Table 1.
Different stimulation protocols used to optimize the WBA.
Fig 1.
(A) Viability of lymphocytes (% live cells in the single cell gate) in healthy blood samples (Median and IQR for n = 5 of biological replicates) stimulated with and without SEB using three different WBA stimulation protocols (SET1, SET2 and SET3, see details in Table 1) followed by ICS and flow cytometry. Difference in % live cells in healthy samples with or without SEB stimulation across the three WBA stimulation protocols were analysed with one-way ANOVA (Kruskal Wallis) followed by Tukey’s multiple comparisons test. Only significant 2-tailed p values (p<0.05) are shown in the bar graphs. (B) Representative flow cytometry density plots showing CD4 and CD8 expression on CD3+ T cells stimulated with (c, d) and without SEB (a, b) using protocol SET1 and subsequently stained with (b, d) and without (a, c) a fixable live-dead dye.
Fig 2.
Functional characterization of distinct T cell subsets from healthy donors (Median and IQR for n = 5 of biological replicates) using three different WBA stimulation protocols.
Frequency of IFN-γ, TNF and IL-2 single (1+), double (2+) or triple (3+) positive CD4+ (A) and CD8+ (E) T cells after stimulation with SEB according to three different protocols (SET1-3) as described in Table 1. Difference in % cytokine secreting in parent population in healthy samples stimulated with SEB across the three WBA stimulation protocols were analysed with one-way ANOVA followed by Tukey’s comparisons test, only significant 2-tailed p values (p<0.05) are shown on the bar graphs. Pie charts show the proportion of cytokine-producing CD4+ (B-D) and CD8+ (F-H) T cells according to their polyfunctionality. Percentage under each pie chart indicates the proportion of total cytokine secreting cells.
Fig 3.
Procedure to perform the optimised WBA for patients with scrub typhus at a field site laboratory.
HI-WCA-OT = heat inactivated whole cell antigen O. tsutsugamushi, SEB = staphylococcal enterotoxin B, RBC = red blood cell, ICS = intracellular cytokine staining. Figure was created using Biorender.com (https://biorender.com/).
Fig 4.
Functional characterization of CD4+ (A) and CD8+ (C) T cell subsets in patients with acute scrub typhus (Median and IQR for n = 5 of biological replicates). Frequency of IFN-γ, TNF and IL-2 single (1+), double (2+) or triple (3+) positive CD4+ and CD8+ T cells after stimulation with HI-WCA-OT or without stimulation. Respective pie charts show the proportion of cytokine-producing cells in response to HI-OT-WCA in CD4+ (B) and CD8+ (D) T cells according to their polyfunctionality. Percentage under each pie chart indicates the total frequency of cytokine expressing T cells. Difference in % cytokine secreting in parent population between stimulated and unstimulated samples were analysed by Mann-Whitney U-test, only significant 2-tailed p values (p<0.05) are shown in the bar graphs.
Fig 5.
(A) Representative flow cytometry dot plots showing IFN-γ and CD45RA expression on CD4+ and CD8+ T cells following stimulation with HI-WCA-OT or left unstimulated. (B) Frequency of IFN-γ positive CD4 and CD8 T cells amongst CD45RA positive and negative cell subsets when stimulated with HI-WCA-OT and in unstimulated controls (Median IQR for n = 4 of biological replicates). Differences in % IFN-γ secreting cells in each subsets between unstimulated and HI-WCA-OT samples were analysed by Mann Whitney U-test. Only significant 2-tailed p values (p<0.05) are shown in the bar graph.