pH 7.4)). Before each assay, 100 µl of the cell suspension was incubated for 3 min at 37°C in a thermoblock and subsequently stimulated with the following recombinant chemokines (500 ng/ml): RANTES, MIP-1, MIP-1, MIP-3, IL-8, and IP-10 (all from R&D Systems). For comparison, a mixture of human defensins hBD2 (PeproTech, Rocky Hill, NJ) and HNP1 and HNP2 (Sigma-Aldrich, Steinheim, Germany) was used each at a final concentration of 500 ng/ml.

Chemotaxis

Lymphocyte chemotaxis was measured using a 48-well Boyden’s chamber (NeuroProbe, Cabin John, MD). Chemokines were serially diluted in RPMI 1640 (without phenol red) containing 0.1% BSA, 0.9 mM CaCl₂, and 0.5 mM MgCl₂, and 30 µl of the respective solutions were added to the bottom wells of the chamber. These were covered with a polycarbonate membrane (pore size, 5 µm; Costar Nucleopore, Tubingen, Germany), and the top wells received 1 x 10⁵ T cell clone cells suspended in 50 µl RPMI 1640 supplemented with 0.1% BSA, 0.9 mM CaCl₂, and 0.5 mM MgCl₂. After incubation for 2.5 h at 37°C in an atmosphere containing 5% CO₂, the assay was stopped by replacing the cell suspension in the upper well with ice-cold medium for 10 min. Thereafter, fresh cold medium was added for another 10 min to completely detach migrated cells from the bottom of the filters. Then filters were removed, and the migrated cells were transferred from the bottom wells to a microtiter plate. Residual cells in the bottom wells received 20 µl medium, were lysed by adding 5 µl 1% Triton X-100 (v/v) for 10 min, and were combined with the cells transferred to the microtiter plate, and cell lysis was continued for 10 min. Fifty microliters of 0.01 M p-nitrophenyl--glucuronide (Sigma-Aldrich) in 0.1 sodium acetate buffer (pH 4) was added for 40 h at 37°C, and the enzymatic reaction was stopped by adding 100 µl 0.4 M glycine buffer (pH 10). The OD was determined at 405 nm in a microplate reader. The number of migrated cells was calculated from a standard of lysed cells run in parallel.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential expression of chemokine receptors on circulating and T lymphocytes

We analyzed the expression of a panel of CC and CXC chemokine receptors on circulating peripheral blood and T lymphocytes by two-color flow cytometry. The results of a representative experiment are shown in Fig. 1. Compared with T cells, the expression of several CC receptors (CCR1, CCR2, CCR3, CCR6) and CXC receptors (CXCR1, CXCR2, CXCR5) was higher on T cells of this healthy adult blood donor, while other receptors, such as CCR7 and CXCR4, were equally expressed on both T cell subsets. The most striking differences were observed for CCR5 and CXCR3, which were both strongly expressed on T cells, but only at low levels on T lymphocytes. Because the expression of chemokine receptors varies among individuals, we analyzed the expression on and T cells in nine additional healthy donors. The results of the 10 separate experiments are summarized for CCR in Fig. 2. As can be seen, the strongly increased expression of CCR5 compared with that on T cells is a general feature of peripheral blood T cells (p < 0.001). T cells also expressed increased levels of the MCP receptor CCR2 (p < 0.01) and the MCP/eotaxin-receptor CCR3 (p < 0.01; but note the different scales of mean fluorescence intensity in Fig. 2), while differences in CCR6 and CCR7 expression between and T lymphocytes were not statistically significant. T cells are composed of subpopulations with regard to CD4/CD8 expression and naive vs memory (CD45RA vs CD45RO) phenotype, while the vast majority of the dominant T cell population (V2V9) is double negative and CD45RO⁺ (34, 35). Therefore, we compared CCR5 expression on CD45RA and CD45RO subsets of CD4⁺, CD8⁺, and T cells. The results of a representative experiment are shown in Fig. 3A. As can be seen, CCR5 expression was primarily confined to the CD45RO⁺ and CD45RA^- subsets. The relative percentages of CCR5-positive cells within the CD45RA⁺ and CD45RO⁺ subsets of CD4⁺, CD8⁺, and T cells measured in eight healthy individuals are shown Fig. 3B. In all three T cell populations, greater percentages of CD45RO⁺ cells expressed CCR5 compared with CD45RA⁺ subsets. Most T cells were CD45RO⁺ and not CD45RA⁺ (see Fig. 3A); CCR5, however, was also expressed on the few CD45RA⁺ T cells.

View larger version (51K): [in this window] [in a new window]   FIGURE 1. Surface expression of chemokine receptors on peripheral blood and T lymphocytes. Freshly isolated PBMC from a healthy adult donor were stained with FITC-conjugated anti-TCR or anti-TCR mAb plus anti-chemokine receptor mAb (PE-conjugated or unconjugated, followed by PE-labeled goat anti-mouse Ab). Chemokine receptor expression was separately analyzed on gated (solid line) and T (dotted line) cells. Isotype controls are shown as the shaded histograms. The fluorescence intensity scale on the x-axis comprises four log steps.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. CCR expression on peripheral blood T cells. PBMC from 10 adult donors were stained as detailed in Fig. 1. Results are presented as the mean fluorescence intensity on gated and T cells. The mean fluorescence intensity of appropriate isotype control Ig was subtracted.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Three-color analysis of CCR5 expression on CD45RO+ and CD45RA+ subsets of T lymphocytes. PBMC from eight donors were stained with FITC-labeled anti-CD4, anti-CD8, or anti-TCR mAb plus TC-conjugated anti-CD45RA or anti-CD45RO mAb, plus PE-conjugated anti-CCR5 mAb. A gate was set on CD4+, CD8+, and T cells, respectively. A, Dot plot analysis of a representative experiment. B, The fraction of CCR5+ cells among the respective subsets of CD45RA+ and CD45RO+ subsets was calculated.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. CXCR expression on peripheral blood T cells. See Fig. 2 for explanation. The results of experiments with 10 donors are shown.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Three-color analysis of CXCR3 expression on CD45RA+ and CD45RO+ subsets of T lymphocytes. Staining and analysis were performed in analogy to CCR5. See Fig. 3 for details. The results of experiments with eight donors are shown.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Differential expression of chemokine receptors on peripheral blood T cell subsets. PBMC from five adult donors were stained with FITC-conjugated anti-TCR mAb (pan-, V1, V2) and anti-chemokine receptor mAb. Results are presented as the mean fluorescence intensity on gated T cells and V1+ or V2+ T cell subsets.

View larger version (24K): [in this window] [in a new window]   FIGURE 7. CD45RO+ T cells comprise both CCR7+ and CCR7- subsets. PBMC were stained with FITC-conjugated anti-TCR and TC-conjugated anti-CD45RO mAb plus PE-conjugated anti-chemokine receptor mAb. A gate was set on the CD45RO+ TCR+ cell population. The fractions of CCR5+, CXCR3+, and CCR7+ cells determined in eight individuals are shown.

Chemokine receptor expression is known to be modulated (up- or down-regulated) in response to cellular activation or ligand binding. To investigate the possible modulation of chemokine receptor expression on T cells in response to cellular activation, PBMC were stimulated with PHA, M. tb., or phosphoantigen IPP, and chemokine receptors were analyzed after 24 h on gated T cells. As illustrated in Fig. 8 (top), ex vivo expressed CXCR4 and CCR5 were strongly down-regulated by PHA, while other chemokine receptors with little expression on freshly isolated T cells, including CXCR1 and CXCR2, were up-regulated. We also analyzed chemokine receptor modulation on T cells in response to M. tb. and IPP, two well-known ligands for TCR-dependent recognition by V2V9 T cells (22, 32). Interestingly, there was a dramatic down-regulation of CCR5 on T cells in response to M. tb., whereas much less down-modulation was observed in the presence of IPP (Fig. 8, bottom). As also shown in Fig. 8, there was moderate modulation of other chemokine receptors on T cells in response to M. tb. and/or IPP, which, however, was clearly less pronounced than the effect of PHA and also was more variable when analyzed in different donors (marked with an asterisk in Fig. 8). The finding that M. tb. induced a much stronger CCR5 down-modulation than IPP on T cells when PBMC were used as responder cells suggested that the modulation triggered by M. tb. might be an indirect effect, in part due to the M. tb.-induced macrophage activation and subsequent cytokine and/or chemokine production by macrophages (36). To address this issue, we compared the CCR5 modulation on gated T cells when PBMC (containing 30% monocytes) or E-rosette-purified T cells were used as responder cells. As illustrated in Fig. 9, the V2V9 ligand IPP triggered comparable CCR5 down-modulation on T cells in both cases, as did a mixture of CCR5 ligands RANTES, MIP-1, and MIP-1. In contrast, M. tb. induced strong CCR5 down-modulation on T cells only when PBMC were used as responder cells and not with purified responder T cells, suggesting a significant contribution of M. tb.-activated macrophages. This was further supported by the finding that stimulation of PBMC with LPS triggered CCR5 down-modulation on T cells as efficiently as did M.tb., whereas LPS did not have any effect when purified responder T cells were used (Fig. 9). Moreover, the down-modulation of CCR5 on T cells in response to M. tb. or IPP stimulation of PBMC was completely inhibited by a mixture of neutralizing Ab against CCR5 ligands (anti-RANTES, anti-MIP-1, and anti-MIP-1), suggesting that CC chemokine production by macrophages (in response to M. tb.) and/or T cells (in response to IPP) was critically involved (Fig. 10).

View larger version (38K): [in this window] [in a new window]   FIGURE 8. Modulation of chemokine receptor expression on T cells by M. tb. extracts and phosphoantigen IPP. PBMC were cultured with PHA, M. tb., or IPP. After 24 h, cells were washed and stained with FITC-conjugated anti-TCR mAb plus anti-chemokine receptor mAb. Overlay histograms show the chemokine receptor expression of nonstimulated control (dotted line) and PHA/M. tb./IPP-stimulated cells (solid line) on the gated T cell population. Isotype controls are shown as shaded histograms. Comparable effects were seen in four additional experiments. Asterisks indicate variable results observed in individual experiments with different donors (up- or down-regulation of chemokine receptors).

View larger version (25K): [in this window] [in a new window]   FIGURE 9. Modulation of CCR5 on T cells. PBMC or E-rosette-purified T cells (E+) were cultured with M. tb., IPP (10 µg/ml), LPS (1 µg/ml), or a mixture of RANTES, MIP-1, and MIP-1 (50 ng/ml each). After 24 h CCR5 expression was analyzed on gated TCR+ cells.

View larger version (36K): [in this window] [in a new window]   FIGURE 10. Down-modulation of CCR5 is due to M. tb./IPP-induced chemokines. PBMC were stimulated with M. tb. (left) or IPP (right) in the absence or the presence of neutralizing mAb against CCR5 ligands (anti-RANTES, 10 µg/ml; anti-MIP-1, 5 µg/ml; and anti-MIP-1, 10 µg/ml) or irrelevant mouse Ig as indicated. After 24 h, CCR5 expression was analyzed on the gated TCR+ cells.

Functional chemokine receptor expression on T cell clones

In contrast to the uniformly strong ex vivo expression of CCR5 on V2V9 T cells, chemokine receptor expression was more variable on established T cell clones and varied with the activation status (not shown). To demonstrate that the cell surface chemokine receptors on T cells are functional, we measured the Ca²⁺ influx in Fluo-3/AM-loaded T cell clones in response to the corresponding ligands. The results of a representative experiment with a V1 clone are illustrated in Fig. 11. This clone expressed CCR5, CCR6, and CXCR3 (Fig. 11B) and responded to the respective ligands MIP-1, MIP-1, RANTES (for CCR5), MIP-3 (for CCR6), and IP-10 (for CXCR3) with rapid Ca²⁺ influx (Fig. 11A), whereas no Ca²⁺ influx was elicited by IL-8, in line with the absent expression of IL-8R CXCR1 and CXCR2. Comparable results were obtained with other T cell clones displaying different V/V TCR (not shown). In addition, T cells expressing the relevant chemokine receptors migrated in response to the corresponding ligands in a chemotactic assay, as illustrated for a representative CCR5⁺ and CXCR3⁺ V2V9 clone in response to RANTES in Fig. 12. A clear chemotactic response was also seen with IP-10, although the intensity of the response was more variable than that obtained for RANTES (not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 11. Ca2+ mobilization in T cell clone in response to chemokines. Ca2+ mobilization was measured in Fluo-3/AM-loaded T cell clone K937 expressing V1 paired with V3, as evidenced by anti-V mAb (19 ) in response to 500 ng/ml recombinant chemokines (A). The expression of the corresponding CCR and CXCR is shown in B, where isotype controls are displayed as shaded histograms.

View larger version (11K): [in this window] [in a new window]   FIGURE 12. Chemotactic response of T cell clone to RANTES. The V2V9 T cell clone D 768/3 expressing CCR5 and CXCR3 was stimulated with titrated concentrations of RANTES. Migration was monitored as detailed in Materials and Methods. The values shown are the mean ± SD of two experiments. Similar results were obtained with two additional and one T cell clone.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have investigated the expression of chemokine receptors on human T cells with two goals in mind. First, we aimed at a comparative analysis of chemokine receptor expression on peripheral blood vs T cells to determine whether major differences in the expression patterns exist. Secondly, we asked whether the expression of cell surface chemokine receptors on the dominant V2V9 T cell population would correlate with their previous classification as memory and Th1 polarized cells (based on CD45RO expression and cytokine pattern) (34, 41). Our results reveal striking differences in the expressed chemokine receptor repertoire between peripheral blood and T cells and their CD4/CD8 subpopulations. Despite substantial interindividual heterogeneity, T cells expressed increased levels of some of the analyzed CCR (CCR1, CCR2, CCR3), but most significantly of CCR5, while there was no significant difference in the expression of other CCR such as CCR6 and the CCL19/CCL21 receptor CCR7. Further analysis revealed that CCR5 was expressed on the vast majority of the CD45RO⁺ T cells as well as on most of the few CD45RA⁺ T cells. Although CCR5 was also preferentially expressed on the CD45RO⁺ compared with CD45RA⁺ subsets when CD4⁺ and CD8⁺ cells were investigated (Fig. 3), our results clearly identify T cells as the subset within ex vivo-analyzed CD45RO⁺ peripheral blood T cells with the highest fraction of CCR5-expressing cells. Most CD45RO⁺ T cells expressed CCR5 and CXCR3, while, on the average, <50% also expressed CCR7 (Fig. 7). On the basis of their CCR7 expression, memory T cells have been subdivided into effector memory (CCR7^-) and central memory (CCR7⁺) T cells (13), and CCR5 has been reported to be preferentially expressed on CCR7^- effector memory T cells. Our present results indicate that CCR5 is also expressed on at least subsets of CCR7^- CD45RO⁺ T cells. Functional studies with cell sorter-purified CCR5⁺ CD45RO⁺ T cells coexpressing, or not, CCR7 are required to define their functional phenotype in terms of the proposed effector memory and central memory classification (7, 13). In accordance with CCR5, a more detailed analysis of CXCR3 expression also revealed the strongest expression (in terms of the percentage of positive cells) within CD45RO⁺ (and the few CD45RA⁺) T cells compared with the respective subsets of CD4⁺ and CD8⁺ cells (Fig. 5). The expression of the RANTES/MIP-1/MIP-1 receptor CCR5 together with CCR1 and CXCR3 has been associated with polarized Th1 cells (9, 10, 11). Although the studies of deletion mutants indicate that CCR5 is not absolutely essential for Th1 function in vivo (43), the high level expression of CCR5 on ex vivo-analyzed T cells and the increased expression of CXCR3 clearly support the idea that circulating T cells are primed Th1-type effector cells. Importantly, our results show that it is the dominant population of V2V9 cells that strongly expresses CCR5 (and less so CXCR3), while the minor population of circulating V1 T cells does not significantly differ from T cells in this respect; both display rather low levels of CCR5 on their surface. Taken together, the strong expression of CCR5 reported in this study together with the known expression of CD45RO (34, 35) and the constitutive expression of serine esterase (40) all support the assumption that circulating V2V9 (but not V1) T cells in the peripheral blood of healthy adults are experienced cells, perhaps due to chronic exposure to microbial ligands, and are ready to rapidly respond to TCR-dependent ligand recognition by Th1-like cytokine production (41) and cytotoxic effector activity (44).

Although CCR5 was expressed on almost all V2 T cells, we observed a preferential expression of CXCR1 on the minor T cell subset of V1 cells, suggesting that the CXCR1 ligand IL-8 might preferentially act on this subpopulation. Although no chemotactic migration of T cells in response to IL-8 was observed in a previous study, the TCR V usage of the analyzed T cell populations (total peripheral blood T cells or established clones) was not reported (28).

The expression of chemokine receptors is modulated by cellular activation, which can result in up-regulation (45, 46, 47, 48, 49) or down-modulation (50). Not unexpectedly (50), CCR5 expression on ex vivo-analyzed T cells was dramatically down-regulated by activation of PBMC with PHA or M. tb. and less strikingly by exposure to IPP. We speculated that the difference between the effects of the T cell ligands M. tb. and IPP (Fig. 8) might be due to the fact that IPP is recognized exclusively by V2V9 T cells within the PBMC, while the M. tb. lysate used in this study also activates monocytes/macrophages (36). Indeed, our further analysis with E-rosette-purified T cells confirmed this assumption, because CCR5 down-modulation on T cells in response to M. tb. was much less dramatic when purified T cells were analyzed, while down-modulation induced by IPP was unchanged (which, in fact, was comparable to chemokine-induced down-modulation; Fig. 9). Moreover, LPS stimulation of PBMC, but not of purified T cells, also triggered CCR5 down-modulation on T cells, again suggesting that macrophages activated by M. tb. (or LPS) contribute to CCR5 down-modulation on T cells when unseparated PBMC are used as responder cells. Down-modulation of CCR5 by IPP (and M. tb.) involved the production of CC chemokines by T cells (and monocytes in the case of M. tb.) as it could be completely prevented by a cocktail of neutralizing anti-RANTES/MIP-1/MIP-1 mAb, well in line with the reported CC chemokine production of phosphoantigen-stimulated V2V9 T cells (29, 51). In addition to CCR5, other chemokine receptors were also up-regulated or down-modulated by cellular activation, but with considerable variation among individual donors, thereby excluding definitive conclusions about the significance of these observations.

To address the functionality of chemokine receptors on T cells, we measured Ca²⁺ influx and chemotactic migration in response to chemokines and observed a clear correlation between chemokine receptor expression and responsiveness to the respective ligands. Specifically, we observed Ca²⁺ mobilization with RANTES, MIP-1, and MIP-1 in established CCR5⁺ clones, with MIP-3 in CCR6⁺ clones, and with IP-10 in CXCR3⁺ clones, while no response was obtained with IL-8, in line with the absence of CXCR1 and CXCR2 on the analyzed T cell clones. In this regard our results extend a recent report showing that human thymic T cells expressing CXCR3 migrate in response to IP-10, monokine-induced by IFN-, or IFN-inducible T cell -chemoattractant, which all are produced by thymic epithelial cells (30), while no transendothelial chemotaxis of freshly isolated peripheral blood T cells in response to IP-10 was observed in a previous study (28) where chemokine receptor expression was not analyzed. The functional significance of CCR5 expression was further confirmed in our experiments by the demonstration that CCR5⁺ T cell clones showed a chemotactic response to RANTES. Similarly, CXCR3⁺ T cell clones migrated in response to IP-10, even though there was more variability than with the chemotactic response of CCR5⁺ cells to RANTES.

In conclusion, our studies have identified strong CCR5 expression as a selective feature of ex vivo-analyzed peripheral blood V2V9 T cells, which distinguishes these cells from NK cells and most other circulating T lymphocytes (52), including the majority of T cells and other subsets (V1) of T cells. Future studies will address the functional significance of high level CCR5 expression on ex vivo-isolated V2V9 cells by analyzing the consequences of MIP-1/MIP-1/RANTES binding at the level of cellular activation and signal transduction of V2V9 T cells.

	Footnotes

 
¹ This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ka 502/7-2).

² This work forms part of the Ph.D. thesis of A.G.

³ Address correspondence and reprint requests to Dr. Dieter Kabelitz, Institute of Immunology, University of Kiel, Michaelisstrasse 5, D-24105 Kiel, Germany. E-mail address: kabelitz{at}immunologie.uni-kiel.de

⁴ Abbreviations used in this paper: MCP, monocyte chemoattractant protein; IP-10, IFN-inducible protein 10; IPP, isopentenyl pyrophosphate; MIP, macrophage inflammatory protein; M. tb., Mycobacterium tuberculosis.

Received for publication September 24, 2001. Accepted for publication March 11, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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