建立表達單一第一型白血球抗原的人造抗原呈現細胞以刺激T細胞及抗原表位定位

Abstract

T細胞在後天性免疫中扮演重要的角色，現今有許多癌症免疫療法，即是希望可藉由刺激更多的T細胞，使其活化、擴增，最後有效清除癌細胞而得到治療的成效。而刺激更多的T細胞有許多方法，其中一項就是分離人體內的樹突細胞，讓此抗原呈現細胞在體外受到抗原刺激，再將活化的抗原呈現細胞打回體內，因而可在體內達到刺激 T 細胞的效果，但此方法十分耗時且昂貴，因此，逐漸發展出利用人造的抗原呈現細胞在體外刺激T細胞。 而抗原呈現細胞刺激T細胞需要三種訊號，其一就是需要主要組織相容性複合體將抗原的抗原表位呈現給T細胞的受體；其二，需要一些協同刺激的分子放大刺激 T 細胞的訊號及延長刺激T細胞的時間，這些分子包含 CD86、CD70、4-1BBL 等等。其三，需要細胞激素如白血球介素-2 (IL-2)等的協助，以增加T細胞的擴增、活化。因此，本研究目的即是要做出一種人造的抗原呈現細胞，在人造的抗原呈現細胞上給與刺激T細胞所需的主要組織相容性複合體，並利用帶有4-1BBL、CD86 等協同刺激分子的質體使此細胞過度表現這些分子，最後在體外給予細胞激素作為額外的刺激，期望能在體外更有效率、快速地刺激並活化T細胞。 除了好的抗原呈現細胞可以更有效率刺激T細胞外，一個好的抗原表位(Epitope) 也很重要。抗原表位被T細胞受體辨識後即可活化T細胞，因此對於了解哪些抗原表位會引起T 細胞反應是必要的。 目前較常使用T2細胞作為抗原表位定位的工具，由於此細胞缺乏抗原加工相關轉運體(transporter associated with antigen processing; TAP protein)而無法將內源性的胜肽運至表面與主要組織相容性複合體結合，因此可利用外加胜肽來測試此抗原表位是否會與細胞表面的主要組織相容性複合體結合而穩定其結構，若偵測到的主要組織相容性複合體表現量有上升則代表可成功結合。但T2細胞表面並不是表現單一等位基因的主要組織相容性複合體，因此即使胜肽結合上去使偵測到的主要組織相容性複合體表現量有升高，也不清楚此胜肽是與哪個主要組織相容性複合體的等位基因結合。基於此，我們利用群聚且有規律間隔的短回文重複序列(CRISPR)技術建立一株缺乏抗原加工相關轉運體的細胞，並只表達單一等位基因主要組織相容性複合體，相比於以往使用的T2細胞株，可以更精確知道此抗原表位會與哪個主要組織相容性複合體的等位基因結合，因此可使用此細胞株作為抗原表位定位的工具。 有了人造抗原呈現細胞可以更有效率地刺激毒殺性T細胞，再加上可以更精確知道抗原表位的主要組織相容性複合體基因型，對於未來免疫療法會有極大的貢獻，希冀可以造福更多需要被治療的病患們。

T cells play a vital role in adaptive immunity. With enough functional T cells, cancer cells can be effectively eliminated to achieve cancer cure. One strategy to stimulate more functional T cells is to isolate patients’ dendritic cells, a kind of antigen presenting cells, which are then activated in vitro and subsequently transferred to patients. However, this approach is very time-consuming and expensive; therefore, artificial antigen presenting cells (APCs) have gradually gained wide interest, and are also used to stimulate T cells in vitro. APCs activate antigen-specific T cells require three distinct signals. Signal one is antigen-specific signaling mediated by T-cell receptor engagement of pathogenic peptides presented by major histocompatibility complex (MHC) molecules, also named human leukocyte antigens (HLA) in humans. Signal two is costimulatory molecules expressing on APCs, which mainly function to amplify signals and prolong the time of T cell stimulation and activation. Signal 3 is polarizing signaling mediated by various cytokine milieus to enhance T cell activation and expansion. Accordingly, in my thesis study, the first research purpose is to establish an artificial APC with robust ability for T cell stimulation. First of all, we used the technique of clustered, regularly interspaced, short palindromic repeats (CRISPR) to knock out the endogenous class I MHC and then transferred mono-allelic one on this artificial APC. Second, we generated the cells overexpress the costimulatory molecules that are critical for T cell activation. Last, we supple the cells with cytokine in vitro additionally as another stimulator. The ability of this artificial APC in activating and proliferating T cells will be examined and optimized. On the other hand, APCs can present epitopes to T cells and stimulate them. As a result, epitopes are important for stimulating T cells. However, it remains a time-consuming and labor-intensive work for epitope mapping. It is known that T2 cells lack the transporter associated with antigen processing (TAP) protein, so can be utilized for epitope mapping. Sometimes, the results of epitope mapping are ambiguous due to the presence of multiple HLA alleles. For the unambiguous identification of class I HLA-restricted epitopes, the cells expressing mono-allelic class I HLA that we established previously were used to knock out the TAP genes by CRISPR. After generating these mono-allelic HLA expressing TAP Knock out cell lines, we can precisely confirm the class I HLA-restricted epitopes to T cells. In the end, it is possible to develop powerful T cell-based immunotherapies against many diseases using epitope mapping and artificial APCs.