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.