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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^¶, Alfred E. Chang^¶, Zimin Sun^*, Jin Youxin and Tan Jinquan^3,*,,

^* Department of Hematology, Anhui Medical University Affiliated Provincial Hospital, Hefei, China; Departments of Immunology and Pathphysiology 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 ^|| Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI 48109

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 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-T cell 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 cytokine expressions. IL-10 may be one of the critical cellular factors conferring B-ALL CD23⁺CD5⁺ B cells to escape from host immune surveillance.

	Introduction

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It has been highlighted in several recent investigations that B cells produce cytokines in response to a diverse array of stimuli including microbial products, Ags, and T cells (1, 2). Naive B cells can differentiate into discrete B cell effector subsets to produce differential arrays of cytokines upon restimulation (3, 4, 5). These cytokine-producing effector B cells can interact with T cells and modulate their immune responses (6). Cytokine-producing B cells, seen in draining lymph nodes and spleens of autoimmune and pathogen-infected mice (1, 2, 3, 4, 5, 6, 7), play critical protective roles in several autoimmune disease models (3, 4, 5). However, the knowledge of interactions between malignant B cells (leukemic B 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 development and maintenance of lymphoid tissue microarchitecture (9). CXCR5 is expressed on mature recirculating B cells, small subsets of normal CD4⁺ and CD8⁺ T cells, and skin-derived migratory dendritic cells (DC)⁴ (10, 11, 12, 13). CXCR5 is responsible for guiding B cells into the B cell zone of secondary lymphoid organs (14, 15, 16). CXCR5 on a subset of T cells plays an additional role in T cell migration (16, 17). CCR7, highly expressed on naive T cells (18), is closely related to T cell differentiation toward effector cells (19, 20, 21). The synergy of CCL19/CCR7 and CXCL13/CXCR5 has been shown in both physiological and pathological situations, such as controlling T cells and DCs homing to secondary lymphoid organs (19, 20, 21) and determining the positioning and proper function of follicular Th cells (18). CXCR5- and CCR7-deficient mice lack lymphoid follicles due to an impaired migration of B cells (22). Overexpression of CCR7 and CXCR5 on tumor lymphocytes was closely related to the apoptosis of cells as well as their migration and infiltration (23, 24, 25, 26). CCR7 and CXCR5 are expressed on B cell acute lymphocytic leukemia (B-ALL) CD23⁺CD5⁺ B cells at high frequency. CCL19 plus CXCL13 render resistance to apoptosis in B-ALL CD23⁺CD5⁺ B cells (8). However, the functions of CCR7/CCL19 and CXCR5/CXCL13 receptor-ligand pairs are not fully elucidated in the pathophysiological events of malignant B cells interacting with T cells.

Paternally expressed gene 10 (PEG10) is identified as a paternally expressed gene from a newly defined imprinted region at human chromosome 7q21 (27). PEG10^–/– mice show early embryonic lethality owing to defects in the placenta, indicating a critical role of PEG10 in mouse parthenogenetic development (28). An elevated level of PEG10 expression has been found in the majority of the human hepatocellular carcinoma cells (29, 30). Exogenous expression of PEG10 confers oncogenic activity and transfection of hepatoma cells (29). In addition, overexpression of PEG10 protects the cells from death mediated by SIAH1 (29). PEG10 knockdown inhibits the proliferation of pancreatic carcinoma and HepG2 hepatocellular carcinoma cells (30). Elevated PEG10 expression, caused by ligation of CCL19-CCR7/CXCL13-CXCR5, is involved in the induction of apoptotic resistance in B-ALL CD23⁺CD5⁺ B cells (8).

In this study, we report that CCL19 plus CXCL13 regulate interaction between B-ALL CD23⁺CD5⁺ B cells and CD8⁺ T cells by inducing activation of PEG10, in turn, resulting in IL-10 overexpression and impairment of tumor-specific cytotoxicity in syngeneic CD8⁺ T cells.

	Materials and Methods

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Patients, cell purification, and reagents

All patients with B cell lineage B-ALL and chronic lymphocytic leukemia (B-CLL) fulfilled The French-American-British Cooperative Group criteria (31) and the guidelines of the National Cancer Institute Working Group (32, 33). The clinical data, immunophenotype and cytogenetic characteristics of B-ALL and B-CLL cases included in this study were listed in Table I. All patients gave informed consent according to institutional guidelines. CD19⁺ (from normal periphery), CD23⁺, or CD23⁺CD5⁺ B cells were purified from PBMCs from peripheral blood of normal subjects, cord blood (CB) of uncomplicated births (IgM undetectable) or patients with B-ALL or B-CLL using FACStar^PLUS sorting (34, 35). The viability of all cultured cells >95% tested by trypan blue exclusion. Human chronic myelogenous leukemia K562 cell line was obtained from the American Type Culture Collection. The anti-CXCR5, anti-CCR7, and chemokines (CXCL13 and CCL19) were purchased from R&D Systems. Mouse anti-human IFN- receptor I mAb (IFNGR1 mAb, GIR-94), mouse anti-human 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 Abcam. All Abs (IL-2, IFN-, IL-4, IL-5, IL-10, TGF-β1, CXCR5, and CCR7) were from R&D Systems, and anti-β-actin was from Sigma-Aldrich. For cytogenetic study, chromosome analysis was performed in both unstimulated and LPS and PMA mitogen-stimulated metaphase B cells from overnight cultures of bone marrow aspirate samples.

Freshly isolated syngeneic CD4⁺ or CD8⁺ T cells (2 x 10⁵ cells/well) were used for primary stimulation in the presence of optimal numbers of syngeneic DCs in 24-well plates (400 µl/well) for 2 days (36). A secondary stimulation was performed in the addition of different numbers of purified syngeneic CD23⁺CD5⁺ B cells as indicated in the figure legends for 4 days in the presence of tetanus toxoid (2.5 µg/ml). A total of 6 days after the onset of the primary culture, CD3⁺ T cells were harvested using a positive selection procedure of anti-CD3 mAb-coated MACS bead assay (Miltenyi Biotec), followed by real-time quantitative RT-PCR (Q-PCR) or intracellular cytokine flow cytometry. In some cases, transwell experiments were done in 24-well plates as described previously (37). Briefly, primary stimulation systems of CD3⁺ T cells in the presence of optimal numbers of syngeneic DCs (after 2 days) were placed in transwell chambers (Millicell, 0.4 µM; Millipore) in the presence of different numbers of purified syngeneic CD23⁺CD5⁺ B cells and tetanus toxoid (2.5 µg/ml), as indicated in the figure legends, for 4 days. After 4 days of culture, activated CD3⁺ T cells were harvested using MACS beads before further investigation.

Flow cytometry

For immunophenotyping, PBMCs were stained with appropriate combinations of fluorochrome-labeled Abs for 20 min, followed by washing twice in staining buffer as previously described (8). For intracellular cytokine detection by immunofluorescence staining as described elsewhere (38), based on the protocol from Cytofix/Cytoperm Plug with a Golgiplug Kit (BD Pharmingen), the purified B or T cells were stimulated without or with endogenous and exogenous stimuli as indicated in the presence of 0.2 µl of Golgiplug at 37°C for duration as indicated. The analyses were performed with flow cytometer (COULTER XL; Coulter). Data were analyzed by means of the WinList program (Scripps Research Institute).

Q-PCR

All Q-PCR were performed as described elsewhere (35, 39). Briefly, Q-PCR was performed in special optical tubes in a 96-well microtiter plate (Applied Biosystems) with an ABI PRISM 7700 Sequence Detector System (Applied Biosystems). By using a SYBR Green PCR Core Reagents Kit, fluorescence signals were generated during each PCR cycle via the 5'- to 3'-endonuclease activity of AmpliTaq Gold to provide Q-PCR information. The sequences of the specific primers are as follows. IFN-: sense, 5'-GCTAAAACAGGGA AGCGAAAAA-3'; antisense, 5'-GGACAACCATTACTGGGATGCT-3'. IL-2: sense, 5'-TGCAAGGGACTCAGGTGATG-3'; antisense, 5'-TGCTGCTTATTTAGGATACCTATTAACTCA-3'. IL-4: sense, 5'-CACAGGCACAAGCAGCTGAT-3'; antisense, 5'-GCCAGGCCCCAGAGGTT-3'. IL-5: sense, 5'-ACGCAGTCTTGTACTATGCACTTTCT-3'; antisense, 5'-AGAAGCATCCTCATGGCTCTGA-3'. IL-10: sense, 5'-GTGATGCCCCAAGCTGAGA-3'; antisense, 5'-TCCCCCAGGGAGTTCACA-3'. TGF-β1: sense, 5'-TCAGAGCCACAAATCCTGAAAG-3'; antisense, 5'-CACCAAGTGTACCCCGAAAGA-3'. CXCR5: sense, 5'-GGTCTTCATCTTGCCCTTTG-3'; antisense, 5'-ATGCGTTTCTGCTTGGTTCT-3', CCR7: sense, 5'-GCTCCAGGCACGCAACTTT-3'; antisense, 5'-ACCACGACCACAGCGATGA-3'. PEG10: sense, 5'-ATGATGACATCGAGCTCCG-3'; antisense, 5'-GCT GGGTAGTTGTGCATCA-3'.

All unknown cDNAs were diluted to contain equal amounts of β-actin cDNA. PCR retain conditions were 2 min at 50°C, 10 min at 95°C, 40 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 is substituted by dUTP.

Western blot assay

For protein detection, the cells were lysed in lysis buffer. Cell lysis was performed for 30 min at 4°C with lysis buffer. Expression of target proteins was semiquantified after Western blot analysis (40, 41). Lysates were centrifuged at 10,000 rpm for 5 min at 4°C. Protein concentration was measured by Bio-Rad protein assay. Protein (around 40 µg) was loaded onto 16% SDS-PAGE, transferred onto polyvinylidene difluoride membranes after electrophoresis, and incubated with the appropriate Abs at 0.5 µg/ml. Analyses were conducted using ECL detection (Amersham Pharmacia Biotech).

ELISPOT assay

For ELISPOT cytokine detection, the purified B or T cells were stimulated with different stimuli and purified as indicated. The purified cells were seeded, cultured in the plats according to the manufacturers’ instructions. The plates were stained with streptavidin-HRP (Mabtech), diluted 1/100, and Nova Red Substrate according to the manufacturers’ instructions and then read manually.

DC generation and in vitro cytotoxicity assay

Monocytes were isolated from PBLs from healthy donors, B-CLL patients, or B-ALL patients with CD14⁺ magnetic beads (Miltenyi Biotech) as described (42). The cells were cultured for 6 days in 24-well plates at 5 x 10⁵ cells/well in GM-CSF and IL-4 (both 1000 IU/ml; R&D Systems). Maturation was induced by addition of 1 µg/ml LPS and 50 ng/ml TNF- (R&D Systems) for the last 40 h of cultures. For CTL induction, CD8⁺ T cells (96–98% pure) were negatively isolated with the StemSep system (StemCell Technologies). The cells (5 x 10⁵ cells) were then sensitized by autologous DCs (5 x 10⁴ cells) pulsed with 5 x 10⁴ irradiated CD4⁺ T cells and target cells (K562 cells) (43). At day 14, differentially treated syngeneic CD23⁺CD5⁺ or CD23⁺CD5^– B cells (5 x 10⁴ cells) were added into culture. At day 28, the cytotoxic activity of human CTL incubated under various effector-target ratios was assessed by a ⁵¹Cr release assay (44). CD8⁺ CTLs were added to the wells containing the ⁵¹Cr-labeled target K562 cells. The percentage of specific cell lysis was calculated as the proportion of released radioactivity vs 100% released radioactivity by the supernatants of cells incubated with 0.1 N HCl and after subtracting the spontaneous ⁵¹Cr release.

Gene silencing assay

A gene silencing assay was conducted as described early (8). Short hairpin RNAs (shRNAs) were produced in vitro using chemically synthesized DNA oligonucleotide templates (Sigma-Aldrich) as described early (45).

Statistical analysis

Statistical significance was assessed by a paired or unpaired Student t test. Values of p < 0.05 were considered statistically significant.

	Results

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B-ALL CD23⁺CD5⁺ B cells express IL-10 at high level

We previously reported that B-ALL CD23⁺CD5⁺ B cells expressed CXCR5 and CCR7 at high frequency. Ligation of CCR7-CCL19 and CXCR5-CXCL13 up-regulated the expression and function of PEG10, and rendered a significant apoptotic resistance in B-ALL CD23⁺CD5⁺ B cells (8).

We further investigated the expression of cytokines in different CD23⁺CD5⁺ B cells and CD19⁺CD5⁺ B cells in normal periphery by intracellular cytokine flow cytometry, Q-PCR at protein and mRNA levels, respectively. Normal B cells (including normal peripheral CD19⁺CD5⁺ and cord blood CD23⁺CD5⁺ B cells) expressed IFN-, IL-2, IL-5, IL-10, and TGF-β1 at low levels (Fig. 1A). However, unstimulated B-ALL CD23⁺CD5⁺ B cells expressed IFN- and IL-10 at very high levels (Fig. 1A), whereas B-CLL CD23⁺CD5⁺ B cells expressed these two cytokines at moderate levels. Normal CD23⁺CD5⁺ B cells expressed IL-4 at slightly higher level than leukemic cells, whereas both normal and leukemic B cells expressed IL-5 at similar levels (Fig. 1A). Given that CCR7 and CXCR5 were expressed on B-ALL CD23⁺CD5⁺ B cells at high frequency (8), we treated the B-ALL CD23⁺CD5⁺ B cells in vitro either with CCL19 (CCR7 ligand) or/and CXCL13 (CXCR5 ligand), consequently followed by measuring the cytokine expression in the cells. CCL19 and CXCL13 together, but not alone, up-regulated expressions of IFN- and IL-10 in B-ALL CD23⁺CD5⁺ B cells (Fig. 1B). The expressions of other cytokines (IL-2, IL-4, IL-5, and TGF-β1) were not altered in B-CLL CD23⁺CD5⁺ 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 results indicated that B-ALL CD23⁺CD5⁺ B cells expressed IFN- and IL-10 at very high level, which could be further up-regulated by stimulation with CCL19 plus CXCL13.

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Cytokine expressions in different B cells. IFN-, IL-2, IL-4, IL-5, IL-10, and TGF-β1 were measured in freshly isolated normal CD19+ B cells, CB, B-ALL, or B-CLL CD23+CD5+ B cells by intracellular cytokine flow cytometry (tops) and Q-PCR (bottoms). Data were mean ± SD (three for normal peripheral and CB; four for B-ALL and B-CLL blood). Statistically significant differences as compared with normal periphery were indicated. *, p < 0.05; **, p < 0.001. A, Cells were freshly isolated normal peripheral CD19+ B cells (white bars), normal CB (black bars), B-ALL (brick format bars), and B-CLL (gray bars) CD23+CD5+ B cells. B, B-ALL CD23+CD5+ B cells were treated either 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 (each 1 µg/ml) before stimulation with CCL19 plus CXCL13 (gray bars).

By knowing that discrete B cell effectors could produce various arrays of cytokines, and modulated T cell responses (3, 6), we proceeded by examining the expression of cytokines in CD4⁺ or CD8⁺ T cells cocultured with syngeneic CD23⁺CD5⁺ B cells from normal cord blood, or from B-ALL and B-CLL patients. IFN-, IL-2, IL-4, IL-5, IL-10, and TGF-β1 were expressed at similar levels in the CD4⁺ T cells cocultured with different CD23⁺CD5⁺ B cells either from normal CB or from B-ALL or B-CLL patients (Fig. 2A; some data not shown). Nevertheless, IL-10 was markedly up-regulated in CD8⁺ T cells cocultured with syngeneic B-ALL CD23⁺CD5⁺ B cells (Fig. 2A), even though other cytokines (e.g., IFN-, IL-2, IL-4, IL-5, and TGF-β1) were not changed in CD8⁺ T cells after similar coculture (Fig. 2A; some data not shown). In an attempt to explore the mechanism involved in up-regulation of IL-10 expression in syngeneic CD8⁺ T cells by B-ALL CD23⁺CD5⁺ B cells, we pretreated the B-ALL CD23⁺CD5⁺ B cells with CCL19 or/and CXCL13 before coculture with CD4⁺ or CD8⁺ T cells and then measured the cytokine expression profile in CD4⁺ or CD8⁺ T cells. Treatment of CCL19 and CXCL13 together, but not alone, further up-regulated IL-10 expression in CD8⁺ T cells isolated from the B-ALL patients after coculture (Fig. 2B). In contrast, the other cytokines (e.g., IFN-, IL-2, IL-4, IL-5, and TGF-β1) were not altered in these syngeneic CD8⁺ T cells by coculture with B-ALL CD23⁺CD5⁺ B cells pretreated with CCL19 or/and CXCL13 (Fig. 2B; some data not shown). IFN-, IL-2, IL-4, IL-5, and TGF-β1 as well as IL-10 were not altered in syngeneic CD4⁺ T cells by coculture with B-ALL CD23⁺CD5⁺ B cells pretreated with CCL19 or/and CXCL13 (Fig. 2B; some data not shown). mAbs against CCR7 and CXCR5 completely blocked the effects of CCL19 and CXCL13 on up-regulation of IL-10 production in CD8⁺ T cells (Fig. 2B), whereas isotype Ab controls had no such blockade function (data not shown). The results were further confirmed by ELISPOT assay and Western blot (data not shown).

View larger version (45K): [in this window] [in a new window]   FIGURE 2. Cytokine expressions in CD4+ and CD8+ T cells. The syngeneic T cells were cocultured with normal peripheral CD19+ B cells, normal CB, B-ALL, or B-CLL CD23+CD5+ B cells. IFN-, IL-4, and IL-10 were measured in CD4+ or CD8+ T cells cocultured with different B cells in the presence of tetanus toxoid (2.5 µg/ml) by intracellular cytokine flow cytometry (tops) and Q-PCR (bottoms). DC:T:B cells = 1:10:2. CD4+ or CD8+ T cells were harvested using MACS beads. Data were mean ± SD (three for normal periphery and CB; five for B-ALL and B-CLL patients). Statistically significant differences as compared with normal periphery were indicated. *, p < 0.05; **, p < 0.001. A, Syngeneic CD4+ or CD8+ T cells were cocultured with normal peripheral CD19+ B cells (white bars), normal CB (black bars), B-ALL (brick format bars), or B-CLL (gray bars) CD23+CD5+ B cells for 48 h. B, B-ALL CD23+CD5+ B cells were pretreated either 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 before coculture with syngeneic CD4+ or CD8+ T cells, or pretreated with anti-CCR7 plus anti-CXCR5 mAbs for 12 h (each 1 µg/ml) before stimulation with CCL19 plus CXCL13 (gray bars).

View larger version (30K): [in this window] [in a new window]   FIGURE 3. IL-10 expression in syngeneic CD8+ T cells. The cells were cocultured with B-ALL CD23+CD5+ B cells. IL-10 expressions were measured in syngeneic CD8+ T cells cocultured with B-ALL CD23+CD5+ B cells in the presence of tetanus toxoid (2.5 µg/ml) by intracellular cytokine flow cytometry (all left panels), Q-PCR (all middle panels), and Western blots (all right panels). DC:T:B cells = 1:10:2. CD8+ T cells were then harvested using MACS beads. Data were mean ± SD (n = 3). Statistically significant differences as compared with different Ab treatment were indicated. *, p < 0.05; **, p < 0.001. Actins in each lower picture indicated the quantity of total cellular protein from the tested samples loaded in each lane. Arrows indicate appropriate cytokines identified by markers of equivalent molecular weights in each lane. A, Syngeneic CD8+ T cells were cocultured with B-ALL CD23+CD5+ B cells for 48 h. The syngeneic CD8+ T cells were pretreated with IFNGR1 mAb (5 µg/ml; white bars), IL-10RA mAb plus IL10RB pAb (each 5 µg/ml; black bars), or with all three Abs (each 5 µg/ml; brick format bars) for 12 h before coculture or pretreated with isotype Abs for 12 h (each 5 µg/ml; gray bars). Superscript a, Isotype Ab treatment. B, Cells were treated as described in A, but cocultures were conducted in a transwell manner as stated in Materials and Methods.

We further investigated the effect of B-ALL CD23⁺CD5⁺ B cells on the cytotoxicity of syngeneic CD8⁺ T cells. We previously reported that although CXCR5 and CCR7 were functionally expressed on B-ALL CD23⁺CD5⁺ B cells at high frequency, they were not found on CD23⁺CD5^– B cells (8). In the following experiments, we compared their regulatory functions. Each of different CD23⁺CD5^– and CD23⁺CD5⁺ B cells were added into coculture system during the tumor-specific CTL killing mediated by syngeneic CD8⁺ T cells. To measure CTL activity, we generated tumor-specific CTL CD8⁺ T cells to human chronic myelogenous leukemia K562 cells and set up the coculture in the presence of CD23⁺CD5^– and CD23⁺CD5⁺ B cells, respectively. To our surprise, B-ALL CD23⁺CD5⁺ B cells, but not CD23⁺CD5^– B cells, showed strong inhibitory effect on specific CTL responses of syngeneic CD8⁺ T cells (Fig. 4A). Furthermore, only B-ALL, but not normal or B-CLL, CD23⁺CD5⁺ B cells selectively and significantly inhibited the specific CTL responses of CD8⁺ T cells (Fig. 4A). Considering the abundance of CCR7 and CXCR5 on B-ALL CD23⁺CD5⁺ B cells (8), we pretreated these cells with CCL19 and/or CXCL13 for 24 h before CTL activity analysis. B-ALL CD23⁺CD5⁺ B cells cultured with CCL19 and CXCL13 together, but not with CCL19 or CXCL13 alone, completely abolished tumor-specific CTL responses of syngeneic CD8⁺ T cells (Fig. 4B). In this regard, Abs against CCR7 and CXCR5 together blocked the function of CCL19 and CXCL13 on B-ALL CD23⁺CD5⁺ B cells (Fig. 4B), whereas isotype Abs had no such blocking function (data not shown). To explore potential mechanisms of underlying the phenomena, we pretreated the syngeneic CD8⁺ 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 impairment of syngeneic CD8⁺ 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 CD23⁺CD5⁺ B cells in vitro were able to impair tumor-specific cytotoxicity of syngeneic CD8⁺ T cells, in which the IL-10 was an essential mediator.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Regulation of CTL activity of syngeneic CD8+ T cells by different B cells. To measure CTL activity, specific CTL CD8+ T cells were generated as described in Materials and Methods. Different 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 14. The CTL activity was assessed by a 51Cr release assay with various E:T ratios. A, CD23+CD5– or CD23+CD5+ B cells were purified from healthy CB (), B-CLL (), or B-ALL () patients and cocultured with syngenic CD8+ T cells. B, Purified B-ALL CD23+CD5+ B cells were either pretreated with ligand CCL19 (), CXCL13 (), or CCL19 plus CXCL13 () each at 100 ng/ml for 24 h (L; right), or pretreated with anti-CCR7 Ab (), anti-CXCR5 Ab (), anti-CCR7 Ab plus anti-CXCR5 Ab () each at 5 µg/ml for 12 h before treatment with CCL19 and CXCL13 (A+L; left); subsequently CTL activity assay was conducted. C, Syngenic CD8+ T cells in CTL system were pretreated either with IFNGR1 mAb (5 µg/ml; ), IL-10RA mAb plus IL10RB pAb (each 5 µg/ml; ), or with all three Abs (each 5 µg/ml; ) for 12 h before CTL assay. The dashed lines represented the maximal extent of spontaneous cell lysis. Statistically significant differences as compared among different pretreatments were indicated. n = 6; *, p < 0.05; **, p < 0.001.

We previously showed that PEG10 was highly up-regulated and involved in the resistance to TNF--mediated apoptosis in B-ALL CD23⁺CD5⁺ B cells (8). In this study, we further evaluated the role of PEG10 in the expression of cytokines in B-ALL CD23⁺CD5⁺ 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 levels, respectively. As expected, shRNAPEG10 at a high concentration (2 µg/ml) sequence-specifically knocked down the expression of PEG10 in B-ALL CD23⁺CD5⁺ B cells (Ref. 8 and data not shown). shRNAPEG10 (2 µg/ml) significantly and sequence-specifically down-regulated the expression of IL-10, whereas the expressions of IL-2, IL-4, IL-5, and TGF-β1 were not altered in B-ALL CD23⁺CD5⁺ B cells (data not shown). The shRNAPEG10 at low concentration (0.02 µg/ml), DNAPEG10, and vector showed no such effects (data not shown). As we described above (Fig. 1B), CCL19 plus CXCL13 could selectively up-regulate IL-10 expressions in B-ALL CD23⁺CD5⁺ B cells. In further studies, we first transfected B-ALL CD23⁺CD5⁺ B cells with shRNAPEG10 (2 µg/ml) and consequently treated the cells with CCL19 or/and CXCL13. CCL19 and CXCL13, either alone or together, could not alter the expression patterns of IL-10 and other cytokines (e.g., IL-2, IL-4, IL-5, and TGF-β1) in these B-ALL CD23⁺CD5⁺ B cells pretreated with shRNAPEG10 (data not shown).

Expression of cytokine (IL-10) in syngeneic CD8⁺ T cells was regulated by coculture with B-ALL CD23⁺CD5⁺ B cells (Fig. 2). We used the same setting to further investigate the involvement of PEG10 in cytokine production in syngeneic CD8⁺ T cells. The B-ALL CD23⁺CD5⁺ B cells transfected with shRNAPEG10 (2 µg/ml) significantly and sequence-specifically down-regulated the expression of IL-10 in syngeneic CD8⁺ T cells in the coculture system (Fig. 5A). As controls, shRNAPEG10 at a low concentration (0.02 µg/ml), DNAPEG10 and vector alone showed no such effects (Fig. 5A). The expression of other cytokines (e.g., IL-2, IL-4, IL-5, and TGF-β1) were not altered in the syngeneic CD8⁺ T cells in the same setting system (data not shown). We next tested the effect of CCL19 or/and CXCL13 on shRNAPEG10-transfected (2 µg/ml) B-ALL CD23⁺CD5⁺ B cells in the coculture system. CCL19 and CXCL13, either alone or together, could not alter the patterns of IL-10 expression in the syngeneic CD8⁺ T cells (Fig. 5B). The expressions of other cytokines (e.g., IL-2, IL-4, IL-5, and TGF-β1) were not altered in the syngeneic CD8⁺ T cells either (data not shown). These data were support the conclusion that PEG10 was indeed involved in the up-regulation of IL-10 in the described B-T cell interaction.

View larger version (29K): [in this window] [in a new window]   FIGURE 5. IL-10 expression in syngenic CD8+ T cells. IL-10 expression was measured in syngenic CD8+ T cells cocultured with CD23+CD5+ B cells pretreated with shRNAPEG10 in the presence of tetanus toxoid (2.5 µg/ml) by intracellular cytokine flow cytometry (all left panels), Q-PCR (all middle panels), Western blots (all right panels). DC:T:B cells = 1:10:2. Data were mean ± SD (n = 4). Statistically significant differences as compared among different shRNA treatments were indicated. *, p < 0.05; **, p < 0.001. Actins in each lower picture indicated the quantity of total cellular protein from the tested samples loaded in each lane. Arrows indicated markers used to verify equivalent molecular weights of appropriate cytokine in each lane. A, shRNAPEG10 was transfected into B-ALL CD23+CD5+ B cells with appropriate concentration following the manufacturer’s instructions for 2 days, and the cells were then added into coculture with syngenic CD8+ T cells. Vector, empty vectors (white bars); shDNA, shDNA with irrelevant sequence (2 µg/ml; black bars); shRNAPEG10l, low concentration (0.02 µg/ml; brick format bars); shRNAPEG10h, high concentration (2 µg/ml; gray bars). B, B-ALL CD23+CD5+ B cells were transfected with high concentration of shRNAPEG10 (2 µg/ml) as described in A. Consequently, the cells were treated either 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 before cytokine assays, or pretreated with anti-CCR7 plus anti-CXCR5 mAbs for 12 h (each 1 µg/ml) before stimulation with CCL19 plus CXCL13 (gray bars). Superscript a, Anti-CCR7 plus anti-CXCR5 mAbs treatment.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Regulation of CTL activity of syngeneic CD8+ T cells by B-ALL CD23+CD5+ B cells. To measure CTL activity, specific CTL syngeneic CD8+ T cells were generated. B-ALL CD23+CD5– (upper left panel) and CD23+CD5+ (all other panels) B cells were added into culture at day 14. The CTL activity was assessed by a 51Cr release assay with various E:T ratios. A, B-ALL CD23+CD5– or CD23+CD5+ B cells were pretreated with empty vectors (), low concentration (0.02 µg/ml; ), or high concentration (2 µg/ml; ) shRNAPEG10. B, Purified B-ALL CD23+CD5+ B cells were first transfected with a high concentration of shRNAPEG10 (2 µg/ml) as described in A. Consequently, the cells were treated with ligand CCL19 (), CXCL13 (), or CCL19 plus CXCL13 (), each at 100 ng/ml for 24 h before CTL activity (left; L). In some cases, syngeneic CD8+ T cells were pretreated with exogenous IL-10 (100 ng/ml; ), IFN- (10 ng/ml; ), IL-10 plus IFN- () for 12 h before CTL activity (Exo-IL; right). In the CTL system, the purified B-ALL CD23+CD5+ B cells were transfected with high concentrations of shRNAPEG10 (2 µg/ml) as described in A. The dashed lines represented the maximal extent of spontaneous cell lysis. Statistically significant differences as compared among different pretreatments were indicated. n = 6; *, p < 0.05; **, p < 0.001.

	Discussion

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Dynamic and productive interaction between T and B cells is required for the humoral immune responses to many foreign protein Ags and production of pathogenic Abs characteristic of several autoimmune conditions (46). Although a number of recent researches have shown that B cells produce immunoregulatory cytokines such as IFN- in several infections and autoimmune diseases (3, 4, 5, 6), the mechanisms that control cytokine production by B cells, particularly by malignant B cells, are still needed to be further clarified. In this study, we have observed that B-ALL CD23⁺CD5⁺ B cells express IFN- and IL-10 at high levels, which can be further up-regulated by CCL19 plus CXCL13 (Fig. 1). IL-10 secreted from B-ALL CD23⁺CD5⁺ B cells is able to up-regulate expression of IL-10 in syngeneic CD8⁺ T cells (Fig. 2). To the best of our knowledge, this is the first report that malignant CD23⁺CD5⁺ B cells play an immunoregulatory role to differentially regulate inflammatory cytokine expression through a CXCR5/CXCL13-CCR7/CCL19-PEG10 axis in the cells.

In physiological situations, CCL19 is detected in T zone stromal cells, DCs, and tonsillar perivascular cells in vivo (47, 48); meanwhile, CXCL13 is mainly found in stromal cells in B cell follicles (23). CCL19/CCR7 has been identified as the gatekeepers for both naive T cells and DCs to control their entry and exit from secondary lymphoid tissues organs. In contrast, in pathophysiological situations, it has been reported that chemokine receptors are expressed on neoplastic cells of hemopoietic and nonhematopoietic origin, and that overexpression of some of these receptors is closely related to tumor progression and metastasis (49, 50, 51). As for B cell-derived lymphoproliferative disorders, CXCR5 have been detected in neoplastic B cells from B-ALL (8, 52) and B-CLL (8, 53). CCR7 has been found in B-CLL (8, 54) and in tumor cells from classical Hodgkin’s disease with lymphocyte predominance (55). In our previous study, we have reported that CXCR5 and CCR7 are frequently and functionally expressed on B-ALL CD23⁺CD5⁺ B cells. Coactivation of CXCR5 and CCR7 has displayed a novel function to induce resistance to TNF--mediated apoptosis in B-ALL CD23⁺CD5⁺ B cells (8). In this study, we have extended our investigation on B-ALL CD23⁺CD5⁺ B cells concerning the aberrations of their immune functions. We have observed that, through the ligation of CCR7 and CXCR5, B-ALL CD23⁺CD5⁺ B cells significantly inhibit tumor-specific cytotoxicity of syngeneic CD8⁺ T cells, where IL-10 plays functions as a critical mediator (Fig. 4). Innate and adaptive immune responses can be induced against tumors, and the protective and therapeutic effector cells include CD8⁺ CTL, IFN--producing CD4⁺ and CD8⁺ T cells, NK cells, and macrophages (56). Based on the fact that CTL can directly kill tumor cells, cancer-directed and immune-based therapies have focused on eliciting a CTL response. An increasing important focus is being given to the stimulation of a CD4⁺ Th cell response in cancer development immunology and its immunotherapy (57). Th1 cells, characterized by secretion of IFN- and TNF-, are primarily responsible for activating and regulating the development and persistence of CTL. B cells are important producers of cytokines such as IL-2, TNF-, IL-4, IL-6, IL-10, and IL-12 (3, 6, 58). B cells can be differentiated into distinct cytokine-producing effector subsets (3, 6). The cytokines produced by B cells regulate the activity or function of other cell types, such as regulating the differentiation of naive CD4⁺ T cells into Th1 and Th2 cells through production of polarizing cytokines (IL-4 and TNF-). This function has obvious importance, particularly as the cytokine environment in which a naive T cell first encounters Ag presented by an APC. Our observations that overexpression of IL-10 in B-ALL CD23⁺CD5⁺ B cells impairs tumor-specific cytotoxicity of syngeneic CD8⁺ T cells indicate a novel pathway of malignant cells to escape immune surveillance. Overexpression of CCR7 and CXCR5 on the cells can be an important element in the chain of escaping events. Our findings, together with the results from other groups, are suggesting that IL-10 may be one of the critical cellular factors whose increased expression confers tumor escaping from immune surveillance, thereby contributing to the pathogenesis and progression of malignant cells such as B-ALL CD23⁺CD5⁺ B cells.

PEG10 is identified on human chromosome 7q21 (28, 59). Mouse homolog PEG10 has recently been located in a large imprinted gene cluster on mouse proximal chromosome 6 and was confirmed to be imprinted (60). Because the protein products from the predicted open reading frames (open reading frame 1 and open reading frame 2) of PEG10 show homology to the gag and pol proteins of vertebrate retrotransposon Ty3/Gypsy, PEG10 is speculated to be a retrotransposon-derived gene. Distinct expression of PEG10 is found in the brain, kidney, lung, testis, and placenta but not in the liver and a number of other tissues (27). In contrast to this, expression of PEG10 is detected only in the placenta among the 14 adult mouse tissues (60). Some data suggest a role for preferential expression of PEG10 in regulating growth control of liver and pancreatic carcinoma cells (30). Exogenous PEG10 promotes growth of certain hepatocellular carcinoma cell lines that does not manifest endogenous PEG10. The interaction of PEG10 with SIAH plays important roles in resistance to apoptosis (29). We have found that B-ALL CD23⁺CD5⁺ B cells overexpresses PEG10 significantly up-regulated by costimulation with CCL19 and CXCL13 (8). Functionally, CCL19 and CXCL13 cooperatively up-regulate expression and function of PEG10 to confer resistance to apoptosis in B-ALL CD23⁺CD5⁺ B cells (8). In this study, shRNA of PEG10 significantly down-regulated the expression of IL-10 in B-ALL CD23⁺CD5⁺ B cells. Pretreatment with shRNAPEG10 has selectively down-regulated the expression of IL-10 in syngeneic CD8⁺ T cells in coculture with B-ALL CD23⁺CD5⁺ B cells. The shRNAPEG10 has blocked the impairment of tumor-specific CTL responses of syngeneic CD8⁺ T cells by B-ALL CD23⁺CD5⁺ B cells (Fig. 5). Collectively, these data are demonstrating that PEG10 is indeed involved in the up-regulation of IL-10 during the interaction of B-ALL CD23⁺CD5⁺ B cells with T cells. It is worthwhile to investigate the molecular basis of CCR7 and CXCR5 in vivo as how the PEG10 gene is activated in human B-ALL CD23⁺CD5⁺ B cells, which may lead to further understanding of PEG10 function and mechanisms involved in the process of malignant lymphoproliferative disorders.

	Disclosures

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The authors have no financial conflict of interest.

	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 This work was supported by the National Natural Science Foundation of China (Grants 39870674, 30572119, 30030130, 30471509, and 30670937); Science Foundation of Anhui Province, China (Grant 98436630), and Education and Research Foundation of Anhui Province, China (Grant 98JL063) and Research Foundation from Health Department of Hubei Provincial Government, China (Grant 301140344); and a special grant from the Personnel Department of Wuhan University, China. T.J. is a Chang Jiang Scholar supported by Chang Jiang Scholars Program from Ministry 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.

³ Address correspondence and reprint requests to Dr. Tan Jinquan, Department of Hematology, Anhui Medical University Affiliated Provincial Hospital, Hefei, People’s Republic of China or Department of Immunology, Wuhan University School of Medicine, Wuhan University, Dong Hu Road 115, Wuchang, Wuhan, People’s Republic of China. E-mail address: jinquan_tan{at}hotmail.com

⁴ Abbreviations used in this paper: DC, dendritic cell; B-ALL, B cell acute lymphocytic leukemia; B-CLL, B cell chronic lymphocytic leukemia; CB, cord blood; IL10RB pAb, IL-10 receptor B polyclonal Ab; Q-PCR, quantitative RT-PCR; shRNA, short hairpin RNA.

Received for publication November 21, 2006. Accepted for publication June 20, 2007.

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