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Over-expression of Hlx homeobox gene in DC2.4 dendritic cell enhances its maturation and antigen presentation
Cellular Immunology 275 (2012) 61–68
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Over-expression of Hlx homeobox gene in DC2.4 dendritic cell enhances its maturation and antigen presentation Siamak Sandoghchian Shotorbani a, Zhiqiang He a,b, Heng Yang a, Qiang Sun a, Yan Xu a, Zhaoliang Su a, Yuan Xue a,c, Dong Zheng a, Yun Zhang a, Shengjun Wang a,⇑, Qixiang Shao a, Liwei Lu d, Huaxi Xu a,⇑ a
Department of Immunology, Institute of Laboratory Medicine, Jiangsu University, Zhenjiang, PR China Department of Laboratory Medicine, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou, PR China Department of Physical Testing and Chemical Analysis, Zhenjiang Center for Disease Control and Prevention, Zhenjiang, PR China d Department of Pathology, Hong Kong University, Hongkong, PR China b c
a r t i c l e
i n f o
Article history: Received 21 November 2011 Accepted 23 February 2012 Available online 14 March 2012 Keywords: H2.0-like homebox Genetic modification Dendritic cell Antigen presenting
a b s t r a c t Hlx as a Th1-specific transcription factor, it appears to drive maturation of Th1 and IFN-c secretion in cooperation with T-bet. In this study, we established a stable Hlx-over-expressed dendritic cell line (DC2.4/Hlx), and investigated the possible effect of Hlx gene on maturation of dendritic cell-line (DC2.4). Results shown that over-expressed Hlx in DC2.4 up-regulated the transcription and expression of IFN-c, increased the expression of maturation makers including CD40, CD80, CD86, MHC-I and MHC-II. Functional assay for DC2.4/Hlx showed that over-expressed Hlx increased the expression level of interleukin-12 in the supernatant and decreased DC endocytosis when cells were incubated in vitro. Furthermore, using a syngeneic T cell activation model, we found that DC2.4/Hlx could obviously present ovalbumin (OVA) antigen to T cell in OVA pre-immunized mice. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Hlx is a homeobox gene, was originally isolated from a murine pre-B-lymphocyte cell line [1,2]. Hlx is very important not only in hematopoietic cells, but also in normal intestinal and hepatic development in mice [3]. In immune system, Hlx is a Th1-special transcription factor that interacts specifically with T-bet. Hlx and T-bet synergistically promote IFN-c expression when co-expressed. Thus, as a cofactor of T-bet, Hlx appears to enhance the activities of T-bet and then favor Th1 differentiation [4]. The co-engagement of T-bet and Hlx actively suppressed Th2 commitment. Hlx down-regulates the IL-4 receptor expression in naive CD4+ T cells [5]. Over-expressed Hlx resulted in the aberrant expression of IFN-c in normal CD4+ T cells during the differentiation under Th2-polarizing conditions [6,7]. Therefore, Hlx played an important role in the regulation of IFN-c production by T cell. Interestingly, in NK cells Hlx was a negative regulator of IFN-c production and its inhibitory function was achieved at least in part through the proteasomal degradation of STAT4 [8]. Therefore, Hlx may play the different role in different systems or cells. Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for the initiation of antigen (Ag)-specific immune re⇑ Corresponding authors. Address: Department of Immunology, Institute of Laboratory Medicine, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu Province, China. Fax: +86 511 85038449. E-mail address: [email protected] (H. Xu). 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.02.016
sponses as well as maintenance of tolerance to self-antigens [9]. In addition, DCs was also directly involved in regulating other innate immunity such as promoting activation of NK cells and their effector function [10,11]. However, the role of Hlx in DCs functions remains unknown. In the present study, we introduced exogenous Hlx gene into a dendritic cell line DC2.4, established a stable DC2.4/ Hlx cell-line. Our results showed that over-expression of Hlx in DC2.4 cells up-regulated production of IFN-c, enhanced their maturation and antigen-presenting function. 2. Materials and methods 2.1. Cell culture The immature murine dendritic cell line DC2.4 (H-2b), DC2.4/EGFP and DC2.4/Hlx were cultured in RPMI1640 medium, supplemented with 100 U/ml penicillin, 100 mg/L streptomycin, 2 mmol/L L-glutamine, and 10% FBS (GIBCO). The transfected cell lines were pulsed with 0.6 g/L G418 to eliminate negative cells. 2.2. Transfection and selection of stable clones The DC2.4 cells were transfected by Hlx with liposomes (lipofectamine™ 2000, Invitrogen). 4 105 DC2.4 cells in 500 ll complete RPMI1640 medium without antibiotics were plated in 24-well plates. Next day when the cells were at 60–70% confluent, Hlx was transfected according to the instruction of the manufac-
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turer. The medium was changed into 100 ll serum-free medium before transfection. The 100 ll DNA/lipofectamine complexes that were prepared at the ratio of 1:3 (ll) were added to each well and then mixed gently by rocking the plate back and forth, followed by incubating at 37 °C in a CO2 incubator for 6 h. The medium was changed by 200 ll new complete RPMI1640 medium. The cells were passaged at 1:10 with fresh growth medium 24 h after transfection, and then selected by adding selective medium (contain 600 mg/L G418) in the following day. The target clones were selected on the basis of their resistance to G418 and expression of EGFP. The Hlx high-expressed DC2.4 was assessed by Real-time PCR and Western blot. 2.3. Total RNA isolation and Real-time PCR Total RNA was prepared using Trizol (Invitrogen), and reversely transcribed with first strand cDNA synthesis kit ReverTraAce-a-™ (TOYOBO Co., Ltd., Life Science Department) according to the manufacturer’s instructions. RT-PCR was performed by the comparative threshold cycle (DCT) method and normalized to b-actin. Sequences of the primers are as follows: 50 -CTC GTG GTC CCG TGC TGT CTT TTC-30 and 50 -GTT CCC TCA GTC CGT TCC GTG TCG-30 for mouse Hlx, 50 -ATG CCA GGG AAC CGC TTA T-30 and 50 -CAG ATG CGT ACA
TGG ACT CAA A-30 for mouse T-bet, 50 -CTG TGG GCT GTA CTA CAA GCT TCA-30 and 50 -ACC CAT GGC GGT GAC CAT GC-30 for mouse GATA3, 50 -AAG CGT CAT TGA ATC ACA CC-30 and 50 -CGA AAT CAG CAG CGA CTC CTT AT-30 for mouse IFN-c, 50 -CGA GGT CAC AGG AGA AGG-30 and 50 -TGC TCT TTA GGC TTT CCA G-30 for mouse IL4, 50 -CTC ACC TGT GAC ACG CCT GA-30 and 50 -CAG GAC ACT GAA TAC TTC TC-30 for mouse Il-12p40, 50 -CCT CAG TTT GGC CAG GGT C-30 and 50 -CAG GTT TCG GGA CTG GCT AAG-30 for mouse Il12p35, 50 -TGG AAT CCT GTG GCA TCC ATG AAA C-30 and 50 -TAA AAC GCA GCT CAG TAA CAG TCC G-30 for mouse b-actin. PCR were set up with the SYBR Premix Ex Taq™ II (TaKaRa Biotechnology Co., Ltd., Dalian, China). Data were collected using the Rotor-gene 6000 real-time system (Corbett Life Science) and analyzed using the relative quantification (based on the relative expression of target genes vs. b-actin reference genes). Transcripts of each gene relative to the control gene were determined with the 2DCt method. 2.4. Western blot Whole cells were lysed in lysis buffer (150 mM NaCl, 20 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM sodium fluoride, 0.5% Doc, 1% Triton X-100, and 1% Nonidet P40). The cell lysates were boiled with 2 loading buffer for 5 min and
Fig. 1. Establishment of stable Hlx-overexpressed dendritic cell line (DC2.4-Hlx). (A) Expression and location of EGFP in stable DC2.4-Hlx clone, a and c showed the fluorescence intensity and localization of EGFP expression in DC2.4-Hlx detected by confocal microscopy; b and d appeared original state of DC2.4-Hlx. (B) The fluorescence ratio expressed in DC2.4-Hlx was identified by FACS. (C and D) Indicated the Hlx high-expressed in DC2.4-Hlx assessed by Real-time PCR and Western blot, upper half of figure C showed the Hlx expression in different cell clones, lane 1 Dl2000 marker, lane 2 DC2.4, lane 3 DC2.4/EGFP, lane 4–7 different DC2.4/Hlx clone, and the lower half showed the statistical analysis of Hlx mRNA (mean ± SD, n = 5), #represent p < 0.01.
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Fig. 2. The levels of IFN-c, T-bet and GATA3 in DC2.4-Hlx cell line. The secretion level of IFN-c in supernatant, and the mRNA expression of IFN-c, T-bet and GATA3 in DC2.4-Hlx cell line were detected by ELISA and RT-PCR, respectively. Compared with DC2.4/EGFP and wild DC2.4, the levels of IFN-c and T-bet in DC2.4/Hlx were enhanced (#p < 0.01), but GATA3 no significant change. (A, C and D) Showed the relative mRNA of IFN-c, T-bet and GATA3, respectively; (B) indicated the protein level of IFN-c in supernatant detected by ELISA after DC2.4/Hlx cell was cultured for 48 h in vitro.
analyzed by Western blot. Rabbit anti-mouse T-bet, rabbit antimouse b-actin and goat anti-mouse Hlx used as primary antibodies (Abs) were purchased from Santa Cruz Biotechnology. After PAGE, the protein was transferred into cellular membrane. The membrane was incubated with the primary antibody followed by incubating with HRP-conjugated goat anti-rabbit IgG (Sigma–Aldrich) or rabbit anti-goat IgG (Santa Cruz Biotechnology). After extensive washing in TBS-T, the blots were processed for detection of Ag using the ECL plus Western blotting detection system (GE Healthcare Life Sciences).
was performed and isotype matched monoclonal antibody was used as a negative control. 2.6. Enzyme-linked immunosorbent assays (ELISAs) for detecting IFN-c and IL-12p70 in supernatant The concentration of cytokines in supernatants was detected by ELISA. The cells were cultured in complete RPMI1640 for 48 h and cell-free supernatants were collected. The levels of IFN-c and IL12p70 in supernatants were measured by ELISA kit (eBiosence, USA). All samples were measured in triplicate.
2.5. FACS for phenotype analysis 2.7. Endocytosis assay For phenotypic analysis, 1 106 cells were resuspended in 100 ll of staining buffer (PBS containing 0.1% BSA) and incubated for 30 min on ice with PE-conjugated Abs specific for H-2Kb, I-Ab, CD80, CD86, CD40 and TLR4 (each, 5 lg/ml). After washing three times with staining buffer, the cells were analyzed for surface phenotype by FACSCalibur with Cell Quest software. Each cell type was treated with 10 lg/ml LPS (Sigma) for 24 h before the analysis
The endocytic capacity of DC2.4/Hlx, DC2.4/EGFP and DC2.4 was determined by an energy-dependent uptake of a special Escherichia coli which expressed red fluorescent protein HcRed. Three different lines of DCs (2 105) were incubated in the presence of HcRed-expressed dead E. coli (5 108/ml) at 37 °C for 2 h. The cells were washed three times with cold FACS buffer (PBS containing 1% FBS). HcRed-expressed E. coli
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Fig. 3. High expression of Hlx up-regulated maturation markers on DC2.4/Hlx cell. Phenotypical changes in DC2.4/Hlx cell was examined by flow cytometry analysis. The horizontal coordinate indicates the fluorescence intensity, the number on figure show the fluorescence positive cell percentage and vertical coordinate indicates the amount of cells. The fluorescence intensity and numbers of fluorescence-positive cells were related to the expression of associated-membrane molecules. The expression levels of CD80, MHC-II, CD86 and CD40 were markedly enhanced in DC2.4/Hlx compared with wild DC2.4 and DC2.4/EGFP.
uptaked by DC was determined by a FACSCalibur. The parallel experiments were conduced at 4 °C to determine the nonspecific adherence as background. 2.8. Antigen presenting analysis Antigen-presenting function of DC was assessed by allogeneic T cell activation responding to a specific antigen. The DC2.4, DC2.4/ EGFP and DC2.4/Hlx were cultured with 100 mg/L ovalbumin (OVA; Sigma) for 24 h, respectively. Spleens from C57BL/6 mice immunized by OVA were aseptically obtained and the single cell suspensions were prepared. Purified CD4+ T cells were prepared by negative selection with magnetic activated cell sorting (MACS).
T cells were stained using 3 mmol/L fluorescein diacetate succinimidyl ester (CFSE; Santa Cruz Biotechnology, Inc.) for 10 min at 37 °C and washed three times by complete RPMI medium with 10% FBS. In a 24-well plate, 2 106 CD4+ T cell per well were cocultured 72 h with 2 105 different lines of DC that were pre-treated by 50 lg/ ml mitomycin C at 37 °C for 20 min, respectively. The cells were harvested and green fluorescence from CFSE was analyzed by a FACSCalibur. In the mean time, the method of MTT was also used to detect the ability of cellular proliferation. Briefly, 2 105 CD4+ T cells were cocultured with 2 104 DCs as described above in 96-well flat-bottom plates for 72 h, and then 20 ll 5 mg/ml MTT3-(4,5)dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumro-mide solu-
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Fig. 4. The levels of IL-12 mRNA and protein in DC2.4-Hlx cell line. (A) The relative mRNA of IL-12p40, it was increased in DC2.4/Hlx than that in wild DC2.4 and DC2.4/EGFP. (B) The IL-12p35 produced by DC2.4/Hlx was enhanced. (C) The protein level of IFN-c in supernatant detected by ELISA after DC2.4/Hlx cell was cultured for 48 h in vitro, it was significant increased than that in cell-cultured supernatant of DC2.4 and DC2.4/EGFP (#p < 0.01; ⁄p < 0.05).
tion (Y-S Biotechnology, China) was added to each well and incubated another 4 h, the untransformed MTT was removed carefully by pipetting and 150 ll DMSO was added. Ten minutes later, optical density (OD) was determined in an ELISA reader at 570 nm and the value of optical density reflected the number of living cells.
microscopy (Fig. 1C and D). The rate of EGFP expression was 99% identified by FACS (Fig. 1B). The transfected DC2.4/Hlx cells were screened, and the clone of high transcription and expression of Hlx was identified by Real-time PCR and Western-blot (Fig. 1C and D). The rate of EGFP expression in the DC2.4/Hlx clone was more than 90% (Fig. 1B).
2.9. Statistical analysis
3.2. Enhanced IFN-c secretion in DC2.4/Hlx cell-line
All statistical analyses were performed using the Prism program (GraphPad) statistical software. Data were expressed as means ± SEM. The difference in mRNA expression of the target genes between DC2.4/Hlx, DC2.4/EGFP and DC2.4 was assessed by t-test respectively. P < 0.05 was considered to be statistically significant.
Hlx, as a Th1-specific transcription factor, it appears to drive secretion of IFN-c in cooperation with T-bet. We detected the mRNA of transcription factors T-bet, GATA3 and cytokines IFN-c, IL-4 in DC2.4/Hlx, DC2.4/EGFP or wild DC2.4. The RT-PCR data showed that IFN-c mRNA level in DC2.4/Hlx was enhanced about 7-folds than that in DC2.4/EGFP or wild DC2.4 (Fig. 2A). But, the mRNA levels of T-bet and GATA3 were not significant change in different DC cell lines (Fig. 2C and D). ELISA results also confirmed that a greater production of IFN-c was found in the supernatant of cultured DC2.4/Hlx cells (18 ng/ml) in comparison with that in DC2.4/EGFP (4 ng/ml) and DC2.4 cells (5 ng/ml) (Fig. 2B).
3. Results 3.1. DC2.4/Hlx cell-line was established To evaluate the effects of Hlx in DCs, we generated a DC cell line that stably express Hlx gene. An empty vector PIRES2-EGFP was used as control. After transfection the DC2.4/Hlx and DC2.4/EGFP cell-line were selected by subcloning in selection medium based on EGFP expression (Fig. 1A and B). The EGFP distributed in whole cell, but expressed abundantly in the nucleus, as seen in confocal
3.3. Increased expression of maturation markers in DC2.4/Hlx cell-line Phenotypical changes in three different DC2.4 cells were examined by FACS analysis using specific antibodies against MHC mole-
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cules and co-stimulatory molecules (Fig. 3). The expression of CD80, MHC-II, MHC-I, CD86 and CD40 were markedly enhanced in DC2.4/Hlx compared with wild DC2.4, suggesting that the DC2.4 maturation was markedly promoted by high-expression of Hlx gene (Fig. 3).
with OVA. When co-cultured with OVA-loaded DC2.4/Hlx cells, the T cells showed a significantly faster cell cycle division indicated by CFSE, while that was not obvious when co-cultured with the wild DC2.4 or DC2.4/EGFP (Fig. 6A and B). 4. Discussion
3.4. Increased expression of IL-12p40 and IL-12p35 in DC2.4/Hlx cellline The expression levels of IL-12 mRNA in three different DC cell-lines were detected by qRT-PCR. The results shown that the transcription of IL-12p40 and IL-12p35 in DC2.4/Hlx cells were up-regulated compared with DC2.4/EGFP and wild DC2.4 (Fig. 4A), and the level of IL-12p70 in the cell-culture supernatant of DC2.4/Hlx was 8-folds more than DC2.4/EGFP and wild DC2.4 (Fig. 4B). However, the endocytosis of bacteria by DC2.4/Hlx was decreased (Fig. 5). 3.5. Up-regulated antigen-presenting functions in DC2.4/Hlx cell-line Antigen-presenting function of DC is often used as a key index to further confirm the extent of DC maturation. In this study, we used the CD4+ T cells from C57/BL6 mice that were immunized
The Hlx gene is a member of the homeobox family of genes, with homology to the Drosophila homeobox gene H2.0. During Th1 cell differentiation, the transcription factor Hlx and T-bet are induced. These two transcription factors control Th1 cell differentiation and IFN-c gene expression [12]. In addition, Hlx has been implicated in cell proliferation in vitro in several different tissues, including placental extravillous trophoblasts and CD34 positive bone marrow cells [13,14]. Hlx expression can be stimulated by many cytokines or other growth factors in these cell types, and thus is implicated in immature stem cell and progenitor stem cell development [14,15]. Hlx is also necessary for the development of the enteric nervous system [16,17]. DCs are the most potent antigen-presenting cells for the initiation of Ag-specific immune responses as well as maintaining tolerance to self-antigens [18–20]. In the present study, we transfected
Fig. 5. The endocytic capacity of DC2.4/Hlx cell-line. DC2.4/Hlx, DC2.4/EGFP and wild DC2.4 were incubated in the presence of HcRed-expressed dead E. coli (5 108/ml) at 37 °C for 2 h. HcRed-expressed E. coli uptaked by DCs was determined using a FACSCalibur, respectively. The parallel experiments were conduced at 4 °C to determine the nonspecific adherence as background. The number in figure shown the fluorescence positive cell percentage, it represented the endocytic capacity of dendritic cells. HcRedexpressed E. coli uptaked by DC2.4/Hlx cell-line was decreased compared with wild DC2.4 or DC2.4/EGFP. The experiment was repeated three times and the same trend was observed.
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Fig. 6. Analysis of antigen-presenting functions in DC2.4/Hlx cell-line. (A) After OVA-loaded DC2.4 cells were incubated with OVA-specific T cells, compared with wild DC2.4 and DC2.4/EGFP, a significantly faster cell cycle division was indicated by CFSE while DC2.4/Hlx presence. Anti-CD3 and anti-CD28 were used for positive control in this experiment. The peak shape on Fig. 6A was used for analyzing the cell proliferation, peak more wide showed the proliferative ability stronger. The lower half of Fig. 6A indicated what the control panels from each group (it was not stimulated by OVA). (B) The proliferation of OVA-specific T cells indicated by absorbance was detected using MTT method after they were incubated with OVA-loaded DC2.4/Hlx, wild DC2.4 or DC2.4/EGFP cells, respectively, it showed that the ability of DC2.4/Hlx cell proliferation stimulated by OVA was stronger than wild DC2.4 or DC2.4/EGFP cells (#p < 0.01; ⁄p < 0.05).
exogenous Hlx gene into DC2.4 cells, and established a stable DC2.4/Hlx cell-line, which could strongly express HLX. The data also showed that IFN-c mRNA level in DC2.4/Hlx was enhanced about 7-folds than that in wild DC2.4 cell. The protein of IFN-c in the cell-cultivated supernatant was quantified by ELISA, a greater production of IFN-c was found in DC2.4/Hlx, it indicated that the Hlx gene transfected into DC2.4 cells was activated and functional. The T-bet expression was not significantly enhanced in DC2.4/Hlx, it might due to Hlx as a cofactor for T-bet just appeared to enhance the activities of T-bet on promoting the production of IFN-c, but could not enhance T-bet transcription. In addition, the mature feature and antigen-presenting function of DC2.4/Hlx were analyzed, it showed that DC2.4 maturation was markedly promoted by high-expression of Hlx gene, the expression levels of IL-12p40
and IL-12p35 in DC2.4/Hlx cell-line were up-regulated obviously, these alteration suggested that the ability of antigen-presenting function in DC2.4/Hlx cell-line might be enhanced. Antigen-presenting function of DC is often used as a key index to further confirm the extent of DC maturation. Hereby, we used OVA to stimulate CD4+ T cells from C57/BL6 and established an OVA-specific T cells, which partly expressed TCR for recognizing OVA peptide. After OVA-loaded DC2.4/Hlx cells were incubated with the T cells, a significantly cell proliferation was found. This phenomenon could not be found in wild DC2.4. In summary, our results demonstrated that over-expressed Hlx up-regulated the production of IFN-c in DC2.4, enhanced DC2.4 maturation and antigen-presenting function. Our data generated will serve as a source for further investigation of Hlx over-expressed DC differentiation pathways
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and related biological responses including anti-tumor or microbial invasion. [9]
Acknowledgments This work was supported by the National Natural Science Foundation of China (30972748, 30910103084, 81072453, 81001319), Natural Science Foundation of Jiangsu Province (BK2004405), Natural Science Foundation of Colleges and Universities in Jiangsu Province and Innovation Fund for candidate of doctor in Jiangsu Province (Grant Nos. 09KJB310001 and CX09B_217Z, respectively). References [1] J.D. Allen, J.M. Adams, Enforced expression of Hlx homeobox gene prompts myeloid cell maturation and altered adherence properties of T cells, Blood 81 (12) (1993) 3242–3251. [2] L. Cao, J.D. Gibson, S. Miyamoto, V. Sail, R. Verma, D.W. Rosenberg, C.E. Nelson, C. Giardina, Intestinal lineage commitment of embryonic stem cells, Differentiation 81 (1) (2011) 1–10. [3] P. Hollington, P. Neufing, B. Kalionis, P. Waring, J. Bentel, D. Wattchow, W.D. Tilley, Expression and localization of homeodomain proteins DLX4, HB9 and HB24 in malignant and benign human colorectal tissues, Anticancer Res. 24 (2B) (2004) 955–962. [4] W.P. Zheng, Q. Zhao, X. Zhao, B. Li, M. Hubank, D.G. Schatz, R.A. Flavell, Upregulation of Hlx in immature Th cells induces IFN-gamma expression, J. Immunol. 172 (1) (2004) 114–122. [5] N. Mikhalkevich, B. Becknell, M.A. Caligiuri, M.D. Bates, R. Harvey, W.P. Zheng, Responsiveness of naive CD4 T cells to polarizing cytokine determines the ratio of Th1 and Th2 cell differentiation, J. Immunol. 176 (3) (2006) 1553–1560. [6] T. Kaneko, H. Hosokawa, M. Yamashita, C.R. Wang, A. Hasegawa, M.Y. Kimura, M. Kitajiama, F. Kimura, M. Miyazaki, T. Nakayama, Chromatin remodeling at the Th2 cytokine gene loci in human type 2 helper T cells, Mol. Immunol. 44 (9) (2007) 2249–2256. [7] A.C. Mullen, A.S. Hutchins, F.A. High, H.W. Lee, K.J. Sykes, L.A. Chodosh, S.L. Reiner, Hlx is induced by and genetically interacts with T-bet to promote heritable T(H)1 gene induction, Nat. Immunol. 3 (7) (2002) 652–658. [8] B. Becknell, T.L. Hughes, A.G. Freud, B.W. Blaser, J. Yu, R. Trotta, H.C. Mao, M.L. Caligiuri de Jesús, M. Alghothani, D.M. Benson, A. Lehman Jr., D. Jarjoura, D.
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Perrotti, M.D. Bates, M.A. Caligiuri, Hlx homeobox transcription factor negatively regulates interferon-gamma production in monokine-activated natural killer cells, Blood 109 (6) (2007) 2481–2487. L. Lois, Cavanagh, W. Weninger, Dendritic cell behaviour in vivo: lessons learned from intravital two-photon microscopy, Immunol. Cell Biol. 86 (2008) 428–438. K. Onoé, Y. Yanagawa, K. Minami, N. Iijima, K. Iwabuchi, Th1 or Th2 balance regulated by interaction between dendritic cells and NKT cells, Immunol. Res. 38 (1–3) (2007) 319–332. M. Yang, S. Wang, S. Wang, J. Ma, X. Xu, C. Mao, B. Ma, J. Tong, G. Qiu, Q. Shao, Q. Ding, H. Xu, Tat-mediated intracellular delivery of T-bet protein into THP-1 cells can induce Th1-type response, Immunol. Invest. 37 (2008) 97–111. E.G. Schulz, L. Mariani, A. Radbruch, T. Höfer, Sequential polarization and imprinting of type 1 T helper lymphocytes by interferon-gamma and interleukin-12, Immunity 30 (5) (2009) 673–683. P. Murthi, V. Doherty, J. Said, S. Donath, S.P. Brennecke, B. Kalionis, Homeobox gene HLX1 expression is decreased in idiopathic human fetal growth restriction, Am. J. Pathol. 168 (2006) 511–518. G. Rajaraman, P. Murthi, B. Leo, S.P. Brennecke, B. Kalionis, Homeobox gene HLX1 is a regulator of colony stimulating factor-1 dependent trophoblast cell proliferation, Placenta 28 (2007) 991–998. S. Halene, P. Gaines, H. Sun, T. Zibello, S. Lin, A. Khanna-Gupta, S.C. Williams, A. Perkins, D. Krause, N. Berliner, C/EBPepsilon directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via Hlx, Exp. Hematol. 38 (2) (2010) 90–103. M.D. Bates, D.T. Dunagan, L.C. Welch, A. Kaul, R.P. Harvey, The Hlx homeobox transcription factor is required early in enteric nervous system development, BMC Dev. Biol. 6 (2006) 33. Z. Zhang, J.E. Balmer, A. Lovlie, S.H. Fromm, R. Blomhoff, Specific teratogenic effects of different retinoic acid isomers and analogs in the developing anterior central nervous system of zebrafish, Dev. Dyn. 206 (1) (1996) 73–86. M.S. Longhi, M.J. Hussain, W.W. Kwok, G. Mieli-Vergani, Y. Ma, D. Vergani, Autoantigen-specific regulatory T cells, a potential tool for immune-tolerance reconstitution in type-2 autoimmune hepatitis, Hepatology 53 (2) (2011) 536– 547. L. Bar-On, T. Birnberg, K.W. Kim, S. Jung, Dendritic cell-restricted CD80/86 deficiency results in peripheral regulatory T-cell reduction but is not associated with lymphocyte hyperactivation, Eur. J. Immunol. 41 (2) (2011) 291–298. V. Wijewardana, A.C. Soloff, X. Liu, K.N. Brown, S.M. Barratt-Boyes, Early myeloid dendritic cell dysregulation is predictive of disease progression in simian immunodeficiency virus infection, PLoS Pathog. 6 (12) (2010) e1001235.
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Over-expression of Hlx homeobox gene in DC2.4 dendritic cell enhances its maturation and antigen presentation Siamak Sandoghchian Shotorbani a, Zhiqiang He a,b, Heng Yang a, Qiang Sun a, Yan Xu a, Zhaoliang Su a, Yuan Xue a,c, Dong Zheng a, Yun Zhang a, Shengjun Wang a,⇑, Qixiang Shao a, Liwei Lu d, Huaxi Xu a,⇑ a
Department of Immunology, Institute of Laboratory Medicine, Jiangsu University, Zhenjiang, PR China Department of Laboratory Medicine, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou, PR China Department of Physical Testing and Chemical Analysis, Zhenjiang Center for Disease Control and Prevention, Zhenjiang, PR China d Department of Pathology, Hong Kong University, Hongkong, PR China b c
a r t i c l e
i n f o
Article history: Received 21 November 2011 Accepted 23 February 2012 Available online 14 March 2012 Keywords: H2.0-like homebox Genetic modification Dendritic cell Antigen presenting
a b s t r a c t Hlx as a Th1-specific transcription factor, it appears to drive maturation of Th1 and IFN-c secretion in cooperation with T-bet. In this study, we established a stable Hlx-over-expressed dendritic cell line (DC2.4/Hlx), and investigated the possible effect of Hlx gene on maturation of dendritic cell-line (DC2.4). Results shown that over-expressed Hlx in DC2.4 up-regulated the transcription and expression of IFN-c, increased the expression of maturation makers including CD40, CD80, CD86, MHC-I and MHC-II. Functional assay for DC2.4/Hlx showed that over-expressed Hlx increased the expression level of interleukin-12 in the supernatant and decreased DC endocytosis when cells were incubated in vitro. Furthermore, using a syngeneic T cell activation model, we found that DC2.4/Hlx could obviously present ovalbumin (OVA) antigen to T cell in OVA pre-immunized mice. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Hlx is a homeobox gene, was originally isolated from a murine pre-B-lymphocyte cell line [1,2]. Hlx is very important not only in hematopoietic cells, but also in normal intestinal and hepatic development in mice [3]. In immune system, Hlx is a Th1-special transcription factor that interacts specifically with T-bet. Hlx and T-bet synergistically promote IFN-c expression when co-expressed. Thus, as a cofactor of T-bet, Hlx appears to enhance the activities of T-bet and then favor Th1 differentiation [4]. The co-engagement of T-bet and Hlx actively suppressed Th2 commitment. Hlx down-regulates the IL-4 receptor expression in naive CD4+ T cells [5]. Over-expressed Hlx resulted in the aberrant expression of IFN-c in normal CD4+ T cells during the differentiation under Th2-polarizing conditions [6,7]. Therefore, Hlx played an important role in the regulation of IFN-c production by T cell. Interestingly, in NK cells Hlx was a negative regulator of IFN-c production and its inhibitory function was achieved at least in part through the proteasomal degradation of STAT4 [8]. Therefore, Hlx may play the different role in different systems or cells. Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for the initiation of antigen (Ag)-specific immune re⇑ Corresponding authors. Address: Department of Immunology, Institute of Laboratory Medicine, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu Province, China. Fax: +86 511 85038449. E-mail address: [email protected] (H. Xu). 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.02.016
sponses as well as maintenance of tolerance to self-antigens [9]. In addition, DCs was also directly involved in regulating other innate immunity such as promoting activation of NK cells and their effector function [10,11]. However, the role of Hlx in DCs functions remains unknown. In the present study, we introduced exogenous Hlx gene into a dendritic cell line DC2.4, established a stable DC2.4/ Hlx cell-line. Our results showed that over-expression of Hlx in DC2.4 cells up-regulated production of IFN-c, enhanced their maturation and antigen-presenting function. 2. Materials and methods 2.1. Cell culture The immature murine dendritic cell line DC2.4 (H-2b), DC2.4/EGFP and DC2.4/Hlx were cultured in RPMI1640 medium, supplemented with 100 U/ml penicillin, 100 mg/L streptomycin, 2 mmol/L L-glutamine, and 10% FBS (GIBCO). The transfected cell lines were pulsed with 0.6 g/L G418 to eliminate negative cells. 2.2. Transfection and selection of stable clones The DC2.4 cells were transfected by Hlx with liposomes (lipofectamine™ 2000, Invitrogen). 4 105 DC2.4 cells in 500 ll complete RPMI1640 medium without antibiotics were plated in 24-well plates. Next day when the cells were at 60–70% confluent, Hlx was transfected according to the instruction of the manufac-
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turer. The medium was changed into 100 ll serum-free medium before transfection. The 100 ll DNA/lipofectamine complexes that were prepared at the ratio of 1:3 (ll) were added to each well and then mixed gently by rocking the plate back and forth, followed by incubating at 37 °C in a CO2 incubator for 6 h. The medium was changed by 200 ll new complete RPMI1640 medium. The cells were passaged at 1:10 with fresh growth medium 24 h after transfection, and then selected by adding selective medium (contain 600 mg/L G418) in the following day. The target clones were selected on the basis of their resistance to G418 and expression of EGFP. The Hlx high-expressed DC2.4 was assessed by Real-time PCR and Western blot. 2.3. Total RNA isolation and Real-time PCR Total RNA was prepared using Trizol (Invitrogen), and reversely transcribed with first strand cDNA synthesis kit ReverTraAce-a-™ (TOYOBO Co., Ltd., Life Science Department) according to the manufacturer’s instructions. RT-PCR was performed by the comparative threshold cycle (DCT) method and normalized to b-actin. Sequences of the primers are as follows: 50 -CTC GTG GTC CCG TGC TGT CTT TTC-30 and 50 -GTT CCC TCA GTC CGT TCC GTG TCG-30 for mouse Hlx, 50 -ATG CCA GGG AAC CGC TTA T-30 and 50 -CAG ATG CGT ACA
TGG ACT CAA A-30 for mouse T-bet, 50 -CTG TGG GCT GTA CTA CAA GCT TCA-30 and 50 -ACC CAT GGC GGT GAC CAT GC-30 for mouse GATA3, 50 -AAG CGT CAT TGA ATC ACA CC-30 and 50 -CGA AAT CAG CAG CGA CTC CTT AT-30 for mouse IFN-c, 50 -CGA GGT CAC AGG AGA AGG-30 and 50 -TGC TCT TTA GGC TTT CCA G-30 for mouse IL4, 50 -CTC ACC TGT GAC ACG CCT GA-30 and 50 -CAG GAC ACT GAA TAC TTC TC-30 for mouse Il-12p40, 50 -CCT CAG TTT GGC CAG GGT C-30 and 50 -CAG GTT TCG GGA CTG GCT AAG-30 for mouse Il12p35, 50 -TGG AAT CCT GTG GCA TCC ATG AAA C-30 and 50 -TAA AAC GCA GCT CAG TAA CAG TCC G-30 for mouse b-actin. PCR were set up with the SYBR Premix Ex Taq™ II (TaKaRa Biotechnology Co., Ltd., Dalian, China). Data were collected using the Rotor-gene 6000 real-time system (Corbett Life Science) and analyzed using the relative quantification (based on the relative expression of target genes vs. b-actin reference genes). Transcripts of each gene relative to the control gene were determined with the 2DCt method. 2.4. Western blot Whole cells were lysed in lysis buffer (150 mM NaCl, 20 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM sodium fluoride, 0.5% Doc, 1% Triton X-100, and 1% Nonidet P40). The cell lysates were boiled with 2 loading buffer for 5 min and
Fig. 1. Establishment of stable Hlx-overexpressed dendritic cell line (DC2.4-Hlx). (A) Expression and location of EGFP in stable DC2.4-Hlx clone, a and c showed the fluorescence intensity and localization of EGFP expression in DC2.4-Hlx detected by confocal microscopy; b and d appeared original state of DC2.4-Hlx. (B) The fluorescence ratio expressed in DC2.4-Hlx was identified by FACS. (C and D) Indicated the Hlx high-expressed in DC2.4-Hlx assessed by Real-time PCR and Western blot, upper half of figure C showed the Hlx expression in different cell clones, lane 1 Dl2000 marker, lane 2 DC2.4, lane 3 DC2.4/EGFP, lane 4–7 different DC2.4/Hlx clone, and the lower half showed the statistical analysis of Hlx mRNA (mean ± SD, n = 5), #represent p < 0.01.
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Fig. 2. The levels of IFN-c, T-bet and GATA3 in DC2.4-Hlx cell line. The secretion level of IFN-c in supernatant, and the mRNA expression of IFN-c, T-bet and GATA3 in DC2.4-Hlx cell line were detected by ELISA and RT-PCR, respectively. Compared with DC2.4/EGFP and wild DC2.4, the levels of IFN-c and T-bet in DC2.4/Hlx were enhanced (#p < 0.01), but GATA3 no significant change. (A, C and D) Showed the relative mRNA of IFN-c, T-bet and GATA3, respectively; (B) indicated the protein level of IFN-c in supernatant detected by ELISA after DC2.4/Hlx cell was cultured for 48 h in vitro.
analyzed by Western blot. Rabbit anti-mouse T-bet, rabbit antimouse b-actin and goat anti-mouse Hlx used as primary antibodies (Abs) were purchased from Santa Cruz Biotechnology. After PAGE, the protein was transferred into cellular membrane. The membrane was incubated with the primary antibody followed by incubating with HRP-conjugated goat anti-rabbit IgG (Sigma–Aldrich) or rabbit anti-goat IgG (Santa Cruz Biotechnology). After extensive washing in TBS-T, the blots were processed for detection of Ag using the ECL plus Western blotting detection system (GE Healthcare Life Sciences).
was performed and isotype matched monoclonal antibody was used as a negative control. 2.6. Enzyme-linked immunosorbent assays (ELISAs) for detecting IFN-c and IL-12p70 in supernatant The concentration of cytokines in supernatants was detected by ELISA. The cells were cultured in complete RPMI1640 for 48 h and cell-free supernatants were collected. The levels of IFN-c and IL12p70 in supernatants were measured by ELISA kit (eBiosence, USA). All samples were measured in triplicate.
2.5. FACS for phenotype analysis 2.7. Endocytosis assay For phenotypic analysis, 1 106 cells were resuspended in 100 ll of staining buffer (PBS containing 0.1% BSA) and incubated for 30 min on ice with PE-conjugated Abs specific for H-2Kb, I-Ab, CD80, CD86, CD40 and TLR4 (each, 5 lg/ml). After washing three times with staining buffer, the cells were analyzed for surface phenotype by FACSCalibur with Cell Quest software. Each cell type was treated with 10 lg/ml LPS (Sigma) for 24 h before the analysis
The endocytic capacity of DC2.4/Hlx, DC2.4/EGFP and DC2.4 was determined by an energy-dependent uptake of a special Escherichia coli which expressed red fluorescent protein HcRed. Three different lines of DCs (2 105) were incubated in the presence of HcRed-expressed dead E. coli (5 108/ml) at 37 °C for 2 h. The cells were washed three times with cold FACS buffer (PBS containing 1% FBS). HcRed-expressed E. coli
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Fig. 3. High expression of Hlx up-regulated maturation markers on DC2.4/Hlx cell. Phenotypical changes in DC2.4/Hlx cell was examined by flow cytometry analysis. The horizontal coordinate indicates the fluorescence intensity, the number on figure show the fluorescence positive cell percentage and vertical coordinate indicates the amount of cells. The fluorescence intensity and numbers of fluorescence-positive cells were related to the expression of associated-membrane molecules. The expression levels of CD80, MHC-II, CD86 and CD40 were markedly enhanced in DC2.4/Hlx compared with wild DC2.4 and DC2.4/EGFP.
uptaked by DC was determined by a FACSCalibur. The parallel experiments were conduced at 4 °C to determine the nonspecific adherence as background. 2.8. Antigen presenting analysis Antigen-presenting function of DC was assessed by allogeneic T cell activation responding to a specific antigen. The DC2.4, DC2.4/ EGFP and DC2.4/Hlx were cultured with 100 mg/L ovalbumin (OVA; Sigma) for 24 h, respectively. Spleens from C57BL/6 mice immunized by OVA were aseptically obtained and the single cell suspensions were prepared. Purified CD4+ T cells were prepared by negative selection with magnetic activated cell sorting (MACS).
T cells were stained using 3 mmol/L fluorescein diacetate succinimidyl ester (CFSE; Santa Cruz Biotechnology, Inc.) for 10 min at 37 °C and washed three times by complete RPMI medium with 10% FBS. In a 24-well plate, 2 106 CD4+ T cell per well were cocultured 72 h with 2 105 different lines of DC that were pre-treated by 50 lg/ ml mitomycin C at 37 °C for 20 min, respectively. The cells were harvested and green fluorescence from CFSE was analyzed by a FACSCalibur. In the mean time, the method of MTT was also used to detect the ability of cellular proliferation. Briefly, 2 105 CD4+ T cells were cocultured with 2 104 DCs as described above in 96-well flat-bottom plates for 72 h, and then 20 ll 5 mg/ml MTT3-(4,5)dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumro-mide solu-
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Fig. 4. The levels of IL-12 mRNA and protein in DC2.4-Hlx cell line. (A) The relative mRNA of IL-12p40, it was increased in DC2.4/Hlx than that in wild DC2.4 and DC2.4/EGFP. (B) The IL-12p35 produced by DC2.4/Hlx was enhanced. (C) The protein level of IFN-c in supernatant detected by ELISA after DC2.4/Hlx cell was cultured for 48 h in vitro, it was significant increased than that in cell-cultured supernatant of DC2.4 and DC2.4/EGFP (#p < 0.01; ⁄p < 0.05).
tion (Y-S Biotechnology, China) was added to each well and incubated another 4 h, the untransformed MTT was removed carefully by pipetting and 150 ll DMSO was added. Ten minutes later, optical density (OD) was determined in an ELISA reader at 570 nm and the value of optical density reflected the number of living cells.
microscopy (Fig. 1C and D). The rate of EGFP expression was 99% identified by FACS (Fig. 1B). The transfected DC2.4/Hlx cells were screened, and the clone of high transcription and expression of Hlx was identified by Real-time PCR and Western-blot (Fig. 1C and D). The rate of EGFP expression in the DC2.4/Hlx clone was more than 90% (Fig. 1B).
2.9. Statistical analysis
3.2. Enhanced IFN-c secretion in DC2.4/Hlx cell-line
All statistical analyses were performed using the Prism program (GraphPad) statistical software. Data were expressed as means ± SEM. The difference in mRNA expression of the target genes between DC2.4/Hlx, DC2.4/EGFP and DC2.4 was assessed by t-test respectively. P < 0.05 was considered to be statistically significant.
Hlx, as a Th1-specific transcription factor, it appears to drive secretion of IFN-c in cooperation with T-bet. We detected the mRNA of transcription factors T-bet, GATA3 and cytokines IFN-c, IL-4 in DC2.4/Hlx, DC2.4/EGFP or wild DC2.4. The RT-PCR data showed that IFN-c mRNA level in DC2.4/Hlx was enhanced about 7-folds than that in DC2.4/EGFP or wild DC2.4 (Fig. 2A). But, the mRNA levels of T-bet and GATA3 were not significant change in different DC cell lines (Fig. 2C and D). ELISA results also confirmed that a greater production of IFN-c was found in the supernatant of cultured DC2.4/Hlx cells (18 ng/ml) in comparison with that in DC2.4/EGFP (4 ng/ml) and DC2.4 cells (5 ng/ml) (Fig. 2B).
3. Results 3.1. DC2.4/Hlx cell-line was established To evaluate the effects of Hlx in DCs, we generated a DC cell line that stably express Hlx gene. An empty vector PIRES2-EGFP was used as control. After transfection the DC2.4/Hlx and DC2.4/EGFP cell-line were selected by subcloning in selection medium based on EGFP expression (Fig. 1A and B). The EGFP distributed in whole cell, but expressed abundantly in the nucleus, as seen in confocal
3.3. Increased expression of maturation markers in DC2.4/Hlx cell-line Phenotypical changes in three different DC2.4 cells were examined by FACS analysis using specific antibodies against MHC mole-
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cules and co-stimulatory molecules (Fig. 3). The expression of CD80, MHC-II, MHC-I, CD86 and CD40 were markedly enhanced in DC2.4/Hlx compared with wild DC2.4, suggesting that the DC2.4 maturation was markedly promoted by high-expression of Hlx gene (Fig. 3).
with OVA. When co-cultured with OVA-loaded DC2.4/Hlx cells, the T cells showed a significantly faster cell cycle division indicated by CFSE, while that was not obvious when co-cultured with the wild DC2.4 or DC2.4/EGFP (Fig. 6A and B). 4. Discussion
3.4. Increased expression of IL-12p40 and IL-12p35 in DC2.4/Hlx cellline The expression levels of IL-12 mRNA in three different DC cell-lines were detected by qRT-PCR. The results shown that the transcription of IL-12p40 and IL-12p35 in DC2.4/Hlx cells were up-regulated compared with DC2.4/EGFP and wild DC2.4 (Fig. 4A), and the level of IL-12p70 in the cell-culture supernatant of DC2.4/Hlx was 8-folds more than DC2.4/EGFP and wild DC2.4 (Fig. 4B). However, the endocytosis of bacteria by DC2.4/Hlx was decreased (Fig. 5). 3.5. Up-regulated antigen-presenting functions in DC2.4/Hlx cell-line Antigen-presenting function of DC is often used as a key index to further confirm the extent of DC maturation. In this study, we used the CD4+ T cells from C57/BL6 mice that were immunized
The Hlx gene is a member of the homeobox family of genes, with homology to the Drosophila homeobox gene H2.0. During Th1 cell differentiation, the transcription factor Hlx and T-bet are induced. These two transcription factors control Th1 cell differentiation and IFN-c gene expression [12]. In addition, Hlx has been implicated in cell proliferation in vitro in several different tissues, including placental extravillous trophoblasts and CD34 positive bone marrow cells [13,14]. Hlx expression can be stimulated by many cytokines or other growth factors in these cell types, and thus is implicated in immature stem cell and progenitor stem cell development [14,15]. Hlx is also necessary for the development of the enteric nervous system [16,17]. DCs are the most potent antigen-presenting cells for the initiation of Ag-specific immune responses as well as maintaining tolerance to self-antigens [18–20]. In the present study, we transfected
Fig. 5. The endocytic capacity of DC2.4/Hlx cell-line. DC2.4/Hlx, DC2.4/EGFP and wild DC2.4 were incubated in the presence of HcRed-expressed dead E. coli (5 108/ml) at 37 °C for 2 h. HcRed-expressed E. coli uptaked by DCs was determined using a FACSCalibur, respectively. The parallel experiments were conduced at 4 °C to determine the nonspecific adherence as background. The number in figure shown the fluorescence positive cell percentage, it represented the endocytic capacity of dendritic cells. HcRedexpressed E. coli uptaked by DC2.4/Hlx cell-line was decreased compared with wild DC2.4 or DC2.4/EGFP. The experiment was repeated three times and the same trend was observed.
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Fig. 6. Analysis of antigen-presenting functions in DC2.4/Hlx cell-line. (A) After OVA-loaded DC2.4 cells were incubated with OVA-specific T cells, compared with wild DC2.4 and DC2.4/EGFP, a significantly faster cell cycle division was indicated by CFSE while DC2.4/Hlx presence. Anti-CD3 and anti-CD28 were used for positive control in this experiment. The peak shape on Fig. 6A was used for analyzing the cell proliferation, peak more wide showed the proliferative ability stronger. The lower half of Fig. 6A indicated what the control panels from each group (it was not stimulated by OVA). (B) The proliferation of OVA-specific T cells indicated by absorbance was detected using MTT method after they were incubated with OVA-loaded DC2.4/Hlx, wild DC2.4 or DC2.4/EGFP cells, respectively, it showed that the ability of DC2.4/Hlx cell proliferation stimulated by OVA was stronger than wild DC2.4 or DC2.4/EGFP cells (#p < 0.01; ⁄p < 0.05).
exogenous Hlx gene into DC2.4 cells, and established a stable DC2.4/Hlx cell-line, which could strongly express HLX. The data also showed that IFN-c mRNA level in DC2.4/Hlx was enhanced about 7-folds than that in wild DC2.4 cell. The protein of IFN-c in the cell-cultivated supernatant was quantified by ELISA, a greater production of IFN-c was found in DC2.4/Hlx, it indicated that the Hlx gene transfected into DC2.4 cells was activated and functional. The T-bet expression was not significantly enhanced in DC2.4/Hlx, it might due to Hlx as a cofactor for T-bet just appeared to enhance the activities of T-bet on promoting the production of IFN-c, but could not enhance T-bet transcription. In addition, the mature feature and antigen-presenting function of DC2.4/Hlx were analyzed, it showed that DC2.4 maturation was markedly promoted by high-expression of Hlx gene, the expression levels of IL-12p40
and IL-12p35 in DC2.4/Hlx cell-line were up-regulated obviously, these alteration suggested that the ability of antigen-presenting function in DC2.4/Hlx cell-line might be enhanced. Antigen-presenting function of DC is often used as a key index to further confirm the extent of DC maturation. Hereby, we used OVA to stimulate CD4+ T cells from C57/BL6 and established an OVA-specific T cells, which partly expressed TCR for recognizing OVA peptide. After OVA-loaded DC2.4/Hlx cells were incubated with the T cells, a significantly cell proliferation was found. This phenomenon could not be found in wild DC2.4. In summary, our results demonstrated that over-expressed Hlx up-regulated the production of IFN-c in DC2.4, enhanced DC2.4 maturation and antigen-presenting function. Our data generated will serve as a source for further investigation of Hlx over-expressed DC differentiation pathways
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and related biological responses including anti-tumor or microbial invasion. [9]
Acknowledgments This work was supported by the National Natural Science Foundation of China (30972748, 30910103084, 81072453, 81001319), Natural Science Foundation of Jiangsu Province (BK2004405), Natural Science Foundation of Colleges and Universities in Jiangsu Province and Innovation Fund for candidate of doctor in Jiangsu Province (Grant Nos. 09KJB310001 and CX09B_217Z, respectively). References [1] J.D. Allen, J.M. Adams, Enforced expression of Hlx homeobox gene prompts myeloid cell maturation and altered adherence properties of T cells, Blood 81 (12) (1993) 3242–3251. [2] L. Cao, J.D. Gibson, S. Miyamoto, V. Sail, R. Verma, D.W. Rosenberg, C.E. Nelson, C. Giardina, Intestinal lineage commitment of embryonic stem cells, Differentiation 81 (1) (2011) 1–10. [3] P. Hollington, P. Neufing, B. Kalionis, P. Waring, J. Bentel, D. Wattchow, W.D. Tilley, Expression and localization of homeodomain proteins DLX4, HB9 and HB24 in malignant and benign human colorectal tissues, Anticancer Res. 24 (2B) (2004) 955–962. [4] W.P. Zheng, Q. Zhao, X. Zhao, B. Li, M. Hubank, D.G. Schatz, R.A. Flavell, Upregulation of Hlx in immature Th cells induces IFN-gamma expression, J. Immunol. 172 (1) (2004) 114–122. [5] N. Mikhalkevich, B. Becknell, M.A. Caligiuri, M.D. Bates, R. Harvey, W.P. Zheng, Responsiveness of naive CD4 T cells to polarizing cytokine determines the ratio of Th1 and Th2 cell differentiation, J. Immunol. 176 (3) (2006) 1553–1560. [6] T. Kaneko, H. Hosokawa, M. Yamashita, C.R. Wang, A. Hasegawa, M.Y. Kimura, M. Kitajiama, F. Kimura, M. Miyazaki, T. Nakayama, Chromatin remodeling at the Th2 cytokine gene loci in human type 2 helper T cells, Mol. Immunol. 44 (9) (2007) 2249–2256. [7] A.C. Mullen, A.S. Hutchins, F.A. High, H.W. Lee, K.J. Sykes, L.A. Chodosh, S.L. Reiner, Hlx is induced by and genetically interacts with T-bet to promote heritable T(H)1 gene induction, Nat. Immunol. 3 (7) (2002) 652–658. [8] B. Becknell, T.L. Hughes, A.G. Freud, B.W. Blaser, J. Yu, R. Trotta, H.C. Mao, M.L. Caligiuri de Jesús, M. Alghothani, D.M. Benson, A. Lehman Jr., D. Jarjoura, D.
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Perrotti, M.D. Bates, M.A. Caligiuri, Hlx homeobox transcription factor negatively regulates interferon-gamma production in monokine-activated natural killer cells, Blood 109 (6) (2007) 2481–2487. L. Lois, Cavanagh, W. Weninger, Dendritic cell behaviour in vivo: lessons learned from intravital two-photon microscopy, Immunol. Cell Biol. 86 (2008) 428–438. K. Onoé, Y. Yanagawa, K. Minami, N. Iijima, K. Iwabuchi, Th1 or Th2 balance regulated by interaction between dendritic cells and NKT cells, Immunol. Res. 38 (1–3) (2007) 319–332. M. Yang, S. Wang, S. Wang, J. Ma, X. Xu, C. Mao, B. Ma, J. Tong, G. Qiu, Q. Shao, Q. Ding, H. Xu, Tat-mediated intracellular delivery of T-bet protein into THP-1 cells can induce Th1-type response, Immunol. Invest. 37 (2008) 97–111. E.G. Schulz, L. Mariani, A. Radbruch, T. Höfer, Sequential polarization and imprinting of type 1 T helper lymphocytes by interferon-gamma and interleukin-12, Immunity 30 (5) (2009) 673–683. P. Murthi, V. Doherty, J. Said, S. Donath, S.P. Brennecke, B. Kalionis, Homeobox gene HLX1 expression is decreased in idiopathic human fetal growth restriction, Am. J. Pathol. 168 (2006) 511–518. G. Rajaraman, P. Murthi, B. Leo, S.P. Brennecke, B. Kalionis, Homeobox gene HLX1 is a regulator of colony stimulating factor-1 dependent trophoblast cell proliferation, Placenta 28 (2007) 991–998. S. Halene, P. Gaines, H. Sun, T. Zibello, S. Lin, A. Khanna-Gupta, S.C. Williams, A. Perkins, D. Krause, N. Berliner, C/EBPepsilon directs granulocytic-vs-monocytic lineage determination and confers chemotactic function via Hlx, Exp. Hematol. 38 (2) (2010) 90–103. M.D. Bates, D.T. Dunagan, L.C. Welch, A. Kaul, R.P. Harvey, The Hlx homeobox transcription factor is required early in enteric nervous system development, BMC Dev. Biol. 6 (2006) 33. Z. Zhang, J.E. Balmer, A. Lovlie, S.H. Fromm, R. Blomhoff, Specific teratogenic effects of different retinoic acid isomers and analogs in the developing anterior central nervous system of zebrafish, Dev. Dyn. 206 (1) (1996) 73–86. M.S. Longhi, M.J. Hussain, W.W. Kwok, G. Mieli-Vergani, Y. Ma, D. Vergani, Autoantigen-specific regulatory T cells, a potential tool for immune-tolerance reconstitution in type-2 autoimmune hepatitis, Hepatology 53 (2) (2011) 536– 547. L. Bar-On, T. Birnberg, K.W. Kim, S. Jung, Dendritic cell-restricted CD80/86 deficiency results in peripheral regulatory T-cell reduction but is not associated with lymphocyte hyperactivation, Eur. J. Immunol. 41 (2) (2011) 291–298. V. Wijewardana, A.C. Soloff, X. Liu, K.N. Brown, S.M. Barratt-Boyes, Early myeloid dendritic cell dysregulation is predictive of disease progression in simian immunodeficiency virus infection, PLoS Pathog. 6 (12) (2010) e1001235.