李建國臺灣大學：免疫學研究所黃雅婷Huang, Ya-TingYa-TingHuang2007-11-292018-07-092007-11-292018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/63318STAT1是第一型與第二型干擾素訊息傳遞中的重要訊息調控分子，然而STAT1在體液免疫中扮演何種角色仍然未知。在此我們證明STAT1是調控體液免疫反應的重要分子。我們經實驗觀察發現STAT1基因剔除小鼠在受到T-dependent抗原TNP-OVA的刺激後其體液免疫反應出現嚴重的缺陷，這些包括TNP專一性抗體效價降低、同型抗體轉換(isotype switching)反應不全、濾泡生發中心( germinal center)數量減少，而且負責濾泡生發中心形成的BCL6基因表現量降低。此外刺激後的STAT1基因剔除小鼠的脾臟與骨髓中之TNP專一性漿細胞數目大量減少，有趣的是sXBP1而非Blimp1基因表現量有明顯降低。 為了更進一步探討STAT1調控體液免疫的可能機制，我們使用了T-dependent抗原進行的體外刺激。STAT1基因剔除小鼠的脾臟細胞和B淋巴球在受到抗CD40單株抗體與IL-4的刺激後，出現了細胞分裂能力降低與漿細胞分化不全之現象。經流式細胞儀分析結果顯示經刺激後表現CD138+與IgG+細胞數目有明顯的減少，而這些細胞中的Blimp1、sXBP1、AID等基因之表現量均有降低情形。綜合以上結果，我們發現在STAT1基因剔除老鼠體內與體外培養系統中均出現體液免疫反應缺陷，這些現象都顯示STAT1在B淋巴球分化過程中扮演重要的角色。Signal transducer and activator of transcription (STAT) 1 is a critical signal mediator for type I and type II interferon. However, the role of STAT1 in humoral immunity remains unclear. Here, we demonstrate that STAT1 was required for humoral immune response. Severely impaired humoral immunity, including reduced titer of TNP-specific antibodies, impaired isotype switching, and decreased numbers of germinal center in the spleen were observed in STAT1KO mice in response to TNP-OVA, a T-dependent antigen. Interestingly, STAT1KO mice displayed reduced level of BCL6, which is important for germinal center formation. In addition, the TNP-specific plasma cells in the spleen and bone marrow of STAT1KO mice were significantly reduced after immunization. Coincidentally, we found that the level of sXBP-1, but not Blimp1 was reduced. To further elucidate the underlying mechanisms, we did in vitro stimulation using T-dependent antigen. Splenocytes or purified B cells of STAT1KO mice treated with anti-CD40 plus IL-4 in vitro also showed reduced proliferation activity and plasma cell formation. Flow cytometric analysis showed that CD138 + cells and IgG1 + cells after stimulation were dramatically reduced in STAT1KO cells, which was consistent with reduced level of Blimp1, sXBP-1 and AID in these cells. Taken together, the results from STAT1KO mice revealed defective humoral responses in vivo and in vitro, indicating that STAT1 plays an important role in B cell differentiation.致謝 i Abbreviations ii 摘要 iv Abstract v Table of Contents vii Chapter I Introduction 1 1.1 Antigen-dependent B cell differentiation 1 1.2 Transcriptional control of plasma cell formation 2 1.3 STAT1 4 1.4 Rationale 6 1.5 Specific aims 6 Chapter II Materials and Methods 8 2.1 Mice 8 2.2 Immunization 8 2.3 Cell culture 9 2.4 Purification of B cells 9 2.5 Flow cytometry 9 2.6 BrdU incorporation assay 10 2.7 Quantitative Real-Time PCR 11 2.8 ELISA 12 2.9 Cryosection and Immunohistochemistry 13 Chapter III Results 14 3.1 Impaired TNP-specific IgG production and isotype switching in STAT1KO mice in response to T-dependent antigen in vivo 14 3.2 Impaired germinal center formation in STAT1KO mice in response to T-dependent antigen 15 3.4 Decreased expression of BCL6 and sXBP1 in spleen of STAT1KO mice in response to T-dependent antigen in vivo 16 3.5 Reduced TNP+ CD138+ cells in spleen and bone marrow in T-dependent antigen immunized STAT1KO mice 18 3.6 Reduced proliferation and proliferation of splenocytes and purified B cells of STAT1KO in response to TD stimulation in vitro 19 3.7 Decreased expression of AID, Blimp1, sXBP1 expression in splenocytes and purified B cells of STAT1KO in response to TD stimulation in vitro 20 Chapter IV Discussion 22 4.1 Impaired Ig secretion, germinal center formation, istype switching, and plasma cell differentiation in response to TD antigen in STAT1KO mice in vivo 22 4.2 Impaired in vitro proliferation and plasma cell differentiation in response to TD antigen in STAT1KO mice 25 Figure 28 Fig. 1 Slightly decreased percentage of mature B cell in peripheral blood in STAT1 KO mice 29 Fig. 2 Comparable basal level of IgM and IgG in WT and STAT1KO mice 30 Fig. 3 Impaired T-dependent response in STAT1KO mice 31 Fig. 4 Reduced production of TNP-specific IgG subclass from STAT1KO mice in response to T-dependent antigen 32 Fig. 5 Reduced germinal centers number in spleen of STAT1KO mice in response to TD stimulation in vivo 33 Fig. 6 Reduced high affinity TNP-specific IgG level in WT and STAT1KO mice 34 Fig. 7 A-D Decreased gene expression of BCL6 in STAT1KO mice 35 Fig.7 E-G Decreased gene expression of sXBP1 in STAT1KO mice 36 Fig. 8 Decreased percentage of TNP+ CD138+ cell in the spleen of STAT1KO mice 37 Fig. 9 Decreased percentage of TNP+ CD138+ cell in the spleen and bone marrow in STAT1KO mice after immunization for 21 days 38 Fig. 10 Reduced titer of TNP-specific IgG and decreased percentage of TNP+ CD138+ cell in the bone marrow of STAT1KO mice after secondary immunization 39 Fig. 11A-B Decreased cellularity of STAT1KO cells in response to T-dependent antigen stimulation in vitro 40 Fig. 11C-D Decreased proliferation of purified STAT1KO B cells in response to T-dependent antigen stimulation in vitro 41 Fig. 12 Decreased CD138+ cell in STAT1KO mice in response to T-dependent antigen stimulation in vitro 42 Fig. 13 Decreased IgG1 producing cells in STAT1KO mice in response to T-dependent antigen stimulation in vitro 43 Fig. 14 Decrease expression of genes that are critical for plasma cell formation in splenocytes of STAT1KO mice in response to T-dependent antigen stimulation in vitro 44 Fig. 15 Decreased expression of genes that are critical for plasma cell formation in purified B cells of STAT1KO mice in response to T-dependent antigen stimulation in vitro 45 Reference 46977004 bytesapplication/pdfen-USB 淋巴球分化STAT1B cell differentiationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63318/1/ntu-96-R94449007-1.pdf