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. 2019 Aug:471:46-56.
doi: 10.1016/j.jim.2019.05.006. Epub 2019 May 25.

A versatile high-throughput assay to characterize antibody-mediated neutrophil phagocytosis

Christina B Karsten  1 , Nickita Mehta  1 , Sally A Shin  1 , Thomas J Diefenbach  1 , Matthew D Slein  1 , Wiktor Karpinski  1 , Edward B Irvine  2 , Thomas Broge  1 , Todd J Suscovich  1 , Galit Alter  3
Affiliations

A versatile high-throughput assay to characterize antibody-mediated neutrophil phagocytosis

Christina B Karsten et al. J Immunol Methods. 2019 Aug.

Abstract

Neutrophils, the most abundant white blood cell, play a critical role in anti-pathogen immunity via phagocytic clearance, secretion of enzymes and immunomodulators, and the release of extracellular traps. Neutrophils non-specifically sense infection through an array of innate immune receptors and inflammatory sensors, but are also able to respond in a pathogen/antigen-specific manner when leveraged by antibodies via Fc-receptors. Among neutrophil functions, antibody-dependent neutrophil phagocytosis (ADNP) results in antibody-mediated opsonization, enabling neutrophils to sense and respond to infection in a pathogen-appropriate manner. Here, we describe a high-throughput flow cytometric approach to effectively visualize and quantify ADNP and its downstream consequences. The assay is easily adaptable, supporting both the use of purified neutrophils or white blood cells, the use of purified Ig or serum, and the broad utility of any target antigen. Thus, this ADNP assay represents a high-throughput platform for the in-depth characterization of neutrophil function.

Keywords: Antibody-dependent neutrophil phagocytosis; High-throughput assay; Immune correlates of protection; Non-neutralizing antibody; Systems serology.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Schematic representation and gating strategy of the ADNP (antibody-dependent neutrophil phagocytosis) assay. (A) Fluorescent NeutrAvidin beads were coated with biotinylated human immunodeficiency virus 1 (HIV-1) YU2 gp120 and immune complexes were formed with purified antibody or serum. White blood cells (WBCs) were collected by ammonium-chloride-potassium (ACK) lysis from human blood using acid citrate dextrose (ACD) as a coagulant. WBCs were stimulated with immune complexes, stained for CD66b and fixed. Cell fluorescence was measured by flow cytometry and data were analyzed using FlowJo. (B) Granulocytes were gated based on SSC-A and FSC-A and the neutrophil subpopulation was determined using CD66b-PacBlue as a positive marker. Uptake of fluorescent immune complexes was quantified by measuring the percentage and gMFI (geometric mean fluorescence intensity) of FITC+ cells within the neutrophil population. Phagocytosis expressed as a “phagoscore” was calculated as described in the Materials and Methods. (C, D) Depiction of the data presented in B for the bead+ neutrophil population as histograms (C) and an overlay histogram (D).
Fig. 2
Fig. 2
Effect of neutrophil source and cell number on phagocytosis. (A) After purification, CD66b+ neutrophils were gated. (B-D) ADNP assays were performed using HIVIG (pooled Ig from HIV-1+ subjects serving as a positive control), IVIG (pooled Ig from HIV− subjects serving as a negative control) and HIV-1 gp120 to assess the phagocytic ability of neutrophils present in WBCs or purified neutrophils (B). The graphs depicts the influence of the anti-coagulants, ACD or ethylenediaminetetraacetic acid (EDTA), on WBCs or purified neutrophils (C), or the impact of WBC number for phagocytosis (D). For all presented assay results, phagoscores are displayed as mean ± SD of at least two independent experiments.
Fig. 3
Fig. 3
Optimization of antigen input and incubation times for the formation of immune complexes. (A) Sera from 27 HIV+, and 2 HIV− patients were used to obtain a range of background, low, medium and high phagocytic responses using HIV-1 gp120 as an antigen. No antibody, IVIG and HIV− serum were used as a negative control and HIVIG was used as a positive control. Phagoscores were measured and area under the curve values of 1:10, 1:100 and 1:1000 diluted samples were calculated. We chose donor 4 and 23 to represent low, donor 16 and 19 to represent medium and donor 5 and 24 to represent high phagocytic activity and these were used in the following experiments. The impact of (B) antigen amounts and (C) incubation time on phagocytosis was assessed. Results are shown as the mean ± SD phagoscore from two independent experiments. (D) The correlation plots depicts the non-parametric Spearman correlation of IgG1 titers and ADNP reactivity aginst HIV-1 gp120-specific antibodies in the tested serum samples. The samples selected for high, medium and low activity are indicated.
Fig. 4
Fig. 4
The influence of incubation time on phagocytosis and cellular quality. (A) HIV-1 gp120 immune complexes generated using a 1:100 serum dilution were incubated with neutrophils for 1, 2, 4, 16, 18 and 24 h. The mean, minimum and maximum values are displayed. (B) The same data presented in (A) is expressed as fold over background over the HIV− serum sample in the bar graph. (C) The same assay as in (A) was performed with titrated samples and performed at incubation times of 1, 2 or 4 h. The mean ± SD phagoscore is displayed. (D) Neutrophil quality after a 1-4 h stimulation with HIV-1 gp120 immune complexes in an ADNP assay was assessed by labeling the cells with live/dead stain and Annexin V in addition to the standard staining. The median phagoscore or percent apoptotic or dead cells are shown.
Fig. 5
Fig. 5
Characterization of neutrophil-immune complex interactions in the ADNP assay and assay reproducibility. (A-C) Neutrophil phagocytic uptake or attachment of HIV-1 gp120-immune complexes to neutrophils was measured by imaging flow cytometry after 1 h of incubation at 4 °C or 37 °C. Neutrophils were labeled with anti-CD66b (red), nuclei with DAPI (purple) and immune complexes were measured in the FITC channel (green). A total of 1000 cells from 500,000 pooled replicate wells from a single donor were analyzed. (A) Representative images of neutrophils are shown in the following order from top to bottom: no bead internalization, surface bead attachment, single bead internalization, and internalization of two or three beads by neutrophils. (B) The total percentage of neutrophils associated with fluorescent beads was calculated for the different conditions tested. (C) The data obtained for HIVIG and IVIG after an incubation of the immune complexes with the neutrophils at 37 °C is further analyzed to show the % of attached immune complexes as well as the % of phagocytosed immune complexes within the bead-associated neutrophil population. (D) Whether or not immune complex uptake was mediated by Fc gamma receptors (FcgRs) was assessed by performing an ADNP assay using HIVIG and IVIG with HIV-1 gp120-coated beads, in which single or all FcRs were blocked. Four independent experiments were performed. Displayed is the mean ± SEM phagoscore of HIVIG-containing samples normalized to the results in the absence of FcgR block. P values were calculated using a paired T-Test. (E) Donor-to-donor variability of neutrophils was assessed by conducting an ADNP assay using 5 different WBC donors and HIV-1 gp120 coated beads. (F) The robustness of the ADNP assay was assessed across different users. For this purpose, 3 different experimenters performed the same ADNP assay using HIV-1 gp120-coated beads, HIV-1+ samples, controls, and the same cell donors. For E-F, the results of a single experiment are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Neutrophil phagocytosis is antigen-specific. (A) ADNP assay specificity was analyzed by comparing the phagocytosis of immune complexes formed using HIV-1+ or HIV-1− samples with HIV-1 gp120-, influenza HA1- or Ebola virus GP-antigen coated beads. (B-D) The results obtained in (A) show the phagocytic profiles obtained for each serum donor for HIV-1 gp120 (B), influenza HA1 (C) or tetanus toxoid (D). Two independent experiments were performed and the mean ± SD phagoscore is displayed without (A) or after (B-D) subtraction of the background (no antibody).
Fig. 7
Fig. 7
Secondary ADNP assays provide additional insights into antibody-mediated neutrophil activation. (A) Secondary readouts were performed on supernatants collected following incubation of 27 HIV+ sera as well as 2 HIV− sera and HIV-1 gp120-coated beads with neutrophils at 1 or 4 h. (B) Neutrophil degranulation in the supernatant was assessed using human lactoferrin and myeloperoxidase (MPO) ELISAs. (D) Cytokine secretion into the supernatants was analyzed by luminex. Results are displayed for granulocyte-colony stimulating factor (G-CSF), tumor necrosis factor alpha (TNF-alpha), CD40 ligand (CD40L) and interleukin-1 receptor antagonist protein (IL-1RA). Figs. A, B and D show the averaged results of two independent experiments after subtraction of the background (HIV− samples) with lines representing the median cytokine concentrations. (C) Non-parametric Spearman correlation of the results from A, B, and D.
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