Inthawong et al 2022 Whole blood assay Scrub Typhus

Using whole blood provides the possibility to stimulate peripheral T cells without the need for prior processing, is more suitable when sample volume is limited and can be performed in resource limited settings. The aim of this study was to optimise a whole blood assay using intracellular cytokine staining as a readout in order provide a robust tool for the characterisation of antigen-specific T cell responses to Orientia tsutsugamushi (OT), the causative agent of Scrub typhus with an emphasis to make the protocol feasible to run at field study sites. The protocol was optimised using whole blood from healthy volunteers and stimulating with staphylococcal enterotoxin B (SEB). The optimal stimulation conditions were 18hs of antigen stimulation followed by another 4 hours in the presence of brefeldin A (BFA) to block cytokine secretion. This protocol was subsequently used to study polyfunctional CD4+ and CD8+ T cell responses to heat killed whole cell antigen derived from OT and shows polyfunctional CD4+ T cell responses in acute scrub typhus patients. Flow cytometry data was acquired on a MACSQuant 10 Analyzer (Miltenyi Biotec) and analysed using FlowJo Version 10.5.3 (BD Biosciences). ELISpot data was generated using a Human IFN-g ELISpot Basic Kit (ALP) from Mabtech AB, 3420-2A and plates were read on a CTL ELISPot reader (Cellular Technology Limited, USA) using Immunospot 3.1 software and SmartCount settings.

使用全血可无需预处理即可刺激外周T细胞，在样本体积受限的场景下更具适用性，且可在资源匮乏的环境中开展实验。本研究旨在优化以细胞内细胞因子染色（intracellular cytokine staining）为读出指标的全血检测方法，以期构建一款稳健的工具，用于表征针对恙虫病（Scrub typhus）致病病原体——恙虫病东方体（Orientia tsutsugamushi, OT）的抗原特异性T细胞应答，重点在于使该实验方案可在野外研究现场顺利实施。本研究采用健康志愿者的全血，以葡萄球菌肠毒素B（staphylococcal enterotoxin B, SEB）进行刺激，对该实验方案进行了优化。最佳刺激条件为：抗原刺激18小时后，加入布雷菲德菌素A（brefeldin A, BFA）继续培养4小时以阻断细胞因子分泌。随后，该方案被用于探究针对OT来源的热灭活全细胞抗原的多功能CD4+及CD8+ T细胞应答，并在急性恙虫病患者样本中检测到了多功能CD4+ T细胞应答。流式细胞术数据采用MACSQuant 10分析仪（Miltenyi Biotec）采集，并使用FlowJo 10.5.3版本（BD Biosciences）进行分析。ELISpot检测数据采用Mabtech AB公司生产的人类干扰素γ（IFN-γ）ELISpot基础试剂盒（ALP型，货号3420-2A）制备，并使用CTL ELISpot读板仪（美国Cellular Technology Limited）搭配Immunospot 3.1软件及SmartCount分析参数完成读板。