CCL19 and CXCL13 Synergistically Regulate Interaction between Acute Lymphocytic CD23+CD5+ B Cells and CD8 + T Cells This information is current as of September 24, 2021. Xingbing Wang, He Yuling, Jiang Yanping, Tan Xinti, Yang Yaofang, Yu Feng, Xiao Ruijin, Wang Li, Chen Lang, Liu Jingyi, Tang Zhiqing, Ouyang Jingping, Xia Bing, Qiao Li, Alfred E. Chang, Zimin Sun, Jin Youxin and Tan Jinquan

J Immunol 2007; 179:2880-2888; ; Downloaded from doi: 10.4049/jimmunol.179.5.2880 http://www.jimmunol.org/content/179/5/2880 http://www.jimmunol.org/ References This article cites 60 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/179/5/2880.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CCL19 and CXCL13 Synergistically Regulate Interaction between B Cell Acute Lymphocytic Leukemia CD23؉CD5؉ B Cells and CD8؉ T Cells1

Xingbing Wang,2*† He Yuling,2† Jiang Yanping,2† Tan Xinti,2† Yang Yaofang,2‡ Yu Feng,2† Xiao Ruijin,†§ Wang Li,† Chen Lang,† Liu Jingyi,† Tang Zhiqing,† Ouyang Jingping,† Xia Bing,¶ Qiao Li,ʈ Alfred E. Chang,ʈ Zimin Sun,* Jin Youxin,§ and Tan Jinquan3*†‡

Interacting with T cells, cytokine-producing B cells play a critical protective role in autoimmune diseases. However, the interaction between malignant B and T cells remains to be fully elucidated. In a previous study, we have reported that ligation of CCL19- CCR7 and CXCL13-CXCR5 activates paternally expressed gene 10 (PEG10), resulting in an enhancement of apoptotic resistance in B-cell acute lymphocytic leukemia (B-ALL) CD23؉CD5؉ B cells. Here, we report that B-ALL CD23؉CD5؉ B cells produce Downloaded from IL-10 at high level, which can be further elevated by costimulation with CCL19 and CXCL13. CCL19/CXCL13-activated B-ALL CD23؉CD5؉ B cells, in turn, increase IL-10 expression in syngeneic CD8؉ T cells in a B cell-derived IL-10-dependent manner and requiring a cell-cell contact. IL-10 secreted from B-ALL CD23؉CD5؉ B cells in vitro impairs tumor-specific CTL responses of syngeneic CD8؉ T cells. The impairment of cytotoxicity of syngeneic CD8؉ T cells is escalated by means of CCL19/CXCL13- ,induced up-regulation of IL-10 from B-ALL CD23؉CD5؉ B cells. Moreover, using a short hairpin RNA to knockdown PEG10 /we provide direct evidence that increased expression of PEG10 in B-ALL CD23؉CD5؉ B cells is involved in malignant B- http://www.jimmunol.org interaction, contributing to the up-regulation of IL-10 expression, as well as to the impairment of cytotoxicity of syngeneic CD8؉ -T cells. Thus, malignant B-ALL CD23؉CD5؉ B cells play an immunoregulatory role in controlling different inflammatory cyto kine expressions. IL-10 may be one of the critical cellular factors conferring B-ALL CD23؉CD5؉ B cells to escape from host immune surveillance. The Journal of Immunology, 2007, 179: 2880–2888.

t has been highlighted in several recent investigations that B and modulate their immune responses (6). Cytokine-producing B cells produce cytokines in response to a diverse array of stim- cells, seen in draining lymph nodes and spleens of autoimmune

I uli including microbial products, Ags, and T cells (1, 2). Na- and pathogen-infected mice (1–7), play critical protective roles in by guest on September 24, 2021 ive B cells can differentiate into discrete B cell effector subsets to several autoimmune disease models (3–5). However, the knowl- produce differential arrays of cytokines upon restimulation (3–5). edge of interactions between malignant B cells (leukemic B cells) These cytokine-producing effector B cells can interact with T cells and syngeneic T cells is not fully established. CCL19 and CCL21 are ligands for CCR7, whereas CXCL13 is the only ligand for CXCR5 (8, 9). They closely cooperate in the *Department of Hematology, Anhui Medical University Affiliated Provincial Hospi- tal, Hefei, China; †Departments of Immunology and Pathphysiology and Laboratory development and maintenance of lymphoid tissue microarchitec- of Allergy and Clinical Immunology, Institute of Allergy and Immune-Related Dis- ture (9). CXCR5 is expressed on mature recirculating B cells, eases and Center for Medical Research, Wuhan University School of Medicine, Wu- ϩ ϩ ‡ small subsets of normal CD4 and CD8 T cells, and skin-derived han, China; Department of Anatomy, Jiujiang University Medical College, Jiujiang 4 University, Jiujiang, China; §The State Key Laboratory of Molecular Biology, Insti- migratory dendritic cells (DC) (10–13). CXCR5 is responsible tute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, for guiding B cells into the B cell zone of secondary lymphoid ʈ Chinese Academy of Science, Shanghai, China; Departments of Internal Medicine and organs (14–16). CXCR5 on a subset of T cells plays an additional Geriatrics, The Zongnan University Hospital, Wuhan University, Wuhan, China; and ¶Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109 role in T cell migration (16, 17). CCR7, highly expressed on naive Received for publication November 21, 2006. Accepted for publication June T cells (18), is closely related to T cell differentiation toward ef- 20, 2007. fector cells (19–21). The synergy of CCL19/CCR7 and CXCL13/ The costs of publication of this article were defrayed in part by the payment of page CXCR5 has been shown in both physiological and pathological charges. This article must therefore be hereby marked advertisement in accordance situations, such as controlling T cells and DCs homing to second- with 18 U.S.C. Section 1734 solely to indicate this fact. ary lymphoid organs (19–21) and determining the positioning and 1 This work was supported by the National Natural Science Foundation of China (Grants 39870674, 30572119, 30030130, 30471509, and 30670937); Science Foun- proper function of follicular Th cells (18). CXCR5- and CCR7- dation of Anhui Province, China (Grant 98436630), and Education and Research deficient mice lack lymphoid follicles due to an impaired migra- Foundation of Anhui Province, China (Grant 98JL063) and Research Foundation tion of B cells (22). Overexpression of CCR7 and CXCR5 on from Health Department of Hubei Provincial Government, China (Grant 301140344); and a special grant from the Personnel Department of Wuhan University, China. T.J. tumor was closely related to the apoptosis of cells as is a Chang Jiang Scholar supported by Chang Jiang Scholars Program from Ministry well as their migration and infiltration (23–26). CCR7 and CXCR5 of Education, People’s Republic of China and Li Ka Shing Foundation, Hong Kong, China. 2 X.W., H.Y., J.Y., T.X., Y.Y., and Y.F. contributed equally to this work. 4 Abbreviations used in this paper: DC, ; B-ALL, B cell acute lympho- 3 Address correspondence and reprint requests to Dr. Tan Jinquan, Department of cytic leukemia; B-CLL, B cell chronic lymphocytic leukemia; CB, cord blood; Hematology, Anhui Medical University Affiliated Provincial Hospital, Hefei, Peo- IL10RB pAb, IL-10 B polyclonal Ab; Q-PCR, quantitative RT-PCR; ple’s Republic of China or Department of Immunology, Wuhan University School of shRNA, short hairpin RNA. Medicine, Wuhan University, Dong Hu Road 115, Wuchang, Wuhan, People’s Re- public of China. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 2881

Table I. Clinical, immunophenotypic and genotypic information for all patients investigated

Phenotyping CD19ϩ B Genotyping Case Sex/Age WBCa Cellsb (%) CD20d CD23 CD5 CD10 CD34 CD38 CD45 (major cyto)c Diagnosis

1 M/3 156 9 98 8 17 68 29 28 54 t(8;14)(q24;q32) Ae 2 F/4 33 22 99 10 11 59 35 19 31 t(12;21)(q24;q11) A 3 F/11 54 14 97 9 9 78 19 18 26 t(11;14)(q13;q32) A 4 M/14 123 29 95 10 12 72 18 17 38 t(8;14)(q24;q32) A 5 F/14 51 15 96 15 9 71 21 16 37 t(12;21)(q24;q32) A 6 M/15 98 11 98 11 13 81 16 21 53 t(12;21)(q24;q32) A 7 F/15 49 28 94 18 17 83 23 23 55 del(7)(q11) A 8 F/16 22 12 98 10 9 58 39 28 42 t(8;22)(q24;q11) A 9 M/22 128 18 95 16 11 67 33 35 36 t(9;22)(q24;q11) A 10 M/23 62 16 91 12 10 71 24 31 34 t(2;8)(p12;q24) A 11 M/23 18 18 96 11 16 66 20 26 44 t(8;14)(q24;q32) A 12 M/24 23 21 99 10 19 64 22 29 35 del(1)(p36) A 13 F/31 31 14 97 14 18 84 19 27 32 t(2;8)(p12;q24) A 14 M/33 82 19 95 9 15 66 10 25 35 t(12;21)(q24;q32) A 15 F/38 19 39 99 6 19 76 17 27 31 t(8;22)(q24;q11) A 16 M/39 35 21 94 15 10 70 14 50 36 t(9;22)(q24;q32) A 17 M/42 218 27 97 9 10 77 19 37 28 t(9;22)(p12;q24) A Downloaded from 18 F/43 22 14 99 15 9 79 22 49 45 t(2;8)(p12;q24) A 19 M/45 88 18 95 7 11 68 30 35 31 t(12;21)(q24;q32) A 20 F/49 85 25 94 9 15 75 22 34 37 t(8;22)(q24;q11) A 21 M/52 31 12 97 17 12 76 21 36 28 NDg A 22 M/53 25 15 99 12 9 69 18 29 41 del(1)(p36) A 23 M/53 49 9 97 14 15 56 20 37 35 t(12;21)(q24;q32) A

24 F/54 64 12 91 16 17 70 26 41 24 t(12;21)(q24;q32) A http://www.jimmunol.org/ 25 M/55 36 16 96 12 15 81 15 32 28 t(9;22)(p12;q24) A 26 F/10 12 42 95 27 46 40 38 91 34 Trisomy(12) C 27 M/11 14 34 97 38 50 67 47 66 35 t(11;14)(q13;q32) C 28 M/17 40 41 98 40 47 38 40 70 25 t(11;14)(q13;q32) C 29 F/20 25 35 96 45 41 31 45 69 29 ND C 30 M/24 10 20 90 47 28 20 26 60 33 Trisomy(12) C 31 F/35 28 28 96 61 53 41 24 65 27 del(13)(q14q32) C 32 M/36 71 29 94 53 58 40 27 70 32 Trisomy(12) C 33 F/36 25 45 97 29 34 41 29 69 24 del(13)(q22) C 34 M/38 49 40 95 37 51 25 30 62 25 Trisomy(12) C

35 M/39 20 41 98 48 16 48 20 63 20 ND C by guest on September 24, 2021 36 M/40 31 36 96 33 37 60 31 67 3.8 t(11;14)(q13;q32) C 37 F/44 16 44 96 46 29 36 28 56 2.8 del(13)(q22q32) C 38 M/44 21 51 98 29 37 42 33 65 3.8 Trisomy(12) C 39 F/51 20 40 96 60 27 63 27 56 2.8 ND C 40 M/64 18 36 99 38 53 29 35 58 2.3 Trisomy(12) C

a WBC, White blood cells counting, 1 ϫ 109 cells/L; ND, no determination. b Total CD19ϩ B cells in PBMC. c Major findings of cytogenetic characteristics. d Percentages in gated CD19ϩ B cells from PBMC. Three-color flow cytometry was used for phenotyping by follow combinations: CD19/CD20/CD10, CD19/CD23/CD5, CD19/CD34/CD38, CD19/CD45/CD22, CD19/CD25/HLA-DR, CD19/CD45RO/MCF7. Data of CD22, CD25, HLA-DR, CD45RO, and MCF7 were not shown. e A, B-ALL; C, B-CLL. are expressed on B cell acute lymphocytic leukemia (B-ALL) CXCR5, is involved in the induction of apoptotic resistance in CD23ϩCD5ϩ B cells at high frequency. CCL19 plus CXCL13 B-ALL CD23ϩCD5ϩ B cells (8). render resistance to apoptosis in B-ALL CD23ϩCD5ϩ B cells (8). In this study, we report that CCL19 plus CXCL13 regulate in- However, the functions of CCR7/CCL19 and CXCR5/CXCL13 teraction between B-ALL CD23ϩCD5ϩ B cells and CD8ϩ T cells receptor-ligand pairs are not fully elucidated in the pathophysio- by inducing activation of PEG10, in turn, resulting in IL-10 over- logical events of malignant B cells interacting with T cells. expression and impairment of tumor-specific cytotoxicity in syn- Paternally expressed gene 10 (PEG10) is identified as a pater- geneic CD8ϩ T cells. nally expressed gene from a newly defined imprinted region at Ϫ Ϫ human chromosome 7q21 (27). PEG10 / mice show early em- Materials and Methods bryonic lethality owing to defects in the placenta, indicating a crit- Patients, cell purification, and reagents ical role of PEG10 in mouse parthenogenetic development (28). An elevated level of PEG10 expression has been found in the All patients with B cell lineage B-ALL and chronic lymphocytic leukemia (B-CLL) fulfilled The French-American-British Cooperative Group criteria majority of the human hepatocellular carcinoma cells (29, 30). (31) and the guidelines of the National Cancer Institute Working Group Exogenous expression of PEG10 confers oncogenic activity and (32, 33). The clinical data, immunophenotype and cytogenetic character- transfection of hepatoma cells (29). In addition, overexpression of istics of B-ALL and B-CLL cases included in this study were listed in PEG10 protects the cells from death mediated by SIAH1 (29). Table I. All patients gave informed consent according to institutional guidelines. CD19ϩ (from normal periphery), CD23ϩ, or CD23ϩCD5ϩ B PEG10 knockdown inhibits the proliferation of pancreatic carci- cells were purified from PBMCs from peripheral blood of normal subjects, noma and HepG2 hepatocellular carcinoma cells (30). Elevated cord blood (CB) of uncomplicated births (IgM undetectable) or patients PEG10 expression, caused by ligation of CCL19-CCR7/CXCL13- with B-ALL or B-CLL using FACStarPLUS sorting (34, 35). The viability 2882 MALIGNANT B-T CELL INTERACTION

FIGURE 1. Cytokine expressions in different B cells. IFN-␥, IL-2, IL-4, IL-5, IL-10, and TGF-␤1 were mea- sured in freshly isolated normal CD19ϩ B cells, CB, B-ALL, or B- CLL CD23ϩCD5ϩ B cells by intra- cellular cytokine flow cytometry (tops) and Q-PCR (bottoms). Data were mean Ϯ SD (three for normal peripheral and CB; four for B-ALL and B-CLL blood). Statistically sig- nificant differences as compared with normal periphery were indicated. p Ͻ 0.001. A, Cells ,ءء ;p Ͻ 0.05 ,ء were freshly isolated normal periph- eral CD19ϩ B cells (white bars), nor- mal CB (black bars), B-ALL (brick format bars), and B-CLL (gray bars) ϩ ϩ CD23 CD5 B cells. B, B-ALL Downloaded from CD23ϩCD5ϩ B cells were treated ei- ther with CCL19 (100 ng/ml; white bars), CXCL13 (100 ng/ml; black bars), with CCL19 plus CXCL13 (each 100 ng/ml; brick format bars) for 24 h or pretreated with anti-CCR7 plus anti-CXCR5 mAbs for 12 h http://www.jimmunol.org/ (each 1 ␮g/ml) before stimulation with CCL19 plus CXCL13 (gray bars).

of all cultured cells Ͼ95% tested by trypan blue exclusion. Human chronic (BD Pharmingen), the purified B or T cells were stimulated without or with myelogenous leukemia K562 cell line was obtained from the American endogenous and exogenous stimuli as indicated in the presence of 0.2 ␮lof by guest on September 24, 2021 Type Culture Collection. The anti-CXCR5, anti-CCR7, and chemokines Golgiplug at 37°C for duration as indicated. The analyses were performed (CXCL13 and CCL19) were purchased from R&D Systems. Mouse anti- with flow cytometer (COULTER XL; Coulter). Data were analyzed by human IFN-␥ receptor I mAb (IFNGR1 mAb, GIR-94), mouse anti-human means of the WinList program (Scripps Research Institute). IL-10 receptor A mAb (IL-10RA mAb, 37607.11), and goat anti-human IL-10 receptor B polyclonal Ab (IL10RB pAb) were purchased from Ab- cam. All Abs (IL-2, IFN-␥, IL-4, IL-5, IL-10, TGF-␤1, CXCR5, and Q-PCR CCR7) were from R&D Systems, and anti-␤-actin was from Sigma- Aldrich. For cytogenetic study, chromosome analysis was performed in All Q-PCR were performed as described elsewhere (35, 39). Briefly, Q-PCR both unstimulated and LPS and PMA mitogen-stimulated metaphase B was performed in special optical tubes in a 96-well microtiter plate (Applied cells from overnight cultures of bone marrow aspirate samples. Biosystems) with an ABI PRISM 7700 Sequence Detector System (Applied Biosystems). By using a SYBR Green PCR Core Reagents Kit, fluorescence T cell stimulation signals were generated during each PCR cycle via the 5Ј-to3Ј-endonuclease activity of AmpliTaq Gold to provide Q-PCR information. The sequences of Freshly isolated syngeneic CD4ϩ or CD8ϩ T cells (2 ϫ 105 cells/well) the specific primers are as follows. IFN-␥: sense, 5Ј-GCTAAAACAGGGA were used for primary stimulation in the presence of optimal numbers of AGCGAAAAA-3Ј; antisense, 5Ј-GGACAACCATTACTGGGATGCT-3Ј. syngeneic DCs in 24-well plates (400 ␮l/well) for 2 days (36). A secondary IL-2: sense, 5Ј-TGCAAGGGACTCAGGTGATG-3Ј; antisense, 5Ј-TGCTGC stimulation was performed in the addition of different numbers of purified TTATTTAGGATACCTATTAACTCA-3Ј. IL-4: sense, 5Ј-CACAGGCACA syngeneic CD23ϩCD5ϩ B cells as indicated in the figure legends for 4 AGCAGCTGAT-3Ј; antisense, 5Ј-GCCAGGCCCCAGAGGTT-3Ј. IL-5: days in the presence of tetanus toxoid (2.5 ␮g/ml). A total of 6 days after sense, 5Ј-ACGCAGTCTTGTACTATGCACTTTCT-3Ј; antisense, 5Ј-AGAA the onset of the primary culture, CD3ϩ T cells were harvested using a GCATCCTCATGGCTCTGA-3Ј. IL-10: sense, 5Ј-GTGATGCCCCAAGCTG positive selection procedure of anti-CD3 mAb-coated MACS bead assay AGA-3Ј; antisense, 5Ј-TCCCCCAGGGAGTTCACA-3Ј. TGF-␤1: sense, 5Ј- (Miltenyi Biotec), followed by real-time quantitative RT-PCR (Q-PCR) or TCAGAGCCACAAATCCTGAAAG-3Ј; antisense, 5Ј-CACCAAGTGTAC intracellular cytokine flow cytometry. In some cases, transwell experiments CCCGAAAGA-3Ј. CXCR5: sense, 5Ј-GGTCTTCATCTTGCCCTTTG-3Ј; were done in 24-well plates as described previously (37). Briefly, primary antisense, 5Ј-ATGCGTTTCTGCTTGGTTCT-3Ј, CCR7: sense, 5Ј-GCTCCA stimulation systems of CD3ϩ T cells in the presence of optimal numbers of GGCACGCAACTTT-3Ј; antisense, 5Ј-ACCACGACCACAGCGATGA-3Ј. syngeneic DCs (after 2 days) were placed in transwell chambers (Millicell, PEG10: sense, 5Ј-ATGATGACATCGAGCTCCG-3Ј; antisense, 5Ј-GCT 0.4 ␮M; Millipore) in the presence of different numbers of purified syn- GGGTAGTTGTGCATCA-3Ј. geneic CD23ϩCD5ϩ B cells and tetanus toxoid (2.5 ␮g/ml), as indicated in All unknown cDNAs were diluted to contain equal amounts of ␤-actin the figure legends, for 4 days. After 4 days of culture, activated CD3ϩ T cDNA. PCR retain conditions were 2 min at 50°C, 10 min at 95°C, 40 cells were harvested using MACS beads before further investigation. cycles with 15 s at 95°C, 60 s at 60°C for amplifications. Potential PCR product contamination was digested by uracil-N-glycosylase because dTTP Flow cytometry is substituted by dUTP. For immunophenotyping, PBMCs were stained with appropriate combina- tions of fluorochrome-labeled Abs for 20 min, followed by washing twice Western blot assay in staining buffer as previously described (8). For intracellular cytokine detection by immunofluorescence staining as described elsewhere (38), For detection, the cells were lysed in lysis buffer. Cell lysis was based on the protocol from Cytofix/Cytoperm Plug with a Golgiplug Kit performed for 30 min at 4°C with lysis buffer. Expression of target The Journal of Immunology 2883

FIGURE 2. Cytokine expressions in CD4ϩ and CD8ϩ T cells. The syn- geneic T cells were cocultured with normal peripheral CD19ϩ B cells, nor- mal CB, B-ALL, or B-CLL CD23ϩCD5ϩ B cells. IFN-␥, IL-4, and IL-10 were measured in CD4ϩ or CD8ϩ T cells cocultured with different B cells in the presence of tetanus toxoid (2.5 ␮g/ml) by intracellular cytokine flow cytometry (tops) and Q-PCR (bot- toms). DC:T:B cells ϭ 1:10:2. CD4ϩ or CD8ϩ T cells were harvested using MACS beads. Data were mean Ϯ SD (three for normal periphery and CB; five for B-ALL and B-CLL patients). Statistically significant differences as compared with normal periphery were .p Ͻ 0.001 ,ءء ;p Ͻ 0.05 ,ء .indicated A, Syngeneic CD4ϩ or CD8ϩ T cells Downloaded from were cocultured with normal peripheral CD19ϩ B cells (white bars), normal CB (black bars), B-ALL (brick format bars), or B-CLL (gray bars) CD23ϩ CD5ϩ B cells for 48 h. B, B-ALL CD23ϩCD5ϩ B cells were pretreated

either with CCL19 (100 ng/ml; white http://www.jimmunol.org/ bars), CXCL13 (100 ng/ml; black bars), with CCL19 plus CXCL13 (each 100 ng/ml; brick format bars) for 24 h before coculture with syngeneic CD4ϩ or CD8ϩ T cells, or pretreated with anti-CCR7 plus anti-CXCR5 mAbs for 12 h (each 1 ␮g/ml) before stimulation with CCL19 plus CXCL13 (gray bars). by guest on September 24, 2021 was semiquantified after Western blot analysis (40, 41). Lysates were cen- Gene silencing assay trifuged at 10,000 rpm for 5 min at 4°C. Protein concentration was mea- sured by Bio-Rad protein assay. Protein (around 40 ␮g) was loaded onto A gene silencing assay was conducted as described early (8). Short hairpin 16% SDS-PAGE, transferred onto polyvinylidene difluoride membranes RNAs (shRNAs) were produced in vitro using chemically synthesized after electrophoresis, and incubated with the appropriate Abs at 0.5 ␮g/ml. DNA oligonucleotide templates (Sigma-Aldrich) as described early (45). Analyses were conducted using ECL detection (Amersham Pharmacia Statistical analysis Biotech). Statistical significance was assessed by a paired or unpaired Student t test. ELISPOT assay Values of p Ͻ 0.05 were considered statistically significant. For ELISPOT cytokine detection, the purified B or T cells were stimulated Results with different stimuli and purified as indicated. The purified cells were ϩ ϩ seeded, cultured in the plats according to the manufacturers’ instructions. B-ALL CD23 CD5 B cells express IL-10 at high level The plates were stained with streptavidin-HRP (Mabtech), diluted 1/100, ϩ ϩ and Nova Red Substrate according to the manufacturers’ instructions and We previously reported that B-ALL CD23 CD5 B cells ex- then read manually. pressed CXCR5 and CCR7 at high frequency. Ligation of CCR7- CCL19 and CXCR5-CXCL13 up-regulated the expression and DC generation and in vitro cytotoxicity assay function of PEG10, and rendered a significant apoptotic resistance ϩ ϩ Monocytes were isolated from PBLs from healthy donors, B-CLL patients, in B-ALL CD23 CD5 B cells (8). or B-ALL patients with CD14ϩ magnetic beads (Miltenyi Biotech) as de- We further investigated the expression of cytokines in different 5 scribed (42). The cells were cultured for 6 days in 24-well plates at 5 ϫ 10 CD23ϩCD5ϩ B cells and CD19ϩCD5ϩ B cells in normal periph- cells/well in GM-CSF and IL-4 (both 1000 IU/ml; R&D Systems). Matu- ration was induced by addition of 1 ␮g/ml LPS and 50 ng/ml TNF-␣ (R&D ery by intracellular cytokine flow cytometry, Q-PCR at protein and Systems) for the last 40 h of cultures. For CTL induction, CD8ϩ T cells mRNA levels, respectively. Normal B cells (including normal pe- ϩ ϩ ϩ ϩ (96–98% pure) were negatively isolated with the StemSep system (Stem- ripheral CD19 CD5 and cord blood CD23 CD5 B cells) ex- Cell Technologies). The cells (5 ϫ 105 cells) were then sensitized by au- pressed IFN-␥, IL-2, IL-5, IL-10, and TGF-␤1 at low levels (Fig. ϫ 4 ϫ 4 ϩ tologous DCs (5 10 cells) pulsed with 5 10 irradiated CD4 T cells 1A). However, unstimulated B-ALL CD23ϩCD5ϩ B cells ex- and target cells (K562 cells) (43). At day 14, differentially treated synge- ϩ ϩ ϩ Ϫ ϫ 4 pressed IFN-␥ and IL-10 at very high levels (Fig. 1A), whereas neic CD23 CD5 or CD23 CD5 B cells (5 10 cells) were added into ϩ ϩ culture. At day 28, the cytotoxic activity of human CTL incubated under B-CLL CD23 CD5 B cells expressed these two cytokines at various effector-target ratios was assessed by a 51Cr release assay (44). moderate levels. Normal CD23ϩCD5ϩ B cells expressed IL-4 at ϩ 51 CD8 CTLs were added to the wells containing the Cr-labeled target slightly higher level than leukemic cells, whereas both normal and K562 cells. The percentage of specific cell lysis was calculated as the propor- leukemic B cells expressed IL-5 at similar levels (Fig. 1A). Given tion of released radioactivity vs 100% released radioactivity by the superna- ϩ ϩ tants of cells incubated with 0.1 N HCl and after subtracting the spontaneous that CCR7 and CXCR5 were expressed on B-ALL CD23 CD5 ϩ ϩ 51Cr release. B cells at high frequency (8), we treated the B-ALL CD23 CD5 2884 MALIGNANT B-T CELL INTERACTION

B cells in vitro either with CCL19 (CCR7 ligand) or/and CXCL13 (CXCR5 ligand), consequently followed by measuring the cyto- kine expression in the cells. CCL19 and CXCL13 together, but not alone, up-regulated expressions of IFN-␥ and IL-10 in B-ALL CD23ϩCD5ϩ B cells (Fig. 1B). The expressions of other cytokines (IL-2, IL-4, IL-5, and TGF-␤1) were not altered in B-CLL CD23ϩCD5ϩ B cells by stimulation with CCL19 or/and CXCL13 (Fig. 1B). These observations were further confirmed by ELISPOT assay and Western blot (data not shown). Collectively, these re- sults indicated that B-ALL CD23ϩCD5ϩ B cells expressed IFN-␥ and IL-10 at very high level, which could be further up-regulated by stimulation with CCL19 plus CXCL13. B-ALL CD23ϩCD5ϩ B cells regulate IL-10 expression in syngeneic CD8ϩ T cells By knowing that discrete B cell effectors could produce various FIGURE 3. IL-10 expression in syngeneic CD8ϩ T cells. The cells were arrays of cytokines, and modulated T cell responses (3, 6), we ϩ ϩ ϩ cocultured with B-ALL CD23 CD5 B cells. IL-10 expressions were proceeded by examining the expression of cytokines in CD4 or ϩ ϩ ϩ ϩ measured in syngeneic CD8 T cells cocultured with B-ALL CD8 T cells cocultured with syngeneic CD23 CD5 B cells CD23ϩCD5ϩ B cells in the presence of tetanus toxoid (2.5 ␮g/ml) by Downloaded from from normal cord blood, or from B-ALL and B-CLL patients. intracellular cytokine flow cytometry (all left panels), Q-PCR (all middle IFN-␥, IL-2, IL-4, IL-5, IL-10, and TGF-␤1 were expressed at panels), and Western blots (all right panels). DC:T:B cells ϭ 1:10:2. ϩ similar levels in the CD4 T cells cocultured with different CD8ϩ T cells were then harvested using MACS beads. Data were mean Ϯ CD23ϩCD5ϩ B cells either from normal CB or from B-ALL or SD (n ϭ 3). Statistically significant differences as compared with different p Ͻ 0.001. Actins in each ,ءء ;p Ͻ 0.05 ,ء .B-CLL patients (Fig. 2A; some data not shown). Nevertheless, Ab treatment were indicated ϩ lower picture indicated the quantity of total cellular protein from the tested IL-10 was markedly up-regulated in CD8 T cells cocultured with http://www.jimmunol.org/ syngeneic B-ALL CD23ϩCD5ϩ B cells (Fig. 2A), even though samples loaded in each lane. Arrows indicate appropriate cytokines iden- tified by markers of equivalent molecular weights in each lane. A, Synge- other cytokines (e.g., IFN-␥, IL-2, IL-4, IL-5, and TGF-␤1) were ϩ ϩ ϩ ϩ neic CD8 T cells were cocultured with B-ALL CD23 CD5 B cells for not changed in CD8 T cells after similar coculture (Fig. 2A; some ϩ 48 h. The syngeneic CD8 T cells were pretreated with IFNGR1 mAb (5 data not shown). In an attempt to explore the mechanism involved ␮ ␮ ϩ g/ml; white bars), IL-10RA mAb plus IL10RB pAb (each 5 g/ml; black in up-regulation of IL-10 expression in syngeneic CD8 T cells bars), or with all three Abs (each 5 ␮g/ml; brick format bars) for 12 h ϩ ϩ by B-ALL CD23 CD5 B cells, we pretreated the B-ALL before coculture or pretreated with isotype Abs for 12 h (each 5 ␮g/ml; ϩ ϩ CD23 CD5 B cells with CCL19 or/and CXCL13 before cocul- gray bars). Superscript a, Isotype Ab treatment. B, Cells were treated as ture with CD4ϩ or CD8ϩ T cells and then measured the cytokine described in A, but cocultures were conducted in a transwell manner as expression profile in CD4ϩ or CD8ϩ T cells. Treatment of CCL19 stated in Materials and Methods. by guest on September 24, 2021 and CXCL13 together, but not alone, further up-regulated IL-10 ϩ ϩ expression in CD8ϩ T cells isolated from the B-ALL patients after CD23 CD5 B cells (Fig. 3A), whereas, isotype-matching control coculture (Fig. 2B). In contrast, the other cytokines (e.g., IFN-␥, Abs had no such function. Other cytokines (e.g., IFN-␥, IL-2, IL-4, ϩ IL-2, IL-4, IL-5, and TGF-␤1) were not altered in these syngeneic IL-5, and TGF-␤1) were not altered in the B-ALL CD8 T cells CD8ϩ T cells by coculture with B-ALL CD23ϩCD5ϩ B cells after the blockage of IL-10 receptors (data not shown). These data ϩ ϩ pretreated with CCL19 or/and CXCL13 (Fig. 2B; some data not indicated that IL-10 from B-ALL CD23 CD5 B cells was shown). IFN-␥, IL-2, IL-4, IL-5, and TGF-␤1 as well as IL-10 mainly responsible for the up-regulated expression of IL-10 in syn- ϩ were not altered in syngeneic CD4ϩ T cells by coculture with geneic CD8 T cells during the B-T cell interaction. To explore B-ALL CD23ϩCD5ϩ B cells pretreated with CCL19 or/and whether cell-cell contact was necessary for IL-10 expression in ϩ CXCL13 (Fig. 2B; some data not shown). mAbs against CCR7 and B-ALL CD8 T cells in our B-T cell coculture system, we used a ϩ CXCR5 completely blocked the effects of CCL19 and CXCL13 on transwell culture system to separate B cells from T cells. CD8 T up-regulation of IL-10 production in CD8ϩ T cells (Fig. 2B), cells were pretreated with three Abs against IFN-␥ and IL-10 re- whereas isotype Ab controls had no such blockade function (data ceptors in some samples as described above. Cytokine analysis not shown). The results were further confirmed by ELISPOT assay revealed that IFN-␥, IL-2, IL-4, IL-5, and TGF-␤1 as well as IL-10 ϩ and Western blot (data not shown). expressions were not altered at all in syngeneic CD8 T cells in To determine which factor secreted by B-ALL CD23ϩCD5ϩ B the coculture of B-T cells in trans-well system (Fig. 3B; some cells, IFN-␥ or IL-10, is the key factor to enhance production of data not shown), indicating that cell-cell contact of B and T IL-10 in B-ALL CD8ϩ T cells during B-T cell interaction, we cells was required to up-regulate IL-10 production in syngeneic ϩ pretreated CD8ϩ T cells either with IFNGR1 mAb, IL-10RA mAb CD8 T cells in addition to secreted factor (IL-10) from B-ALL ϩ ϩ plus IL10RB pAb, or with all three Abs for 12 h before coculture CD23 CD5 B cells. ϩ ϩ with the syngeneic CD23 CD5 B cells. We then examined the ϩ ϩ ϩ B-ALL CD23 CD5 B cells impair the cytotoxicity of syngeneic expression of cytokines in CD8 T cells by intracellular cytokine ϩ flow cytometry, Q-PCR assay, and Western blots at protein and CD8 T cells mRNA levels. Pretreatment of CD8ϩ T cells with IFNGR1 mAb We further investigated the effect of B-ALL CD23ϩCD5ϩ B cells did not change the expression of IL-10 in these cells (Fig. 3A). on the cytotoxicity of syngeneic CD8ϩ T cells. We previously Other cytokines (e.g., IFN-␥, IL-2, IL-4, IL-5, and TGF-␤1) were reported that although CXCR5 and CCR7 were functionally ex- not altered in B-ALL CD8ϩ T cells either by IFNGR1 mAb (data pressed on B-ALL CD23ϩCD5ϩ B cells at high frequency, they not shown). Interestingly, pretreatment of CD8ϩ T cells with IL- were not found on CD23ϩCD5Ϫ B cells (8). In the following ex- 10RA mAb plus IL10RB pAb significantly down-regulated the periments, we compared their regulatory functions. Each of dif- expression of IL-10 in these cells after coculture with ALL ferent CD23ϩCD5Ϫ and CD23ϩCD5ϩ B cells were added into The Journal of Immunology 2885

(Fig. 4A). Considering the abundance of CCR7 and CXCR5 on B-ALL CD23ϩCD5ϩ B cells (8), we pretreated these cells with CCL19 and/or CXCL13 for 24 h before CTL activity analysis. B-ALL CD23ϩCD5ϩ B cells cultured with CCL19 and CXCL13 together, but not with CCL19 or CXCL13 alone, completely abol- ished tumor-specific CTL responses of syngeneic CD8ϩ T cells (Fig. 4B). In this regard, Abs against CCR7 and CXCR5 together blocked the function of CCL19 and CXCL13 on B-ALL CD23ϩCD5ϩ B cells (Fig. 4B), whereas isotype Abs had no such blocking function (data not shown). To explore potential mecha- nisms of underlying the phenomena, we pretreated the syngeneic CD8ϩ T cells with IFNGR1 mAb, IL-10RA mAb plus IL10RB pAb, or all three Abs before CTL assay. Abs against IL-10RA and IL10RB together, but not Ab against IFNGR1, reversed the im- pairment of syngeneic CD8ϩ CTL tumor-specific killing (Fig. 4C), whereas isotype-matching control Abs showed no such rescue effect (data not shown). These data suggested that B-ALL CD23ϩCD5ϩ B cells in vitro were able to impair tumor-specific ϩ cytotoxicity of syngeneic CD8 T cells, in which the IL-10 was an Downloaded from essential mediator. Involvement of PEG10 in B-ALL B-T cell interaction We previously showed that PEG10 was highly up-regulated and involved in the resistance to TNF-␣-mediated apoptosis in B-ALL ϩ ϩ

CD23 CD5 B cells (8). In this study, we further evaluated the http://www.jimmunol.org/ role of PEG10 in the expression of cytokines in B-ALL CD23ϩCD5ϩ B cells. We used shRNA of PEG10 (shRNAPEG10) to knockdown PEG10 expression and then measured expression of cytokines by intracellular cytokine flow cytometry, Q-PCR, ELISPOT assay, and Western blots at protein and mRNA lev- els, respectively. As expected, shRNAPEG10 at a high concen- tration (2 ␮g/ml) sequence-specifically knocked down the ex- pression of PEG10 in B-ALL CD23ϩCD5ϩ B cells (Ref. 8 and data not shown). shRNAPEG10 (2 ␮g/ml) significantly and se- by guest on September 24, 2021 FIGURE 4. Regulation of CTL activity of syngeneic CD8ϩ T cells by ϩ quence-specifically down-regulated the expression of IL-10, different B cells. To measure CTL activity, specific CTL CD8 T cells whereas the expressions of IL-2, IL-4, IL-5, and TGF-␤1 were were generated as described in Materials and Methods. Different ϩ ϩ ϩ Ϫ ϩ ϩ not altered in B-ALL CD23 CD5 B cells (data not shown). CD23 CD5 (upper left panel) and CD23 CD5 (all other panels) B cells ␮ from healthy CB, B-CLL, or B-ALL patients were added into culture at day The shRNAPEG10 at low concentration (0.02 g/ml), 14. The CTL activity was assessed by a 51Cr release assay with various E:T DNAPEG10, and vector showed no such effects (data not ratios. A, CD23ϩCD5Ϫ or CD23ϩCD5ϩ B cells were purified from healthy shown). As we described above (Fig. 1B), CCL19 plus CXCL13 CB (ࡗ), B-CLL (Ⅺ), or B-ALL (f) patients and cocultured with syngenic could selectively up-regulate IL-10 expressions in B-ALL ϩ ϩ CD8ϩ T cells. B, Purified B-ALL CD23ϩCD5ϩ B cells were either pre- CD23 CD5 B cells. In further studies, we first transfected B- treated with ligand CCL19 (ࡗ), CXCL13 (Ⅺ), or CCL19 plus CXCL13 ALL CD23ϩCD5ϩ B cells with shRNAPEG10 (2 ␮g/ml) and con- (f) each at 100 ng/ml for 24 h (L; right), or pretreated with anti-CCR7 Ab sequently treated the cells with CCL19 or/and CXCL13. CCL19 and ࡗ Ⅺ f ( ), anti-CXCR5 Ab ( ), anti-CCR7 Ab plus anti-CXCR5 Ab ( ) each CXCL13, either alone or together, could not alter the expression at 5 ␮g/ml for 12 h before treatment with CCL19 and CXCL13 (AϩL; ϩ patterns of IL-10 and other cytokines (e.g., IL-2, IL-4, IL-5, and left); subsequently CTL activity assay was conducted. C, Syngenic CD8 ϩ ϩ TGF-␤1) in these B-ALL CD23 CD5 B cells pretreated with T cells in CTL system were pretreated either with IFNGR1 mAb (5 ␮g/ml; ࡗ ␮ Ⅺ shRNAPEG10 (data not shown). ), IL-10RA mAb plus IL10RB pAb (each 5 g/ml; ), or with all three ϩ ␮ f Expression of cytokine (IL-10) in syngeneic CD8 T cells was Abs (each 5 g/ml; ) for 12 h before CTL assay. The dashed lines rep- ϩ ϩ resented the maximal extent of spontaneous cell lysis. Statistically signif- regulated by coculture with B-ALL CD23 CD5 B cells (Fig. 2). icant differences as compared among different pretreatments were indi- We used the same setting to further investigate the involvement of ϩ p Ͻ 0.001. PEG10 in cytokine production in syngeneic CD8 T cells. The ,ءء ;p Ͻ 0.05 ,ء ;cated. n ϭ 6 B-ALL CD23ϩCD5ϩ B cells transfected with shRNAPEG10 (2 ␮g/ml) significantly and sequence-specifically down-regulated the coculture system during the tumor-specific CTL killing mediated expression of IL-10 in syngeneic CD8ϩ T cells in the coculture by syngeneic CD8ϩ T cells. To measure CTL activity, we gener- system (Fig. 5A). As controls, shRNAPEG10 at a low concentra- ated tumor-specific CTL CD8ϩ T cells to human chronic myelog- tion (0.02 ␮g/ml), DNAPEG10 and vector alone showed no such enous leukemia K562 cells and set up the coculture in the presence effects (Fig. 5A). The expression of other cytokines (e.g., IL-2, of CD23ϩCD5Ϫ and CD23ϩCD5ϩ B cells, respectively. To our IL-4, IL-5, and TGF-␤1) were not altered in the syngeneic CD8ϩ surprise, B-ALL CD23ϩCD5ϩ B cells, but not CD23ϩCD5Ϫ B T cells in the same setting system (data not shown). We next tested cells, showed strong inhibitory effect on specific CTL responses of the effect of CCL19 or/and CXCL13 on shRNAPEG10-transfected syngeneic CD8ϩ T cells (Fig. 4A). Furthermore, only B-ALL, but (2 ␮g/ml) B-ALL CD23ϩCD5ϩ B cells in the coculture system. not normal or B-CLL, CD23ϩCD5ϩ B cells selectively and sig- CCL19 and CXCL13, either alone or together, could not alter the nificantly inhibited the specific CTL responses of CD8ϩ T cells patterns of IL-10 expression in the syngeneic CD8ϩ T cells (Fig. 2886 MALIGNANT B-T CELL INTERACTION

FIGURE 5. IL-10 expression in syngenic CD8ϩ T cells. IL-10 expres- sion was measured in syngenic CD8ϩ T cells cocultured with ϩ ϩ

CD23 CD5 B cells pretreated with shRNAPEG10 in the presence of Downloaded from tetanus toxoid (2.5 ␮g/ml) by intracellular cytokine flow cytometry (all left panels), Q-PCR (all middle panels), Western blots (all right panels). DC: T:B cells ϭ 1:10:2. Data were mean Ϯ SD (n ϭ 4). Statistically significant differences as compared among different shRNA treatments were indicated. FIGURE 6. Regulation of CTL activity of syngeneic CD8ϩ T cells by -p Ͻ 0.001. Actins in each lower picture indicated the B-ALL CD23ϩCD5ϩ B cells. To measure CTL activity, specific CTL syn ,ءء ;p Ͻ 0.05 ,ء quantity of total cellular protein from the tested samples loaded in each geneic CD8ϩ T cells were generated. B-ALL CD23ϩCD5Ϫ (upper left ϩ ϩ lane. Arrows indicated markers used to verify equivalent molecular panel) and CD23 CD5 (all other panels) B cells were added into culture http://www.jimmunol.org/ weights of appropriate cytokine in each lane. A, shRNAPEG10 was trans- at day 14. The CTL activity was assessed by a 51Cr release assay with ϩ ϩ fected into B-ALL CD23 CD5 B cells with appropriate concentration various E:T ratios. A, B-ALL CD23ϩCD5Ϫ or CD23ϩCD5ϩ B cells were following the manufacturer’s instructions for 2 days, and the cells were pretreated with empty vectors (ࡗ), low concentration (0.02 ␮g/ml; Ⅺ), or ϩ then added into coculture with syngenic CD8 T cells. Vector, empty high concentration (2 ␮g/ml; f) shRNAPEG10. B, Purified B-ALL vectors (white bars); shDNA, shDNA with irrelevant sequence (2 ␮g/ml; black CD23ϩCD5ϩ B cells were first transfected with a high concentration of bars); shRNAPEG10l, low concentration (0.02 ␮g/ml; brick format bars); shRNAPEG10 (2 ␮g/ml) as described in A. Consequently, the cells were shRNAPEG10h, high concentration (2 ␮g/ml; gray bars). B, B-ALL treated with ligand CCL19 (ࡗ), CXCL13 (Ⅺ), or CCL19 plus CXCL13 ϩ ϩ CD23 CD5 B cells were transfected with high concentration of (f), each at 100 ng/ml for 24 h before CTL activity (left; L). In some cases, shRNAPEG10 (2 ␮g/ml) as described in A. Consequently, the cells were syngeneic CD8ϩ T cells were pretreated with exogenous IL-10 (100 ng/ml; by guest on September 24, 2021 treated either with CCL19 (100 ng/ml; white bars), CXCL13 (100 ng/ml; black ࡗ), IFN-␥ (10 ng/ml; Ⅺ), IL-10 plus IFN-␥ (f) for 12 h before CTL bars), with CCL19 plus CXCL13 (each 100 ng/ml; brick format bars) for 24 h activity (Exo-IL; right). In the CTL system, the purified B-ALL before cytokine assays, or pretreated with anti-CCR7 plus anti-CXCR5 mAbs CD23ϩCD5ϩ B cells were transfected with high concentrations of for 12 h (each 1 ␮g/ml) before stimulation with CCL19 plus CXCL13 (gray shRNAPEG10 (2 ␮g/ml) as described in A. The dashed lines represented bars). Superscript a, Anti-CCR7 plus anti-CXCR5 mAbs treatment. the maximal extent of spontaneous cell lysis. Statistically significant dif- ferences as compared among different pretreatments were indicated. n ϭ 6; .p Ͻ 0.001 ,ءء ;p Ͻ 0.05 ,ء 5B). The expressions of other cytokines (e.g., IL-2, IL-4, IL-5, and ϩ TGF-␤1) were not altered in the syngeneic CD8ϩ T cells either to preculture syngeneic CD8 T cells before CTL activity. In this ϩ ϩ (data not shown). These data were support the conclusion that CTL assay setting, B-ALL CD23 CD5 B cells were also trans- PEG10 was indeed involved in the up-regulation of IL-10 in the fected with shRNAPEG10 (2 ␮g). Exogenous IL-10, but not described B-T cell interaction. IFN-␥, induced the impairment of tumor-specific CTL responses ϩ ϩ ϩ As observed early, B-ALL CD23ϩCD5ϩ B cells in vitro were of CD8 T cells by B-ALL CD23 CD5 B cells (Fig. 6B), al- able to impair tumor-specific cytotoxicity of syngeneic CD8ϩ T though B-ALL B cells were pretreated with shRNAPEG10. In the ϩ ϩ cells in an IL-10 secretion-dependent manner (Fig. 4). In the fol- CTL system without B-ALL CD23 CD5 B cells, exogenous lowing CTL assay, tumor-specific killing was measured in the IL-10 could not significantly inhibit tumor-specific killing of syn- ϩ presence of shRNAPEG10-transfected (2 ␮g/ml) B-ALL geneic CD8 T cells (data not shown). CD23ϩCD5ϩ B cells (Fig. 6). As expected, B-ALL CD23ϩCD5Ϫ Thus, this group of experiments suggests that CCL19/CCR7- ϩ ϩ B cells showed no effect on tumor-specific killing of CD8ϩ T cells CXCL13/CXCR5-PEG10 pathway in B-ALL CD23 CD5 B no matter what concentrations of shRNAPEG10 were applied (Fig. cells was involved in B-T cell interaction in terms of regulations of 6A). However, the shRNAPEG10 (2 ␮g/ml) applied in B-ALL both cytokine expression (IL-10) and tumor-specific CTL re- ϩ CD23ϩCD5ϩ B cells selectively and sequence-specifically sponses of syngeneic CD8 T cells. blocked the impairment of tumor-specific CTL responses of syn- geneic CD8ϩ T cells (Fig. 6A). Neither shRNAPEG10 at low con- Discussion centrations, nor DNAPEG10 (Fig. 6A), nor vector (data not shown) Dynamic and productive interaction between T and B cells is re- had such a function. We next transfected B-ALL CD23ϩCD5ϩ B quired for the humoral immune responses to many foreign protein cells with shRNAPEG10 (2 ␮g/ml) and consequently treated the Ags and production of pathogenic Abs characteristic of several cells either with CCL19 and/or CXCL13 before the tumor-specific autoimmune conditions (46). Although a number of recent re- CTL system. CCL19 and CXCL13, either alone or together, could searches have shown that B cells produce immunoregulatory cy- not alter the pattern of tumor-specific killing of syngeneic CD8ϩ T tokines such as IFN-␥ in several infections and autoimmune dis- cells (Fig. 6B). We further applied exogenous IL-10 and/or IFN-␥ eases (3–6), the mechanisms that control cytokine production by B The Journal of Immunology 2887 cells, particularly by malignant B cells, are still needed to be fur- PEG10 is identified on human chromosome 7q21 (28, 59). ther clarified. In this study, we have observed that B-ALL Mouse homolog PEG10 has recently been located in a large im- CD23ϩCD5ϩ B cells express IFN-␥ and IL-10 at high levels, printed gene cluster on mouse proximal chromosome 6 and was which can be further up-regulated by CCL19 plus CXCL13 (Fig. confirmed to be imprinted (60). Because the protein products from 1). IL-10 secreted from B-ALL CD23ϩCD5ϩ B cells is able to the predicted open reading frames (open reading frame 1 and open up-regulate expression of IL-10 in syngeneic CD8ϩ T cells (Fig. reading frame 2) of PEG10 show homology to the gag and pol 2). To the best of our knowledge, this is the first report that ma- proteins of vertebrate retrotransposon Ty3/Gypsy, PEG10 is spec- lignant CD23ϩCD5ϩ B cells play an immunoregulatory role to ulated to be a retrotransposon-derived gene. Distinct expression of differentially regulate inflammatory cytokine expression through a PEG10 is found in the brain, kidney, lung, testis, and placenta but CXCR5/CXCL13-CCR7/CCL19-PEG10 axis in the cells. not in the liver and a number of other tissues (27). In contrast to In physiological situations, CCL19 is detected in T zone stromal this, expression of PEG10 is detected only in the placenta among cells, DCs, and tonsillar perivascular cells in vivo (47, 48); mean- the 14 adult mouse tissues (60). Some data suggest a role for pref- while, CXCL13 is mainly found in stromal cells in B cell follicles erential expression of PEG10 in regulating growth control of liver (23). CCL19/CCR7 has been identified as the gatekeepers for both and pancreatic carcinoma cells (30). Exogenous PEG10 promotes naive T cells and DCs to control their entry and exit from second- growth of certain hepatocellular carcinoma cell lines that does not ary lymphoid tissues organs. In contrast, in pathophysiological sit- manifest endogenous PEG10. The interaction of PEG10 with uations, it has been reported that chemokine receptors are ex- SIAH plays important roles in resistance to apoptosis (29). We pressed on neoplastic cells of hemopoietic and nonhematopoietic have found that B-ALL CD23ϩCD5ϩ B cells overexpresses origin, and that overexpression of some of these receptors is PEG10 significantly up-regulated by costimulation with CCL19 Downloaded from closely related to tumor progression and metastasis (49–51). As and CXCL13 (8). Functionally, CCL19 and CXCL13 coopera- for B cell-derived lymphoproliferative disorders, CXCR5 have tively up-regulate expression and function of PEG10 to confer been detected in neoplastic B cells from B-ALL (8, 52) and B-CLL resistance to apoptosis in B-ALL CD23ϩCD5ϩ B cells (8). In this (8, 53). CCR7 has been found in B-CLL (8, 54) and in tumor cells study, shRNA of PEG10 significantly down-regulated the expres- from classical Hodgkin’s disease with predominance sion of IL-10 in B-ALL CD23ϩCD5ϩ B cells. Pretreatment with

(55). In our previous study, we have reported that CXCR5 and shRNAPEG10 has selectively down-regulated the expression of http://www.jimmunol.org/ CCR7 are frequently and functionally expressed on B-ALL IL-10 in syngeneic CD8ϩ T cells in coculture with B-ALL CD23ϩCD5ϩ B cells. Coactivation of CXCR5 and CCR7 has dis- CD23ϩCD5ϩ B cells. The shRNAPEG10 has blocked the impair- played a novel function to induce resistance to TNF-␣-mediated ment of tumor-specific CTL responses of syngeneic CD8ϩ T cells apoptosis in B-ALL CD23ϩCD5ϩ B cells (8). In this study, we by B-ALL CD23ϩCD5ϩ B cells (Fig. 5). Collectively, these data have extended our investigation on B-ALL CD23ϩCD5ϩ B cells are demonstrating that PEG10 is indeed involved in the up-regu- concerning the aberrations of their immune functions. We have lation of IL-10 during the interaction of B-ALL CD23ϩCD5ϩ B observed that, through the ligation of CCR7 and CXCR5, B-ALL cells with T cells. It is worthwhile to investigate the molecular CD23ϩCD5ϩ B cells significantly inhibit tumor-specific cytotox- basis of CCR7 and CXCR5 in vivo as how the PEG10 gene is icity of syngeneic CD8ϩ T cells, where IL-10 plays functions as a activated in human B-ALL CD23ϩCD5ϩ B cells, which may lead by guest on September 24, 2021 critical mediator (Fig. 4). Innate and adaptive immune responses to further understanding of PEG10 function and mechanisms in- can be induced against tumors, and the protective and therapeutic volved in the process of malignant lymphoproliferative disorders. effector cells include CD8ϩ CTL, IFN-␥-producing CD4ϩ and ϩ CD8 T cells, NK cells, and macrophages (56). Based on the fact Disclosures that CTL can directly kill tumor cells, cancer-directed and im- The authors have no financial conflict of interest. mune-based therapies have focused on eliciting a CTL response. An increasing important focus is being given to the stimulation of a CD4ϩ Th cell response in cancer development immunology and References 1. Pistoia, V. 1997. Production of cytokines by human B cells in health and disease. its immunotherapy (57). Th1 cells, characterized by secretion of Immunol. Today 18: 343–350. IFN-␥ and TNF-␣, are primarily responsible for activating and 2. Lund, F. E., B. A. Garvy, T. D. Randall, and D. P. Harris. 2005. Regulatory roles regulating the development and persistence of CTL. B cells are for cytokine-producing B cells in infection and autoimmune disease. Curr. Dir. ␣ Autoimmun. 8: 25–25. important producers of cytokines such as IL-2, TNF- , IL-4, IL-6, 3. Mizoguchi, A., E. Mizoguchi, H. Takedatsu, R. S. Blumberg, and A. K. Bhan. 2002. IL-10, and IL-12 (3, 6, 58). B cells can be differentiated into dis- Chronic intestinal inflammatory condition generates IL-10-producing regulatory B tinct cytokine-producing effector subsets (3, 6). 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CORRECTIONS

Anderson, M. J., K. Shafer-Weaver, N. M. Greenberg, and A. A. Hurwitz. 2007. Tolerization of tumor-specific T cells despite efficient initial priming in a primary murine model of prostate cancer. J. Immunol. 178: 1268–1276.

The authors revised the Footnotes to include additional funding information. The corrected footnote is shown below.

1 This research was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, by the Department of Defense Congressionally Directed Medical Research Program/Prostate Cancer Research Program Award DAMD17-01-1-0085, and by the Prostate Cancer Foundation (CaP CURE). This work is in partial fulfillment of the degree of Doctor of Philosophy at State University of New York, Upstate Medical University for Michael J. Anderson.

Wang, W., M. Milani, N. Ostlie, D. Okita, R. K. Agarwal, R. Caspi, and B. M. Conti-Fine. 2007. C57BL/6 mice genetically deficient in IL-12/IL-23 and IFN-␥ are susceptible to experimental autoimmune myasthenia gravis, suggesting a pathogenic role of non-Th1 cells. J. Immunol. 178: 7072–7080.

The sixth author’s middle initial was omitted. The correct name is Rachel R. Caspi.

Wang, X., H. Yuling, J. Yanping, T. Xinti, Y. Yaofang, Y. Feng, X. Ruijin, W. Li, C. Lang, L. Jingyi, et al. 2007. CCL19 and CXCL13 synergistically regulate interaction between B cell acute lymphocytic leukemia CD23ϩCD25ϩ B cells and CD8ϩ T cells. J. Immunol. 179: 2880–2888.

The institution for the 13th author and the institution for the 14th and 15th authors are reversed. Xia Bing is from the Departments of Internal Medicine and Geriatrics, The Zongnan University Hospital, Wuhan University, Wuhan, China. Qiao Li and Alfred E. Chang are from the Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109. The corrected author and affiliation lines are shown below.

Xingbing Wang,*† He Yuling,† Jiang Yanping,† Tan Xinti,† Yang Yaofang,‡ Yu Feng,† Xiao Ruijin,†§ Wang Li,† Chen Lang,† Liu Jingyi,† Tang Zhiqing,† Ouyang Jingping,† Xia Bing,¶ Qiao Li,ʈ Alfred E. Chang,ʈ Zimin Sun,* Jin Youxin,§ and Tan Jinquan*†‡

*Department of Hematology, Anhui Medical University Affiliated Provincial Hospital, Hefei, China; †Departments of Immunology and Pathophysiology and Laboratory of Allergy and Clinical Immunology, Institute of Allergy and Immune- Related Diseases and Center for Medical Research, Wuhan University School of Medicine, Wuhan, China; ‡Department of Anatomy, Jiujiang University Medical College, Jiujiang University, Jiujiang, China; §The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, China; ¶Departments of Internal Medicine and Geriatrics, The Zongnan University Hospital, Wuhan University, Wuhan, China; and ʈDepartment of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109

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