Antibody-dependent cell-mediated cytotoxicity (ADCC) assay with natural killer cells in malaria immunity

doi:10.21203/rs.3.pex-2513/v1

Method Article

Antibody-dependent cell-mediated cytotoxicity (ADCC) assay with natural killer cells in malaria immunity

https://doi.org/10.21203/rs.3.pex-2513/v1

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Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells describes the release of cytotoxic granules against a pathogen or infected cells. This can be mediated by antibodies opsonising a pathogen or infected cell and interacting with the cell surface receptor CD16 (FcγRIIIa) on NK cells. Studies suggest that NK ADCC activity plays a role in acquired and vaccine-induced immunity to malaria. This protocol measures ADCC using CD107 as a marker for cytotoxic granule release following incubation with antigen-coated beads or malaria parasites. Briefly, antigen-coated beads or malaria parasites are opsonised by antigen-specific antibodies and incubated with PBMCs and a fluorescently tagged anti-CD107 antibody. Interactions between the Fc-region of antibodies bound in immune complexes and CD16 expressed by NK cells promotes ADCC results in activation of NK cells with granule release and surface expression of CD107. The level of ADCC is assessed by measuring CD107 expression on NK cells (defined as CD3^-CD56⁺ lymphocytes) with flow cytometry.

NK cell

ADCC

FCγRIIIa

CD16

CD107

malaria

antibodies

This protocol describes the use of beads coated with Plasmodium falciparum circumsporozoite protein (CSP), a surface protein expressed by sporozoites of malaria, but it can be adapted for use with other protein antigens and whole malaria parasites (merozoites, sporozoites). Isolated PBMCs are also required, and can be harvested from buffy coats or whole blood.

Reagents and equipment:

§ 96 well plate U-bottom

§ Incubator (37°C in 5% CO₂)

§ PBMCs, isolated

§ RF10: 10% fetal calf serum (FCS) in RPMI-1640

§ Interleukin-2 (IL-2) stock (100 µg/mL) (2.1 x 10⁶ IU/mL)

§ CSP-coated beads (5 x 10⁷ beads/mL) (e.g. 2.0 µm Amine-Modified Polystyrene microspheres (Sigma L9529-1ML), coated with CSP or other protein antigen)

§ Brefeldin A (BFA) (Sigma) (Stock solution 5 mg/ml in DMSO)

§ Monensin (GolgiStop, BD Biosciences)

§ FACS buffer: 2% FCS in 1X PBS

§ Labelled antibodies: Anti-CD107a-AF647, Anti-CD16-BV450, Anti-CD56-PE-Cy7, Anti-CD3-APC-H7

Procedure:

1. Resuspend isolated PBMCs at 2x10⁶/mL and culture in RF10 with 100 IU/mL of IL-2 overnight.

2. To opsonise CSP-coated beads, add 40 µL of beads and 50 µL human sera (diluted 1:50 in flow cytometry/FACS buffer) to each well. Incubate in the dark for 1 hr at room temperature (RT).

3. To wash opsonised beads, add 150 µL of RPMI-1640 to each well and centrifuge at 400 x g for 5 min. Discard supernatant and repeat for a total of 3 washes. After the final wash, resuspend beads in 20 µL of RF10.

4. Spin down PBMCs at 300 x g for 5 min at RT and resuspend the cells at 5 x 10⁶/mL.

5. Add 100 µL of PBMCs and 5 µL of anti-CD107a-AF647 to each well of opsonised beads. Incubate for 60 min at 37°C in 5% CO₂ incubator.

5. Meanwhile, prepare BFA (1/100) and monensin (1/150) working stocks. Following PBMC incubation, add 13 µL of each to each well. Incubate for a further 3 hours.

6. Stop reaction by spinning down cells at 300 x g for 4 min at 4°C and resuspend cells in 50 µL of FACS buffer.

7. Stain with anti-CD16-BV450 (2.5 μL per well), anti-CD56-PE-Cy7 (1.5 μL per well) and anti-CD3-APC-H7 (1.5 μL per well) for 30 min on ice.

8. Wash 3 times with FACS buffer.

9. Analyse cells by FACS.

10. Natural killer (NK) cells are defined as CD3^-, CD56⁺ lymphocytes and the level of ADCC was quantified as the percentage of NK cells with CD107a staining.

Lichtfuss, G.F., et al. HIV inhibits early signal transduction events triggered by CD16 cross-875 linking on NK cells, which are important for antibody-dependent cellular cytotoxicity. J Leukoc Biol 89, 149-158 (2011).

Feng, G., Wines, B.D., Kurtovic, L. et al. Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites. Nat Commun 12, 1742 (2021). https://doi.org/10.1038/s41467-021-21998-4

Thanks to Jessica Horton for editing and formatting this protocol

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Antibody-dependent cell-mediated cytotoxicity (ADCC) assay with natural killer cells in malaria immunity

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Version 1

Abstract

Introduction

Reagents

Reagents and equipment:

Procedure

Procedure:

References

Acknowledgements

Associated Publications

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Antibody-dependent cell-mediated cytotoxicity (ADCC) assay with natural killer cells in malaria immunity

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Version 1

Abstract

Introduction

Reagents

Reagents and equipment:

Procedure

Procedure:

References

Acknowledgements

Associated Publications

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