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: Cell reports

Article Title: Maternal Anti-Dengue IgG Fucosylation Predicts Susceptibility to Dengue Disease in Infants

doi: 10.1016/j.celrep.2020.107642

Figure Lengend Snippet:

Article Snippet: To assess FcγR expression cells were stained at 1:100 with a FITC conjugated anti-human FcγRIIa antibody, clone IV.3 (STEMCELL Technologies; 60012FI); Brilliant Violet 711 conjugated anti-human FcγRIII, clone 3G8 (Biolegend; 302044); an FcγRIIb antibody clone 2B6 (gifted by the Ravetch lab, Rockefeller University) conjugated to Alexa Fluor 647 (ThermoFisher; A20186) and an APC conjugated anti-human FcγRI, clone 10.1 (Biolegend, 305014) for 30 minutes at 4°C.

Techniques: Virus, Recombinant, Plasmid Preparation, Software

Journal: The FASEB Journal

Article Title: Identification of cyclic peptides able to mimic the functional epitope of IgG1-Fc for human FcγRI

doi: 10.1096/fj.08-117069

Figure Lengend Snippet: Peptide binding specificity for FcγRs assessed by AlphaScreen and rosette formation on FcγR bearing cells. A ) Each biotinylated peptide was incubated with streptavidin donor beads, while the FcγRs were preincubated with nickel chelate acceptor beads. Signal due to peptide complexes added over a range of concentrations of 37, 111, and 333 nM was normalized relative to the signal due to IgG1 binding (equivalent to 100%) at 5, 15, and 46 nM for FcγRI, FcγRIIa, and FcγRIIb; and 15, 46, and 137 nM for FcγRIIIa and FcγRIIIb. Graph shows binding results for FcγRI, with results for FcγRIIIa shown as an example. Results shown were means of 4 independent experiments. B ) Microbeads were sensitized with 10 μM peptide (22, 29, 30 or 33) or 400 nM IgG1 and incubated with 2 × 10 5 primary NK, Daudi, K562, or U937 cells at a ratio of 100:1 (molecule-bead complexes:leukocyte cells). Peptide or IgG-bead complex binding to cells was measured as rosette formation using a fluorescence microscope. Rosettes were defined as at least 5 beads clustered around 1 cell. Percentage rosetting indicates the ratio of rosetted cells to total cells counted. Control beads were not conjugated with any peptide. Results shown are 1 of 5 comparable experiments. C ) Rosette formation with peptide 22 and a U937 cell visualized by light microscopy (×400).

Article Snippet: To express the other human FcγR in soluble form, DNA was purchased that encoded human FcγRIIa, FcγRIIb, and FcγRIIIa extracellular domains (Geneservice, Cambridge, UK), cloned in fusion with a Flag-His-tag DNA sequence in pDEST12.2 (Invitrogen, Paisley, UK) and transfected into HEK-293 EBNA cells (6×10 6 cells).

Techniques: Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Incubation, Fluorescence, Microscopy, Control, Light Microscopy

Journal: Nature Communications

Article Title: Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites

doi: 10.1038/s41467-021-21998-4

Figure Lengend Snippet: A Effect of blocking-specific FcγRs on phagocytosis of sporozoites by neutrophils. P. falciparum sporozoites were opsonized with a pool of Kenyan adult sera. Bars represent mean and range from two experiments in duplicate ( P < 0.001, one-way ANOVA). B Effect of blocking-specific FcγRs on phagocytosis of CSP-beads by neutrophils. CSP-coated beads were opsonized with a pool of serum from Kenyan adults. Bars represent mean and standard error from five experiments conducted in duplicate ( P = 0.001, one-way ANOVA). C Effect of blocking FcγR receptors on phagocytosis of CSP beads by THP-1 cells. Bars represent mean and standard error from two experiments ( P < 0.001 for FcγRI, one-way ANOVA). D Opsonic phagocytosis of CSP-coated beads by THP-1 cells was much lower in the presence of heat-inactivated human serum (light-blue bars) compared to the inclusion of FCS alone (dark-blue bars). Bars and error bars represent the mean and range from two independent experiments. E Antibodies from selected Kenyan serum samples induced ADCC activity using CSP-coated beads and an ADCC reporter cell line. Selected individuals ( n = 13, AR1–AR13) are shown as examples to demonstrate the variation of activity observed (Kruskal–Wallis test P = 0.008). Sera from malaria-naive Melbourne donors were used as negative controls (MC), which were lower than the Kenyan serum samples (Mann–Whitney test P = 0.023). Bars and error bars represent the mean and range of the duplicates. ADCC values on the Y axis are luminescence units. F Correlation between antibody activity in FcγRIII binding and ADCC assays (reporter cell line) among selected Kenyan adult serum samples ( n = 31) with high, intermediate or low total IgG against CSP ( r = 0.405, P = 0.022, linear regression). The solid and dotted lines represent the linear regression line and 95% confidence intervals, respectively. G Antibodies from the Kenyan sera were tested for ADCC activity using CSP-coated beads and primary NK cells. Selected individuals ( n = 13 S1–S13) are shown as examples of the variation of responses observed (Kruskal–Wallis test P = 0.015). Sera pool from malaria-naive Melbourne donors was used as negative controls (MC), which were lower than the Kenyan serum samples (Mann–Whitney test P = 0.03 for FcγRIIa and P = 0.05 for FcγRIII). Bars and error bars represent the mean and range of the duplicates. ADCC values on the Y axis represent the percentage of NK cells that are positive for CD107 staining by flow cytometry. H Correlation between antibody-mediated FcγRIII binding and ADCC activity (using primary NK cells) among selected Kenyan adult serum samples ( n = 31) with high, intermediate or low total IgG against CSP ( r = 0.538, P = 0.002, linear regression). The solid and dotted lines represent the linear regression line and 95% confidence intervals, respectively. * indicates significant difference ( P < 0.05; ** indicates P < 0.01); ns not significant, RPI relative phagocytosis index.

Article Snippet: Cells were phenotyped for FcγR expression using antibodies to FcγRI (Clone 10.1 BD Bioscience, 1:50), FcγRIIa (Clone 8.26, BD Bioscience, 1:25) and FcγRIII (Clone 3G8 BD Bioscience, 1:50).

Techniques: Blocking Assay, Activity Assay, MANN-WHITNEY, Binding Assay, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites

doi: 10.1038/s41467-021-21998-4

Figure Lengend Snippet: A Antibodies to CSP among Kenyan adults were tested for their ability to promote binding to FcγRIIa and FcγRIII. Bars and error bars represent the mean and standard deviation for each sample tested in duplicate. Representative individuals ( n = 10) are shown to reflect the range of activities observed. Sera from malaria-naive Melbourne donors were used as negative controls (MC), which were lower than the Kenyan serum samples (Mann–Whitney test P = 0.026). B Correlation between FcγRIIa binding and FcγRIII binding in the Kenyan adult cohort. There was a significant correlation between FcγRIIa binding and FcγRIII binding ( n = 104, r = 0.71, P < 0.001, linear regression). The solid and dotted lines represent the linear regression line and 95% confidence intervals, respectively. C Correlation between antibody-mediated binding of two different alleles of FcγRIIa (R131 and H131) using samples from the Kenyan adult cohort ( n = 104, Spearman’s rho = 0.837, P < 0.001). The solid and dotted lines represent the linear regression line and 95% confidence intervals, respectively. D Correlation between antibody-mediated binding of two different alleles of FcγRIII (F158 and V158) using samples from the Kenyan adult cohort ( n = 104, Spearman’s rho = 0.777, P < 0.001). The solid and dotted lines represent the linear regression line and 95% confidence intervals, respectively.

Article Snippet: Cells were phenotyped for FcγR expression using antibodies to FcγRI (Clone 10.1 BD Bioscience, 1:50), FcγRIIa (Clone 8.26, BD Bioscience, 1:25) and FcγRIII (Clone 3G8 BD Bioscience, 1:50).

Techniques: Binding Assay, Standard Deviation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites

doi: 10.1038/s41467-021-21998-4

Figure Lengend Snippet: A The level of opsonic phagocytosis by neutrophils of beads coated with different regions of CSP. Beads coated with full-length CSP (FL), the CT region, the NT region and the NANP repeat region (repeats) were opsonized with antibodies from a pool of Kenyan adult donors. Unopsonized beads were used as a negative control for assay background. Data show mean and standard error from six experiments ( P = 0.051 and P = 0.187 for comparisons between NT and NANP or CT domains, respectively; one-way ANOVA). The level of phagocytosis activity was expressed relative to the IgG reactivity to each region (see Supplementary Fig. ). B Antibodies from the Kenyan adult pool were tested for their ability to promote the binding of FcγRIIa and FcγRIII binding activity with each region of CSP. The level of FcγR binding was standardised according to the level of total IgG binding to each CSP construct (FcR-binding efficiency; see Supplementary Fig. ). Data show mean and standard error based on three experiments. FcγRIIa and FcγRIII binding to the NT region was significantly higher compared to the NANP-repeat region or CT region ( P < 0.001, two-way ANOVA). C Opsonic phagocytosis of P. falciparum sporozoites by neutrophils mediated by rabbit polyclonal antibody to CSP, compared to no-antibody (no Ab) control ( P < 0.001, unpaired t test). Data show mean and standard errors from four experiments. D Opsonic phagocytosis of P. falciparum sporozoites by neutrophils mediated by mouse monoclonal antibody (2H8) against CSP at different concentration, compared to mouse monoclonal antibody (3D11) against P. berghei CSP as a control ( P = 0.0025, one-way ANOVA). Data shown are mean and standard errors from four experiments. E Rabbit IgG to the NT region was more efficient in promoting phagocytosis of PfCSP- P. berghei sporozoites by neutrophil compared to rabbit IgG to the CT region ( P = 0.0079, unpaired t test). Data shown are mean and standard error based on two experiments. Phagocytosis activity was standardised relative to the level of IgG to CSP of each rabbit antibody. F Rabbit antibodies generated against the NT region were tested for reactivity against an array of overlapping peptides (15 aa with 12 aa overlap) including the NT region, NANP/NVDP repeat and the junctional region. Data show IgG reactivity to different peptides; the reactive peptides include a 21-aa region corresponding to CSP residues 76–96 ( P = 0.036 for differences in peptide reactivity, Kruskal–Wallis test, two experiments). Error bars represent the range from two experimental repeats. Peptide sequences are provided in Supplementary Fig. .

Article Snippet: Cells were phenotyped for FcγR expression using antibodies to FcγRI (Clone 10.1 BD Bioscience, 1:50), FcγRIIa (Clone 8.26, BD Bioscience, 1:25) and FcγRIII (Clone 3G8 BD Bioscience, 1:50).

Techniques: Negative Control, Activity Assay, Binding Assay, Construct, Concentration Assay, Generated

Journal: Nature Communications

Article Title: Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites

doi: 10.1038/s41467-021-21998-4

Figure Lengend Snippet: Samples from Kenyan children and adults ( n = 75) were tested for total IgG binding to CSP ( A ), opsonic phagocytosis of CSP beads (blue bar) or whole merozoites (purple bars) by neutrophils ( B ), FcγRIIa binding ( C ) and FcγRIII binding ( D ) against CSP (blue bars) and FcγRIIa binding ( E ) and FcγRIII binding ( F ) against merozoites (purple bars). Individuals were categorised into age groups of 0–1 (0.5), 1–2 (1), 2–4(2), 4–18 (5) 18 or more years (adults). The total level of IgG to CSP was significantly lower among children compared to adults ( P < 0.001, Kruskal–Wallis Test). Opsonic phagocytosis of merozoites and CSP-coated beads was also lower among children compared to adults ( P < 0.001, respectively, Kruskal–Wallis Test), with a higher level of opsonic phagocytosis against merozoites compared to CSP-coated beads ( P < 0.001, Kruskal–Wallis Test). Similar trends were seen for FcγRIIa and FcγRIII binding to CSP ( P < 0.001, respectively, Kruskal–Wallis Test). The level of FcγRIIa and FcγRIII binding to merozoites was also varied among age groups ( P = 0.018 for FcγRIIa and P = 0.003 for FcγRIII, respectively, Kruskal–Wallis Test). A – F Boxes and whiskers indicate the medians and interquartile range (IQR) and 1.5 × IQR of each group. Values exceeding this range are presented as dots. G Opsonic phagocytosis activity by neutrophils was expressed relative to IgG reactivity against CSP (phagocytosis efficiency) for each individual. Phagocytosis efficiency was significantly higher for adults compared to children (Mann–Whitney U test, P = 0.022). Bars and error bars represent the median and interquartile range for each group.

Article Snippet: Cells were phenotyped for FcγR expression using antibodies to FcγRI (Clone 10.1 BD Bioscience, 1:50), FcγRIIa (Clone 8.26, BD Bioscience, 1:25) and FcγRIII (Clone 3G8 BD Bioscience, 1:50).

Techniques: Binding Assay, Activity Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites

doi: 10.1038/s41467-021-21998-4

Figure Lengend Snippet: A Among Kenyan samples ( n = 104), levels of opsonic phagocytosis were significantly correlated with FcγRIIa binding and FcγRIII binding. B – D Antibody-mediated FcγRIIa binding, FcγRIII binding and opsonic phagocytosis were positively correlated with IgG ( B ), IgG1 ( C ) and IgG3 ( D ) reactivity to CSP among samples. All correlations were statistically significant ( P < 0.001, Spearman’s correlation). E Samples ( n = 104) with higher opsonic phagocytosis activity (defined as greater than the median) showed higher levels of total IgG to full-length CSP ( P < 0.001, two-way ANOVA), the NANP repeat region ( P = 0.001, two-way ANOVA), the C-terminal region ( P < 0.001, two-way ANOVA) and the N-terminal region ( P < 0.001, two-way ANOVA) compared to samples with lower opsonic phagocytosis. FcγRIIa and FcγRIII binding by antibodies to CSP was also significantly higher among individuals with high opsonic phagocytosis ( P < 0.001 and P < 0.001 respectively, two-way ANOVA). The levels of CSP-specific IgG2 and IgG3 against were significantly higher in samples with high opsonic phagocytosis ( P = 0.0464 and P < 0.001 respectively, two-way ANOVA), but not clearly higher for IgG1 ( P = 0.106, two-way ANOVA) and IgG4 ( P > 0.999, two-way ANOVA). The solid and dotted lines represent the linear regression line and 95% confidence intervals. Boxes and whiskers indicate the medians and interquartile range (IQR) and 1.5 × IQR of each group. Values exceeding this range are presented as dots. Asterisks indicate statistically significant levels (* P < 0.05, ** P < 0.01).

Article Snippet: Cells were phenotyped for FcγR expression using antibodies to FcγRI (Clone 10.1 BD Bioscience, 1:50), FcγRIIa (Clone 8.26, BD Bioscience, 1:25) and FcγRIII (Clone 3G8 BD Bioscience, 1:50).

Techniques: Binding Assay, Activity Assay

Journal: Nature Communications

Article Title: Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites

doi: 10.1038/s41467-021-21998-4

Figure Lengend Snippet: Multivariate analysis of the association of opsonic phagocytosis of CSP-coated beads by neutrophils with FcγRIIa, FcγRIII, IgG subclasses and in the Kenyan adult cohort.

Article Snippet: Cells were phenotyped for FcγR expression using antibodies to FcγRI (Clone 10.1 BD Bioscience, 1:50), FcγRIIa (Clone 8.26, BD Bioscience, 1:25) and FcγRIII (Clone 3G8 BD Bioscience, 1:50).

Techniques: Binding Assay