探討抗原贈與細胞凋亡在組織胞漿菌感染所引起CD8 T細胞活化之角色

Abstract

組織胞漿菌是一種寄生在巨噬細胞吞噬小體內之病原菌，在體外的研究中發現，酵母型組織胞漿菌可以引發巨噬細胞的凋亡。在我們之前的實驗中發現，樹突狀細胞吞噬了凋亡小體後，可以刺激經抗原致敏之CD8 T細胞活化。另一方面，CD8 T細胞在組織胞漿菌感染的動物實驗中也被活化。然而，體內之CD8 T細胞是如何被活化的目前並不清楚。根據以上研究的結果，我們假設組織胞漿菌進入宿主後，會誘發巨噬細胞的凋亡，而且在凋亡小體內之胞漿菌抗原被樹突狀細胞交叉呈獻後，而造成CD8 T細胞的活化。因此，組織胞漿菌所引起之宿主細胞的死亡對於感染後宿主體內CD8 T 細胞的活化是很重要的。 本論文中，我證明皮下注射活的組織胞漿菌會引發，噬中性球及巨噬細胞聚集到注射之部位並且這兩種細胞都有細胞凋亡的現象，而且在局部淋巴結中CD4 T細胞及CD8 T細胞也都被活化，表現活化之標記抗原及產生IFN-γ。為了研究巨噬細胞凋亡與CD8 T細胞活化有關，我們利用皮下注射內含死的組織胞漿菌的凋亡巨噬細胞之方法來誘發老鼠體內CD8 T細胞的活化，此實驗結果顯示CD8 T細胞及CD4 T細胞都有被活化的現象，但是活化CD8 T細胞比例大於CD4 T細胞活化 比例。相反的，注射入內含死的組織胞漿菌的活巨噬細胞只引起微弱的T細胞反應。這結果指出抗原贈與細胞的凋亡對誘發T細胞，尤其是CD8 T細胞的免疫反應很重要。此外，抗原贈與細胞若有β2m缺失並不影響誘發CD8 T細胞活化的效果。這結果也暗示了CD8 T細胞的活化是因為宿主的抗原呈獻細胞從抗原贈與的凋亡巨噬細胞中獲得抗原，而引起的抗原交叉呈獻所導致。有趣的是，接種帶有抗原的凋亡巨噬細胞可以保護老鼠抵抗組織胞漿菌的感染。這結果也指出這些被活化的 T細胞是具有保護的功能。此研究結果的貢獻在於了解組織胞漿菌宿主細胞凋亡對組織胞漿菌感染後引發的CD8 T 細胞活化的重要性。

Histoplasma capsulatum (Hc) is an intracellular pathogen residing in the phagosomal compartment of the macrophages. In vitro studies showed that the yeast cells induce macrophage apoptosis. We have also reported that DCs which have engulfed Hc-infected apoptotic macrophages stimulate sensitized CD8 T cells in vitro and CD8 T cells are activated in animals after Hc infection. However, how CD8 T cell are activated in vivo remains unclear. Based on results of the above studies, I hypothesized that after entering the host, Histoplasma yeast cells induce macrophage apoptosis and the fungal antigen contained within the apoptotic macrophages are cross-presented by DCs to activate CD8 T cell. Thus, Histoplasma-induced host cell apoptosis would be key to CD8 T cell activation in the infected host. In this study, I showed that subcutaneous inoculation of live Histoplasma yeasts induced neutrophil and macrophage infiltration into the injection site and both of them underwent apoptotic death. Both CD8 and CD4 T cells in the draining lymph nodes were activated to express phenotypic activation markers as well as IFN-γ. To investigate whether macrophage apoptosis is related to T cell activation, mice were injected subcutaneously with heat-killed Histoplasma-containing apoptotic macrophages. The results showed that both CD8 and CD4 T cell were activated but the magnitude of CD8 T cell activation was greater than that of CD4 T cells. In contrast, HKHc-containing nonapoptotic macrophages only triggered a minimal T cell response. These results indicate that antigen-donor cell apoptosis is important to T cell, especially CD8 T cell, activation. Furthermore, β2m-/- apoptotic macrophages were as efficient as wild-type cells in inducing CD8 T cell activation in the immunized host. It suggests that CD8 T cell activation is the result of cross-presentation by host APCs that acquired Histoplasma antigens contained within donor apoptotic macrophages. Interestingly, inoculation of HKHc-containing apoptotic macrophages protected mice against Histoplasma infection, indicating that the activated T cells are functionally activated. The results of my work contribute to the understanding of how Histoplasma host cell apoptosis is important to CD8 T cell cross-priming after Histoplasma infection.