College of Veterinary Medicine, Chonbuk National University, Iksan 54596, Republic of Korea.
College of Veterinary Medicine, Chonbuk National University, Iksan 54596, Republic of Korea.
J Immunol Methods. 2019 Jul;470:20-26. doi: 10.1016/j.jim.2019.04.006. Epub 2019 Apr 25.
This study aimed to investigate whether the human antigen presenting cells (APCs) can process and present Salmonella expressing H7N9 hemagglutinin (Sal-HA), neuraminidase (Sal-NA) or M2 ectodomain (Sal-M2e) to T cells and subsequently activate CD4 T cell responses in vitro. In this study, APCs generated from human peripheral blood mononuclear cells (PBMCs) were first treated with mitomycin-C, followed by stimulation with Sal-HA, Sal-M2e, Sal-NA or Salmonella alone for 24 h. Subsequently, stimulated APCs were coincubated with untreated PBMCs (1:10) of the same individual for 24 or 72 h and then analysed for cytokine induction and T cell proliferations by qRT-PCR assay and flow cytometry, respectively. Our results demonstrated that APCs stimulated with Sal-HA, Sal-M2e or Sal-NA induced significantly (p < .05) higher CD3CD4 T cell proliferations compared to the APCs treated with Salmonella alone. Our data further revealved that APCs treated with Sal-HA induced significantly (p < .05) higher CD3CD4 T cell responses compared to the APCs treated with either Sal-M2e or Sal-NA, which both induced almost comparable levels. The T cell proliferation responses were further measured by lymphocyte proliferation assay and the results showed that Sal-HA and Sal-M2e stimulated APCs induced significantly (p < .05) higher proliferations in T cells compared to the APCs stimulated with either Sal-NA or Salmonella alone. With respect to cytokine inductions, APCs treated with either Sal-HA or Sal-M2e induced significantly (p < .05) higher mRNA transcription levels of proinflammatory (IL-1β, IL-6, IL-12 and IL-23), Th1 (IFN-γ), Th17 (IL-17 and IL-21) and Th2 (IL-10 and TGF-β) cytokines in T cells compared to Sal-NA or Salmonella alone treated APCs. In conclusion, we show that Salmonella system can efficiently deliver vaccine antigens to APCs and is, thus, capable to elicit heterologous antigen-specific adaptive immunity.
本研究旨在探讨人类抗原呈递细胞 (APCs) 是否可以加工和呈递表达 H7N9 血凝素 (Sal-HA)、神经氨酸酶 (Sal-NA) 或 M2 胞外结构域 (Sal-M2e) 的沙门氏菌，从而在体外激活 CD4 T 细胞反应。在这项研究中，首先用丝裂霉素 C 处理来自人外周血单核细胞 (PBMCs) 的 APCs，然后用 Sal-HA、Sal-M2e、Sal-NA 或沙门氏菌单独刺激 24 小时。随后，将刺激的 APCs 与同一个体未经处理的 PBMCs（1:10）共孵育 24 或 72 小时，然后通过 qRT-PCR 分析和流式细胞术分别分析细胞因子诱导和 T 细胞增殖。我们的结果表明，与用沙门氏菌单独处理的 APCs 相比，用 Sal-HA、Sal-M2e 或 Sal-NA 刺激的 APCs 诱导的 CD3CD4 T 细胞增殖明显更高（p<0.05）。我们的数据进一步揭示，用 Sal-HA 处理的 APCs 诱导的 CD3CD4 T 细胞反应明显高于用 Sal-M2e 或 Sal-NA 处理的 APCs（p<0.05），而用 Sal-M2e 或 Sal-NA 处理的 APCs 诱导的 CD3CD4 T 细胞反应几乎相当。通过淋巴细胞增殖试验进一步测量 T 细胞增殖反应，结果表明，与用 Sal-NA 或沙门氏菌单独刺激的 APCs 相比，Sal-HA 和 Sal-M2e 刺激的 APCs 诱导的 T 细胞增殖明显更高（p<0.05）。关于细胞因子诱导，与用 Sal-NA 或沙门氏菌单独处理的 APCs 相比，用 Sal-HA 或 Sal-M2e 处理的 APCs 诱导的促炎（IL-1β、IL-6、IL-12 和 IL-23）、Th1（IFN-γ）、Th17（IL-17 和 IL-21）和 Th2（IL-10 和 TGF-β）细胞因子的 mRNA 转录水平明显更高（p<0.05）。总之，我们表明沙门氏菌系统可以有效地将疫苗抗原递送至 APCs，从而能够引发异源抗原特异性适应性免疫。
