2013-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658891摘要：B細胞在移植排斥的免疫反應中扮演重要的角色，B細胞可以分化為漿細胞進而產生抗體，是抗體性排斥居中的重要媒介。而且，對人體器官移植而言，HLA 抗體與慢性移植腎病變有關。最近，我們發現去除 B細胞可以減少中心記憶 T細胞的比率而且抑制高敏感性移植之記憶 T細胞反應；該論文已發表於 Transplant Immunology 2009。先前，我們進行前膽性之臨床試驗，探討去除 B細胞是否有助於降低急性排斥率與改善腎功能。我們發現：移植前去除 B細胞可以降低排斥率，而且改善移植後之腎功能。所以，目前正進行第二年動物實驗，研究去除 B細胞抑制記憶 T細胞之免疫機制，我們提出假說：B細胞藉由促進 T細胞凋亡抑制 T細胞記憶反應。希望後續第三年計畫可以得到補助，進一步研究改善移植病患預後之方法。第一年的研究發現：去除 B細胞後的脾細胞在混合淋巴球試驗中的增殖反應變強，而且不同時間點細胞激素 IL-2 和 IFN-γ的表現增加。另外我們也發現去除 B細胞後再刺激脾細胞，促進 T細胞表現較多與凋亡相關之 Bim 基因與 caspase-3，顯示去除 B細胞可能透過促進 T細胞之凋亡抑制 T細胞之記憶反應第二年：我們正分析在不同時間點去除 B細胞與否對 Bcl-2，Bim 在 Q-PCR 與西方點墨法上之表現之差異，並且進一步以流式細胞儀分析 T細胞在刺激後，凋亡的現象是否受去除 B細胞與否影響。另外，為進一步探討細胞激素對 T細胞凋亡之影響，計畫在混合淋巴球試驗中加入細胞激素抗體(anti-IL-2, anti-IL-7 and anti-IFN-γ)然後再次分析Bcl-2 與 Bim 之基因表現。目前發現去除 B 細胞後接受刺激 96 小時，caspase-3 陽性的CD8+T 細胞比例明顯比未去除 B 細胞的對照組增加。流逝細胞儀進一步發現: 去除 B細胞之實驗組 Annex V 陽性的凋亡細胞，佔 CD4+與 CD8+之 T 細胞比例明顯比對照組增加。而且，上述現象明顯受到 IL-2 抗體的抑制，但是 IFN-γ抗體卻沒有抑制作用。第三年：我們計劃以心移植模型探討去除 B細胞之後移植心臟中是否存在比較多的凋亡 T細胞，以免疫化學染色和流式細胞儀分析心移植浸潤細胞凋亡相關分子的表現。研究成果預期可以進一步確認去除 B細胞在移植後免疫抑制策略上的定位，而且可望為臨床器官移植的病人，找尋與去除 B細胞的策略，相互搭配的免疫調控方式，進一步改善臨床移植成果。<br> Abstract: B cells play an important role in allogeneic immune responses. B cells, whichdifferentiate into plasma cells to produce antibodies, are central to antibody-mediatedallograft rejection. And, for human renal allograft transplantation, anti-HLA antibodieswere found to be associated with chronic allograft nephropathy and graft failure. Recentlywe found that B-cell depletion reduced the percentage of central memory T cells in thespleen and inhibited the memory T-cell allogeneic immune responses in sensitized recipients;the results have been published in Transplant Immunology 2009. Then we conducted aprospective randomized clinical trial in renal transplant patients looking at if B-cell depletionwould reduce acute rejection and improve renal function (NSC: 97-2314-B-002-175-MY3).We found that rituximab induction therapy could provide additional immunosuppressiveeffect and benefit to renal transplant patients: the incidence of acute rejection was reducedand the renal function improved. Therefore we are now working on the immune responses ofthe leftover T cells after B-cell depletion (NSC:100-2314-B-002-150-). We hypothesize thatB cell depletion lessens T cell memory responses by enhancement of T cell apoptosis. Webelieved that further investigation into the mechanisms responsible for the suppression ofmemory T-cell immune response by B cell depletion in animals would help to improveclinical outcomes of transplant patients.In the first year, we have completed the assays for proliferation and cytokine productionof T cells in the presence of B cells or not. We found enhanced proliferation responses of theB-cell depleted leftover BALB/c splenocytes when stimulated by allogeneic C57BL/6splenocytes (p<0.001). Moreover, the B-cell depleted leftover BALB/c splenocytes alsosecreted much more IL-2 and IFN-γthan the splenocytes without depletion (p<0.0001).Additionally, the gene expression of proapoptotic Bim and caspase-3 increased when T cellsreceived allogeneic stimulation in absence of B cells, suggesting that B cell depletion couldbe lessening T cell memory response by enhancement of T cell apoptosis. We felt that ourfindings were extremely interesting and could contribute to the advancement oftransplantation science significantly. We would accordingly apply for a follow-up researchgrants for the next two years.In the second year, we are working on the apoptosis-related gene and expression in vitro.Q-PCR of Bcl-2 and Bim will be measured on 48, 96 and 168 hours in mixed lymphocytecultures with or without B cell depletion. So far, we found that a higher percentage ofcaspase-3 positive CD8+ cells were identified at 96 hours in the B-cell depleted splenocytes,as compared to the splenocytes without depletion. Flow cytometry further revealed that thepercentages of CD4+ and CD8+ memory cells decreased if B cells were depleted duringallo-stimulation. And, at the same time, more apoptotic CD4+ and CD8+ T cells could bedetected by Annex V staining. Additionally, we found that anti-IL-2 antibody inhibited theapoptosis of the alloreactive T cells in both CD4+ and CD8+ populations, but the controlantibody did not. Interestingly, IFN-γ did not inhibit T cell apoptosis as Il-2 did; thepercentages of apoptotic T cells were quite the same between those with anti- IFN-γandcontrol antibody.In the third year, we would like to demonstrate that more apoptosis happens in the hearttransplant grafts when B cells depleted in the recipients. A heart transplant model with Bcell depletion will be set up using ex vivo B cell depletion and adoptive transfer to SCIDmouse. C57BL/6 heart grafts will be removed from the recipients at various time points forimmunohistochemistry study of Bcl-2 and Bim. Graft-infiltrating cells will also beharvested for flow cytometric analysis of apoptosis markers including annexin-V, 7-AADand caspase-3.