M-DC8⁺ monocytes from viremic patients produce greater amounts of TNFα than those from healthy donors and are responsible for a large part of the TNFα overproduction after LPS stimulation. (A-D) After 18-hour stimulation with or without LPS, TNFα concentrations were measured in culture supernatants from (A) total PBMCs (1 × 10⁶ cells/mL; 8 healthy donors and 7 viremic patients), (B) total vs [M-DC8⁺ cell]–depleted PBMC (5 × 10⁵ cells/mL; 6 healthy donors and 5 viremic patients), (C) FACS-sorted M-DC8⁺ CD11c⁺CD14⁺CD16⁺⁺ monocytes from 4 healthy donors and 4 viremic patients (2.5 × 10⁵ cells/mL), and (D) monocyte subsets from 1 healthy donor (5 × 10⁵ cells/mL; representative of 3 independent experiments). (E) Plasma TNFα concentrations from 16 healthy donors (HIV⁻, cART⁻, open circles), 8 virologically suppressed patients (HIV⁺, cART⁺, gray circles) and 15 viremic patients (HIV⁺, cART⁻, filled circles). Patients with M-DC8⁺ monocyte counts > 50/μL as defined in Figure 2B are shown (red circles). (F-H) PBMCs were cultured for 7 hours with or without LPS. (F) Dot plot of 9-color intracellular TNFα FACS analysis from (left panels) 1 representative healthy donor and (right panels) 1 representative viremic patient showing TNFα production in different monocyte (top panels), DC, and lymphocyte populations (bottom panels) after stimulation of PBMCs with LPS. Percentages of TNFα-positive cells among the parent population and MFI of the TNFα-positive population (in brackets) are indicated. (G-H) After LPS stimulation, comparison of 6 healthy donors and 8 viremic patients for (G) the percentages of TNFα-positive cells among different monocyte subsets, and (I) of the MFI of the TNFα-positive populations in the same experiments as panel G. P values calculated using the Mann-Whitney test; bars indicate medians. ND indicates not determined; and Unst, unstimulated.