Hu Paul Q, Tuma-Warrino Renee J, Bryan Marianne A, Mitchell Kathleen G, Higgins Darren E, Watkins Simon C, Salter Russell D
Department of Immunology and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
J Immunol. 2004 Feb 1;172(3):1595-601. doi: 10.4049/jimmunol.172.3.1595.
Vaccination against cancer or intracellular pathogens requires stimulation of class I-restricted CD8(+) T cells. It is therefore important to develop Ag delivery vectors that will promote cross-presentation by APCs and stimulate appropriate inflammatory responses. Toward this goal, we tested the potential of Escherichia coli as an Ag delivery vector in in vitro human culture. Bacteria expressing enhanced green fluorescent protein were internalized efficiently by dendritic cells, as shown by flow cytometry and fluorescence microscopy. Phenotypic changes in DC were observed, including up-regulation of costimulatory molecules and IL-12p40 production. We tested whether bacteria expressing recombinant Ags could stimulate human T cells using the influenza matrix protein as a model Ag. Specific responses against an immunodominant epitope were seen using IFN-gamma ELISPOT assays when the matrix protein was coexpressed with listeriolysin O, but not when expressed alone. THP-1 macrophages were also capable of stimulating T cells after uptake of bacteria, but showed slower kinetics and lower overall levels of T cell stimulation than dendritic cells. Increased phagocytosis of bacteria induced by differentiation of THP-1 increased their ability to stimulate T cells, as did opsonization. Presentation was blocked by proteasome inhibitors, but not by lysosomal protease inhibitors leupeptin and E64. These results demonstrate that recombinant E. coli can be engineered to direct Ags to the cytosol of human phagocytic APCs, and suggest possible vaccine strategies for generating CD8(+) T cell responses against pathogens or tumors.
针对癌症或细胞内病原体的疫苗接种需要刺激I类限制性CD8(+) T细胞。因此，开发能够促进抗原呈递细胞交叉呈递并刺激适当炎症反应的抗原递送载体非常重要。为了实现这一目标，我们在体外人类培养中测试了大肠杆菌作为抗原递送载体的潜力。通过流式细胞术和荧光显微镜观察发现，表达增强型绿色荧光蛋白的细菌能被树突状细胞有效内化。观察到树突状细胞的表型变化，包括共刺激分子上调和IL-12p40产生。我们以流感病毒基质蛋白作为模型抗原，测试了表达重组抗原的细菌是否能刺激人类T细胞。当基质蛋白与李斯特菌溶血素O共表达时，使用IFN-γ ELISPOT检测可观察到针对免疫显性表位的特异性反应，而单独表达时则未观察到。THP-1巨噬细胞在摄取细菌后也能够刺激T细胞，但与树突状细胞相比，其动力学较慢且T细胞刺激的总体水平较低。THP-1分化诱导的细菌吞噬作用增强，其刺激T细胞的能力也增强，调理作用也是如此。蛋白酶体抑制剂可阻断抗原呈递，但溶酶体蛋白酶抑制剂亮抑酶肽和E64则不能。这些结果表明，重组大肠杆菌可被设计用于将抗原导向人类吞噬性抗原呈递细胞的胞质溶胶，并提示了针对病原体或肿瘤产生CD8(+) T细胞反应的可能疫苗策略。
