胞內抗原位置對於肝臟T細胞反應之影響

Abstract

過敏性疾病是因為過敏原引起的免疫反應所造成的一群疾病統稱,包含在全球造成三億多人受害的氣喘。為了了更更好的管控氣喘,有一種方式是針對過敏原專一性所研發的長效免疫療療法。另外對於肝臟免疫耐受性效應的了了解,有可能使我們利利用肝臟環境的特性應用到氣喘治療療上。我們推論論在肝臟的抗原表現有可能造成周邊免疫耐受性,且機會成為有效的氣喘療療法。另外肝細胞抗原的位置對於的位置對於免疫活化或免疫耐受性的影響仍不不清楚,本論論文將一併討論論。 為進行不同抗原位置的研究,我們先做出可表現在不不同位置的質體並確認其 表現位置正確。將正確的質體包裹至Adeno-associated virus (AAV) 並將抗原卵卵白蛋白Ovalbumin, OVA)傳感至老老鼠肝細胞,待表現一個月後分別將具抗原特異異性的 CD4+和 CD8+ T 淋淋巴球以繼承性轉移(Adoptive transfer)方式去追蹤淋淋巴球表型變 化和免疫病理理現象。此外,我們也以complete Freund’s adjuvant immunization探討抗 體生成是否有受到影響,並期望能使用氣喘小鼠模型觀察此一影響對 Liver tolerance 的應用性是否有所延展。從實驗中可發現,Antigen-specific CD8+ T cells 碰到胞內抗原都都會有強烈烈的分裂裂反應,從淋淋巴球上表面標記蛋白(Surface marker) 可以看到在Adoptive transfer後第一天就能看到細胞活化,包括CD62L表現量量下降降 和CD69表現量量上升。其活化造成短期的 ALT 上升,並能在肝臟病理理切切片中明顯 發現有淋淋巴球浸潤的現象,但十天後開始回歸正常值。一個月後以活體毒殺殺性淋淋 巴球試驗測試發現,胞內抗原對 CD8+ T cells 的反應雖在初期有造成活化並且有 effector的功能,但在一個月後再次碰到抗原時無法進行行清除,此並非一完整的 CD8+ T cells 活化。其中表現在粒粒線體的抗原造成的免疫反應較表現在細胞質中的 抗原略略強,此現象仍待進一步的實驗證明,是否抗原表現位置會對免疫反應造成 影響。另一方面,胞內抗原並無法造成 Antigen-specific CD4+ T cells 增生。 Immunization的實驗中,在對 B6 或是 BALB/c 品系老老鼠進行行AAV transduction後 一個月做抗原免疫試驗,發現胞內抗原都都無法抑制專一性抗體的產生。 合併來來看,我們的結果進一步了了解肝臟環境內調控T cell的機制,而這些訊息 對於日後發展過敏性疾病或自體免疫疾病的新型免疫調控療療法可能有所助益。

It is now known that the ‘liver tolerance effect’ mediates local and systemic tolerance to self as well as foreign antigens. We hypothesized that ectopic expression of autoantigens in the liver might be an effective approach for induction of peripheral tolerance to autoreactive lymphocytes. Besides, the influence of antigen localization in hepatocytes on immune activation/ tolerance is not clear and was investigated in this study. We expressed ovalbumin (OVA) at different subcellular localizations, including cytoplasmic (cOVA), mitochondrial (mtOVA), nuclear (nOVA) and membrane-bound (mbOVA) OVA. ELISA assays showed that only sOVA but not the other forms was present in the culture supernatant. mbOVA was the unique form present at the cell surface on a significant fraction of transfected cells. Besides, we confirmed OVA expression in the cytoplasm, nucleus, and mitochondria, respectively, in cOVA, nOVA, and mtOVA transfected cells by immunofluorescence assay (IFA). In vivo expression of OVA proteins was confirmed by analyzing hepatocytes isolated from AAV-OVA-transduced mice. IFA analysis showed that the different forms of OVA were expressed at the expected subcellular localizations. When OVA-specific OT-1 CD8+ T cells encountered with the hepatic OVA by adoptive transferring, there was a significant increase in the absolute numbers of OT-1 cells in the liver of the cOVA and mtOVA mice but not in the GFP mice. Strong OT-1 proliferation was further confirmed by the diluted CFSE intensity in these OVA-expressing mice. These OT-1 cells were activated as indicated by their lower CD62L expression in the livers of cOVA mice compared with that in the GFP control mice. Consistently, CD69, another activation marker of lymphocytes, was uniformly up-regulated on OT-1 T cells in the OVA-expressing mice versus mice expressing GFP. In an in vivo cytotoxicity assay, peptide-pulsed splenocytes were eliminated favorably in cOVA and mtOVA mice versus the GFP mice. The serum ALT levels, an indicator of liver damage, was significantly increased in cOVA and mtOVA mice but not in the GFP mice. Histological analysis revealed foci of mononuclear cell infiltration in cOVA and mtOVA mice but not in the control GFP mice. In contrast, the hepatic OVA did not stimulate OT-2 CD4+ T cells. In AAV/cOVA- and AAV/mtOVA-transduced mice, the transferred OT-2 cells maintained the naive T cell phenotypes, CD62Lhigh and CD69low, similar to what was observed in the GFP mice. Our preliminary data showed that hepatic expression of cOVA and mtOVA did not induce immune tolerance to inhibit antibody responses to OVA vaccination in two strains of mice. Together, our data provide insight into mechanisms that regulate T cell function in the liver environment. This information might be useful in future development of new immunomodulatory treatment for allergic and other autoimmune diseases.