An In Vitro Artificial Activation Assay for Studying Fc Receptor Activation by Antibodies

Overview

This video demonstrates FcγRIIIa-driven events initiated by therapeutic antibodies in human natural killer cells. The artificial stimulation platform facilitates the exploration of downstream effector functions, encompassing cytoskeletal rearrangement, degranulation, chemokine/cytokine production, and signaling pathways mediated by the FcγRIIIa and Fc portions of antibodies involved in binding.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Isolation of PBMCs and enrichment/purification of NK cells

NOTE: Other methods for the isolation of peripheral blood mononuclear cells (PBMCs) and enrichment/purification can also be performed.

1. Draw 200 mL of blood under regulated conditions into a tube containing heparin.
2. Add 15 mL of the density gradient medium into a 50 mL tube with a porous barrier incorporated. Spin the tube at 1000 x g for 30 s.
3. Add 12.5 mL of blood into the tube, followed by another 12.5 mL of PBS, and gently invert 3x. Spin the tube at 800 x g for 15 min at room temperature (RT) with no breaks.
4. Prepare a 50 mL conical tube with 40 mL of PBS for each tube with a porous barrier while the cells are spinning.
5. Carefully remove the tubes and inspect after centrifugation. Check for a thin, visibly white layer of PBMCs above the porous barrier, between the 20 mL and 25 mL demarcations on the 50 mL tube.
6. Carefully remove as much liquid as possible from the top using a pipette without disturbing the thin, white PBMC layer.
7. With a clean 10 mL serological pipette, gently remove the PBMC layer in addition to the clear, yellowish liquid layer down to the filter of the tube.
8. Expel the PBMCs and liquid into the 50 mL conical containing PBS prepared in step 1.4. Spin tubes at RT and 300 x g for 5 min.
9. Aspirate the PBS wash and add an additional 40 mL of PBS. Spin tubes at RT and 300 x g for 5 min.
10. After washing, count the cells and resuspend them in 40 µL of 2% BSA/PBS per 1 x 10⁷ cells.
11. Proceed to the isolation of NK cells using the method of choice. Choices include manual or automated magnetic bead-based sorting. Fluorescence-activated cell sorting can also be performed.
12. Remove a small aliquot of cells to determine the purity of NK cells by flow cytometry. Gating strategy includes the following: gate on live cells based on forward vs. side scatter profile, then assess the purity based on NK markers (e.g., CD3, CD56) in the specific live population. Typical purity is >95%.
13. After isolation is completed, spin cells at RT and 300 x g for 5 min to pellet and aspirate.
14. Resuspend the cell pellet to 1 x 10⁷ cells/mL in RPMI 1640 with nutrients, 10% heat-inactivated FBS, 1 mM sodium pyruvate, 55 mM 2-ME, and 10 mM HEPES (pH = 7.2).

2. Antibody-mediated activation of NK cells via FcγRIIIa

1. Set a refrigerated microcentrifuge to 4 °C.
2. Dispense 1 x 10⁶ (100 µL of) resuspended NK cells into 1.5 mL tube(s) and place on ice.
   NOTE: Cells can be dispensed into a 96 well U-bottom plate if there are numerous samples.
3. Prepare 1 µL of 100 µg/mL rituximab (or antibody of interest) per sample and add 1 µL to the cells for a final concentration of 1 μg/mL antibody.
   NOTE: Other molecules can be added to the cells at this point or earlier for the assessment of combination effects. Here, ritixumab was used for stimulation. In prior studies, small molecule inhibitors have been added to stimulations.
4. Incubate the sample on ice for 30 min. During incubation, prepare 50 μL of 50 µg/mL anti-human κ light chain antibody per sample in media and warm to 37 °C on a heat block or in a water bath.
5. After incubation of cells on ice, add 1 mL of ice-cold media and spin at 135 x g for 5 min at 4 °C. Wash again with 1 mL of ice-cold media.
6. Aspirate the supernatant after the last wash and add 50 µL of the anti-human κ light chain mixture prepared in step 2.4.
7. Immediately incubate samples for the end-user-determined timepoints at 37 °C on a heat block or in a water bath.
8. Stop stimulation by adding 1 mL of ice-cold media and immediately spin samples in a refrigerated centrifuge at 135 x g for 5 min. Wash once more with 1 mL of ice-cold media.

Materials

Name	Company	Catalog Number	Comments
16% paraformaldehyde	Thermo Fisher Scientific	50-980-487
Alexa Fluor 488 phalloidin	Thermo Fisher Scientific	A12379
anti-mouse HRP antibody	Cell Signaling Technologies	7076
AutoMACS instrument	Miltenyi		NK cell isolation method; another isolation instrument may be used
CD107a APC antibody	Biolegend	328620
Bovine Serum Albumin Fraction V, fatty acid free	Millipore Sigma	10775835001
Cytokine 30-Plex Human Panel	Thermo Fisher Scientific	LHC6003M	chemokine/cytokine method; another chemokine/cytokine analysis method may be used
FBS (Fetal Bovine Serum)	Hyclone	SH30071.01
Goat anti-human κ light chain antibody	Millipore Sigma	AP502
Halt Protease and Phosphatase Inhibitor Cocktail	Thermo Fisher Scientific	78440
pAKT (S473) antibody	Cell Signaling Technologies
pERK1/2 antibody	Cell Signaling Technologies
pPRAS40 antibody	Cell Signaling Technologies	13175
β-actin HRP antibody	Abcam	ab6721
NK cell isolation kit	Miltenyi	130-092-657	NK cell isolation kit; another isolation kit may be used