Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Large expansion of phenotypically defined sx-MDSCs comprised mainly of M-MDSC and PMN-MDSCs immediately after surgery (POD1). ( A ) Representative flow plots showing CD33 versus Lineage (CD3/CD56/CD19) for baseline (left) and POD1 (right) PBMCs. ( B ) Representative flow plots gated down on CD33 + Lin − cells showing CD15 versus CD14 expression. ( C ) Healthy controls (n=15), Baseline, and POD1 patients (n=44) proportion of CD33 + Lin − cells (left) and MDSC subsets (right). ( D ) The MFI of HLA-DR gated on M-MDSCs (left) and the proportion of M-MDSCs that are HLA-DR lo (right) in healthy controls, baseline and POD1 patients with cancer. ( E ) Representative histograms of common M-MDSC and PMN-MDSC markers, before and after surgery. Statistical analysis was performed using Wilcoxon matched pairs signed-rank test or Kruskal-Wallis with Dunn’s post test. **** p≤0.0001. FMO, fluorescence minus one control; HLA-DR, human leukocyte antigen - DR isotype; E-MDSC, early stage-MDSC; M-MDSC, monocytic-MDSC; MDSC, myeloid derived suppressor cell; sx-MDSC, surgery-induced MDSC, MFI; mean fluorescence instensity; PBMC, peripheral blood mononuclear cell; PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Expressing, Fluorescence, Control, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Sx-MDSCs suppress NK92-MI cytotoxicity. ( A ) Schematic of MDSC-NK92 co-culture suppression assay with CP450-labeled K562 target cells. ( B ) NK92-MIs were co-cultured at increasing ratios with CD33+ cells for 6 or 24 hours (in the absence of K562s) to assess CD33+ cell death by NK92-MI (left). Sx-MDSCs from POD1 patient samples (n=10) were co-cultured with NK92-MI cells and NK cytotoxicity was measured as % dead K562 (right). ( C ) % suppression was calculated as the reduction of NK cytotoxicity, normalized to NK cells alone (ratio=0). CD33+ cells isolated from baseline and patient-matched POD1 blood were co-cultured with NK92-MI and K562 targets to measure their suppressive capacity (n=5). ( D ) Representative images of sorted cells stained with Giemsa Wright. ( E ) Bulk Sx-MDSCs, M-MDSCs, PMN-MDSCs, and HDN from POD1 patients (n=3) co-cultured with NK92-MI to measure effect on NK cytotoxicity. The NK92-MI alone control group is indicated by the broken horizontal line. Statistical analysis used Kruskal-Wallis with Dunn’s post-test. ****p≤0.0001. HDN, high-density neutrophils; M-MDSC, monocytic-MDSC; MDSC, myeloid-derived suppressor cell; NK, natural killer; sx-MDSC, surgery-induced MDSC, PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Co-Culture Assay, Suppression Assay, Labeling, Cell Culture, Isolation, Staining, Control, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: scRNA-seq of cryopreserved PBMCs before and after surgery reveals drastically altered monocyte/myeloid cell expression profiles on POD1. ( A ) Schematic showing PBMCs (cryopreserved) from six matched baseline and POD1 patients were processed for multiplexed scRNA-seq with the 10x Genomics Chromium platform. ( B ) Dot plot displaying the relative expression of the top three marker genes (x-axis) of each cluster (y-axis). ( C ) UMAP plot of scRNA-seq data. Each point corresponds to a single cell and is colored by cluster. ( D ) Identical UMAP embedding as in ( C ), with Baseline and POD1 cells labeled. ( E ) UMAP plot of the CD14+ and CD16+ monocyte population at baseline and POD1. ( F ) GSEA showing the NES of the top upregulated and downregulated pathways. All gene sets are significantly enriched (FDR<0.05). ( G ) UMAP plot showing activity of an M-MDSC gene set from Alshetaiwai et al ; in monocyte populations in Baseline and POD1 samples. ( H ) NMF plots of gene expression programs for NMF 1, 2, 3 and 4 (see for all NMF programs). ( I ) A heatmap of selected genes driving the various NMF gene expression programs (z-score transformed; ranked according to NMF1). FDR, false discovery rate; GSEA, gene set enrichment analysis; M-MDSC, monocytic-myeloid-derived suppressor cell; NES, normalized enrichment scores; NMF, non-negative matrix factorization; PBMC, peripheral blood mononuclear cell; POD1, postoperative day 1; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Expressing, Marker, Labeling, Activity Assay, Gene Expression, Transformation Assay, Derivative Assay, RNA Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: PI3K inhibitors reverse the suppressive effects of sx-MDSCs on NK cells. ( A ) A library of 147 small molecules covering the major cellular signaling pathways was screened (n=4 experiments) at 1 µM in the MDSC-NK suppression assay. Compounds that improved NK cell cytotoxicity (blue, left y-axis) by >50% from DMSO control (black dotted line), without impacting NK cell viability (red, right y-axis), were considered hits. LY294002 (purple circle) improved cytotoxicity in all screens and was the top hit in 3/4 screens. ( B ) LY294002 improves NKC (n=6). ( C ) Dose response of LY294002 and the effect on NKC and K562 viability. ( D ) pan-PI3K and ( E ) PI3K-γ specific inhibitors (IPI-549, TG100-115) in NK92—MDSC suppression assays. ( F ) pan-PI3K and ( G ) PI3K-γ specific inhibitors (IPI-549, TG100-115) in MDSC suppression assays with primary healthy donor NK cells. Statistical analysis used one-way ANOVA with Dunnett’s multiple comparisons test. ***p<0.0001. ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; MDSC, myeloid-derived suppressor cell; NK, natural killer; sx-MDSC, surgery-induced MDSC.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Protein-Protein interactions, Suppression Assay, Control, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: PI3K pathway activation in postoperative MDSCs and functional impact of PI3K inhibition. ( A ) Cohort of differentially expressed genes from baseline versus POD1 MDSCs. ( B ) Box plot inference of PI3K pathway activity in baseline versus POD1 MDSCs. ( C ) Pathway diagram of PI3K signaling and Akt phosphorylation. Phosphorylation status of AKT at the T308 (left) and S473 residue (right) (n=5). ( D ) Effect of PI3K inhibitors on pAKT (T308) MFI (top), MDSC-NK suppression (middle), and NKC (bottom). ( E ) Effect of IPI-549 on POD1 expression of anti-inflammatory versus pro-inflammatory mRNA transcripts, normalized to baseline. Statistical analysis used Wilcoxon matched pairs signed-rank tests and one-way ANOVA with Dunnett’s multiple comparisons test (vs DMSO control). ****p>0.0001. ANOVA, analysis of variance; ARG1, arginase-1; CCL, CC chemokine ligand; DMSO, dimethyl sulfoxide; IL, interleukin; MDSC, myeloid-derived suppressor cell; mRNA, messenger RNA; MFI, mean fluorescence intensity; NK, natural killer; POD1, postoperative day 1; TGF, transforming growth factor; TNF, tumor necrosis factor.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Activation Assay, Functional Assay, Inhibition, Activity Assay, Phospho-proteomics, Residue, Expressing, Control, Derivative Assay, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Blockade of PI3Kγ signaling in sx-MDSCs reduces NK cell suppression and metastatic disease in mouse models of surgical stress. ( A ) Ex vivo effect of PI3K inhibitors on pAKT (T308) phosphorylation in splenic MDSCs from C57Bl/6 mice (n=4). ( B ) Effect of preoperative in vivo administration of PI3K inhibitors on pAKT (T308) phosphorylation measured on POD1 in splenic MDSCs (n=4). ( C ) Ex vivo effect of PI3K inhibitors on MDSC-NK %suppression (n=3). ( D ) Effect of preoperative in vivo administration of PI3K-γ inhibitors on sx-MDSC suppressive capacity. ( E ) Adoptive transfer of representative images of lungs is shown. One-way ANOVA with Holm-Sidak or two-way ANOVA with Dunnett’s multiple comparisons tests were performed. ****p<0.0001. ANOVA, analysis of variance; M-MDSC, monocytic-MDSC; MDSC, myeloid-derived suppressor cell; MFI, mean fluorescence intensity; NK, natural killer; sx-MDSC, surgery-induced MDSC, PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Ex Vivo, Phospho-proteomics, In Vivo, Adoptive Transfer Assay, Derivative Assay, Fluorescence

Journal: Acta Neuropathologica Communications

Article Title: Spatial characterisation of TREM2 expression in actively demyelinating multiple sclerosis lesions supports its key roles in lipid metabolic pathways

doi: 10.1186/s40478-026-02241-x

Figure Lengend Snippet: Spatial transcriptomics in hippocampal tissue containing chronic active lesions from two cases (MS13C and MS15A) of secondary progressive MS. a Schematic of spatial transcriptomics workflow using sample integration, clustering and deconvolution functions. b LFB staining and MHC class II immunohistochemistry were used to identify lesioned (yellow outline) areas (2 mm scale bar; left panels). c Cell populations were estimated by deconvolution analysis of spatial gene expression profiles in each cluster. Clusters 0, 4, 11, 14, 15, and 16 contained a higher proportion of glial cells relative to other clusters associated with neuronal cell types in the hippocampus. d Quantitative analysis of spatial distributions of total TREM2 + spots and TREM2 in relation to selected myeloid cell markers ( CD68, CD163, IBA1 ). OPC = Oligodendrocyte progenitor cell, vSMC = Vascular smooth muscle cell, OLG = Oligodendrocyte

Article Snippet: Rabbit anti-human TREM2 (Polyclonal) , Nonspecific , ProSci, USA (13-679) , 1:100.

Techniques: Spatial Transcriptomics, Staining, Immunohistochemistry, Gene Expression

Journal: Acta Neuropathologica Communications

Article Title: Spatial characterisation of TREM2 expression in actively demyelinating multiple sclerosis lesions supports its key roles in lipid metabolic pathways

doi: 10.1186/s40478-026-02241-x

Figure Lengend Snippet: Gene enrichment pathway analysis of module B revealed lesion-associated submodules, with TREM2 enriched exclusively in submodule B. a Submodules of original module B for the two cases MS13C and MS15A allowed the identification of submodules associated with distinct glial cell states. b Heat map of the cellular composition of each submodule for MS13C and MS15A. c Submodule B hub gene network for MS13C revealed TREM2 and proximal genes. d Gene ontology biological process enrichment analysis for each submodule for MS15A and MS13C. e The results of enrichment analysis within the KEGG gene sets database for co-expressed glial submodules for MS13C and MS15A are shown. OPC = Oligodendrocyte progenitor cell, vSMC = Vascular smooth muscle cell, OLG = Oligodendrocyte, GO = Gene ontology

Article Snippet: Rabbit anti-human TREM2 (Polyclonal) , Nonspecific , ProSci, USA (13-679) , 1:100.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Spatial characterisation of TREM2 expression in actively demyelinating multiple sclerosis lesions supports its key roles in lipid metabolic pathways

doi: 10.1186/s40478-026-02241-x

Figure Lengend Snippet: Heat map highlighting heterogeneity of TREM2 protein expression in myeloid cells in MS lesions, MS and control NAWM. NAWM and lesion sub-areas were analysed (lesion centres, PLWM and lesion rims). Staining intensity for ORO, CD68+, MHC class II+ and, MBP+ for each tissue are shown. ORO+ cells per ROI (314.74 × 236.06 microns) ranged from 0 to 30 + cells. CD68+ or MHC class II+ cells per ROI (500 × 500 microns) ranged from 0 to 100+ cells. Relative MBP immunohistochemistry was characterised as “light”, “med” and “dark”. The three main cell types analysed were: PVMs (left), parenchymal macrophages (middle) and microglia (right). Images used to calculate the average number of immunolabeled cells per ROI were 312.39 × 312.39 µm. *Tissue lesions provided by Washington University Repository were identified by LFB and MHC class II staining. **MS3E was a case with active MS lesions, which subsequently was identified to have progressive multifocal leukoencephalopathy. This case was not included in subsequent overall analyses of the cohort of MS lesion cases. ***MS15A was a chronic active lesion confirmed by a neuropathologist, however the section provided did not have a clearly identifiable rim. ORO = Oil Red O, MBP = Myelin Basic Protein, NAWM = Normal-appearing white matter, PLWM = Perilesional white matter, n.a = not applicable, ROI = Region of Interest, MHC = MHC class II

Article Snippet: Rabbit anti-human TREM2 (Polyclonal) , Nonspecific , ProSci, USA (13-679) , 1:100.

Techniques: Expressing, Control, Staining, Immunohistochemistry, Immunolabeling

Journal: Acta Neuropathologica Communications

Article Title: Spatial characterisation of TREM2 expression in actively demyelinating multiple sclerosis lesions supports its key roles in lipid metabolic pathways

doi: 10.1186/s40478-026-02241-x

Figure Lengend Snippet: Characterisation of TREM2 expression on microglia and macrophages in active lesions. a Representative Luxol Fast Blue (LFB) and Oil Red O (ORO) images from MS2A highlight the histopathology of an active MS lesion. Lipid-filled cells in the active lesion are highlighted by LFB staining (left; 25 µm scale bar) and ORO staining (middle; yellow dotted box highlights the area of the right image; 10 µm scale bar). Arrows point to LFB staining inside PVMs (possibly indicative of intracellular myelin products—white arrow), ORO + cells in the CNS parenchyma (white star) and lining the vessel (white arrow) (×40 magnification). b Representative immunofluorescence images of macrophages (left), reactive microglia (centre), and PVMs (right) are from active lesions (from MS2A and MS4A) (×40 magnification; 30 µm scale bar). Cropped images are from the highlighted area in the merged overview (yellow square). The area has been split into separate channels: TREM2 (red), CD163 (green) and Iba1 (white). DAPI-stained nuclei (blue) are only seen in the merged overview images. The white arrow marks a triple-labelled TREM2+ CD163+ Iba1+ macrophage. The star marks a rare putative triple-labelled TREM2+ CD163+ Iba1+ microglia. In this panel, other reactive microglia can be seen that are negative for CD163. c TREM2 (red), CD163 (green) and Iba1 (white) triple-labelled macrophages in the choroid plexus (white arrow) from MS15A (20 µm scale bar)

Article Snippet: Rabbit anti-human TREM2 (Polyclonal) , Nonspecific , ProSci, USA (13-679) , 1:100.

Techniques: Expressing, Histopathology, Staining, Immunofluorescence

Journal: Acta Neuropathologica Communications

Article Title: Spatial characterisation of TREM2 expression in actively demyelinating multiple sclerosis lesions supports its key roles in lipid metabolic pathways

doi: 10.1186/s40478-026-02241-x

Figure Lengend Snippet: Perinuclear and plasma membrane distribution of TREM2 respectively in microglia and macrophages in MS lesions. Actively demyelinating tissues were immunolabelled with TREM2 (red), CD163 (green), Iba1 (white), counter-stained with DAPI (blue; merged images), and imaged using a 100× objective with 6-step confocal z-stacks. a Triple labelled PVM–CD163+ TREM2+ Iba1+ (case MS4A). Punctuate discontinuous labelling with the AF1828 antibody in a thin line around the inner surface of the vessel can also be seen, indicative of endothelium (arrow). b TREM2+ CD163-negative microglia (MS2A), c TREM2+ CD163+ putative microglia (MS2A), d TREM2-negative CD163+ putative microglia (MS4A), e CD163+ TREM2+ macrophage (MS2A). f A rare TREM2+ CD163-negative Iba1-negative cell (white arrow) (MS4A). Scale bars = 5 µm

Article Snippet: Rabbit anti-human TREM2 (Polyclonal) , Nonspecific , ProSci, USA (13-679) , 1:100.

Techniques: Clinical Proteomics, Membrane, Staining

Journal: Acta Neuropathologica Communications

Article Title: Spatial characterisation of TREM2 expression in actively demyelinating multiple sclerosis lesions supports its key roles in lipid metabolic pathways

doi: 10.1186/s40478-026-02241-x

Figure Lengend Snippet: Quantification of TREM2+ cells in control NAWM, MS NAWM and MS lesions. a Number of cells (total DAPI nuclei) per mm 2 , b number of microglia/macrophages (Iba1+ and/or CD163+) per mm 2 , c number of TREM2+ microglia/macrophages per mm 2 , and d percentage of TREM2+ microglia/macrophages in distinct tissue areas as indicated. e Proportions of single, double, and triple labelled TREM2+ CD163+ Iba1+ cell populations in distinct tissue areas. The four most abundant cell phenotypes for each area are listed below each chart. Each cell phenotype has been referenced with the same colour across different lesion types as indicate in figure legend. f Immunofluorescence intensity of TREM2 (pink), Iba1 (blue) and CD163 (green) in distinct tissue areas. The size of the individual cell region of interest used to determine intensity ranged from 64 to 295 µm 2 . * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001. NAWM = normal-appearing white matter (from MS cases or controls as indicated), LC = lesion centre, PLWM = perilesional white matter, LR = lesion rim

Article Snippet: Rabbit anti-human TREM2 (Polyclonal) , Nonspecific , ProSci, USA (13-679) , 1:100.

Techniques: Control, Immunofluorescence

Journal: Acta Neuropathologica Communications

Article Title: Spatial characterisation of TREM2 expression in actively demyelinating multiple sclerosis lesions supports its key roles in lipid metabolic pathways

doi: 10.1186/s40478-026-02241-x

Figure Lengend Snippet: TREM2 co-expression with lipid metabolism and activation markers. a TREM2 (red), MHC class II (green), and PLIN2 (white) immunolabelling in parenchymal macrophages (top) and perivascular macrophages (bottom) from an active lesion centre (MS2A). b Quantification per mm 2 of number of PLIN2+ cells, PLIN2+ TREM2+ cells and the proportion of all PLIN2+ cells that are TREM2+ in different tissue areas. All TREM2+ cells were PLIN2+. c TREM2 (red), CD68 (green) and TMEM119 (white) indicate TREM2 expression in active microglia in the lesion centre (top) and PLWM (bottom). DAPI nuclei counterstain is shown in the merged channels (100× objective; 10 µm scale bar) (MS4A). NAWM = Normal-appearing white matter, LC = lesion centre, PLWM = Perilesional white matter, LR = lesion rim

Article Snippet: Rabbit anti-human TREM2 (Polyclonal) , Nonspecific , ProSci, USA (13-679) , 1:100.

Techniques: Expressing, Activation Assay

Journal: Acta Neuropathologica Communications

Article Title: Spatial characterisation of TREM2 expression in actively demyelinating multiple sclerosis lesions supports its key roles in lipid metabolic pathways

doi: 10.1186/s40478-026-02241-x

Figure Lengend Snippet: TREM2 and MS4A4A expression in a PML and active MS lesion. a ORO staining of a PML lesion (20× objective; 200 µm scale bar). The insert (left) shows lipid-filled round macrophage infiltrates characteristic of PML (40× objective; 20 µm scale bar). b TREM2 (red), CD163 (green) and Iba1 (white) immunofluorescence with DAPI nuclei counterstain (blue), highlights abundant CD163+ TREM2+ Iba1+ triple labelled macrophages in the PML lesion with TREM2 distributed to the plasma membrane of foamy macrophages (merge panel 30 µm scale bar; right panels 5 µm scale bar). c TREM2 (red), TMEM119 (white), CD163 (green) and DAPI (blue), 20 µm scale bar. d TREM2 (red), MS4A4A (white) and DAPI (blue), 20 µm scale bar. e TREM2 (red), TMEM119 (white), CD163 (green) and DAPI (blue; merged image) indicate TREM2+ microglia in peri-lesional white matter in PML (10 µm scale bar). f TREM2 (red), CD68 (green) and MS4A4A (white) indicate TREM2 and MS4A4A co-expression in PVMs from an active MS lesion centre (MS4A). DAPI nuclei counterstain is shown in the merged channels (100× objective; 10 µm scale bar). Representative images in a–e are of tissue from PML case MS3E. PLWM = Perilesional white matter, PML = Progressive multifocal leukoencephalopathy

Article Snippet: Rabbit anti-human TREM2 (Polyclonal) , Nonspecific , ProSci, USA (13-679) , 1:100.

Techniques: Expressing, Staining, Immunofluorescence, Clinical Proteomics, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: PD-L1 expression positively correlates with proinflammatory markers in M1-polarized macrophages. (A-C) PD-L1-overexpressing (PD-L1 HI ) and control (PD-L1 NC ) THP-1-M cells were stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml) for 12 h followed by RNA sequencing. A Heatmap of inflammatory gene expression in PD-L1 HI vs. PD-L1 NC cells. B MA plot showing differential gene expression between PD-L1 HI and PD-L1 NC cells. C TPM expression values of IL-6, IL-27 and NOS2. D - E PD-L1 HI and PD-L1 NC cells were stimulated with LPS + IFN-γ for 24 h. ( n = 3 independent biological replicates) ( D ) qRT-PCR analysis of IL-6, IL-27 and NOS2 mRNA levels ( n = 3 independent biological replicates). E ELISA for IL-6/IL-27 and Griess reagent assay for NO in supernatants ( n = 3 independent biological replicates). (F-G) PD-L1-knockout THP-1-M (PD-L1 KO /THP-1-M) and PD-L1 NC /THP-1-M were stimulated with LPS + IFN-γ for 24 h. F qRT-PCR analysis of IL-6, IL-27 and NOS2 mRNA levels ( n = 3 independent biological replicates). G ELISA and Griess reagent detection of IL-6/IL-27 proteins and NO in supernatants ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C, D, E, F and G was performed by Student’s t-test. * p < 0.05

Article Snippet: IL-27, IL-6, IL-18, and IFN-γ concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm.

Techniques: Expressing, Control, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: Triple-verification strategy (gene silencing/antibody neutralization/site-directed mutagenesis) confirms regulatory hierarchy of PD-L1/AIM2/IL-18/STAT1 signaling axis in macrophage M1 polarization. A PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were pretreated with AIM2-targeting siRNA or scramble siRNA (scr siRNA) prior to 24 h LPS (100 ng/ml) + IFN-γ (20 ng/ml) co-stimulation, with subsequent Western blotting analysis of STAT1 phosphorylation. B - C Anti-IL-18 neutralizing antibody (1 µg/ml) and isotype control (1 µg/ml) were pretreated for 24 h followed by LPS + IFN-γ 24 h co-stimulation. B qRT-PCR quantification of IFN-γ mRNA ( n = 3 independent biological replicates). C STAT1 phosphorylation profiling by Western blotting. D Truncation mutant validation: LPS + IFN-γ-stimulated (24 h) PD-L1 Δ35–90 cells analyzed for STAT1 activation vs. full length type. E Following AIM2 silencing in PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells, qRT-PCR quantification of IL-6, IL-27 and NOS2 mRNA after 24 h LPS + IFN-γ stimulation ( n = 3 independent biological replicates). F Post IL-18 neutralization, mRNA levels of IL-6, IL-27 and NOS2 were assessed by qRT-PCR under LPS + IFN-γ 24 h stimulation ( n = 3 independent biological replicates). G STAT1-silenced cells were analyzed for IL-6/IL-27/NOS2 transcriptional changes post 24 h LPS + IFN-γ exposure ( n = 3 independent biological replicates). H Truncated PD-L1 Δ35–90 mutants were evaluated for IL-6, IL-27 and NOS2 mRNA expression following LPS + IFN-γ 24 h challenge ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for B, E, F, G and H was performed by Student’s t-test. * p < 0.05

Article Snippet: IL-27, IL-6, IL-18, and IFN-γ concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm.

Techniques: Neutralization, Mutagenesis, Western Blot, Phospho-proteomics, Control, Quantitative RT-PCR, Biomarker Discovery, Activation Assay, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: PD-L1 knockout reduces septic mortality dampens systemic inflammation and alleviates vital organ (cardiac/hepatic/renal) injury in murine models. A PD-L1 wild-type (WT) and knockout (KO) mice were subjected to CLP followed by survival monitoring, inflammatory marker assessment, and multi-organ functional evaluation. B Survival rates of PD-L1 WT and PD-L1 KO mice at indicated time points post-CLP ( n = 15 mice per group). Results were compared by log-rank test. C mRNA levels of IL-6, IL-27, and NOS2 in monocytes analyzed by qRT-PCR at 24 h post-CLP ( n = 3 mice per group). D Serum concentrations of IL-6 and IL-27 proteins measured by ELISA, and NO levels determined by Griess assay at 24 h post-CLP ( n = 3 mice per group). E Serum biomarkers of organ function including ALT and AST for hepatic injury, BUN for renal dysfunction, and cTnI for cardiac damage at 24 h post-CLP ( n = 3 mice per group). The data are presented as the mean ± SEM. Statistical analysis for C, D and E was performed by Student’s t-test. * p < 0.05

Article Snippet: IL-27, IL-6, IL-18, and IFN-γ concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm.

Techniques: Knock-Out, Marker, Functional Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Griess Assay