Journal: Frontiers in Immunology

Article Title: Sub-neutralizing levels of antibodies against RSV F protein enhance RSV infection via Fc-FcγR interactions

doi: 10.3389/fimmu.2025.1594937

Figure Lengend Snippet: ADE requires the co-engagement of FcγRI and TLR4. (A) The percentage of RFP+ THP-1 cells at 12 h post-infection was detected by flow cytometry after blocking FcγRI, FcγRIIa and TLR4. (B) The expression of FcγRI, FcγRIIa and FcγRIIb in THP-1 cells at 24 h post-infection using qPCR. ** for p ≤ 0.01; ^^^ for p ≤ 0.001; ****, ^^^^ for p ≤ 0.0001. ns means no statistical significance compared to the T0 group.

Article Snippet: THP-1 cells were pre-incubated for 1 h with 20 μg/mL neutralization antibodies against TLR4 (clone HTA125) (Invitrogen,14-9917-82), or blocking antibodies (10 μg/mL) against FcγRI (10.1, BD), or FcγRIIa (BioXcell, BE0224-1MG).

Techniques: Infection, Flow Cytometry, Blocking Assay, Expressing

Journal: Nature Communications

Article Title: Cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 limits antibody-mediated neutrophil recruitment

doi: 10.1038/s41467-018-07506-1

Figure Lengend Snippet: CD18 integrins, Mac-1 and LFA-1 in cis inhibit FcγRIIA mediated interaction with ICs under flow. Cells were perfused under physiological flow conditions over plate immobilized ICs ( a , b ) or TNF activated human dermal microvascular endothelial cells (HDMEC) with or without ICs generated by incubating cells with mouse anti-endoglin mAb (anti CD105) and rabbit anti-mouse IgG ( c , d ). The number of adherent cells was assessed and averaged. a Jurkat cells expressing FcγRIIA (J-IIA) with Mac-1 (J-IIA Mac1) or with LFA-1 (J-IIA LFA1) were perfused over immobilized ICs or ICs co-immobilized with ICAM with or without prior PMA treatment. Left panel shows cell adhesion to immobilized ICs at the indicated shear stress. Data is presented as average ± SD of one representative of four experiments with duplicate coverslips per condition. Right panel, bar graphs represent fold change compared to J-IIA cells at 1.0 dyne/cm 2 under indicated conditions. Data is presented as average ± SEM. b After perfusing cells over immobilized ICs, the coverslips were fixed, permeabilized, and stained with rhodamine-phalloidin to visualize the actin cytoskeleton. Bar graphs represent cell area. Data is presented as average ± SEM of n = 3. c , d Indicated Jurkat cells were perfused over TNFα-activated HDMEC with or without deposited ICs. The number of adherent cells was assessed. No rolling was observed. Cells were pre-treated where indicated with anti-FcγRIIA (IV.3) or isotype controls for 30 min at 37 °C. Bar graphs represent fold change compared to J-IIA TNF/IC. Data is presented as average ± SEM of n = 3–5. e Isolated human peripheral blood PMNs from 3 normal donors (control) and 3 LAD1 patients were pre-treated where indicated with anti-FcγRIIA (IV.3) and perfused across TNFα activated HDMEC with or without IC coating and analyzed as in c . Results for each of the three LAD1 patients with 15.3, 17.9, and 22.7% CD18 compared to their respective healthy donors (Con) is shown. Data is average fold change compared to TNF alone control ± SD from duplicate coverslips. For a , b * p < 0.05; *** p < 0.001 using the Student’s unpaired t -test. For c – e * p < 0.05; *** p < 0.001 using the one way ANOVA followed by Sidak’s Multiple comparison test

Article Snippet: Neutrophil FcγRIIA was isolated from ~5 million neutrophils from blood drawn from 5 healthy donors as part of the Momenta Pharmaceuticals blood donor program.

Techniques: Generated, Expressing, Shear, Staining, Isolation, Control, Comparison

Journal: Nature Communications

Article Title: Cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 limits antibody-mediated neutrophil recruitment

doi: 10.1038/s41467-018-07506-1

Figure Lengend Snippet: Mac-1’s αI domain interacts in cis with FcγRIIA’s ectodomain to lower FcγRIIA’s affinity for IgG. a Model of FcγRIIA, IgG, and Mac-1 in bent/closed and extended/open conformation based on published crystal structures. b FLIM was conducted on Jurkat cells expressing Mac-1 and FcγRIIA by staining with the donor (D) antibody for anti-FcγRIIA (IV.3 AF488) and the acceptor (A) antibody for Mac-1 (ICRF44 AF568) or an isotype control. FLIM images were taken and presented in pseudocolors from red to green. The fluorescence lifetimes of the donor in the absence (D) or presence (A + D) of the acceptor antibody were calculated. n = 3 experiments. * p < 0.01; unpaired t -test. c – f BFP measurements of adhesion frequency-versus-contact time of the indicated Jurkat cells (J-IIA) alone or with wild-type Mac-1 (WT), or Mac-1 lacking E 253 -R 261 (E MT) with or without NIF treatment against bead immobilized Mac-1. c bead immobilized FcγRIIA ( d ) or bead immobilized IgG ( e , f ). The absence or presence of binding after a contact of given duration was analyzed to obtain the adhesion frequency. Bar graphs show effective 2D affinity (A c K a , E) and off-rate (k off , F) for each cell line. The adhesion frequencies of J-IIA-WT were lower than those of J-IIA, J-IIA-E MT, and NIF treatment of J-IIA-WT increased the effective FcγRIIA 2D affinity. Data is average ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, by unpaired, two-tailed Student’s t -test

Article Snippet: Neutrophil FcγRIIA was isolated from ~5 million neutrophils from blood drawn from 5 healthy donors as part of the Momenta Pharmaceuticals blood donor program.

Techniques: Expressing, Staining, Control, Fluorescence, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 limits antibody-mediated neutrophil recruitment

doi: 10.1038/s41467-018-07506-1

Figure Lengend Snippet: Mac-1’s αI-domain reduces FcγRIIA binding to ICs, but does not affect cell spreading or FcγRIIIB binding to ICs. Jurkat cells expressing either FcγRIIA or FcγRIIA and Mac-1 with or without pretreatment with recombinant αI-domain (100 μg/ml) for 30 min at 37 °C ( a ), without or with pretreatment with recombinant NIF (10 nM) ( b ), or J-IIA cells expressing Mac-1-E 253 -R 261 mutant ( b ) were perfused at 1 dyne/cm 2 over IC-coated coverslips ( a and b , left panels) or TNF activated and IC coated HDMEC on coverslips ( a and b , right panels). The number of adherent Jurkats was assessed and averaged. Data are represented as fold change compared to J-IIA cells of an average of 3 experiments ± SEM. c Indicated Jurkat cell lines were seeded onto immobilized ICs on coverslips for 30 min under static conditions. Coverslips were fixed, permeabilized, and stained with Alexa 568 phalloidin to visualize the actin cytoskeleton. Bar graphs represent average adherent cell area and representative images are shown. Data is presented as average ± SEM of n = 3. d Jurkat cells expressing FcγRIIIB without (J-IIIB) or with Mac-1 (J-IIIB Mac1) were perfused over coverslip immobilized ICs at 1 dyne/cm 2 and the number of adherent cells were determined. Data are represented as fold change compared to J-IIIB. Data is average of 3 experiments ± SEM ** p < 0.01, *** p < 0.001 using the One way ANOVA followed by Sidak’s Multiple comparison test

Article Snippet: Neutrophil FcγRIIA was isolated from ~5 million neutrophils from blood drawn from 5 healthy donors as part of the Momenta Pharmaceuticals blood donor program.

Techniques: Binding Assay, Expressing, Recombinant, Mutagenesis, Staining, Comparison

Journal: Nature Communications

Article Title: Cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 limits antibody-mediated neutrophil recruitment

doi: 10.1038/s41467-018-07506-1

Figure Lengend Snippet: The lupus variant, Mac-1 R77H does not interact with FcγRIIA in cis , and fails to reduce FcγRIIA affinity for IgG and binding to ICs under flow. a FLIM was conducted on Jurkat cells expressing Mac-1 WT or R77H variant and FcγRIIA by staining with the donor (D) antibody for anti-FcγRIIA and the acceptor (A) antibody for Mac-1 or isotype control and the fluorescence lifetimes were calculated as described in Fig. . n = 3 experiments. ** p < 0.01; unpaired t -test. b – d BFP measurements of adhesion frequency-versus-contact time (left panels) and kinetic parameters (right panels) of the indicated Jurkat cells (J-IIA) alone or with wild-type Mac-1 (J-IIA Mac1), or Mac-1 R77H variant (J-IIA-R77H) against beads immobilized FcγRIIA ( b ), IgG ( c ), or Mac-1 ( d ). Data is presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, by unpaired, two-tailed Student’s t -test. e Indicated Jurkat cells were perfused at 1 dyne/cm over IC-coated glass coverslips (left) or TNF activated and IC coated HDMEC plated coverslips (right). The number of adherent Jurkats was assessed and averaged. Data is represented as fold change compared to J-IIA cells at 1 dyne/cm of an average of 4 experiments ± SEM. *** p < 0.001, using the One way ANOVA followed by Sidak’s Multiple comparison test

Article Snippet: Neutrophil FcγRIIA was isolated from ~5 million neutrophils from blood drawn from 5 healthy donors as part of the Momenta Pharmaceuticals blood donor program.

Techniques: Variant Assay, Binding Assay, Expressing, Staining, Control, Fluorescence, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 limits antibody-mediated neutrophil recruitment

doi: 10.1038/s41467-018-07506-1

Figure Lengend Snippet: Mac-1 αI domain interaction with FcγRIIA requires FcγRIIA sialylation and divalent cations. a A model of glycosylated FcγRIIA and bent Mac-1 with high rotational ability and flexibility of the αI domain is depicted. Spheres are Mg 2+ (green) and Ca 2+ (magenta). The electrostatic surface (−5 [red] to + 5 [blue] kT/e) was calculated using the Adaptive Poisson-Boltzmann Solver software plug-in for PyMOL. b Site-specific glycopeptide analysis of human neutrophil derived FcγRIIA. Average relative abundance for n = 5 donors are shown for both sites in the context of the crystal structure of the FcγRIIA-R131 variant. c Site-specific glycopeptide analysis of FcγRIIA-R131 variant and FcγRIIIB NA2 allele expressed in Jurkat cells. d Plates coated with GST-αI domain or GST were incubated with FcγRIIA or neuraminidase A treated FcγRIIA (asialylated). As indicated, EDTA was included in the buffer to chelate divalent cations. For experimental controls, GST was incubated with FcγRIIA and GST-αI domain was incubated with NIF to show αI-domain specificity. Lectin blots were performed to detect terminal α2,6 sialic acid on FcγRIIA in the absence (−) and presence ( + ) of neuraminidase (Neur). e J-IIA and J-IIA Mac1 cells were incubated with neuraminidase for 30 min at 37 °C. The cells were diluted 100-fold in flow buffer and drawn over IC-coated coverslips or TNF stimulated anti-endoglin (IC) coated HDMEC coverslips at 1.0 dynes/cm 2 and the number of adherent cells were determined as in Fig. . Data is represented as fold change compared to J-IIA cells (-Neur) of an average of 3 experiments ± SEM; ** p < 0.01 using Student’s unpaired t -test. f Indicated Jurkat cells were incubated ± neuraminidase as in e and seeded onto immobilized ICs for 30 min. Coverslips were fixed, permeabilized, and stained with Alexa 568 phalloidin to visualize the actin cytoskeleton. Bar graphs represent average adherent cell area. g Human neutrophils ± neuraminidase (Neur) were drawn across TNF-stimulated HDMEC coated with or without anti-endoglin (IC) at 1 dynes/cm 2 and the number of adherent cells were evaluated. Data is average of 3 experiments ± SEM. Student’s unpaired t -test. For c – e ), ** p < 0.05; *** p < 0.001 using the One way ANOVA followed by Sidak’s Multiple comparison test

Article Snippet: Neutrophil FcγRIIA was isolated from ~5 million neutrophils from blood drawn from 5 healthy donors as part of the Momenta Pharmaceuticals blood donor program.

Techniques: Software, Glycoproteomics, Derivative Assay, Variant Assay, Incubation, Staining, Comparison

Journal: Bioanalysis

Article Title: Assessing prognosis by quantifying FcγRIIa on fixed platelets

doi: 10.1080/17576180.2024.2395706

Figure Lengend Snippet: The pFCG test was performed with the addition of clone FL18.26 to nonfixed platelets and the addition of clone 5G1 to fixed platelets (n = 100 patients with MI). Patients with MI were chosen for this analysis because the range of expression (∼400->5000 molecules/platelet) is greater than that seen in healthy subjects (<1000 molecules/platelet). The graph on the left shows a comparison of the two methods across a broad range of FcγRIIa expression. The graph in the middle shows the absolute difference between results obtained with the two methods (results with 5G1 minus results with FL18.26) over a broad range of expression. The image on the right shows results from western blot analysis. Lane 1 shows results obtained with probing of the lysate from CHO cells (that do not express FCγRIIa). Lane 2 shows results from CHO cells that have been stably transfected to express FCγRIIa. Lane 3 shows results from a platelet lysate. Similar specificity of 5G1 and FL18.26 is demonstrated. The greater intensity associated with the 5G1 clone is consistent with greater affinity. CHO: Chines Hamster Ovary; MI: Myocardial infarction; pFCG: Platelet FcγRIIa.

Article Snippet: Both biologic specimens (platelets from patients) and TruCytesTM synthetic cells conjugated with FcγRIIa (Slingshot Biosciences) were used to assess the precision and accuracy of the test performed on fixed platelets.

Techniques: Expressing, Comparison, Western Blot, Stable Transfection, Transfection

Journal: Bioanalysis

Article Title: Assessing prognosis by quantifying FcγRIIa on fixed platelets

doi: 10.1080/17576180.2024.2395706

Figure Lengend Snippet: FcγRIIa was conjugated to Slingshot synthetic cells (TruCytes™). The pFCG test was performed and results were compared with the expected results (left). Linearity of the pFCG test (right) was assessed with the use of Slingshot synthetic cells (TruCytes™). The pFCG test was performed (n = 765 determinations) on samples that ranged from 591 to 7640 molecules of FcγRIIa/bead. This range encompasses values seen in healthy subjects and patients with MI. Linear regression demonstrated R 2 = 0.984. MI: Myocardial infarction; pFCG: Platelet FcγRIIa.

Article Snippet: Both biologic specimens (platelets from patients) and TruCytesTM synthetic cells conjugated with FcγRIIa (Slingshot Biosciences) were used to assess the precision and accuracy of the test performed on fixed platelets.

Techniques:

Journal: Bioanalysis

Article Title: Assessing prognosis by quantifying FcγRIIa on fixed platelets

doi: 10.1080/17576180.2024.2395706

Figure Lengend Snippet: The interassay CV was assessed with TruCytes™ synthetic cells conjugated with FcγRIIa. Intraday samples were performed on the same day. The second pFCG test was performed at least 2 h after the first test. interday samples were performed on 20 nonconsecutive days. CV: Coefficient of variation.

Article Snippet: Both biologic specimens (platelets from patients) and TruCytesTM synthetic cells conjugated with FcγRIIa (Slingshot Biosciences) were used to assess the precision and accuracy of the test performed on fixed platelets.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Cartilage-binding antibodies induce pain through immune complex–mediated activation of neurons

doi: 10.1084/jem.20181657

Figure Lengend Snippet: List of primary antibodies used for both IHC and immunocytochemistry (ICC) assays

Article Snippet: Human DRG (IHC) , PFA 2 h 4°C , Mouse anti-FcγRIIa (1:100, hCD32a, clone IV.3; Stem Cell Technologies) , Not shown.

Techniques: Immunocytochemistry, Immunohistochemistry-Fresh tissue

Journal: Cell reports

Article Title: Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.celrep.2024.114922

Figure Lengend Snippet: (A) FcγRIIIa reporter activity of the RBD-, NTD-, S2-, and unknown-epitope-binding mAbs. (B) FcγRIIa reporter activity of the RBD-, NTD-, S2-, and unknown-epitope-binding mAbs. (C) Binding of complement protein C1q to the Fc portion of the mAbs in the presence of wild-type SARS-CoV-2 spike protein. Dotted lines denote the limit of detection. Each assay was performed at least twice, and the geometric mean and geometric standard deviation of these replicates is shown. The control groups for each assay are shared and are displayed in all panels belonging to the respective assay.

Article Snippet: 3×10 6 ADCC and ADCP Bioassay Effector Cells expressing the human FcγRIIIa or FcγRIIa receptors (Promega) were added per well and the cells were incubated at 37°C with 5% CO 2 for 6 h. 75 μL of Bio-Glo luciferase reagent (Promega) was then added to each well and the luminescence was read in a Synergy 4 (BioTek) plate reader.

Techniques: Activity Assay, Binding Assay, Standard Deviation, Control

Journal: Cell reports

Article Title: Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.celrep.2024.114922

Figure Lengend Snippet: (A) FcγRIIIa reporter activity of the seven most protective mAbs and their LALA and LALAPG mutant counterparts. (B) FcγRIIa reporter activity of the wild-type, LALA, and LALAPG mAbs. (C) Binding of complement protein C1q by the wild-type, LALA, and LALAPG mAbs in the presence of wild-type SARS-CoV-2 spike protein. In (A)–(C), dotted lines denote the limit of detection. Each assay was performed twice, and the geometric mean and geometric standard deviation of these replicates is shown. (D) Weight loss and survival of BALB/cAnNHsd mice ( n = 4, mean ± SD) treated intraperitoneally with a 10 mg/kg dose of the seven most protective mAbs and their LALA and LALAPG counterparts following lethal challenge with a 5×LD 50 dose of mouse-adapted SARS-CoV-2. The negative control was shared across experimental groups and is shown in each respective panel.

Article Snippet: 3×10 6 ADCC and ADCP Bioassay Effector Cells expressing the human FcγRIIIa or FcγRIIa receptors (Promega) were added per well and the cells were incubated at 37°C with 5% CO 2 for 6 h. 75 μL of Bio-Glo luciferase reagent (Promega) was then added to each well and the luminescence was read in a Synergy 4 (BioTek) plate reader.

Techniques: Activity Assay, Mutagenesis, Binding Assay, Standard Deviation, Negative Control

Journal: Cell reports

Article Title: Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.celrep.2024.114922

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 3×10 6 ADCC and ADCP Bioassay Effector Cells expressing the human FcγRIIIa or FcγRIIa receptors (Promega) were added per well and the cells were incubated at 37°C with 5% CO 2 for 6 h. 75 μL of Bio-Glo luciferase reagent (Promega) was then added to each well and the luminescence was read in a Synergy 4 (BioTek) plate reader.

Techniques: Virus, Recombinant, Luciferase, Electron Microscopy, Enzyme-linked Immunosorbent Assay, Microneutralization Assay, Bioassay, Variant Assay, Software

Journal: Nature

Article Title: Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection

doi: 10.1038/s41586-020-2838-z

Figure Lengend Snippet: Fc domain variants with differential FcγR binding affinity were generated through the introduction of amino acid substitutions at the hinge proximal region of the CH2 domain of human IgG1. a , The positions of the mutated residues for the different Fc domain variants are highlighted. b , The affinity of these human IgG1 Fc variants for the different human FcγRs was assessed by surface plasmon resonance and the dissociation constant ( K d ) (in M) is presented. The following references are cited in the table: refs. , . c , d , HPLC analysis of Fc domain variants using size-exclusion chromatography (SEC) columns was performed to determine whether mutations at the Fc domain are associated with increased antibody aggregation. The SEC profiles ( c : overlay; d : individual Fc variants) and the abundance (percentage) of monomeric IgG is presented for the different Fc variants. e , Fc variants for the anti-NP monoclonal antibody 3B62 were generated and their binding (monomeric for FcγRI or as IgG immune complexes using NP-BSA for all other FcγRs) to immobilized human FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa was assessed by ELISA. Results are from one experiment performed in duplicates. The Fc effector activity of anti-influenza monoclonal antibody FY1 Fc variants was assessed in vitro using FcγRIIa-expressing ( f : n = 2 independent experiments for wild type, n = 1 for other groups, i : n = 2 independent experiments) and FcγRIIIa-expressing ( g (F 158 allele), j (V 158 allele); n = 1 for each variant, except for GAALIE–LS ( n = 2 independent experiments)) NFAT reporter cell lines. Fc variants with enhanced affinity for FcγRIIa or FcγRIIIa demonstrated increased capacity to induce NFAT reporter activation. h , Similarly, FY1 Fc variants engineered for FcγRIIIa binding exhibited improved primary human natural killer cell-mediated ADCC activity against HA-expressing cells. Results are the mean from two independent experiments using different natural killer cell donors.

Article Snippet: FcγRIIa- and FcγRIIIa-expressing NFAT reporter cells were purchased from Promega and have been authenticated by the vendor by STR analysis.

Techniques: Binding Assay, Generated, SPR Assay, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay, Activity Assay, In Vitro, Expressing, Variant Assay, Activation Assay

Journal: Nature

Article Title: Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection

doi: 10.1038/s41586-020-2838-z

Figure Lengend Snippet: a – f , To determine the abundance and FcγR expression profile of lung resident and infiltrated leukocytes during the course of influenza infection, cohorts of FcγR-humanized mice were infected (intranasally with H1N1 PR8; 5 mLD 50 ) and euthanized at different time points after infection (day 0 to 6). Lungs were homogenized and analysed by flow cytometry ( a : gating strategy) to determine the frequency ( b , c ) and FcγR expression profile ( f ) of innate effector leukocytes. n = 3 and n = 4 mice per time point for day 0–2 and day 3–6 time points, respectively. Influenza infection was associated with the recruitment of natural killer (NK) cells, neutrophils and monocytes, whereas the number of alveolar macrophages was reduced at the later stages of infection. Owing to the high degree of sequence similarity between FcγRIIa and FcγRIIb, expression of these FcγRs was assessed using antibody clones ( d : IV.3 for FcγRIIa; e : 2B6 for FcγRIIb) that exhibit high specificity, as assessed by ELISA using recombinant FcγRs ( n = 1 experiment performed in duplicates). Analysis of the FcγR expression profile (MFI) revealed that influenza infection had no effect on the levels of FcγRs expressed by the various leukocyte types. With the exception of natural killer cells, which expressed only FcγRIIIa, most innate effector leukocytes co-expressed multiple FcγRs, including the activating FcγRs, FcγRIIa and FcγRIIIa/b, as well as the inhibitory FcγRIIb. n = 3 and n = 4 mice per time point for day 0–2 and day 3–6 time points, respectively.

Article Snippet: FcγRIIa- and FcγRIIIa-expressing NFAT reporter cells were purchased from Promega and have been authenticated by the vendor by STR analysis.

Techniques: Expressing, Infection, Flow Cytometry, Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Nature

Article Title: Fc-optimized antibodies elicit CD8 immunity to viral respiratory infection

doi: 10.1038/s41586-020-2838-z

Figure Lengend Snippet: a – f , To determine the abundance and FcγR expression profile of dendritic cell subsets during the course of influenza infection, cohorts of FcγR-humanized mice were infected (intranasally with H1N1 PR8; 5 mLD 50 ) and euthanized at different time points after infection (day 0 to 6). Lungs were homogenized and analysed by flow cytometry ( a : gating strategy) to determine the frequency ( b ) and FcγR expression profile ( c : representative flow cytometry overlay; d – f : MFI) of the three major dendritic cell subsets identified: cDC1, cDC2 and tipDCs. Influenza infection was not associated with any major changes in the number of lung-resident cDC1 and cDC2, whereas tipDCs were almost absent at baseline, but their number increased markedly after infection. cDC1 and cDC2 expressed FcγRIIa and FcγRIIb, but they were negative for FcγRIIIa. By contrast, tipDCs expressed FcγRIIa and FcγRIIIa, along with the inhibitory FcγRIIb. Owing to the very low number of tipDCs at baseline, FcγR expression (MFI) was omitted. n = 4 mice per time point assessed. g – i , To investigate the effect of enhanced FcγRIIa engagement by GAALIE variants on the maturation status of dendritic cells, FcγR-humanized mice were treated with Fc domain variants of the anti-HA stalk antibody FI6v3, exhibiting differential FcγR affinity—wild type IgG1 (baseline FcγR affinity), GRLR (diminished binding to all classes of FcγRs), and GAALIE (increased FcγRIIa and FcγRIIIa affinity). Fc domain variants were administered intraperitoneally (3 mg kg −1 ) to FcγR-humanized mice ( n = 4 mice per treatment group in two independent experiments) 4 h before lethal challenge with H1N1 (PR8; 5 mLD 50 ). Mice were euthanized on day 4 and lung-resident dendritic cells were analysed by flow cytometry. The abundance of mature (defined as CD80 high CD86 high ) cDC1 ( g ), cDC2 ( h ), and tipDCs ( i ) was compared between mice treated with the various Fc domain variants of FI6v3. Representative flow cytometry plots from data presented in Fig. . In contrast to cDC1 and cDC2, no differences were observed in the maturation status of tipDCs among mice treated with different FI6v3 Fc variants (one-way ANOVA). j , k , In vitro differentiated monocyte-derived dendritic cells ( n = 4 peripheral blood mononuclear cell donors performed in two independent experiments) were stimulated overnight with IgG immune complexes (anti-NP–NP-BSA immune complexes ( j ) and heat-aggregated IgG complexes ( k )). The abundance (percentage) of mature dendritic cells (defined as CD80 high CD86 high ) was assessed by multicolour flow cytometry and compared against the corresponding wild-type-treated group by one-way ANOVA (Bonferroni post hoc analysis adjusted for multiple comparisons) * P = 0.0417, ** P = 0.0134, *** P < 0.0001, ^ P = 0.0049. l – n , Cluster analysis of dendritic cell populations present in the lungs of influenza-infected mice treated with Fc domain variants of anti-HA monoclonal antibodies. Mice ( n = 4 mice per group in two independent experiments) were treated as described in g – i , euthanized on day 4 after infection and dendritic cells (Lin + MHCII + CD11c + ) were analysed by multicolour flow cytometry. Data were dimensionally reduced and visualized using the UMAP algorithm. l , UMAP plots of dendritic cells in mice treated with the different Fc domain variants are presented. Populations were identified by X-shift using KNN density estimation and assigned IDs (A-J). m , The abundance of the various dendritic cell clusters in the different treatment groups was plotted and populations that are enriched or reduced in GAALIE-treated mice were identified. n , Histogram plots of the expression of CD80, CD86, CD40 and MHCII in dendritic cell populations that are enriched (red, A; and orange, H) or reduced (cyan, D; purple, E; and blue, F) in GAALIE-treated mice. GAALIE treatment was associated with the enrichment of dendritic cell populations characterized by high levels of CD86 and CD40 expression. Results are from four mice per treatment condition in two independent experiments.

Article Snippet: FcγRIIa- and FcγRIIIa-expressing NFAT reporter cells were purchased from Promega and have been authenticated by the vendor by STR analysis.

Techniques: Expressing, Infection, Flow Cytometry, Binding Assay, In Vitro, Derivative Assay