GBM是一種高度惡性的膠質母細胞瘤，即使結合化療、放射線治療等眾多治療方式，GBM患者的存活率平均仍約15個月，而且三年內的死亡率高達88%；forkhead box P3 (Foxp3)被認為在來自胸線的調節性T細胞中扮演著重要功能的角色，調節性T細胞具有調控免疫反應之能力，然而腫瘤細胞卻有能力逃避免疫系統的攻擊，在最近的研究中指出幾種腫瘤細胞表現Foxp3轉錄因子，並有可能因此逃避T細胞的攻擊，目前的工作就是要研究並探討在GBM中表達出的Foxp3轉錄因子對於病人是否有意義，Foxp3 轉錄因子在一定程度上控制著調節性T細胞的發展，在最近的研究中指出Foxp3轉錄因子至少具有3種亞型(Isoforms)，分別為full length (Foxp3-WT)、exon 2 deletion (Foxp3-△2)以及exons 2 and 7 deletion (Foxp3-△2△7)，而且也已經被表明有表現在調節性T細胞中；WT具有抑制NFAT，以及由NFκB所調控的基因在轉錄過程中也受到WT的抑制，Foxp3 -△2會參與抑制轉錄因子retinoic acid-related orphan receptors alpha (ROR??)和gamma-t (ROR?莰)，Foxp3-△2△7則是在最近的研究證實Foxp3-△2△7能負向的調控WT及Foxp3-△2的功能；我們從Glioblastoma Multiform(GBM)以及Tumor Infiltrating Lymphocytes (TILs)中分離出調節性T細胞並藉由FACS來分析，接著我們將分析調節性T細胞的功能關鍵分子的表現在GBM和TILs中以及與DC-therapy反應的關聯。我們發現GBM病患中存活時間較短的病人在TILs整個CD4+的T細胞中調節性T細胞的比例較高；儘管如此，存活時間較短的病人在調節性T細胞中Foxp3 不同isoform的表現情形卻沒有一個明顯的差異性，但是另一方面我們也觀察到一個趨勢，在GBM的病患中存活時間超過兩年的病人，會有FOXP3-△2△7 dominant negative的表現，因此Foxp3 不同Isoform表現量的差異在GBM的預後發展或許可以做為一個重要的臨床評估要點。另外，由於調節性T細胞可能會限制DC-therapy對抗GBM的功效，然而利用改變剪接來調控Foxp3 Isoforms表現量也許可以提供新的方向去抑制調節性T細胞的活化，並改善DC-therapy的效果。
As themost malignant high-grade glioma, prognosis for patients with glioblastoma multiforme (GBM) has only a median survival of 15 month, and 88% mortality within 3 years. Forkhead box protein 3 (Foxp3) is considered to be an important gene for thymically derived and naturally occurring regulatoryTcells (Tregs),whichregulate the immunosuppressive response. Recent studies described the expression of forkhead box P3 (Foxp3) in several tumor cells for providing the possibility of effector T-cell evasion. The present work was to investigate the significance of Foxp3 expression in glioblastoma multiforme (GBM) patients. FOXP3 largely controls the development into Treg cells, and three isoforms Foxp3, Foxp3-WT, FOXP3-△2 and FOXP3-△2△7, have been shown to be expressed in human Treg cells. FOXP3-WT represses NFAT- and NF?羠-mediated gene transcription process , while FOXP3-△2 is involved in repressing the transcriptional activity of the retinoic acid-related orphan receptors alpha (RORα) and gamma-t (RORγt) that regulates the development of antigen-stimulated naive CD4+ T cells into T-helper 17 (Th17) cells . FOXP3-△2△7 has been identified to negatively regulate the function of FOXP3-WT, and FOXP3-△2. We isolated Tregs from infiltrating lymphocytes (TILs) of GBM by FACS analysis. Then, we analyzed the expression of key molecules of Treg function in lymphocytes and tumor cells and correlate with the DC-therapy response of the corresponding patient. Both Tregs and tumor cells express 3 different isoforms of the FOXP3 gene by alternative splicing, one of which represents a naturally occurring dominant negative version of the Foxp3 protein.The proportion of Tregs in CD4+ TILs had been observed the increasing percentage in GBM patients with short survival. However, there was no significant difference in three isoforms of Foxp3 expressed in Tregs comparing with survival of GBM patients. But, three patients with high survival (over 2 years) had shown the expression of dominant negative FOXP3-△2△7 isoform.The differential expression level of Foxp3 isoforms may constitute a potent prognosis factor for GBM patients. Tregs may limit DC-therapy against GBM, while the differential expression of Foxp3 isoforms could provide a basis for the evaluation of the effectiveness of DC-therapy. Functional abrogation of Foxp3 by tuning the splicing preference to its dominant negative isoform may offer an attractive window to inhibit Treg activity.
