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The Transient Expression of C-C Chemokine Receptor 8 in Thymus Identifies a Thymocyte Subset Committed to Become CD4⁺ Single-Positive T Cells¹

Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Developing T cells journey through the different thymic microenvironments while receiving signals that eventually will allow some of them to become mature naive T cells exported to the periphery. This maturation can be visualized by the phenotype of the developing cells. CCR8 is a -chemokine receptor preferentially expressed in the thymus. We have developed 8F4, an anti-mouse CCR8 mAb that is able to neutralize the ligand-induced activation of CCR8, and used it to characterize the CCR8 protein expression in the different thymocyte subsets. Taking into account the intrathymic lineage relationships, our data showed that CCR8 expression in thymus followed two transient waves along T cell maturation. The first one took place in CD4^- CD8^- double-negative thymocytes, which showed a low CCR8 expression, and the second wave occurred after TCR activation by the Ag-dependent positive selection in CD4⁺ CD8⁺ double-positive cells. From that maturation stage, CCR8 expression gradually increased as the CD4⁺ cell differentiation proceeded, reaching a maximum at the CD4⁺ CD8^- single-positive stage. These CD4⁺ cells expressing CCR8 were also CD69^high CD62L^low thymocytes, suggesting that they still needed to undergo some differentiation step before becoming functionally competent naive T cells ready to be exported from the thymus. Interestingly, no significant amounts of CCR8 protein were detectable in CD4^- CD8⁺ thymocytes. Our data showing a clear regulation of the CCR8 protein in thymus suggest a relevant role for CCR8 in this lymphoid organ, and identify CCR8 as a possible marker of thymocyte subsets recently committed to the CD4⁺ lineage.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemokines are a family of structurally related, small chemoattractant proteins that interact with specific seven-transmembrane, G protein-coupled receptors (1, 2, 3, 4). Initially considered proinflammatory proteins, it has now become evident that chemokines also have other roles, such as the control of angiogenesis (5), regulation of hemopoietic progenitors (6), control of tumor growth (7, 8), fetal development (9, 10, 11), and HIV infection (12).

Chemokines act on different types of leukocytes regulating their trafficking. From a functional point of view, chemokines can be divided into inflammatory and homeostatic proteins. Inflammatory chemokines are those ones strongly up-regulated by inflammatory and immune stimuli in affected tissues, whereas homeostatic chemokines are produced constitutively and seem to be implicated in the control of the constitutive trafficking of leukocytes. Using chemokine receptor null mice, two clear examples of the in vivo role of homeostatic chemokines have recently been shown. B cells from mice deficient in CXC chemokine receptor 5 (CXCR5)³ fail to migrate from the T cell-rich zone into B cell follicles in the spleen, and these mice lack inguinal lymph nodes and have few, and phenotypically abnormal, Peyer’s patches (13). These results strongly suggest an important role for CXC chemokine receptor ligand 13 (CXCL13) (B cell attracting chemokine 1/B lymphocyte chemoattractant), the CXCR5 ligand (14, 15), in the control of B cell migration to defined positions within lymphoid organs. The second example was provided by studies performed with CCR7-null mice. These animals have profound morphological alterations in all secondary lymphoid organs, as a consequence of an impaired migration of lymphocytes and activated dendritic cells (16). Again, these results underscore the importance of CCR7 ligands, CCL19 (macrophage-inflammatory protein (MIP)-3/EBI 1 ligand chemokine) (17) and CCL21 (6 conserved-cystine chemokine/secondary lymphoid tissue chemoattractant) (18, 19), in controlling cell migration processes to and through secondary lymphoid organs.

CCR8, the receptor for CCL1 (human I-309/mouse T cell activation gene 3 (TCA3)) (20, 21, 22, 23), might have a constitutive role in the thymus, where it is preferentially expressed (22, 23, 24, 25). T cell maturation in thymus is a process by which thymocytes bearing the appropriate TCR are selected by a complex process that requires their controlled migration through the different thymic compartments (26, 27). Different reports implicate some thymus-expressed chemokines and their receptors in the control of this intrathymic migration of maturing thymocytes (28, 29, 30). The special features of CCR8 prompted us to study in more detail its expression in thymus. Using 8F4, a neutralizing anti-mouse CCR8 mAb we have developed, we have characterized those thymocyte subsets expressing CCR8. Our findings show that CCR8 expression is modulated along the process of thymocyte maturation, with two transient expression waves. The first one takes place in CD4^- CD8^- double-negative (DN) thymocytes, which showed a relatively low CCR8 expression, whereas the second wave occurs after the TCR activation by the Ag-dependent positive selection in CD4⁺ CD8⁺ double-positive (DP) cells. From that maturation stage, CCR8 expression increases along the pathway leading to CD4⁺ single-positive (SP) cells, in which CCR8 expression is maximal. Conversely, no significant amounts of CCR8 protein were found in CD8⁺ SP thymocytes. These results suggest a relevant role for CCR8 in T cell maturation, and the possibility of using this -chemokine receptor as a marker for the identification of thymocyte subpopulations recently committed to the CD4⁺ lineage.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Primary cell isolation

Thymi were obtained from 4- to 6-wk-old BALB/c or C57BL/6 mice; the organs were gently disrupted and filtered through nylon mesh to remove aggregates. Thymocytes were depleted of CD8⁺ cells using an anti-CD8 FITC Ab and paramagnetic anti-FITC MACS microbeads, and MACS separation columns (Miltenyi Biotec, Auburn, CA). The cell fraction isolated was <1% CD8⁺ in all experiments (data not shown). For the anti-CD3 plus anti-CD28 treatment, cells (2 x 10⁶ cells/ml) were cultivated in 24-well plates coated with 5 µg/well of hamster anti-mouse CD3 (clone 145-2C11; PharMingen, San Diego, CA); then, hamster anti-mouse CD28 mAb (clone 37.51; PharMingen) was added at 5 µg/well. Cells were collected at different times, washed, and stained for three-color experiments.

Cell lines and reagents

Human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown in DMEM supplemented with 10% FCS (Life Technologies, Paisley, U.K.) and antibiotics, at 37°C in a humidified 5% CO₂ atmosphere. The murine IL-3-dependent Ba/F3 cell line was obtained from Dr. D. Milligan (Arris Pharmaceuticals, San Francisco, CA) and cultured in RPMI 1640 with 10% FCS, 10% conditioned medium from the IL-3-producing cell line WEHI.3B, and supplements. The mouse thymic lymphoma BW5147 (TIB-47), the rat hybridoma YB2/0 (CRL-1662), and the murine myeloma P3X63Ag8.653 (CRL 1580) cells were obtained from the ATCC and cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% FCS (Eurobio, Paris, France), 1 mM sodium pyruvate, and 2 mM L-glutamine.

Plasmid constructs were prepared using the expression vector pCIneo (Promega, Madison, WI) and human CCR5, CCR6, and CCR8, and murine CCR6, CCR8, and CCR9 full-length coding sequences. The resulting plasmids were used to transfect Ba/F3 cells by electroporation. The expression of human and mouse chemokine receptors in those transfectants was verified with the appropriate Abs and/or calcium mobilization assays (data not shown). A 5'-end Myc-tagged version of mouse CCR8 cDNA was cloned in pCIneo, and stable YB2/0 transfectants were obtained after G418 selection of cells transfected with the resulting plasmid by electroporation. Recombinant mouse CCL1 (TCA3), CCL4 (MIP-1), CCL17 (thymus and activation-regulated cytokine), CCL19 (MIP-3), and CCL22 (macrophage-derived chemokine (MDC)), and human CCL1 (I-309), CCL2 (monocyte chemoattractant protein-1), and CXCL12 (stromal cell-derived factor (SDF)-1) were bought from PharMingen (mouse CCL1) and R&D Systems (Minneapolis, MN); mouse CCL20 (MIP-3) was chemically synthesized, as described elsewhere (31). The peptide SFLLR-amide was obtained from Bachem (Torrence, CA).

Generation of mAb specific to mouse CCR8

Rat mAb against mouse CCR8 were raised by i.p. immunization of 10-wk-old LOU rats with stable transfected irradiated (10 Gy) YB2/0 cells, expressing Myc-tagged CCR8 (20 x 10⁶ cells/0.3 ml of sterile PBS). Rats were boosted on days 30 and 60 with the same amount of cells. Serum from immunized rats was collected 7–10 days after each boost, and the presence of specific Abs was tested in flow cytometry using CCR8-transfected HEK 293 cells; as a negative control, HEK 293 cells transfected with the void vector were used. Selected rats were boosted i.v. with 20 x 10⁶ cells in 0.2 ml sterile PBS on days -3 and -2 before fusion of spleen lymphocytes with the P3X63Ag8.653 murine plasmacytoma, using polyethylene glycol 4000 (Merck, West Point, PA) essentially as described (32, 33). Two weeks after fusion, culture supernatants were screened in flow cytometry for the presence of CCR8-specific Abs using the CCR8-transfected HEK 293 cells. Positive supernatants were confirmed by flow cytometry using BW5147 cells and mock-transfected cells. Positive hybridomas were cloned by limiting dilution. mAb were produced in tissue culture supernatants and in ascites fluids induced in Pristane (Sigma, St. Louis, MO)-primed irradiated (4.5 Gy) BALB/c mice (34). mAb were partially purified from ascites fluid by ammonium sulfate precipitation (33) or from tissue culture medium by affinity chromatography using protein G-Sepharose (Pharmacia, Piscataway, NJ). The mAb isotypes were determined by radial immunodiffusion using a rat mAb-typing kit (ICN Pharmaceuticals, Costa Mesa, CA); the selected Ab 8F4 is IgG2b. Purified 8F4 was biotinylated using sulfo-NHS-LC-biotin (Pierce, Rockford, IL).

Flow cytometry

In this study, the following murine mAb from PharMingen were used: rat anti-mouse CD4 FITC and tricolor (H129.19), CD5 FITC (53-7.3), CD8-R FITC and tricolor (53-6.7), CD24 FITC (M1/69), CD62L FITC (Mel-14), and CD90 FITC (53-2.1), and hamster anti-mouse CD69 FITC (H1.2F3), TCR FITC (H57-597), and CD3 FITC (145-2C11). In addition, streptavidin-PE from Southern Biotechnology Associates (Birmingham, AL), and rat anti-mouse CD4 Red-613 from Life Technologies were used. Biotinylated anti-CCR8 mAb 8F4 was used at 10 µg/ml. For staining, 1–10 x 10⁵ cells were centrifuged in V-bottom 96-well plates and washed with PBS containing 2% BSA, 2% FCS, and 0.05% sodium azide (PBSst). To avoid nonspecific binding, cells were preincubated with mouse IgG (40 µg/ml) for 20 min at 4°C, and the same concentration of IgG was present together with Ab during staining. The cells were incubated with primary mAb at 4°C for 40 min, washed twice with PBSst, and incubated for 30 min with streptavidin-PE. Four-color stainings were conducted with biotinylated anti-CCR8 mAb 8F4, followed by streptavidin-PE, anti-mouse CD4 Red-613, SpectraRed-labeled anti-mouse CD8, and FITC-labeled mAb. Two- and three-color experiments used biotin-labeled mAb 8F4, followed by streptavidin-PE. After blocking with rat Igs, labeled anti-CD were added. For competition experiments, cells were preincubated (40 min, 4°C) with chemokines before their staining with the biotin-labeled mAb 8F4. Control stainings with a biotinylated isotype-matched rat mAb were routinely performed throughout this study. Samples were analyzed in an EPICS XL flow cytometer (Coulter, Palo Alto, CA). Events corresponding to more than 2.5 x 10⁵ viable cells were collected for total thymus samples and for more than 5 x 10⁴ viable cells when CD8-depleted cells were studied.

Chemotaxis and calcium mobilization assays

Migration assays with thymocytes and BW5147 cells were performed in Transwell inserts (Costar, Cambridge, MA) with a 5-µm-pore diameter. Cells were resuspended in RPMI with 1% BSA and 25 mM HEPES, pH 7.3 (10⁷ cells/ml), and 100-µl aliquots were loaded into upper inserts. Samples of 0.2 nM mouse CCL1, prepared in 600 µl of the same medium, were placed in the lower wells. After 2-h incubation, inserts were removed and migrated cells were counted in an EPICS XL flow cytometer (Coulter). For the chemotaxis assays with HEK 293/CCR8 cells, a 48-well microchamber (Neuroprobe, Cabin John, MD) was used. Lower wells were loaded with 1 nM mouse CCL1 (27 µl/well), and cells (50 µl/well, 10⁶ cells/ml) were placed in the upper wells. Polyvinylpyrrolidone-free filters with 10-µm pores (Poretics; Osmonics, Livermore, CA), precoated for 2 h at 37°C with type VI collagen (Sigma), were used. The chamber was incubated for 5–6 h at 37°C in a humidified atmosphere with 5% CO₂. Cells in the migration assays were first preincubated (20 min, 4°C) with increasing amounts of the anti-mouse CCR8 mAb 8F4, or an isotype-matched irrelevant Ab. Quadruplicate wells were used for each point. A migration index was established as the ratio between cell number migrated in response to the chemokine/cell number migrated to buffer. This migration index was used to estimate the percentage of migration inhibition caused by the preincubation with the Abs. Variations in the intracellular concentration of calcium in stable transfected HEK 293/CCR8 cells were analyzed by fluorometry essentially as described elsewhere (22).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Derivation of mAbs that recognize mouse CCR8 specifically

To generate rat anti-mouse CCR8 mAbs, a Myc-tagged version of the mouse CCR8 cDNA was first cloned into pCIneo and then used to transfect rat YB2/0 cells. Stable transfectant clones expressing mouse CCR8 were selected by flow cytometry using an anti-Myc mAb (not shown), and one of them was used for rat immunization. The recognition specificity of the mAbs obtained was checked by flow cytometry analysis performed on mouse pro-B BaF/3 cells stably transfected to express different chemokine receptors. The mAb 8F4 recognized CCR8 on BaF/3 cells transfected with this receptor cDNA, whereas BaF/3 transfectant cells expressing the void pCIneo vector, human CCR6, or mouse CCR9 cDNAs were not stained by this mAb (Fig. 1). In addition, staining assays were performed with transfectant cells expressing mouse CCR6, and human CCR5 or CCR8, the latter being 71% identical with mouse CCR8 (22), all of them with negative results (data not shown). Additional control stainings using an isotype-matched nonrelevant rat mAb were performed on BaF/3 cells expressing CCR8, also with negative results (not shown).

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Rat mAb 8F4 specifically recognizes mouse CCR8. Murine Ba/F3 cells transfected with different human or mouse chemokine receptors were stained with the rat anti-mouse CCR8 mAb 8F4, and the results analyzed by flow cytometry. Ba/F3 transfectants expressing the following chemokine receptors were used: mouse CCR8 (bold histogram), mouse CCR9 (- - -), and human CCR6 () A control staining of Ba/F3 cells transfected with the void vector pCIneo (—) is also shown.

To investigate the possible use of the mAb 8F4 to interfere with CCR8 signaling, some activity assays were performed with a HEK 293 clone stably transfected with mouse CCR8 (HEK 293/CCR8) and BW5147 cells, a mouse thymic lymphoma cell line we have previously shown to express high amounts of CCR8 mRNA (22). Intracytoplasmic calcium mobilization assays with Indo-1-loaded HEK 293/CCR8 cells were conducted, incubating the cells with increasing amounts of the mAb 8F4 before their stimulation with 1 nM mouse CCL1. As shown in Fig. 2A, in the absence of 8F4, or the presence of 100 nM of an isotype-matched nonrelevant mAb, mouse CCL1 was able to provoke a wave of calcium mobilization on HEK 293/CCR8 cells. This calcium wave diminished when cells were preincubated with 1 nM 8F4, practically disappeared with 10 nM 8F4, and was completely abolished by a preincubation with 100 nM 8F4. Similar experiments in which the chemokine and the mAb were added simultaneously were also performed. Fig. 2B shows that the magnitude of the mouse CCL1-mediated calcium mobilization peak was reduced when the chemokine and the mAb 8F4 were added together, thus suggesting that some kind of competition between both types of molecules for CCR8 binding was taking place. This also happened when 100 nM 8F4 was added to the cells a few seconds before the addition of mouse CCL1; the mAb prevented the clear calcium mobilization that 65 nM mouse CCL1 would have otherwise provoked in the HEK 293/CCR8 cells (Fig. 2C). This effect of 8F4 was specific for CCR8 and, therefore, it was not detected in HEK 293 cells expressing other -chemokine receptor such as mouse CCR6, stimulated with mouse CCL20 (MIP-3), the CCR6 ligand (Fig. 2D). The neutralizing activity of the mAb 8F4 was also tested in migration assays using both the HEK 293/CCR8 transfectants and BW5147 cells. As shown in Fig. 3, preincubation of cells with increasing amounts of the mAb 8F4 was able to inhibit more than 80% their mouse CCL1-induced chemotaxis. As in the calcium mobilization assays, the CCL20-mediated migration of HEK 293 transfectants expressing CCR6 was not affected (not shown). As depicted in Figs. 2 and 3, the mAb 8F4 did not display any agonist activity.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. The mAb 8F4 blocks the mouse CCL1 (TCA3)-mediated calcium mobilization on HEK 293/CCR8 transfectant cells. A, Indo-1 AM-loaded HEK 293/CCR8 cells were preincubated for 20 min at 4°C with the indicated amounts of 8F4, prior to their stimulation with 1 nM mouse CCL1 (TCA3). As controls, incubations without any Ab (none) and an isotype-matched, irrelevant mAb (100 nM, irrel.) were performed. Additions of 1.4 µM SFLLR-amide were also conducted to check the cell ability to mobilize Ca2+ by a chemokine-independent mechanism. B, HEK 293/CCR8 cells were simultaneously added 1 nM mouse CCL1 (TCA3) and 100 nM 8F4, and their Ca2+ mobilization was monitored. C, The previous addition of 100 nM 8F4 to HEK 293/CCR8 cells prevents their ability to mobilize Ca2+ in response to 1 or 65 nM mouse CCL1. D, The 8F4-blocking effect on CCR8 signaling is specific. Indo-1 AM-loaded HEK 293/CCR6 transfectant cells were preincubated with or without 100 nM 8F4; then they were stimulated with 10 nM CCL20 (MIP-3), the CCR6 ligand, and the induced Ca2+ mobilization was registered. Arrows indicate the time of additions. Changes in Ca2+ were monitored as alterations in the F395/F>500 ratio. The data shown are representative of two experiments.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. The mAb 8F4 blocks the mouse CCL1 (TCA3)-induced migration of HEK 293/CCR8 transfectants and BW5147 T cell lymphoma. In vitro migration experiments were performed in the Boyden microchamber (HEK 293/CCR8 transfectants, circles), or Transwell inserts (BW5147 cells, squares). Before being set to migrate, cells were preincubated with the indicated amounts of 8F4 (filled symbols) or an irrelevant isotype-matched mAb (empty symbols). As chemoattractant, 1 nM (HEK 293/CCR8 transfectants) or 0.2 nM (BW5147 cells) mouse CCL1 (TCA3) was used. For every point, migration was done in quadruplicate. The data shown are representative of three experiments.

Previous analysis by Northern blotting showed that the highest expression of both human (24, 25) and mouse CCR8 (22) RNA was found in the thymus. Using the anti-mouse CCR8 mAb 8F4, we performed a more detailed study of the murine thymocyte subpopulations expressing CCR8 protein. Phenotypic analysis of total thymic cells showed that 2%–4% of them expressed CCR8, both in BALB/c and in C57BL/6 mouse strains. Next, flow cytometry analysis of CCR8 expression was conducted on different phenotypically well-defined thymocyte stages according to their expression of the CD4 and CD8 markers. Representative results obtained with thymocytes from BALB/c mice are depicted in Fig. 4. Following the intrathymic lineage relationships, we detected a low percentage of CCR8⁺ thymocytes in CD4^- CD8^- DN cells (3.8%, gate 1) that decreased in the CD4⁺ CD8⁺ DP thymocyte subpopulation (0.5%, gate 4); however, CCR8 expression was higher in CD4^low CD8^low cells (4.3%, gate 2), an intermediate subpopulation from which both CD4⁺ and CD8⁺ SP cells are derived. Interestingly, CCR8 levels increased dramatically in cells committed to the CD4⁺ lineage (gates 7–9), the highest expression of the CCR8 protein being among the CD4⁺ CD8^- cells (20.5%, gate 9). Conversely, the analysis performed on the CD8⁺ SP thymocytes showed practically no detectable expression of this receptor (gates 5 and 6). Analysis of CCR8 expression in thymocyte subpopulations from C57BL/6 mice was also done, with similar results (data not shown).

View larger version (44K): [in this window] [in a new window]   FIGURE 4. Analysis of CCR8 expression on different thymocyte subsets. Mouse thymocytes were stained in three-color assays with the anti-mouse CCR8 mAb 8F4, followed by anti-CD4 and anti-CD8. Different gates were established in the CD4/CD8 plot (A) as indicated, and the CCR8 expression of the gated thymocyte subpopulations was analyzed (B). Control stainings with an isotype-matched, irrelevant mAb were also performed (dotted line). Gates in A are indicated by numbers in the bottom right in B; percentages of cells expressing CCR8 are shown on the top right. The data shown are representative of more than 10 independent experiments.

To further substantiate the specificity of the signal that the mAb 8F4 was giving in thymocyte subsets, we studied the binding of mouse CCL1 to CCR8 in thymocytes and how it affected CCR8 recognition by the mAb 8F4. For this, total mouse thymocytes were preincubated (40 min, 4°C) with 100 nM mouse CCL1, stained in three-color experiments, and then analyzed for the binding of mAb 8F4 to their membranes. Fig. 5A depicts the results obtained with gated CD4⁺ SP and CD4^- CD8^- DN thymocyte subpopulations, showing that in both thymocyte subsets preincubation with mouse CCL1 prevented the 8F4 staining; indeed, the 8F4 signal decreased to the levels shown by the control isotype-matched irrelevant mAb. Because cell preincubations with mouse CCL1 were done at 4°C in the presence of sodium azide, the mouse CCL1 blocking of 8F4 binding to CCR8 could not be due to receptor internalization. As previously suggested by the results obtained with HEK 293/CCR8 transfectants and BW5147 cells, mouse CCL1 and 8F4 binding sites on CCR8 appear to be at least partially overlapping; therefore, once mouse CCL1 has occupied its binding site in CCR8, the epitope recognized by 8F4 seems not to be accessible. Alternatively, the binding of mouse CCL1 could provoke a conformational change affecting the 8F4 binding, and vice versa.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Mouse CCL1 (TCA3) blocks the binding of the mAb 8F4 to CCR8 in thymocytes. A, Total mouse thymocytes were preincubated (40 min, 4°C) with 100 nM mouse CCL1 (TCA3), and then stained with anti-CD4, anti-CD8 mAb, and the mAb 8F4. Gated CD4+ SP and CD4- CD8- DN thymocytes were analyzed for the presence of the CCR8 protein. The results obtained in control stainings with an isotype-matched irrelevant mAb, or the mAb 8F4 when cells were not preincubated with chemokines are also shown. B, Similar experiments were performed in which total thymocytes were preincubated with chemokines, as shown. Analysis of the CCR8 protein on the membranes of gated CD4+ SP thymocytes was then performed. Preincubations were done with 100 nM chemokines; for mouse CCL1, 1 nM was also tested (mCCL1*). Data are presented as percentage of inhibition of the 8F4 binding to thymocytes that were not preincubated with chemokines. The data shown are representative of three different experiments. C, In vitro migration experiments with total mouse thymocytes were performed in Transwell inserts to study the mouse CCL1-induced cell chemoattraction. Control mouse chemokines were used at 100 nM, except CCL25 that was used at 300 nM. The data shown are representative of four experiments.

Migration assays in Transwell inserts and the Boyden microchamber were performed with total thymocytes to study the mouse CCL1-mediated migration of these cells. As depicted in Fig. 5C, no mouse CCL1-induced chemoattraction was detected in the chemokine concentration range studied. Nevertheless, thymocytes were able to migrate toward control chemokine gradients of murine CXCL12, CCL17, CCL22, and CCL25, as expected. Similar experiments were performed with CD8-depleted thymocytes to enrich in CCR8-expressing cells, with identical negative results (not shown).

CCR8 expression is modulated concomitantly with the commitment to the CD4⁺ lineage, but it is not expressed in fully mature CD4⁺ cells

The results obtained on the expression of CCR8 in different thymocyte subsets prompted us to investigate in more detail variations in CCR8 expression along thymocyte maturation. Total thymocytes were stained in four-color assays, and gated subpopulations differing in their CD4 and CD8 levels were analyzed for their expression of CCR8 and different thymocyte maturation markers, such as TCR, CD3, and CD24. The same gates as in Fig. 4 were used. The results obtained (Fig. 6) showed that among CD4^- CD8^- DN cells their low CCR8 expression was mainly associated to TCR^- CD3^int/high CD24^int cells (gate 1). Concerning the CD4^- CD8⁺ SP thymocytes, practically no significant CCR8 expression was detected (gate 6). Interestingly, a modulation of CCR8 expression was evident in cells committed to the CD4⁺ lineage. CCR8 expression was already detected in CD4⁺ CD8^int cells (gate 7), increased in CD4⁺ CD8^low cells (gate 8), and was maximal in CD4⁺ CD8^- thymocytes (gate 9). Concomitantly with the CCR8 up-regulation, and in agreement with their progressive maturation stage, CD4⁺ thymocytes showed increased TCR and CD3, and decreased CD24.

View larger version (49K): [in this window] [in a new window]   FIGURE 6. Modulation of CCR8 expression within the CD4+ cell lineage. Total thymocytes were stained in four-color experiments, and gates on cell subsets showing different expression levels of CD4 and CD8 were established and numbered as in Fig. 4 (boxed numbers on the top). Gated subpopulations were analyzed for their CCR8 expression and that of different thymocyte maturation markers, as indicated. Control stainings with an isotype-matched irrelevant mAb instead of the 8F4 anti-CCR8 mAb were also performed (not shown), and this staining signal was taken as the reference for establishing the axis in the plots. Percentages of CCR8+ cells (shown in right quadrants) were estimated by deducing the control staining from the mAb 8F4 signal. The data shown are representative of at least three independent experiments.

View larger version (27K): [in this window] [in a new window]   FIGURE 7. The phenotype of the CD4+ SP thymocytes expressing CCR8 is not that of cells that have completed their maturation process. CD8+ cells were removed from total mouse thymocytes, as described in Materials and Methods. CD8-depleted thymocytes were then stained in three-color experiments, and gated CD4+ cells were analyzed for their expression of CCR8 and that of different thymocyte maturation markers, as indicated. Control stainings with an isotype-matched, irrelevant mAb were also performed (not shown). The data shown are representative of four different experiments.

Analysis of mRNA levels showed that CCR8 expression is mainly associated to activated polarized Th2 cells (23, 38). The expression of CCR8 mRNA in CD4⁺ thymocytes led Zingoni et al. (23) to postulate that this chemokine receptor may be part of a Th2-specific genetic program shared by CD4⁺ and Th2 cells. We studied whether the CD4⁺ thymocytes could up-regulate CCR8 expression when activated as described for Th2 cells (38). For this, CD8-depleted thymocyte samples were prepared and treated with anti-CD3 and anti-CD28 for different times. Then, gated CD4⁺ cells were examined for their CCR8 expression. As depicted in Fig. 8, the results obtained showed that, similarly to what was described for CCR8 mRNA in human Th2 cells (38), the activation treatment caused a transient CCR8 up-regulation. The increase of CCR8 protein on the membranes of CD4⁺ cells was moderate at 2 h, clearly increased by 4 h, and then decreased by 12 h. Consistent with the activation process induced by the anti-CD3 and anti-CD28 treatment, CD69 expression was increased, whereas that of CD62L decreased with time. Concerning the untreated thymocytes, a down-regulation of the CCR8 expression was clearly noticed by 8 h of culture.

View larger version (33K): [in this window] [in a new window]   FIGURE 8. Transient up-regulation of CCR8 expression in CD4+ thymocytes treated with anti-CD3 and anti-CD28. CD8-depleted thymocytes were prepared and treated with anti-CD3 and anti-CD28, as described in Materials and Methods. At the times indicated, gated CD4+ cells were analyzed for their CCR8 expression with mAb 8F4, and those of CD69 and CD62L (bold histograms). In parallel, untreated CD8-depleted thymocytes were also analyzed (shaded histograms). Control stainings with an isotype-matched, irrelevant mAb were also performed (not shown). The data shown are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The specific recognition of CCR8 by the mAb 8F4 has been studied in transfectant cells expressing different chemokine receptors, BW5147 thymoma cells, and thymocytes. Importantly, studies performed on thymocytes in which the 8F4 binding to CCR8 was competed with different chemokines clearly showed that this binding was only prevented by mouse CCL1, the CCR8 ligand (22). Human CCL1, which is able to act as a mouse CCR8 ligand (22), was also able to partially block the 8F4 binding. The mouse CCL1 and human CCL1 concentrations at which these effects were observed are consistent with the K_d reported for the binding of mouse CCL1 to mouse CCR8, and the human CCL1 competition of that binding (22). All together, the results obtained strongly support that 8F4 is a specific and neutralizing anti-mouse CCR8 mAb. Moreover, it can be concluded that the binding sites for mouse and human CCL1 on mouse CCR8 are at least partially overlapping the region where the 8F4 epitope is located. Alternatively, the binding of the chemokine to CCR8 could provoke a conformational change that prevents the binding of 8F4, and vice versa. CCL17 is a controversial CCR8 ligand (35, 36, 39). We have also tested CCL17 ability to compete with 8F4 for its binding to CCR8. Our results suggest that if CCL17 binds to CCR8, either it requires CCR8 domains different from those necessary for CCL1 binding, or the CCL17 binding affinity is clearly lower.

The specificity of 8F4 has allowed us to track the expression of CCR8 during T cell differentiation in the thymus. Our data show that CCR8 expression in thymus follows two transient waves along T cell maturation. The first one takes place in CD4^- CD8^- DN thymocytes, which showed a low CCR8 expression. These CCR8⁺ DN thymocytes were cells that have lowered their CD24 and up-regulated their CD3. The second wave occurs after the TCR activation by the Ag-dependent positive selection in CD4⁺ CD8⁺ DP cells. From that maturation stage, CCR8 expression increased along the pathway leading to CD4⁺ SP cells, in which CCR8 expression was maximal. The transition from a DP to a CD4⁺ CD8^- stage is accompanied by a gradual up-regulation of surface markers, such as TCR, CD3, CD5, CD69, and CD90 (40, 41). Then, SP thymocytes partially down-regulate CD24, CD69, and CD90, and modulate their CD62L levels from CD62L^low to CD62L^high before leaving the thymus as naive T cells (40). The data from the four-color staining assays analyzing the concomitant expression of CCR8 and the cited markers evidenced that CCR8 expression is gradually increased as the CD4⁺ cell differentiation proceeds. Conversely, no significant amounts of CCR8 protein were detectable in CD8⁺ thymocytes, thus suggesting that during the process of commitment to this lineage, the expression of this -chemokine receptor is shut off.

In addition, the data shown in Fig. 7, showing that the CD4⁺ cells expressing CCR8 are also CD69^high CD62L^low thymocytes, suggest that CD4⁺ cells expressing CCR8 still need to undergo some differentiation step(s) before becoming the functionally competent naive T cells that are exported from the thymus to the periphery, where CCR8 expression is practically lost. These results are consistent with the reported very low CCR8 expression in peripheral lymphoid organs, where CCR8 is most abundantly expressed by activated Th2-polarized cells (23, 38).

Presently, it is not known why only 20% of the CD4⁺ thymocytes express CCR8 (Fig. 4B). A possible explanation is that CCR8 expression is time restricted, creating a temporal window of expression through which all CD4⁺-committed cells have to pass. In this case, our results would indicate that, on average, 20% of the CD4⁺ thymocytes are expressing CCR8 at a given moment of their differentiation process. In relation with this idea, treatment of CD4⁺ thymocytes in a way that mimics Ag-dependent activation provoked a transient up-regulation of CCR8 that lasted only some hours, but affected most of the cells (Fig. 8).

The clear regulation of CCR8 expression in thymus suggests a relevant role for CCR8 in this lymphoid organ. Thymocyte precursors arrive to the thymus through the corticomedullary junction, migrate to the subcapsular region, and return to the thymic medulla, concomitantly with the fulfillment of their maturation program (26, 27). Reports on in vitro migration assays performed with total mouse thymocytes or different thymocyte subsets strongly suggest that some thymus-expressed chemokine/receptor pairs are implicated in the control of that intrathymic migration. In the mouse, this has been shown for CCL25 (TECK)/CCR9 (Refs. 28, 30 , and our unpublished observations), CXCL12 (SDF-1)/CXCR4 and CCL19 (MIP-3)/CCR7 (29, 30), and CCL21 (SLC)/CCR7 and CCL22 (MDC)/CCR4 (30). We have investigated whether the mouse CCL1/CCR8 axis plays a similar role in mouse thymocytes. Assays in which the chemoattraction of total or CD8-depleted thymocytes was studied repeatedly failed to detect any mouse CCL1-mediated migration of these cells, although they were able to migrate to control mouse chemokine gradients (Fig. 5C). Despite this, the data shown in Fig. 5, A and B, demonstrate that there is a specific binding of mouse CCL1 to CCR8 in thymocytes. There are several possible explanations to these results. First, it has been reported that chemokine receptor responses are not only regulated by receptor expression, but also by regulating the signaling (42). Indeed, examples of cells expressing a chemokine receptor that are unable to migrate in response to the specific ligand have previously been reported (43). Second, we are not aware of any previous report showing a mouse CCL1-induced chemoattraction of thymocytes, so it might be that the role of mouse CCL1/CCR8 in the thymus is different from promoting thymocyte migration. In this regard, mouse CCL1 has been reported to exert a specific protective effect on murine T cell lymphomas against dexamethasone-induced apoptosis (44). This is of special relevance, taking into account the regulated CCR8 expression in the thymus, where 90–95% of thymocytes are eliminated by apoptosis during T cell repertoire selection (45). The CCR8 signaling could be a protective mechanism participating in the developmental program leading some thymocytes to become CD4⁺ SP cells. In other cases, chemokines as CCL3 (MIP-1) have been reported to promote a strong adhesion reaction on cells expressing CCR1 and CCR5 (46). It could be speculated that in thymocytes expressing CCR8, such an activity could help to retain them in specific thymic niches until receiving signals appropriate for their maturation. Third, the in vivo role of CCR8 in thymus could be mediated by its interaction with another ligand, different from mouse CCL1. In this regard, it has already been commented that human CCL4 and CCL17, which are chemokines produced in the thymus, are claimed to be other CCR8 ligands (35, 36).

Interestingly, an anti-CD3 and anti-CD28 treatment of CD4⁺ thymocytes up-regulates CCR8 expression (Fig. 8), in a similar way to that described for CCR8 mRNA in human Th2 cells, when they were similarly stimulated (38). These data support the proposed role of CCR8 as a part of a Th2-specific genetic program shared by CD4⁺ thymocytes and Th2 cells (23). Human Tc2 cells generated from cord blood have also been reported to preferentially express a functional CCR8 after being stimulated (38). Because CD8⁺ thymocytes do not express significant amounts of CCR8, in this case the connection between thymocytes and peripheral CD8⁺ cells based on CCR8 expression is not as evident as for CD4⁺ cells. In any case, the reported preferential CCR8 expression on Th2 cells in the periphery suggests that this receptor, apart from its role in the thymus, is also an interesting potential target for the treatment of allergic diseases. The neutralizing ability of 8F4 could make this mAb useful in these investigations.

Summarizing, this study shows for the first time that the expression of CCR8 protein on the cell surface of mouse thymocytes is regulated and mainly restricted to cells committed to the CD4⁺ cell compartment. Great efforts have been recently devoted to the elucidation of the molecular mechanisms responsible for T cell commitment to one or the other lineage. In this regard, CCR8 may be a very useful marker for the identification of thymocyte subsets recently committed to the CD4⁺ lineage.

	Acknowledgments

 
We thank Dr. R. Villares for his critical reading of the manuscript. We also thank M. Lozano for excellent technical assistance, L. Gómez for help with animal care, and I. López-Vidriero for help with flow cytometry analysis.

	Footnotes

 
¹ The Departamento de Inmunología y Oncología was founded and is supported by the Spanish Research Council (Consejo Superior de Investigaciones Cientificas) and by Pharmacia. Í.G. is the recipient of a Fellowship from the Programa de Formación de Investigadores del Gobierno Vasco (Spain).

² Address correspondence and reprint requests to Dr. Gabriel Márquez, Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid Spain.

³ Abbreviations used in this paper: CXCR, CXC chemokine receptor; CCL, CC chemokine receptor ligand, CCR ligand; CXCL, CXC chemokine receptor ligand; DN, double-negative; DP, double-positive; HEK, human embryonic kidney; MDC, macrophage-derived chemokine; MIP, macrophage-inflammatory protein; SDF, stromal cell-derived factor; TCA3, T cell activation gene 3.

Received for publication July 13, 2000. Accepted for publication October 4, 2000.

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