臺灣博碩士論文加值系統

造血細胞的數量規模在生物體的生命過程中是非常穩定的，即使在生物體產生免疫反應後其數量也必須被嚴密的調控，這樣的能力反應著生物體內精確的細胞增生、細胞自裁、細胞分化與細胞募集的能力。細胞內部有許多已知的分子參與上述的精密調控，但是其各分子的功能角色仍待釐清。顆粒性血球細胞/巨噬細胞聚落刺激因子/第三介白素/第五介白素受體共同乙型鏈結合蛋白（CBAP）是一個新穎的膜蛋白，已被證實可與沒有受體結合的顆粒性血球細胞/巨噬細胞聚落刺激因子/第三介白素/第五介白素共同乙型鏈結合而驅動細胞自裁死亡反應。在本研究中，我們發現CBAP基因剔除小鼠的造血細胞恆定性受到改變，例如T細胞、B細胞、單核白血球細胞、嗜鹼性細胞的數量改變。在缺乏細胞介白素的培養下，CBAP基因剔除的T細胞的死亡會比正常T細胞慢一點，這表示CBAP具有促進細胞死亡的功能。再者，當同時剔除CBAP與Bim基因時，則會影響部分造血細胞的恆定而在數量上有輕微的加成作用，這顯示CBAP與Bim在調控造血細胞死亡的恆定上是平行的角色。除了參與細胞死亡調控，剔除CBAP基因的T細胞回巢到淋巴結的能力明顯下降，這與此細胞對於細胞趨化激素CXCL12與CCL21依賴性的細胞移動與粘附的能力變弱有一致性的關係。在CBAP基因默化的人類Jurkat T細胞株中，此細胞的CXCL12-依賴性細胞移動與integrin α4β1- 或 αLβ2-依賴性粘附能力也都下降，而這些缺失的功能可在大量表現小鼠CBAP蛋白後重新回復其能力。在進一步研究中發現，CBAP可以持續的與integrin β1和ZAP70結合在一起，這個重要的結合有助於促進在細胞趨化素刺激引起的起始結合蛋白群talin-Vav1與integrin β1的結合，並且有利於後續的ZAP70依賴性talin-Vav1蛋白群的分離及Vav1的磷酸化。在這樣的integrin 訊息蛋白群當中，CBAP蛋白的作用像是一個適配器來引導並增進integrin 與Rac1的活化。最後，我們的研究發現缺乏CBAP的小鼠其呈現較弱的接觸性超敏反應，剔除CBAP基因的T細胞導致接受者小鼠較趨緩的移植物抗宿主病，以及發現在CBAP基因默化的T細胞株會緩和其導致的小鼠血癌模式之病理發展。這些證據都足以進一步強化CBAP對於調控免疫反應與一些T細胞相關的疾病中所扮演的重要角色。

The size of hematopoietic cell pool is remarkably stable throughout life and tightly regulated after immune responses, reflecting precise control of cellular proliferation, apoptosis, differentiation and recruitment. A variety of proteins are known to be involved in these processes, but the understanding about the roles of these components is still not well characterized. GM-CSF/IL-3/IL-5 receptor common beta-chain-associated protein (CBAP) is a newly annotated transmembrane protein, which associates with un-linganded common beta chain (βc) of GM-CSF/IL-3/IL-5 receptor to trigger apoptosis. In our current study, we found that some of hematopoietic populations in CBAP-deficient mice display minor abnormalities, such as monocytes, basophiles, T and B lymphocytes. The loss of CBAP delayed apoptosis of T lymphocytes induced by stress of death-by-neglect, suggesting a proapoptotic role of CBAP in T cells. Moreover, combined loss of proapoptotic genes Bim and CBAP caused a modestly summated effect in some of hematopoietic cell populations, suggesting a parallel cooperation of CBAP and Bim in controlling hematopoietic cell homeostasis. Besides regulation of cell death, CBAP-deficient T lymphocytes manifested defective homing to lymph nodes, which correlated with attenuated chemotaxis toward CC chemokine ligand-21 (CCL21) and CXC chemokine ligand-12 (CXCL12), and these chemokines-induced T-cell adhesions. Knockdown of CBAP expression in human Jurkat T cells resulted in attenuated CXCL12-induced cell migration and adhesion to integrin α4β1- or αLβ2-specific ligand coated surface, which could be efficiently rescued by expression of murine CBAP proteins. Further analysis revealed that CBAP constitutively associated with both integrin β1 and ZAP70 and that CBAP is required for chemokine-induced initial binding of the talin-Vav1 complex to integrin β1 and to facilitate subsequent ZAP70-mediated dissociation of the talin-Vav1 complex and Vav1 phosphorylation. Within such an integrin signaling complex, CBAP likely functions as an adaptor and ultimately leads to activation of both integrin α4β1 and Rac1. Last, we demonstrated that loss of CBAP manifested diminished ear swelling and T-cell infiltration in a contact hypersensitivity model and blunt pathological progression of graft-versus-host disease (GVHD) and leukemogenesis, further strengthening the important role of CBAP in modulating immune responses and development of T-cell-mediated diseases.

Abstract (Chinese)……………..………………………….……………………………… i
Abstract (English)..…………………………………………..……………………………ii
Table of contents……………………………………………..………………………….. iv
List of figures…………………………….…………………..…………………………...vi
Chapter I: General introduction …………………..…………………….………………... 1
Introduction ………………………………….………………………..…………..... 2
Figures …...………………………………….……………………….……….…......13
Chapter II: Materials and methods ……………………………………………………….15
Chapter III: Investigating on the pro-apoptotic function of CBAP in hematopoietic cell homeostasis …………………………………………………………………….………. 24
Introduction ………………………………….………………………..…………... 25
Results …...………………………………….……………………….……….…... 26
Discussion ………………………………….…………………………..……….... 31
Figures ……………………………………………………………………………. 33
Chapter IV: CBAP functions in controlling chemokine-induced ZAP70-mediated T cell adhesion and migration ……………………………………………………………...… 45
Introduction ………………………………………………..……………….……... 46
Results …...……………………………………………………………...………... 47
Discussion ……………………………………….……………….……….…….... 55
Figures …………………………………………………….……........................... 58
Chapter V: Revealing the physiological and pathological functions of CBAP by using animal models…………………………………………………………………..……… 71
Introduction ……………………………………………….……….……………... 72
Results …...……………………………………………..……………………….... 74
Discussion ………………………………………………………..…………….... 77
Figures ……………………………………………………………………........... 79
Chapter VI: References………………………………..…………………..…………… 83
Chapter VII: Appendix……………………………………..……………………………99


List of figures
Figure 1.1 Amino acid sequence alignment of CBAP from different mammals. ……….. 13
Figure 1.2 The output of human CBAP transmembrane regions from TOPCONS webserver. ……………………………………………………………………………………………..14

Figure 3.1 Analysis of complete blood counts in CBAP–/– mice. ………………………..33
Figure 3.2 Phenotypic analysis of lymphoid organs from CBAP+/+ and CBAP-/- mice....34
Figure 3.3 Representative FACS analysis of hematopoietic cells from CBAP knockout mice......................................................................................................................................35
Figure 3.4 Quantitative analysis of hematopoietic populations in CBAP-/- mice as determined by flow cytometry………………………………………………………….…36
Figure 3.5 T lymphocyte subpopulations in lymph node of CBAP- deficient mouse…….38
Figure 3.6 In vivo BrdU incorporation in thymocytes and bone marrow cells……...…….39
Figure 3.7 Comparable TCR-induced T cell proliferation between CBAP+/+ and CBAP-/- T cells……………………………………………………………………………………...40
Figure 3.8 in vitro death-by-neglect assay and IL-7 whthdral-induced cell death……...…41
Figure 3.9 In vivo survival assay of CBAP-deficient T cells……………………………...42
Figure 3.10 Phenotype analysis of Bim/CBAP double-knockout mice …………………...43

Figure 4.1 CBAP is required for CXCL12-induced migration and adhesion and activation of integrins in Jurkat T cells……………………………………………………………….58
Figure 4.2 CBAP–/– primary T cells display reduced chemokine-dependent cell migration and adhesion in vitro………………………………………………………………………60
Figure 4.3 In vivo T-cell homing to LNs and in vitro S1p-induced chemotaxis……….….62
Figure 4.4 CBAP is important for chemokine-dependent activation of Vav1 and Rac1 in T cells………………………………………………………………………………………..63
Figure 4.5 CBAP pre-associates with integrin β1 and ZAP70 and facilitates chemokine-induced ZAP70-mediated dissociation of the Vav1-talin complex……………………….65
Figure 4.6 Complex formation among CBAP, ZAP70 and integrin β1…………………..66
Figure 4.7 Mapping of interaction domains on CBAP with Vav1 and ZAP70…………...67
Figure 4.8 CBAP–/– activated T cells display reduced chemotaxis toward each of several chemokines………………………………………………………………………………..68
Figure 4.9 CBAP is a modulator for formation of FLNa/Rac1 complex………………....69
Figure 4.10 Formation of CBAP homodimer……………………………………………..70

Figure 5.1 Reduced inflammatory response of CBAP-deficient mice in a contact hypersensitivity model…………………………………………………………………….79
Figure 5.2 Survival and GVHD scores of animals receiving T cell-depleted (TCD) BM cells, CBAP+/+ or CBAP-/- T cells……………………………………………………….80
Figure 5.3 CBAP is important for the development of leukemogenesis…………………..81
Figure 5.4 Methylcellulose colony-forming assay of leukemia cell lines…………………82

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