Fig 2: Bone marrow monocytes increased prior to adipose tissue macrophages.Analysis of monocytes and macrophages in the stromal vascular fraction (SVF) of adipose tissue via flow cytometry analysis, identified first by size and granularity through forward and side scatter and then using the following markers: CD11b CD115 F4/80 Ly6C GR1 CD11c (n=7–10 mice in each dietary group at each feeding duration). (A) Monocytes, (B) Total macrophages, (C) Pro-inflammatory macrophages. (D–F) Whole adipose tissue gene expression of the myeloid marker Itgam, macrophage marker Adgre and pro-inflammatory myeloid marker Itgax by RTqPCR. Each symbol represents a biological replicate analyzed in duplicate and normalized to average of a group of housekeeping genes. (n=5 mice in each dietary group at each feeding duration). (G–H) Gene expression analysis of embryonic derived tissue resident macrophage markers Timd4 and Marco by RTqPCR. Each symbol represents a biological replicate analyzed in duplicate and normalized to average of a group of housekeeping genes (n=3–5 mice in each dietary group at each feeding duration). (I) Flow cytometry analysis of total BM monocytes (n=7 mice in each dietary group at each feeding duration). (J) Monocyte subsets in the bone marrow with HFD-feeding (n=7 mice in each dietary group at each feeding duration). Results are expressed as mean ± SEM. One-way ANOVA was used for statistical analysis at each dietary group and across feeding periods. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 and ns = not significant p>0.05. Figure 2—source data 1.Bone marrow monocytes increased prior to adipose tissue macrophages.

Fig 3: Most border-associated macrophages and a subset of parenchymal microglia are derived from the Hoxb8 lineage, with Hoxb8 microglia being selectively sensitive to loss of Myb function. (A) Representative images of brain sections of frontal cortex from E14.5 Myb+/+ and Myb-/- embryos. Scale bars: 50 µm. (B,C) Number of (B) Hoxb8 (B8L) microglia and (C) non-Hoxb8 (nB8L) microglia in the brains of E14.5 Myb+/+ and Myb-/- embryos. n=4 biological replicates per group. Unpaired t-test comparing Myb+/+ with Myb-/- in B8L microglia (P=0.0238) and nB8L microglia (P=0.2704). (D) Representative image of a brain section of the frontal cortex from a 2-month-old Cx3cr1GFP/+; Hoxb8IRES-Cre/+; Rosa26CAG-LSL-tdTomato/+ mouse stained with anti-CD206. White arrows indicate nB8L and B8L microglia (MG). Magenta arrows indicate mMF cells (a type of BAM) that are tdTomato+ GFP+ CD206+. Scale bar: 50 µm. (E) Representative flow cytometry plots examining tdTomato and GFP signals in the border-associated macrophage (BAMs: F4/80+ CD11b+ CD206+) and parenchymal microglia (MG: F4/80+ CD11b+ CD206-) populations from a brain of a 2-month-old mouse. The FACS analysis of BAMs include the mMF, pvMF and cpMF subsets. (F) Percentage of B8L and nB8L cells in the BAM and microglia populations. n=5 mice. Two-way ANOVA with post-hoc analysis comparing B8L cells with nB8L cells in both BAMs and MG populations (P<0.0001). Gray circles are individual data points. Data are mean±s.e.m. n.s. non-significant, *P<0.05, ****P<0.0001. Data represented in all graphs are from two independent experiments.

Fig 4: Effects of combinatorial blockade of PD-1, CTLA-4, and LAG-3 on Treg and MDSC population in ovarian TME. (A) Dual and triple antibody blockade treatment decreased the frequency of CD4+CD25+FoxP3+ cells at early time point of ovarian tumor progression. TALs and TILs were isolated as described above and stained for the expression of CD4+, CD25, and FoxP3 protein and analyzed using flow cytometry. (B) Arginase 1 (ARG1)-expressing population was increased in the ovarian TME as compared with that in spleen. Splenocytes and TALs were isolated as described in Fig. 5. Frequency of ARG1+ population in the CD45+CD11b+ cells was determined based on the gating shown in Fig. S3. (C–E) Various checkpoint blockade combinations reduced the frequencies of ARG1+-MDSCs. The frequency of ARG1+-Ly6G+6C+ cells was significantly reduced by PD-1/CTLA-4 dual blockade (C). All three dual blockades of PD-1/LAG-3, PD-1/CTLA-4, and LAG-3/CTLA-4 significantly reduced the frequency of ARG1+-Ly6G-6C+ (D) and ARG1+-F4/80+ cells (E). Data were obtained from 5 mice per group, analyzed using GraphPad Prism 6, and are representative of two independent experiments. Error bars represent SD. Statistical significance was determined by Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5: STIM1/STIM2 Deficiency Prevents Leukemia-Associated Inflammation(A) Wright-Giemsa-stained blood smears from healthy WT control mice (untreated) and mice with WT and Stim1/2-/- leukemia. Arrows indicate neutrophils. Magnification 4003. Data are representative of three mice per group.(B) H&E-stainedBMsections of mice with WT and Stim1/2-/- leukemia at day 24 of disease. Magnification 400×. Inset for WTBMshows a macrophage engulfing red blood cells. Images are representative of 7–10 mice per cohort.(C and D) Percentages and absolute numbers of F4/80+CD11b+ macrophages in the BM (C) and spleen (D) of healthy WT control mice (untreated, UT) and mice withWT and Stim1/2-/- leukemia. Representative flow cytometry plots at days 14 and 24 of disease (left) and quantification of macrophage numbers (right). Data are representative of 3–5 mice per cohort and time point.(E) Serum levels of cytokines in healthy WT control mice (UT) and mice with WT and Stim1/2-/- leukemia measured by multiplex assay. Data are representative of 3–5 mice per cohort.All values represented by bar graphs (C–E) are mean ± SEM. Statistical analysis in all experiments was performed using Student’s t test. *p < 0.05. nd, not detected; ns, not significant; nt, not tested (no cells or mice available).