Fig 2: Representative gating strategy from flow cytometry analysis of intrahepatic neutrophils and monocyte/macrophage populations performed on total liver lysates of c-metfx/fx (a), c-met?hepa (b), and met/Keap1?hepa (c) after 4 weeks of MCD feeding. Neutrophils were gated by FSC/SSC, CD45+/alive CD11b+, and Ly6G+ cells. Macrophages were gated by FSC/SSC, CD45+/alive, Ly6G-, CD11b+, F4/80+. Quantification of the gated populations reported on histogram (d, f). (e) **p values < 0.01. *Data are expressed as mean ± SE, ANOVA test with p < 0.05 (N = 3 - 5).

Fig 3: The expansion and activation of MDSCs in patients with MALT lymphoma. (A) The proportion of myeloid-derived suppressor cells (MDSCs) in CD45+ peripheral blood mononuclear cells (PBMCs) is higher in newly diagnosed mucosa-associated lymphoid tissue (MALT) lymphoma patients (n = 8) compared to age-matched controls (n = 12); **P < 0.01. (B) Representative flow cytometric dot plots showing MDSCs in the peripheral blood of a patient and age-matched healthy individual. (C) Representative immunofluorescence images of CD11b+CD33+ MDSCs in gastric MALT lymphoma and paired normal tissues. Red, CD11b; green, CD33; and blue, 4' ,6-diamidino-2-phenylindole (DAPI); original magnification, 400×. (D) Quantitative analysis of MDSCs (CD11b+CD33+) in gastric MALT lymphoma and paired normal tissues; **P < 0.01. (E) Representative immunohistochemistry (IHC) images of arginase 1 (Arg-1) and inducible nitric oxide synthase (iNOS) in gastric MALT lymphoma and paired normal tissues. Original magnification, 400×. (F) Histoscores of Arg-1 and iNOS were higher in gastric MALT lymphoma tissues compared to control; **P < 0.01, *P < 0.05.

Fig 4: Cell isolation method affects microglial surface and intracellular marker expression in flow cytometric analysis. Flow cytometric analysis of surface and intracellular marker expression levels on microglia after ED37° versus MD4°. (a) Representative 2D-flow cytometric plot overlay with adjunct histograms and (b) scatterplots comparing forward scatter (FSC-A, t8 = 5.439, p = 0.0006) and side scatter (SSC-A, t8 = 18, p < 0.0001). (c) Photomicrographs illustrating the cell shape after MD4° or ED4° (20x magnification). (d–g) Representative 2D-flow cytometric plot overlays with adjunct histograms and scatter plots comparing geometric mean fluorescence intensities (gMFIs) of surface and intracellular expression of (d) CD45 (t8 = 13.28, p < 0.0001), (e) CD11b (t8 = 5.288, p = 0.0007), (f) SIRPa (t8 = 5.213, p = 0.0004) and (g) Fc?RI, (t8 = 1.030, p = 0.3). (h) A t-SNE clustering algorithm was used to depict CD11b expression in different populations of microglia. Left row showing relative intracellular CD11b expression levels for both conditions. The right row shows an overlay of the two conditions, as well as all populations overlaid with high CD11b ICS expression. (i) Scatterplot for the quantification of the CD11b intracellular signal for microglia isolated via MD4° versus ED37° (t8 = 24.43, p < 0.0001) Summary of two independent experiments, n = 4 and 5 biological replicates/group respectively. Unpaired two-tailed Student t-tests were used to compare the means. *** p < 0.001, **** p < 0.0001. Error bars represent the mean ± standard deviation.

Fig 5: LILRB4 deficiency causes changes in NK cell maturation and metabolism during ZIKV infection.(A) NK cell maturation determined by CD27 and CD11b staining. The stacked bar graphs show the percentage of NK cell population based on CD27 and CD11b expression. (B) RNA expression of Il-12a, Il-12ß, Il-15, Il-18, Il-2, Il-21, Il-27, and Il-7 in the brains of ZIKV-infected mice at 15 dpi. Data presented as fold induction over age-matched uninfected controls for each strain. (C–E) NK cells were isolated from pooled spleens of naive (P16; n = 5 per strain) or ZIKV-infected (15 dpi; n = 10–16 per strain) mice. ECAR and real-time changes of OCR were measured in IL-2–stimulated NK cells from naive mice (C) or nonstimulated NK cells from ZIKV-infected mice (D). Bar graphs indicate basal respiration (Basal), ATP production (ATP), and spare respiratory capacity (Spare). Cytotoxicity assay was performed against YAC-1 target cells. The graphs summarize the percentage of 7-aminoactinomycin D–positive (7-AAD+) YAC-1 cells (E). Data are representative of 3 independent experiments, each performed in triplicate or quadruplicate. Data were analyzed using 2-tailed unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.