anti-ccl2 neutralizing antibodies be0185  (Bio X Cell)

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: Primers for qPCR

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: LILRB4 is associated with microglial inflammatory phenotypes and morphology after tMCAO. ( A ) Gene expression of M1-associated phenotype markers (MCP-1, TNF-α, IL-1β, and CD32) and M2-associated phenotype markers (Arg-1, TGF-β, and CD206). ( n = 6; ** p = 0.0061, *** p = 0.0007, **** p < 0.0001, * p = 0.0222; ns p >0.05). ( B , C ) Fluorescence imaging of microglia in the infarct border region in Control, LILRB4-KO, and LILRB4-TG mice. The lower shows Sholl analysis, where the cell body is the center, and the number of points intersecting several concentric circles is calculated. Shows the number, length of microglia processes (branches), scale bar 10 μm. ( n = 10, * p = 0.0397/0.0306/0.0285)

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques: Gene Expression, Fluorescence, Imaging, Control

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: Microglia LILRB4 deficiency increases the CCL2 production. ( A ) qPCR analysis of CCL2, CCL5, CXCL1, CXCL5, CXCL10 in Control and LILRB4-KO mice 1 day after tMCAO. ( n = 6; * p = 0.0109). ( B ) UMAP plots of 19 cell populations identified by single-cell spatial transcriptomics analysis and the expression level of CCL2 among each cell type in mice after stroke. ( C ) Spatially transcriptome heatmaps of expression patterns of CCL2 across tissue sections from sham or stroke mouse. ( D ) qPCR analysis of CCL2 in microglia of Control and LILRB4-KO mice 1 day after tMCAO. ( n = 6; * p = 0.0229)

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques: Control, Expressing

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: Blockade of CCL2 or addition of Arg1 suppress CD8 + T cell activation and migration in co-culture with LILRB4-KD microglia. ( A , B ) Differential expression of LILRB4 in BV2 microglia transfected by knockdown and negative control lentiviral vectors. The expression of LILRB4 in BV2 was detected by PCR ( A ) and Flow cytometry assay ( B ) ( n = 3; ** p = 0.0025/0.0065/0.0057). ( C ) Experimental procedure. Transwell-placed, Control, and LILRB4-KD microglia (without or with CCL2 inhibitor or IgG) were cultured for 4 h under OGD conditions. During reoxygenation, the t-cell-containing Transwell device was placed on a 24-well plate and exposed to medium on its lower surface for 24 h, and the levels of t-cell migration to the lower layer were measured by Flow cytometry. In another experiment, microglia cells were co-cultured with CD8 + T cells. Control and LILRB4-KD microglia were collected and cultured under OGD conditions for 4 h, and CD8 + T cells were added directly to the medium during reoxygenation. One group was added recombinant Arg-1 and another group was not. After 24 h, CD69 and IFN-γ expression in CD8 + T cells were detected by flow cytometry. ( D ) T cell migration after exposure to OGD/R, measured by the number and type of T cells microglia into 24-well plates, with or without CCL2 inhibition. Flow cytometry tests for T cell migration and ratio of CD8 + T cell. ( n = 4; * p = 0.0187/0.0383/0.0104, ** p = 0.0029). ( E ) Control or LILRB4-KD microglia were exposed to OGD/R and co-cultured with CD8 + T cells with or without the addition of recombinant Arg-1. CD8 + T cells were collected for flow cytometry detection of CD69 and IFN-γ expression. ( F ) Quantitation and statistical evaluation of data in ( E ). ( n = 6; * p = 0.0106/0.0427, *** p = 0.0006, ** p = 0.0024/0.0024/0.0029). ( G ) Control or LILRB4-KD microglia were exposed to OGD/R and co-cultured with CD8 + T cells with or without the addition of recombinant Arg-1. Flow cytometry tests for T cell proliferation. ( n = 5; * p = 0.0348/0.0487/0.0153)

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques: Activation Assay, Migration, Co-Culture Assay, Quantitative Proteomics, Transfection, Knockdown, Negative Control, Expressing, Flow Cytometry, Control, Cell Culture, Recombinant, Inhibition, Quantitation Assay

Journal: Cellular and Molecular Immunology

Article Title: Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2

doi: 10.1038/s41423-024-01142-0

Figure Lengend Snippet: Desialylation of MDSCs downregulates MDSC functional markers and cytokines at the transcript level. Suppressive MDSC-like cells were generated in vitro by coculture with A549 or A549-sia cancer cell lines as described in Fig. . CD33 + cells were isolated on Day 7 and processed for single-cell RNA sequencing (scRNAseq). A Seurat analysis of the scRNAseq dataset projected in UMAP colored by cluster. n = 4 donors per treatment group . B The dataset was subdivided into individual groups showing MDSC-like cells generated by A549 (green, left) or A549-sia (pink, right) coculture. C Stacked bar plots showing the frequency of each cluster annotated in ( A ) subclustered in A549- and A549-sia-generated MDSC-like cells. D Gene Ontology (GO) enrichment analysis of the top 10 upregulated gene sets found in Cluster 2. Dot plot showing the mean normalized enrichment score (NES) of the GO gene sets. The color coding indicates the adjusted p values, and the dot size is proportional to the gene count found in the listed pathway. E Heatmap of selected genes per patient divided into A549- and A549-sia-generated MDSC-like cells. The genes were functionally categorized into 5 groups: (i) chemokines and chemotaxis genes; (ii) MDSC and macrophage marker genes; (iii) protumor function MDSC genes; (iv) other genes; and (v) adhesion, attachment and ECM-related genes. F Gene expression of selected markers (IL1B, S100A9, S100A8, MKi67 and CCL2) as a density plot. The expression density is shown as a scale from blue (low) to yellow (high). G Expression of CCL2 was normalized to that of the donor.The data are presented as the mean ± SD. A two-tailed paired t test ( F , E ) was used. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For neutralization of CCL2 in vivo, mice were injected intraperitoneally 3 times a week with 200 µg/mouse of an anti-CCL2 neutralizing antibody (clone: 2H5, BioXCell) in PBS.

Techniques: Functional Assay, Generated, In Vitro, Isolation, RNA Sequencing Assay, Chemotaxis Assay, Marker, Expressing, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2

doi: 10.1038/s41423-024-01142-0

Figure Lengend Snippet: CCL2 is involved in T-cell suppression via the Siglec-sialoglycan axis in suppressive myeloid cells. A MCP-1/CCL2 found in the supernatant of murine MDSC:T-cell cocultures at the endpoint of the experiment from Fig. . MDSCs were untreated, or were pretreated with sialidase or a Siglec-E blocking antibody . n = 3 donors per group . B Correlation of MCP-1/CCL2 levels measured in supernatants of murine MDSC:T-cell cocultures at the endpoint and percentage of proliferating CD8 + T cells from the same conditions from ( A ). n = 3 donors per group . C Experimental setup: Neutralization of CCL2 using a neutralization antibody in SigE ΔLysM mice and SigE WT littermates bearing B16F10 tumors. Mice were injected with a CCL2 neutralization antibody up to 3 times a week (gray arrow) starting 1 day after subcutaneous B16F10 tumor injection (black arrow). Tumor growth and survival were monitored, and the suppressive capacity of MDSCs was analyzed in vitro. D Kaplan‒Meier survival curves from pooled data from 2 independent experiments. n = 5–8 mice per group . E B16F10 tumors at the endpoint ( C ) were digested, cocultured with pervanadate and analyzed by phospho-flow cytometry for phosphorylated STAT3 (pSTAT3). The percentage of pSTAT3 gated on total MDSCs is shown. Representative histograms for each condition are shown on the right. n = 3 mice per group . F Suppressive capacity of MDSCs against naïve T cells. Percentage of proliferating CD8 + T cells cocultured without MDSCs, ( G ) with MDSCs from SigE WT mice or ( H ) MDSCs from SigE ΔLysM mice with or without addition of CCL2 blocking antibody. N = 3–5 mice from N = 3 experiments . I Cytokine expression found in the supernatant of human primary CD33 + :CD8 + cell cocultures at the endpoint of the experiment from Fig. . CD33 + cells were left untreated or pretreated with sialidase. Z scores were calculated for each cytokine and are shown on a color scale from blue (low) to red (high). n = 3 donors per group . The data are presented as mean ± SD. Two-tailed paired t tests or unpaired t tests ( E ) were used. R shows the Pearson correlation coefficient. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For neutralization of CCL2 in vivo, mice were injected intraperitoneally 3 times a week with 200 µg/mouse of an anti-CCL2 neutralizing antibody (clone: 2H5, BioXCell) in PBS.

Techniques: Blocking Assay, Neutralization, Injection, In Vitro, Flow Cytometry, Expressing, Two Tailed Test