neutrophil-depleting antibody anti-ly6g (clone 1a8, rat igg2a/k)  (Bio X Cell)

Journal: Cell reports

Article Title: Wnt5 controls splenic myelopoiesis and neutrophil functional ambivalency during DSS-induced colitis

doi: 10.1016/j.celrep.2024.113934

Figure Lengend Snippet: (A–F) DKO mice and WT control mice were treated as . The percentages of LK and LSK cells in Lineage − and their absolute numbers were determined by flow cytometry (A and B). The percentages of GMPs and MKPs in LK and their absolute numbers were determined by flow cytometry (C and D). (E) Uniform maniform approximation and projection (UMAP) plots of splenic CD45 + cells from DSS-treated Wnt5 DKO mice and WT mice. (F) Percentages of the major cell types identified in (E). (G) UMAP plots of the subclusters of cells expressing Ly6g + , S100a8 + , and/or Cxcr2 + neutrophil markers from DSS-treated Wnt5 DKO mice and WT mice. (H) Percentages of the subclusters identified in (G). (I) WT mice were treated with normal water or DSS water for 7 days, and Wnt5a and Wnt5b mRNA expressions were determined by RT-qPCR in splenic CD45 + cells and CD45PDGFRα + PDGFRβ + cells. (J) WT mice and Wnt5 DKO mice were treated as in . Spleen CD45 − cells were collected and subjected to single-cell RNA sequencing (scRNA-seq). Expressions of Cxcl12 , Jag1 , and Tgfb in the PDGFRα/PDGFRβ + cells of WT mice and DKO mice are shown in (J). (K) WT and Wnt5 DKO mice were treated as in . Spleen CD45 − PDGFRα + PDGFRβ + cells were collected, and Cxcl12 mRNA expression was detected. Results in (A)–(D), (I), and (K) are shown as means ± SEM with p values (two-tailed Student’s t test). Each datum point represents one mouse. Each independent experiment consists of at least three technical replicates. See also and and .

Article Snippet: Anti-Ly6G mAB depletion antibody , Bio X Cell , Cat##BE0075-1; RRID:AB_1107721.

Techniques: Control, Flow Cytometry, Expressing, Quantitative RT-PCR, RNA Sequencing, Two Tailed Test

Journal: Cell reports

Article Title: Wnt5 controls splenic myelopoiesis and neutrophil functional ambivalency during DSS-induced colitis

doi: 10.1016/j.celrep.2024.113934

Figure Lengend Snippet: DKO mice and WT control mice were treated as in . (A) Common upregulated (red) and downregulated (blue) genes (log2 fold change > 0.1 or < −0.1, adjusted p value [p value adj] < 0.05) from transcriptomic and proteomic analyses of blood neutrophils from DKO mice and WT mice are denoted on the volcano plot. (B) CD101 was detected by flow cytometry in blood neutrophils from DKO mice and WT control mice treated with water or DSS. (C–E) CD8 + T cells were cocultured with blood neutrophils sorted from WT water, WT-DSS, and DKO-DSS mice for 72 h. GZMB, interferon γ (IFNγ), and T cell expansion were determined by flow cytometry. (F–I) CD8 + T cells were cocultured with WT-DSS blood neutrophils and DKO-DSS CD101-low and DKO-DSS CD101-high blood neutrophils, respectively, for 72 h. GZMB, IFNγ, and T cell expansion were determined by flow cytometry. Colored shades denote fluorescence-activated cell sorting (FACS) gating. (J) CD101 expression was determined in blood Ly6G + CFSE + and Ly6G + Far-Red + cells by flow cytometry. (K–N) Splenic neutrophils from WT-DSS mice and DKO-DSS mice were isolated by FACS and transfected with the Cd101 small interfering RNA (siRNA) for 48 h and then cocultured with CD8 + T cells for 72 h. The knockdown efficiency of Cd101 is shown in (K). GZMB, IFNγ, and T cell expansion were determined by flow cytometry. Data in (C)–(E), (G)–(I) and (L)–(N) are presented as means ± SEM with p values (two-tailed one-way ANOVA), and each datum point represents one mouse. Each independent experiment consists of at least three technical replicates. See also , , , , and .

Article Snippet: Anti-Ly6G mAB depletion antibody , Bio X Cell , Cat##BE0075-1; RRID:AB_1107721.

Techniques: Control, Flow Cytometry, Fluorescence, FACS, Expressing, Isolation, Transfection, Small Interfering RNA, Knockdown, Two Tailed Test

Journal: Cell reports

Article Title: Wnt5 controls splenic myelopoiesis and neutrophil functional ambivalency during DSS-induced colitis

doi: 10.1016/j.celrep.2024.113934

Figure Lengend Snippet:

Article Snippet: Anti-Ly6G mAB depletion antibody , Bio X Cell , Cat##BE0075-1; RRID:AB_1107721.

Techniques: Control, Purification, Recombinant, Red Blood Cell Lysis, Cell Isolation, Flow Cytometry, Staining, Blocking Assay, cDNA Synthesis, SYBR Green Assay, RNA Sequencing, Software

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: Myeloid cells express Siglec receptors in humans and mice. A Percentages of Lin - HLA-DR low CD33 + CD11b + cells expressing Siglec-5, Siglec-7, Siglec-9 and Siglec-10 in the peripheral blood (PB) from lung cancer patients detected by flow cytometry. n = 9–12 donors per group . B MFI of Siglec-9 and ( C ) Siglec-10 in CD45 + Lin - HLA-DR low CD33 + CD11b + cells derived from healthy donor and lung cancer patient PB from ( A ). Exemplary results for each condition, including the fluorescence minus one (FMO) control, are shown on the right. The MFI is shown as the change in FMO and was determined by flow cytometry. n = 7–10 donors per group . D Subcutaneously injected endpoint tumors from B16F10 melanoma engrafted mice were harvested and digested, and immune cell infiltration was assessed via multiparameter flow cytometry. Siglec-E, Siglec-F and Siglec-G expression was assessed on CD45 + CD11b + Ly6G + and CD45 + CD11b + Ly6C cells. n = 5 mice . E T-distributed stochastic neighbor embedding (t-SNE) projection of multicolor flow cytometry immunophenotyping of pooled infiltrating immune cells from B16F10 tumors. n = 5 mice . F The Siglec-E expression intensity is shown as a color gradient from blue (low) to red (high). G Spleens from naïve and B16F10 melanoma tumor-bearing mice at the endpoint were collected and analyzed for Siglec-E expression via flow cytometry. n = 3–9 mice per group . The data are presented as the mean ± SD. Two-tailed unpaired Student’s t test or multiple unpaired t tests ( G ) were used. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For in vivo Ly6G or Gr1 depletion, mice were injected intraperitoneally twice per week with 100 μg/mouse of anti-Ly6G depletion antibody (clone: 1A8, BioXCell) or 300 μg/mouse of anti-Gr1 depletion antibody (clone: RB6-8C5, BioXCell) in PBS.

Techniques: Expressing, Flow Cytometry, Derivative Assay, Fluorescence, Control, Injection, 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: Myeloid cells in cancer are highly sialylated. A MFI of SNA or ( B ) MALII on PB-derived Lin - HLADR low CD33 + CD11b + cells from lung cancer patients and healthy controls. Exemplary results for each condition, including the fluorescence minus one (FMO) control, are shown on the right. The MFI is shown as the change in FMO and was determined by flow cytometry. n = 9 donors per group . C MALDI-TOF mass spectra (m/z 1200–5000) of N-glycans isolated from fresh PB-derived CD33 + cells from healthy donors and lung cancer patients. The N-glycans were released by PNGaseF and permethylated prior to MALDI-TOF-TOF profiling. The main structures are depicted above the corresponding peaks. Assignments are based on the composition and knowledge of biosynthetic pathways. All molecular ions are [M + Na] + . The locations of residues above a bracket have not been clearly defined. D Relative quantification of N-glycan levels detected in cancer patient-derived and healthy donor-derived CD33 + cells from ( C ). N = 1 . E Fresh blood from B16F10 tumor-bearing mice and naïve wild-type mice was collected on Day 14 after tumor inoculation and analyzed for SNA gated on ( E ) CD45 + CD11b + Ly6C + or ( F ) CD45 + CD11b + Ly6G + cells. Representative results for each condition, including the FMO control, are shown on the right. The MFI is shown as the change in FMO. 7–8 mice per group . The data are presented as the mean ± SD. Two-tailed unpaired Student’s t test was used. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For in vivo Ly6G or Gr1 depletion, mice were injected intraperitoneally twice per week with 100 μg/mouse of anti-Ly6G depletion antibody (clone: 1A8, BioXCell) or 300 μg/mouse of anti-Gr1 depletion antibody (clone: RB6-8C5, BioXCell) in PBS.

Techniques: Derivative Assay, Fluorescence, Control, Flow Cytometry, Isolation, Quantitative Proteomics, Glycoproteomics, 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: Siglec-E depletion on myeloid cells decreases tumor growth in mice. A Experimental setup: Siglec-ExLysM-Cre mice (SigE ΔLysM ) and Siglec-E wild-type (SigE WT ) littermates were subcutaneously injected with B16F10 or EL4 cells. Tumor growth, survival probability, tumor immune cell infiltration and suppressive capacity of Gr1 + CD11b + cells in vitro were analyzed. B The MFI of Siglec-E expression was assessed in myeloid cells in the tumor homogenates at the endpoint in SigE ΔLysM mice and SigE WT littermates. The MFI of Siglec-E is shown as the change in fluorescence relative to that of the control (FMO). The cell populations were identified as gMDSCs (CD45 + CD11b + Ly6G + ), mMDSCs (CD45 + CD11b + Ly6C + ), macrophages (CD45 + CD11b + F4/80 + ), or dendritic cells (DCs) (CD45 + CD11c + MHCII + F4/80 - ). n = 4–5 mice per group . C Representative results showing Siglec-E staining of the cell populations depicted in ( B ) as a histogram. Siglec-E expression was assessed in SigE ΔLysM (pink) and SigE WT (green) littermates and compared to that in the FMO control (gray). D Tumor growth according to pooled data from mice subcutaneously injected with B10F10 and ( E ) EL4 cells. n = 9–12 mice per group . F Kaplan‒Meier survival curves from pooled data of mice injected subcutaneously with B16F10 cells. n = 9–12 mice per group . G Kaplan‒Meier survival curves from pooled data from 2 experiments in which cells were injected with EL4. n = 9 mice per group . H B16F10 tumors at the endpoint ( D ) were digested and analyzed by flow cytometry. Intratumoral CD8 + cells (CD45 + CD19 - NKp46 - CD3 + CD8 + ), ( I ) Ki67 + CD8 + T cells, ( J ) Granzyme B + CD8 + T cells + (GzmB + ), and ( K ) CD25 + CD8 + T cells were quantified as cells per gram of tumor at the endpoint of the experiment. Exemplary results for intratumoral CD8 + cells in SigE ΔLysM (pink) and SigE WT (green) littermates are shown on the right ( H ). n = 13–16 mice per group . The data are presented as the mean ± SD or SEM ( D , E ). Two-tailed unpaired Student’s t test or multiple unpaired t tests ( B ) were used. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. Tumor growth was compared by mixed-effects analysis followed by Bonferroni’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For in vivo Ly6G or Gr1 depletion, mice were injected intraperitoneally twice per week with 100 μg/mouse of anti-Ly6G depletion antibody (clone: 1A8, BioXCell) or 300 μg/mouse of anti-Gr1 depletion antibody (clone: RB6-8C5, BioXCell) in PBS.

Techniques: Injection, In Vitro, Expressing, Fluorescence, Control, Staining, Flow Cytometry, 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: Reduced suppressive function of MDSCs lacking Siglec-E upon sialidase or Siglec-E blocking antibody treatment. A Experimental setup: Depletion of Ly6G-positive cells in SigE ΔLysM mice and SigE WT littermates bearing B16F10 tumors using a depleting antibody. Mice were injected up to 6 times (gray arrow) with the anti-Ly6G depletion antibody starting 1 day before subcutaneous B16F10 tumor injection (black arrow). Tumor growth and survival were monitored. B Kaplan‒Meier survival curves or ( C ) tumor growth curves from pooled experiments from ( A ). n = 9–11 mice per group . D Experimental setup for assessing the effect of the DR5 antibody on SigE ΔLysM mice and SigE WT littermates subcutaneously injected with B16F10 tumors. E Kaplan‒Meier survival curves and ( F ) tumor growth curves from ( D ) with n = 6–7 mice per group . G Experimental setup for assessing the suppressive effect of Gr1 + CD11b + (MDSC) cells on naïve CD3 + (T cell) cells. MDSCs were isolated from the spleens of B16F10 tumor-bearing SigE ΔLysM mice and SigE WT littermates. T cells were isolated from naïve littermates and stained with CellTrace Violet (CTV) to track T-cell proliferation by flow cytometry. Stained T cells were cocultured with MDSCs for 48 h in the presence of aCD3, aCD28 and IL-2. MDSCs were used immediately or pretreated with sialidase. A Siglec-E blocking antibody or rat IgG2a, κ isotype control was added to the cocultures as indicated. H Percentage of proliferating CD8 + T cells cocultured without MDSCs, ( I ) with MDSCs from SigE WT mice or ( J ) MDSCs from SigE ΔLysM mice. Exemplary results for each untreated condition are shown on the right, indicating untreated T cells alone (J, gray), SigE WT (K, green), and SigE ΔLysM (L, pink). n = 3–9 mice per condition . The data are presented as the mean ± SD or SEM ( C , F ). Multiple paired t tests were used. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. Tumor growth was compared by mixed-effects analysis followed by Bonferroni’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For in vivo Ly6G or Gr1 depletion, mice were injected intraperitoneally twice per week with 100 μg/mouse of anti-Ly6G depletion antibody (clone: 1A8, BioXCell) or 300 μg/mouse of anti-Gr1 depletion antibody (clone: RB6-8C5, BioXCell) in PBS.

Techniques: Blocking Assay, Injection, Isolation, Staining, Flow Cytometry, Control

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: Siglec-E depletion on myeloid cells decreases tumor growth in mice. A Experimental setup: Siglec-ExLysM-Cre mice (SigE ΔLysM ) and Siglec-E wild-type (SigE WT ) littermates were subcutaneously injected with B16F10 or EL4 cells. Tumor growth, survival probability, tumor immune cell infiltration and suppressive capacity of Gr1 + CD11b + cells in vitro were analyzed. B The MFI of Siglec-E expression was assessed in myeloid cells in the tumor homogenates at the endpoint in SigE ΔLysM mice and SigE WT littermates. The MFI of Siglec-E is shown as the change in fluorescence relative to that of the control (FMO). The cell populations were identified as gMDSCs (CD45 + CD11b + Ly6G + ), mMDSCs (CD45 + CD11b + Ly6C + ), macrophages (CD45 + CD11b + F4/80 + ), or dendritic cells (DCs) (CD45 + CD11c + MHCII + F4/80 - ). n = 4–5 mice per group . C Representative results showing Siglec-E staining of the cell populations depicted in ( B ) as a histogram. Siglec-E expression was assessed in SigE ΔLysM (pink) and SigE WT (green) littermates and compared to that in the FMO control (gray). D Tumor growth according to pooled data from mice subcutaneously injected with B10F10 and ( E ) EL4 cells. n = 9–12 mice per group . F Kaplan‒Meier survival curves from pooled data of mice injected subcutaneously with B16F10 cells. n = 9–12 mice per group . G Kaplan‒Meier survival curves from pooled data from 2 experiments in which cells were injected with EL4. n = 9 mice per group . H B16F10 tumors at the endpoint ( D ) were digested and analyzed by flow cytometry. Intratumoral CD8 + cells (CD45 + CD19 - NKp46 - CD3 + CD8 + ), ( I ) Ki67 + CD8 + T cells, ( J ) Granzyme B + CD8 + T cells + (GzmB + ), and ( K ) CD25 + CD8 + T cells were quantified as cells per gram of tumor at the endpoint of the experiment. Exemplary results for intratumoral CD8 + cells in SigE ΔLysM (pink) and SigE WT (green) littermates are shown on the right ( H ). n = 13–16 mice per group . The data are presented as the mean ± SD or SEM ( D , E ). Two-tailed unpaired Student’s t test or multiple unpaired t tests ( B ) were used. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. Tumor growth was compared by mixed-effects analysis followed by Bonferroni’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For in vivo Ly6G or Gr1 depletion, mice were injected intraperitoneally twice per week with 100 μg/mouse of anti-Ly6G depletion antibody (clone: 1A8, BioXCell) or 300 μg/mouse of anti-Gr1 depletion antibody (clone: RB6-8C5, BioXCell) in PBS.

Techniques: Injection, In Vitro, Expressing, Fluorescence, Control, Staining, Flow Cytometry, 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: Reduced suppressive function of MDSCs lacking Siglec-E upon sialidase or Siglec-E blocking antibody treatment. A Experimental setup: Depletion of Ly6G-positive cells in SigE ΔLysM mice and SigE WT littermates bearing B16F10 tumors using a depleting antibody. Mice were injected up to 6 times (gray arrow) with the anti-Ly6G depletion antibody starting 1 day before subcutaneous B16F10 tumor injection (black arrow). Tumor growth and survival were monitored. B Kaplan‒Meier survival curves or ( C ) tumor growth curves from pooled experiments from ( A ). n = 9–11 mice per group . D Experimental setup for assessing the effect of the DR5 antibody on SigE ΔLysM mice and SigE WT littermates subcutaneously injected with B16F10 tumors. E Kaplan‒Meier survival curves and ( F ) tumor growth curves from ( D ) with n = 6–7 mice per group . G Experimental setup for assessing the suppressive effect of Gr1 + CD11b + (MDSC) cells on naïve CD3 + (T cell) cells. MDSCs were isolated from the spleens of B16F10 tumor-bearing SigE ΔLysM mice and SigE WT littermates. T cells were isolated from naïve littermates and stained with CellTrace Violet (CTV) to track T-cell proliferation by flow cytometry. Stained T cells were cocultured with MDSCs for 48 h in the presence of aCD3, aCD28 and IL-2. MDSCs were used immediately or pretreated with sialidase. A Siglec-E blocking antibody or rat IgG2a, κ isotype control was added to the cocultures as indicated. H Percentage of proliferating CD8 + T cells cocultured without MDSCs, ( I ) with MDSCs from SigE WT mice or ( J ) MDSCs from SigE ΔLysM mice. Exemplary results for each untreated condition are shown on the right, indicating untreated T cells alone (J, gray), SigE WT (K, green), and SigE ΔLysM (L, pink). n = 3–9 mice per condition . The data are presented as the mean ± SD or SEM ( C , F ). Multiple paired t tests were used. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. Tumor growth was compared by mixed-effects analysis followed by Bonferroni’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For in vivo Ly6G or Gr1 depletion, mice were injected intraperitoneally twice per week with 100 μg/mouse of anti-Ly6G depletion antibody (clone: 1A8, BioXCell) or 300 μg/mouse of anti-Gr1 depletion antibody (clone: RB6-8C5, BioXCell) in PBS.

Techniques: Blocking Assay, Injection, Isolation, Staining, Flow Cytometry, Control