Inthawong et al 2022 Whole blood assay Scrub Typhus
Description
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.
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Datasets for each figure are available in separate tabs within the excel file. SET1, 2 and 3 are the different optimisation protocols used for the whole blood assay (Set1: 18hs with antigen plus 4hs with BFA=22hs total, Set2: 24hs with antigen plus 24hs with BFA=48hs total, Set3: 44hs with antigen plus 4hs with BFA=48hs total). The whole blood assay is using intracellular flow cytometry staining as a readout. Figure 1 data: Relative frequency of live cells in unstimulated and SEB stimulated whole blood of n=5 healthy volunteers comparing protocols Set1, Set2 and Set3. Figure 2 data: Relative frequency of cytokine expressing T cells (in parent population, either CD4+ or CD8+ T cells) in whole blood (n=5 healthy) stimulated with SEB comparing protocols Set1, Set2 and Set3. Figure 4 data: Relative frequency of cytokine expressing T cells (in parent population, either CD4+ or CD8+ T cells) in whole blood of acute scrub typhus patients (n=5) stimulated with heat inactivated whole cell Orientia tsutsugamushi antigen (HI-WCA-OT), SEB or unstimulated using the optimal WBA protocol (Set1). Figure 5 data: Relative frequency of IFN-g secreting memory T cells (in parent population, either CD4+ or CD8+ T cells) in whole blood of acute scrub typhus patients (n=5) stimulated with HI-WCA-OT or left unstimulated. Figure S2: Data for correlation analysis of IFN-g secretion by T cells determined by WBA intracellular cytokine staining and ELISpot upon stimulation with HI-WCA-OT. ELISpot data: spot forming units/million PBMC, WBA ICS data: Relative frequency of IFN-g expressing T cells (in parent population, either CD4+ or CD8+ T cells).