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Keywords:

  • HTLV-1;
  • ATL;
  • Tax;
  • CCR9

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Adult T-cell leukemia (ATL) is a highly aggressive mature CD4+ T-cell malignancy that is etiologically associated with human T-lymphotropic virus Type 1 (HTLV-1). ATL is characterized by frequent infiltration of lymph nodes, spleen, liver, skin and gut. Previously, we and others have shown that the majority of ATL cases are strongly positive for CCR4, which may explain the frequent skin invasion of ATL. Here, we examined whether ATL cells express CCR9, which is involved in T-cell homing to the gastrointestinal tract. Human T cell lines carrying HTLV-1 consistently expressed CCR9 together with the HTLV-1-encoded transcriptional activator Tax. Although ATL cells freshly isolated from peripheral blood hardly expressed CCR9, ATL cells cultured for 1 day consistently expressed CCR9 in parallel with the upregulation of Tax. Induction of Tax by Cd2+ in JPX-9, a subline of Jurkat human T cell line carrying Tax under the control of metallothionein promoter, led to upregulation of CCR9. A luciferase reporter gene under the control of the CCR9 promoter was expressed by cotransfection of an expression vector for Tax or in Cd2+-treated JPX-9 cells. Furthermore, immunohistochemical staining demonstrated that ATL cells infiltrating gastrointestinal tract were frequently positive for CCR9. Collectively, CCR9 is inducible in ATL cells expressing Tax and may play a role in the gastrointestinal involvement of ATL. © 2006 Wiley-Liss, Inc.

Adult T-cell leukemia (ATL) is a highly aggressive malignancy of mature CD4+CD25+ T cells that is etiologically associated with human T-lymphotropic virus Type 1 (HTLV-1).1, 2, 3 HTLV-1 encodes a potent viral transactivator Tax that activates HTLV-1 long terminal repeat (LTR) and also induces the expression of various cellular target genes that include cytokines, cytokine receptors, chemokines, cell adhesion molecules and nuclear transcriptional factors.4 The net effect of Tax on HTLV-1-infected T cells is a strong promotion of cell proliferation and cell activation.4 However, ATL develops only after a long period of latency, usually several decades, during which progression is likely to occur through accumulation of multiple genetic alterations.5, 6, 7, 8 Thus, circulating ATL cells usually do not express Tax and are likely to have become independent of the growth-promoting effects of Tax.9, 10, 11 However, circulating ATL cells still express the Tax gene at low levels in most cases.12 Furthermore, there are reports showing that ATL cells in tissues express Tax mRNA.8, 13 Moreover, freshly isolated ATL cells readily upregulate the expression of Tax upon brief in vitro culture.13 Thus, Tax may still play a significant role in the pathophysiology of ATL cells.

ATL is notorious for frequent infiltration of leukemic cells into lymph nodes, spleen, liver, skin and gastrointestinal tract.1, 3, 8, 14, 15 Previous studies have shown possible roles of matrix metalloproteases (MMPs), growth factors and cell adhesion molecules in enhanced tissue infiltration of ATL.16, 17, 18, 19 Chemokines are a family of structurally related cytokines that induce directed migration of various leukocyte populations through interactions with a group of 7 transmembrane G protein-coupled receptors.20 It is now known that migration and tissue microenvironmental localization of lymphocytes are finely regulated by the expression of certain cell adhesion molecules and chemokine receptors.20, 21 Previously, we have shown that increased surface expression of CCR7, whose ligands CCL19 and CCL21 are abundantly expressed in the secondary lymphoid tissues,20, 21 positively correlates with lymphoid organ involvement of ATL.22 Furthermore, we have shown that the majority of ATL cases (>90%) are positive for CCR4.23 Given that CCR4 is known to be critically involved in skin-homing of memory T cells,20, 21 our results suggested a possible role of CCR4 in skin invasion of ATL cells.23 Subsequently, Ishida et al. have demonstrated a significant correlation of CCR4 expression with skin involvement and poor prognosis in ATL patients.24 Furthermore, CCR4 is known to be selectively expressed by Th2 cells and regulatory T cells.23 Thus, our results have suggested a predominant origin of ATL from Th2 cells or regulatory T cells.23 Indeed, a highly frequent expression of FOXP3 in ATL was subsequently reported.25, 26

ATL is also known to frequently invade the gastrointestinal tract,14, 15 even though the exact incidence has not been determined yet. CCR9 is known to be involved in T cell homing to the gastrointestinal tract.20, 27 In the present study, we have demonstrated that CCR9 is expressed by HTLV-1+ T cells and ATL cells expressing Tax and may play a role in the gastrointestinal involvement of ATL.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Cells

All HTLV-1+ T cell lines and adult T-cell leukemia (ATL)-derived cell lines were described previously.23 JPX-9 and JPX-M are the sublines of a human T cell line Jurkat that express HTLV-1 Tax and Tax-mutant (Tax-Arg63), respectively, under the control of the metallothionein gene promoter.28 Blood samples were obtained from ATL patients and healthy adult donors with a written informed consent. PBMC were isolated from heparinized venous blood samples using Ficoll-Paque (Amersham Biosciences, Piscataway, NJ) and Leucosep (Greiner Bio-One GmbH, Frickenhausen, Germany). Cells were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% (v/v) nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μM 2-ME (the culture medium). For PBMC, recombinant IL-2 was further added to the culture medium at 100 U/ml. This study was approved by the local ethical committee.

RT-PCR analysis

Total RNA was prepared from cells by using Trizol reagent (GIBCO-BRL, Gaithersburg, MD). RNA was further purified by using RNeasy (Qiagen, Hilden, Germany). Total RNA (1 μg) was reverse transcribed using oligo (dT)18 primer and SuperScript II reverse transcriptase (Gibco-BRL). Resulting first-strand cDNA (20 ng total RNA equivalent) and original total RNA (20 ng) were placed in a final volume of 20-μl buffer containing 10 pmol of each primer and 1 U of Ex-Taq polymerase (Takara Shuzo, Kyoto, Japan). The primers used were +5′-AAGAAGAACAAGGCGGTGAAGATG-3′ and −5′-AGGCCCCTGCAGGTTTTGAAG-3′ for CCR4; +5′-CACTGTCCTGACCGTCTTTGTCT-3′ and −5′-CTTCAAGCTTCCCTCTCTCCTTG-3′ for CCR9; +5′-AAAAAGCGGGTCACTCTATATGCTC-3′ and -5′-CCACTGCTACCTGGTACTCTGTTGT-3′ for CD25; +5′-ATCGGCTCAGCTCTACAGTTCCT-3′ and −5′-ATTCGCTTGTAGGGAACATTGGT-3′ for JPX-9 and JPX-M Tax; +5′-CCGGCGCTGCTCTCATCCCGGT-3′ and −5′-GGCCGAACATAGTCCCCCAGAG-3′ for ATL Tax29; +5′-GCCAAGGTCATCCATGACAACTTTGG-3′ and −5′-GCCTGCTTCACCACCTTCTTGATGTC-3′ for GAPDH. Amplification conditions were denaturation at 94°C for 30 sec (5 min for the first cycle), annealing at 60°C for 30 sec and extension at 72°C for 30 sec (5 min for the last cycle) for 38 cycles for CCR9, 35 cycles for CCR4 and CD25, 33 cycles for Tax and 27 cycles for GAPDH. Amplification products were subjected to electrophoresis on a 2% agarose and stained with ethidium bromide.

Flow cytometric analysis

FITC- and PE-labeled monoclonal anti-CCR9 (112509) were purchased from R&D Systems (Minneapolis, MN). FITC-labeled monoclonal anti-CD4 (MT310), PE-labeled monoclonal anti-CD25 (ACT-1) and unlabeled mouse IgG1 were purchased from DAKO Japan (Kyoto, Japan). Unlabeled monoclonal anti-CCR4 (1G1), unlabeled mouse IgG3 (isotype control) and streptavidin-allophycocyanin (APC) were purchased from BD Biosciences (Mountain View, CA). APC-labeled monoclonal anti-CD4 (13B8.2), FITC-labeled mouse IgG2a (isotype control) and PE-labeled mouse IgG1 (isotype control) were purchased from Beckman Coulter (Fullerton, CA). FITC-labeled sheep anti-mouse IgG F(ab′)2 was purchased from Sigma-Aldrich (St. Louis, MO). Biotin-labeled goat anti-mouse IgG3 F(ab′)2 was purchased from Southern Biotechnology Associates (Birmingham, AL). Monoclonal anti-HTLV-1 Tax (Lt-4) was described previously.30 PBMC prepared from ATL patients and normal donors were suspended in ice-cold PBS containing 2% FBS and 0.05% sodium azide (the staining buffer). The following steps were all conducted on ice. Cells were first treated with normal human serum to block Fc receptors for 30 min. After washing, cells were incubated for 30 min with a mixture of an FITC-labeled mAb, a PE-labeled mAb and an APC-labeled mAb. In some experiments, cells were stained with a nonlabeled primary mAb first and then with an FITC-labeled secondary antibody. After washing, cells were immediately analyzed on FACSCalibur (BD Biosciences), using appropriate gatings in comparison with isotype control Abs. Dead cells were gated out by staining with propidium iodide. Intracellular staining of Tax and CCR9 was performed as described previously.30

Confocal microscopy

For double staining of CCR4 and CCR9, cells were fixed with 4% paraformaldehyde in PBS. After washing, cells were blocked with normal human serum and normal goat serum. After that, cells were reacted with monoclonal anti-CCR4 (1G1) and then stained with Alexa Fluor 546-labeled goat anti-mouse IgG (Invitrogen, Carlsbad, CA). Then, the cells were treated with normal mouse serum. And then, cells were further stained with FITC-labeled monoclonal anti-CCR9 (112509). For double staining of Tax and CCR9, cells were blocked, fixed and permeabilized in PBS containing 7% normal goat serum and 0.2% saponin (Sigma-Aldrich) for 10 min at room temperature. Permeabilized cells were reacted with monoclonal anti-Tax (Lt-4)30 or isotype control. Then, cells were reacted with biotin-labeled goat anti-mouse IgG3 F(ab′)2 (Southern Biotechnology Associates, Birmingham, AL). And then, cells were stained with Alexa Fluor 546-labeled streptavidin (Invitrogen) and blocked with normal mouse serum. After that, cells were stained with FITC-labeled monoclonal anti-CCR9 (112509). Finally, cells were resuspended in ProLong Antifade (Invitrogen). Fluorescence images were taken on LSM-5 (Carl Zeiss, Jena, Germany).

Luciferase reporter assay

The expression vector for Tax (pHβPr.1-Tax MT2) and the control vector (pHβPr.1) were described previously.31 To generate a promoter–reporter construct, the 1-kb promoter region of human CCR9 gene32 was amplified from the genomic DNA by PCR using primers: +5′-CATCTCGAGACATCTCACAGGCAGACTTTCTAAAG-3′ and −5′-CATAAGCTTAGGCTTTGTGGGTTCTGAGCAGGCAG-3′. The amplification products were digested with XhoI and HindIII and cloned into XhoI-HindIII sites of the reporter plasmid pGL3-Basic (Promega, Madison, WI), generating pGL3-CCR9p. Jurkat cells were cotransfected with pGL3-CCR9p and pHβPr.1 or pHβPr.1-Tax MT2 using DMRIE-C (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. After 48 hr, luciferase activity was determined by using Luciferase Assay kit (Promega). Luciferase activity was normalized by total cellular protein determined by BCA protein assay kit (Pierce, Rockford, IL). For analysis of the CCR9 promoter activity in JPX-9 and JPX-M, cells were tranfected with pGL3-CCR9p using DMRIE-C as described earlier and then treated with 20 μM of Cd2+ for 3 days. Luciferase activity was determined as described earlier and then normalized by living cell numbers.

Chemotaxis assay

Recombinant human chemokines were purchased from R&D Systems. Chemoataxis assays were carried out using CHEMOTX chemotaxis chamber (Neuro Probe, Gaithersburg, MD) as described previously.33 After 4 hr at 37°C, the membrane was removed and a known number of counting beads (BD Biosciences) were added to lower wells. The content of each well was transferred to a polypropylene pointed-bottom tube. The beads and cells were pelleted by centrifugation at 200g for 5 min, resuspended in the staining buffer (see above) and stained with FITC-labeled monoclonal anti-CD4 (MT310), and PE-labeled monoclonal anti-CD25 (MT310) as described earlier. After washing, cells were analyzed on FACSCalibur. Migration of the CD4+CD25+ cells was shown as a percentage of input cells. All assays were done by in triplicate.

Immunohistochemistry

Archived biopsy tissue samples were used. Tissue sections were made from formalin-fixed and paraffin-embedded tissue blocks, and treated with microwave for 5 min 3 times in Target Retrieval Solution (DAKO). Sections were then incubated at 4°C overnight with affinity purified goat polyclonal anti-CCR9 (Capralogics, Hardwick, MA) or mouse monoclonal anti-CCR4 (KM-2160; Kyowa Hakko, Tokyo, Japan). Normal goat IgG (IBL, Gunma, Japan) or isotype-matched mouse IgG1 (DAKO) were used as negative controls. After washing, the sections were incubated with biotin-labeled rabbit anti-goat IgG or biotin-labeled horse anti-mouse IgG (both from Vector Laboratories, Burlingame, CA). Finally, sections were treated with Vectastain ABC/HRP kit (Vector) according to the manufacturer's instructions. Peroxidase enzymatic development was performed using 3,3′-diaminobenzidine (DAB) and H2O2, resulting in dark brown products in positive cells. Sections were counterstained with Gill's hematoxylin (Polysciences, Warrington, PA) before dehydration and mounted in nonaqueous mounting medium (Muto Pure Chemicals, Tokyo, Japan).

Results and discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

CCR9 plays an important role in T cell homing and localization in the gastrointestinal tract.20, 27 We first examined 5 HTLV-1-transformed T cell lines and 5 ATL-derived cell lines for expression of CCR9 and CCR4 by RT-PCR (Fig. 1a). HTLV-1-transformed T cell lines and ATL-derived cell lines consistently expressed CCR4 as reported previously.23 Even though at lower levels, these T cell lines also consistently expressed CCR9. We also confirmed that all these T cell lines expressed HTLV-1 Tax.

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Figure 1. RT-PCR analysis for expression of CCR4, CCR9 and Tax in HTLV-1+ T cell lines and primary ATL cells. RT-PCR analysis was carried out for CCR4, CCR9, Tax and GAPDH (loading control). Normal PBMC treated with phytohemagglutinin (PHA) for 3 days served as positive control (PHA-PBMC). The representative results from 2 separate experiments are shown. (a) 5 HTLV-1-transformed T cell lines (HTLV-1+) and 5 ATL-derived T cell lines (ATL). (b) PBMC samples from 3 normal donors (Normal cells) and 5 ATL patients (ATL cells) without (Day 0) or with 1 day culture (Day 1).

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We next examined expression of CCR9 and CCR4 mRNA in PBMC samples from ATL patients containing leukemic cells at high levels (Table I) and those from normal donors (Fig. 1b). As reported previously,23 PBMC samples from most ATL patients expressed CCR4 mRNA at high levels compared to those from normal donors without culture (Day 0), and no changes in CCR4 mRNA expression were seen after 1-day culture (Day 1). As for CCR9, PBMC samples from normal donors were virtually negative for CCR9 mRNA before (Day 0) or after 1-day culture (Day 1). Similarly, PBMC samples from ATL patients were mostly negative for CCR9 mRNA before culture (Day 0). However, ATL cells from all donors became positive for CCR9 mRNA after 1 day culture (Day 1) together with expression of Tax mRNA.13

Table I. Summary of ATL Patients from whom PBMC Samples were Obtained for RT-PCR and Flow Cytometric Analysis
CaseSexAgeType% ATL cells
1F42Chronic90
2F61Acute50
3M71Acute68
4F66Acute94
5F57Acute85
6M58Acute91
7M53Acute95
8M70Chronic91
9M57Acute50
10M68Chronic93

We next examined surface expression of CCR9 and CCR4 proteins in PBMC from normal donors (n = 3) and ATL patients (n = 5) (Table I). The representative results are shown in Figure 2a. Since ATL cells are known to coexpress CD4 and CD25, we examined the CD4+ fraction of PBMC for surface expression of CD25 and CCR9 or CCR4. PBMC from normal donors contained average (11.7 ± 1.4)% of CD4+ T cells that coexpressed CD25 and CCR4 without culture (Day 0), and the frequencies were slightly reduced upon 1 day culture (6.9 ± 2.0)% (Day 1). On the other hand, PBMC from ATL patients contained average (89.6 ± 9.3)% of CD4+ T cells that coexpressed CD25 and CCR4 at high levels without culture (Day 0) as reported previously,23 and there were no changes in these frequencies after 1 day culture (88.7 ± 12.3)% (Day 1). In the case of CCR9, average (1.0 ± 0.3)% of CD4+ T cells from normal donors coexpressed CD25 and CCR9 without culture (Day 0), and there were no significant changes after 1 day culture [average (0.5 ± 0.5)%; (Day 1)]. As for ATL patients, while average (3.6 ± 0.4)% of CD4+ T cells coexpressed CD25 and CCR9 before culture (Day 0), average (21.1 ± 13.4)% coexpressed CD25 and CCR9 after 1 day culture (Day 1). This was consistent with the expression of CCR9 mRNA in ATL samples after 1 day culture (Fig. 1).

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Figure 2. Flow cytometric analysis and confocal microscopy of PBMC derived from normal donors and ATL patients. (a) Flow cytometric analysis: PBMC samples from normal healthy donors and ATL patients (Table I) were analyzed without (Day 0) or with 1 day culture (Day 1). Cells were triple stained for CD4, CD25 and CCR9 or CCR4, using APC-labeled monoclonal anti-CD4 (MT310), PE-labeled monoclonal anti-CD25 (ACT-1), FITC-labeled monoclonal anti-CCR9 (11259) and monoclonal anti-CCR4 (1G1) followed by FITC-labeled anti-mouse IgG. Data shown are gated on CD4+ cells. The percentages of cells are depicted. (b) Confocal microscopy: PBMC from an ATL patient (No. 7; Table I) were double stained for CCR9 and Tax or CCR4, using FITC-labeled monoclonal anti-CCR9 (112509), monoclonal anti-Tax (Lt-4) followed by biotin-labeled anti-mouse IgG and Alexa Fluor 546-labeled streptavidin, and monoclonal anti-CCR4 (1G1) followed by Alexa Fluor 546-labeled anti-mouse IgG. Separate and merged confocal pictures are shown. The bars indicate 10 μm.

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We further examined the coexpression of CCR9 and Tax or CCR4 proteins in ATL cells cultured for 1 day by confocal microscopy. As shown in Figure 2b, essentially all CCR9+ cells overlapped with Tax+ cells. Similarly, essentially all CCR9+ cells overlapped with CCR4+ cells. Thus, ATL cells, at least a substantial fraction, can express CCR9 together with CCR4, an unusual combination of chemokine receptors never seen in normal circulating T cells.

The above-mentioned results suggested that Tax induced CCR9 expression. To test this possibility, we used JPX-9 and JPX-M, the Jurkat sublines carrying HTLV-1 Tax and mutant-Tax (Tax-Arg63), respectively, under the control of the metallothionein gene promoter.28 These cell lines have been widely used to examine the effect of Tax on expression of various cellular genes.16, 28, 34 The results are shown in Figure 3a. The treatment of JPX-9 and JPX-M with Cd2+ rapidly induced expression of Tax and mutant-Tax, respectively. Accordingly, CD25, one of the known target genes of Tax,35 was induced in JPX-9 expressing wild-type Tax but not in JPX-M expressing mutant Tax as expected. Similarly, CCR9 was upregulated in JPX-9, but not in JPX-M, upon treatment with Cd2+. On the other hand, no such upregulation was seen for CCR4 in Cd2+-treated JPX-9 as reported previously.23

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Figure 3. Induction of CCR9 by Tax. (a) RT-PCR analysis: JPX-9 and JPX-M are the sublines of a human T cell line Jurkat carrying wild-type Tax and mutant Tax (Tax-Arg63), respectively, under the control of the metallothionein gene promoter.28 Cells were treated with 20 μM of Cd2+ for 1, 3 and 5 days. RT-PCR was carried out for CCR4, CCR9, CD25, Tax and GAPDH (loading control). PHA-treated normal PBMC were used as positive control. Representative results from 2 separate experiments are shown. (b) Flow cytometric analysis: JPX-9 cells without treatment (Day 0) or JPX-9 cells treated with 20 μM of Cd2+ for 3 days (Day 3) were double stained for Tax and CCR9, using monoclonal anti-Tax (Lt-4) followed by biotin-labeled anti-mouse IgG and APC-labeled streptavidin and PE-labeled monoclonal anti-CCR9 (112509). (c) Luciferase reporter assay: pGL3-CCR9p was the construct of luciferase reporter under the control of 1-kb promoter region of CCR9, while pHβPr.1-Tax MT2 was an expression vector for Tax. Jurkat cells (5 × 105 cells/well) were cotransfected with pGL-3-CCR9p and pHβPr.1 (vector) or pHβPr.1-Tax MT2 (Tax). pGL3-CCR9p was used at 3 μg/5 × 105 cells. pHβPr.1 and pHβPr.1-Tax MT2 were used at 1 μg/5 × 105 cells. After 48 hr, luciferase assays were done in triplicate to obtain mean ± SD. Representative results from 3 separate experiments are shown. *p < 0.01. (d) Luciferase reporter assay: parental Jurkat cells, JPX-9 cells and JPX-M cells (5 × 105 cells/well) were transfected with pGL-3-CCR9p and cultured with 20 μM of Cd2+ for 3 days. Luciferase assays were done in triplicate to obtain mean ± SD. Representative results from 3 separate experiments are shown. *p < 0.01.

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We next performed double staining of JPX-9 for CCR9 and Tax before and after treatment with Cd2+ for 3 days. As shown in Figure 3b, while untreated JPX-9 cells (Day 0) were virtually negative for Tax, ∼40% of Cd2+-treated JPX-9 cells (Day 3) became positive for Tax. Among Tax+ cells, ∼22% became positive for CCR9.

We further examined induction of a luciferase reporter gene under the control of the 1-kb promoter region of CCR932 by Tax. As shown in Figure 3c, expression of the luciferase reporter gene in Jurkat was significantly induced by cotransfection of an expression vector for Tax. We also examined activation of the CCR9 promoter by Tax using JPX-9 and JPX-M. As shown in Figure 3d, the luciferase activity was significantly induced in Cd2+-treated JPX-9 but not in Cd2+-treated JPX-M or Cd2+-treated Jurkat. Collectively, Tax is capable of activating, either directly or indirectly, the 1-kb promoter region of CCR9. However, the transcriptional activation of the CCR9 promoter by Tax is only modest. TFSEARCH (http://mbs.cbrc.jp/research/db/TFSEARCH.html) has revealed that the 1-kb promoter region of CCR9 contains several potential AP-1 binding sites but not those for NF-κB, CREB/ATF or SRF, the major Tax-responsible elements.4 Furthermore, mutant Tax (Tax-Arg63) expressed in Cd2+-treated JPX-M is completely devoid of the ability to activate NF-κB or SRF and only weakly (∼20%) activates CREB.36 However, it is fully capable of activating AP-1.36 Thus, Tax may activate the CCR9 promoter via a new element(s) or by an indirect mechanism.

To examine functional significance of CCR9 expression in ATL cells, we next performed chemotaxis assays. Since ATL cells are known to coexpress CD4 and CD25, we monitored chemotactic responses of CD4+CD25+ cells in PBMC from normal donors (n = 4) and ATL patients (n = 4) (Table I) to CCL22 (the CCR4 ligand) and CCL25 (the CCR9 ligand).20 The results are shown in Figure 4. As reported previously,23 CD4+CD25+ cells from all ATL patients vigorously responded to CCL22 with a peak at 1 nM. On the other hand, CD4+CD25+ cells from normal donors responded to CCL22 less vigorously and also with a peak at 100–1,000 nM. Thus, compared to normal T cells, chemotactic responses of ATL cells to CCL22 were highly elevated not only in efficiency but also in potency. Even though modestly and variably, CD4+CD25+ cells from all ATL patients significantly responded to CCL25 at concentrations in the range of 100–1,000 nM. On the other hand, CD4+CD25+ cells from normal donors hardly showed chemotactic responses to CCL25.

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Figure 4. Chemotaxis assay of primary ATL cells. PBMC obtained from normal donors and ATL patients were cultured for 1 day, and tested for chemotactic responses to CCL22 (CCR4 ligand) and CCL25 (CCR9 ligand) in CHEMOTX chambers. CD4+CD25+ cells in input cells and cells migrated to lower wells were counted by flow cytometry. The data are expressed as percentage input cells. The assays were done at least 2 times and the representative results are shown. Each point represents mean ± SD from triplicate assays. *p < 0.05; **p < 0.01.

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To see a possible role of CCR9 in ATL infiltration of the gastrointestinal tract, we performed immunohistochemical staining of CCR9 and CCR4 for ATL cells invading gastrointestinal tissues using archived biopsy tissue blocks (n = 10) (Table II). Because of the easy accessibility for diagnostic biopsy, the archived samples were limited to those from stomach and colon. The representative results are shown in Figure 5, and the whole results are summarized in Table II. ATL cells were strongly positive for CCR4 in all the tissue samples examined. ATL cells in 7 out of 10 gastrointestinal tissue samples were also scored positive for CCR9. The staining intensity of CCR9 was, however, variable from case to case and generally much weaker than that of CCR4. Furthermore, in contrast to relatively homogeneous staining intensity of CCR4 in ATL cells, staining intensity of CCR9 in ATL cells appeared to be heterogeneous. We also tried to detect Tax protein in these tissue sections by immunohistochemistry but could not detect specific signals above background levels (not shown).

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Figure 5. Immunohistochemical staining of CCR4 and CCR9 in ATL cells infiltrating gastrointestinal tissues. Tissue sections were immunologically stained as described in Materials and Methods Section. All staining experiments were repeated at least twice. (a) affinity-purified goat polyclonal anti-CCR9 followed by biotin-labeled rabbit anti-goat IgG and Vectastain ABC/HRP kit, (b) monoclonal anti-CCR4 (KM-2160) followed by biotin-labeled horse anti-mouse IgG and Vectastain ABC/HRP kit, (c) normal goat IgG followed by biotin-labeled rabbit anti-goat IgG and Vectastain ABC/HRP kit, and (d) hematoxillin-eosin. Peroxidase enzymatic development was performed using 3,3′-diaminobenzidine (DAB) and H2O2. The representative results from case 11 (Table II) are shown. No positive staining was observed with normal mouse IgG1 (the isotype control of anti-CCR4) (not shown). Original magnifications, ×400.

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Table II. Summary of ATL Cases whose Archived Biopsy Tissue Samples were Examined by Immunohistochemistry
CaseSexAgeTypeBiopsy siteCCR4CCR9
  1. +++, strongly positive; ++, moderately positive; +, weakly positive; −, negative.

11F64AcuteStomach+++++
12M54AcuteColon+++
13M63AcuteStomach++++
14F81AcuteColon+++
15M49AcuteStomach+++++
16M70AcuteStomach++++
17F72LymphomaStomach++++
18M69AcuteStomach+++++
19M69LymphomaStomach+++
20M51ChronicStomach++++

In the present study, we have first demonstrated expression of CCR9 in T cells expressing HTLV-1 Tax. Furthermore, we have shown that Tax activates the CCR9 promoter. However, the activation of the CCR9 promoter by Tax is relatively weak and the element(s) in the CCR9 promoter responsible for the effect of Tax is not known. Thus, Tax may indirectly activate the CCR9 promoter via its potent effects on T cells. Second, we have shown that CCR9 is frequently positive in ATL cells invading the gastrointestinal tract. Thus, CCR9 may play a role in invasion and/or localization of ATL cells in the gastrointestinal tract where CCL25 is abundantly produced.27, 37 However, given that CCR9 was not always positive in ATL cells invading the gastrointestinal tract (Table II), CCR9 may not be essential for ATL invasion of the gastrointestinal tract. Previously, Tanaka et al.38 as well as Chen et al.39 have suggested the role of α4β7 integrin, which interacts with mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on the vascular endothelial cells of the gastrointestinal tract,21 in ATL invasion of the gastrointestinal tract. The expression of α4β7 integrin in ATL cases is clonal and only seen in a fraction of cases38 (data not shown). Thus, the expression of α4β7 integrin may be a more critical determinant, while that of CCR9 is supportive. Furthermore, since circulating ATL cells hardly express surface CCR9, ATL cells may be induced to express CCR9 either in lymphoid tissues such as Peyer's patches or after infiltration into the gastrointestinal tract through upregulation of Tax in tissues8, 13 or by other stimulatory factors such as cytokines and cell adhesion molecules present in the local milieu. In this context, Iwata et al. have recently shown that all-trans retinoic acid is a potent inducer of CCR9 in memory T cells.40 Given that CCL25 has also been shown to be antiapoptotic for CCR9-expressing cells,41 expression of CCR9 may also promote survival of ATL cells infiltrating the gastrointestinal tract.

In conclusion, HTLV-1 Tax is either directly or indirectly capable of inducing CCR9. Furthermore, CCR9 is frequently expressed in ATL cells invading the gastrointestinal tract. Thus, CCR9, induced either by Tax or by other stimuli, may be involved in infiltration and/or localization of ATL cells and HTLV-1+ T cells in the gastrointestinal tract.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

We thank Dr. Masahiro Fujii at Niigata University Graduate School of Medical and Dental Sciences for providing the Tax expression vector (pHβPr.1-Tax) and control vector (pHβPr.1), Dr. Masataka Nakamura at Tokyo Medical and Dental University for providing JPX-9 and JPX-M, and Namie Sakiyama for her excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References
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