- 學位論文
金針菇免疫調節蛋白FVE活化小鼠T淋巴細胞機制之研究
Study on the Mechanisms of FVE-mediated Murine T Cell Activation
摘要
文獻指出金針菇免疫調節蛋白 FVE 具有刺激免疫細胞增生，抑制系統性過敏反應以及增加細胞激素 IL-2 與 IFN-γ 表現之功效，同時 FVE 可能具有裂質素 (mitogen) 的特質，能夠誘導人類週邊血細胞進行有絲分裂，且 FVE 活化小鼠T細胞之增生作用，需要抗原呈獻細胞 (antigen presenting cell, APC) 協助。但是裂質素活化淋巴胞分裂的能力超過 FVE，而 FVE 刺激淋巴細胞增生的能力又比一般抗原的活性高，這些結果排除 FVE 為裂質素的可能性，同時我們懷疑FVE 可能透過類似超抗原 (superantigen) 之方式活化 T 細胞，因此本研究之目的在於探討 FVE 活化小鼠 T 細胞之機制，並證明 FVE 是一種超抗原。 在證明 FVE 活化小鼠 T 細胞不需要 processing 的部分，實驗發現以經三聚甲醛 (paraformaldehyde) 固定之脾臟細胞作為抗原呈獻細胞，與 T細胞及 FVE 共同培養後，可引起 T 細胞之增生，然而純 T 細胞之對照組則不會被活化增生，此結果除了說明 FVE 活化細胞增生必須藉由抗原呈獻細胞與 T 細胞間相互作用才能達成，同時 FVE 呈現之機制與一般抗原不同，不需要被細胞進行 processing，即可以作為抗原呈現細胞與 T 細胞連接橋樑活化 T 細胞。 其次，大部分超抗原之特性是會與抗原呈獻細胞上之MHC分子鍵結後，再呈現給具有特定變異β鏈 (Vβ) 的 T 細胞受器，本研究於是利用免疫螢光染色法 (FACS) 與即時定量聚合酶反應法 (Real-time PCR) 進行分析，發現以 FVE 處理不會使特定TCR Vβ於 T 細胞中所佔之比例改變，但是以 real-time PCR 檢測基因層級之結果顯示，以 FVE 刺激後能提升表現 TCR Vα 19 之T細胞所佔之比例，以 FVE 活化後之比例較未活化組高 6 倍。因此我們推測 FVE 可藉由連接 TCR Vα 19 活化 T 細胞，進而刺激具有TCR Vα 19的T細胞增生。 此外，實驗亦發現 MHC class I與MHC class II 中和性之抗體 (anti-H-2Kd以及 anti-IA/IE)，具有抑制由 FVE 及抗原呈獻細胞引起之 T 細胞增生效應，且具濃度趨勢，同時抗體也會抑制 FVE 促進抗原呈獻細胞表現細胞激素 IL-12 p35、p40 與 IL-18，因此這些結果除了顯示 FVE 能夠活化 CD4＋ 與 CD8＋ 兩種 T 細胞增生外，其活化機制可能是藉由抗原呈獻細胞之MHC class II 與 MHC class I 分子，而 FVE 活化抗原呈獻細胞產生 IL-12 與 IL-18 可能也與MHC 分子相關。 綜合上述結果，本研究清楚證明 FVE 活化 T 細胞之機制類似超抗原，須藉由抗原呈獻細胞的 MHC 分子協助，並經由與 TCR Vα 19 連結而活化 T 細胞；另一方面，此結果亦闡明 FVE調節免疫調節之機制，支持未來 FVE於免疫方面醫藥用途之應用。
並列摘要
FVE, a fungal immunomodulatory protein purified from Flammulina velutipes, is capable to stimulate lymphocyte proliferation, to suppress systemic anaphylaxis responses, and to increase IL-2 and IFN-γ production. It is known that FVE may exhibit potent mitogenic effects on human peripheral blood lymphocytes and murine splenic T cells only in the presence of antigen presenting cells (APCs). However, cell proliferation stimulated by mitogens is greater than FVE, and the activity of FVE is much better than conventional antigens. These information exclude the possibility that FVE is a mitogen and suggest FVE might activate T cells throuth a pathway similar to what superantigen does. The objective of this study is to study the mechanisms of FVE-mediated murine T cell activation and to argue FVE a superantigen. To demonstrate that APCs are necessary in FVE-mediated T cell activation, result shows that FVE incubated paraformaldehyde-fixed murine splenocytes are able to induce cell proliferation of MACS-purified murine T cells, and this effect is not found in the T cell alone control cells This evidence suggests that FVE-mediated T cell activation performs only in a synergistic incubation of both APCs and FVE is directly presented by the receptor on APCs without processing procedure that common antigens need. On the other hand, most superantigens are a class of antigens presented by MHC molecules on APCs and they recognized the variable region of the β-chain (Vβ) of the T cell receptors (TCR). In this study, fluorescent-activated cell sorting (FACS) and real-time PCR methods are carried out to determine the expression of variable regions within TCR. Result shows that no T cell bearing specific Vβ extension and a remarked Vα enhancement, especially Vα 19 mRNA (above 6 folds of the enhancement), are observed, suggesting that FVE could bind to Vα 19 and further enhance its expression. Furthermore, the cross-linking between FVE and MHC molecules is also investigated. And the result shows that both anti-MHC class I and class II neutralizing antibodies (anti-H-2Kd and anti-IA/IE, respectively) are capable to inhibit FVE-stimulated cell proliferation in a dose-dependent manner. The addition of both the antibodies also reduces IL-12 p35, IL-12 p40, and IL-18 gene expression stimulated by FVE. These results indicate that FVE can not only activate CD4+ T cell but also CD8+ T cell proliferation, and MHC class II and MHC class I on APCs are probably responsible for presenting FVE to further activate T cells. The pathway that FVE activates cytokine gene expansion within APCs might also through MHC class I and class II molecules. Taken together, this study clearly demonstrates that FVE, which activates T cells through the binding of MHC molecules in APCs and Vα 19 TCR region in T cells, is a superantigen-like immunomodulatory protein. This finding clarifies the molecular mechanism of FVE-mediated T cell activation and supports the further utilization of FVE in immune-related pharmaceutical and medicinal applications.