自然殺手細胞活化致死之研究

1. 自然殺手細胞活化致死之研究 • 自然殺手細胞是人類先天免疫系統中重要的作用細胞﹐並可調控後天免疫反應。目前已知, 在一免疫反應中, 大多數被活化淋巴細胞最終都會死亡。尤其經由CD3/TCR複合物的啟動, 再刺激已被活化的T細胞, 其後會造成此細胞進行細胞凋零, 此一過程被稱為活化致死( Activation-inducedcell death: AICD)。 因此, 本論文主要在探討自然殺手細胞活化致死的現象及調控其活化致死的機制。首先, 利用PMA/Ionophore, 抗活化型接受體CD16抗體, 及抗抑制型接受體Kp43抗體, 去刺激細胞。發現PMA/Ionophore或抗CD16抗體對細胞刺激12-15小時後, 約有60-70%的細胞死於細胞凋零, 但抗Kp43抗體對細胞的刺激, 則無此現象。我們進一步檢測TNF接受體超族群(TNF receptor superfamily)中Fas及Lymphotoxin-b(LTbR)在上述自然殺手細胞活化致死現象之角色。經由流式細胞分析儀針對細胞表面分子的分析, 發現剛從健康人血分離出的自然殺手細胞, 可表達少數Fas分子和極高量的LTbR及LTb分子, 但不表達Fas ligand。 經過兩禮拜的體外培養後, 則Fas的表現量增加﹐其餘三者不變。之後在PMA/Ionophore或抗CD16抗體的刺激下, Fas ligand被表達出來, Fas的表現量不變, 而LTb和LTbR的表達卻降為原本的30-50%。至於抗Kp43抗體的刺激對於這四種表面分子的表達﹐與刺激前比較﹐並無明顯不同。同時﹐我們利用桿狀病毒蛋白質表達系統( Baculovirus protein expressionsystem)製備溶解性Fas-Fc融合蛋白及溶解性LTbR-Fc融合蛋白後﹐發現溶解性Fas-Fc融合蛋白可抑制PMA/Ionophore或抗CD16抗體所引起的細胞凋零, 而溶解性LTbR-Fc融合蛋白則否。接著,利用介白素-4號, 發現在一濃度依存趨勢下(dose dependent ), 也可抑制細胞的活化致死. 綜合以上資料, 我們證實了自然殺手細胞在經由其表面活化型接受體CD16的刺激後﹐可進行"活化致死"的現象, 且Fas所導致的細胞凋零為調控此"活化致死"的主要機制, 而LTbR所導致之細胞凋零則不明顯。相反地, 我們認為LTbR可傳遞一抑制細胞凋零的訊號, 因當我們利用溶解性LTbR-Fc融合蛋白去抑制LTb和LTbR的交互作用時, 反而會造成Fas ligand的產生並進而促進細胞凋零. 最後, 我們也證明介白素-4號有調控此活化致死現象的能力.

2. Study of Activation-induced Cell Death of Natural Killer Cell • Human natural killer cells (NK) play important roles in innateimmune system and have the potential to regulate the adaptedimmune responses. During a immune response, most activatedlymphocytes eventually died. Especially, restimulating activatedT cells by triggering CD3/TCR complex would make them undergoapoptosis, a process termed as activation-induced cell death:AICD. Thus, we intend to determine the occurrence of AICD on NKcells and mechanisms directing the AICD. First of all, westimulated cells by the treatment of PMA/Ionophore orcrosslinking CD16 or Kp43 molecules on the surface of NK cells,and found that by the treatment of PMA /Ionophore orcrosslinking CD16 molecules for 12-15 hours, 60-70% of short-term cultured NK cells died in the form of apoptosis but not forthe crosslinking of Kp43 molecules. We further determine theroles of Fas and LTbR, two members of TNF receptor super-familyin the AICD of NK cells. Through analysis toward surfacemolecules on NK cells by Flow Cytometer, we have found thatfreshly isolated NK cells could express low level of Fasmolecules, pretty high amounts of LTb and LTbR molecules but noFas ligand. Two-week in-vitro culture later, the only differenceis the enrichment of Fas molecules. By the treatment of PMA/Ionophore or crosslinking CD16 molecules, Fas ligand moleculeswere induced, Fas molecules were with same expressive patternbut the expressive amounts of LTb and LTbR molecules weresignificantly reduced to 30-50% of the original ones.Stimulation upon crosslinking Kp43 molecules have no significanteffects on the expressive patterns of the four molecules. Thenwe took advantage of Baculovirus protein expression system tosynthesize soluble Fas-Fc and soluble LTbR-Fc proteins and foundthat s-Fas-Fc could prevent cells from apoptosis which resultedfrom stimulation of PMA/Ionophore or cross- linking CD16 ,but s-LTbR-Fc could not. Finally, the usage of IL-4 has been shown toblock the AICD of NK cells in a dose-dependent manner. Insummary, we proved that NK cells could undergo " AICD" uponcrosslinking the activation-receptor, CD16 and Fas mediatedapoptosis played the major roles in regulating AICD, but not forLTbR mediated apoptosis. In contrast, signals delivered fromLTbR would inhibit apoptosis and therefor the interactionsbetweem LTb and LTbR were essential for survival of human NKcells.