Fig 2: The expanded ITM subset in dengue patients originates from the CL subset. (A) CD16–CD14 profile of monocytes in a representative healthy donor, a dengue patient, and a recovered patient. (B) Analysis of monocytes from a dengue patient using the new gating strategy. (C) Analysis of conventionally-gated subsets with the new gating strategy. (D) Percentage (%) common ITM subset in healthy donors, dengue patients and recovered patients. Data represent the means ± SD. (E) Proportion of each subset as a percentage of total monocytes using the conventional (a,c,e) and new gating strategies (b,d,f). Data represent the means ± SD.

Fig 3: Phagocytosis of SNECs in human blood neutrophils and monocytes is negatively correlated with the oxidative burst. (A, B) Peripheral blood from individuals with systemic lupus erythematosus (SLE; n = 13), chronic granulomatous disease (CGD; n = 3) and from normal healthy donors (NHD; n = 8) was incubated with human PBMC-derived SNECs for 3 h. Phagocytosis indices (PhIx) of CD14−CD16+CCR2- neutrophils (A) and CD14+CD16-/LOCCR2+ monocytes (B) were determined by flow cytometry. Patients with SLE exhibited significant accumulation of SNECs. Graphs show individual values, means ± S.E.M. **p < 0.01, ***p < 0.001, as determined by Student's t-test. (C, D) Correlation analysis between spontaneous intracellular production of ROS (DHR-123 MFI) and phagocytosis indices in blood CD14−CD16+CCR2- neutrophils (C) and CD14+CD16-/LOCCR2+ monocytes (D). (E, F) Correlation analysis between levels of anti-dsDNA autoantibodies and PhIx in blood CD14−CD16+CCR2- neutrophils (E) and CD14+CD16-/LOCCR2+ monocytes (F). Pearson coefficients (r) and p values (determined by 2-tailed Student's t-test) in C-F are depicted within the graphs.

Fig 4: The CD16+ monocytes differ phenotypically from CD16- monocytes. (A) t-SNE clustering of monocytes using all 34 markers (a) and with the three monocyte subsets overlaid (b). (c) Identification of the three subsets based on the conventional CD16–CD14 plot. (B) t-SNE clustering of monocytes without CD16. (C) t-SNE clustering of monocytes without CD16 and CD14.

Fig 5: A new marker combination objectively identifies monocyte subsets. (A) Expression heatmaps of the five selected markers on total monocytes on t-SNE plots using data from CyTOF (top panel) and on CD16-CD14 plots using data from flow cytometry (bottom panel). (B) The new gating strategy for the three subsets using the five selected markers. (C) Analyzing the conventionally-gated subsets with the new gating strategy. (D) Purity of the New subset gates. Data represent the means ± SD. (E) Proportion of each subset as a percentage of total monocytes using the two different gating strategies. Data represent the means ± SD.