摘要
L121-adjuvant (L121-adj.)為一含有Pluronic L121 block copolymer、Tween 80與squalane等成分之疫苗佐劑。本論文研究目的為探討此L121-adj.所引發之免疫反應，包含毒殺T細胞(cytotoxicity T lymphocyte, CTL)反應、對抗腫瘤增生，與其所造成之發炎反應對於顆粒性白血球、B細胞、T細胞、血液幹細胞、前驅細胞[hematopoietic stem/progenitor cell (HSPC)]等免疫細胞分化的影響。 在第一部分的研究中，我們證實皮下注射L121-adj.與抗原能夠有效產生抗原專一性CTL反應，與可以做為治療性疫苗，抑制B16F10黑色素細胞腫瘤的增生。為了瞭解L121-adj.產生CTL的機制，我們以流式細胞儀分析了不同組織中的抗原呈現細胞，發現注射L121-adj.會導致局部注射皮下位置產生發炎反應，吸引顆粒性白血球與樹突細胞至注射部位，並且促進顆粒性白血球與樹突細胞吞噬攜帶抗原至淋巴結中，同時導致骨髓中的顆粒性白血球釋放減少，增加脾臟中的顆粒性白血球。除此之外，我們發現注射位置中的樹突細胞能夠透過MHC-I呈現抗原，使CD8+ T細胞活化。為了瞭解顆粒性白血球是否能夠活化CD8+ T細胞，我們以抗體去除體內顆粒性白血球後，然而卻不會降低L121-adj.所產生的CTL反應。以上實驗結果證明注射部位中的抗原呈現細胞是活化CTL之重要機制。 第二部分的研究裡，為了分析L121-adj.對於T細胞與B細胞發育分化的影響，我們以流式細胞儀分析骨髓與脾臟中的B細胞，與胸腺中的T細胞的CD marker表現型。我們發現注射L121-adj.之後的發炎反應會驅使骨髓中的前驅B細胞減少，並轉移至脾臟中分化為transitional B細胞，以產生更多的marginal zone B細胞以及follicular B細胞，進一步使脾臟中產生germinal center B細胞與plasma B細胞。L121-adj.亦加速胸腺中T細胞之發育成熟，使更高比例的前驅T細胞通過positive selection，成熟為CD4+ T細胞。 第三部分的研究中，為了分析L121-adj.對於HSPCs分化的影響，我們以流式細胞儀分析骨髓中的HSPCs，我們發現注射L121-adj.後會改變血液系統的恆定狀態(homeostasis)，造成血液幹細胞分化為更多顆粒性白血球與巨噬細胞，並且減少common lymphoid progenitors (CLPs)之分化。表示L121-adj.所造成之發炎反應能夠調控血液幹細胞的分化。 綜合以上結果，證明注射L121-adj.會造成多層次的影響，包含增加樹突細胞的抗原呈現能力，並且活化毒殺T細胞，抑制腫瘤增生。與促進T細胞、B細胞的分化成熟。另一方面，透過血液調控骨髓中血液幹細胞分化，以產生更多的顆粒性白血球，補償顆粒性白血球的消耗。
並列摘要
The objective of this study was to examine the immunological effect induced by the L121-adjvant (L121-adj.), an emulsion vaccine adjuvant consisting of Pluronic L121, Tween 80, and squalane, including the cytotoxicity T lymphocyte (CTL) response and the differentiation of B cells, T cells, and the hematopoietic stem/progenitor cells (HSPCs). Vaccination of B16F10 melanoma-bearing mice with L121-adj. containing ovalbumin (OVA) induced an antigen-specific CTL response, resulted in an inhibition of tumor growth with an increased the survival rate. Flow cytometric analysis illustrated both dendritic cells and granulocytes were recruited to the injection sites after vaccination, and antigens were transported to draining lymph nodes by the antigen presenting cells (APCs). Accelerated production of granulocytes was observed in the bone marrow, in response to inflammation induced by vaccination, followed by moving into the spleen. Dendritic cells were able to effectively cross-present antigen in vivo via MHC-I molecule and activate the CD8+ T cells. Depletion of granulocytes prior to immunization resulted in an enhanced CTL response. Injection of L121-adj. promoted the translocation of B cell precursors from bone marrow to the spleen, resulted in the differentiation of transitional B cells into marginal zone B cells and follicular B cells, followed by the formation of germinal center and plasma B cells. Positive selection of developing T cells in the thymus was accelerated by the treatment of L121-adj., resulted in a significant production of mature CD4+ T cells. Modification of hematopoietic homeostasis was also noticed after vaccination of animals with L121-adj. Flow cytometric analysis showed marked increase of Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem cells (HSCs) and consequently the granulocyte-macrophage progenitors (GMPs), presumably at an expense of the common myeloid progenitors (CMPs). Generation of F4/80+ and Ly6G+ cells from LSK, CMPs, and GMPs was profoundly increased due to the presence of granulocyte colony-stimulating factor (G-CSF) in the serum of immunized mice. Taken together, injection of mice with L121-adj. exhibited several immunological effects, including the recruitment of dendric cells for the activation of CTL, promoting the differentiation and maturation of progenitors of B and T progenitors into functional lymphocytes. L121-adj. also triggers the differentiation and proliferation of hematopoietic stem/progenitor cells (HSPCs), giving rise to the generation of granulocytes in the bone marrow of immunized animals.