Journal: Journal for Immunotherapy of Cancer

Article Title: p38 MAPK signaling in M1 macrophages results in selective elimination of M2 macrophages by MEK inhibition

doi: 10.1136/jitc-2020-002319

Figure Lengend Snippet: M2 macrophages are highly sensitive to MEK inhibition in vivo and ex vivo. (A) Immunohistochemistry of a PDA30364 tumor using an F4/80-specific antibody. Scale bar: 100 µm. (B) Flow cytometric identification of iNOS+ M1 and CD206+ M2 macrophages in a freshly dissociated tumor sample upon gating on the live CD45+, CD11b+, F4/80+ cell subset. (C) MHC-II (IA/IE) and CD86 surface levels of macrophages gated in (B). Mean±SEM, n=4. (D) Mice bearing PDA30364 tumors were treated with MEKi 30 mg kg −1 GDC-0623 (daily), anti-CSF1R antibody (1 mg once, followed by 0.5 mg every other day), or anti-CD40 antibody (200 µg on days 10, 12, 14, and 17 after tumor inoculation). Mean±SEM, n=5. Two-way ANOVA with post hoc Tukey test. (E) Flow cytometry-based quantification of M2 (CD206 + ) and M1 (iNOS + ) macrophages in PDA30364 tumors from mice treated as described in (C). Mean±SEM, n=4. One-way ANOVA with post hoc Dunnett test. (F) Dose–response curves of PDA30364 and M1 (20 ng mL −1 M-CSF+1 ng mL −1 IFNγ/LPS) and M2 (20 ng mL −1 M-CSF+2.5 ng mL −1 IL-4) polarized murine macrophages treated with GDC-0623. Mean±SEM, n=3. Numbers indicate best-fit inhibitory concentration (IC)50 values. (G) Viability of PDA30364 and murine M1 and M2 macrophages treated with GDC-0623 at 1 µM. Mean±SEM, n>4. One-way ANOVA with post hoc Tukey test. Significance levels are indicated by asterisks (*p≤0.05; **p≤0.01; ***p≤0.001: ****p≤0.0001). ANOVA, analysis of variance; IFNγ, interferon-γ; IL, interleukin; LPS, lipopolysaccharide; M-CSF, macrophage-colony stimulating factor; MEK, mitogen-activated protein kinase kinase.

Article Snippet: MEKi GDC-0623 (A-1181; Chemgood), p38 MAPKi BIRB 796 (S1574; DP, Selleckchem), murine multimeric CD40L (AG-40B-0020; Adipogen), poly I:C (tlrl-pic; InvivoGen), lipopolysaccharide (LPS) (L4391; Sigma), recombinant murine interferon-γ (IFNγ) (12343536; ImmunoTools), murine macrophage-colony stimulating factor (M-CSF) (12343115; ImmunoTools), murine interleukin (IL)-4 (12340042; ImmunoTools), murine IL-10 (12340105; ImmunoTools), anti-mouse CSF1R (BP0213; BioXCell), recombinant human M-CSF (11343115; ImmunoTools), human IFNγ (11343536; ImmunoTools), human IL-4 (11340045; ImmunoTools), human transforming growth factor-β1 (TGFβ1) (11343160; ImmunoTools), and human IL-10 (11340103; ImmunoTools).

Techniques: Inhibition, In Vivo, Ex Vivo, Immunohistochemistry, Flow Cytometry, Concentration Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Immune signatures of megakaryocytes in persistent inflammation-immunosuppression and catabolism syndrome

doi: 10.3724/abbs.2025087

Figure Lengend Snippet: MKs in the peripheral blood, bone marrow, and spleen of PICS and sepsis model mice and control sham mice (A) Schematic diagram showing the timeline of the experimental design. (B,C) Proportions of monocytes, B cells, plasma cells, and MKs in (B) PICS and (C) sepsis model mice after injection with anti-CD115, anti-B220, or anti-CD25 antibodies (n = 5–6 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant by one-way ANOVA. The error bars represent the range of the changes. (D) The proportions of MKs in the bone marrow and spleen of PICS model mice and sham mice. (E) CD41 staining of MKs in the bone marrow of PICS model mice and sham mice. (F) Size of two representative spleens from sham and PICS model mice. The error bars represent the SDs.

Article Snippet: To deplete specific cell types, PICS mice were injected intraperitoneally (i.p.) with anti-mouse CSF1R (CD115) (clone AFS98, which is usually used as a marker of bone marrow cells, such as monocytes, macrophages, and DCs) [ – ], anti-mouse B220 (clone AFS98, anti-B cell) , or anti-mouse CD25 (clone AFS98, anti-Treg) monoclonal antibodies (BioXCell, West Lebanon, USA).

Techniques: Control, Clinical Proteomics, Injection, Staining

Journal: bioRxiv

Article Title: Macrophage Antigen Presentation Is Unleashed by Pan-RAS Inhibition to Promote Antitumor Immunity

doi: 10.64898/2026.01.30.702886

Figure Lengend Snippet: A) Experimental scheme for B16-OVA transplantation in nude mice followed by RMC-6236 oral gavage (10 mg/kg, daily) ; B) C omparison of tumor size between vehicle and RMC-6236–treated nude mice ; C) Tumor growth curves ; D,E) Flow-cytometry analysis of TAM CD206 (D) and CD86 (E) with vehicle or RMC-6236 ; F) Immunofluorescence showing macrophages (F4/80, green) and MHC-I (red) with DAPI (blue) ; G–I) Flow-cytometry quantification in TAMs of MHC-I (H-2K b ) (G) , OVA-specific presentation (SIINFEKL) (H) , and MHC-II (I-A/I-E) (I); J) Schematic of anti-CSF1R– mediated monocyte/macrophage depletion during RMC-6236 treatment; K) Tumor growth with RMC-6236 plus IgG or anti-CSF1R ; L, M) Intratumoral CD8 + T-cell frequency after monocyte depletion ; N,O) Granzyme B (N) and perforin (O) in tumor CD8 + T-cells. Data are shown as mean ± SEM. *p < 0.05, **p< 0.01, ***p < 0.001 and ****p < 0.0001 using unpaired two-tailed t-tests and Two-way ANOVA followed by Sidak’s multiple comparisons test (n≥6).

Article Snippet: To deplete monocytes/macrophages, mice were treated with 300 μg anti-CSF1R antibody (clone AFS98, Bio X Cell, Cat. No. BP0213) via intraperitoneal injection every 3 days for the indicated pre-treatment period before tumor challenge , .

Techniques: Transplantation Assay, Flow Cytometry, Immunofluorescence, Two Tailed Test

Journal: STAR Protocols

Article Title: Murine brain tumor microenvironment immunophenotyping using mass cytometry

doi: 10.1016/j.xpro.2022.101357

Figure Lengend Snippet: Antibody master mix

Article Snippet: Anti-Mouse CD115 (AFS98)-144Nd—100 Tests , Fluidigm , SKU# 3144012B.

Techniques:

Journal: STAR Protocols

Article Title: Murine brain tumor microenvironment immunophenotyping using mass cytometry

doi: 10.1016/j.xpro.2022.101357

Figure Lengend Snippet:

Article Snippet: Anti-Mouse CD115 (AFS98)-144Nd—100 Tests , Fluidigm , SKU# 3144012B.

Techniques: Purification, Recombinant, Red Blood Cell Lysis, Centrifugation, Staining, Electron Microscopy, Antibody Labeling, Software, Sterility, Spectrophotometry

Journal: Cell

Article Title: Nr4a1 -Dependent Ly6C low Monocytes Monitor Endothelial Cells and Orchestrate Their Disposal

doi: 10.1016/j.cell.2013.03.010

Figure Lengend Snippet: Neutrophils Kill Endothelial Cells (A) Endothelial cell microscopic features of chimeric mice described in Figure 6 D expressed as the percentage of mononuclear cells (mono) or PMN-containing fields that present with the indicated lesions. (B) Representative FACS dot plots of peripheral blood cells of mice treated 8 hr earlier with Ly6G-depleting Ab (1A8) or isotype control (2A3). The arrow in the FSC/SSC panel indicates granulocytic cells, the percentage of Lin − CD115 − granulocytes are indicated in red. n = 3 mice per group, mean ± SEM. (C) Presence of intravascular mononuclear (left) and polymorphonuclear cells (right) as quantified by TEM in mice treated with 1A8 or 2A3 8 hr before kidney painting with R848. n = 3 mice per group, mean ± SEM. (D) Endothelial cell microscopic features of granulocyte-depleted and control mice. n = 3 mice per group, mean ± SEM. (E) Representative peritubular capillary containing a monocyte from a 1A8-treated mouse. (F) Proinflammatory cytokine production in vitro by sorted Ly6C low and Ly6C + monocytes after 24 hr stimulation with medium alone or R848 (top) in the absence or presence of a MEK inhibitor (PD) or for medium alone or LPS (bottom) in the absence or presence of the MEK inhibitor (PD) (bottom). Multiplexed ELISA, n = 3 mice per condition. (G) Schematic representation of the molecular and cellular features of the interaction of Ly6C low monocytes with the endothelium in a steady state and TLR7-mediated endothelial “safe disposal.” Also see Figure S3 .

Article Snippet: Rat IgG2a isotype control (2A3; Bio X Cell or BD PharMingen) anti-mouse Ly6G PE (1A8; BD Pharmingen), Anti-mouse CD115 FITC (CSF1R; AFS98 BD PharMingen), Anti-mouse CD16/32 (2.4G2BD Pharmingen), Anti-mouse CD3 Biotin (145 2C-11; BD PharMingen), Anti-mouse NK1.1Biotin (PK136; BD Pharmingen), Anti-mouse CD19 Biotin (1D3; BD PharMingen).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Cancer cell

Article Title: Extensive Remodeling of the Immune Microenvironment in B Cell Acute Lymphoblastic Leukemia.

doi: 10.1016/j.ccell.2020.04.015

Figure Lengend Snippet: Figure 3. Bone Marrow Immune B-ALL Microenvironment throughout Conventional Chemotherapy (A) UMAP visualization of 97,456 individual cells from seven individual B-ALL patients with matched diagnosis and relapse bone marrow samples—ETV6/RUNX1 B-ALL (ETV, n = 5) and Ph+ B-ALL (PH, n = 2)—as well as matched remission samples in all patients except ETV001. (B) Marker-based cell type identification of six broad immune cell types across all profiled cells. (C) Six transcriptionally distinct myeloid cell clusters overlaid on the UMAP representation. Cluster abbreviations, for example, DRR-M1 is based on diagnosis/ remission/relapse myeloid cluster 1. (D) Gene expression heatmap of top myeloid cluster-specific marker genes. (E) Expression levels of CD14, CSF1R, FCGR3A, CD1C, CDKN1C, and FLT3 in the myeloid cells overlaid on the UMAP representation. (F) Boxplot showing fraction of myeloid clusters in the myeloid fraction of individual patients. Wilcoxon rank-sum test performed to measure pairwise differences in representation between diagnosis, remission and relapse groups, with p values indicated on plots. Horizontal lines in the boxplots represent the median, the

Article Snippet: Individual leukemia recipient mice were treated every two days following transplantation with either 300 mg anti-mouse CSF1R (CD115) monoclonal antibody (AFS98 clone, BioXCell) or 300 mg rat IgG2a isotype control (BioXCell) for 30 days post-transplantation.

Techniques: Biomarker Discovery, Marker, Gene Expression, Expressing

Journal: Cancer cell

Article Title: Extensive Remodeling of the Immune Microenvironment in B Cell Acute Lymphoblastic Leukemia.

doi: 10.1016/j.ccell.2020.04.015

Figure Lengend Snippet: Figure 7. Leukemia-Associated Monocyte Targeting Enhances TKI Responsiveness In Vivo (A) Marker-based cell type identification analysis allowed prediction of six broad murine immune cell types across 27,162 profiled single cells from both bone marrow and peripheral blood of leukemia-bearing B-ALL recipients and healthy littermates. (B) Heatmap of select differentially expressed genes (based on KEGG pathways) distinguishing B-ALL recipient and healthy littermate non-classical monocytes as measured by log-transformed fold-change. (C) Csf1r mRNA expression overlaid on UMAP representation split into bone marrow or peripheral blood, and healthy or B-ALL conditions. (D) Kaplan-Meier analysis of Ph+GFP+ B-ALL transplant recipient survival after TKI and mAb treatment. Treatment regimen and number of recipients per condition are indicated. Three primary B-ALL were each transplanted into n > 4 recipient mice per condition. *p < 0.05, ****p < 0.0001, log rank test.

Article Snippet: Individual leukemia recipient mice were treated every two days following transplantation with either 300 mg anti-mouse CSF1R (CD115) monoclonal antibody (AFS98 clone, BioXCell) or 300 mg rat IgG2a isotype control (BioXCell) for 30 days post-transplantation.

Techniques: In Vivo, Marker, Transformation Assay, Expressing