疫苗誘導之調節性T細胞和第一介白素接受器的訊息在建立流感病毒專一性記憶型T細胞的角色

Abstract

因為流感病毒的高突變率，造成目前去活化疫苗刺激所產生的中和性抗體，無法有效的中和不同型流感病毒。T細胞是辨認流感病毒的高保留性抗原，所以可以有效的對抗不同型流感病毒。之前的研究指出，流感專一性記憶型T細胞有潛力對抗不同型或抗原差異大之流感病毒株，但其會隨著時間減少，因此如何建立良好的流感專一性T細胞免疫系統，顯得相當重要。先天免疫系統和後天免疫系統對於建立好的T細胞免疫扮演著重要角色，因此本篇論文分為兩部分去探討: (1) 調節型T細胞對於流感病毒專一性T細胞的影響。(2)介白素-1接受器對於流感病毒專一性記憶T細胞的影響。第一部份研究疫苗誘發的具病毒專一性之調節性T細胞對抗病毒能力之影響。胜肽疫苗可以針對病毒的高保留抗原產生專一性T細胞免疫，但因其免疫刺激效果不強，若無添加佐劑，不易有保護效果，且會產生抗原專一性調節型T細胞。目前對於疫苗誘導具病毒抗原專一性之調節型T細胞如何影響抗病毒免疫反應目前尚不清楚。因此，我們發展一特殊流感病毒之小鼠感染模式，利用過繼轉移給予小鼠OT-II CD4 T細胞的方式，發現OVA323-339胜肽可誘導產生具抗原專一性調節性T細胞，且專一性地去除這群調節性T細胞會提升小鼠抗病毒免疫反應。且流感病毒的感染，會使原先存在的疫苗誘導之調節性T細胞顯著增加。如果搭配不同佐劑，尤其是CpG，會抑制疫苗誘導調節性T細胞的產生，且在第二次免疫時，CpG也會抑制先前疫苗誘導調節性T細胞的增加。此外，疫苗搭配CpG並透過peripheral-priming-local-boosting方式給予，除了減少疫苗誘導調節性T細胞進入到肺中，也增加了effector在肺中的量。最後，我們發現，此種免疫方式，除了可用在胜肽疫苗外(OVAI/II或NPI/II 胜肽)和蛋白質疫苗(OVA蛋白、去活化流感病毒)之外，也能明顯的增加小鼠對抗異型流感病毒的感染。第二部份研究介白素-1接受器對於流感病毒專一性記憶T細胞的影響，之前的研究指出，介白素-1乙型可以經由NLR-發炎體路徑來產生，除了影響到先天免疫細胞的功能之外，也會影響到T細胞的功能和增生，且發現在初次流感病毒感染時，介白素-1接受器缺陷小鼠的病毒專一性T細胞相較野生型小鼠來的少，無法有效的清除流感病毒，使小鼠存活率下降。但我們發現，在初次感染後期，介白素-1接受器缺陷小鼠相較於野生型小鼠，卻有較多的病毒專一性T細胞。所以當介白素-1接受器缺陷小鼠在第二次異型流感病毒感染時，反而有較高的存活率和抗病毒能力。我們進一步發現，介白素-1接受器缺陷小鼠肺中的常駐型T細胞多於野生型小鼠，且不管是CD4或CD8肺常駐型T細胞，對於幫助介白素-1接受器缺陷小鼠的抗病毒能力和存活率都扮演著重要角色。最後，我們發現，介白素-1接受器缺陷小鼠有較多的調節性T細胞，且病毒抗原較晚清除，這可能與其有較多的肺常駐型T細胞有關。綜合以上結果，我們證明了，疫苗誘導調節型T細胞會抑制抗流感病毒的免疫反應，且含有CpG的疫苗，可以藉由抑制調節型T細胞的發展和刺激T細胞免疫反應，進而增加對抗異型流感病毒感染的能力。我們也證明了缺少介白素-1，會因為調節型T細胞的增加或抗原清除的太晚，使的肺常駐型T細胞的量增加，進而讓IL-1R1缺陷小鼠可以對抗異型流感病毒的感染。

Because of the high mutation rate of influenza virus, current inactivated influenza vaccine inducing neutralizing antibody against viral surface antigens cannot efficiently neutralize different subtypes of influenza virus. T cell immunity that recognizes the conserved epitopes derived from the internal proteins of influenza virus has the potential to protect against heterosubtypic and antigenically distinct influenza virus infection. Nevertheless, previous studies have shown memory T cells decrease along with time. Thus, establishment of a robust and long-lasting T cell immunity is important to protect from heterosubtypic influenza virus infection. Several factors have been shown to affect T cell responses induced by vaccination or infection. Therefore, this thesis aims to explore the factors that shape the antiviral T cell immunity against influenza virus infection in two parts. We have studied two specific factors: (1) the role of vaccine-induced antigen-specific regulatory T (Treg) cells in virus-specific T cell immunity. (2) the role of interleukin 1-recepter (IL-1R) signaling in the development of virus-specific memory T cells following acute influenza virus infection. In the first part, we aim to explore the influence of vaccine-induced antigen-specific Treg cells on antiviral T cell immunity. It has been known that peptide-based vaccines are subimmunogenic, and often induce Treg cells. However, it remains unclear about how vaccine-induced antigen-specific Treg cells affect antiviral immunity, and how repeated vaccination and infection influence antigen-specific Treg cells. Using an adoptive transfer system, we found that vaccination with the OVA323-339 peptide induced antigen-specific Treg cells and repeated vaccination further expanded them. Depletion of vaccine-induced antigen-specific Treg cells enhanced antiviral immunity against influenza virus infection. We also found that influenza virus infection drove the expansion of pre-existing vaccine-induced Treg cells in a dose-dependent manner. In addition, vaccination combined with certain adjuvants, especially with CpG, suppressed the generation and expansion of vaccine-induced antigen-specific Treg cells. Furthermore, CpG-adjuvanted vaccines by using the peripheral-priming-local-boosting strategy not only decreased vaccine-induced antigen-specific Treg cells but also increased effector cells in lung, so conferred a more effective viral control than unadjuvanted vaccines. Collectively, antigen-specific Treg cells induced by peptide vaccines attenuated the antiviral immunity against influenza virus infection. CpG-adjuvanted peptide vaccines provide influenza protection probably by inhibiting Treg development and enhancing T cell immunity. In the second part, IL-1, including IL-1α and IL-1β influences the survival and function of immune cells, and regulates certain adaptive immune responses directly and indirectly. Previous studies have shown IL-1R signaling promotes the development of antiviral T cell immunity, which confers a better viral control in acute viral infection. Surprisingly, we found that compared to wildtype (WT) mice, IL-1R type 1 (IL-1R1)-deficient mice had a higher survival rate and more effective viral control when they had primary infection with sublethal dose of HKx31 influenza virus and then re-challenged by heterosubtypic PR8 influenza virus infection. We also found that IL-1R1-/- mice had more resident and circulatory memory T cells in late phase of infection up to 28 days post infection. Both resident CD4 and CD8 T cells contributed to the better control of heterosubtypic influenza virus infection in IL-1R1-/- mice. Finally, we also observed longer stimulation of viral antigen and more regulatory T cells in lung of IL-1R1-/- mice, which may contribute to more resident T cells in the lung of IL-1R1-/- mice. In summary, we discovered the role of vaccine-induced antigen-specific Treg cells and the detrimental effect of IL-1R signaling and in the development of antiviral T cell immunity against acute influenza virus infection.