clone 1a8  (Novus Biologicals)

Journal: Cancer Research Communications

Article Title: Neutrophil Antigen Presentation Reprograms the Tumor Microenvironment and Elicits Durable and Broad Antitumor Immunity

doi: 10.1158/2767-9764.CRC-25-0509

Figure Lengend Snippet: αCD16-Ova–induced T-cell expansion in the spleen requires neutrophils and MHC on neutrophils, whereas Batf3 + cDC1s and splenic macrophages are dispensable. Mice were treated as in , and the role of neutrophils ( A and B ), MHC-I on neutrophils ( C and D ), and the contribution of cDC1 ( E and F ) and splenic macrophages ( G and H ) in αCD16-Ova–mediated T-cell proliferation was evaluated. A and B, Role of neutrophils. Treatment with 1A8 resulted in marked neutrophil depletion in blood but only partial depletion in the spleen ( A ), with a corresponding partial reduction in splenic CD8 OT-I and CD4 OT-II T-cell proliferation ( B ). C and D, Requirement for neutrophil MHC-I. In CD16B/γ −/− mice with neutrophil-specific MHC-I deletion (Ly6G-Cre/MHC-I fl/fl ), MHC-I expression was partially reduced on splenic neutrophils but intact on cDC1s ( C ); isotype control was used to set gates. CD8 OT-I proliferation was proportionally reduced in Ly6G-Cre/MHC-I fl/fl , consistent with incomplete MHC-I deletion in neutrophils ( D ). E and F, Requirement for Batf3 + cDC1s. In CD16B/γ −/− mice with a deletion in Batf3 (CD16B/γ −/− Batf3 −/− ), there was a marked loss of cDC1 but intact cDC2 populations ( E ). cDC1 loss did not affect αCD16-Ova induced CD8 OT-I or CD4 OT-II T-cell proliferation ( F ). G and H, Anti-CD115 antibody–mediated immunodepletion resulted in a significant but partial reduction of F4/80 + macrophages in the spleen compared with isotype control I (Ctr) treatment ( G ). CD115 antibody treatment did not alter αCD16-Ova–induced CD8 T-cell proliferation ( H ). Sp, spleen. Data are presented as mean ± SEM. Two-group comparisons were done using an unpaired two-tailed Student t test; comparisons among ≥3 groups were done using one-way ANOVA with Tukey post hoc test. Significance was defined as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Ab, antibody; KO, knockout.

Article Snippet: CD16B-CD32A +/− /γ +/− male mice received 350 μg/mouse anti-Ly6G (clone 1A8, Bio X Cell, cat. #BP0075-1) for neutrophil depletion or isotype control rat IgG2a, κ (clone 2A3, Bio X Cell, cat. #BP0089) intraperitoneally on days −2 and −1 prior to OTI/OTII injection, then every 2 days thereafter.

Techniques: Expressing, Control, Immunodepletion, Two Tailed Test, Knock-Out

Journal: Frontiers in Immunology

Article Title: Patrolling monocytes mediate virus neutralizing IgG effector functions: beyond neutralization capacity

doi: 10.3389/fimmu.2025.1600056

Figure Lengend Snippet: Macrophages and inflammatory monocytes are dispensable for nAbs-mediated LCMV control. (A) Flow cytometric analysis of monocytes subsets, CD45 + Lin - (CD45R + CD8 + CD4 + NK + ), CD11b + Ly6G - , in the peripheral blood of MC21 or isotype (rat IgG2a)-treated mice 3 days pot infection (d.p.i). Mice were infected with LCMV-WE (2x10 5 PFU) on day 0, preceded by treatment with 25 µg MC21 or isotype (rat IgG2b) on day -1 and again on day 1. See ( Supplementary Figure S2 ) for gating strategy of the monocyte subsets. (B) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, and lymphocytes in the peripheral blood of MC21 or isotype (rat IgG2a)-treated mice 3 d.p.i. Results show the pooled data from two independent experiments with similar results. (n=3–5 mice/group/experiment). (C) Virus titres (3 d.p.i) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), On day -1 and day 1 mice were treated with 25 µg MC21 or isotype (rat IgG2b). Results show the pooled data from two independent experiments with similar results. (n=3–5 mice/group/experiment). (D) Schematic presentation of the experiments in (E-G) . (E) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, and lymphocytes in the peripheral blood and spleen (3 d.p.i.) of mice treated with 10 µL/g bodyweight control or clodronate liposomes one day before LCMV infection (2x10 5 PFU). Results show the pooled data from two independent experiments with similar results. Experiment was repeated three times with similar results (n=4–5 mice/group/experiment). (F) Shown are the virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a). On day -1 mice were treated with 10µL/g bodyweight control or clodronate liposomes. Results show the pooled data from two independent experiments with similar results. Experiment was repeated three time with similar results (n=3–5 mice/group/experiment). (G) Spleen sections collected 3 d.p.i. were stained for F4/80 (blue), CD169 (green), and LCMV nucleoprotein (−NP) (red). Scale bar 300 µm. Shown are representative pictures from three independent experiments (n=2–4 mice/group/experiment). Statistical analysis was performed by using the One-way ANOVA test (C, F) or Student’s t-test (B, E) . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, ns, non-significant. Horizontal dotted lines indicating the detection limit.

Article Snippet: Depletion antibodies utilized were CD115 (Clone: AFS98), Ly6G (Clone: 1A8), NK (Clone: PK136), all procured from BioXcell, and each was administered at a dosage of 200 μg per mouse (except CD115 100 μg).

Techniques: Control, Infection, Virus, Liposomes, Staining

Journal: Frontiers in Immunology

Article Title: Patrolling monocytes mediate virus neutralizing IgG effector functions: beyond neutralization capacity

doi: 10.3389/fimmu.2025.1600056

Figure Lengend Snippet: Contribution of patrolling monocytes for antiviral activity of Wen3. (A) Schematic presentation of the experiments in (B-E) . (B) Flow cytometric analysis of monocyte subsets in the peripheral blood and spleen of control or clodronate liposomes-treated mice 3 days post infection (d.p.i.). Animals were treated with 0.5 µL/g control or clodronate liposomes (Clo. (low) ) on day -1 and 1. Mice were infected with LCMV-WE (2x10 5 PFU) on day 0. See ( Supplementary Figure S2 ) for gating strategy of the monocyte subsets. (C) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, and lymphocytes/macrophages in the peripheral blood and spleen of control or clodronate liposome-treated mice 3 d.p.i. Animals were treated with 0.5µL/g control or clodronate liposomes (Clo. (low) ) on day -1 and 1. mice were infected with LCMV-WE (2x10 5 PFU) on day 0. Results show the pooled data from two independent experiments with similar results. (n=3–5 mice/group/experiment). (D) Virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), On day -1 and day 1 mice were treated with 0.5µL/g bodyweight control or clodronate liposomes (Clo. (low) ). Results show the pooled data from three independent experiments with similar results (n=3–4 mice/group/experiment). (E) Spleen sections collected 3 d.p.i. were stained for CD169 (green) and LCMV nucleoprotein (−NP) (red). Shown are the representative merged images from three independent experiments. Scale bar= 300 µm. (F) Virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with neutralizing serum (Neutralizing S.) or naïve serum (see methods). On day -1 and day 1 mice were treated with 0.5µL/g control or clodronate liposomes (Clo. (low) ). Results show the pooled data from two independent experiments with similar results (n=3–5 mice/group/experiment). (G) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, along with lymphocytes in the peripheral blood or macrophages in spleen (3 d.p.i.) of mice treated with anti-CD115 or Isotype. Mice were infected with LCMV-WE (2x10 5 PFU) on day 0, preceded by treatment with 100 µg anti-CD115 or isotype (rat IgG2b) on day -1 and again on day 1. Results show the pooled data from two independent experiments with similar results (n=3 mice/group/experiment). (H) Shown are the virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a). On day -1 and day 1 mice were treated with 100 µg anti-CD115 or isotype. Results show the pooled data from two independent experiments with similar results (n=3 mice/group/experiment). (I) Shown are the virus titres (3 d.p.i.) in spleen of CD11c-cre +/+ /iDTR and CD11c-cre tg/+ /iDTR mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), on day -1 and day 1 mice were treated with diphtheria toxin 12ng/g bodyweight. Results show the pooled data from two independent experiments with similar results (n=3–4 mice/group/experiment). (J) Shown are the virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), mice were treated with 200 µg 9E9 or isotype for blocking FcγRIV. Results show the pooled data from three independent experiments with similar results (n=3–4 mice/group/experiment). (K) Heat map showing the expression of FcγRIV on CD11b + Ly6G - population gated from CD45 + Lin - (CD45R + CD8 + CD4 + ), 24 hour following infection with LCMV. Statistical analysis was performed by using the Student’s t-test (C, G) or One-way ANOVA test (D, F, H–J) . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, ns, non-significant. Horizontal dotted lines indicating the detection limit.

Article Snippet: Depletion antibodies utilized were CD115 (Clone: AFS98), Ly6G (Clone: 1A8), NK (Clone: PK136), all procured from BioXcell, and each was administered at a dosage of 200 μg per mouse (except CD115 100 μg).

Techniques: Activity Assay, Control, Liposomes, Infection, Virus, Staining, Blocking Assay, Expressing