A protocol for anti-CD4 IgG antibody purification using plasma samples from people with HIV and antibody-mediated cytotoxicity

Background. Up to 20% of people with HIV (PWH) fail to recover their CD4+ T cell counts to levels similar to healthy controls after suppressive antiretroviral therapy (ART). Immune non-responders (INRs) are PWH on suppressive ART with CD4+ T cell counts lower than 350 cells/mL, whereas their CD8+ T cell counts are higher than healthy controls. We are the first group to report that increased anti-CD4 autoantibody IgGs in INRs are responsible for blunted CD4+ T cell reconstitution in PWH with ART and viral suppression through anti-CD4 IgG-induced antibody-mediated cytotoxicity (ADCC) against CD4+ T cells in vitro. Notably, anti-CD4 IgG-mediated poor CD4+ T cell recovery from suppressive ART is the only mechanism targeting CD4+ T cells, specifically. Results. We provide a detailed one-by-one step protocol from antigen-specific antibody isolation using plasma samples, to ADCC assay. Conclusions. To promote reproducible research, a detailed protocol for isolating anti-CD4 IgG autoantibodies from plasma samples of PWH and evaluating ADCC effects is reported here.• Antigen-specific antibody isolation using human plasma samples• Antibody-mediated cytotoxicity (ADCC)


Method details
After long-term antiretroviral therapy (ART) and peripheral viral suppression, up to 20% of people with HIV (PWH) fail to recover their CD4 + T cell counts to levels similar to healthy controls.CD4 + T cell decline characterizes these immune non-responders (INRs, aviremic/ART + , CD4 + T cell counts < 350 cells/mL); moreover, their CD8 + T cell counts are higher than healthy controls (1).We are the first group to report that plasma levels of anti-CD4 autoantibody IgGs are increased in INRs compared to immune responders (IRs) and uninfected controls, and purified anti-CD4 IgGs from plasma samples of INRs induced CD4 + T cell death through antibodymediated cytotoxicity (ADCC) in vitro (1-7).Notably, anti-CD4 IgG-mediated poor CD4 + T cell recovery from suppressive ART is the only mechanism targeting CD4 + T cells, specifically.Other known mechanisms (e.g., thymic and lymph node fibrosis, chronic immune activation, leaky gut, and increased microbial translocation) may not account for decreased CD4 + T cell counts specifically in INRs.
Recently, there are studies and interests related to autoimmunity and anti-CD4 autoantibodies in HIV, though there is a debate about whether anti-CD4 autoantibodies mediate CD4 + T cell death through antibody-mediated cytotoxicity (8), which has conflicting results of our previous studies (1, 3, 9, 10)mainly due to the challenging technologies (antigen-specific antibody isolation from plasma and ADCC).To increase transparency and research reproducibility, we show methodology and explain the details of key steps in the anti-CD4 autoantibody isolation from plasma samples and the ADCC assay to allow reproducibility in the future.This protocol for isolating anti-CD4 antibodies also can be used to isolate any antigen-specific antibodies from plasma or from antibody containing fluids, using the desired antigens.
3. Add 400 μL PBS and cap it, spin the device at 14,000 × g for approximately 7 min ( Fig. 1 ).4. Repeat step 3 two times.5. To recover the concentrated solute, place the Amicon® Ultra filter device upside down in a clean microcentrifuge tube.Spin for 2 min at 1000 × g to transfer the concentrated sample from the device to the tube ( Fig. 2 ).
Step 2 recombinant soluble CD4 protein binding to the magnetic bead ➢ Add the protein from step 1 to a 1.5 mL Eppendorf tube, then add the coupling buffer from the kit, and keep it on ice.➢ Prepare 1 mM HCl and make sure it is ice-cold before use.

Magnetic bead preparation
A. Dispense the 100uL magnetic beads into a 1.5 mL Eppendorf tube.Step 3 purify the isolated anti-CD4 antibodies from plasma 1. Binding of the target protein A. Add 100 uL labeled beads to a 50 mL tube, add 20 mL plasma containing the anti-CD4 antibodies to the 50 mL tube.Add 20 mL wash/bind buffer containing 2 M Urea.B. Incubate with slow end-over-end mixing (slowly rotate at 200 rpm on a rotator to prevent beads precipitation) for 4 h at 4 °C or room temperature for 2 h.C. Briefly spin at 10 g for 1 min, remove the supernatant (containing non-bound antibodies, which may be stored for future usage) and collect pellets.2. Wash (perform this step 3 times totally) A. Add 500 μL wash buffer, mix, then place on the magnetic rack.B. Remove the liquid.

Elution (perform this step 2 times totally)
A. Add 500 uL volumes of elution buffer for each the 50 μL magnetic bead volume.B. Fully resuspend the medium and incubate for at least 2 min.C. Remove and collect the elution fraction.
Step 4 exchange buffer and concentrate protein 1. Insert an Amicon® Ultra-0.5 device into one microcentrifuge tube provided in the kit.
2. Add the 500 uL purified anti-CD4 antibodies to an Amicon® Ultra filter device.
3. Spin the device at 14,000 × g for approximately 5 min.Decant the liquid that flowed through the filter.4. Repeat steps 2-3 until all purified anti-CD4 antibodies are concentrated.5. Add 400 uL PBS, spin the device at 14,000 × g for approximately 5 min.Decant the liquid that flowed through the filter.6. Repeat step 5 two times.7. To recover the concentrated solute, place the Amicon® Ultra filter device upside down in a clean microcentrifuge tube.Spin for 2 min at 1000 × g to transfer the concentrated sample from the device to the tube.
Step 5 isolation of anti-CD4 IgGs The isolated anti-CD4 antibodies contain all classes of antibodies (e.g., IgA, IgM, IgG).Since IgG is the main class of antibodies presenting with ADCC activity, next we will isolate IgG from the anti-CD4 antibodies.
➢ Protein A IgG purification kit (Catalog number: 44667, ThermoScientific) ➢ Amicon Ultra-0.5 Centrifugal Filter Unit (Catolog number: UFC503096, Millipore) 1. Remove the end write cover from the column.2. Insert the column into a 15 ml tube.3. Add 1 mL binding buffer to wash the column, perform 3 times, do not let the column dry completely.4. When column is almost dry, add 200-500ul plasma; let IgG bind to the column 5. Add 1 mL binding buffer to wash the unbounded antibodies, perform 5 times.6. Add 1 mL elution buffer to wash the column; because the first wash buffer has the most IgG, the second wash can occur with 0.5 ml elution buffer.7. Add nanodrop to calculate protein concentration; wash nanodrop, add 2uL elution buffer to adjust for blank; add sample 2uL to measure protein concentration in the sample.8. To reuse the column, release all binding IgGs; add 1 mL elution buffer, perform 3 times to release the binding IgGs.Then add 1 mL binding buffer, l wash 3 times, leave 1 ml binding buffer; cap and store for future use.9. Next to concentrate IgGs and change buffer to PBS since elution buffer could impair IgGs.
9a. Use Amicon Ultra-0.5 ml centrifugal filters 30k (IgG is about 150k) 9b.Add the filter into a 2 ml vial provided in the kit 9c.Add 0.5 mL isolated IgGs in elution buffer from step 8 to the column, then 14,000 g 10 min, repeat until all elution buffer uses.9d.Add PBS 0.5 ml, 14,000 g 10 min, 3 times 9e.Reverse the column in a new tube, then centrifuge 1000 g for 2 min.This produces the concentrated IgGs, then add PBS to 0.5-1 mg/mL, aliquot and store at − 80C to avoid repeated freezing and thawing.
Step 6 ADCC 1. Target cells (CD4 + T cells) and Effect cells (NK cells) obtained from non-immune responder HIV patients (undetectable plasma viral load, CD4 + T cell counts < 350 cells/μL) on antiretroviral therapy.Use the enrichment kit, strictly following the protocol provided in the kit.This procedure is used for processing 250 μL -2 mL of sample (up to 1 × 10 8 cells).
A. Prepare a mononuclear cell suspension at a concentration of 5 × 10 7 cells/mL in the Separation Buffer.Cells were placed in a 4. ➢ Use the R10 buffer in all these steps, unless a different specified buffer is needed.

CD4 + T cells depletion
A. Dilute the cell at 5 × 10 5 /mL in R10, add 50 μL to the 96 well V bottom plate, with CD4 + T cells 2.5 × 10 4 per well.R10: RPMI + 10% FBS B. Using the R10 at all of steps, unless the specific explain C. Add the anti-CD4 antibody (from 1 μg/mL, 2.5 μg/mL, to 5 μg/mL, you may need to titrate your isolated anti-CD4 IgGs from 1 to 10 μg/mL), and purified IgG or positive control (hIgG OKT3, 0.5 μg/mL) to the CD4 + T cells, culture the cells at 37 °C for 30 min.D. Dilute the NK cells at 1.5 × 106/mL in R10, add 50 μL to the 96 well V bottom plate, NK cells 7.5 × 104 per well, NK cells: CD4 + T cells at a ratio of 3:1, with each well containing 1 × 105 total cells per well.E. Culture the cells at room temperature for 15 min, then spin the plate at 300 g for 1 min.F. Incubate plates for 6 h at 37 °C.G. Stain the cells with CD4 and CD3, then Annexin V in annexin V buffer (BD, Catalog number: 556454) H. Run on flow cytometry within 30 min.

Antibody dependent NK activation.
A. Dilute the cells at 5 × 10 5 /mL in R10, add 50 μL to the 96 well V bottom plate, with CD4 + T cells 2.5 × 10 4 per well.R10: RPMI + 10% FBS B. Add the anti-CD4 antibody and purified IgG or positive control (hIgG OKT3) to the CD4 + T cells; culture the cells at 37 °C for 15-30 min.C. Dilute the NK cells at 5 × 105/mL in R10, add 50 μL to the 96 well U bottom plate, NK cells 2.5 × 104 per well, NK cells: CD4 + T cells at 1:1, with each well containing 5 × 104 total cells.D. Add the CD107a-BV 510 antibody (Catalog number: 563078, BD) 2 μL/well, the GolgiStop TM Protein Transport Inhibitor (brefeldin A, Catalog number: 554724, BD) and GolgiPlug TM Protein Transport Inhibitor (monensin, Catalog number: 555029, BD) at the final concentration of 1 μL/mL.E. Culture the cells at room temperature for 15 min, then spin the plate at 250 g for 4 min.F. Incubate plates for 6 h at 37 °C.G. Stain the cells with Ghost Red 780 and CD3.H. Add 500 μL 1x PBS to wash the cells, 350 g, 5 min, decant the supernatant.Vortex the cells for 5 s.

Fig. 2 .
Fig. 2. Transfer the concentrated sample from the device to the tube.
5 or 5 mL polystyrene tube to properly fit into the Purple EasySep TM Magnet.a. Separation buffer: PBS PH7.2, supplemented with 0.5%BSA and 2 mM EDTA b.Falcon TM 5 mL Polystyrene Round-Bottom Tubes (BD Biosciences, Catalog number: 352058) or 4.5 mL polystyrene tubes (Catalog number: 120161, 4.5 mL PS tube, Greiner bio-one) are recommended.B. Add the EasySep TM Human CD4 or NK Cell Enrichment Cocktail at 50 μL/mL cells (e.g. for 2 mL of cells, add 100 μL of cocktail).Mix well and incubate at room temperature (15-25 °C) for 10 min.C. Vortex the EasySep TM D Magnetic Particles for 30 s. Ensure that the particles are in a uniform suspension with no visible aggregates.D. Add the EasySep TM D Magnetic Particles at 100 μL/mL cells (e.g. for 2 mL of cells, add 200 μL of magnetic particles).Mix well and incubate at room temperature (15-25 °C) for 5 min.E. Bring the cell suspension up to a total volume of 2.5 mL by adding separation buffer.Mix the cells in the tube by gently pipetting up and down 2 -3 times.Place the tube (without cap) into the magnet.Set aside for 5 min.F. Pick up the EasySep TM Magnet, and in one continuous motion invert the magnet and tube, pouring off the desired fraction into a new 4.5 mL polystyrene tube.The magnetically labeled unwanted cells will remain bound inside the original tube, held by the magnetic field of the EasySep TM Magnet.Leave the magnet and tube in an inverted position for 2-3 s, then return to upright position.a. Do not shake or blot off any drops that may remain hanging from the mouth of the tube.b.From the following step, the separated NK cells should be kept at 4 °C, until the NK cells have been added in the 96 wells plate.G. Wash the cells with pre-cold separation buffer 2 times; the negatively selected, enriched cells in the new tube in R10 are now ready for use.R10: RPMI + 10% FBS