Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: N-glycan analysis of the anti-PD-L1 variants αPDL1 WT and αPDL1 GE

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques:

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody shows an enhanced binding to FcγRIIIa. A competitive FcγRIIIa AlphaLISA was performed for the three anti-PD-L1 variants. Thereby the test antibody competes with antibody-conjugated acceptor beads for binding to FcγRIIIa-conjugated donor beads. The chemiluminescent signal as a result of close proximity of the donor and acceptor beads was plotted against increasing concentrations of αPDL1 NG (open circles), αPDL1 WT (gray triangles), and αPDL1 GE (black squares). Statistics: mean and SD were plotted in the graph. Data are representative of two independent experiments.

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody and its normal and non-glycosylated counterparts show comparable antigen binding characteristics. The three anti-PD-L1 variants αPDL1 NG (open circles), αPDL1 WT (gray triangle), and αPDL1 GE (black squares) were tested for PD-L1 antigen binding and their capacity to block interaction with PD-L1 ligands in enzyme-linked immunosorbent assays (ELISA). (A) PD-L1 antigen binding ELISA. OD 450–620 values were plotted against increasing concentrations of test antibody to assess binding to plate-bound human PD-L1. (B) Competitive ELISA measuring binding of soluble programmed death 1 to plate-bound PD-L1 in presence of test antibody. OD4 450–620 values were plotted against increasing concentrations of test antibody. (C) Competitive ELISA measuring binding of soluble CD80 to plate-bound PD-L1 in presence of test antibody. OD 450–620 values were plotted against increasing concentrations of test antibody. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments.

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Competitive ELISA

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces strongest NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) against PD-L1 + cancer cells, but none against B cells and monocytes. (A) The NK cell line KHYG-1-CD16aV as effector cells was incubated with europium-loaded PD-L1 + DU-145 cancer cells as target cells in an effector to target ratio of 10:1 in the presence of increasing concentrations of αPDL1 NG (white circles), αPDL1 WT (gray triangles), or αPDL1 GE (black squares) for 5 h to determine the lysis of target cells in an in vitro cytotoxicity assay. The percentage of specific target cell lysis is plotted against the antibody concentration used. The dashed line indicates % of lysis in the medium control. (B,C) The NK cell line KHYG-1-CD16aV as effector cells were incubated with calcein-labeled primary B cells (B) or monocytes (C) as target cells in an effector to target ratio of 10:1 in the presence of increasing concentrations of αPDL1 WT (gray bars) or αPDL1 GE (black bars) for 4 h to determine the killing of target cells in a flow cytometry based in vitro cytotoxicity assay. The relative frequency of dead 7-AAD + of calcein + target cells was plotted against the antibody concentration used. As a positive control (striped bars) either obinutuzumab (αCD20) was used to induce B cell lysis or staurosporine was used to induce monocyte lysis. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments. Significance was tested against the medium control (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. Abbreviation: ns., not significant).

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Incubation, Lysis, In Vitro, Cytotoxicity Assay, Concentration Assay, Control, Labeling, Flow Cytometry, Positive Control

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces strong CD8 T cell activation in a mixed leukocyte reaction. The three anti-PD-L1 variants αPDL1 NG (open circles), αPDL1 WT (gray triangles), and αPDL1 GE (black squares) were tested for their effect on T cell activation in a mixed leukocyte reaction (MLR). The medium control (black crosses) served as a negative control. T cells as responder cells were isolated from a single healthy donor (donor A). Monocyte-derived dendritic cells as stimulator cells were generated from different healthy donors. (A) IL-2 enzyme-linked immunosorbent assays on day 2 of MLR. The determined concentrations of IL-2 in the culture supernatants were plotted. (B) The activation status of CD8 and CD4 T cells in the MLR was determined on day 5 by flow cytometric analysis. The relative frequencies of CD25 + and CD137 + in CD8 and CD4 T cells were plotted. (C) The proliferation of CFSE-labeled CD8 T cells in the MLR was determined on day 5 by CFSE dilution measured by flow cytometric analysis. Representative plots of CD25 expression and CFSE signal intensity of CD8 T cells are shown. Statistics for (A,B) : besides individual data points ( n = 8 for IL-2 secretion, n = 16 for CD25 expression, and n = 7 for CD137), mean and SEM were plotted in all graphs (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001. Abbreviation: ns, not significant).

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Activation Assay, Control, Negative Control, Isolation, Derivative Assay, Generated, Labeling, Expressing

Journal: Frontiers in Immunology

Article Title: Glyco-Engineered Anti-Human Programmed Death-Ligand 1 Antibody Mediates Stronger CD8 T Cell Activation Than Its Normal Glycosylated and Non-Glycosylated Counterparts

doi: 10.3389/fimmu.2018.01614

Figure Lengend Snippet: Glyco-engineered anti-human programmed death-ligand 1 (PD-L1) antibody induces increased CD8 T cell activation in presence of cancer cells. The three anti-PD-L1 variants αPDL1 NG (light gray bar), αPDL1 WT (dark gray bar), and αPDL1 GE (black bar) were tested for their effect on T cell activation (donor A) in allogeneic mixed leukocyte reaction (MLRs) in absence or presence of the cancer cell lines HSC-4 (horizontal stripes), ZR-75-1 (plaid), and Ramos (vertical stripes). MLR without addition of test antibody (medium; white bar) served as negative control. The relative frequencies of CD25 + in CD8 T cells were plotted. Statistics: mean and SD were plotted in all graphs. Data are representative of two independent experiments. Significance was tested against the medium control without presence of cancer wells (* p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001).

Article Snippet: This work was supported by Glycotope GmbH who provided the anti-PD-L1 antibodies and financial support for the conduct of experiments.

Techniques: Activation Assay, Negative Control, Control

Journal: EMBO Molecular Medicine

Article Title: Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

doi: 10.15252/emmm.202114182

Figure Lengend Snippet: BW5147 reporter cells stably expressing human FcγR‐ζ chain chimeras or BW5147 parental cells (grey/dashed) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry. Bar graphs show means of three independent experiments performed in technical duplicates. Error bars = SD. FCS coating of an ELISA microtiter plate allows for suspension of subsequently added IgG. Plate bound IgG was quantified via ELISA. Bar graph shows means of one experiment performed in technical triplicates. Error bars = SD. Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X‐Axis shows sample concentration determined by antibody molarity. Y‐Axis shows FcγR activation determined by reporter cell mouse IL‐2 production (OD 450 nm). Two independent experiments performed in technical duplicates. Error bars = SD. Schematic of used assay setups. BW5147 reporter cells expressing chimeric human FcγR receptors express endogenous CD69 or secrete mouse IL‐2 in response to FcγR activation by clustered IgG. sICs are generated using mAbs and multivalent antigens. sIC suspension requires pre‐blocking of an ELISA plate using PBS supplemented with 10% FCS (FCS coat, grey‐dashed).

Article Snippet: FcγR‐specific mAbs were obtained from Stem Cell technologies (CD16: clone 3G8; CD32: IV.3).

Techniques: Stable Transfection, Expressing, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, Activation Assay, Generated, Blocking Assay

Journal: EMBO Molecular Medicine

Article Title: Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

doi: 10.15252/emmm.202114182

Figure Lengend Snippet: Ultra‐pure antigen (Ag, TNF‐α) mixed with therapy‐grade mAb (infliximab, Ifx) was used to generate sICs. X‐Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. Immobilized IgG (Rtx) or immobilized FcγR‐specific mAbs served as positive controls. Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown. Left panel: IL‐2 quantification 16 h after reporter cell activation. Background (blank) was subtracted (dashed line). IL‐2 was measured via anti‐IL‐2 ELISA (A 450nm ) and IL‐2 concentrations were calculated from an IL‐2 standard. Right panel: Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry. Mean florescence intensity (MFI) were normalized to untreated cells (ctrl.) and are presented as fold‐change increase.

Article Snippet: FcγR‐specific mAbs were obtained from Stem Cell technologies (CD16: clone 3G8; CD32: IV.3).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

doi: 10.15252/emmm.202114182

Figure Lengend Snippet: Two different multivalent ultra‐pure antigens (Ag) mixed with respective therapy‐grade mAbs were used to generate sICs as indicated for each set of graphs (top to bottom). IC pairs: mepolizumab (Mpz) and rhIL‐5; bevacizumab (Bvz) and rhVEGFA. X‐Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. FcγR responses were normalized to immobilized rituximab (Rtx) at 1 µg/well (set to 1) and a medium control (set to 0). Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown.

Article Snippet: FcγR‐specific mAbs were obtained from Stem Cell technologies (CD16: clone 3G8; CD32: IV.3).

Techniques:

Journal: Nature microbiology

Article Title: Fc-γR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2

doi: 10.1038/s41564-023-01359-1

Figure Lengend Snippet: (a-c) Levels of IgG1 (a), IgG2b (b), IgG2c (c) that bind to SARS-CoV-2 spike [Wuhan-1, B.1.617.2, BA.1, and BA.4/5], or influenza hemagglutinin (HA) in naïve and vaccine-induced immune sera. (d-f) Levels of spike- or HA-binding IgG antibodies that engage FcγR IIb (d), FcγR III (e), or FcγR IV (f) in naïve and vaccine-induced immune sera. (g-j) Antibody effector functions. Antibody-mediated cellular phagocytosis with monocytes (ADCP, g) or neutrophils (ADNP, h) activity using vaccine-induced immune (squares) or naïve (circles) sera and beads coated with SARS-CoV-2 Wuhan-1 and BA.4/5 spike proteins and murine monocytes (g-h) or human donors (i) (bars indicate mean ± SEM; n = 4 (g); n = 3 (h) mice per group, one experiment (g), two experiments (h); one-way ANOVA with Tukey’s post-test; ns, not significant; in order left to right **P = 0.0042, **P = 0.0083 (g); **P = 0.0012, *P = 0.0106 (h)). (i) CD107a surface expression on natural killer cells (ADNKA) after incubation with beads encoded with Wuhan-1 or BA4/5 spike proteins and immune sera (bars indicate median values; n = 2 donors per group, one experiment; one-way ANOVA with Tukey’s post-test; ns, not significant). (j) Deposition of complement (ADCD) on beads coated with indicated SARS-CoV-2 spike or influenza HA proteins after treatment with naïve or immune sera.

Article Snippet: FcγR I KO 49 , FcγR II KO (Taconic Biosciences; Cat # 580), FcγR III KO (Jackson Laboratory; Cat # 009637), FcγR I/III/IV (common γ-chain) KO (Taconic Biosciences; Cat # 583), and C1q KO 50 mice were obtained commercially or from collaborators and then backcrossed onto a C57BL/6J background (>99%) using Speed Congenics (Charles River Laboratories) and single nucleotide polymorphism analysis.

Techniques: Binding Assay, Activity Assay, Expressing, Incubation

Journal: Nature microbiology

Article Title: Fc-γR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2

doi: 10.1038/s41564-023-01359-1

Figure Lengend Snippet: (a) Scheme of immunization, serum sampling, virus challenge, and tissue harvest. (b-c) Anti-Wuhan-1 (b) and BA.4/5 (c) RBD IgG responses from serum of mice immunized with control or mRNA-1273 vaccines. (d-e) Neutralizing antibody responses against WA1/2020 N501Y/D614G (d) and BA.5 (e) from serum collected from wild-type, FcγR I KO, FcγR III KO, and FcγR I/III/IV KO mice 25 days after completion of a two-dose primary vaccination series with control (closed circles) or mRNA-1273 (open circles). (f-g) Nine-week-old male wild-type, FcγR I KO, FcγR III KO, and FcγR I/III/IV KO mice were immunized twice at four-week intervals with control or mRNA-1273 vaccine via intramuscular route. Four weeks after the primary vaccination series, mice were challenged via intranasal route with 104 FFU of BA.5. At 3 dpi, infectious virus in the nasal turbinates (f) and lungs (g) was determined (boxes illustrate geometric mean titers [GMT], dotted lines show LOD, bars indicate mean ± SEM; in order left to right n = 10, 10, 10, 10, 25, 10, 10, 14 (b); n = 12, 10, 10, 10, 22, 10, 10, 15 (c); n = 22, 9, 9, 12, 30, 15, 15, 15 (d); n = 22, 9, 9, 12, 25, 10, 10, 15 (e); n = 19, 10, 8, 8, 19, 9, 10, 10 (f); n = 19, 10, 8, 8, 19, 9, 10, 10 (g) mice per group, one experiment (b-e), two experiments (f-g), dotted lines show LOD, one-way ANOVA with Dunnett’s test (ns, not significant, ***P = 0.002, ****P < 0.0001 (f); **P = 0.0044, ****P < 0.0001 (g)); additional statistical comparisons are shown in Supplementary Table 3.

Article Snippet: FcγR I KO 49 , FcγR II KO (Taconic Biosciences; Cat # 580), FcγR III KO (Jackson Laboratory; Cat # 009637), FcγR I/III/IV (common γ-chain) KO (Taconic Biosciences; Cat # 583), and C1q KO 50 mice were obtained commercially or from collaborators and then backcrossed onto a C57BL/6J background (>99%) using Speed Congenics (Charles River Laboratories) and single nucleotide polymorphism analysis.

Techniques: Sampling, Virus, Vaccines

Journal: Nature microbiology

Article Title: Fc-γR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2

doi: 10.1038/s41564-023-01359-1

Figure Lengend Snippet: (a) Scheme of passive transfer, virus challenge and tissue harvest. (b) Neutralizing antibody responses against SARS-CoV-2 MA-10 using sera from naïve (circles) or Wuhan-1 spike protein vaccinated mice (pooled from animals immunized and boosted with mRNA-1273 or ChAd-SARS-CoV-2-S) (squares). Also shown is serum neutralizing antibody activity from recipient wild-type and FcγR I/III/IV KO mice one day after transfer of immune sera. (c-g) Twelve-week-old male wild-type, C1q KO, and FcγR I/III/IV KO C57BL/6 mice were passively transferred by intraperitoneal injection 60 μL of naïve or vaccine-induced immune sera one day before intranasal challenge with 103 FFU of SARS-CoV-2 MA-10. At 4 dpi, viral RNA in the nasal wash (c), nasal turbinates (d), and lungs (f) was quantified, and infectious virus in the nasal turbinates (e) and lungs (g) was determined (bars indicate mean ± SEM; in order left to right n = 5 (b); n = 6, 6, 7, 7, 6, 7 (c); n = 6, 6, 7, 7, 6, 7 (d); n = 6, 6, 7, 7, 6, 7 (e); n = 6, 6, 7, 7, 6, 7 (f); n = 6, 6, 7, 7, 6, 7 (g) mice per group, one experiment (b), two experiments (c-g), dotted lines show limit of detection [LOD]). One-way ANOVA with Tukey’s post-test; ns, not significant; *P = 0.0188 (f); ****P < 0.0001 (g)); additional statistical comparisons are shown in Supplementary Table 3. In c-g, the LOD are weight and volume based, and vary based on the amount of material collected for RNA extraction.

Article Snippet: FcγR I KO 49 , FcγR II KO (Taconic Biosciences; Cat # 580), FcγR III KO (Jackson Laboratory; Cat # 009637), FcγR I/III/IV (common γ-chain) KO (Taconic Biosciences; Cat # 583), and C1q KO 50 mice were obtained commercially or from collaborators and then backcrossed onto a C57BL/6J background (>99%) using Speed Congenics (Charles River Laboratories) and single nucleotide polymorphism analysis.

Techniques: Virus, Activity Assay, Injection, RNA Extraction

Journal: Nature microbiology

Article Title: Fc-γR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2

doi: 10.1038/s41564-023-01359-1

Figure Lengend Snippet: (a) Scheme of passive transfer, virus challenge, and tissue harvest. (b) Neutralizing antibody response against SARS-CoV-2 WA1/2020 N501Y/D614G using sera from naïve (circles) or Wuhan-1 spike protein vaccinated (squares) mice. Also shown is serum neutralizing antibody activity from recipient wild-type and FcγR I/III/IV KO mice one day after transfer of immune sera. (c-g) Twelve-week-old male wild-type, FcγR I KO, FcγR II KO, FcγR III KO, and FcγR I/III/IV KO mice were passively transferred by intraperitoneal injection 60 μL of naïve or vaccine-immune sera one day before intranasal challenge with 104 FFU of WA1/2020 N501Y/D614G. At 4 dpi, viral RNA and infectious virus were measured in the upper respiratory tract (nasal wash, c; nasal turbinates, d-e; or lungs, f-g). Panels c-e: wild-type mice only; panels f-g: wild-type, FcγR I KO, FcγR II KO, FcγR III KO, and FcγR I/III/IV KO mice (bars indicate mean ± SEM; in order left to right n =5 (b); n = 18, 11 (c); n = 18, 11 (d); n = 18, 11 (e); n = 18, 9, 9, 12, 10, 11, 8, 10, 11, 11 (f); n = 18, 9, 9, 12, 10, 11, 8, 10, 11, 11 (g) mice per group, one experiment (b), three experiments (c-g), dotted lines show LOD). One-way ANOVA with Tukey’s post-test (ns, not significant; **P = 0.0068, ****P < 0.0001 (f); ***P = 0.0002, ****P < 0.0001 (g)); additional statistical comparisons are shown in Supplementary Table 3.

Article Snippet: FcγR I KO 49 , FcγR II KO (Taconic Biosciences; Cat # 580), FcγR III KO (Jackson Laboratory; Cat # 009637), FcγR I/III/IV (common γ-chain) KO (Taconic Biosciences; Cat # 583), and C1q KO 50 mice were obtained commercially or from collaborators and then backcrossed onto a C57BL/6J background (>99%) using Speed Congenics (Charles River Laboratories) and single nucleotide polymorphism analysis.

Techniques: Virus, Activity Assay, Injection

Journal: Nature microbiology

Article Title: Fc-γR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2

doi: 10.1038/s41564-023-01359-1

Figure Lengend Snippet: (a) Representative flow cytometry plots show gating scheme for quantification of spike-specific CD4+ and CD8+ T cell responses in the spleen of wild-type, FcγR III KO, and FcγR I/III/IV KO mice at day 10 after boosting with control or mRNA-1273 vaccines. (b-c) At day 10 after boosting, the spleen of wild-type and FcγR KO mice were harvested, and T cell responses were measured after spike peptide re-stimulation. Splenocytes were incubated overnight with class I MHC (b) or class II MHC (c) immunodominant spike peptides, and the percentages and numbers of IFNγ and TNFα positive CD8+ (b) or CD4+ (c) T cells were quantified by intracellular staining and flow cytometry. Data are pooled from two experiments (in order left to right n = 10, 10, 8, 10, 10, 9 (b-c)). Comparisons were made between groups that received the mRNA 1273 vaccine (one-way ANOVA with Tukey’s post-test; all comparisons were not significant; column height indicates mean values).

Article Snippet: FcγR I KO 49 , FcγR II KO (Taconic Biosciences; Cat # 580), FcγR III KO (Jackson Laboratory; Cat # 009637), FcγR I/III/IV (common γ-chain) KO (Taconic Biosciences; Cat # 583), and C1q KO 50 mice were obtained commercially or from collaborators and then backcrossed onto a C57BL/6J background (>99%) using Speed Congenics (Charles River Laboratories) and single nucleotide polymorphism analysis.

Techniques: Flow Cytometry, Vaccines, Incubation, Staining

Journal: Nature microbiology

Article Title: Fc-γR-dependent antibody effector functions are required for vaccine-mediated protection against antigen-shifted variants of SARS-CoV-2

doi: 10.1038/s41564-023-01359-1

Figure Lengend Snippet: Lung cells from wild-type, FcγR I KO, FcγR III KO, and FcγR I/III/IV KO mice were stained with antibodies for FcγR I, FcγR III, or FcγR IV. After gating on live cells, alveolar macrophages, neutrophils, and monocytes were defined (see Extended Data Fig 5). The data are representative of results with n = 3 mice per group, and histograms are shown.

Article Snippet: FcγR I KO 49 , FcγR II KO (Taconic Biosciences; Cat # 580), FcγR III KO (Jackson Laboratory; Cat # 009637), FcγR I/III/IV (common γ-chain) KO (Taconic Biosciences; Cat # 583), and C1q KO 50 mice were obtained commercially or from collaborators and then backcrossed onto a C57BL/6J background (>99%) using Speed Congenics (Charles River Laboratories) and single nucleotide polymorphism analysis.

Techniques: Staining