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研究生：	蔡進焜 Tsai, Chin-Kun
論文名稱：	探討肝醣合成酶激酶3參與在TPA/Ionomycin活化人類CD4 T淋巴球中調控細胞激素的生成 Investigating the role of glycogen synthase kinase-3 for regulating cytokine production in TPA/ionomycin-activated human CD4+ T lymphocytes
指導教授：	林秋烽 Lin, Chiou-Feng
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	62
中文關鍵詞：	12-O-tetradecanoylphorbol-13-acetate/Ionomycin 、肝醣合成酶激酶3 、細胞激素、鈣調磷酸酶2B 、CD4+ T淋巴球
外文關鍵詞：	12-O-tetradecanoylphorbol-13-acetate/ionomycin, Glycogen synthase kinase-3, Cytokine, Calcineurin, CD4+ T lymphocyte
相關次數：	點閱：115 下載：1
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CD4+ T淋巴球對免疫系統監控病原體、腫瘤及同種異體移植組織扮演著重要的防禦角色。CD4+ T淋巴球藉由製造細胞激素以傳遞訊息調控免疫系統，而細胞內肝醣合成酶激酶3 (GSK-3) 被認為可以調控胞內轉錄因子進而參與細胞激素的製造。本研究以12-O-tetradecanoylphorbol-13-acetate (TPA)/ionomycin (T/I) 模式係利用TPA活化蛋白質激酶C (PKC) 及ionomycin促使細胞內鈣離子的釋放共同造成CD4+ T淋巴球的活化作用產生細胞激素。我們發現在T/I的處理下可促使人類T細胞Jurkat T分泌丙型干擾素 (IFN-γ)、甲型腫瘤壞死因子 (TNF-α) 及介白素2 (IL-2)，但這些現象在另一株人類T細胞MOLT-4卻無法證實。利用流式細胞儀分析免疫染色發現MOLT-4細胞表面具有CD4+及CD8+ (double positive) 蛋白質標記，而Jurkat T細胞則呈現CD4lowCD8-。以T/I誘導小鼠胸腺細胞的活化實驗中發現，只要帶有CD4的胸腺細胞都會產生丙型干擾素。為求實驗的精準性，遂以純化人類周邊血液中的T淋巴球作為標的細胞。結果顯示T/I處理可顯著地誘導人類CD4+ T淋巴球產生丙型干擾素、甲型腫瘤壞死因子及介白素2，進一步使用蛋白質激素C抑制劑和鈣離子螯合劑均可抑制T/I誘導細胞激素的產生。值得注意的是當以肝醣合成酶激酶3抑制劑處理細胞則會減少T/I誘導丙型干擾素、甲型腫瘤壞死因子及介白素2的產生。這些結果顯示肝醣合成酶激酶3可能參與在T/I活化人類CD4+ T淋巴球產生細胞激素的訊息傳遞。此外，以proline-rich tyrosine kinase 2 (Pyk2) 抑制劑和鈣調磷酸酶2B (PP2B) 抑制劑同樣地可干擾T/I誘導人類CD4+ T淋巴球細胞激素的產生並影響肝醣合成酶激酶3的活化，以上結果顯示在T/I誘導人類CD4+ T淋巴球細胞中Pyk2和鈣調磷酸酶2B參與正向調控肝醣合成酶激酶3的角色。更進一步的實驗發現在T/I刺激下肝醣合成酶激酶3可以調控轉錄因子T-bet的活化進而調控這些細胞激素。最後，在小鼠動物實驗中發現處理肝醣合成酶激酶3抑制劑可以顯著的減緩T/I誘發小鼠死亡率及周邊血液中細胞激素的表現。根據結果我們可以推測肝醣合成酶激酶3可做為治療性標靶蛋白質以抑制CD4+ T細胞活化生成細胞激素。

CD4+ T lymphocytes are important for immunity to monitor pathogens, tumors, and allografts. Cytokine production is the major regulators released from CD4+ T lymphocytes. While glycogen synthase kinase (GSK)-3, a serine/threonine kinase, has been speculated for facilitating cytokine production probably by controlling several transcription factors, this study aimed to investigate the involvement of GSK-3 for cytokine production in CD4+ T lymphocytes. In an experimental model of CD4+ T lymphocyte activation, a combination of 12-O-tetradecanoylphorbol-13-acetate (TPA), used as an activator of protein kinase C (PKC), and ionomycin, used for calcium influx, so called T/I model, was utilized for this work. Here we showed that T/I treatment induced the production of cytokines IFN-γ, TNF-α, and IL-2 only in human Jurkat T cells but not in another human MOLT-4 cells. Immunostaining followed by flow cytometry analysis showed that MOLT-4 cells were mostly CD4+CD8+ double positive, whereas Jurkat T cells were CD4lowCD8-. However, T/I treatment was able to activate all mouse CD4-bearing thymocytes to produce IFN-γ ex vivo. For this study, purified human T lymphocytes from peripheral blood were specifically utilized. T/I treatment significantly increased the production of IFN-γ, TNF-α, and IL-2 in human CD4+ T lymphocytes. Treating cells with PKC inhibitor bisindolymaleimide or calcium chelator BAPTA blocked T/I-induced cytokine production. It is notable that treatment of GSK-3 inhibitor BIO and short hairpin RNA against GSK-3β decreased T/I-induced IFN-γ, TNF-α, and IL-2. These results demonstrate the common upstream role of GSK-3β for IFN-γ, TNF-α, and IL-2 production in T/I-activated human CD4+ T lymphocytes. Moreover, proline-rich tyrosine kinase 2 (Pyk2) inhibitor Tyrphostin A9 and calcineurin (PP2B) inhibitor cyclosporine A treatment also blocked cytokine production as well as GSK-3 activation in T/I-activated human CD4+ T lymphocytes. It is speculated that Pyk2 and PP2B positively regulate GSK-3β activation in T/I-stimulated human CD4+ T lymphocytes. Activated GSK-3β regulated transcription factor T-bet, which was specifically and individually regulated for these cytokines under T/I stimulation. In a mice model, BIO treatment significantly inhibited T/I-induced mortality and the serum levels of cytokines. According to these results obtained from this work, targeting GSK-3 confers the therapeutic efficacy against CD4+ T cell activation and cytokine production.

Abstract in Chinese	I
Abstract in English	II
Acknowledgement	III
Abbreviations	IV
Contents	VI
I-1. T lymphocyte differentiation	1
I-2. T lymphocyte function	1
I-3. T lymphocyte activation	2
I-4. 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ionomycin	3
I-5. Glycogen synthase kinase-3β	3
I-6. Cytokines	5
I-6-1. IFN-γ	5
I-6-2. Tumor necrosis factor (TNF)-α	5
I-6-3. IL-2	5
I-7. T-bet	6
II. Study Objective and Specific Aims	7
II-1. Objective	7
II-2. Specific Aims	7
Specific Aim 1：To establish an in vitro model of T/I-induced human T cells cytokine production.	7
Specific Aim 2：To clarify the role of GSK-3β invoved in T/I-induced cytokine production.	7
Specific Aim 3：To establish an in vivo model of T/I-induced cytokine production and mortality in mice and to test the protective effect of GSK-3 inhibition.	7
III. Materials and Methods	8
III-1. Mice	8
III-2. Cell Cultures	8
III-3. Reagents and Antibodies	8
III-4. Primary Cells Isolation	9
III-5. Separation of mouse thymocytes	10
III-6. Animal treatment	11
III-7. Cytotoxicity Assay	11
III-8. Western Blotting	11
III-9. Immunostainning and Cell Imaging	12
III-10. Enzyme-Linked Immunosorbent Assay (ELISA)	13
III-11. Calcineurin cellular activity assay	13
III-12. RNA Interference	13
III-13. RNA interference	14
III-14. Statistical Analysis	15
IV. Results	16
IV-1. T/I treatment induces IFN-γ production in different T lymphocyte lineages.	16
IV-2. Jurkat T cells secrete cytokine after T/I stimulation.	16
IV-3. Jurkat T cells express CD4 molecules, but MOLT-4 cells are CD4+CD8+ double positive (DP) cells.	17
IV-4. Mouse CD4-bearing thymocytes produce IFN-γ after T/I treatment.	17
IV-5. Primary CD4 T lymphocytes secrete IFN-γ, TNF-α, and IL-2 after T/I stimulation.	17
IV-6. T/I treatment induces cytokine production through PKC- and calcium-regulated pathways.	18
IV-7. GSK-3β regulates T/I-activated cytokine production.	18
IV-8. T/I treatment induces GSK-3β-mediated cytokine production through Pyk2-regulated pathway.	19
IV-9. PP2B regulates GSK-3β activation and cytokine production after T/I stimulation.	19
IV-10. T/I treatment induces GSK-3β-regulated T-bet nuclear translocation followed by T-bet-mediated cytokine production.	20
IV-11. GSK-3 inhibitor BIO treatment inhibits the T/I-induced mortality and cytokine production in mice.	20
V. Discussion	22
VI. Conclusion and Implication	26
References	27
Figures and Figure Legends	35
Figure 1. T/I treatment induces IFN-γ production greatly in CD4+ T lymphocytes.	35
Figure 2. T/I treatment induces production of IFN-γ, TNF-α, and IL-2 in Jurkat T cells, but not MOLT-4.	36
Figure 3. Jurkat T cells are CD4+-bearing cells.	37
Figure 4. IFN-γ production in T/I-treated murine thymocytes.	38
Figure 5. T/I treatment induces production of IFN-γ, TNF-α, and IL-2 in primary CD4+ T lymphocytes.	39
Figure 6. Either PKC inhibitor bisindolylmaleimide or calcium chelator BAPTA blocks T/I-induced cytokine production.	40
Figure 7. T/I activates GSK-3β and induces GSK-3-regulated cytokine production.	41
Figure 8. T/I induces Pyk-2-regulated GSK-3β activation and cytokine production.	43
Figure 9. PP2B determinesT/I induced and cytokine production.	44
Figure 10. T/I treatment causes T-bet nuclear translocation and T-bet-mediated cytokine production.	45
Figure 11. BIO increases survival and blocks cytokine production in T/I-stimulated mice.	46
Figure 12. A model of GSK-3β-regulated cytokine production in T/I-activated human CD4+ T lymphocytes.	47
Appendix	48
A. Figures	48
B. Materials	51
B-1 Chemicals	51
B-2 Antibodies	52
B-3 Kits	53
B-4 Consumables	53
B-5 Apparatus	54
C. Methods	55
C-1 Cell culture	55
C-1.1 Cell culture medium	55
C-1.2 Cell passage	55
C-1.4 Cell defreeze	56
C-2 Western blot	57
C-2.1 Lysis buffer	57
C-2.2 5× loading dye and TBS-T	57
C-2.3 Running gel preparation	57
C-2.4 Stacking gel preparation	58
C-2.5 Cell lysate preparation	58
C-2.6 SDS-PAGE	58
C-3 Lentiviral-based shRNA knockdown	59
C-3.1 Plasmid preparation	59
C-3.2 Lentiviral production	59
.C-3.3 Lentiviral concentration	60
C-3.4 Lentiviral infection	60
C-3.5 Plasmid transfection	61
CURRICULUM VITAE	62
                                    

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