核小體和調節性T細胞在紅斑性狼瘡所扮演的角色(2/3)

行政院國家科學委員會專題研究計畫 期中進度報告

核小體和調節性 T 細胞在紅斑性狼瘡所扮演的角色(2/3)

期中進度報告(精簡版)

中 華 民 國 96 年 05 月 31 日

行政院國家科學委員會補助專題研究計畫期中進度報告

核小體和調節性 T 細胞在紅斑性狼瘡所扮演的角色(2/3)

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中文摘要 關鍵詞：紅斑狼瘡、調節性 T 細胞、治療 自體免疫疾病在過去幾年來似乎有逐漸增加的趨勢，如何來研究自體免疫疾病的 機轉和進一步研發出更新的治療方法，一直是個刻不容緩的課題。這幾年來利用樹 突細胞來誘發一個較好的免疫反應，並加以來調節一些特定的免疫疾病，在許多疾 病尤其是腫瘤疾病的治療上得到相當不錯的效果。本研究計畫便計畫更進一步利用 樹突細胞來研究全身性紅斑狼瘡的 T 細胞抗原決定位，以進一步設計出更有效的免 疫療法。而最近的研究更顯示凋亡小體(apoptotic body)可能在全身性紅斑狼瘡的發 病機轉中扮演著一個重要的角色， histone、nucleosome 和凋亡小體來當成首要的目 標，本研究計畫將分成三年來加以執行： 第一年：由 NZB/W F1 小鼠骨髓的幹細胞培養出樹突細胞，我們將由小鼠的脾臟細 胞將 T 細胞分離出來後，再跟自體抗原如凋亡小體、histone 或是 nucleosome 等培養 過的樹突細胞一起培養，進一步測定其增殖反應。同時，一但發現這些 T 細胞能夠 被刺激後，我們將進一步分析 T 細胞分泌淋巴介質的情形。在第一年的計畫中，我 們計畫先找出對小鼠體內自體反應性 T 細胞有最佳刺激反應的抗原。我們將進一步 利用合成的 peptides 來分析這些自體反應性 T 細胞的抗原決定位，所以需要合成 onerlapping peptides。同樣的，我們將利用 T 細胞增殖反應及淋巴介質分泌的情形來 測定抗原決定位。同時，我們也將培養樹突細胞與這些主要的自體抗原培養，再注 射入正常的 DBA-2/NZW F1 小鼠體內，看是否可以誘發自體免疫疾病。 第二年：建立能夠表現 Fas-L 的腺病毒載體，並將這些載體送入樹突細胞內，利用 自體抗原呈現的樹突細胞來誘發動物體內自體反應性 T 細胞的死亡。我們有興趣的 是此類能夠表現 Fas-L 的樹突細胞是否能夠專一性地誘發抗原特異性 T 細胞的凋 亡。如果此種帶有 Fas-L 的樹突細胞具有這樣的功能，是否能夠將已經建立好的過 敏動物模式體內的過敏原特異性 T 細胞除掉，而達到治療的目的。一但找到 T 細胞 的重要抗原決定位，我們也將利用 peptides 來嘗試治療自體免疫疾病的小鼠。 第三年：在今年度的計畫中，我們將著重在調節性 T 細胞(regulatory T cells)的研究 上。主要是因為目前有愈來愈多的證據顯示調節性 T 細胞可能在免疫疾病如自體免 疫疾病和器官移植上扮演了一個非常重要的角色。由於我們在前兩年的研究已經顯 示 nucleosome 內的 histone 蛋白是重要的 T 細胞抗原，我們將進一步來分離出與 nucleosome 特異性 T 細胞相關的調節性 T 細胞，以期能夠應用到自體免疫疾病治療 上的應用。 多年來，研究者認為在全身性紅斑狼瘡的致病機轉中 T 細胞抗原扮演著一個 重要的角色，但是對如何來定義出這些抗原決定位卻一直沒有較好的方法，本計畫 便是描述一個簡單的方法利用樹突細胞來進行此一工作。同時，我們也可以利用表 現 Fas 的樹突細胞及 peptide 療法來抑制體內的自體反應性 T 細胞，也許可以達到一 個治療自體免疫疾病的機會。在第三年的計畫中，我們將研究這些自體免疫傾向小 鼠體內的調節性 T 細胞活性，並培養出能夠抑制自體反應性 T 細胞的調節性 T 細 胞。這些研究的方向和未來的成果都是目前尚未探討過的研究題目，也因此，本計 畫在整個紅斑性狼瘡的致病機轉和治療的研發上具有非常重要的意義。

Abstract

Keywoeds: SLE, regulatory T cells, therapy

Systemic lupus erythematosus (SLE) is characterized by persistent production of autoantibodies against DNA, nucleosome and the small nuclear ribonucleoproteins (snRNPs). However, only few studies about the self-T cells have been reported. The information on the self-T epitopes might provide us the key to solve the critical pathogenic mechanisms of lupus. Therefore, in this

proposal, we like to use the potent bone marrow-derived dendritic cells (BM-DCs) to define the T cell epitopes of self-reactive T cells in murine lupus. Furthermore, advanced therapeutic

approaches such as DCs expressing Fas L or peptides therapy will be used to delete self-reactive T cells and prevent the progress of the lupus. In addition, the role of apoptotic body in the

pathogenesis of SLE will be investigated. We also like to investigate the role of regulatory T cells in the regulation of autoimmune response in systemic lupus erythematosus.

First year: In vitro culture of dendritic cells isolated from bone marrow stem cells and apoptotic

body, histone and nucleosome will also be used as the major self-antigen in the study. DCs pulsed with these self-antigens will be used to characterize the self-reactive T cell response. With this novel approach, we could identify the most critical self-antigen involved in the activation of autoreactive T cells. In addition,synthetic peptides of the self-antigens will be used to further characterize the epitopes recognized by self-reactive T cells. We also like to study if dendritic cells pulsed with apoptotic body could break tolerance in non-autoimmune mice in vivo.

Second year: In the second year, we like to explore the possible treatments for the lupus.

Establishment of expressing dendritic cells pulsed with the major self-antigen or self peptides will be used to induce apoptosis of self-reactive T cells in murine lupus. Further, the major peptides will also be used to treat the NZB/W F1 mice by following up both the level of autoantibody and life span.

Third year: We like to study the generation of regulatory T cells and for the application of

treatment for the lupus. It has been documented that regulatory T cells might play a critical role in the down-regulation of autoimmune process in murine lupus. We plan to study the interaction between regulatory T cells and nucleosome-specific T cells.

In general, we believe the approaches mentioned here will become the pioneer studies on autoimmune diseases for the years to come. In addition, the approaches might be applied for other diseases such as tumor, transplantation and infectious diseases.

前言 此一研究計畫主要是要研究利用樹突細胞當成抗原呈現細胞來定出 T 細胞反 應性的抗原決定位，再利用這些抗原決定位來研究是否可能作為治療的方法。我們 在之前的研究已經發現利用樹突細胞的確可以在狼瘡小鼠的 T 細胞找到特定的抗原 決定位，而將此一抗原決定位的 peptide 利用來注射在小鼠體內，可以有效地降低這 些狼瘡小鼠體內的抗 DNA 抗體濃度，而且可以延長狼瘡小鼠的壽命，減輕其腎臟發 炎。而在我們第二年的研究計畫中，我們進一步研究樹突細胞應用到表現凋亡的細 胞來刺激自體反應性 T 細胞的增生，而且也研究出利用凋亡的細胞可以在小鼠體內 誘發出抗 DNA 抗體。而且，我們也發現在狼瘡小鼠體內的調節性 T 細胞數目有較低 的情形，如果將其加以抑制，可以觀察到自體抗體的製造增加。 目的 此一研究計畫的主要目的是研究是否能夠在狼瘡小鼠研發出其 T 細胞的抗原 決定位，而能夠進一步研究其是否能夠誘發調節性 T 細胞。 Background

Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease characterized by the production of autoantibodies directed against ubiquitously expressed self-antigens. The hallmark of SLE is the production of autoantibodies directed against nuclear components, including dsDNA, ssDNA, histones, small nuclear ribonucleoproteins (snRNPs) as well as nucleosomes. It is known that during the progression of SLE, the serum level of anti-nucleosome and anti-DNA Abs correlates with the severity of the disease. In addition, nucleosomes have been found in the circulation of patients with SLE; however, the source of autoantigens, such as nucleosomes that can induce pathogenic autoantibodies is still unclear.

Previous studies have shown that autoantigens can be presented on the surface of apoptotic bodies, while some others accumulate inside the apoptotic bodies. For example, during the apoptosis of keratinocyte, the 52kDa ribosomal protein, Ro, accumulates in the ER. In addition, the autoantigens, such as La, Sm, U1-70kDa, PARP, NUMA, and

nucleosome can be detected as well in the apoptotic bodies. It has also been suggested that relocation of the cell components occurrs during apoptosis; for example, nuclear RNPs translocate from the nucleus to the cytosol during apoptosis. In addition, previous work has demonstrated that SLE might result from increased apoptotic neutrophils or impaired clearance of apoptotic cells by macrophages. Many studies have shown that defects in the clearance of apoptotic cells may underlie SLE. For example, characteristic autoantibodies and lupus-like pathology arise in mice lacking the complement protein C1q, a protein that binds apoptotic cells. Similarly, mice with deficiencies in the serum amyloid P or

mutations in the Mer tyrosine kinase develop autoantibodies to DNA and manifest autoimmune disease. These autoantigens may be captured by dendritic cells (DCs) and may activate autoreactive T cells, which in turn provide help for B cells in recognizing nuclear autoantigens. Therefore, it has been proposed that apoptotic cells may be the major autoantigenic target in lupus.

Several studies have identified the critical autoepitopes of the nucleosome in human lupus or lupus in animal models, such as SWR X NZB F1 (SNF1) or NZB X NZW F1 (BWF1) mice. As described above, nucleosomes can be detected in apoptotic bodies. However, there is no direct evidence demonstrating that apoptotic cells provide

self-antigen, such as nucleosome to stimulate autoreactive T cells in lupus. We have previously shown that bone marrow-derived dendritic cells (BMDCs) are able to determine the

self-reactive epitopes in freshly isolated CD4+ T cells from unprimed lupus mice. In this study, BMDCs were used to pulse with apoptotic cells to test for the presence of CD4+ T cells, which would identify apoptotic cells in vitro. Furthermore, we also used BMDCs pulsed with apoptotic cells, intravenously injected into non-autoimmune mice, to explore the immune response directed against apoptotic cells induced by DCs.

The results of this study show that BMDCs are capable of processing and presenting apoptotic cells, resulting in the stimulation of autoreactive CD4+ T cells from unprimed BWF1 mice to proliferate in vitro. Furthermore, histone peptide–specific T cell lines from BWF1 mice can respond to apoptotic cell-pulsed BMDCs. This suggests that BMDCs can process and present autoantigens, such as nucleosomes, which are contained in apoptotic cells. In addition, apoptotic cell-pulsed BMDCs are able to induce persistent Ab responses to ds and ssDNA in normal mice in vivo. Ab (IgG) and complement C3 are deposited in the glomeruli of the kidneys in the immunized mice. The data from our study will help in understanding how pathogenic autoimmune responses develop in spontaneous SLE.

Mice

Female NZB X NZW F1 (BWF1) mice were purchased from Jackson Laboratories (Bar Harbor, ME). DBA-2 X NZW F1 mice are from a non-autoimmune strain with identical major histocompatability complex (MHC) II molecules to BWF1 (H-2d/u) mice. At the age of 6-8 wks, female BWF1 mice and DBA-2 X NZW F1 mice were used as the source of BMDCs. The mice (young BWF1 and DBA-2 X NZW F1 mice) were obtained from and maintained by the Animal Centre of the College of Medicine of National Taiwan

University in a pathogen-free facility.

Generation of dendritic cells from Bone marrow cells

BMDCs were prepared as described previously (18). In brief, DCs were generated from bone marrow cell-depleted red cells by treating the cells with ACK lysis buffer and culturing cells for 4-6 days in a medium supplemented with murine rGM-CSF (750 U/ml) and rIL-4 (IL-4) (1000 U/ml) (Pepro Tech Inc. Rocky Hill, NJ). Approximately one million of the cells were placed in 24-well plates in 1 ml of RPMI 1640 medium that was supplemented with 5% of heat-inactivated FCS, 4 mM L-glutamine, 25 mM HEPES (pH 7.2), 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml

amphotericin. Every other day, the medium was removed by aspiration to remove the lymphocytes, and fresh medium containing GM-CSF and IL-4 was added. Apoptotic cells were added to the BMDC cultures on day 4 or day 6. After 48 h, non-adherent cells (BMDCs) were collected and washed extensively to remove the free apoptotic cells, and then a MACS method was used to enrich the CD11c+ DCs. CD11c+ DCs were positively selected by anti-CD11c-coated magnetic microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. The purity of CD11c+ DCs was (> 93%) analysed by flow cytometry, examining the expressions of MHC class II, B7-1, B7-2 and CD11c.

Generation of apoptotic cells

Thymocytes were retrieved from six to 10-wk-old BWF1 or DBA-2 X NZW F1 mice. Apoptotic cells were generated by treating single-cell suspensions of thymocytes in a medium with dexamethasone 1.2×10-6 M for 12-15 h. The culture medium was RPMI-1640 medium supplemented with 2% heat-inactivated FCS, 4 mM L-glutamine, 25 mM HEPES (pH 7.2), 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin. We used FITC-labeled annexin V (BD PharMingen, San Diego, CA) to evaluate the exposure of phosphatidylserine and DNA-labeled 7-AAD (BD PharMingen) to access plasma membrane integrity. Cells were analyzed by flow cytometry, and the double positive cells were > 82% after treating with dexamethasone for 15 h.

Phagocytosis assay

The phagocytosis of apoptotic cells was quantified by flow cytometry. 7-AAD-labeled apoptotic cells (2x106 per well) were cocultured with day-4 DCs (2x105 per well) for 6 h. BMDCs were further purified by anti-CD11c-coated magnetic microbeads (Miltenyi Biotec) according to manufacturer’s instructions. Purified BMDCs were stained with FITC-conjugated anti-MHC class I or anti-MHC class II Ab (BD PharMingen).

Phagocytosis was quantified by flow cytometry as the percentage of double positive cells (MHC I+7-AAD+ or MHC II+7-AAD+).

Phagocytosis of apoptotic cells was also visualized by confocal microscopy. CFDASE (Molecular probe, Inc., Eugene, OR) labeled apoptotic cells were incubated with BMDCs for 6 h, and then were prepared on a slide with a cytocentrifuge (Cytospin) and fixed in acetone. The slides were washed with PBS and then stained in PBS with 20 µg/ml I-Ad

-PE (BD PharMingen) and 0.5% BSA (19). The slides were washed three times with PBS after staining for 90 min in 37oC, and mounted with 90% glycerol in PBS, observed under a Confocal Spectral Microscope (Leica Microsystems, Wetzlar, Germany).

Cytokine secretion by dendritic cells

For cytokine secretion, day-4 or day-6 BMDCs (2x105 per well) from BWF1 mice or from DBA-2×NZW F1 mice were cocultured with an increasing number of syngenic apoptotic cells (1x106, 2x106, 4x106 per well). BMDCs stimulated with LPS (1 µg/ml) were

considered as the positive control. The supernatants were collected after 24-48 h and assayed for IL-12p70, IL-12p40, IL10, and TGF-　. The concentration of cytokines in culture supernatants was detected by sandwich-ELISA (R&D, Minneapoils, MN).

T cell Proliferation assays

All of the BWF1 mice used in this study developed anti-dsDNA IgG. CD4+ T cells were positively selected from splenocytes by anti-CD4-coated magnetic microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. On the other hand, day-4 and day-6 BMDCs from BWF1 mice or DBA-2 X NZW F1 mice were incubated with syngenic or allogenic apoptotic cells for 48 h. CD11c+ DCs were further positively

selected by anti-CD11c-coated magnetic microbeads (Miltenyi Biotec). Purified T cells (1-2×105 per well) were co-cultured with purified CD11c+ BMDCs (2500-7500 per well) in the presence or absence of anti-I-Ad/Ed (2G9, BD PharMingen) or anti-I-Ak (clone:11-5.2, BD PharMingen) for 4-5 days. During the last 4-6 h of culture, 1 µCi of [3_H]thymidine

was added to each well. The cells were harvested onto glass fiber filters using an

automated multisample harvester. [3_{H]thymidine incorporation was then measured in a dry}

scintillation counter (Packard Instrument Co., Meridan, CT). A proliferative response is defined as stimulation index (SI), and SI > 3 indicates a significant proliferative response. SI was evaluated by dividing the mean cpm incorporated in cultures of T cells plus apoptotic cell-pulsed BMDCs by the mean cpm of T cells plus non-antigen-pulsed BMDCs.

Immunization of mice

DBA-2 X NZW F1 mice were intravenously injected with 1.5x105 syngenic BMDCs, which uptaked or not syngenic apoptotic cells, or with PBS at 8 wks of age. Five days later, the mice received an intravenous boost of 2x106 cells/mice, 1x107 cells /mice of apoptotic cells or PBS respectively for the primary (first) and secondary (2nd) response, as indicated in Figure 6. For the tertiary, quaternary response, all groups of mice received an

intravenous injection of apoptotic cells or PBS. The mice were treated every 3 wks, and all mice were bled seven days after treatment with apoptotic cells or the PBS boost to

evaluate the titer of anti-DNA Abs. In this study, the mice were sacrified to examine the renal pathology four mo after their initial treatment.

ELISA for anti-DNA Ab production from normal mice

Abs specific for dsDNA and ssDNA were evaluated in serum samples by a standard ELISA assay as previously described (17, 20). Briefly, ELISA plates were coated with 10 µg/ml methylated bovine serum albumin (mBSA; Sigma). Native DNA was prepared by phenol and chloroform extraction from calf thymus DNA (Sigma). Native DNA was denatured by boiling for 20 min and incubated on ice for 20 min to generate ssDNA. DsDNA and ssDNA were coated overnight at 4oC, then washed and blocked with gelatin post-coating solution for 2 h. Serum diluted 100 times for IgG was applied to each well at 37oC for 45 min and then moved to room temperature for 15 min. After washing the plates, HRP-conjugated goat anti-mouse 　-chain specific Abs (Sigma) was added at 37oC for 45 min. ABTS solution was used as a substrate, and the OD value was evaluated at 405nm. The levels of anti-IgG are presented as ELISA units (EU/ml) compared with mAb 10F10 (17). This mAb is specific to dsDNA or ssDNA. The OD value generated by 37 ng/ml of 10F10 Ab was defined as 1 EU/ml.

Generation of peptide-specific T cell lines

The generation of peptide-specific T cell lines were prepared as described previously (17). In brief, CD4+ T cells (3x106/well) from disease-developing BWF1 mice were cultured with histone peptide (H2B81-100, H3111-130, H491-110) and U1A201-220 peptide pulsed

syngeneic BMDCs (1x105/well) in serum-free medium (AIM-5 containing TCM) for 2-3 days. On day 2, 50 U/ml rIL-2 was added to each well. On day 3, the culture medium was replaced by RPMI-1640 containing 10% FCS and IL-2 (50 U/ml). After 10-14 days of coculture, T-cell lines were positively selected by anti-Thy1.2-coated magnetic

microbeads (Miltenyi Biotec) and then restimulated with apoptotic cells or peptide pulsed BMDCs (2500 per well) for five days. During the last 4-6 h of culture, 1 µCi of

[3_{H]thymidine was added to each well.}

Renal deposition of IgG

Histological assessment was performed at 6 mo of age. Kidneys were fixed in formalin for haematoxylin and eosin staining as previously described (21). IgG deposits were studied by embedded kidneys in OCT (Sakura Finetek, Torrance, CA), and 4-µm thick cryostat sections were prepared on poly L-lysine-coated slides (Dako, Kyoto, Japan) at -20oC. Kidney sections for fluorescence microscopy were fixed in an acetone-chloroform (1:1) solution and washed with PBS three times. The slides were stained with 10 µg/ml of fluorescein goat anti-mouse IgG (H+L) (F(ab,)2) (Molecular Probes, Inc., Eugene, OR) or fluorescein-conjugated anti-mouse complement C3(F(ab,)2) (ICN Pharmaceuticals, Inc., Auroua, Ohio), and then were counterstained with Evan’s Blue, mounted with glycergel (Dako, Japan). Images were generated using fluorescence microscopy (Olympus IX 70).

Results

We have published two papers concerning this project in year 2006

Tzeng, T.-C., Suen, J.-L. and Chiang, B.-L. Dendritic cells pulsed with apoptotic body activate self-reactive T cells of lupus mice. Rheumatology 2006; 45:1230-1237.*

Hsu, W.-T., Suen, J.-L. and Chiang, B.-L. The role of CD4+ CD25+ T cells in the autoantibody production in murine lupus. Clin Exp Immunol 2006;145: 513-519.*

Amoura, Z., J.C. Piette, H. Chabre, P. Cacoub, T. Papo, B. Wechsler, J.F. Bach, and S. Koutouzov. 1997. Circulating plasma levels of nucleosomes in patients with systemic lupus erythematosus: correlation with serum antinucleosome antibody titers and absence of clear association with disease activity. Arthritis Rheum 40:2217-2225.

Anderson, H.A., C.A. Maylock, J.A. Williams, C.P. Paweletz, H. Shu, and E. Shacter. 2003. Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells. Nat Immunol 4:87-91.

Balomenos, D., J. Martin-Caballero, M.I. Garcia, I. Prieto, J.M. Flores, M. Serrano, and A.C. Martinez. 2000. The cell cycle inhibitor p21 controls T-cell proliferation and sex-linked lupus development. Nat Med 6:171-176.

Berden, J.H., S. Fournel, S. Neichel, H. Dali, S. Farci, B. Maillere, J.P. Briand, S. Muller, N. Jiang, C.F. Reich, 3rd, D.S. Pisetsky, and S.K. Datta. 2003. Lupus nephritis: consequence of disturbed removal of apoptotic cells? CD4+ T cells from (New Zealand Black x New Zealand White)F1 lupus mice and normal mice immunized against apoptotic nucleosomes recognize similar Th cell epitopes in the C terminus of histone H3 Role of macrophages in the generation of circulating blood

nucleosomes from dead and dying cells. Major peptide autoepitopes for

nucleosome-centered T and B cell interaction in human and murine lupus. Neth J Med 61:233-238.

Biggiogera, M., M.G. Bottone, T.E. Martin, T. Uchiumi, and Pellicciari. 1997. Still

immunodetectable nuclear RNPs are extruded from the cytoplasm of spontaneously apoptotic thymocytes. Exp Cell Res 234:512-520.

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