HIVGKO: A Tool to Assess HIV-1 Latency Reversal Agents in Human Primary CD4+ T Cells

While able to suppress HIV replication in HIV infected individuals, combination antiretroviral therapy (ART) fails to eliminate viral latent reservoir, which consists in integrated transcriptional silenced HIV provirus. So far, identification of latently-infected cells has relied on activating cells to induce expression of HIV proteins which can then be detected. Unfortunately, this activation significantly changed the cell phenotype. We developed a novel HIV reporter, named HIVGKO, that allows the purification of latently-infected cells in absence of reactivation. Indeed, latent cells can be identified by expression of the EF1a-driven mKO2 and lack of expression of the LTR-driven csGFP. This protocol can be used to study the effectiveness of LRAs (Latency Reversal Agents) in reactivating latent HIV in primary cells.


D.
Sorting cells

Production of HIV GKO viral particles in HEK293T cells
Note: HIV GKO particles are produced after calcium phosphate transfection in HEK293T cells.

b.
For maintenance of the HEK293T culture, when approaching confluence (~80%) cells, aspirate media, wash once with PBS, then trypsinize (0.05% trypsin) and plate cells after 1/12 dilution in complete DMEM, in 182 cm 2 tissue culture flasks. HEK293T cells are split every 3 days.

c.
One day before transfection, plate approximately 4 × 10 6 of HEK293T cells per 182 cm 2 tissue culture flasks in 18 ml of complete DMEM.
Note: After thawing frozen cell vials, HEK293T cells are cultured for at least one week before transfecting them for virus production. In order to maximize viral particle production, HEK293T cells are never kept more than 4 weeks in culture. HEK293T cells should never be grown to 100% confluency as they lose their ability to be transfected.

k.
Add 17 ml of DMEM (supplemented with 10% FBS, 1% penicillin/streptomycin) medium (add at the bottom of the flask, as shown in Figure 1, to avoid detaching cells).

l.
Incubate for 48 h at 37 °C to allow viral production.

m.
Collect the supernatant into a 50 ml Falcon tube.

n.
Centrifuge for 20 min at 800 × g at RT or 4 °C.

o.
Filter supernatant through a 50 ml centrifuge tube filtration.

p.
Transfer filtered supernatant into UltraClear Centrifuge Tubes (To avoid the collapse of the tube, make sure it is filled with at least 34 ml-add media or PBS to viral supernatant if necessary to reach that volume).

q.
Spin viral supernatant in the ultracentrifuge for 2 h at 100,000 × g at 4 °C.

r.
Pour out supernatant, dry as much as you can the inside of the tube and resuspend the pellet with 50 μl of cold RPMI (supplemented with 10% FBS, 1% penicillin/streptomycin) medium or FBS, make aliquots in 1.5 ml Eppendorf tubes and freeze at −80 °C. Notes:

a.
When scaling up the viral production, you should be able to see a pellet. Also, scale up the volume used to resuspend the pellet. Do not make air bubbles when resuspending the pellet and, pipet up and down at least 50 times.

b.
Concentrated viral supernatant can also be titered and used fresh. However, even though the p24 content will not change, fresh versus frozen virus will give different infection outcomes, which will require different viral input to start with (described in Step C2).

s.
Thaw one aliquot, make viral dilutions up to 10,000 to 1 million and, titer virus for p24 content using the FlaQ assay protocol (Gesner et al., 2014).
Note: In addition of tittering p24 content of the virus with the FlaQ assay, I would recommend directly titering the infection rate of your viral stock on activated cells using 4 or 5 viral dilutions, before proceeding with big experiments. You want to avoid total infection greater than 15%.

B.
Isolation of human primary CD4 + T cells

a.
Human primary CD4 + T cells are cultured in RPMI medium (supplemented with 10% FBS, 1% penicillin/streptomycin) + 20 U/ml of IL-2 in tissue culture flasks or plates at a concentration of 5 × 10 6 /ml, in a CO 2 incubator at 37 °C.

b.
Half of the medium is replaced with fresh RPMI medium (supplemented with 10% FBS, 1% penicillin/streptomycin) + 20 U/ml of IL-2 every other day.

2.
Isolation of human primary CD4 + T cells Note: This protocol describes CD4 + T cells isolation using the RosetteSep Human CD4 + T Cell Enrichment Cocktail. Any other CD4 + T cells isolation kit can be used, but the protocol might slightly differ. Always follow the manufacturer's protocol.

a.
Order fresh blood or LRC in advance to have it delivered on the day of the experiment.
Note: One blood/LRC corresponds to one donor. Three donors should be tested at least, in two different experiments at least.

b.
Transfer blood into a 50 ml Falcon tube (if using LRC, cut both extremities of the chamber with clean scissors and let the blood drops into the tube).

c.
Add 1,800 μl of RosetteSep Cocktail to sample, incubate at RT for 20 min, and mix sample by swirling the tube every 5 min.

d.
In a new 50 ml Falcon tube, add 10 ml of Histopaque-1077.

e.
Dilute sample with equal volume of PBS containing 2% FBS.

f.
Slowly and carefully layer diluted sample on density gradient medium to minimize their mixing.

g.
Centrifuge sample for 20 min at 800 × g, RT, with brake off.

h.
CD4 + T cells are contained in the white ring, below the plasma phase ( Figure 2). Pipet the ring and transfer it to a new 50 ml Falcon tube.

i.
Wash cells by filling up the tube with PBS containing 2% FBS.

j.
Centrifuge for 3 min at 800 × g and discard supernatant.

k.
If pellet appears red, resuspend pellet with 15 ml of cold AKC lysis buffer (see Recipes), incubate for 2 min at RT.

l.
Fill the tube up to 50 ml with PBS containing 2% fetal bovine serum, centrifuge for 3 min at 800 × g, and get rid of the supernatant.

m.
If the pellet is still red, repeat Steps B2k-B2l, if not, proceed to Step B2n.

o.
Count cells using an automated cell counter or a hemacytometer.
Note: Isolated CD4 + T cells are mainly resting and can be kept in culture as such for several days. Medium can be changed every 4 to 5 days.

Infection of human primary CD4 + T-cells with HIV GKO
Note: This protocol describes the activation of 250 million CD4 + T cells using Dynabeads Human T-Activator CD3/CD28. Any other human CD4 + T cells activators kit can be used, but the protocol might slightly differ. Always follow the manufacturer's protocol.

1.
Activation of human primary CD4 + T cells

a.
Vortex the Dynabeads Human T-Activator CD3/CD28 in the vial.

b.
Transfer 3.125 ml of Dynabeads (1 bead for 2 cells, which is half of the manufacturer's protocol) to a 15 ml tube.

c.
Place the tube on a magnet for 1 min and discard the supernatant.

d.
Remove the tube from the magnet and resuspend the washed Dynabeads with 10 ml of the CD4 + T cells to activate (at a concentration of 5 × 10 6 /ml), and add those 10 ml to the rest of the CD4 + T cells to activate. Transfer the whole suspension to an appropriate vessel (5 millions of activated cells can be cultured in one well of a 24-well plate).

e.
Culture in a CO 2 incubator at 37 °C for three days as such (you should see cells aggregates due to activation).

Infection of human primary CD4 + T cells with HIV GKO
Note: The spin-infection of 1 million activated CD4 + T cells requires 30 μl of a 10,000 ng p24 Gag /ml viral dilution to reach a total infection rate of 9%-12% (to keep the ratios of latent versus productive infections consistent, avoid infection rates greater than 15%). However, the total infection rate (productive + latent infections) might slightly change according to fresh versus frozen stocks, viral stocks themselves and donors. Thus, in addition of titering the virus with the FlaQ assay, I would recommend directly titering your viral stock on activated cells using 4 or 5 viral dilutions, before proceeding with big experiments.
Using frozen viral stocks requires the use of bigger amount of virus to reach 9%-12% infection rate.

a.
Three days post-activation, mix cells and beads and, transfer cells to 15 ml tubes.

b.
Place the tube on a magnet for 1 min and transfer the supernatant into a new 50 ml tube.

c.
Centrifuge cells for 3 min at RT at 800 × g.

d.
Discard supernatant and resuspend the cell pellet of 250 million cells with media containing the viral preparation at 10,000 ng p24 Gag /ml.

e.
Transfer resuspended cells to a pipetting reservoir and distribute 30 μl of the cells/virus suspension per one well of a 96 well V-bottom plate using a multi-channel pipetman.

g.
Add 100 μl of fresh RPMI medium (supplemented with 10% FBS, 1% penicillin/streptomycin) + 20 U/ml of IL-2 in each well and pool cells back together. Resuspend cells in a final volume of 100 ml of fresh RPMI medium (supplemented with 10% FBS, 1% penicillin/streptomycin) + 20 U/ml of IL-2 and, transfer into a 182 cm 2 flask.
Note: Viral solution stays in, but it is possible to wash it away after spin-infection.

h.
Culture as such for 4 to 5 days in a CO 2 incubator at 37 °C.

i.
Replace half of the medium by centrifuging down cells, with fresh RPMI medium (supplemented with 10% FBS, 1% penicillin/streptomycin) + 20 U/ml of IL-2 every other day.

D.
Sorting cells

1.
Five days post-infection, collect and transfer cells to 50 ml tubes.

2.
Centrifuge cells for 3 min at RT at 800 × g.

3.
Discard supernatant and resuspend cells in 2 ml of FACS buffer (see Recipes).

4.
Pipette resuspended cells through a 5 ml 35 μm cell strainer capped tube and place on ice.

5.
Prepare several 15 ml collection tubes with 2 ml of FBS and place on ice. Do not forget to label your tubes.

6.
Then proceed directly to cell sorting using the flow cytometer FACS Ariall (PE channel for mkO2, and FITC channel for GFP) (see Figure 3 for gating strategy).
Note: The sort can take up to 10 h, thus sort cells at 4 °C. Use the 85 μm nozzle to prevent spontaneous reactivation of latently infected cells. When collecting populations into 15 ml tubes, you can only collect 2 different populations at once. Keep sorting the latent population at all time, and exchange tubes for uninfected and productively infected cells when you have reached 5 million cells per population. You should be able to collect 1.5-2.5 million latently infected cells depending on your infection rate.

7.
Spin down sorted cells for 3 min at RT, 800 × g.
Note: To test 5 LRAs, sorted populations should be divided equally into 5 wells. Given that each well contains 200 μl, the pellet should be resuspended in 1 ml final of RPMI media. Note that a few hundred thousand cells/well are enough to assay LRAs activity.

a.
Avoid fixing samples with PFA since it decreases fluorescence intensity.

b.
For flow cytometry, Ariall sorter was used to run samples since fluorescence intensity is higher. However, other flow cytometers such as LSRII or Calibur are also suitable for these experiments as long as they have the right filters. Steps A2b and A2k require to add fresh media with and without chloroquine, respectively. First, aspirate media, and then add fresh media directly at the bottom of the flask to prevent detaching the cells. Once the media is in the bottom of the flask, slowly swirl the flask to distribute the solution evenly and place the flask back into the incubator. Battivelli   A. Slowly layer the blood (containing the RosetteSep Cocktail and diluted to half with PBS + 2% FBS) on top of histopaque density gradient. Centrifuge for 20 min at 800 × g, RT, with brake off. B. CD4 + T cells are contained in the white ring, below the plasma phase, but above the histopaque and red cell phases. Carefully pipet out the ring. Battivelli   A. Set the gate on live cells. Cell viability is monitored by forward (FSC-Area) and side scatter (SSC-Area) analysis. B and C. Gate successively on singlets FSC-Area vs. FSC-Width, and SSC-Area vs. SSC-Width. D. Set the gate on GFP/FITC-Area + to sort productively infected cells, or on GFP/FITC-Area − vs. mKO2/PE-Area + to sort latently infected cells, or to GFP/FITC-Area − vs. mKO2/PE-Area − to sort uninfected cells. Run briefly each sorted sample when the sort is over to check purity (usually > 90%). Battivelli