Fig. 7
High frequencies of circulating CD4low non-classical monocytes predict increased odds of DHF. A–C PBMCs collected at first presentation from DENV-positive individuals progressing to DF (n = 5) or DHF (n = 4) after a primary infection were stained with a panel of metal-labelled antibodies and analysed by CyTOF. tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub-populations within gated CD3−CD19−CD14+ monocytes (A) and CD3−CD19−CD14−CD56+ NK cells (B). The tSNE plots in top panels display cell density and represent the pooled data for each group, while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot. Heatmaps show the median marker expression for each FlowSOM cluster (C). Differentially abundant populations were identified by CITRUS among gated monocytes. The top panels show differentially abundant populations identified in colours on a tSNE overlay, and the viSNE plots on the right-hand side from each top panel depict relevant marker expression on tSNE overlays. The lower left panels show the frequency of differentially abundant cell populations identified by CITRUS. Boxes represent the 25th to 75th percentiles, whiskers show the range (minimum to maximum), and lines represent the median of 6 (DF) and 6 (DHF) biological replicates. The lower right panels illustrate marker expression in differentially abundant populations (pink histograms), relative to background expression (lilac histograms). D Odds ratios as determined by logistic regression showing associations between cell frequencies and the risk of progressing to DHF. Symbols represent the odds ratio estimated using 10 patients progressing to DHF, and 11 patients progressing to DF after primary or secondary infection. The vertical lines depict the 95% confidence interval. *p < 0.05, **p < 0.01. E–F ROC curve (E) and predicted probability plot (F) classifying individuals progressing to DF (n = 11) or DHF (n = 10) at first presentation based on the frequency of CD4+ classical monocytes and CD4low non-classical monocytes