Journal: Immunity

Article Title: PD-L1:CD80 Cis -Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

doi: 10.1016/j.immuni.2019.11.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human peripheral blood CD14+ monocytes , iXCells , Cat # 10HU-008.

Techniques: Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Cell Isolation, Software, Imaging

Journal: The EMBO Journal

Article Title: A G1‐like state allows HIV ‐1 to bypass SAMHD 1 restriction in macrophages

doi: 10.15252/embj.201696025

Figure Lengend Snippet: A, B Stimulated or unstimulated MDM were stained for classical macrophage markers (CD68, CD14) and M1 and M2 macrophage markers (CD163, CD80, CD86 and CD40) and analysed by FACS.

Article Snippet: Antibodies used were as follows: anti‐cdc2 (Cell Signaling Technology, Beverly, MA, USA); anti‐CDK2 (H‐298, Santa Cruz Biotechnology); anti‐pCDK2(Thr160) (Bioss Inc., MA, USA); anti‐CDK4 (DCS156, Cell Signaling Technology); anti‐CDK6 (B‐10, Santa Cruz Biotechnology); anti‐SAMHD1 (ab67820, Abcam, UK), beta‐actin (ab6276, abcam, UK); mouse anti‐MCM2 (BM‐28, BD Biosciences, UK); and rabbit anti‐MCM2 (SP85) from Sigma; pSAMHD1 (a kind gift from M. Benkirane) and ProSci (Poway, CA, USA); anti‐Geminin (NCL‐L‐Geminin, Leica); anti‐mouse F4/80 and CD11b (kind gift from S. Yona, UCL); anti‐human CD68, CD14, CD163, CD80, CD86, CD40 (kind gift from M. Noursadeghi).

Techniques: Staining

Journal: Molecular Therapy Oncolytics

Article Title: Myxoma Virus Optimizes Cisplatin for the Treatment of Ovarian Cancer In Vitro and in a Syngeneic Murine Dissemination Model

doi: 10.1016/j.omto.2017.08.002

Figure Lengend Snippet: MYXV Infection in OC Ascites-Associated CD14 + Monocytes Remodels the Intracellular Signaling Pathway (A) OC patient ascites-associated CD14 + monocytes/macrophages display non-canonical STAT3 signaling. CD14 + monocytes were enriched from ascites fluid of patient (e.g., OvCa37) and tested for phosphorylation of STAT3 on both Y705 and S727 by flow cytometry. The signature of non-canonical STAT3 signaling is characterized by minimal phosphorylation at the Y705 site of STAT3. (B) MYXV infection in ascites-associated CD14 + monocytes causes inhibition of STAT3 phosphorylation at the Ser727 residue. CD14 + monocytes were enriched from patient-ascites fluid and tested for purity via flow cytometry. Monocytes were immediately mock treated and infected with WT or M062R -null MYXV at an MOI of 10 for 1 h; washing with PBS followed before the cells were cultured for 18 hr. Media was harvested for multiplex array ( Figure 5 ). Cells were fixed and permeablized for intracellular staining as described in the to examine the level of STAT3 phosphorylation at the Ser727 residue. (C) Infection by MYXV does not cause general change in cell viability in ascites CD14 + cells. Patient CD14 + ascites-associated monocytes were mock treated or infected with WT or M062R -null MYXV for 18 hr before the cells were stained with propidium iodide (PI). Live cells were gated to examine GFP (infection) and the presence of PI staining.

Article Snippet: Human CD14 + monocytes are from healthy female donors (Lonza).

Techniques: Infection, Phospho-proteomics, Flow Cytometry, Inhibition, Residue, Cell Culture, Multiplex Assay, Staining

Journal: Molecular Therapy Oncolytics

Article Title: Myxoma Virus Optimizes Cisplatin for the Treatment of Ovarian Cancer In Vitro and in a Syngeneic Murine Dissemination Model

doi: 10.1016/j.omto.2017.08.002

Figure Lengend Snippet: MYXV Infection in Patient-Ascites-Associated CD14 + Monocytes Inhibits Cytokine Secretion that Is the Signature of Immunosuppressive Tumor Environment Patient-ascites-associated CD14 + monocytes or CD14 + monocytes from a healthy female donor were mock treated or infected with WT or M062R null MYXV for 18 hr before supernatant was collected for multiplex array. Comparisons in levels of IL-10 (A) and IL-6 (B) are shown for mock treatment and infection in patients and healthy monocytes. To test the effect of STAT3 in cytokine secretion, patient-ascites-associated CD14 + monocytes were treated with 5 μM Stattic, a STAT3 inhibitor, for 48 hr before supernatant was collected for multiplex array. (C and D) The effect of Stattic on the levels of IL-10 (C) and IL-6 (D) in patient monocytes. The error bars represents SD, and the mean is calculated from the quantification in triplicate.

Article Snippet: Human CD14 + monocytes are from healthy female donors (Lonza).

Techniques: Infection, Multiplex Assay

Journal: Molecular Metabolism

Article Title: Activated macrophages control human adipocyte mitochondrial bioenergetics via secreted factors

doi: 10.1016/j.molmet.2017.07.008

Figure Lengend Snippet: Effects of LPS/IFNγ- and IL10/TGFβ-activated primary MΦ-CM on bioenergetics of human SGBS and primary adipocytes . Primary human CD14 + monocytes from 4 healthy donors were cultured, differentiated to macrophages (naïve MΦs) and activated with LPS/IFNγ or IL10/TGFβ as described in . (A) Morphological changes detected by bright field microscopy (one representative experiment shown). (B) mRNA expression of CD163 and CD40 analyzed with ΔΔCT method using naïve MΦs as Calibrator (n = 4). (C) CD163 and CD40 mRNA expression presented as ratio. (B, C) *p < 0.05 vs. LPS/IFNγ-activated MΦ, ***p < 0.001 vs. LPS/IFNγ-activated MΦ, ###p < 0.001 vs. naïve MΦs. (D) mRNA expression of UQCRC2 and NDUFB8 analyzed with ΔΔCT method using naïve THP1 macrophages as Calibrator (n = 4). ( E–G) SGBS adipocytes were treated with either cell-free control media, LPS/IFNγ-activated or IL10/TGFβ-activated MΦ-CM collected from 3 different donors for 48 h as described in and analyzed as described in Figure 4 , Figure 5 . Data are normalized to cell number (DNA content) and are the mean + SEM of 5 independent experiments each performed in 2–8 wells condition. OCR and ECAR traces vs time are shown in ( Figure S4B and C ). (E) ATP-linked respiration of SGBS adipocytes after treatment for 48 h with indicated MΦ-CM. (F) ATP-linked respiration of SGBS adipocytes presented as relative change in OCR in percent of cell-free control. (G) ECARs due to glycolysis of SGBS adipocytes after treatment for 48 h with indicated MΦ-CM. (H – J) Human primary subcutaneous adipocytes were treated with either cell-free control media, LPS/IFNγ-activated or IL10/TGFβ-activated MΦ-CM from 3 different donors as described in . Bioenergetic profile was analyzed as described in Figure 4 , Figure 5 . Data are presented as fold change to the mean rates of cell-free control for each donor and are the mean + SEM of experiments from 3 adipocyte donors performed in 6–8 wells per condition and donor. OCR and ECAR traces vs time are shown in ( Figure S4E and F ) (H) ATP-linked respiration of human primary adipocytes after treatment for 48 h with indicated MΦ-CM. (I) ATP-linked respiration of human primary adipocytes presented as relative change in OCR in percent of cell-free control. (J) ECARs due to glycolysis of human primary adipocytes after treatment for 48 h with indicated MΦ-CM. (E)–(J) *p < 0.05 vs. LPS/IFNγ-activated MΦ-CM; **p < 0.01 vs. LPS/IFNγ-activated MΦ-CM; ***p < 0.001 vs. LPS/IFNγ-activated MΦ-CM; #p < 0.05 vs. cell-free control media.

Article Snippet: Human CD14 + blood monocytes from 4 healthy donors were purchased from Zenbio (Research Triangle Park, NC, USA) and differentiated with 10 ng/ml CSF1 for 7 days to adherent macrophages (MΦ) before same stimulation as for THP1 cells was performed.

Techniques: Cell Culture, Microscopy, Expressing, Control