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Clinical Cancer Research Vol. 11, 2427-2435, March 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Production of Thymus and Activation-Regulated Chemokine and Macrophage-Derived Chemokine by CCR4+ Adult T-Cell Leukemia Cells

Takatoshi Shimauchi, Satoshi Imai, Ryosuke Hino and Yoshiki Tokura

Department of Dermatology, University of Occupational and Environmental Health, Kitakyusyu, Japan

Requests for reprints: Takatoshi Shimauchi, Department of Dermatology, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyusyu 807-8555, Japan. Phone: 81-93-691-7445; Fax: 81-93-691-0907; E-mail: t-shima{at}med.uoeh-u.ac.jp.


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Adult T-cell leukemia/lymphoma (ATL) is a peripheral CD4+CD25+ T-cell malignancy caused by human T-cell leukemia virus type I. The tumor cells frequently infiltrate in the skin, lymph nodes and other organs and especially form prominent cutaneous masses. Recently, ATL cells have been shown to express Th2 chemokine receptor CCR4. The aim of this study is to investigate the possibility that CCR4 ligands, thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), are produced by CCR4+ ATL cells per se.

Experimental Design: CD4+ or CD4+CD14– cells were purified from peripheral blood mononuclear cells of 11 ATL patients with cutaneous involvement and normal healthy volunteers. Tissue-infiltrating cells were isolated from skin tumors. The expression of chemokine receptors on these cells were analyzed by flow cytometry. The production of chemokines and cytokines by the neoplastic cells was assessed by ELISA and reverse transcription-PCR after cultivation for 96 hours in the presence or absence of anti-CD3/CD28 monoclonal antibodies. Finally, TARC and CCR4 expressions were examined by immunohistochemistry.

Results: ATL cells highly expressed CCR4 but did not necessarily exhibit the Th2 cytokine profile. The cells also produced TARC and MDC. The production level of MDC was higher in the skin tumor formation group than that in the nontumor group. Immunohistochemically, both CCR4 and TARC were expressed by the tumor cells in the lesional skin.

Conclusions: ATL cells not only express CCR4 but also produce TARC and MDC. The skin tumor formation as well as the monoclonal integration of proviral DNA are the factors that are associated with the high production of Th2 chemokines by ATL cells.

Key Words: Chemokine • Chemokine receptor • Tumor formation


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature CD4+ T cells caused by the human T-cell leukemia virus type I (HTLV-I; refs. 1, 2). This endemic hematologic neoplasm develops in 1% to 5% of individuals infected with HTLV-I after more than two decades of viral persistence (3, 4). ATL malignant T cells are usually positive for CD3, CD4, CD25, and CD45RO but negative for CD7, CD8, CD19, and CD20 (5). Based on the organ involvement and severity, ATL is divided into four clinical categories: acute, chronic, lymphoma, and smoldering types (5). Cutaneous involvement is seen in up to 50% of patients, and lymph nodes, liver, and spleen are the other predilection sites. Although patients with ATL exhibit various cutaneous manifestations, the most frequent type of eruption is nodules/tumors (33.9%) in which tumor cells aggregate massively (6).

T-cell migration and activation driven by the interaction between chemokines and chemokine receptors play a pivotal role in the pathogenesis of various neoplastic as well as inflammatory disorders (7–10). ATL cells have been shown to produce several chemokines, monocyte chemoattractant protein-1 (MIP-1; ref. 11), macrophage inflammatory protein-1{alpha}, macrophage inflammatory protein-1ß (12, 13), and I-309 (14), and to express chemokine receptors, CCR4 (15, 16), CCR7 (17), and CCR8 (14). Of importance is the finding that overexpression of chemokine I-309 and their receptor CCR8 contributes to antiapoptotic autocrine loops in ATL cells (14).

Among chemokines, thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are known as Th2 chemokines that bind to their receptor CCR4 on Th2 cells (18–20), whereas IFN-{gamma} inducible protein 10 (IP-10) and monokine induced by IFN-{gamma} (MIG) are Th1 chemokines with affinity to CXCR3 on Th1 cells (21, 22). It has been shown recently that ATL malignant cells express CCR4 (15, 16).

In this study, we investigated whether TARC and/or MDC are produced by ATL cells per se by using malignant T cells isolated freshly from both patients' peripheral blood and skin tumors. Results suggest that ATL cells not only express CCR4 but also secrete TARC and MDC.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients. Eleven patients with ATL (6 men and 5 women ages 42-87 years) listed in Table 1 and 5 healthy donors as control were enrolled in this study. ATL was diagnosed based on clinical features and laboratory findings according to the criteria (5). The patients were divided into two groups: group A (eight patients designated cases A1-A8) had peripheral blood mononuclear cells (PBMC) integrated monoclonally with HTLV-I proviral DNA as assessed by the standard Southern blot analysis (2) and group B (three patients designated cases B1-B3) had polyclonal integration in PBMC but monoclonal integration in biopsy specimens from skin lesions.


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  		Table 1 Clinical and laboratory features of ATL patients

 
Cell Preparation. Peripheral blood was obtained by vein puncture from the subjects with heparin as anticoagulant. PBMCs were isolated by the standard Ficoll-Paque (Pharmacia, Uppsala, Sweden) method. CD4+ cells were separated from PBMC with anti-CD4 monoclonal antibody (mAb)–conjugated magnetic beads (Dynal, Inc., Oslo, Norway) and subsequent DETACHaBEAD (Dynal) according to the manufacturer's directions. CD4– cells were negatively selected with anti-CD4 mAb-conjugated magnetic beads. Single cell suspensions of skin tumor-infiltrating cells were prepared by teasing of skin biopsy specimens of cases A2 to A5 and B1 and were used after Ficoll-Paque separation.

To eliminate monocytes contaminated in the CD4+ fraction, we prepared CD4+CD14– and CD14+CD4+ cells in cases A2 and A3 and a normal healthy donor. CD14+ cells were separated from PBMC with anti-human CD14 mAb-conjugated magnetic beads (BD Biosciences PharMingen, San Diego, CA). Subsequently, CD4+ cells were obtained from this CD14– fraction with anti-CD4 mAb-conjugated magnetic beads (BD Biosciences PharMingen) according to the manufacturer's directions.

Cell Culture. Cells were cultured in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated FCS, 2 mmol/L L-glutamine, 5 x 10–5 mol/L 2-mercaptoethanol, 10–5 mol/L sodium pyruvate, 25 mmol/L HEPES, 1% nonessential amino acids, 100 units/mL penicillin, and 100 µg/mL streptomycin (all from Life Technologies). To test the production of chemokines and cytokines by purified CD4+, CD4–, CD14+CD4+, or CD14–CD4+ PBMCs and skin tumor cells, they were cultured in 24-well plates (Corning, Inc., Corning, NY, 1 x 106/mL culture medium) at 37°C in 5% CO2 in air in the presence or absence of anti-CD3 mAb (soluble form stimulatory for T cells, BD Biosciences PharMingen) at 2 µg/mL and anti-CD28 mAb (Immunotech, Marseilles, France) at 2 µg/mL. After 96-hour culture, the supernatants were collected and the cells were washed thrice with PBS (pH 7.4). They were stored at –80°C until use.

Flow Cytometric Analysis. Crude or variously purified PBMCs and skin tumor-infiltrating cells were washed with PBS containing 2% FCS. HBSS containing 0.1% NaN3 and 1% FCS was used as the staining buffer. After incubation for 30 minutes with mAbs or control isotype-matched controls, 10,000 labeled cells were analyzed on a FACSCalibur (Becton Dickinson, Mountain View, CA) in each sample. FITC-labeled mAbs to CD3 (SK7), CD4 (SK3), CD25 (2A3), and IgG1 (X40) and phycoerythrin-labeled mAbs to CD14 (M{phi}P9), CD25 (2A3), CD28 (CD28.2), and IgG1 (X40) were purchased from BD Immunocytometry Systems (San Jose, CA). FITC-labeled anti–cutaneous leukocyte antigen (CLA; HECA-452) and phycoerythrin-labeled anti-CCR4 (1G1), CXCR3 (1C6), and CTL-associated antigen-4 (CTLA-4; BNI3) were also purchased from BD Biosciences PharMingen. FITC-labeled mouse IgG1 or phycoerythrin-labeled mouse IgG1 was used as isotype-matched control.

Cytokine and Chemokine Assays. TARC and MDC concentrations in the culture supernatants and sera were measured by ELISA with ANALYZA Immunoassay System (Techne Corp., Minneapolis, MN). The minimum detectable doses of TARC and MDC were 7 and 62.5 pg/mL, respectively.

To quantify cytokines [IFN-{gamma}, tumor necrosis factor-{alpha}, interleukin (IL)-2, IL-4, IL-5, and IL-10] and chemokines (CXCL10/IP-10, CCL2/MIP-1, CXCL9/MIG, CCL5/RANTES, and CXCL8/IL-8), Cytometric Beads Array kits (Human Th1/Th2 Cytokine CBA and Human Chemokine Kit I, BD Biosciences PharMingen) were used with the manufacturer's directions. Briefly, 50 µL of each sample were added to an equal volume of the cytokine or chemokine bead mixture and detection reagent followed by a 3-hour incubation at room temperature in the dark. A standard curve for each cytokine or chemokine was generated in parallel. Unstained, FITC-labeled, or phycoerythrin-labeled cytometer setup beads were prepared toward the end of the sample incubation period. After washing, the beads were assayed immediately on the FACSCalibur. Cytokine and chemokine concentrations were determined using the software provided. The sensitivities for cytokines and chemokines using these quantification kits were as follows: IL-2, 2.6 pg/mL; IL-4, 2.6 pg/mL; IL-5, 2.4 pg/mL; IL-10, 2.8 pg/mL; tumor necrosis factor-{alpha}, 2.8 pg/mL; IFN-{gamma}, 7.1 pg/mL; CXCL8/IL-8, 0.2 pg/mL; CCL5/RANTES, 1.0 pg/mL; CXCL9/MIG, 2.5 pg/mL; CCL2/MIP-1, 2.7 pg/mL; and CXCL10/IP-10, 2.8 pg/mL.

RNA Preparation and Reverse Transcription-PCR. Total RNA was extracted from CD4+ cells and skin tumor-infiltrating cells unstimulated or stimulated with anti-CD3/CD28 mAbs by using the SV Total RNA Isolation System (Promega Co., Madison, WI). First-strand cDNA was prepared according to the manufacturer's directions, and the MOR gene was amplified in 50 µL of a PCR solution containing 0.8 mmol/L MgCl2, deoxynucleotide triphosphate mix, and DNA polymerase with either synthesized primers of MDC (sense 5'-CTGAGCCAATGAAGAGCCTACT-3' and antisense 5'-GCGGAGACTGTGACTAGGGTTA-3') or TARC (sense 5'-GAAGATGCTGGCCCTGGTC-3' and antisense 5'-TCACTCTCTTGTTGTTGGGG-3'). Samples were heated to 95°C for 2 minutes, 55°C for 2 minutes, and 72°C for 3 minutes and cycled 39 times through 95°C for 1 minutes, 55°C for 2 minutes, and 72°C for 3 minutes. The final incubation was at 72°C for 7 minutes. The mixture was subjected to 1% agarose gel that for electrophoresis with the indicated markers and primers for the internal standard ß-actin (sense 5'-GGCACCACACCTTCTACAATGAF-3' and antisense 5'-CGTCATACTCCTGCTTGCTGATC-3'). Each sample was applied more than two lanes in the same gel. The agarose gel was stained with ethidium bromide and photographed with UV transillumination.

Immunohistochemical Staining. Biopsy specimens from skin lesions were fixed in 10% formaldehyde and embedded in paraffin. Immunohistochemical stainings for CCR4 and TARC were done on deparaffinized 5 µm sections by using the avidin-biotin system according to the manufacturer's instructions (Vectastain ABC-Apkit, Vector Laboratories, Burlingame, CA). Specimens from all cases, except for case A7, were stained along with a control specimen from natural killer (NK) cell lymphoma. As the primary antibodies, murine mAb against CCR4 [KM2160; recognizing the NH2-terminal portion (amino acids 12-29) of CCR4 and kindly provided by Dr. Kouji Matsushima and Kyowa Hakko Kogyo, Inc., Tokyo, Japan] and TARC (6SN; Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom) were used. The final concentration of both antibodies were 40 µg/mL.

Statistical Analysis. Student's t test was employed to evaluated significance of differences, and P < 0.05 was considered as significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Background Study of the Patients. Clinical and laboratory information on 11 patients are summarized in Table 1. The clinical type was determined by the widely accepted criteria (5). All patients exhibited skin eruptions in which atypical lymphocytes infiltrated histologically, and six cases (cases A1-A5 and B1) had apparent skin tumors showing massive aggregation of tumor cells. Cases A1 to A8 had monoclonal integration with HTLV-I proviral DNA in PBMC, whereas cases B1 to B3 had polyclonal integration in PBMC but did monoclonal integration in skin tumors. By flow cytometry, the percentages of circulating T cells expressing the ATL phenotype (i.e., CD4+CD25+ or CD25+CCR4+) were high in almost all the patients to various degrees compared with normal healthy control (Table 1), whereas that of CD25+CXCR3+ cells were very low. Only cases A6 and A8 had high percentages of CD25+CLA+ cells. CD28 was expressed on ATL cells in all the patients tested (cases A2, A4, A5 A6, and B2). Soluble IL-2 receptor and lactate dehydrogenase were elevated largely in the acute type but not in the smoldering or lymphoma type. Serum TARC levels were increased in cases A8, B1, and B2.

Purification of Adult T-Cell Leukemia Cells and Their CCR4 Expression. CD4+ cells were purified from the patients' and normal subjects' PBMC with immunomagnetic beads, and tissue-infiltrating cells were isolated from skin tumors of five patients. They were examined in the expression of surface molecules by flow cytometry. The results are summarized in Table 2. The purity of CD4+ cells from PBMC was >96%, except for case A4. Dual staining with anti-CD4 and anti-CD25 mAbs showed that the double-positive cells, corresponding to ATL cells, ranged from 45% to 97% (monoclonally integrated group A, 82.1 ± 18.2%; polyclonally integrated group B, 61.0 ± 14.5%). Normal volunteers had lower percentages and lower expression levels of these CD25+ cells. In ATL patients, especially in group A, ~90% of CD25+ cells were positive for CCR4 but not CXCR3. In skin tumors, 45% to 82% of the infiltrating cells were the tumor cell as determined by the CD4+CD25+ or CD25+CCR4+ phenotype (Table 2). Thus, ATL cells in both PBMC and skin tumors expressed high levels of Th2 chemokine receptor CCR4. We also found that some of the cases expressed CTLA-4 (cases A4 and A5), a functional molecule on CD4+CD25+ regulatory T cells (23), or CLA (cases A5, A7, and A8), a skin-homing molecule on T cells.


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  		Table 2 Flow cytometric analysis of PBMC purified with anti-CD4 immunomagnetic beads and skin tumor-infiltrating lymphocytes

 
Lack of Th2 Cytokine Profile in Adult T-Cell Leukemia Cells. Because ATL cells expressed both CD3 and CD28, CD4+ PBMCs were stimulated with anti-CD3/CD28 mAbs, and the concentration of various cytokines in 96-hour culture supernatants was measured. Despite the expression of Th2 chemokine receptor CCR4, none of Th2 cytokine (IL-4, IL-5, or IL-10) was highly produced by the patients' CD4+ PBMC compared with normal CD4+ cells (Table 3). On the other hand, Th1 cytokines (IFN-{gamma} and IL-2) in cases A3 and A5 were secreted at higher levels than normal CD4+ cells. The production of tumor necrosis factor-{alpha} was also enhanced in cases A2, A5, and A7. The increased production of these cytokines has been reported in the previous studies (24, 25).


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  		Table 3 Cytokine profiles of CD4+ PBMC and tumor-infiltrating cells in stimulation with anti-CD3/CD28 mAbs

 
Thymus and Activation-Regulated Chemokine and Macrophage-Derived Chemokine Production by Circulating Adult T-Cell Leukemia Cells. Purified CD4+ and CD4– PBMCs were cultured for 96 hours in the presence or absence of anti-CD3/CD28 mAbs, and the concentration of TARC and MDC in the supernatants was measured by ELISA. CD4+ cells from cases A1 and A2 produced higher amounts of TARC than did those from the control subjects (Fig. 1A). Especially in case A1, even unstimulated CD4+ cells secreted a comparable level of TARC to the stimulated cells. Although to lesser degrees, CD4+ cells in cases A3, A4, A6, and B1 produced higher levels of TARC than did those in the control on stimulation with anti-CD3/CD28 mAbs. Cases A5, A7, and A8 were nonproducers for TARC. In contrast, CD4– cells did not produce substantial amounts of TARC compared with the control (Fig. 1B). It is notable that TARC was elaborated by CD4– cells rather than CD4+ cells in the control subjects.



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  		Fig. 1 TARC and MDC production by CD4+, CD4– PBMC, and skin tumor cells. A-D, CD4+ and CD4– populations were separated from PBMC of the patients and normal subjects (control) and were cultured for 96 hours in the presence or absence of anti-CD3/anti-CD28 mAbs. Culture supernatants were subjected to ELISA for TARC and MDC. E and F, lymphocytes were isolated from skin lesions and cultured for 96 hours with or without CD3/CD28 stimulation. Culture supernatants were tested for TARC and MDC concentrations. Columns, mean of duplicate cultures; bars, SD.

 
MDC production by stimulated or unstimulated CD4+ cells in 6 (cases A1-A4, A6, and B1) of 11 ATL patients was also apparently greater than that in the normal control (Fig. 1C). In both ATL patients and control subjects, however, CD4– cells secreted high levels of MDC (Fig. 1D). Therefore, MDC seemed to be produced by both ATL cells and nonneoplastic CD4– cells.

The two highly TARC-producing cases (cases A1 and A2) were monoclonally proviral DNA-integrated and skin tumor-forming ones, and similarly, the six highly MDC-producing cases (cases A1-A4, A6, and B1) were monoclonally integrated and/or skin tumor forming. This suggested that the ability of ATL cells to produce TARC and MDC is associated with the tumor formation. On our statistical analysis, there was no statistical difference in TARC production in the patients with skin tumors (cases A1-A5 and B1) and the nontumor group (cases A6-A8, B2, and B3; mean ± SD, 679 ± 850 versus 41.0 ± 74.0; P = 0.13), although the average was higher in the former than the latter. More convincingly, MDC was significantly elevated in the tumor group compared with the nontumor one (3,722 ± 1,907 versus 877 ± 1,185; P < 0.018). Therefore, the skin tumor formation as well as the monoclonal integration of proviral DNA are the factors that are associated with the high TARC and MDC levels.

Thymus and Activation-Regulated Chemokine and Macrophage-Derived Chemokine Production by Skin Tumor Adult T-Cell Leukemia Cells. Lymphocytes isolated from skin tumors of cases A2 to A5 contained of 45% to 82% ATL cells as determined by the CD4+CD25+ or CD25+CCR4+ phenotype (Table 2). TARC and MDC in 96-hour culture supernatants were quantified by ELISA. As shown in Fig. 1E and F, the skin-infiltrating cells in cases A2 to A4 produced TARC and MDC at greatly higher levels than did peripheral lymphocytes from control subjects. Thus, ATL tumor cells infiltrating in the skin also functionally produced TARC and MDC.

No Substantial Production of Thymus and Activation-Regulated Chemokine and Macrophage-Derived Chemokine by Bystander Monocytes. In general, monocytes are producers of TARC and MDC (26, 27). To exclude the possibility that monocytes contaminated in the CD4+ fraction produced TARC and MDC, CD14+ and CD4+CD14– populations were purified in cases A2 and A3 and normal subjects. As shown in Fig. 2A, CD14+ cells were successfully eliminated in the CD4+CD14– fraction. The purified CD14+ and CD4+CD14– cells were cultured for 96 hours in the presence or absence of anti-CD3/CD28 mAbs. CD4+CD14– cells from case A2 produced high amounts of TARC and MDC and so did those from case A3 at moderate levels (Fig. 2B and C). In contrast, CD14+ cells did not produce any substantial amount of TARC or MDC. Therefore, TARC and MDC were produced more markedly by ATL cells than monocytes.



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  		Fig. 2 Flow cytometric analysis of CD14+ and CD4+CD14– cells purified from PBMC and their production of TARC and MDC. A, as represented by case A3, CD14+ or CD4+CD14– cells were purified from cases A2 and A3 and normal subjects' PBMC with immunomagnetic beads. The purity of each fraction was examined by flow cytometry (CD14+ cells: case A2, 86.0%; case A3, 82.4%; control, 92.3%; CD4+CD14– cells: case A2, 94.4%; case A3, 98.3%; control 96.1%). B and C, purified cells were cultured for 96 hours with or without CD3/CD28 stimulation. Culture supernatants were measured for TARC and MDC. Columns, mean of duplicate cultures; bars, SD.

 
Thymus and Activation-Regulated Chemokine and Macrophage-Derived Chemokine mRNA Expression by Adult T-Cell Leukemia Cells. The expression of mRNA for TARC and MDC by CD4+ cells purified from PBMC was examined by reverse transcription-PCR (RT-PCR) in cases A1 to A3 along with five normal control volunteers. When cultured for 96 hours in the presence of anti-CD3/CD28 mAbs, CD4+ cells from cases A1 to A3 expressed mRNA for TARC (Fig. 3A), whereas no expression was found in any of the five control subjects (representative data in Fig. 3A). Although CD4+ cells from the normal subjects gave only weak signals for MDC, the intensities of MDC in cases A1 to A3 were markedly higher. Because CD4+ cells from these group A patients contained high percentages of ATL cells monoclonally integrated with HTLV-I proviral DNA, ATL cells were again confirmed to produce TARC and MDC.



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  		Fig. 3 RT-PCR analysis of CD4+ cells and skin tumor cells for the expression of TARC and MDC. Total RNA was prepared from CD4+ cells obtained from three patients (cases A1-A3) and normal control subjects (A) and skin tumor cells of case A3 (B). RT-PCR was done for TARC, MDC, and ß-actin.

 
Lymphocytes were isolated from a skin tumor of case A3 and subjected to RT-PCR. The tumor-infiltrating cells strongly expressed mRNA for MDC even when isolated freshly (Fig. 3B). After 96-hour culture, the cells yielded clear signals for both TARC and MDC. The addition of anti-CD3/CD28 mAbs augmented TARC expression. Normal PBMC (control) expressed no and very weak mRNA for TARC and MDC, respectively. These data further supported the TARC and MDC production by ATL cells.

Chemokines Other Than Thymus and Activation-Regulated Chemokine and Macrophage-Derived Chemokine Produced by Adult T-Cell Leukemia Cells. We also examined chemokines other than TARC and MDC produced by CD4+ PBMC and skin tumor cells on stimulation with anti-CD3/CD28 mAbs. Data are summarized in Table 4 and the values above the mean ± 2 SD of the controls are italicized. In some of the monoclonally integrated cases, high production levels of CXCL10/IP-10, CCL5/RANTES, and CXCL8/IL-8 were observed. On the other hand, CXCL9/MIG or CCL2/MIP-1 was not significantly elevated in most of the cases. None of the polyclonally integrated cases exhibited high values. The skin tumor cells produced IP-10, MIG, and RANTES at high levels, but this might stem from the coinfiltrating cells. Thus, not only TARC or MDC but also those chemokines were possibly elaborated by ATL cells variously from case to case. However, the production of TARC and MDC was important because the cells concomitantly expressed CCR4.


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  		Table 4 Chemokine profiles in CD4+ PBMC and tumor-infiltrating cells in stimulation with anti-CD3/CD28 mAbs

 
Immunostaining for Thymus and Activation-Regulated Chemokine and CCR4 in Adult T-Cell Leukemia Skin Lesions. Skin specimens from all the patients, except for case A7 and one patient with extranodal NK/T-cell lymphoma (nasal type) as control, were immunohistochemically stained for TARC and CCR4. As shown representatively in cases A1 to A4 (Fig. 4), skin-infiltrating large tumor cells had a strong immunoreactivity for CCR4 in all the patients tested. ATL tumor cells were also positively stained for TARC in cases A1 to A4, B1, and B2. This result was consistent with the ELISA-assessed TARC production by skin-infiltrating cells (see Fig. 1E). In NK/T-cell lymphoma, tumor cells were negative for both TARC and CCR4.



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  		Fig. 4 Immunohistochemical staining for TARC and CCR4 in lesional skin. Deparaffinized sections of specimens from skin lesions were immunohistochemically stained with anti-CCR4 or anti-TARC mAb. NK/T-cell lymphoma (NK-T) was employed as negative control.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study showed that neoplastic T cells present in PBMC and lesional skin of ATL patients highly expressed Th2 chemokine receptor CCR4 as has been reported recently by other groups (15, 16). Despite the CCR4 expression, ATL cells were not functional Th2 cells because they mostly secreted low levels of IL-4, IL-5, and IL-10. The increased production of IFN-{gamma}, IL-2, and tumor necrosis factor-{alpha} observed in some cases is consistent with the previous studies on the cytokine production pattern of ATL cell lines and HTLV-I-immortalized T cells, which showed no apparent Th1 or Th2 cytokine profile (24, 25). On the other hand, ATL cells share the CD4+CD25+ phenotype with regulatory T cells (23). In two cases, we found that ATL cells expressed CTLA-4, a marker for regulatory T cells (28). Another group of investigators also detected the expression of forkhead/winged helix transcription factor (Foxp3), a specific molecule important for the function of regulatory T cells, by using quantitative RT-PCR and immunostaining of ATL cells. However, the expression was relatively down-regulated compared with normal subjects (29). Therefore, it is unlikely that ATL cells serve as immunologically functional Th2 or regulatory T cells.

Of particular interest, in our study, is the finding that ATL cells expressed and produced high amounts of CCR4 ligands, TARC and MDC, as assessed by ELISA, RT-PCR, and immunohistochemistry. Although CD4 purification was not a complete method for isolation of ATL cells, the production of TARC and MDC by the tumor cell was convinced by the following observations: (a) CD4+ PBMC from case A1 having 97% of CD25+ and 90% of CCR4+ cells, respectively, apparently produced high levels of TARC and MDC; (b) CD4+ PBMC monoclonally integrated with proviral DNA secreted higher amounts of TARC and MDC than polyclonally integrated PBMC, although the monoclonally integrated cases were not necessarily high producers of these chemokines; (c) CD4+CD14– cells, but not CD14+ cells, produced TARC and MDC, indicating that the main producer of TARC and MDC are the ATL cells but not monocytes; (d) lymphocytes isolated from skin tumors produced TARC and MDC at high levels; and (e) skin-infiltrating large tumor cells were immunohistochemically positive for TARC.

Several cellular sources of TARC and MDC have been identified, including macrophages, dendritic cells, NK cells, bronchial epithelial cells (30–32), and even several malignant tumors (33–35). In cutaneous T-cell lymphoma (CTCL), the skin infiltration of malignant T cells expressing CCR4 and CLA is associated with the abundant expression of TARC and MDC by keratinocytes (7, 8). Thus, several authors have speculated that malignant T cells migrate into skin tissues by the guidance of TARC and MDC produced by keratinocytes (7, 8). Accordingly, Yoshie et al. (15) and Ishida et al. (16) have detected mRNA for TARC, MDC, and CCR4 in ATL skin lesions, suggesting that the source of TARC and MDC is, at least, local skin tissues. However, it has been shown recently that normal human keratinocytes are incapable of producing TARC in vitro even on stimulation with the effective combination of cytokines (36, 37). Our study strongly suggests that the main source of TARC in the lesional skin of ATL is the tumor cell per se.

About 50% of patients with ATL have skin involvement (5). Although the skin eruptions seen in ATL resemble those in CTCL, the most frequent type in ATL is nodular/tumors, which occur in CTCL only at the advanced stage. Similarly to or more remarkably than ATL cells, CTCL malignant T cells have the Th2 nature (38, 39) and the ability to proliferate in response to TCR/CD3-mediated stimuli (40). However, ATL cells seem to be different from CTCL cells in that they produce TARC and MDC when compared with the reported immunostaining results in CTCL (8). In comparison with CTCL, the production of these Th2 chemokines may lead to the preponderant formation of tumors in ATL.


   ACKNOWLEDGMENTS
 
We thank Dr. Kouji Matsushima and Kyowa Hakko Kogyo (Tokyo, Japan) for providing anti-CCR4 mAb (KM2160) and Dr. Etsushi Kuroda and Junko Nagai for skillful technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3/10/04; revised 12/20/04; accepted 12/29/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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