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[CANCER RESEARCH 63, 6469–6477, October 1, 2003] Selectively Increased Expression and Functions of Chemokine Receptor CCR9 on CD4؉ T Cells from Patients with T-Cell Lineage Acute Lymphocytic Leukemia1

Zhang Qiuping,2 Li Qun,2 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 ment that regulate their survival and proliferation (8). CXCR4 is functionally expressed on primary acute myeloblastic leukemia cells In a total of 38 typical T-cell lineage acute lymphocytic leukemia (9). CXCR3 is selectively expressed in distinct subtypes of malignant (T-ALL) and T-cell lineage chronic lymphocytic leukemia (T-CLL) cases B cells, particularly in CLL (10, 11). CCR4 expression accounts for investigated, we found that CC chemokine receptor CCR9 was selectively and frequently expressed on T-ALL CD4؉ T cells, was moderately ex- frequent infiltration of adult T-cell leukemia (ATL) cells into tissues pressed on T-CLL CD4؉ T cells, and was rarely expressed on normal such as skin and lymph nodes (4). CCR7 has been found to be CD4؉ T cells. These findings were demonstrated at protein and mRNA overexpressed in lymphoid organ infiltration of ATL cells (5). CCR7 levels using flow cytometry and real-time quantitative reverse transcrip- and ␣4 integrin are important for the migration of CLL cells into tion-PCR technique and were verified by digital confocal microscopy and lymph nodes (1). Despite these findings, little is known about the Northern blotting. Thymus-expressed chemokine, a ligand for CCR9, exact mechanisms and molecules that regulate the homing, retention, ؉ selectively induced T-ALL CD4 T-cell chemotaxis and adhesion. Inter- and migration of ALL and CLL cells into organs. leukin (IL)-2 and IL-4, together, down-regulated the expression and func- -؉ CCR9, together with TECK/CCL25, efficaciously induces chemo tions of CCR9 in T-ALL CD4 T cells including chemotaxis and adhesion. taxis of immature CD4ϩCD8ϩ double-positive and mature CD4ϩ and It was also demonstrated that IL-2 and IL-4, together, internalized CCR9 ϩ -؉ CD8 single-positive thymocytes, suggesting that TECK/CCR9 in on T-ALL CD4 T cells and subsequently inhibited functions of CCR9 in these cells. Thymus-expressed chemokine mRNA was highly expressed in teraction play a pivotal role in T-cell migration in the thymus (12, 13). /CD4؉ T cells, involving lymph node and skin in T-ALL patients, and was There are two forms of CCR9: CCR9A and CCR9B (14). TECK expressed at moderate levels in lymph node and skin tissues in T-CLL CCL25 delivers signals through CCR9 for the developing of thymo- Ϫ Ϫ patients. Our findings may provide new clues to understanding various cytes (15) and the developing and/or migrating of both ␣␤ and ␥␦ ␤aspects of T-ALL CD4؉ T cells, such as functional expression of CCR9- T cells (16). CCR9 activation leads to phosphorylation of GSK-3 thymus-expressed chemokine receptor-ligand pairs as well as the effects of and FKHR and provides a cell survival signal (17). In the normal IL-2 and IL-4, which may be especially important in cytokine/chemokine human, CCR9 are restrictedly expressed at high levels on CD4ϩ and ؉ environment for the pathophysiological events of T-ALL CD4 T-cell CD8ϩ T cells in the small intestine but not in other tissues including trafficking. tonsils, lung, inflamed liver, normal, or inflamed skin (18), providing the evidence for distinctive mechanisms of lymphocyte recruitment. INTRODUCTION A common manifestation of T-ALL4 and T-CLL is infiltration of MATERIALS AND METHODS various organs, such as the lymph nodes, liver, spleen, lungs, skin, Patients and Cell Purification. All of the patients with T-ALL fulfilled intestinal tract, and even brain by leukemic cells (1). Malignant the French-American-British (FAB) Cooperative Group criteria (19). Age lymphocyte (particularly T cells) migration into and from organs is an range of patients was 5–52 years with 13 males and 8 females. All of the important aspect of T-ALL and T-CLL because leukemic cell infil- patients with T-CLL were diagnosed according to the guidelines of the Na- tration often causes serious clinical problems for patients, affecting tional Cancer Institute Working Group on B-cell lineage chronic lymphocytic the disease profile and prognosis (1–5). leukemia (B-CLL) and classified according to the FAB classification proposed CLL B cells overexpress functional CXCR4 receptors for the in 1989 (20, 21). Age range of patients was 5–61 years including 10 males and chemokine stromal cell-derived factor-1␣ (SDF-1␣/CXCL12; Refs. 6 7 females. All of the patients gave informed consent according to institutional ϩ and 7). SDF-1␣/CXCL12 and CXCR4 play an important role in guidelines. Overall information of patients is included in Table 1. CD4 and ϩ influencing the localization of ALL cells in marrow microenviron- 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 CD4ϩ and CD8ϩ T cells ranged from 93 to 99% as determined by Received 9/6/02; revised 6/26/03; accepted 7/21/03. flow cytometry. The malignancy of purified T-ALL or T-CLL CD4ϩ T cells The costs of publication of this article were defrayed in part by the payment of page ϩ ϩ charges. This article must therefore be hereby marked advertisement in accordance with is shown in the Table 2. CD4 CD8 T cells (99% pure) were obtained from 18 U.S.C. Section 1734 solely to indicate this fact. PBMCs using fluorescence-activated cell sorting. a-CD25 (2A3), a-CD45RO 1 Supported by the National Science Foundation of China (No. 39870674), a special (UCHL-1), and a-HLA-DR (L243) mAbs were purchased from BD PharMin- grant from the Personnel Department of Wuhan University, China, Science Foundation of gen (San Diego, CA). Statistical information listed in the Text for flow Anhui Province, China (No. 98436630), and Education and Research Foundation of Anhui Province, China (No. 98JL063). cytometry, RT-PCR, chemotaxis, adhesion assay, and immunofluorescence 2 Z. Q. and L. Q. contributed equally to this work. data compare variation of data among sets of patients’ samples (n ϭ number 3 To whom requests for reprints should be addressed, at Department of Immunology, of samples). Medical College, Wuhan University, Dong Hu Road 115, Wuchang 430071, Wuhan, P. R. Flow Cytometry. For the detection of CCR4, CCR9, or CXCR3, the cells China. Phone: 86-27-87331681; Fax: 45-35365326; E-mail: [email protected]. 4 The abbreviations used are: T-ALL, T-cell lineage acute lymphocytic leukemia; were first incubated with PE-labeled a-CCR4 (112509), FITC-labeled a-CCR9 T-CLL, T-cell lineage chronic lymphocytic leukemia; C.I., chemotactic index; CCL, (1G1), or a-CXCR3 mAb (49801.111; R&D Systems, Abingdon, United chemokine ligand; CCR, chemokine receptor; CXCL, CXC chemokine ligand; CXCR, Kingdom) or matched isotype antibody (DAKO) at 5 ␮g/ml in PBS containing ␥ ␥ CXC chemokine receptor; IP-10, IFN- inducible protein 10; MCNC, migrating cells on 2% BSA and 0.1% sodium azide for 20 min, followed by washing twice in negative control; MDC, macrophage-derived chemokine; TECK, thymus-expressed chemokine; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; RT- staining buffer (22). The cells were incubated with a mouse PE-labeled (or PCR, reverse transcription-PCR; IL, interleukin; ATL, adult T-cell leukemia. FITC-labeled) a-CD4 or a-CD8 mAb (DAKO, Glostrup, Denmark) at 5 ␮g/ml 6469

Table 1 Clinical information for all of the patients investigated ϩϩ ϩ ϩ ϩ ϩ Ϫ Ϫ ϩ WBC LDHa Ca CD4 CD8 CD4 CD8 CD4 CD8 CD3 Case Sex/Age (ϫ103) (IU/liter) (mg/ml) (%)b (%)b (%)b (%)b (%)c Type 1 M/5 56 765 8.8 68 28 2.4 0.7 82 A 2 F/6 35 1098 8.9 79 15 3.1 2.4 78 A 3 M/13 64 898 9.6 78 18 2.6 0.8 76 A 4 M/14 22 889 12.6 78 17 2.8 2.0 81 A 5 M/15 56 786 9.9 76 21 2.7 0.4 79 A 6 M/15 88 953 9.7 86 7 5.2 1.6 78 A 7 F/15 39 498 9.8 83 13 0.5 2.8 83 A 8 F/16 25 708 8.4 58 39 2.2 0.4 72 A 9 M/22 28 828 9.4 95 3 0.6 0.6 84 A 10 M/23 32 1065 10.6 71 24 1.4 2.3 73 A 11 M/23 19 797 10.6 66 30 2.4 1.1 72 A 12 M/24 17 1356 13.9 69 26 3.5 1.3 89 A 13 M/30 32 698 14.8 87 10 1.5 1.4 79 A 14 F/35 28 698 11.5 65 30 2.4 1.9 67 A 15 M/37 15 1075 13.9 75 19 5.1 0.3 75 A 16 F/40 45 1267 10.7 90 4 4.6 1.5 82 A 17 F/41 28 760 9.6 87 9 2.6 0.9 82 A 18 M/44 42 599 9.7 69 26 3.5 0.5 85 A 19 M/50 68 650 11.7 65 32 2.1 0.7 75 A 20 F/50 81 398 13.5 74 21 1.7 2.9 84 A 21 F/52 33 974 12.6 75 22 1.8 0.7 87 A 22 M/5 35 798 9.3 59 38 1.3 1.5 73 Ch 23 M/6 46 763 9.5 57 40 1.5 1.1 87 Ch 24 F/8 36 369 12.7 71 26 1.5 1.3 85 Ch 25 M/9 39 579 8.9 82 15 0.8 1.4 83 Ch 26 F/14 52 1432 7.6 90 8 0.7 0.7 80 Ch 27 F/20 34 703 9.0 67 27 3.5 1.8 76 Ch 28 F/23 41 975 13.7 78 20 0.5 1.0 73 Ch 29 M/23 38 1035 12.5 68 29 0.9 1.5 85 Ch 30 M/24 19 650 10.8 70 26 1.3 1.7 76 Ch 31 M/36 48 398 9.3 61 34 2.7 1.6 78 Ch 32 F/36 21 1035 8.8 60 24 3.4 1.3 70 Ch 33 M/36 23 865 8.4 67 29 2.4 1.4 56 Ch 34 M/37 54 597 8.9 65 31 1.5 1.7 74 Ch 35 F/41 27 1165 11.6 68 28 2.0 1.8 69 Ch 36 M/42 20 587 8.7 61 32 3.8 2.3 74 Ch 37 F/54 46 691 9.7 66 29 2.8 1.6 57 Ch 38 M/61 31 698 11.3 59 35 2.3 2.8 85 Ch a-CD25 (2A3), a-CD45RO (UCHL-1) and a-HLA-DR (L243) mAbs were purchased from BD PharMingen (San Diego, CA). a LDH, lactate dehydrogenase; A, acute; Ch, chronic. ϩ b The percentages of different subsets of CD3 T cells. ϩ c Percentages of CD3 T cells in PBMCs.

for 20 min, followed by washing twice. The analyses were performed with a tions, followed by blotting onto Nytran membranes and were cross-linked by flow cytometer (COULTER XL, Coulter, Miami, FL). For CD4, CD8, and UV irradiation (28). CCR4 and CCR9 cDNA probes, labeled by [␣-32P]dCTP, CCR9 triple staining, PE-labeled CD4, PerCP-labeled CD8 (Dako), and FITC- were obtained by PCR amplification of the sequence mentioned above from labeled CCR9 mAbs were applied with the procedure described above. total RNA from PBMCs from normal adults (CCR4) and thymocytes from the For internalization assays, percentage internalization was calculated as specimen of thymusectomy (CCR9). The membranes were hybridized over- Ϫ ϫ ϭ 6 32 [MFIc MFIt)/MFIc] 100, where MFIc mean fluorescence intensity of night with 1 ϫ 10 cpm/ml of P-labeled probe, followed by intensive ϭ anti-CCR9 binding to the cells cultured with medium only, and MFIt mean washing with 0.2ϫ SSC and 0.1% SDS before being autoradiographed. For fluorescence intensity of cells after incubation with IL-2 and/or IL-4 (23, 24). protein detection (Western blot), the cells were lysed in lysis buffer (29). Real-Time Quantitative RT-PCR Assay. Total RNA from purified cells Lysates were centrifuged at 10,000 rpm for 5 min at 4°C. Protein concentration ϫ 6 Ͼ (1 10 , purity 99%) was prepared using Quick Prep total RNA extraction was measured by Bio-Rad protein assay. Total protein (40 ␮g) was loaded onto kit (Pharmacia Biotech; Refs. 25–27), then was reverse transcribed using oligo 16% SDS-PAGE, transferred onto polyvinylidene difluoride membranes after (dT) and Superscript II reverse transcriptase (Life Technologies, Inc., 12–18 electrophoresis, and incubated with the CCR9 mAb (0.5 ␮g/ml). Analyses Grand Island, NY). The real-time quantitative PCR was performed in a 96-well were conducted using enhanced chemiluminescence detection (Amersham microtiter plate (Applied Biosystems, Foster City, CA) with an ABI PRISM Pharmacia Biotech, Little Chalfont, United Kingdom). 7700 Sequence Detector Systems (Applied Biosystems). Using SYBR Green Immunofluroescence Digital Confocal Microscopy. The purified cells PCR Core Reagents kit (Applied Biosystems; P/N 4304886), we generated were spun down on a slide, fixed with a mixture of methanol and acetone, fluorescence signals during each PCR cycle via the 5Ј to 3Ј endonuclease immersed in 1% BSA blocking buffer for 10 min to avoid nonspecific activity of AmpliTaq Gold (26) to provide real-time quantitative PCR information. The target genes were generated by connecting the following se- binding; antibody, either FITC-labeled CCR9 mAb or isotype IgG2a, was ␮ quences of the specific primers: CCR4 sense: 5Ј-ACTGTGGGCTCCTC- added at 10 g/ml, and the cells were incubated overnight at 4°C, followed CAAATTT-3Ј; CCR4 antisense: 5Ј-CATGGTGGACTGCGTGTAAGA-3Ј; by the addition of the PE-labeled CD4 mAb. For internalization detection, CCR9 sense: 5Ј-CATTGACGCCTATGCCATGT-3Ј; CCR9 antisense: 5Ј- cells were permeabilized for 30 min on ice in PBS containing 2% fetal ␮ GACCTGGAAGCAGATGTCAATGT-3Ј; TECK/CCL25 sense: 5Ј-AGCGG- bovine serum and 20 g/ml cholera toxin B Alexa Fluor 594 before GAGCTGCAATCTG-3Ј; TECK/CCL25 antisense: 5Ј-GGGTTCCCACA- staining. Cells were then stained only with FITC-labeled CCR9 mAb or CACCTTCCT-3Ј. isotype IgG2a at 10 ␮g/ml. The preparations were observed using a All unknown cDNAs were diluted to contain equal amounts of ␤-actin fluorescence microscope (model BX60; Olympus, Japan). Confocal mi- cDNA. PCR retained conditions were 2 min at 50°C, 10 min at 95°C, and 40 croscopy analysis was performed using a confocal laser scanning system cycles with 15 s at 95°Cand60sat60°C in each cycle. and an inverted microscope (model LSMSIO; Zeiss, Berlin, Germany). Northern and Western Blot Assays. For mRNA detection (Northern Images of serial cellular section were acquired with the Bio-Rad Comos blot), each 5 ␮g of total RNA were electrophoresed under denaturing condi- graphical user-interface as described previously (23, 24, 30). 6470

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

ϩ Organ involved CD25 CXCR3 ϩ ϩ Case (%)a CD45RO (%)a HLA-DR (%)a CCR4 (%)a CCR9 (%)a (%)a Lnb Lg Sp Li Sk Other Type 179856527817Ϫc ϪϪϩϪ Ϫ Ϫ A 28982ND36453 ϪϪϪϪϪ Ϫ A 3687667ND826 ϩϪϪϪϩ Ϫ A 489847640908 ϩϪϩϪϪ Ϫ A 587966524663 ϪϪϩϪϩ Ϫ A 682897130923 ϩϪϪϪϪ in A 779897318388 ϪϪϪϪϪ Ϫ A 859927829487 ϪϪϪϪϪ Ϫ A 98181753286NDϪϩϪϪϪ Ϫ A 10 78 97 71 51 90 6 ϩϩϪϪϩ Ϫ A 11 82 80 69 29 83 8 ϩϪϪϪϪ Ϫ A 12 98 87 76 28 98 5 ϪϩϩϪϪmϩbA 13 66 88 76 45 65 4 ϩϪϪϪϪ Ϫ A 14 85 95 65 35 75 5 ϪϪϩϪϪ Ϫ A 15 87 84 65 14 77 5 ϪϩϪϪϪ Ϫ A 16 91 97 48 23 76 9 ϩϪϪϪϪ Ϫ A 17 89 95 68 36 72 1 ϪϩϪϪϪ Ϫ A 18 88 87 83 34 46 3 ϪϪϪϪϪ Ϫ A 19 76 89 ND 21 73 4 ϩϪϪϪϪ Ϫ A 20 87 81 87 26 67 11 ϪϪϩϪϪ Ϫ A 21 69 84 82 11 80 8 ϪϩϪϪϪ Ϫ A Av 81 Ϯ 988Ϯ 672Ϯ 929Ϯ 10 73 Ϯ 17 8 Ϯ 3 22 78 86 74 29 31 7 ϩϪϪϪϪ ϩ Ch 23 71 91 ND 51 88 1 ϩϩϩϪϪ Ϫ Ch 24 86 88 68 22 25 3 ϩϪϪϪϪ Ϫ Ch 25 86 76 79 ND 45 4 ϩϪϪϪϪ Ϫ Ch 26 87 92 76 33 29 8 ϩϪϪϪϪ Ϫ Ch 27 54 78 69 38 28 9 ϩϪ ϪϪ Ϫ Ch 28 78 83 ND 30 34 5 ϩϪϪϪϪ Ϫ Ch 29 55 76 65 32 24 10 ϩϪϪϪϪ Ϫ Ch 30 65 74 89 19 9 3 ϪϪϪϪϪ Ϫ Ch 31 68 79 87 19 35 2 ϪϪϪϪϪ Ϫ Ch 32 75 83 72 35 31 5 ϩϪϪϪϪ Ϫ Ch 33 87 99 65 40 12 7 ϪϪϪϪϪ Ϫ Ch 34 75 89 79 20 28 ND ϩϪϪϪϪ Ϫ Ch 35 67 76 83 30 55 4 ϪϩϪϪ Ϫ Ch 36 79 84 64 26 38 ND ϩϪϪϪϪ Ϫ Ch 37 85 87 88 28 45 6 ϪϪϩϪϪ Ϫ Ch 38 78 87 89 27 41 4 ϩϪϪϪϪϪ Ch Av 75 Ϯ 11 84 Ϯ 777Ϯ 930Ϯ 935Ϯ 18 5 Ϯ 3 ϩ a The percentages are indicated marker positive cells in purified peripheral CD4 T cells from ALL or CLL patients. b Ln, lymph nodes; Lg, lung; Sp, spleen; Li, liver; Sk, skin; ND, no determination; mϩb, meninges and brain; in, intestine; A, acute; Ch, chronic; Av, average. c Ϫ, not involved; ϩ, involved.

Chemotaxis Assay. The chemotaxis assay was performed in a 48-well RESULTS microchamber (Neuro Probe, Bethesda, MD) technique (31). Briefly, chemo- ؉ kine (MDC/CCL22, TECK/CCL25 or ␥IP-10/CXCL10; purchased from R&D CCR9 Expression on CD4 Cells from T-ALL Patients Was Systems) in RPMI 1640 with 0.5% BSA was placed in the lower wells (25 ␮l). Selectively Increased. In a total of 38 cases of T-ALL and T-CLL ϩ Twenty-five ␮l of cell suspension (2 ϫ 106 cells/ml) were added to the upper patients (Table 1), T-ALL and T-CLL CD4 T cells expressed CCR4 well of the chamber, which was separated from the lower well by a 5-␮m moderately (Fig. 1A), whereas normal cells expressed CCR4 rarely. pore-size, polycarbonate, polyvinylpyrrolidone-free membrane (Nucleopore, T-ALL CD4ϩ T cells highly expressed CCR9 (91.9%), whereas ϩ Pleasanton, CA). The cells were CD4 T cells positively isolated using CD4 T-CLL CD4ϩ T cells expressed CCR9 moderately, and normal CD4ϩ MACS MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). T cells expressed CCR9 rarely (Fig. 1A). Expression of CXCR3 on The chamber was incubated for 60 min at 37°C and 5% CO . The membrane ϩ ϩ 2 CD4 T cells from all different subjects was very low. CD8 T cells was then carefully removed and was stained for 5 min in 1% Coomassie Brilliant Blue. Approximately 6% of the cells will migrate spontaneously from all different subjects expressed CCR9 at an equally low-level (Fig. 1B). CCR9 expression was much higher on T-ALL CD4ϩCD8ϩ (known as MCNC; Ref. 32). The results were expressed as C.I. with SD (31). Ϫ Ϫ For blocking tests, the cells were preincubated with either anti-CCR9 mAb or T cells compared with CD4 CD8 T cells (92 Ϯ 4%, 4 Ϯ 2%; ␮ ϭ Ͻ IgG2a isotype antibody at 10 g/ml for 120 min at room temperature before n 12; P 0.0001). CCR9 was expressed moderately T-CLL ϩ ϩ Ϫ Ϫ chemotaxis assay. CD4 CD8 T cells compared with CD4 CD8 T cells (36 Ϯ 6%, Adhesion Assays. As described previously (33), 96-well plates were 5 Ϯ 3%; n ϭ 12; P Ͻ 0.01). Expression levels of CCR4 and CCR9 coated with laminin (20 ␮g/ml; Sigma Chemical Co.) in PBS for1hat37°C. on CD4ϩ T cells from patients with acute myeloblastic leukemia were 5 The single-cell suspensions (4 ϫ 10 cells/ml) with 0.2% BSA were added to similar to the levels on the normal cells (data not shown). ␮ the appropriate chemokine. The cell suspension was added at 100 l/well in To be sure that examined CD4ϩ T cells from patients with T-ALL triplicate to the plates, and incubated for 60 min at 37°C. The wells were then and T-CLL were leukemic, the expression of CD25, CD45RO, and washed with 0.2% BSA in PBS, followed by careful aspiration. Subsequently, HLA-DR in these cells was investigated (Refs. 4, 33, 34; Table 2). the adherent cells were fixed with 1% formaldehyde and stained with 1% ϩ crystal violet. Crystal violet was then extracted by the addition of a 1:1 mixture The majority of purified CD4 T cells from patients with T-ALL and of sodium citrate (0.1 M) and ethanol (pH 4.2). The absorbency was then read T-CLL were CD25, CD45RO, and HLA-DR positive. Almost all of at 540 nm. Background cell adhesion to 2% BSA-coated wells was subtracted the T-ALL patients showed highly frequent expression of CCR9 from all readings. (Table 2). 6471

ages in CD4ϩ T cells among normal subjects and T-ALL or T-CLL with detected by immunofluorescence microscopy (Fig. 3D). -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 CD4ϩ cells (C.I. ϭ 3.6 Ϯ 0.72 at 100 ng/ml; n ϭ 8; P Ͻ 0.001; versus normal). MDC/CCL22 (a ligand for CCR4) induced a moderate chemotactic migration of T-ALL CD4ϩ cells (C.I. ϭ 2.2 Ϯ 0.48 at 100 ng/ml; n ϭ 8; P Ͻ 0.01; versus normal; Fig. 4B). ␥IP-10/CXCL10 (a ligand for CXCR3) did not induce significant chemotactic migration in the cells (C.I. ϭ 1.2 Ϯ 0.35 at 100 ng/ml; n ϭ 8; P Ͼ 0.05; versus normal; Fig. 4B). ␥IP-10/CXCL10 did induce a weak chemotaxis in normal CD4ϩ T cells, whereas the other two chemokines mentioned above did not induce significant chemotactic migration in normal CD4ϩ T cells (Fig. 4A). MDC/ CCL22 and TECK/CCL25 induced chemotaxis slightly in T-CLL CD4ϩ T cells (C.I. ϭ 1.5 Ϯ 0.37 and 1.6 Ϯ 0.42 at 100 ng/ml; n ϭ 8; all P Ͼ 0.05; versus normal), whereas ␥IP-10/CXCL10 did not (Fig. 4C). Spontaneous migration negative control (known as MCNC) of different CD4ϩ T cells was ϳ6% of total cells added, indicated as C.I. ϭ 1 (Fig. 4). The a-CCR9 mAb could completely block the

Fig. 1. CCR4 and CCR9 distribution. Double color flow cytometric analysis of the distribution of CCR4, CCR9, or CXCR3 on CD4ϩ (A)orCD8ϩ (B) T cells from normal subjects (T-NOL) and T-ALL and T-CLL patients. The CD4ϩ and CD8ϩ 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 CCR4ϩ, CCR9ϩ, or CXCR3ϩ 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 1 and 2.

CCR4 mRNA was detected at low levels in freshly isolated normal CD4ϩ and CD8ϩ T cells (7.1 Ϯ 0.71 and 8.4 Ϯ 0.65 ϫ 102 copies/50 ng cDNA; n ϭ 8), moderate level in T-ALL CD4ϩ and CD8ϩ T cells (4.3 Ϯ 0.71 and 3.4 Ϯ 0.65 ϫ 103 copies/50 ng cDNA; n ϭ 8; all P Ͻ 0.01; versus normal; Fig. 2A). CCR9 mRNA in T-ALL CD4ϩ T Fig. 2. CCR4, CCR9, and TECK/CCL25 mRNA. The real-time quantitative detection 4 cells was significantly and selectively up-regulated (1.4 Ϯ 0.35 ϫ 10 of RT-PCR for mRNA of CCR4 (A) and CCR9 (B) in freshly isolated CD4ϩ and CD8ϩ copies/50 ng cDNA; n ϭ 8; P Ͻ 0.001; versus normal), and moder- T cells from normal subjects (open bars) and T-ALL (black bars) or T-CLL (gray bars) ϩ Ϯ ϫ 3 patients. The bars are representatives of eight similar experiments. The procedure for ately up-regulated in T-CLL CD4 T cells (5.3 0.42 10 quantitative RT-PCR amplification was described in “Materials and Methods.” The copies/50 ng cDNA; n ϭ 8; P Ͻ 0.03; versus normal; Fig. 2B). statistical error bars are from tests of the same sample in each experiment conducted in ϩ Northern blots confirmed these observations (Fig. 2, C and D). triplicate. Northern blot of CCR4 (C) and CCR9 (D) mRNA in freshly isolated CD4 T ϩ cells from normal subjects (T-NOL) and T-ALL or T-CLL patients. Total RNA from CCR9 was rarely expressed on normal CD4 T cells detected by different cells as indicated were isolated, electrophoresed, and blotted as described in immunofluorescence digital confocal microscopy (Fig. 3A). Only one “Materials and Methods.” Top panels, the hybridization signals for CCR4 or CCR9 ϩ of nine CD4ϩ T cells in a representative field was showing CCR9 mRNA in CD4 T cells from different subjects. The 28S rRNAs in lower panels confirm ϩ the comparable amounts of loaded total RNA. The illustrated data are from a single positive. In contrast, all T-ALL CD4 T cells in a representative field representative experiment (T-ALL patient 8 and T-CLL patient 24) of six performed. The ϩ real-time quantitative detection of RT-PCR for mRNA of TECK/CCL25 (E) in freshly were showing CCR9 positive (Fig. 3B). 3 of 8 T-CLL CD4 T cells ϩ in a representative field were showing CCR9 positive (Fig. 3C). There isolated CD4 T cells, involved lymph nodes (LN) and involved skin from normal ϩ subjects (open bars) and T-ALL (black bars) or T-CLL (gray bars) patients. The bars are were high statistically significant differences of CCR9 cell percent- representatives (T-ALL patient 14 and T-CLL patient 22) of six similar experiments. 6472

CD4ϩ T cells toward TECK/CCL25 (data not shown), whereas the isotype antibody had no blocking effect at all (data not shown). 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 con- trolled 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 CD4ϩ T cells. CCR4 constantly expressed on the T-ALL CD4ϩ T cells under different culture conditions (41 Ϯ 6%; n ϭ 8; all P Ͼ 0.05; Fig. 6A), and CCR3 expression on T-ALL CD4ϩ T cells (date not shown). IL-2 and IL-4, together, significantly reduced CCR9 expression on T-ALL CD4ϩ T cells (Fig. 6B), compared with the freshly isolated cells (Fig. 1) and the cells in culture only, or with IL-2 or IL-4 alone (Fig. 6B). Fresh T-ALL CD4ϩ T cells highly expressed CCR9 (91.9%; Fig. 1B), whereas the cultured CD4ϩ T cells still expressed identical amounts of CCR9 (83 Ϯ 7%; n ϭ 8; Fig. 6B). After culture with IL-2 or IL-4 alone, the CCR9 expression on T-ALL CD4ϩ T cells was 82 Ϯ 8% ϭ Ͼ Ϯ ϭ ϩ (n 8; P 0.05, versus that in culture only) or 80 9% (n 8; Fig. 3. CCR9 distribution. The CCR9 distribution in normal CD4 T cells (T-NOL; A), Ͼ T-ALL (B) or T-CLL (C) patients. The CD4ϩ T cells were collected from different P 0.05, versus that in culture only), respectively. The combination subjects as indicated, and stained as described in “Materials and Methods.” The cells were of IL-2 and IL-4 significantly reduced the CCR9 expression T-ALL photographed under epifluorescent conditions. ϫ1200. Bar,9␮m. Arrows, typical CD4ϩ T cells (16 Ϯ 4%; n ϭ 8; P Ͻ 0.001; versus that in culture CD4ϩCCR9ϩ 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 only). The preincubation of the cells with antibodies toward IL-2 T-CLL and six T-ALL patients. The CCR9ϩ cells percentages in normal CD4ϩ T cells receptor and IL-4 receptor blocked inhibitory function of IL-2 and (T-NOL), T-ALL, or T-CLL patients (D). Cells were counted in hundreds per field in each IL-4 together on CCR9 expression (88 Ϯ 9%; n ϭ 8; P Ͼ 0.05; versus representative field under fluorescent microscope, and positive cells were recorded. The statistically ,ء .showing data were averages of each group of six subjects investigated significant difference in T-ALL patients versus normal subjects (P Ͻ 0.001); in T-CLL patients versus normal subjects (P Ͻ 0.01).

chemotaxis of T-ALL CD4ϩ T cells toward TECK/CCL25 (C.I. ϭ 1.0 Ϯ 0.12 at 100 ng/ml; n ϭ 4; P Ͻ 0.001; versus that without antibody in Fig. 4B), whereas it had no any effect on the chemotaxis of T-ALL CD4ϩ T cells toward MDC/CCL22 (Fig. 4D). The isotype antibody had no blocking effect at all (Fig. 4D). It confirmed that the observed T-ALL CD4ϩ T cell chemotaxis was indeed induced by TECK/CCL25 via CCR9. The checkerboard chemotaxis assays demonstrated that enhanced motility of the T-ALL CD4ϩ T cells toward the chemokine (TECK/CCL25) was caused by chemotaxis, not by chemokinesis (data not shown; 35, 36). Interest- ingly, TECK/CCL25 induced a very strong chemotaxis in purified T-ALL CD4ϩCD8ϩ cells (C.I. ϭ 4.8 Ϯ 1.32 at 100 ng/ml; n ϭ 12). T-CLL CD4ϩCD8ϩ cells showed weak chemotactic response toward TECK/CCL25 (C.I. ϭ 2.1 Ϯ 0.67 at 100 ng/ml; n ϭ 6). Compared with normal subjects, TECK/CCL25 mRNA was highly expressed in purified T-ALL CD4ϩ 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). TECK/CCL25 induced a high adhesion of T-ALL CD4ϩ cells (36 Ϯ 8.2% of cells adhered at 100 ng/ml; n ϭ 8; P Ͻ 0.001; versus normal). MDC/CCL22 induced a moderate adhesion of T-ALL CD4ϩ cells (22 Ϯ 6.7% of cells adhered at 100 ng/ml; n ϭ 8; P Ͻ 0.01; versus normal). ␥IP-10/CXCL10 only slightly induced adhesion in the cells (16 Ϯ 3.1% of cells adhered at 100 ng/ml; n ϭ 8; P Ͻ 0.05; versus normal; Fig. 5B). ␥IP-10/CXCL10 induced a weak adhesion in ϩ normal CD4ϩ T cells, whereas the other two chemokines mentioned Fig. 4. Chemotaxis analysis. The migration of freshly isolated CD4 T cells from ϩ normal subjects (T-NOL, A), T-ALL (B), T-CLL (C), or T-ALL (D; a-CCR9 mAb or above did not induce significant adhesion in normal CD4 T cells isotype antibody blocking) patients toward chemokines as indicated. All of the results (Fig. 5A). MDC/CCL22 and TECK/CCL25 induced moderate adhe- were determined as described in “Materials and Methods” and were expressed as C.I. Ϯ sion in T-CLL CD4ϩ T cells (20 Ϯ 3.7% and 22 Ϯ 3.9% of cells (Chemotactic Index) SD; calculations were based on triplicate determination of chemotaxis on each concentration of chemokine as indicated. The applied chemokine con- adhered at 100 ng/ml; n ϭ 8; all P Ͻ 0.01; versus normal), whereas centrations (ng/ml) are indicated. Open bars, spontaneous migration toward negative ␥IP-10/CXCL10 did not (Fig. 5C). Spontaneous adhesion of different (medium) control (known as MCNC; C.I. ϭ 1) in each experiment. The illustrated data are ϩ from a single representative experiment (T-ALL patient 8 and T-CLL patient 28) of eight CD4 T cells is around 10% of total added cells (Fig. 5). The performed. For blocking experiments, comparisons are D versus B under the same anti-CCR9 mAb could completely block the adhesion of T-ALL chemokine concentration. 6473

DISCUSSION 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 CD4ϩ T cells, and is the first direct evidence of the increased biological activity of T-ALL CD4ϩ T cells induced by TECK/CCL25. We have documented that CCR4 is frequently expressed at moderate levels on T-ALL and T-CLL CD4ϩ and CD8ϩ T cells, and that its ligand, MDC/CCL22, induces freshly isolated T-ALL and T-CLL

Fig. 5. Adhesion analysis. The adhesion of freshly isolated CD4ϩ T cells from normal subjects (T-NOL, A) and T-ALL (B) and T-CLL (C) patients induced by MDC/CCL22, TECK/CCL25, and ␥IP-10/CXCL10. The results were determined as described in “Ma- terials and Methods” and expressed as percentages of adherent cells Ϯ 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.

that in culture only), whereas isotype antibodies did not have such function (14 Ϯ 8%; n ϭ 8; P Ͻ 0.001; versus that in culture only; Fig. 6B). In the normal CD4ϩ T cells, we did not observe such phenom- enon (date not shown). TECK/CCL25 induced a high chemotactic migration of cultured T-ALL CD4ϩ cells. IL-2 and IL-4, together, totally abolished chemotactic migration of T-ALL CD4ϩ cells toward TECK/CCL25 (C.I. ϭ 1.0 Ϯ 0.22 at 100 ng/ml; n ϭ 8; P Ͻ 0.001; versus that in culture only; Fig. 7A). TECK/CCL25 induced significantly high adhesion of cultured T-ALL CD4ϩ T cells, which was identical to the results in the freshly isolated cells (Fig. 5B). IL-2 and IL-4, together, totally inhibited the adhesion of T-ALL CD4ϩ cells induced by TECK/CCL25 (12 Ϯ 4.3% of cells adhered at 100 ng/ml; n ϭ 8; P Ͻ 0.001; versus that in culture only; Fig. 7B). IL-2 or IL-4 alone could affect neither chemotaxis nor adhesion of T-ALL CD4ϩ T cells induced by TECK/CCL25 (data not shown). To our surprise, the expressions of CCR9 mRNA in T-ALL CD4ϩ T cells under different culture conditions were almost identical to each other (Fig. 8A). These CCR9 mRNA expression levels were also identical to the level in freshly isolated T-ALL CD4ϩ T cells (Fig. 2B). Northern blots (Fig. 8B) at mRNA level and Western blots (Fig. 8C) at protein level confirmed these observations. CCR9 only distrib- uted on cellular membrane of medium-cultured T-ALL CD4ϩ T cells detected by confocal microscopy (Fig. 9A). The similar distribution pattern was observed on the cells cultured in the presence of IL-2 (Fig. 9B) or IL-4 (Fig. 9C). CCR9 internalization occurred in T-ALL CD4ϩ T cells cultured with IL-2 and IL-4 together (Fig. 9D). We calculated ϩ the ratio of CCR9 internalization in T-ALL CD4 T cells under Fig. 6. Regulation of CCR4 and CCR9 expression. Double color flow cytometric ϩ different culture conditions (Fig. 9E). CCR9 internalization signifi- analysis of the regulation on CCR4 (A) and CCR9 (B) expression on CD4 T cells from T-ALL patients by cytokine as indicated. The CD4ϩ T cells were isolated according to cantly occurred when the cells were cultured with IL-2 and IL-4 procedure described in “Materials and Methods” and, subsequently, were cultured with or together (78% Ϯ 6%; n ϭ 8; P Ͻ 0.001; versus that in culture only). without IL-2 (10 ng/ml) and/or IL-4 (10 ng/ml) for 24 h as indicated. All of the cytokine ␮ It did not happen under other circumstances as indicated in Fig. 9 receptor antibodies (Abs) were applied at 5 g/ml. The procedure for cell staining with PE-labeled CCR4 or FITC-labeled CCR9 and FITC-labeled or PE-labeled CD4 mAbs was (2% Ϯ 0.8% and 3 Ϯ 0.4%; n ϭ 8; all P Ͼ 0.05; versus that in culture 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 CCR4ϩ and only). Thus, IL-2 and IL-4, together, internalized CCR9 on T-ALL ϩ ϩ CCR9 cells are indicated in the “Results.” The data are from a single experiment (T-ALL CD4 T cells and subsequently inhibited functions of CCR9 in these patient 16 and T-CLL patient 33) that is representative of eight similar experiments cells. performed. 6474

amined. The exact mechanism by which CCR9 and TECK/CCL25 mediate T-ALL CD4ϩ T cell trafficking in vivo should be further investigated. Naõ¬ve T cells enter specialized microenvironments in which antigenic exposure occurs. These primed T cells recirculate to be contin- uously available for potential antigenic rechallenge (43Ð45). Under leukemic condition, however, leukocyte transmigration between cir- culation and the tissues (organs) has significantly been changed (2, 46Ð48). In the present study, T-ALL CD4ϩ T cells express abnor- mally 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. Fig. 7. Chemotaxis and adhesion analysis. The migration (A) and adhesion (B)of CXCR4 binding to its ligand SDF-1 regulate precursor B-cell T-ALL CD4ϩ T cells toward TECK/CCL25. The cells were cultured with or without IL-2 lineage acute lymphocytic leukemia cell survival and proliferation (8). and/or IL-4 as described in the legend for Fig. 6. All of the results were determined as CCR9 activation provides a cell survival signal (17). In the present described in “Materials and Methods” as well as in the legends for Figs. 4 and 5. Open ϩ bars, spontaneous migration or adhesion toward medium. The results are expressed as C.I. study, CCR9 and TECK are highly overexpressed on T-ALL CD4 T or percentages of adherent cells Ϯ SD. The illustrated data are from a single representative cells. A logical implication may be that CCR9 and its ligands promote experiment (T-ALL patient 17 and T-CLL patient 31) of eight similar experiments performed. 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

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 CD4ϩ T cells. The cells were cultured with or without IL-2 and/or IL-4 as described in the legend for Fig. 6. 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. 2. 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.

CD4ϩ and CD8ϩ 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 selec- tive expression of functional CCR9 on T-ALL CD4ϩ, but not CD8ϩ T cells, we have also observed high levels of TECK/CCL25 mRNA expression in CD4ϩ T cells, involving lymph node and skin of T-ALL Fig. 9. CCR9 internalization. Effects of IL-2 and IL-4 (R&D Systems) on CCR9 patients. Our results imply that CCR9, engaged TECK/CCL25, stim- internalization in T-ALL CD4ϩ T cells detected using confocal microscopy (A–D). The ϩ ϩ ulates T-ALL CD4 T cell migration in an autocrine manner, and that CD4 T cells were isolated and were subsequently cultured with medium only (A) or with CCR9 expression is important for this process. TECK/CCL25 expres- IL-2 (B) or IL-4 (C) or IL-2 and IL-4 together (D) for 24 h, described in “Materials and Methods.” ϫ1200. Bar, 9 ␮m. Arrow, typical CCR9 internalized cell. CCR9 internaliza- sion in involved organs was also elevated in the leukemic condition. tion of IL-2 and/or IL-4-treated T-ALL CD4ϩ T cells (E). The cells were cultured without The interpretation could be that a large number of TECK/CCL25 or with IL-2 and/or IL-4 as described in the legend for Fig. 6. The data (T-ALL patient ϩ 11 and T-CLL patient 38) represent eight separate experiments; error bars, SD determined expressing T-ALL CD4 T cell infiltrated into the organs, resulting in as described in “Materials and Methods.” Ten thousand cells were measured in each the elevation of TECK/CCL25 mRNA expression in the organs ex- acquisition. Abs, antibodies. 6475

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Zhang Qiuping, Li Qun, Hu Chunsong, et al.

Cancer Res 2003;63:6469-6477.

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