探討表現IL-15選擇性剪接異構體ENU突變鼠感染第一型疱疹病毒後產生抗原特異性CD8+ T細胞的差異

The deviated outcomes of antigen specific CD8+ T cells generated in ENU mutagenized mice that express alternatively spliced IL-15 after HSV-1 infection

陳建甫(Jian-Fu Chen)

指導教授 ： 顧家綺

國立臺灣大學/醫學院/免疫學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903484

摘要

單純疱疹病毒-1（HSV-1）是感染人類常見的病原體。 HSV-1在原發感染期間。發病於粘膜上皮細胞處並且在神經元中建立終生潛伏期。以HSV-1感染小鼠的側腹皮膚，在接種部位產生潰瘍病變，並且可以在背根神經節（DRG）中建立潛伏感染。抗原特異性CD8+ T細胞對於控制原發性HSV-1感染及其再活化中扮演重要作用。為了幫助CD8+ T細胞的抗病毒活性，IL-15是支持毒殺型T細胞的產生與增殖所需的細胞因子，並維持CD8+記憶T細胞的長期存活。我們利用ENU誘變小鼠（系譜191）進行研究，其在IL-15基因的外顯子7中具有點突變，因此提高選擇性剪切的IL-15異構物（IL-15ΔE7）的表現。根據我們的初步研究，與C57BL / 6野生型（WT）小鼠相比，在P191小鼠中觀察到更嚴重和延長的HSV-1帶狀疱疹病變。此外，在P191小鼠中發現CD44hi記憶型CD8+ T細胞減少。這些結果使我們假設P191小鼠中CD44hi記憶型CD8+ T細胞的減少可能影響抵抗HSV-1免疫反應。在本研究中，我們使用Kb-gB408-505四聚體進行染色，並透過流式細胞儀以研究與WT小鼠相比，gB特異性CD8 + T細胞的產生，輸出和歸巢模式是否可能在P191小鼠中受到影響。我們的結果發現，gB特異性CD8+ T細胞在P191小鼠的引流淋巴結和周邊血中觀察到在感染後第7天減少。此外，在P191小鼠中，gB特異性CD8+ T細胞的皮膚歸巢受體CXCR3的表現顯著降低。在免疫組織化學染色的分析中，CD8+ T細胞的浸潤在感染後第7天的p191小鼠的疱疹傷痂中觀察到減少的現象。我們進一步研究了遷移性樹突細胞 (DC) 和CD8α+ DC的數量以及成熟，我們發現朗格漢斯細胞和CD8α+ DC在P191小鼠中觀察到減少的現象，其可能與IL-15∆E7表現分佈有關。
整體而言，這些數據表明，可能由於受到朗格漢斯細胞和CD8α+ DC減少的影響，gB特異性CD8 + T細胞的產生和歸巢模式在191小鼠中發生偏離，並揭示了IL-15E7可能透過調節DC而導致gB特異性CD8 + T細胞的活化不足而影響對抗HSV-1感染的免疫反應。

關鍵字

單純疱疹病毒 ； 抗原特異性CD8+ T細胞 ； 樹突狀細胞 ； IL-15異構物

並列摘要

Herpes simplex virus-1 (HSV-1) is a common pathogen for human infection. Infection of HSV-1 is initiated at mucosal epithelial cells during primary infection and establishes a lifelong latency in neurons. Cutaneous infection of mouse flank skin with HSV-1 creates an epidermal ulcerated lesion at the inoculated site and may establish latent infection in the innervating dorsal root ganglia (DRG). Viral glycoprotein B (gB498-505) specific CD8+ T cells are known to play an important role in viral clearance and a long-term surveillance against viral reactivation form DRG. Moreover, IL-15 is an essential cytokine to support proliferation and generation of cytotoxic T lymphocyte and maintains long-term survival of CD8+ memory T cell.
Our investigations with an ENU-mutagenized mice pedigree 191 (P191) which has a point mutation in exon 7 of the IL-15 gene and thus expressed an elevated level of an alternatively spliced IL-15 isoform (IL-15ΔE7). According to our preliminary studies, a more severe and prolonged HSV-1 zosteriform lesion was observed in P191 mice compared with C57BL/6 wild type (WT) mice. Furthermore, a reduced expression of CD44hi CD8+ T cells was found in P191 mice. Therefore, we hypothesized that reduced CD44hi CD8+ T cells in P191 mice may bring impacts on effective anti-viral immunity against HSV-1 infection.
In this study, we used Kb-gB408-505 tetramers for flow cytometry to investigate whether the generation, output, and homing pattern of gB specific CD8+ T cells may be affected in P191 compared to WT mice. By flow cytometry analysis, reduced gB specific CD8+ T cells on day 7 post infection were observed in both draining lymph nodes and peripheral blood of P191 mice. Furthermore, the CXCR3 expression of gB specific CD8+ T cells was significantly reduced in P191. Moreover, reduced CD8+ T cell infiltration in lesional skin was observed in p191 mice on day 7 post infection by immunohistochemical staining analysis. We further investigated the profile of migratory DCs and CD8α+ DCs. Reduced Langerhans cells and CD8α+ DCs were observed in P191 mice which may correlate to expression kinetics of IL-15ΔE7. Collectively, these data suggested that generation and homing pattern of gB-specific CD8+ T cells was deviated in 191 mice, which was probably influenced by reduced Langerhans cells and CD8α+ DCs, and revealed the possibilities of IL-15ΔE7 in modulation of DCs biology and lead to an insufficient priming of gB-specific CD8+ cells against HSV-1 infection.

並列關鍵字

HSV-1 ； antigen specific CD8+ T cells ； dendritic cells ； IL-15ΔE7

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