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Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-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 CD69high CD62Llow 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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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.
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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 CD4low
CD8low 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).
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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.
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), CCL19 (MIP-3), and CCL22
(MDC). Total thymocytes were preincubated with chemokines, and the
effects on the 8F4 staining of gated CD4+ SP
cells were analyzed. The results (Fig. 5
B) showed that mouse
CCL1 was the chemokine that most strongly competed for 8F4 staining.
Indeed, 100 nM mouse CCL1 inhibited binding of 8F4 to CCR8 by 92%, and
1 nM mouse CCL1 inhibited by nearly 60%. Consistent with their
reported binding to mouse CCR8, 100 nM human CCL1 was able to inhibit
nearly a 54% of 8F4 binding. The rest of chemokines studied did not
alter significantly the CCR8 binding by 8F4, including CCL17.
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- CD3int/high
CD24int 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+
CD8int cells (gate 7), increased in
CD4+ CD8low 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.
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) showed that
CCR8+ cells were mainly
CD90high CD62Llow
TCRhigh CD69high
CD5high. Concerning CD24, its levels in mature SP
thymocytes are lower that in more immature cells; the
CCR8+ cells had the highest CD24 levels among the
CD4+ SP thymocytes. All together, these cells had
a phenotype that is not compatible with that of fully mature
CD4+ cells, ready to leave the thymus as naive T
cells. The same results were obtained when similar staining assays were
done with total thymocytes (not shown).
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 Kd 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 CD62Llow to
CD62Lhigh 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
CD69high CD62Llow
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.
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Acknowledgments |
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Footnotes |
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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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) A control staining of
Ba/F3 cells transfected with the void vector pCIneo (—) is also
shown.
