SEARCH

SEARCH BY CITATION

Keywords:

  • ATLL;
  • regulatory T cells;
  • CCR4;
  • FOXP3

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Adult T-cell leukemia/lymphoma (ATLL) patients are highly immunocompromised, but the underlying mechanism responsible for this state remains obscure. Recent studies demonstrated that FOXP3, which is a master control gene of naturally occurring regulatory T (Treg) cells, is expressed in the tumor cells from a subset of patients with ATLL. Since most ATLL cells express both CD4 and CD25, these tumors might originate from CD4+CD25+FOXP3+ Treg cells, based on their phenotypic characteristics. However, whether ATLL cells actually function as Treg cells has not yet been clearly demonstrated. Here, we show that ATLL cells from a subset of patients are not only hypo-responsive to T-cell receptor-mediated activation, but also suppress the proliferation of autologous CD4+ non-ATLL cells. Furthermore, ATLL cells from this subset of patients secrete only small amounts of IFN-γ, and suppress IFN-γ production by autologous CD4+ non-ATLL cells. These are the first data showing that ATLL cells from a subset of patients function as Treg cells in an autologous setting. The present study provides novel insights into understanding the immunopathogenesis of ATLL, i.e., how HTLV-1-infected cells can survive in the face of host immune responses. It also adds to our understanding of ATLL patients' severely immunocompromised state. © 2007 Wiley-Liss, Inc.

Adult T-cell leukemia/lymphoma (ATLL), a peripheral T-cell neoplasm, most often composed of highly pleomorphic lymphoid cells, is caused by the retrovirus human T-cell lymphotropic virus type-1 (HTLV-1). ATLL has a very poor prognosis, and the median survival time of patients with either the acute or lymphoma subtype of ATLL is <1 year. ATLL patients are in a severely immunocompromised state, leading to frequent and severe infectious complications and to an unfavorable outcome.1 Although the frequency of opportunistic infections is much higher in patients with ATLL than other hematological malignancies, the underlying mechanisms responsible for this remain obscure. Recently, several investigators including ourselves have reported that FOXP3, which is a master control gene for the development and function of CD4+CD25+ naturally occurring regulatory T (Treg) cells,2, 3, 4 is expressed in the tumor cells from a subset of patients with ATLL.5, 6, 7, 8, 9, 10 Furthermore, we and other investigators have reported that ATLL cells from most patients express CCR4,11, 12 and very recently, Hirahara et al. reported that virtually all peripheral blood CD4+CD25highFoxp3+ Treg cells express high levels of CCR4.13 Collectively, because most ATLL cells express both CD4 and CD25,1 based on their phenotypic characteristics, these tumors might originate from CD4+CD25+FOXP3+CCR4+ Treg cells. Thus, it can be envisaged that ATLL cells would function as Treg cells and give rise to a profound immunosuppressive environment enabling them to escape from the host immune response. In addition, the suppression of the host's normal effector T-cells by the tumor cells could result in a severely immunocompromised state, which is one of the clinical characteristics of patients with ATLL. However, whether ATLL cells do function as Treg cells has not been clearly demonstrated thus far in an autologous setting. Although Kohno et al.8 and Chen et al.9 investigated whether ATLL do function as Treg cells, in their experiments the responding cells and the regulatory cells were obtained from different individuals, i.e. ATLL cells obtained from patients were tested as regulatory cells on PBMC or CD4+ T-cells obtained from healthy individuals. Clearly, this does not reflect the in vivo situation with ATLL patients.

In the present study, we therefore tested ATLL cells as regulators of autologous CD4+ non-ATLL cells in the presence of autologous antigen presenting cells (APC), in response to T-cell receptor (TCR) stimulation.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cells

Peripheral blood mononuclear cells (PBMC) were isolated from ATLL patients or healthy individuals using Ficoll-Paque (Pharmacia, Uppsala, Sweden). All donors provided informed written consent prior to sampling, according to the Declaration of Helsinki, and the present study using human samples was approved by the institutional review board of Nagoya City University Graduate School of Medical Sciences.

Antibodies and flow cytometry

The following monoclonal antibodies (mAbs) were used for flow cytometry: fluorescein isothiocyanate (FITC)-conjugated anti-CCR4 mAb (KM2160), phycoerythrin (PE)-conjugated anti-CD25 mAb (clone M-A251), peridinin chlorophyll protein (PerCP)-conjugated anti-CD4 mAb (clone SK3), and FITC-, PE-, PerCP-conjugated isotype controls (clone MOPC-21). KM2160 was kindly provided by Kyowa Hakko Kogyo (Tokyo, Japan) and the other mAbs were purchased from BD Pharmingen (San Jose, CA). Cells were analyzed by a FACScan (Becton Dickinson, San Jose, CA).

Sorting of lymphocyte populations

Human CD4+ T-cell subsets were purified from PBMC by negative selection using the CD4+ T Cell Biotin-Antibody Cocktail Human Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. CD4+ T-cells were subsequently separated into CCR4-positive and -negative populations, using biotin-conjugated KM2160 and Anti-Biotin MicroBeads (Miltenyi Biotec).

CD4+ T-cell activation/proliferation assays and cytokine measurement

Each CD4+ T-cell population obtained from five ATLL patients and three healthy adults was assessed for proliferation in response to TCR stimulation in the presence of autologous APC. All ATLL cells in the present study expressed CD3 (data not shown). Autologous CD4-negative cells obtained from PBMC were irradiated (25 Gy) and used as APC. Each CD4+ (1 × 104) T-cell population was cultured in the presence of 5 × 104 APC and soluble anti-CD3 mAb (clone Hit3a, BD Biosciences) at a final concentration of 20 ng/mL in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), for 96 hr in a total volume of 150 μL/well (U-bottomed 96-well plate). Wells containing only 5 × 104 APC and soluble anti-CD3 mAb acted as controls. 3H-Thymidine (0.5 μCi) diluted in 50 μL RPMI-1640 medium supplemented with 10% heat-inactivated FBS was added to each well for the last 20 hr of culture. Incorporation of the 3H-Thymidine was measured as an indicator of cell proliferation, using a scintillation counter (PerkinElmer, Wellesley, MA). Data are expressed as average cpm of triplicates ± SD. To determine the regulatory properties of the CD4+CCR4+ T-cells, they were co-cultured with the CD4+CCR4 T-cell population at a ratio of 1:1 (total of 2 × 104 CD4+ T-cells/well) or 0.5:1 (total of 1.5 × 104 CD4+ T-cells/well), in the presence of 5 × 104 APC and soluble anti-CD3 mAb. Each of the populations in the co-culture experiments was also assessed for proliferation in the same manner.

For measurement of IFN-γ production, 50 μL RPMI-1640 medium supplemented with 10% heat-inactivated FBS, but not containing 3H-Thymidine, was added to each well for the last 20 hr, after which cell-free supernatants were collected. The concentrations of IFN-γ in the supernatants were determined by the Cytometric Bead Array Kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions.

Quantitative RT-PCR

Total RNA was prepared from sorted CD4+CCR4+ and CD4+CCR4 cells. Reverse transcription from the RNA to first-strand cDNA was then carried out as described previously.11 DNA segments corresponding to FOXP3 and β-actin were PCR-amplified using primer sets purchased from Roche Molecular Biochemical (Mannheim, Germany), according to the manufacturer's instructions, as described previously.14 Quantitative assessment of FOXP3 was carried out by dividing the number of copies of FOXP3 expressed by the number of β-actin.

Statistical analysis

The significance of the difference between two groups was examined, using the Mann-Whitney U test. Data were analyzed with the aid of StatView software (SAS Institute, version 5.0, Cary, NC). In this study, p < 0.05 was considered as significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Separating CD4+ T-cells obtained from ATLL patients into tumor and non-tumor cell subsets

CD4+ T-cells from five ATLL patients (No. 1, acute type; No. 2–5, chronic type) were divided into a CCR4-positive (indicated as a in Fig. 1a) and a CCR4-negative population (indicated as b in Fig. 1a). Most of the former consisted of ATLL cells and most of the latter of CD4+ non-ATLL cells, because tumor cells obtained from most patients with ATLL express CCR4.11, 12 The percentage of CD4+ cells in the PBMC of each patient are presented above the upper panels (Fig. 1a). The CD4+ T-cells of three healthy controls were divided in the same manner into a CCR4-positive and CCR4-negative population (indicated as a and b, respectively, in Fig. 2a). The percentage of CD4+ cells in the PBMC of each healthy donor are presented above the upper panels (Fig. 2a).

thumbnail image

Figure 1. CD4+CCR4+ ATLL cells from a subset of patients function as Treg cells in an autologous setting. (a) Human CD4+ cells from five ATLL patients consist of two populations: CD4+CCR4+ (population a, most of which are tumor cells), and CD4+CCR4 (population b, most of which are CD4+ non-ATLL cells). The percentage of CD4+ cells in each PBMC is presented above the upper panels. The percentages of CD4+ cells in each quadrant determined by CCR4 and CD25 expressions are presented on the right. (b) cDNA aliquots prepared from CD4+CCR4+ ATLL cells subjected to quantitative RT-PCR for FOXP3 and β-actin. #, not tested. (c) CD4+CCR4+ ATLL cells and non-ATLL CD4+ cells assessed for their proliferation in response to TCR stimulation in the presence of autologous APC. CD4+CCR4+ ATLL cells are also assessed for the ability to suppress proliferation of CD4+ non-ATLL cells in an autologous setting. APC populations alone are also assessed for their responses to TCR stimulation as background. (d) CD4+CCR4+ ATLL cells and CD4+ non-ATLL cells assessed for IFN-γ production in response to TCR stimulation in the presence of autologous APC. CD4+CCR4+ ATLL cells are also assessed for the ability to suppress IFN-γ production by non-ATLL CD4+ cells in an autologous setting. APC populations alone are also assessed for IFN-γ production in responses to TCR stimulation. #, not tested.

Download figure to PowerPoint

thumbnail image

Figure 2. CD4+CCR4+ cells from healthy individuals do not function as Treg cells in an autologous setting. (a) Human CD4+ cells from three healthy individuals divided into CD4+CCR4+ (population a) and CD4+CCR4 (population b). The percentage of CD4+ cells in each PBMC is presented above the upper panels. The percentages of CD4+ cells in each quadrant determined by CCR4 and CD25 expressions are presented on the right. (b) cDNA aliquots prepared from CD4+CCR4+ and CD4+CCR4 cells subjected to quantitative RT-PCR for FOXP3 and β-actin. (c) CD4+CCR4+ and CD4+CCR4 cells assessed for proliferation in response to TCR stimulation in the presence of autologous APC. CD4+CCR4+ cells are also assessed for the ability to suppress proliferation of CD4+CCR4 cells in an autologous setting. APC populations alone are also assessed for their responses to TCR stimulation as background. (d) CD4+CCR4+ and CD4+CCR4 cells assessed for IFN-γ production in response to TCR stimulation in the presence of autologous APC. CD4+CCR4+ cells are also assessed for the ability to suppress IFN-γ production by CD4+CCR4 cells in an autologous setting. APC populations alone are also assessed for IFN-γ production in responses to TCR stimulation.

Download figure to PowerPoint

FOXP3 expression in ATLL cells

Although the level of FOXP3 expression in ATLL cells obtained from 5 patients varied from case to case (FOXP3 copies/β-actin copies × 10−3 = 23.7 ± 12.3, average ± SD, Fig. 1b), overall, it was comparable to the CD4+CCR4+ cells obtained from three healthy controls (28.1 ± 8.5, Fig. 2b). We have previously reported that the level of FOXP3 mRNA in CD4+CCR4+ and CD4+CD25+ cells in healthy individuals is very similar.6 Also as reported previously,6 the level of FOXP3 expression in control CD4+CCR4 cells was low (2.6 ± 0.7, Fig. 2b).

Treg function of ATLL cells from a subset of patients

CD4+CCR4+ ATLL cells and CD4+ non-ATLL cells were assessed for proliferation in response to TCR stimulation in the presence of autologous APC. CD4+CCR4+ ATLL cells from patients No. 1, 2, 3 and 4 responded only weakly to stimulation with soluble anti-CD3 mAb in the presence of autologous APC compared with the CD4+CCR4+ cells from three healthy individuals (44, 3,444, 1,122 and 621 cpm, compared with 8,825 ± 71 cpm, Figs. 1c and 2c). To assay suppressive activity of patients' cells, CD4+CCR4+ ATLL cells were co-cultured with autologous CD4+ non-ATLL cells together with soluble anti-CD3 mAb in the presence of autologous APC. CD4+CCR4+ ATLL cells suppressed the proliferation of CD4+ non-ATLL cells at a ratio of 0.5:1 [{proliferation of (CD4+CCR4+ ATLL cells + CD4+ non-ATLL cells)/proliferation of CD4+ non-ATLL cells alone} × 100 = 47.4%] in patient No. 1 (Fig. 1c). Suppression was even more marked at a ratio of 1:1 (to 37.4% according to the above formula, Fig. 1c). Essentially the same results were obtained from ATLL patient No. 2 (77.1%, at a ratio of 1:1, Fig. 1c). In contrast, the CD4+CCR4+ T-cells from healthy individuals did not clearly suppress autologous CD4+CCR4 T-cell proliferation at a ratio of 1:1. Thus, the regulatory index of the three healthy individuals No. 1, 2 and 3 according to the above formula was 143.8, 91.6 and 85.6%, respectively (Fig. 2c).

Next, IFN-γ production by CD4+CCR4+ ATLL cells and CD4+ non-ATLL cells in response to TCR stimulation in the presence of autologous APC was assessed. The CD4+CCR4+ ATLL cells from all five patients produced significantly less IFN-γ (25.4 ± 18.9 pg/mL) than cells of the same phenotype from three healthy controls (259.4 ± 136.8 pg/mL, p = 0.0253, Figs. 1d and 2d). When the two cell populations were co-cultured at a ratio of 1:1 with soluble anti-CD3 mAb in the presence of autologous APC, the CD4+CCR4+ ATLL cells suppressed IFN-γ production by autologous CD4+ non-ATLL cells [{IFN-γ concentration of (CD4+CCR4+ ATLL cells + CD4+ non-ATLL cells)/IFN-γ concentration of CD4+ non-ATLL cells alone} × 100 = 53.8%] in patient No. 2 (Fig. 1d). Essentially the same results were also obtained from ATLL patients No. 1 (67.6%), 3 (52.3%) and 5 (58.6%) (Fig. 1d). In contrast, CD4+CCR4+ cells from healthy individuals No. 1 and 3 failed to suppress IFN-γ production by autologous CD4+CCR4 cells, while CD4+CCR4+ cells from No. 2 only weakly suppressed (89.8%, Fig. 2d).

Different frequencies of CD4+ subsets from ATLL patients and healthy controls

Three-color flow cytometric analysis using anti-CD4, CCR4 and CD25 mAbs on PBMC obtained from a total of 27 ATLL patients (15 acute, 9 chronic, 3 smoldering) and 11 healthy individuals yielded percentages of CCR4CD25+, CCR4CD25, CCR4+CD25 and CCR4+CD25+ in CD4+ T-cells of 6.9 ± 5.6, 9.6 ± 10.5, 3.7 ± 6.4 and 79.8 ± 14.7 (average ± SD) (Fig. 3a), and 31.0 ± 10.9, 47.0 ± 9.2, 5.4 ± 1.4 and 16.6 ± 4.6 (Fig. 3b), respectively. These findings provide evidence for different frequencies of CD4+ subsets in ATLL patients and healthy individuals, i.e., the CD4+ T-cells in most ATLL patients consisted of a large majority of CCR4+CD25+ ATLL cells.

thumbnail image

Figure 3. Different frequencies of CD4+ cell subsets in ATLL patients and healthy controls. (a) Three-color flow cytometric analysis using anti-CD4, CCR4 and CD25 mAbs on PBMC obtained from a total of 27 ATLL patients (15 acute, 9 chronic, 3 smoldering) give percentages of CCR4CD25+, CCR4CD25, CCR4+CD25 and CCR4+CD25+ in CD4+ T-cells as 6.9 ± 5.6, 9.6 ± 10.5, 3.7 ± 6.4 and 79.8 ± 14.7 (average ± SD). (b) Three-color flow cytometric analysis using anti-CD4, CCR4 and CD25 mAbs on PBMC obtained from a total of 11 healthy individuals give percentages of CCR4CD25+, CCR4CD25, CCR4+CD25 and CCR4+CD25+ in CD4+ T-cells as 31.0 ± 10.9, 47.0 ± 9.2, 5.4 ± 1.4 and 16.6 ± 4.6.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Here, we show that ATLL cells from a subset of patients are not only hypo-responsive to TCR-mediated activation, but also suppress the proliferation of autologous CD4+ non-ATLL cells. Furthermore, ATLL cells from this subset of patients secrete only small amounts of IFN-γ, and suppress IFN-γ production by autologous CD4+ non-ATLL cells. These findings provide the first evidence that ATLL cells from some but not all patients can function as Treg cells in an autologous setting. In the present study, ATLL cells from 2 of 5 patients (No. 1 and 2) clearly demonstrated Treg function in an autologous setting. In the present experimental design, because the APC populations contained CD8-positive T-cells, we could not completely exclude the possibility that these 25 Gy-irradiated cells responded to CD3 stimulation at a low level. However, we considered that these responses of the APC populations were so slight that they would not significantly affect the results and conclusions presented here. We also showed that most of the CD4+CCR4+ cells from ATLL patients also expressed CD25, which is common on ATLL cells.1 Therefore, we believe that separating patients' CD4+ T-cells into ATLL cells and non-ATLL cells using anti-CCR4 mAb is appropriate.

Since FOXP3 expression level in ATLL cells was not associated with Treg activity, this cannot simply be dependent on FOXP3. What types of ATLL cells function as Treg cells remains obscure, and warrants further investigation. In contrast to the findings with ATLL, CD4+CCR4+ cells obtained in the same way from healthy controls did not clearly function as Treg cells. This is probably because of the fact that peripheral CD4+CCR4+ T-cells consist not only of Treg cells13, 15, 16, 17 but also T helper type 2 (Th2) cells,18 with the former making up only a small fraction of the CD4+CCR4+ cells residing within the CD25high population13, 19 It needs to be emphasized that the proportion of CCR4+ cells within the CD4+ population was quite different in ATLL patients and healthy individuals, i.e., as many as 83.5 ± 13.1% in the 27 ATLL patients but only 22.0 ± 5.4% in 11 healthy individuals, in the present study. We showed here that CD4+CCR4+ ATLL cells from a subset of patients were able to suppress proliferation and IFN-γ production of autologous CD4+ non-ATLL cells in vitro at a ratio of 1:1. Because the CD4+ T-cells in most ATLL patients consist of a majority of CCR4+ ATLL cells and a much smaller number of CCR4 non-ATLL cells, the actual ratio of CD4+CCR4+ ATLL cells / CD4+CCR4 non-ATLL cells in vivo is probably much higher than 1:1. Therefore, CD4+CCR4+ ATLL cells could markedly suppress the responses of CD4+ non-ATLL cells in vivo.

Increased frequencies of Treg cells are now accepted as an important mechanism for tumor escape from host immunity in several different types of cancer.16, 17, 20, 21 To the best of our knowledge, this is the first report to clearly demonstrate that tumor cells themselves can function as Treg cells in an autologous setting. The present study will provide novel insights into understanding the immunopathogenesis of ATLL, i.e., how HTLV-1-infected cells can survive for the length of time necessary for ATLL development, in the face of host immune responses to the virus-infected cells. It also adds to our understanding of ATLL patients' severely immunocompromised state, which leads to frequent and severe infectious complications. The present study and the availability of therapeutic anti-CCR4 mAb,6, 22, 23 with which we are currently conducting a phase I clinical trial in patients with CCR4-positive T-cell leukemia/lymphoma11, 14 (ClinicalTrials.gov Identifier: NCT00355472), should offer improved treatment strategies for patients with ATLL.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We are grateful to Kyowa Hakko Kogyo, Inc., (Tokyo, Japan) for providing us with anti-CCR4 mAb (KM2160). We thank Ms. Chiori Fukuyama for her skillful technical assistance. This work was supported by Grant-in-Aids for General Scientific Research (T.I., R.U.), and a Grant-in-Aid for Scientific Research on Priority Areas (R.U.) from the Ministry of Education, Culture, Science, Sports and Technology, and a Grant-in-Aid for Cancer Research (R.U.) from the Ministry of Health, Labor, and Welfare, Japan.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Kikuchi M, Jaffe ES, Ralfkiaer E. Adult T-cell leukaemia/lymphoma. In: JaffeES, HarrisNL, SteinH, VardimanJW, eds. Tumors of haematopoietic and lymphoid tissues. Lyon, France: IARC, 2001. 2003.
  • 2
    Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003; 299: 105761.
  • 3
    O'Garra A, Vieira P. Regulatory T cells and mechanisms of immune system control. Nat Med 2004; 10: 8015.
  • 4
    Yagi H, Nomura T, Nakamura K, Yamazaki S, Kitawaki T, Hori S, Maeda M, Onodera M, Uchiyama T, Fujii S, Sakaguchi S. Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol 2005; 16: 164356.
  • 5
    Karube K, Ohshima K, Tsuchiya T, Yamaguchi T, Kawano R, Suzumiya J, Utsunomiya A, Harada M, Kikuchi M. Expression of FoxP3, a key molecule in CD4CD25 regulatory T cells, in adult T-cell leukemia/lymphoma cells. Br J Hematol 2004; 126: 814.
  • 6
    Ishida T, Iida S, Akatsuka Y, Ishii T, Miyazaki M, Komatsu H, Inagaki H, Okada N, Fujita T, Shitara K, Akinaga S, Takahashi T, et al. The CC chemokine receptor 4 as a novel specific molecular target for immunotherapy in adult T-Cell leukemia/lymphoma. Clin Cancer Res 2004; 10: 752939.
  • 7
    Matsubara Y, Hori T, Morita R, Sakaguchi S, Uchiyama T. Phenotypic and functional relationship between adult T-cell leukemia cells and regulatory T cells. Leukemia 2005; 19: 4823.
  • 8
    Kohno T, Yamada Y, Akamatsu N, Kamihira S, Imaizumi Y, Tomonaga M, Matsuyama T. Possible origin of adult T-cell leukemia/lymphoma cells from human T lymphotropic virus type-1-infected regulatory T cells. Cancer Sci 2005; 96: 52733.
  • 9
    Chen S, Ishii N, Ine S, Ikeda S, Fujimura T, Ndhlovu LC, Soroosh P, Tada K, Harigae H, Kameoka J, Kasai N, Sasaki T, et al. Regulatory T cell-like activity of Foxp3+ adult T cell leukemia cells. Int Immunol 2006; 18: 26977.
  • 10
    Roncador G, Garcia JF, Garcia JF, Maestre L, Lucas E, Menarguez J, Ohshima K, Nakamura S, Banham AH, Piris MA. FOXP3, a selective marker for a subset of adult T-cell leukaemia/lymphoma. Leukemia 2005; 19: 224753.
  • 11
    Ishida T, Utsunomiya A, Iida S, Inagaki H, Takatsuka Y, Kusumoto S, Takeuchi G, Shimizu S, Ito M, Komatsu H, Wakita A, Eimoto T, et al. Clinical significance of CCR4 expression in adult T-cell leukemia/lymphoma: its close association with skin involvement and unfavorable outcome. Clin Cancer Res 2003; 9: 362534.
  • 12
    Yoshie O, Fujisawa R, Nakayama T, Harasawa H, Tago H, Izawa D, Hieshima K, Tatsumi Y, Matsushima K, Hasegawa H, Kanamaru A, Kamihira S, et al. Frequent expression of CCR4 in Adult T-cell leukemia and human T-cell leukemia virus type 1-transformed T cells. Blood 2002; 99: 150511.
  • 13
    Hirahara K, Liu L, Clark RA, Yamanaka K, Fuhlbrigge RC, Kupper TS. The majority of human peripheral blood CD4+CD25highFoxp3+ regulatory T cells bear functional skin-homing receptors. J Immunol 2006; 177: 448894.
  • 14
    Ishida T, Inagaki H, Utsunomiya A, Takatsuka Y, Komatsu H, Iida S, Takeuchi G, Eimoto T, Nakamura S, Ueda R. CXC chemokine receptor 3 and CC chemokine receptor 4 expression in T-cell and NK-cell lymphomas with special reference to clinicopathological significance for peripheral T-cell lymphoma, unspecified. Clin Cancer Res 2004; 10: 5494500.
  • 15
    Iellem A, Mariani M, Lang R, Recalde H, Panina-Bordignon P, Sinigaglia F, D'Ambrosio D. Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4+ CD25+ regulatory T cells. J Exp Med 2001; 194: 84753.
  • 16
    Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M, Zhu Y, Wei S, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004; 10: 9429.
  • 17
    Ishida T, Ishii T, Inagaki A, Yano H, Komatsu H, Iida S, Inagaki H, Ueda R. Specific recruitment of CCR4-positive regulatory T cells in Hodgkin lymphoma fosters immune privilege. Cancer Res 2006; 66: 571622.
  • 18
    Imai T, Nagira M, Takagi S, Kakizaki M, Nishimura M, Wang J, Gray PW, Matsushima K, Yoshie O. Selective recruitment of CCR4-bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine. Int Immunol 1999; 11: 818.
  • 19
    Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA. CD4+CD25high regulatory cells in human peripheral blood. J Immunol 2001; 167: 124553.
  • 20
    Zou W. Regulatory T cells, tumor immunity and immunotherapy. Nat Rev Immunol 2006; 6: 295307.
  • 21
    Zou W. Immunosuppressive networks in the tumor environment and their therapeutic relevance. Nat Rev Cancer 2005; 5: 26374.
  • 22
    Niwa R, Shoji-Hosaka E, Sakurada M, Shinkawa T, Uchida K, Nakamura K, Matsushima K, Ueda R, Hanai N, Shitara K. Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows potent therapeutic activity to T-cell leukemia and lymphoma. Cancer Res 2004; 64: 212733.
  • 23
    Ishida T, Ishii T, Inagaki A, Yano H, Kusumoto S, Ri M, Komatsu H, Iida S, Inagaki H, Ueda R. The CCR4 as a novel specific molecular target for immunotherapy in Hodgkin lymphoma. Leukemia 2006; 20: 21628.