In vitro Antigen-presentation Assay for Self- and Microbial-derived Antigens
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Author:
Date:
2017-06-05
[Abstract] Antigen presenting cells (APC) are able to process and present to T cells antigens from different origins. This mechanism is highly regulated, in particular by Patter Recognition Receptor (PRR) signals. Here, I detail a protocol designed to assess in vitro the capacity of APC to present antigens derived from bacteria, apoptotic and infected apoptotic cells.
[摘要] 抗原呈递细胞(APC)能够处理和呈递来自不同来源的T细胞抗原。这种机制是高度调节的,特别是通过Patter Recognition Receptor(PRR)信号。在这里,我详细说明了一种设计用于评估体外的APC方案,用于展示来源于细菌,凋亡和感染的凋亡细胞的抗原。
背景 T细胞淋巴细胞在其表面上表达T细胞受体(TCR),其允许识别作为与主要组织相容性复合物(MHC)分子结合的抗原加工和呈递的抗原的细胞(自身)或微生物(非自身)抗原)呈递细胞(APC)。 APC能够处理抗原并将其呈递给T细胞,并且MHC-TCR相互作用是感染和自身免疫应答期间T细胞活化的关键步骤。  以前的作品已经描述了基于刺激模式识别受体(PRR),例如toll样受体(TLR)(Blander和Medzhitov,2004和2006)的抗原呈递的调节机制。实际上,特异性地来自含有微生物病原体的吞噬体的TLR信号有利于在MHC-II分子内呈递非自身抗原。另一方面,凋亡细胞吞噬后产生的自身抗原由于不存在TLR刺激而导致溶酶体降解。然而,当两者都来自感染的凋亡细胞并且同时由相同的吞噬体携带时,自身和非自身抗原的分离不会发生,其由针对抗原呈递的TLR信号最佳地定制。已经使用骨髓来源的树突状细胞(BMDC)和凋亡性小鼠B细胞 - ...
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Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cells in vitro and Splenic Dendritic Cells ex vivo Using Antigen-coated Beads
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Author:
Date:
2016-11-20
[Abstract] Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways.
This protocol allows the evaluation of antigen cross-presentation both in vitro (by using bone marrow-derived DC) and ex vivo (by purifying CD8+ DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles. Cross-presentation efficiency is measured by three different readouts: the B3Z hybridoma T cell line (Karttunen et al., 1992) and stimulation of antigen-specific CD8+ T cells (OT-I) (Kurts et al ...
[摘要] 通过MHC I类分子的抗原呈递,也称为交叉呈递,引起细胞毒性免疫应答。特别地,树突细胞(DC)是最熟练的交叉呈递细胞,因为它们已经开发了控制吞噬和降解途径的独特手段。 <此协议允许在体外(通过使用骨髓衍生的DC)和离体(通过纯化CD8 + )评价抗原交叉呈递。 (OVA)偶联的颗粒后,来自脾脏的DC/DC结合颗粒抗原)。通过三种不同的读数测量交叉呈递效率:B3Z杂交瘤T细胞系(Karttunen等人,1992)和抗原特异性CD8 + T细胞的刺激OT-I)(Kurts等人,1996),在用羧基荧光素琥珀酰亚胺酯(CFSE)标记它们之后分析CD69表达或OT-I增殖的OT-I活化。通过使用这种方法,我们可以最近显示DCs能够在TLR4结合时瞬时增加交叉表达效率(Alloatti等人,2015)。 [背景] 在小鼠中,抗原呈递细胞(APC)能够吸收外源抗原以加工它们,并将源自这些外源抗原的肽加载到主要组织相容性复合体(MHC)I类分子上。肽-MHC I复合物随后被转运到质膜,在那里它们可以呈递到CD8 + T细胞,从而促进T细胞活化,这被称为交叉呈递(Joffre等人, al 。,2012)。在不同的APC中,树突状细胞(DC)在交叉呈递方面表现优异,并且包含表达XCR1标记的不同亚群,其已经显示非常有效地交叉呈递抗原(即CD8 + 来自脾的DC和来自皮肤和肺的CD103 ...
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House Dust Mite Extract and Cytokine Instillation of Mouse Airways and Subsequent Cellular Analysis
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Author:
Date:
2016-07-20
[Abstract] Asthma is a complex disease of the airways primarily mediated by T helper 2 cells and innate lymphoid type 2 cells (Licona et al.,2013). Mice do not develop spontaneous asthma and therefore models have been developed for the assessment of key processes that underlie human pathology (Nial et al.,2008). Exposure to House Dust Mite (HDM) extract induces many key features of acute airway inflammation including elevated IgE levels, eosinophilia, goblet cell metaplasia, epithelial hypertrophy and airway hyperresponsiveness (AHR) in response to methacholine (Hammad et al., 2009; Dullaers et al., 2012; Coquet et al., 2015). The exact dose and duration of exposure to HDM can affect the type and extent of inflammation. In our case, we start with a low ...
[摘要] 哮喘是主要由T辅助细胞2和先天淋巴2型细胞介导的气道的复杂疾病(Licona等人,2013)。小鼠不发展自发性哮喘,因此已经开发了用于评估作为人类病理学基础的关键过程的模型(Nial等人,2008)。暴露于房尘螨(HDM)提取物诱导急性气道炎症的许多关键特征,包括升高的IgE水平,嗜酸性粒细胞增多,杯状细胞化生,上皮肥大和响应乙酰甲胆碱的气道高反应性(AHR)(Hammad等人, ,2009; Dullaers等人,2012; Coquet等人,2015)。暴露于HDM的确切剂量和持续时间可以影响炎症的类型和程度。在我们的情况下,我们从在攻击时增加的低致敏剂量开始,而其他的在小鼠致敏期间使用不同的方案或更高的抗原浓度(Hondowicz等人,2016; Trompette < et="" al="" 。,2014; ="" zaiss="" et="" al="" 。,2015)。我们认为使用低敏感剂量更准确地分离初级和次级免疫应答,并降低致敏过程中给予的hdm继续刺激攻击期间的免疫应答的可能性(coquet等人,2015;="" plantinga="" et="" al="" 。,2013)。在这里,我们在文本,图片和视频中概述了如何通过鼻内途径施用hdm提取物或细胞因子,并简要地谈谈随后在气道中的炎症的分析[在han&="">
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