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[Cancer Research 63, 6469-6477, October 1, 2003]
© 2003 American Association for Cancer Research

Immunology

Selectively Increased Expression and Functions of Chemokine Receptor CCR9 on CD4+ T Cells from Patients with T-Cell Lineage Acute Lymphocytic Leukemia1

Zhang Qiuping2, Li Qun2, Hu Chunsong, Zhang Xiaolian, Huang Baojun, Yang Mingzhen, Lao Chengming, He Jinshen, Gao Qingping, Zhang Kejian, Sun Zhimin, Zhang Xuejun, Liu Junyan and Tan Jinquan3

Department of Immunology, Medical College [Z. Q., Z. Xi., L. J., T. J.], and Department of Hematology, The First and Second Affiliated University Hospital [G. Q., Z. K.], Wuhan University, 430071 Wuhan; Department of Immunology, College of Basic Medical Sciences, Anhui Medical University, 230032 Hefei [L. Q., H. C., H. B., L. C., H. J., Z. Xu., T. J.]; Department of Hematology, The Affiliated University Hospital, Anhui Medical University, 230031 Hefei [Y. M.]; and Department of Hematology, The Provincial Hospital of Anhui, 230020 Hefei [S. Z.], Peoples Republic of China


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a total of 38 typical T-cell lineage acute lymphocytic leukemia (T-ALL) and T-cell lineage chronic lymphocytic leukemia (T-CLL) cases investigated, we found that CC chemokine receptor CCR9 was selectively and frequently expressed on T-ALL CD4+ T cells, was moderately expressed on T-CLL CD4+ T cells, and was rarely expressed on normal CD4+ T cells. These findings were demonstrated at protein and mRNA levels using flow cytometry and real-time quantitative reverse transcription-PCR technique and were verified by digital confocal microscopy and Northern blotting. Thymus-expressed chemokine, a ligand for CCR9, selectively induced T-ALL CD4+ T-cell chemotaxis and adhesion. Interleukin (IL)-2 and IL-4, together, down-regulated the expression and functions of CCR9 in T-ALL CD4+ T cells including chemotaxis and adhesion. It was also demonstrated that IL-2 and IL-4, together, internalized CCR9 on T-ALL CD4+ T cells and subsequently inhibited functions of CCR9 in these cells. Thymus-expressed chemokine mRNA was highly expressed in CD4+ T cells, involving lymph node and skin in T-ALL patients, and was expressed at moderate levels in lymph node and skin tissues in T-CLL patients. Our findings may provide new clues to understanding various aspects of T-ALL CD4+ T cells, such as functional expression of CCR9-thymus-expressed chemokine receptor-ligand pairs as well as the effects of IL-2 and IL-4, which may be especially important in cytokine/chemokine environment for the pathophysiological events of T-ALL CD4+ T-cell trafficking.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A common manifestation of T-ALL4 and T-CLL is infiltration of various organs, such as the lymph nodes, liver, spleen, lungs, skin, intestinal tract, and even brain by leukemic cells (1) . Malignant lymphocyte (particularly T cells) migration into and from organs is an important aspect of T-ALL and T-CLL because leukemic cell infiltration often causes serious clinical problems for patients, affecting the disease profile and prognosis (1, 2, 3, 4, 5) .

CLL B cells overexpress functional CXCR4 receptors for the chemokine stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12; Refs. 6 and 7 ). SDF-1{alpha}/CXCL12 and CXCR4 play an important role in influencing the localization of ALL cells in marrow microenvironment that regulate their survival and proliferation (8) . CXCR4 is functionally expressed on primary acute myeloblastic leukemia cells (9) . CXCR3 is selectively expressed in distinct subtypes of malignant B cells, particularly in CLL (10 , 11) . CCR4 expression accounts for frequent infiltration of adult T-cell leukemia (ATL) cells into tissues such as skin and lymph nodes (4) . CCR7 has been found to be overexpressed in lymphoid organ infiltration of ATL cells (5) . CCR7 and {alpha}4 integrin are important for the migration of CLL cells into lymph nodes (1) . Despite these findings, little is known about the exact mechanisms and molecules that regulate the homing, retention, and migration of ALL and CLL cells into organs.

CCR9, together with TECK/CCL25, efficaciously induces chemotaxis of immature CD4+CD8+ double-positive and mature CD4+ and CD8+ single-positive thymocytes, suggesting that TECK/CCR9 interaction play a pivotal role in T-cell migration in the thymus (12 , 13) . There are two forms of CCR9: CCR9A and CCR9B (14) . TECK/CCL25 delivers signals through CCR9 for the developing of thymocytes (15) and the developing and/or migrating of both {alpha}ß- and {gamma}{delta}- T cells (16) . CCR9 activation leads to phosphorylation of GSK-3ß and FKHR and provides a cell survival signal (17) . In the normal human, CCR9 are restrictedly expressed at high levels on CD4+ and CD8+ T cells in the small intestine but not in other tissues including tonsils, lung, inflamed liver, normal, or inflamed skin (18) , providing the evidence for distinctive mechanisms of lymphocyte recruitment.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Cell Purification.
All of the patients with T-ALL fulfilled the French-American-British (FAB) Cooperative Group criteria (19) . Age range of patients was 5–52 years with 13 males and 8 females. All of the patients with T-CLL were diagnosed according to the guidelines of the National Cancer Institute Working Group on B-cell lineage chronic lymphocytic leukemia (B-CLL) and classified according to the FAB classification proposed in 1989 (20 , 21) . Age range of patients was 5–61 years including 10 males and 7 females. All of the patients gave informed consent according to institutional guidelines. Overall information of patients is included in Table 1Citation . CD4+ and CD8+ T cells were purified from PBMCs by a positive selection procedure of a-CD4 or a-CD8 mAb-coated Dynabeads (Dynal A/S, Dynal, Norway). The purity of CD4+ and CD8+ T cells ranged from 93 to 99% as determined by flow cytometry. The malignancy of purified T-ALL or T-CLL CD4+ T cells is shown in the Table 2Citation . CD4+CD8+ T cells (99% pure) were obtained from PBMCs using fluorescence-activated cell sorting. a-CD25 (2A3), a-CD45RO (UCHL-1), and a-HLA-DR (L243) mAbs were purchased from BD PharMingen (San Diego, CA). Statistical information listed in the Text for flow cytometry, RT-PCR, chemotaxis, adhesion assay, and immunofluorescence data compare variation of data among sets of patients’ samples (n = number of samples).


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  		Table 1 Clinical information for all of the patients investigated

 

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  		Table 2 Correlation between CCR9 expression in leukemic CD4+ T cells and organ involvement

 
Flow Cytometry.
For the detection of CCR4, CCR9, or CXCR3, the cells were first incubated with PE-labeled a-CCR4 (112509), FITC-labeled a-CCR9 (1G1), or a-CXCR3 mAb (49801.111; R&D Systems, Abingdon, United Kingdom) or matched isotype antibody (DAKO) at 5 µg/ml in PBS containing 2% BSA and 0.1% sodium azide for 20 min, followed by washing twice in staining buffer (22) . The cells were incubated with a mouse PE-labeled (or FITC-labeled) a-CD4 or a-CD8 mAb (DAKO, Glostrup, Denmark) at 5 µg/ml for 20 min, followed by washing twice. The analyses were performed with a flow cytometer (COULTER XL, Coulter, Miami, FL). For CD4, CD8, and CCR9 triple staining, PE-labeled CD4, PerCP-labeled CD8 (Dako), and FITC-labeled CCR9 mAbs were applied with the procedure described above. For internalization assays, percentage internalization was calculated as [MFIc - MFIt)/MFIc] x 100, where MFIc = mean fluorescence intensity of anti-CCR9 binding to the cells cultured with medium only, and MFIt = mean fluorescence intensity of cells after incubation with IL-2 and/or IL-4 (23 , 24) .

Real-Time Quantitative RT-PCR Assay.
Total RNA from purified cells (1 x 106, purity >99%) was prepared using Quick Prep total RNA extraction kit (Pharmacia Biotech; Refs. 25, 26, 27 ), then was reverse transcribed using oligo (dT)12–18 and Superscript II reverse transcriptase (Life Technologies, Inc., Grand Island, NY). The real-time quantitative PCR was performed in a 96-well microtiter plate (Applied Biosystems, Foster City, CA) with an ABI PRISM 7700 Sequence Detector Systems (Applied Biosystems). Using SYBR Green PCR Core Reagents kit (Applied Biosystems; P/N 4304886), we generated fluorescence signals during each PCR cycle via the 5' to 3' endonuclease activity of AmpliTaq Gold (26) to provide real-time quantitative PCR information. The target genes were generated by connecting the following sequences of the specific primers: CCR4 sense: 5'-ACTGTGGGCTCCTCCAAATTT-3'; CCR4 antisense: 5'-CATGGTGGACTGCGTGTAAGA-3'; CCR9 sense: 5'-CATTGACGCCTATGCCATGT-3'; CCR9 antisense: 5'-GACCTGGAAGCAGATGTCAATGT-3'; TECK/CCL25 sense: 5'-AGCGGGAGCTGCAATCTG-3'; TECK/CCL25 antisense: 5'-GGGTTCCCACACACCTTCCT-3'.

All unknown cDNAs were diluted to contain equal amounts of ß-actin cDNA. PCR retained conditions were 2 min at 50°C, 10 min at 95°C, and 40 cycles with 15 s at 95°C and 60 s at 60°C in each cycle.

Northern and Western Blot Assays.
For mRNA detection (Northern blot), each 5 µg of total RNA were electrophoresed under denaturing conditions, followed by blotting onto Nytran membranes and were cross-linked by UV irradiation (28) . CCR4 and CCR9 cDNA probes, labeled by [{alpha}-32P]dCTP, were obtained by PCR amplification of the sequence mentioned above from total RNA from PBMCs from normal adults (CCR4) and thymocytes from the specimen of thymusectomy (CCR9). The membranes were hybridized overnight with 1 x 106 cpm/ml of 32P-labeled probe, followed by intensive washing with 0.2x SSC and 0.1% SDS before being autoradiographed. For protein detection (Western blot), the cells were lysed in lysis buffer (29) . Lysates were centrifuged at 10,000 rpm for 5 min at 4°C. Protein concentration was measured by Bio-Rad protein assay. Total protein (40 µg) was loaded onto 16% SDS-PAGE, transferred onto polyvinylidene difluoride membranes after electrophoresis, and incubated with the CCR9 mAb (0.5 µg/ml). Analyses were conducted using enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).

Immunofluroescence Digital Confocal Microscopy.
The purified cells were spun down on a slide, fixed with a mixture of methanol and acetone, immersed in 1% BSA blocking buffer for 10 min to avoid nonspecific binding; antibody, either FITC-labeled CCR9 mAb or isotype IgG2a, was added at 10 µg/ml, and the cells were incubated overnight at 4°C, followed by the addition of the PE-labeled CD4 mAb. For internalization detection, cells were permeabilized for 30 min on ice in PBS containing 2% fetal bovine serum and 20 µg/ml cholera toxin B Alexa Fluor 594 before staining. Cells were then stained only with FITC-labeled CCR9 mAb or isotype IgG2a at 10 µg/ml. The preparations were observed using a fluorescence microscope (model BX60; Olympus, Japan). Confocal microscopy analysis was performed using a confocal laser scanning system and an inverted microscope (model LSMSIO; Zeiss, Berlin, Germany). Images of serial cellular section were acquired with the Bio-Rad Comos graphical user-interface as described previously (23 , 24 , 30) .

Chemotaxis Assay.
The chemotaxis assay was performed in a 48-well microchamber (Neuro Probe, Bethesda, MD) technique (31) . Briefly, chemokine (MDC/CCL22, TECK/CCL25 or {gamma}IP-10/CXCL10; purchased from R&D Systems) in RPMI 1640 with 0.5% BSA was placed in the lower wells (25 µl). Twenty-five µl of cell suspension (2 x 106 cells/ml) were added to the upper well of the chamber, which was separated from the lower well by a 5-µm pore-size, polycarbonate, polyvinylpyrrolidone-free membrane (Nucleopore, Pleasanton, CA). The cells were CD4+ T cells positively isolated using CD4 MACS MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The chamber was incubated for 60 min at 37°C and 5% CO2. The membrane was then carefully removed and was stained for 5 min in 1% Coomassie Brilliant Blue. Approximately 6% of the cells will migrate spontaneously (known as MCNC; Ref. 32 ). The results were expressed as C.I. with SD (31) . For blocking tests, the cells were preincubated with either anti-CCR9 mAb or IgG2a isotype antibody at 10 µg/ml for 120 min at room temperature before chemotaxis assay.

Adhesion Assays.
As described previously (33) , 96-well plates were coated with laminin (20 µg/ml; Sigma Chemical Co.) in PBS for 1 h at 37°C. The single-cell suspensions (4 x 105 cells/ml) with 0.2% BSA were added to the appropriate chemokine. The cell suspension was added at 100 µl/well in triplicate to the plates, and incubated for 60 min at 37°C. The wells were then washed with 0.2% BSA in PBS, followed by careful aspiration. Subsequently, the adherent cells were fixed with 1% formaldehyde and stained with 1% crystal violet. Crystal violet was then extracted by the addition of a 1:1 mixture of sodium citrate (0.1 M) and ethanol (pH 4.2). The absorbency was then read at 540 nm. Background cell adhesion to 2% BSA-coated wells was subtracted from all readings.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CCR9 Expression on CD4+ Cells from T-ALL Patients Was Selectively Increased.
In a total of 38 cases of T-ALL and T-CLL patients (Table 1)Citation , T-ALL and T-CLL CD4+ T cells expressed CCR4 moderately (Fig. 1A)Citation , whereas normal cells expressed CCR4 rarely. T-ALL CD4+ T cells highly expressed CCR9 (91.9%), whereas T-CLL CD4+ T cells expressed CCR9 moderately, and normal CD4+ T cells expressed CCR9 rarely (Fig. 1A)Citation . Expression of CXCR3 on CD4+ T cells from all different subjects was very low. CD8+ T cells from all different subjects expressed CCR9 at an equally low-level (Fig. 1B)Citation . CCR9 expression was much higher on T-ALL CD4+CD8+ T cells compared with CD4-CD8- T cells (92 ± 4%, 4 ± 2%; n = 12; P < 0.0001). CCR9 was expressed moderately T-CLL CD4+CD8+ T cells compared with CD4-CD8- T cells (36 ± 6%, 5 ± 3%; n = 12; P < 0.01). Expression levels of CCR4 and CCR9 on CD4+ T cells from patients with acute myeloblastic leukemia were similar to the levels on the normal cells (data not shown).



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  		Fig. 1. CCR4 and CCR9 distribution. Double color flow cytometric analysis of the distribution of CCR4, CCR9, or CXCR3 on CD4+ (A) or CD8+ (B) T cells from normal subjects (T-NOL) and T-ALL and T-CLL patients. The CD4+ and CD8+ T cells were freshly isolated and stained as described in "Materials and Methods." The graphs in the lowest far left panels are isotype controls. The indicated numbers in the graphs are percentages of CCR4+, CCR9+, or CXCR3+ T cells. The data are from a single experiment, which is representative of 17 (T-CLL) and 21 (T-ALL) similar experiments. The illustrated data are from patient 6 (T-ALL) and patient 35 (T-CLL) listed in Tables 1Citation and 2Citation .

 
To be sure that examined CD4+ T cells from patients with T-ALL and T-CLL were leukemic, the expression of CD25, CD45RO, and HLA-DR in these cells was investigated (Refs. 4 , 33 , 34 ; Table 2Citation ). The majority of purified CD4+ T cells from patients with T-ALL and T-CLL were CD25, CD45RO, and HLA-DR positive. Almost all of the T-ALL patients showed highly frequent expression of CCR9 (Table 2)Citation .

CCR4 mRNA was detected at low levels in freshly isolated normal CD4+ and CD8+ T cells (7.1 ± 0.71 and 8.4 ± 0.65 x 102 copies/50 ng cDNA; n = 8), moderate level in T-ALL CD4+ and CD8+ T cells (4.3 ± 0.71 and 3.4 ± 0.65 x 103 copies/50 ng cDNA; n = 8; all P < 0.01; versus normal; Fig. 2ACitation ). CCR9 mRNA in T-ALL CD4+ T cells was significantly and selectively up-regulated (1.4 ± 0.35 x 104 copies/50 ng cDNA; n = 8; P < 0.001; versus normal), and moderately up-regulated in T-CLL CD4+ T cells (5.3 ± 0.42 x 103 copies/50 ng cDNA; n = 8; P < 0.03; versus normal; Fig. 2BCitation ). Northern blots confirmed these observations (Fig. 2, C and D)Citation .



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  		Fig. 2. CCR4, CCR9, and TECK/CCL25 mRNA. The real-time quantitative detection of RT-PCR for mRNA of CCR4 (A) and CCR9 (B) in freshly isolated CD4+ and CD8+ T cells from normal subjects (open bars) and T-ALL (black bars) or T-CLL (gray bars) patients. The bars are representatives of eight similar experiments. The procedure for quantitative RT-PCR amplification was described in "Materials and Methods." The statistical error bars are from tests of the same sample in each experiment conducted in triplicate. Northern blot of CCR4 (C) and CCR9 (D) mRNA in freshly isolated CD4+ T cells from normal subjects (T-NOL) and T-ALL or T-CLL patients. Total RNA from different cells as indicated were isolated, electrophoresed, and blotted as described in "Materials and Methods." Top panels, the hybridization signals for CCR4 or CCR9 mRNA in CD4+ T cells from different subjects. The 28S rRNAs in lower panels confirm the comparable amounts of loaded total RNA. The illustrated data are from a single representative experiment (T-ALL patient 8 and T-CLL patient 24) of six performed. The real-time quantitative detection of RT-PCR for mRNA of TECK/CCL25 (E) in freshly isolated CD4+ T cells, involved lymph nodes (LN) and involved skin from normal subjects (open bars) and T-ALL (black bars) or T-CLL (gray bars) patients. The bars are representatives (T-ALL patient 14 and T-CLL patient 22) of six similar experiments.

 
CCR9 was rarely expressed on normal CD4+ T cells detected by immunofluorescence digital confocal microscopy (Fig. 3A)Citation . Only one of nine CD4+ T cells in a representative field was showing CCR9 positive. In contrast, all T-ALL CD4+ T cells in a representative field were showing CCR9 positive (Fig. 3B)Citation . 3 of 8 T-CLL CD4+ T cells in a representative field were showing CCR9 positive (Fig. 3C)Citation . There were high statistically significant differences of CCR9+ cell percentages in CD4+ T cells among normal subjects and T-ALL or T-CLL with detected by immunofluorescence microscopy (Fig. 3D)Citation .



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  		Fig. 3. CCR9 distribution. The CCR9 distribution in normal CD4+ T cells (T-NOL; A), T-ALL (B) or T-CLL (C) patients. The CD4+ T cells were collected from different subjects as indicated, and stained as described in "Materials and Methods." The cells were photographed under epifluorescent conditions. x1200. Bar, 9 µm. Arrows, typical CD4+CCR9+ double-positive cells. The images were taken in a single experiment (T-ALL patient 20 and T-CLL patient 25), which is a representative of experiments on six T-CLL and six T-ALL patients. The CCR9+ cells percentages in normal CD4+ T cells (T-NOL), T-ALL, or T-CLL patients (D). Cells were counted in hundreds per field in each representative field under fluorescent microscope, and positive cells were recorded. The showing data were averages of each group of six subjects investigated. *, statistically significant difference in T-ALL patients versus normal subjects (P < 0.001); in T-CLL patients versus normal subjects (P < 0.01).

 
Functions of CCR9 in T-ALL CD4+ T Cells Were Also Up-Regulated.
TECK/CCL25 (a ligand for CCR9) induced a high chemotactic migration of T-ALL CD4+ cells (C.I. = 3.6 ± 0.72 at 100 ng/ml; n = 8; P < 0.001; versus normal). MDC/CCL22 (a ligand for CCR4) induced a moderate chemotactic migration of T-ALL CD4+ cells (C.I. = 2.2 ± 0.48 at 100 ng/ml; n = 8; P < 0.01; versus normal; Fig. 4BCitation ). {gamma}IP-10/CXCL10 (a ligand for CXCR3) did not induce significant chemotactic migration in the cells (C.I. = 1.2 ± 0.35 at 100 ng/ml; n = 8; P > 0.05; versus normal; Fig. 4BCitation ). {gamma}IP-10/CXCL10 did induce a weak chemotaxis in normal CD4+ T cells, whereas the other two chemokines mentioned above did not induce significant chemotactic migration in normal CD4+ T cells (Fig. 4A)Citation . MDC/CCL22 and TECK/CCL25 induced chemotaxis slightly in T-CLL CD4+ T cells (C.I. = 1.5 ± 0.37 and 1.6 ± 0.42 at 100 ng/ml; n = 8; all P > 0.05; versus normal), whereas {gamma}IP-10/CXCL10 did not (Fig. 4C)Citation . Spontaneous migration negative control (known as MCNC) of different CD4+ T cells was ~6% of total cells added, indicated as C.I. = 1 (Fig. 4)Citation . The a-CCR9 mAb could completely block the chemotaxis of T-ALL CD4+ T cells toward TECK/CCL25 (C.I. = 1.0 ± 0.12 at 100 ng/ml; n = 4; P < 0.001; versus that without antibody in Fig. 4BCitation ), whereas it had no any effect on the chemotaxis of T-ALL CD4+ T cells toward MDC/CCL22 (Fig. 4D)Citation . The isotype antibody had no blocking effect at all (Fig. 4D)Citation . It confirmed that the observed T-ALL CD4+ T cell chemotaxis was indeed induced by TECK/CCL25 via CCR9. The checkerboard chemotaxis assays demonstrated that enhanced motility of the T-ALL CD4+ T cells toward the chemokine (TECK/CCL25) was caused by chemotaxis, not by chemokinesis (data not shown; 35 , 36 ). Interestingly, TECK/CCL25 induced a very strong chemotaxis in purified T-ALL CD4+CD8+ cells (C.I. = 4.8 ± 1.32 at 100 ng/ml; n = 12). T-CLL CD4+CD8+ cells showed weak chemotactic response toward TECK/CCL25 (C.I. = 2.1 ± 0.67 at 100 ng/ml; n = 6).



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  		Fig. 4. Chemotaxis analysis. The migration of freshly isolated CD4+ T cells from normal subjects (T-NOL, A), T-ALL (B), T-CLL (C), or T-ALL (D; a-CCR9 mAb or isotype antibody blocking) patients toward chemokines as indicated. All of the results were determined as described in "Materials and Methods" and were expressed as C.I. (Chemotactic Index) ± SD; calculations were based on triplicate determination of chemotaxis on each concentration of chemokine as indicated. The applied chemokine concentrations (ng/ml) are indicated. Open bars, spontaneous migration toward negative (medium) control (known as MCNC; C.I. = 1) in each experiment. The illustrated data are from a single representative experiment (T-ALL patient 8 and T-CLL patient 28) of eight performed. For blocking experiments, comparisons are D versus B under the same chemokine concentration.

 
Compared with normal subjects, TECK/CCL25 mRNA was highly expressed in purified T-ALL CD4+ T cells, involved lymph node and skin from T-ALL patients, whereas it was at moderately increased level in these tissues from T-CLL patients (Fig. 2E)Citation .

TECK/CCL25 induced a high adhesion of T-ALL CD4+ cells (36 ± 8.2% of cells adhered at 100 ng/ml; n = 8; P < 0.001; versus normal). MDC/CCL22 induced a moderate adhesion of T-ALL CD4+ cells (22 ± 6.7% of cells adhered at 100 ng/ml; n = 8; P < 0.01; versus normal). {gamma}IP-10/CXCL10 only slightly induced adhesion in the cells (16 ± 3.1% of cells adhered at 100 ng/ml; n = 8; P < 0.05; versus normal; Fig. 5BCitation ). {gamma}IP-10/CXCL10 induced a weak adhesion in normal CD4+ T cells, whereas the other two chemokines mentioned above did not induce significant adhesion in normal CD4+ T cells (Fig. 5A)Citation . MDC/CCL22 and TECK/CCL25 induced moderate adhesion in T-CLL CD4+ T cells (20 ± 3.7% and 22 ± 3.9% of cells adhered at 100 ng/ml; n = 8; all P < 0.01; versus normal), whereas {gamma}IP-10/CXCL10 did not (Fig. 5C)Citation . Spontaneous adhesion of different CD4+ T cells is around 10% of total added cells (Fig. 5)Citation . The anti-CCR9 mAb could completely block the adhesion of T-ALL CD4+ T cells toward TECK/CCL25 (data not shown), whereas the isotype antibody had no blocking effect at all (data not shown).



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  		Fig. 5. Adhesion analysis. The adhesion of freshly isolated CD4+ T cells from normal subjects (T-NOL, A) and T-ALL (B) and T-CLL (C) patients induced by MDC/CCL22, TECK/CCL25, and {gamma}IP-10/CXCL10. The results were determined as described in "Materials and Methods" and expressed as percentages of adherent cells ± SD, and based on triplicate determination of adhesion on each of the chemokines applied. Open bars, spontaneous adhesion in medium control. The illustrated data are from a single representative experiment (T-ALL patient 15 and T-CLL patient 32) of eight similar experiments performed.

 
IL-2 and IL-4, Together, Regulated Expression and Functions of CCR9 on T-ALL CD4+ T Cells.
IL-2 and IL-4 induced the expression of CCR3 on T lymphocytes (33) . The survival, proliferation, and differentiation of leukemia lymphocytes were partially controlled by cytokines such as IL-2 (37) and IL-4 (38) . IL-2 and/or IL-4 did not significantly change CCR4 expression on T-ALL CD4+ T cells. CCR4 constantly expressed on the T-ALL CD4+ T cells under different culture conditions (41 ± 6%; n = 8; all P > 0.05; Fig. 6ACitation ), and CCR3 expression on T-ALL CD4+ T cells (date not shown). IL-2 and IL-4, together, significantly reduced CCR9 expression on T-ALL CD4+ T cells (Fig. 6B)Citation , compared with the freshly isolated cells (Fig. 1)Citation and the cells in culture only, or with IL-2 or IL-4 alone (Fig. 6B)Citation . Fresh T-ALL CD4+ T cells highly expressed CCR9 (91.9%; Fig. 1BCitation ), whereas the cultured CD4+ T cells still expressed identical amounts of CCR9 (83 ± 7%; n = 8; Fig. 6BCitation ). After culture with IL-2 or IL-4 alone, the CCR9 expression on T-ALL CD4+ T cells was 82 ± 8% (n = 8; P > 0.05, versus that in culture only) or 80 ± 9% (n = 8; P > 0.05, versus that in culture only), respectively. The combination of IL-2 and IL-4 significantly reduced the CCR9 expression T-ALL CD4+ T cells (16 ± 4%; n = 8; P < 0.001; versus that in culture only). The preincubation of the cells with antibodies toward IL-2 receptor and IL-4 receptor blocked inhibitory function of IL-2 and IL-4 together on CCR9 expression (88 ± 9%; n = 8; P > 0.05; versus that in culture only), whereas isotype antibodies did not have such function (14 ± 8%; n = 8; P < 0.001; versus that in culture only; Fig. 6BCitation ). In the normal CD4+ T cells, we did not observe such phenomenon (date not shown).



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  		Fig. 6. Regulation of CCR4 and CCR9 expression. Double color flow cytometric analysis of the regulation on CCR4 (A) and CCR9 (B) expression on CD4+ T cells from T-ALL patients by cytokine as indicated. The CD4+ T cells were isolated according to procedure described in "Materials and Methods" and, subsequently, were cultured with or without IL-2 (10 ng/ml) and/or IL-4 (10 ng/ml) for 24 h as indicated. All of the cytokine receptor antibodies (Abs) were applied at 5 µg/ml. The procedure for cell staining with PE-labeled CCR4 or FITC-labeled CCR9 and FITC-labeled or PE-labeled CD4 mAbs was described in "Materials and Methods." The graphs are illustrated as single histograms. The graphs in the far left panels are isotype (Iso) controls. The percentages of CCR4+ and CCR9+ cells are indicated in the "Results." The data are from a single experiment (T-ALL patient 16 and T-CLL patient 33) that is representative of eight similar experiments performed.

 
TECK/CCL25 induced a high chemotactic migration of cultured T-ALL CD4+ cells. IL-2 and IL-4, together, totally abolished chemotactic migration of T-ALL CD4+ cells toward TECK/CCL25 (C.I. = 1.0 ± 0.22 at 100 ng/ml; n = 8; P < 0.001; versus that in culture only; Fig. 7ACitation ). TECK/CCL25 induced significantly high adhesion of cultured T-ALL CD4+ T cells, which was identical to the results in the freshly isolated cells (Fig. 5B)Citation . IL-2 and IL-4, together, totally inhibited the adhesion of T-ALL CD4+ cells induced by TECK/CCL25 (12 ± 4.3% of cells adhered at 100 ng/ml; n = 8; P < 0.001; versus that in culture only; Fig. 7BCitation ). IL-2 or IL-4 alone could affect neither chemotaxis nor adhesion of T-ALL CD4+ T cells induced by TECK/CCL25 (data not shown).



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  		Fig. 7. Chemotaxis and adhesion analysis. The migration (A) and adhesion (B) of T-ALL CD4+ T cells toward TECK/CCL25. The cells were cultured with or without IL-2 and/or IL-4 as described in the legend for Fig. 6Citation . All of the results were determined as described in "Materials and Methods" as well as in the legends for Figs. 4Citation and 5Citation . Open bars, spontaneous migration or adhesion toward medium. The results are expressed as C.I. or percentages of adherent cells ± SD. The illustrated data are from a single representative experiment (T-ALL patient 17 and T-CLL patient 31) of eight similar experiments performed.

 
To our surprise, the expressions of CCR9 mRNA in T-ALL CD4+ T cells under different culture conditions were almost identical to each other (Fig. 8A)Citation . These CCR9 mRNA expression levels were also identical to the level in freshly isolated T-ALL CD4+ T cells (Fig. 2B)Citation . Northern blots (Fig. 8B)Citation at mRNA level and Western blots (Fig. 8C)Citation at protein level confirmed these observations. CCR9 only distributed on cellular membrane of medium-cultured T-ALL CD4+ T cells detected by confocal microscopy (Fig. 9A)Citation . The similar distribution pattern was observed on the cells cultured in the presence of IL-2 (Fig. 9B)Citation or IL-4 (Fig. 9C)Citation . CCR9 internalization occurred in T-ALL CD4+ T cells cultured with IL-2 and IL-4 together (Fig. 9D)Citation . We calculated the ratio of CCR9 internalization in T-ALL CD4+ T cells under different culture conditions (Fig. 9E)Citation . CCR9 internalization significantly occurred when the cells were cultured with IL-2 and IL-4 together (78% ± 6%; n = 8; P < 0.001; versus that in culture only). It did not happen under other circumstances as indicated in Fig. 9Citation (2% ± 0.8% and 3 ± 0.4%; n = 8; all P > 0.05; versus that in culture only). Thus, IL-2 and IL-4, together, internalized CCR9 on T-ALL CD4+ T cells and subsequently inhibited functions of CCR9 in these cells.



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  		Fig. 8. CCR9 mRNA and protein. The real-time quantitative detection of RT-PCR (A), Northern blot (B), and Western blot (C) analyses for mRNA and protein of CCR9 in T-ALL CD4+ T cells. The cells were cultured with or without IL-2 and/or IL-4 as described in the legend for Fig. 6Citation . The bars shown are representative of six similar experiments. The procedure for quantitative RT-PCR amplification and Northern blot were described in "Materials and Methods" as well as in the legend for Fig. 2Citation . CCR9 protein was examined by Western blot analyses (C). The cultured cells were lysed and total protein was obtained, electrophoresed, and blotted as described in "Materials and Methods." The arrows were used to verify equivalent molecular weights of appropriate proteins in each lane. The illustrated data are from a single representative experiment (T-ALL patient 9 and T-CLL patient 23) of four similar experiments performed. KD, Mr in thousands.

 


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  		Fig. 9. CCR9 internalization. Effects of IL-2 and IL-4 (R&D Systems) on CCR9 internalization in T-ALL CD4+ T cells detected using confocal microscopy (A–D). The CD4+ T cells were isolated and were subsequently cultured with medium only (A) or with IL-2 (B) or IL-4 (C) or IL-2 and IL-4 together (D) for 24 h, described in "Materials and Methods." x1200. Bar, 9 µm. Arrow, typical CCR9 internalized cell. CCR9 internalization of IL-2 and/or IL-4-treated T-ALL CD4+ T cells (E). The cells were cultured without or with IL-2 and/or IL-4 as described in the legend for Fig. 6Citation . The data (T-ALL patient 11 and T-CLL patient 38) represent eight separate experiments; error bars, SD determined as described in "Materials and Methods." Ten thousand cells were measured in each acquisition. Abs, antibodies.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A coordinated multistep process is involved during lymphocyte homing into lymphoid organs under the normal condition (39 , 40) . T-ALL, particularly in childhood, is a malignancy with the potential to infiltrate the liver, spleen, lymph nodes, and even brain (41) . The observations on increased migration of malignant lymphoblasts indicate the involvement of chemokine receptors and chemokines (10 , 42) . CXCR4, CCR4, and CCR7, are involved in the increased migration of malignant lymphoblasts (1 , 4 , 5 , 11) . To our knowledge, this study is the first report on CCR9 expression on human T-ALL CD4+ T cells, and is the first direct evidence of the increased biological activity of T-ALL CD4+ T cells induced by TECK/CCL25.

We have documented that CCR4 is frequently expressed at moderate levels on T-ALL and T-CLL CD4+ and CD8+ T cells, and that its ligand, MDC/CCL22, induces freshly isolated T-ALL and T-CLL CD4+ and CD8+ T cell chemotaxis and adhesion. They are in agreement such degree with those of Yoshie et al. (4) who have described that frequent expression of CCR4 in ATL and T-cell leukemia virus type 1-immortalized T cells. In addition to finding selective expression of functional CCR9 on T-ALL CD4+, but not CD8+ T cells, we have also observed high levels of TECK/CCL25 mRNA expression in CD4+ T cells, involving lymph node and skin of T-ALL patients. Our results imply that CCR9, engaged TECK/CCL25, stimulates T-ALL CD4+ T cell migration in an autocrine manner, and that CCR9 expression is important for this process. TECK/CCL25 expression in involved organs was also elevated in the leukemic condition. The interpretation could be that a large number of TECK/CCL25 expressing T-ALL CD4+ T cell infiltrated into the organs, resulting in the elevation of TECK/CCL25 mRNA expression in the organs examined. The exact mechanism by which CCR9 and TECK/CCL25 mediate T-ALL CD4+ T cell trafficking in vivo should be further investigated.

Naïve T cells enter specialized microenvironments in which antigenic exposure occurs. These primed T cells recirculate to be continuously available for potential antigenic rechallenge (43, 44, 45) . Under leukemic condition, however, leukocyte transmigration between circulation and the tissues (organs) has significantly been changed (2 , 46, 47, 48) . In the present study, T-ALL CD4+ T cells express abnormally high CCR9, moderate CCR4, and very low CXCR3. This selectivity in the expression of chemokine receptors in the acute and chronic lymphoproliferative disorders implies the diversity of chemokine receptors in the physiology and pathophysiology of the process of development of T-ALL and T-CLL.

CXCR4 binding to its ligand SDF-1 regulate precursor B-cell lineage acute lymphocytic leukemia cell survival and proliferation (8) . CCR9 activation provides a cell survival signal (17) . In the present study, CCR9 and TECK are highly overexpressed on T-ALL CD4+ T cells. A logical implication may be that CCR9 and its ligands promote survival or proliferation of T-ALL cells. IL-4 inhibits in vitro proliferation of leukemia cells (49 , 50) , and affects acute leukemia patients with severe chemotherapy-induced cytopenia (51) . IL-2 induces significant immunomodulatory effects in pediatric acute leukemia patients (52) . IL-2 and IL-4, together, induce apoptosis of leukemic blasts from childhood T-cell acute lymphoblastic leukemia (53) . One important mechanism for regulating surface chemokine receptor expression is to induce recycling or internalization (23 , 24 , 54) . We have found that IL-2 and IL-4, together, induce internalization of CCR9 on T-ALL CD4+ T cells and, as a subsequence, inhibit chemotactic and adhesive functions of CCR9 in these cells in vitro. When we take into account the possible involvement of CCR9 in supporting the malignant cell proliferation, we conclude that our observations may have important implications in considering the effect of cytokine therapy on the disease process. In view of the data, the in vivo effects of administering a combination of IL-2 and IL-4 on the development of T-ALL in subjects requires further investigation.


   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.

1 Supported by the National Science Foundation of China (No. 39870674), a special grant from the Personnel Department of Wuhan University, China, Science Foundation of Anhui Province, China (No. 98436630), and Education and Research Foundation of Anhui Province, China (No. 98JL063). Back

2 Z. Q. and L. Q. contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Department of Immunology, Medical College, Wuhan University, Dong Hu Road 115, Wuchang 430071, Wuhan, P. R. China. Phone: 86-27-87331681; Fax: 45-35365326; E-mail: jinquan_tan{at}hotmail.com Back

4 The abbreviations used are: T-ALL, T-cell lineage acute lymphocytic leukemia; T-CLL, T-cell lineage chronic lymphocytic leukemia; C.I., chemotactic index; CCL, chemokine ligand; CCR, chemokine receptor; CXCL, CXC chemokine ligand; CXCR, CXC chemokine receptor; {gamma}IP-10, IFN-{gamma} inducible protein 10; MCNC, migrating cells on negative control; MDC, macrophage-derived chemokine; TECK, thymus-expressed chemokine; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; RT-PCR, reverse transcription-PCR; IL, interleukin; ATL, adult T-cell leukemia. Back

Received 9/ 6/02. Revised 6/26/03. Accepted 7/21/03.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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