Protocol for intracellular immunofluorescence measurements of latent HIV reactivation in a primary CD4⁺ T cell model

Summary

Investigating the molecular mechanisms of HIV latency reversal in a proper physiological context can only be done in primary cells. Here, we describe a primary T cell model of HIV latency and a reliable flow cytometry assay to measure latency reversal efficacy by dual immunofluorescence staining for Nef and Tat. We also describe a procedure for identifying latency-reversing agents that effectively induce the biogenesis of P-TEFb, an obligate host transcription factor for HIV, while monitoring their effects on T cell activation.

Graphical abstract

Highlights

• •Ex vivo procedure for generating HIV latently infected primary CD4⁺ T cells
• •Steps for assessing HIV latency reversal efficacy via dual detection of Tat and Nef
• •Flow cytometry measurement of P-TEFb biogenesis induced by HIV latency-reversing agents

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Investigating the molecular mechanisms of HIV latency reversal in a proper physiological context can only be done in primary cells. Here, we describe a primary T cell model of HIV latency and a reliable flow cytometry assay to measure latency reversal efficacy by dual immunofluorescence staining for Nef and Tat. We also describe a procedure for identifying latency-reversing agents that effectively induce the biogenesis of P-TEFb, an obligate host transcription factor for HIV, while monitoring their effects on T cell activation.

Before you begin

Resting memory CD4⁺ T cells are the primary reservoir for persistent HIV in the peripheral circulation. These cells maintain a cellular environment that is non-permissive for HIV transcription. Unfortunately, the rarity of infected cells in the circulation of treated people with HIV (<100 per 1 million memory CD4⁺ T cells) has made it difficult to use these cells to study the mechanistic basis of HIV latency and its reactivation. Therefore, over the last 30 years, the investigation of host transcription factors and signaling pathways essential for regulating the emergence of HIV from latency has largely been conducted using transformed cell lines, such as the Jurkat T-cell model. Although HIV latency can still be established in transformed T-cell lines,¹ these models do not recapitulate the underlying cell biology of infected primary T cells, especially the transition from activated effector cells to quiescent memory cells. Developing a more feasible experimental model for investigating the molecular basis of HIV latency and its reactivation in a proper physiological context has required successfully generating latently infected primary CD4⁺ T cells in vitro using healthy donor-derived naïve CD4⁺ T cells. Here, we describe an optimized procedure for the Quiescent Effector Cell Latency (QUECEL) model,^2,3 which is a refinement of the primary cell model of Bosque and Planelles.⁴ In this updated QUECEL procedure, healthy donor naïve CD4⁺ T cells are first polarized into any of the four major effector T-cell subsets (Th1, Th2, Th17, and Treg) while activating them through the TCR with Concanavalin A (2 μg/mL) to allow the polarized cells to become permissive to infection with a single-round HIV carrying a CD8a-EGFP reporter virus. Acutely infected cells are then positively selected by magnetic bead isolation targeting CD8a expressed on the cell surface and forced into quiescence to acquire a resting memory phenotype that establishes viral latency.

Entry into quiescence of the polarized cells can be monitored by measuring DNA replication through DAPI staining and the incorporation of EdU as well as confirming the restricted expression of cell proliferation markers (Ki67 and Cyclin D3), surface T-cell activation markers (CD25 and CD69) and P-TEFb. We have routinely preferred to generate quiescent latently infected cells with the Th17 polarization phenotype since these cells retain a higher degree of viability than the other effector cell subsets. Moreover, Th17 cells represent the most abundant effector T-cell population in the lamina propria of the GI tract and harbor as much as half of the HIV cellular reservoir in the circulating blood of treated PWH.^5,6,7,8

We also describe two intracellular immunofluorescence flow cytometry procedures that can be reliably utilized to monitor the efficacy of latent HIV reactivation in QUECEL Th17 cells and in any other primary cell model. First, HIV gene expression in acutely infected, latently infected, and reactivated cells can be measured using a sensitive assay that dually detects the expression of Tat and Nef with fluorophore-conjugated monoclonal antibodies. The emergence of HIV from latency is known to be critically dependent on the kinase activity of P-TEFb, whose expression is highly restricted in resting memory CD4⁺ T cells due to the translational repression of its cyclin T1 subunit and sequestration of its CDK9 kinase subunit in the cytoplasm by Hsp90/Cdc37.^9,10,11,12 P-TEFb immuno-flow is a sensitive flow cytometry assay that we have recently developed for detecting the assembly of the transcriptionally active P-TEFb heterodimer through the dual monitoring of cyclin T1 protein expression and phosphorylation of CDK9 on its activation loop at Ser175 (pSer175 CDK9).¹³ As described here, uninfected Th17 cells that have been taken through the QUECEL procedure alongside HIV-infected Th17 cells can be subjected to P-TEFb immuno-flow to identify candidate HIV latency reversal agents capable of inducing P-TEFb biogenesis while also assessing their effects on T-cell activation.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
HIV-1 Nef (antibody conjugate dilution is 1:25; refer to Table 6)	Abcam	ab42355
HIV-1 Tat (antibody conjugate dilution is 1:25; refer to Table 6)	Custom-synthesized by the Cleveland Clinic LRI Hybridoma Core	NT3 2D1.1
Cyclin T1 (antibody conjugate dilution is 1:30; refer to Table 6)	Thermo Fisher Scientific	PA5-77892
pSer175 CDK9 (antibody conjugate dilution is 1:15; refer to Table 6)	Custom-synthesized by Covance Research Products	N/A
CD25-PE (1:7 dilution)	BD Biosciences	555432
CD69-PECy7 (1:20 dilution)	BD Biosciences	557745
Mouse IgG blocking antibody	Thermo Fisher Scientific	10400C
Biological samples
Healthy donor-derived peripheral blood mononuclear cells (PBMCs)	AllCells	N/A
Chemicals, peptides, and recombinant proteins
1X PBS	Corning	21-040-CM
RPMI 1640 media with HEPES and L-glutamine	Cytiva	SH30255.02
Fetal bovine serum	Cytiva	SH30396.03
Penicillin-Streptomycin	Thermo Fisher Scientific	15070063
Opti-MEM reduced serum medium	Thermo Fisher Scientific	31985-070
10X Perm/Wash buffer	BD Biosciences	554723
Triton X-100	MilliporeSigma	T9284
Robosep buffer	STEMCELL	20104
Propidium iodide solution	Cayman Chemical	10008351
Fixable Viability Dye eFluor 450	Thermo Fisher Scientific	65-0863-18
Formaldehyde	Polysciences	04018
Polyethylenimine (PEI)	MilliporeSigma	408727
IL-1β	PeproTech	200-01B
IL-2	NIH BEI Resources	136
IL-6	PeproTech	200-06
IL-7	PeproTech	200-07
IL-8	PeproTech	200-08M
1L-10	PeproTech	200-10
IL-23	PeproTech	200-23
TGF-β	PeproTech	100-21C
Anti-human IL-4	PeproTech	500-M04
Anti-human IFN-γ	PeproTech	500-M90
Concanavalin A	MilliporeSigma	C0412
Critical commercial assays
Human CD4 naive T cell isolation kit	BioLegend	480042
Mouse CD8a positive selection kit	STEMCELL	18953
Pacific Blue antibody labeling kit	Thermo Fisher Scientific	P30013
Alexa Fluor 488 antibody labeling kit	Thermo Fisher Scientific	A20181
Alexa Fluor 555 antibody labeling kit	Thermo Fisher Scientific	A20187
Alexa Fluor 647 antibody labeling kit	Thermo Fisher Scientific	A20186
Experimental models: cell lines
HEK293T	ATCC	CRL-3216
Experimental models: Organisms/strains
Polarized and expanded primary Th17 cells	This paper	N/A
Recombinant DNA
pHR’-d2EGFP-Nef+	Dobrowolski et al.2	Dobrowolski et al.2
pHR’-CD8a-d2EGFP-Nef+	Dobrowolski et al.2	Dobrowolski et al.2
Lentiviral gag/pol pCMV-Delta 8.91r	Dobrowolski et al.2	Dobrowolski et al.2
pMD.G VSVG	Addgene	#8454
Other
Optima L-100K ultracentrifuge	Beckman Coulter	N/A
SW32 Ti rotor	Beckman Coulter	N/A
BD LSR Fortessa	BD Biosciences	N/A
EasySep magnetic tube holder	STEMCELL	18000
Eppendorf 5810R benchtop centrifuge	Eppendorf	022625101

Step-by-step method details

Preparation of VSVG-pseudotyped HIV virus concentrate

Timing: 4 days

This section describes the procedure for generating single-round (replication-incompetent) packaged HIV virus. This VSVG-pseudotyped virus is used to infect polarized and expanded Th17 cells.

Day 1: HEK 293T cell culture

• 1.One day before transfection, HEK 293T cells are split and plated in T150 flasks, equivalent to the number of transfections in 20 mL of the complete medium so that they are ∼80% confluent on the day of transfection.

Note: We prefer to culture the HEK 293T cells in a complete RPMI medium containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin-streptomycin, and 25 mM Hepes, pH 7.2. Alternatively, these cells may be cultured in a complete DMEM medium containing 10% FBS, 100 IU/mL penicillin/streptomycin, and 25 mM Hepes, pH 7.2. 150-mm cell culture dishes may be used instead of T150 flasks to plate HEK 293T cells for transfection.

Day 2: Generation of single-round VSVG-pseudotyped HIV

• 2.To transfect each T150 flask of cells, place 1.5 mL of Opti-MEM medium in a 15-mL conical tube. Add the HIV, packaging, and pseudotyping plasmids as follows: 7.5 μg of pHR’-d2EGFP-Nef+ or pHR’-CD8a-d2EGFP-Nef+ (HIV), 7.5 μg of pCMV-Delta 8.91r (packaging) and 3 μg of pCMV-VSV-G (Envelope VSVG). The genome organization of the pHR’-d2EGFP-Nef+ and pHR’-CD8a-d2EGFP-Nef+ viral constructs is presented in Figure 1A.
• 3.Pass the diluted DNA suspension through a 0.22 μm vacuum-driven filter to ensure sterility of the mix.

Note: Maxiprep preparation of plasmid DNA is usually done on a lab bench, where the procedure's sterility is not assured. Thus, the plasmid DNA is passed through a 0.22 μm filter to ensure the sterility of the DNA used for transfection.

• 4.Thaw a 1 mg/mL stock of polyethylenimine (PEI) to 25°C and add 54 μL to the diluted DNA mix for each transfection. Mix immediately by vortexing and incubate for 15 min at 25°C .
• 5.Take the T150 flasks of HEK293T cells from the incubator and replace the cell culture media with 15 mL of fresh complete medium.
• 6.Add the DNA/PEI mix to cells and place the flasks in the cell culture incubator for 48 h.

Note: The final concentration of PEI in the culture will be 0.0033 mg/mL.

Figure 1.

Generation of quiescent, latently infected primary CD4⁺ T cells in vitro using the QUECEL method

(A) NL4-3-derived single-round EGFP reporter HIV-1 construct was used to infect polarized, expanded Th17 cells. The CD8a-d2EGFP fusion is a transmembrane protein that allows for the enrichment of acutely infected cells. However, unlike d2EGFP, which rapidly degrades with a half-life of 3.6 h, CD8a-d2EGFP is relatively stable and remains expressed even in the quiescent, latently infected cells.

(B) Scheme for generating latently infected polarized CD4⁺ Th17 cells from healthy donor-derived naïve CD4⁺ T cells. This modified procedure uses a five-fold lower concentration of Concanavalin A (2 μg/mL) during the polarization and expansion phase.

(C) Scheme for generating quiescent uninfected CD4⁺ Th17 cells from healthy donor-derived naïve CD4⁺ T cells.

Day 4: Virus concentration

• 7.Harvest the cell culture media containing the packaged virus by placing it in a 50-mL conical tube.
• 8.Clear the harvested media by centrifugation at 300 × g for 3 min and thereafter by passing the viral supernatant through a 0.22 μm vacuum-driven filter.
• 9.Transfer the viral supernatant to sterile polycarbonate ultracentrifuge tubes designed to fit the high-speed SW32Ti rotor. Ensure that the tubes are filled to the brim and balanced.
• 10.To concentrate the packaged virus, the tubes should be centrifuged at 28,000 rpm for 2 h at 4°C in the Optima L-100K Ultracentrifuge.
• 11.After removing the media by decanting or using a 25-mL serological pipette, resuspend the viral pellet(s) in Opti-MEM medium. For a concentrate of viral supernatant obtained from a single T150 flask, 1 mL of Opti-MEM is used for resuspension.
• 12.Transfer the virus concentrates to 1.8 mL cryogenic vials for long-term storage at −80°C.

Generation of expanded and polarized primary Th17 cells

Timing: 1 week

This section describes the procedure for generating polarized and expanded Th17 cells from naïve CD4⁺ T cells isolated from de-identified healthy donor derived PBMCs.

Day 1: Naïve T cell isolation, cell polarization and expansion

• 13.
  Isolate naïve CD4⁺ T cells from a previously frozen, commercially obtained leukopak of de-identified human healthy donor-derived peripheral blood mononuclear cells (PBMCs) using the Mojosort Human CD4 Naïve T cell Isolation Kit.

  Note: This negative selection procedure will generate approximately 6–10 million naïve CD4⁺ T cells from 100 million PBMCs.

  • a.
    Retrieve the vial(s) of frozen 100 million PBMCs from liquid nitrogen storage and thaw in a 37°C water bath.
  • b.
    Transfer the thawed cell suspension to a sterile 50-mL conical tube and add 20 mL of RoboSep buffer.
  • c.
    Mix by gentle pipetting, then centrifuge the cells at 1500 rpm (450 × g) in the Eppendorf 5810R Centrifuge for 3 min.
  • d.
    After aspirating the supernatant, add RoboSep buffer to bring the total volume to 800 μL.
  • e.
    Transfer the cell suspension to a sterile 12 × 75 mm (5 mL) polystyrene tube, then add 100 μL of biotinylated antibody cocktail, resuspend by gentle pipetting, and incubate at 25°C for 15 min.
  • f.
    Add 100 μL of the streptavidin-coated magnetic nanobeads, resuspend by gentle pipetting, and incubate at 25°C for 10 min.
  • g.
    Use an EasySep magnetic tube holder to separate untouched cells from antibody-bound cells.

    • i.
      Add 6 mL of RoboSep buffer, resuspend gently, and then place the polystyrene tube in an EasySep magnet for 5 min.
    • ii.
      Decant the suspension of untouched cells into a 50-mL conical tube by inverting the magnet.
    • iii.
      Remove the polystyrene tube from the magnet and add another 6 mL of Robosep buffer. Gently mix and place the tube back in the magnet for 5 min.
    • iv.
      Combine both cell suspensions and proceed to the next step.
  • h.
    Determine the total yield of naïve CD4⁺ T cells by automated cell counting or using a hemocytometer.
  • i.
    Spin the cell suspension at 450 × g for 3 min, aspirate the supernatant and then resuspend the naïve T cells in complete media at a concentration of 0.5 million cells/mL.
• 14.
  After the cell isolation, stimulate the cells with 2 μg/mL Concanavalin A (Con A) for 72 h while incubating them with polarizing cytokines.

  Note: These cells may be polarized into one of four T cell subsets,^2,3 namely Th1, Th2, Th17 and Treg. For the experimental procedures described below we have preferred to utilize polarized Th17 cells consistently.

  • a.
    Add polarizing cytokines and antibodies to the cell suspension according to the concentrations shown in Table 1. For polarization to Th17 cells, use IL-1β, IL-6, IL-23, TGF-β, anti-IL4 and anti-IFN-γ.
  • b.
    Add Con A at a working concentration of 2 μg/mL.
  • c.
    Mix by gentle pipetting, then transfer the cell suspension to a sterile 25-mL multichannel pipet reservoir.
  • d.
    Using a 200-μL multichannel pipet, transfer 100 μL of the cell suspension to each well of a round-bottomed 96-well plate. The yield of naïve CD4⁺ T cells from 100 million PBMCs will usually require two 96-well plates.
  • e.
    Place the 96-well plates in the cell culture incubator for 72 h.

Table 1.

First round cocktail for generation of polarized and expanded Th17 cells (Days 1 to 4)

Cytokine, antibody or stimulus	Stock conc.	Working dilution	Final conc.
1L-1β	10 μg/mL	1:1000	10 ng/mL
IL-6	30 μg/mL	1:1000	30 ng/mL
IL-23	50 μg/mL	1:1000	50 ng/mL
TGF-β	10 μg/mL	1:2000	5 ng/mL
Anti-human IL-4	500 μg/mL	1:1000	500 ng/mL
Anti-human IFN-γ	10 μg/mL	1:1000	10 ng/mL
Concanavalin A	10 mg/mL	1:5000	2 μg/mL

Day 4: Second round of polarization cocktail and Con A treatment

• 15.Prepare fresh media containing the Th17 polarization cocktail, 2 μg/mL Con A, and 60 IU/mL IL-2, as shown in Table 2. Using a 200-μL multichannel pipet, add 100 μL of the media to each well of the 96-well plates of cells.

Note: With each addition resuspend the cells by pipetting up and down once. Place the 96-well plates back in the cell culture incubator for an additional 72 h.

Table 2.

Second round cocktail for generation of polarized and expanded Th17 cells (Days 4 to 7)

Cytokine, antibody or stimulus	Stock conc.	Working dilution	Final conc.
1L-1β	10 μg/mL	1:1000	10 ng/mL
IL-2	60,000 IU/mL	1:1000	60 IU/mL
IL-6	30 μg/mL	1:1000	30 ng/mL
IL-23	50 μg/mL	1:1000	50 ng/mL
TGF-β	10 μg/mL	1:2000	5 ng/mL
Anti-human IL-4	500 μg/mL	1:1000	500 ng/mL
Anti-human IFN-γ	10 μg/mL	1:1000	10 ng/mL
Concanavalin A	10 mg/mL	1:5000	2 μg/mL

HIV infection of polarized Th17 cells

Timing: 1 week

This section describes the infection of polarized and expanded Th17 cells with the single-round VSVG-pseudotyped packaged HIV.

Day 7

• 16.Determine the cell count. At this point, the yield of expanded and polarized Th17 cells should be between 25 and 40 million cells, from a starting isolation of 6–10 million naïve CD4⁺ T cells.
• 17.For every 5 million cells to be infected use 1 mL of 0.22 μm-filtered pseudotyped HIV concentrate. Begin by centrifuging the cells at 450 × g for 3 min, then resuspend in fresh complete media at 5 million cells per mL.
• 18.Transfer the cell suspension to a 24-well plate (1 mL per well) and add 1 mL of HIV concentrate to each well.
• 19.Add IL-2 and IL-23 to a final concentration of 60 IU/mL and 50 ng/mL, respectively. Mix and place the 24-well plate in the cell culture incubator for 24 h.

Note: Spinoculation of the cells with virus or the utilization of polybrene as a transduction reagent is unnecessary. We have found that neither enhances the percentage of infected cells beyond that observed by simply incubating the cells with the virus as described in this step.

Day 8

• 20.Transfer the culture of infected cells to a T25 flask and dilute with complete media containing 60 IU/mL IL-2, 50 ng/mL IL-23, and 10 ng/mL IL-7 (Table 3) so that the resulting cell concentration is 1 million cells per mL. Place the flask upright in the cell culture incubator and culture for another two days.

Table 3.

Maintenance cytokines for acutely infected Th17 cells

Cytokine	Stock conc.	Working dilution	Final conc.	Days of treatment
IL-2	60,000 IU/mL	1:1000	60 IU/mL	Days 7 to 14
IL-23	50 μg/mL	1:1000	50 ng/mL	Days 7 to 14
IL-7	10 μg/mL	1:1000	10 ng/mL	Days 8 to 14

Day 10

• 21.
  Determine the extent of acute HIV infection. This can be done by fluorescence flow cytometry to monitor GFP reporter expression and following immunostaining to detect Nef and Tat expression.

  Note: The Nef and Tat immunofluorescence staining procedure for flow cytometry is described below.

  • a.
    To assess GFP reporter expression and measure cell viability, collect a small sample (usually 20 μL) of the infected and uninfected cell cultures and transfer to 5-mL polystyrene flow cytometry tubes containing 180 μL 1X PBS.
  • b.
    Add 1 μL of propidium iodide (PI) to each tube and vortex.

    Note: This amounts to a 1:200 addition of the 1 mg/mL stock of PI to achieve a working concentration of 5 μg/mL.

  • c.
    Analyze by flow cytometry using the appropriate voltage settings for the forward/side scatter, GFP, and TRITC laser channels.
• 22.After determining the cell count, transfer the cells to a 15-mL conical tube and spin at 450 × g for 3 min. Aspirate the media containing the virus, then resuspend the cells in fresh, complete media at a concentration of 1 million cells/mL.
• 23.Add IL-2 (at 60 IU/mL), IL-23 (at 50 ng/mL) and IL-7 (at 10 ng/mL). Place the cells in a new T25 flask and culture for four days.

Enrichment of HIV-infected cells by positive CD8a selection

Timing: 2 h

For polarized, expanded Th17 cells infected with the pHR’-CD8a-d2EGFP-Nef+ packaged virus, expression of the CD8a-EGFP transmembrane fusion protein at the cell surface allows for the positive selection of acutely infected cells using the EasySep Mouse CD8a Positive Selection Kit II.

Day 12

• 24.At 4 or 5 days post-infection, spin the cells at 450 × g for 3 min.

Note: The steps outlined below are all done at 25°C.

• 25.Resuspend the cell pellet in 2 mL of Robosep buffer and transfer to a 5 mL polystyrene round-bottom tube. Add 20 μL of FcR blocker reagent to the cell suspension.
• 26.Prepare the selection cocktail by mixing 50 μL of Component A and an equal volume (50 μL) of Component B in a sterile microcentrifuge tube. Incubate for 5 min.
• 27.Add the selection cocktail to the cell suspension. Mix gently by pipetting up and down several times, then incubate for 3 min.
• 28.Vortex the RapidSpheres for 30 s and add 80 μL of the magnetic beads to the cell suspension. Mix and incubate for 3 min.
• 29.Top up the volume of cell suspension to 3 mL with Robosep buffer, then place the tube into the EasySep magnetic tube holder. Incubate for 3 min.
• 30.Remove the supernatant by picking up the magnet and inverting the magnet and tube in one continuous motion to pour off the supernatant. Thereafter, remove the tube from the magnet containing the positively selected cells.
• 31.
  Gently resuspend the cells in 3 mL of Robosep buffer by pipetting up and down several times.

  • a.
    Return the tube to the magnet and incubate for 3 min.
  • b.
    Remove the supernatant as described in Step 30.
  • c.
    Repeat this step one more time.

Note: This step ensures a purer yield of CD8a positively selected cells.

• 32.Following the enrichment of HIV-infected cells, resuspend the cells in 5 mL of complete media.
• 33.Determine the yield by sampling a small quantity for automated cell counting (20 μL) and flow cytometry analysis of EGFP-expressing cells (20 μL). Transfer the cells to a T25 flask and place them in the cell culture incubator for 2 days.

Note: With a decent infection (20%–35% EGFP-positive cells), enrichment by anti-CD8a sorting can generate between 5 and 10 million positively selected cells from an infected mixed population of 25–40 million Th17 cells.

• 34.After 2 days, the anti-CD8a magnetic beads will have dissociated from the cells. To remove these beads, transfer the entire cell suspension to a 5 mL polystyrene round-bottom tube and place the tube into the EasySep magnet for 2 min.
• 35.Transfer the cell suspension with beads removed to a clean T25 flask by inverting the magnet to pour out the supernatant.

Th17 cell quiescence to establish HIV latency

Timing: 2 weeks

Day 14

• 36.Monitor the cell count, viability, and GFP reporter expression as described above.
• 37.Transfer all the cells to a 15-mL conical tube and spin at 450 × g for 3 min. Aspirate the media, leaving one-tenth the original volume.
• 38.Add fresh, complete media so the final cell concentration will be 1 million/mL. Thereafter, as shown in Table 4, add IL-2 (at 15 IU/mL), IL-23 (at 31.25 ng/mL), IL-7 (at 5 ng/mL), TGF-β (at 5 ng/mL), IL-8 (at 50 ng/mL) and IL-10 (at 10 ng/mL).
• 39.Resuspend the cells by gentle pipetting, then transfer to a T25 flask and place in the cell culture incubator for 3 days.

Table 4.

Cytokines for first week of quiescence medium (Days 14 to 21)

Cytokine,	Stock conc.	Working dilution	Final conc.
IL-2	60,000 IU/mL	1:4000	15 IU/mL
IL-23	50 μg/mL	1:1600	31.25 ng/mL
IL-7	10 μg/mL	1:2000	5 ng/mL
TGF-β	10 μg/mL	1:2000	5 ng/mL
IL-8	50 μg/mL	1:1000	50 ng/mL
IL-10	10 μg/mL	1:1000	10 ng/mL

Day 17

• 40.Monitor the cell count, viability, and GFP reporter expression as described above.

Note: If cell concentration is still within 1–2 million/mL, add a second round of cytokine cocktail to the cell culture: IL-2 (at 15 IU/mL), IL-23 (at 31.25 ng/mL), IL-7 (5 ng/mL), TGF-β (at 5 ng/mL), IL-8 (at 50 ng/mL) and IL-10 (at 10 ng/mL). Mix by gentle pipetting and return the flask to the cell culture incubator. Repeat this step on Day 19.

Day 21

• 41.Monitor the cell count, viability, and GFP reporter expression as described above.
• 42.Transfer all the cells to a 15-mL conical tube and spin at 450 × g for 3 min. Aspirate the media, leaving one-tenth the original volume.
• 43.Add fresh, complete media so that the final cell concentration is 1 million/mL.
• 44.Thereafter, add IL-2 (at 15 IU/mL), IL-23 (at 31.25 ng/mL), TGF-β (at 5 ng/mL), IL-8 (at 50 ng/mL) and IL-10 (at 10 ng/mL). Mix by gentle pipetting, then return the flask to the cell culture incubator.

Note: As shown in Table 5, IL-7 addition has been excluded at this step.

Table 5.

Cytokines for second week of quiescence medium (Days 21 to 28)

Cytokine,	Stock conc.	Working dilution	Final conc.
IL-2	60,000 IU/mL	1:4000	15 IU/mL
IL-23	50 μg/mL	1:1600	31.25 ng/mL
TGF-β	10 μg/mL	1:2000	5 ng/mL
IL-8	50 μg/mL	1:1000	50 ng/mL
IL-10	10 μg/mL	1:1000	10 ng/mL

Day 24

• 45.Monitor the cell count, viability, and GFP reporter expression as described above. Cell concentration should be approximately 1 million/mL since the cells are no longer expanding.

Note: A slight cell viability reduction is expected as the cells become quiescent.

• 46.As shown in Table 5, add a second round of IL-2 (at 15 IU/mL), IL-23 (at 31.25 ng/mL), TGF-β (at 5 ng/mL), IL-8 (at 50 ng/mL) and IL-10 (at 10 ng/mL).
• 47.Mix by gentle pipetting, and then return the flask to the cell culture incubator. Repeat this step on Day 26.

Day 28

• 48.Monitor the cell count, viability, and GFP reporter expression as described above. At this point, cells are expected to be fully quiescent.

Note: To assess for quiescence and achievement of HIV latency, unstimulated and re-stimulated cells can be subjected to immunofluorescence staining for flow cytometry of various host and viral factors (P-TEFb, CD25, CD69, Nef and Tat), as described below. Entry into quiescence of the polarized cells can also be monitored by measuring DNA replication through DAPI staining and the incorporation of EdU and confirming the restricted expression of cell proliferation markers (Ki67 and Cyclin D3). This procedure has been described elsewhere.^2,3

Day 29

• 49.Once quiescence and viral latency have been ascertained, immediately use the cells for the intended experiments.

Covalent labeling of antibody with fluorophore

Timing: 2 h

Fluorophore-conjugated antibodies for intracellular immunofluorescence flow cytometry can be prepared using Alexa Fluor and Pacific Blue antibody labeling kits from Thermo Fisher Scientific. Although it is recommended that each conjugation be done with 100 μg of primary antibody at a concentration of 1 mg/mL, we have found that these kits can still be successfully used to label antibody stocks with a concentration as low as 0.5 mg/mL but adjusted for volume (i.e., it is possible to label 200 μL of a 0.5 mg/mL antibody stock with the same procedure).

• 50.Resuspend the lyophilized sodium bicarbonate in 1 mL of deionized water to prepare a 1 M solution.
• 51.Place a volume equivalent to 100 μg of the antibody to be labeled in a microcentrifuge tube, then add a 1/10th volume of 1 M sodium bicarbonate buffer.

Note: Please refer to the manufacturer’s recommendations on the appropriate buffers for dissolving lyophilized antibodies before using these labeling kits.

• 52.Transfer the antibody-bicarbonate mixture to the vial of reactive fluorophore dye. Mix by gentle pipetting.
• 53.Incubate for 1 h at 25°C in the dark with gentle pipetting every 15 min to maximize the labeling efficiency.
• 54.During the labeling incubation, prepare a spin column for purifying the labeled protein by following the manufacturer’s directions.
• 55.Place the spin column and collecting tube in a 15-mL conical tube and centrifuge for 3 min at 1100 × g. Discard the flow-through and return the spin column and empty collection tube to the 15-mL conical tube.
• 56.Load the antibody reaction volume dropwise onto the top of the column resin. Allow the solution to absorb into the resin bed. Centrifuge for 5 min at 1100 × g.
• 57.After centrifugation, the collection tube will contain the labeled antibody.

Note: When stored at 4°C and protected from light, we have found that labeled antibodies are stable for at least 2 months.

Intracellular Nef and Tat immunofluorescence staining of infected Th17 cells for flow cytometry

Timing: 1–2 days (depending on whether a cell stimulation step is included)

Infected primary Th17 cells, with or without an experimental treatment, can be assessed for infection, viral latency, or latent HIV reactivation by immunofluorescence flow cytometry. To phenotype the establishment of HIV latency following cellular quiescence and evaluate responsiveness to reactivating stimuli, primary Th17 cells can be subjected to immunofluorescence staining for Nef and Tat as described below.

Day 1: Antibody conjugation and cell treatments

• 58.Prepare fluorophore conjugates of Nef and Tat antibodies as described above using the Alexa Fluor 647 and Pacific Blue antibody labeling kits, respectively.
• 59.Place the antibody conjugates in light-safe microcentrifuge tubes and store at 4°C until the cells are ready for immunostaining.
• 60.1.2 × 10⁶ viable cells (viability is assessed by PI staining as described above) are harvested and centrifuged at 450 × g for 3 min.
• 61.Thereafter, the cells are resuspended in 6 mL complete media, which is equally distributed into 6 24-wells so that each well will have 2.0 × 10⁵ viable cells.
• 62.In reactivation experiments, latently infected cells can be treated or not with one of the following latency-reversing stimuli: a) T-cell receptor (TCR) activator beads; 50 nM Ingenol-3-angelate; 1 μM Prostratin; 50 nM Bryostatin; 1 μg/mL Ionomycin. Mix by gentle pipetting, and then place the 24-well plate in the cell culture incubator for 24 h.

Day 2: Immunofluorescence staining and flow cytometry

• 63.Transfer the cells to 5-mL polystyrene flow cytometry tubes and centrifuge at 450 × g for 3 min. Decant the media into a 50 mL conical tube and wash the remaining cells once with 1 mL of 1X PBS.
• 64.
  After centrifuging the cells at 450 × g for 3 min, remove the wash by decanting, then dab the rims of the tubes on a paper towel. Empty the contents of the conical tube into a designated liquid biohazard waste container.

  • a.
    Prepare a working solution of the viability dye by adding 1 μL of FVD per 1 mL of 1X PBS.
  • b.
    Add 250 μL of the prepared solution to each tube of cells and resuspend by gentle pipetting.
  • c.
    Incubate the tubes for 30 min at 4°C (wet ice) while shielding them from light.
  • d.
    Centrifuge the cells at 450 × g for 3 min. After washing the cells once with 1X PBS, proceed to the fixation step.

Optional: At this point, the cells that will be subjected to immunostaining can be stained with a fluorescent fixable viability dye (FVD) such as the eBioscience Fixable Viability Dye eFluor 450.

Note: If cells are also immunostained for Tat, whose antibody is conjugated to Pacific Blue, a Fixable Viability Dye in a different color should be used (e.g. eBioscience Fixable Viability Dye eFluor 780).

• 65.Prepare 4% formaldehyde fixative solution in 1X PBS in a chemical fume hood. Resuspend the cells in 500 μL of the fixative, then incubate for 15 min at 25°C while shielding them from light.

CRITICAL: Formaldehyde is classified as a Category 4 carcinogen. Due to its high chemical reactivity, formaldehyde causes local irritation and acute and chronic toxicity after direct contact with target tissues. Please exercise extreme caution during this step.

• 66.Centrifuge the fixed cells at 450 × g for 3 min. Decant the supernatant into a designated formaldehyde waste container in the chemical fume hood, then dab the rim of the tubes on a paper towel to remove as much of the remaining fluid as possible.
• 67.Wash the cells once with 500 μL of 1X PBS. Following centrifugation, decant the wash into the formaldehyde waste container, then dab the rim of the tubes on a paper towel.
• 68.For cell permeabilization, resuspend each tube of cells in 500 μL of a 0.2% Triton X-100 solution prepared in 1X PBS. Incubate for 10 min at 25°C while shielding the cells from light.
• 69.Centrifuge the permeabilized cells at 450 × g for 3 min. Decant the supernatant into a waste container.

Note: From this point forward all liquid waste generated from this procedure can be safely disposed of down the sink.

• 70.After washing each tube of cells once with 0.5 mL of 1X PBS, proceed to the blocking step.
• 71.
  Prepare blocking solution and resuspend cells as follows:

  • a.
    Add 20 μg of purified mouse or rabbit IgG per 1 mL of 1X permeabilization/wash (Perm/Wash) buffer.
  • b.
    Resuspend each tube of cells in 500 μL blocking solution by gentle pipetting.
  • c.
    Incubate for 30 min at 25°C in the dark.
• 72.Centrifuge the cells at 450 × g for 3 min. Decant the supernatant while uniformly dabbing the rim of each tube on a paper towel.

Note: The cells are now in a residual volume of blocking solution (∼50 μL) that is appropriate for the immunostaining step. Only combinations of primary antibodies directly conjugated with fluorophores that exhibit minimal spectral emission overlap are used. Each tube sample is stained with 2 μL of each of the anti-Nef-Alexa Fluor 647 and anti-Tat-Pacific Blue fluorophore-conjugates (Table 6).

• 73.Prepare a master-mix of the appropriate combination of antibody conjugates. Add an equal volume of each master-mix to the corresponding series of tubes then vortex to mix.
• 74.Incubate for 45 min at 25°C in the dark.
• 75.Wash the cells by adding 1 mL of 1X PBS to each tube and vortex to mix. Spin the cells at 450 × g for 3 min and discard the supernatant.
• 76.Resuspend each tube of cells in 200 μL of 1X PBS. The cells are now ready to be examined by flow cytometry using an instrument such as the BD LSR Fortessa that is equipped with the appropriate laser and filters.
• 77.For the Nef and Tat immunostaining series, a new experiment can be set up on the LSR Fortessa with the appropriate compensation controls and following the manufacturer’s recommendations. FCS files generated from samples run using the LSR Fortessa can then be analyzed using Winlist software.

Table 6.

Antibody conjugates used for intracellular immuno-flow of HIV or P-TEFb

Detection target	Fluorophore-conjugated antibodies	Amount of labeled antibody used for IF staining	Approximate final antibody conjugate dilution
Proviral gene expression	Nef-Alexa Fluor 647	2 μL per tube	1:25
	Tat-Pacific Blue	2 μL per tube	1:25
P-TEFb and T-cell activation	Cyclin T1-Alexa Fluor 647	1.75 μL per tube	1:30
	pSer175 CDK9-Alexa Fluor 488	3.5 μL per tube	1:15
	CD25-PE	7.5 μL per tube	1:7
	CD69-PECy7	2.5 μL per tube	1:20

Assessment of HIV latency reversal activity by flow cytometry measurement of P-TEFb biogenesis

Timing: 2 days

For complete details on the use and execution of this section of the protocol, please refer to Mbonye et al.¹³ P-TEFb immuno-flow has proven valuable in enabling the identification of latency reversal treatments that can induce active P-TEFb expression to reverse HIV-1 latency in primary CD4+ T cells and permits the investigation of signaling pathways that mediate their stimulatory effects.

Day 1: Antibody conjugation and cell treatments

• 78.Prepare fluorophore conjugates of Cyclin T1 and pSer175 CDK9 antibodies as described above using the Alexa Fluor 488 and Alexa Fluor 647 antibody labeling kits, respectively.
• 79.Place the antibody conjugates in light-safe microcentrifuge tubes and store at 4°C until the cells are ready for immunostaining.
• 80.After assessing cell viability using PI staining as described above, depending on the number of cell treatments, the appropriate number of quiescent, viable, uninfected Th17 cells are harvested and centrifuged at 450 × g for 3 min.
• 81.Thereafter, the cells are resuspended in complete media, which is equally distributed into 24 wells so that each well will have 2.0 × 10⁵ viable cells in 1 mL of media.
• 82.Following treatment with known or candidate HIV-1 latency-reversing agents, mix the cell suspension by gentle pipetting, then place the 24-well plate in the cell culture incubator for 24 h.

Day 2: Immunofluorescence staining and flow cytometry

• 83.Transfer the cells to 5-mL polystyrene flow cytometry tubes and centrifuge at 450 × g for 3 min.
• 84.
  Decant the media into a 50-mL conical tube and discard it. Resuspend the cells in 1 mL of 1X PBS.

  • a.
    To assess the effect of the treatments on cell viability, a small sample of each cell suspension can be subjected to PI staining. Alternatively, the cells can be stained with a fixable viability dye, as described below in Step 88, prior to fixation and immunofluorescence staining.
  • b.
    Take 20 μL of each cell suspension for PI staining and place it in a fresh 5-mL polystyrene tube containing 180 μL of 1X PBS.
  • c.
    Add 1 μL of PI reagent and immediately vortex the tubes to mix.
  • d.
    Analyze by flow cytometry using the appropriate voltage settings for the forward/side scatter and TRITC laser channels.
• 85.Centrifuge the remainder of the cells at 450 × g for 3 min.
• 86.
  Remove the PBS wash by decanting, then dab the rims of the tubes on a paper towel. Empty the contents of the conical tube into a designated liquid biohazard waste container.

  • a.
    Prepare a working solution of the viability dye by adding 1 μL of FVD per 1 mL of 1X PBS.
  • b.
    Add 250 μL of the prepared solution to each tube of cells and resuspend by gentle pipetting.
  • c.
    Incubate the tubes for 30 min at 4°C (wet ice) while shielding them from light.
  • d.
    Centrifuge the cells at 450 × g for 3 min. After washing the cells once with 1X PBS, proceed to the fixation step.

Optional: As an alternative to assessing cell viability by PI staining, cells can be stained with a fluorescent fixable viability dye (FVD) compatible with the P-TEFb immuno-flow, such as the eBioscience Fixable Viability Dye eFluor 450.

• 87.Prepare 4% formaldehyde fixative solution in 1X PBS in a chemical fume hood. Resuspend the cells in 500 μL of the fixative, then incubate for 15 min at 25°C while shielding them from light.
• 88.Centrifuge the fixed cells at 450 × g for 3 min.
• 89.Decant the supernatant into a designated formaldehyde waste container in the chemical fume hood, then dab the rim of the tubes on a paper towel to remove as much of the remaining fluid as possible.
• 90.Wash the cells once with 500 μL of 1X PBS. Following centrifugation, decant the wash into the formaldehyde waste container, then dab the rim of the tubes on a paper towel.
• 91.For cell permeabilization, resuspend each tube of cells in 500 μL of a 0.2% Triton X-100 solution prepared in 1X PBS. Incubate for 10 min at 25°C while shielding the cells from light.
• 92.Centrifuge the permeabilized cells at 450 × g for 3 min. Decant the supernatant into a waste container.

Note: From this point forward, all liquid waste generated from this procedure can be safely disposed of in the sink.

• 93.After washing each tube of cells once with 0.5 mL of 1X PBS, proceed to the blocking step.
• 94.
  Prepare blocking solution and resuspend cells as follows:

  • a.
    Add 20 μg of purified mouse or rabbit IgG per 1 mL of 1X permeabilization/wash (Perm/Wash) buffer.
  • b.
    Resuspend each tube of cells in 500 μL blocking solution by gentle pipetting.
  • c.
    Incubate for 30 min at 25°C in the dark.
• 95.Centrifuge the cells at 450 × g for 3 min. Decant the supernatant while uniformly dabbing the rim of each tube on a paper towel.

Note: The cells are now in a residual volume of blocking solution (∼50 μL) appropriate for the immunostaining step. For the Cyclin T1 and pSer175 CDK9 fluorophore-conjugated antibodies listed in Table 6, use 1.75 μL of the Cyclin T1 antibody conjugate and 3.5 μL of the pSer175 CDK9 antibody conjugate for staining each sample. These cells can also be simultaneously stained for the T-cell surface activation markers CD25 and CD69 using the following antibody conjugates from BD Pharmingen: 7.5 μL per sample of PE mouse anti-human CD25 and 2.5 μL per sample of PE-Cy7 mouse anti-human CD69.

• 96.Prepare a master mix of the four antibody conjugates. Add an equal volume of each master mix to the corresponding series of tubes, then vortex to mix.
• 97.Incubate for 45 min at 25°C in the dark.
• 98.Wash the cells by adding 1 mL of 1X PBS to each tube and vortex to mix.
• 99.Spin the cells at 450 × g for 3 min and discard the supernatant.
• 100.Resuspend each tube of cells in 200 μL of 1X PBS. The cells are now ready to be examined by flow cytometry using the BD LSR Fortessa.

Note: For P-TEFb:CD25:CD69 immunostaining series, a new experiment can be set up on the LSR Fortessa with the appropriate compensation controls. FCS files generated from samples run using the LSR Fortessa can then be analyzed using Winlist software.

Expected outcomes

The QUECEL Th17 model enables the routine generation of approximately 5–10 million polarized, viable, latently infected CD4⁺ Th17 cells, starting with 10–20 million healthy donor-derived naïve T cells within 1 month. These latently infected cells can be used for proviral DNA assays as well as latency reversal studies involving immunofluorescence flow cytometry, high-resolution fluorescence microscopy, and cell-associated viral RNA measurements.^{2,13,14,15,16}

By using a five-fold lower concentration of Concanavalin A (2 μg/mL) during the polarization phase of the QUECEL procedure (Figure 1) than had previously been utilized,^2,3 we can consistently obtain a well-expanded population of polarized Th17 cells that are both permissive as well as tolerant to infection with any of the packaged single-round HIV constructs. Figure 2 illustrates the generation of latently infected Th17 cells upon infection of the VSV-G pseudotyped d2EGFP single-round viral construct. The viability of the latently infected cells usually drops to 50%–60% due to apoptosis associated with the entry into quiescence. Figure 3 shows the Tat/Nef dual immunofluorescence flow cytometry of Th17 cells infected with the pseudotyped d2EGFP single-round viral construct. Uninfected, acutely infected, latently infected, and reactivated Th17 cells were immunostained for Tat and Nef as described in the text. Figure 4 shows the infection of Th17 cells with the CD8a-d2EGFP single-round viral construct and subsequent enrichment of acutely infected cells by anti-CD8a sorting. After the positive selection, the CD8a-d2EGFP is relatively stable and remains expressed even as the cells enter quiescence. Reactivation profiles of Th17 cells infected with the HIV CD8a-EGFP construct as assessed by Tat/Nef immuno-flow following treatment with latency-reversing stimuli are presented in Figure 5. Reactivation profiles of quiescent, uninfected Th17 cells as assessed by P-TEFb immuno-flow coupled with the examination of T-cell activation are presented in Figure 6. The coupling of P-TEFb immuno-flow with cell viability measurements using a fixable viability dye is shown in Figure 7. Figure 8 is comparative flow data illustrating how the inducible expression of P-TEFb is tightly coupled to the reactivation of latent HIV in primary T cells.

Figure 2.

Monitoring the generation of latently infected Th17 cells upon infection with VSVG-pseudotyped packaged virus of the d2EGFP single-round viral construct

Top panel, Dual assessment of the percentage of EGFP-positive cells, which indicates the extent of proviral gene expression, and their cell viability based on propidium iodide (PI) staining. Bottom panel, Corresponding forward/side scatter plots. pi: post-infection; ps: post-shutdown.

Figure 3.

Tat/Nef dual immunofluorescence flow cytometry of cells infected with VSVG-pseudotyped virus of the d2EGFP single-round viral construct

Uninfected, acutely infected, latently infected, and reactivated Th17 cells were immunostained for Tat and Nef as described in the text. The 2D plots show proviral gene expression as monitored by d2EGFP versus Tat (Top), d2EGFP versus Nef (Middle), and Tat versus Nef (Bottom).

Figure 4.

Infection of polarized Th17 cells with VSVG-pseudotyped virus of the CD8a-d2EGFP single-round viral construct and their subsequent positive selection by anti-CD8a sorting

In this experiment, cells were CD8a-selected at Day 5 post-infection (pi). CD8a-d2EGFP is relatively stable and remains expressed even in the quiescent, latently infected cells.

Figure 5.

Reactivation profiles as assessed by Tat/Nef immuno-flow following treatment of latently infected Th17 cells with latency-reversing stimuli

Polarized Th17 cells were infected with the CD8a-d2EGFP single-round viral construct. Infected cells were then positively sorted by anti-CD8a selection before forcing them into quiescence to establish viral latency. Uninfected, latently infected, and reactivated Th17 cells were immunostained for Tat and Nef as described in the text. The 2D plots show proviral gene expression as monitored by CD8a-d2EGFP versus Tat (Top), CD8a-d2EGFP versus Nef (Middle), and Tat versus Nef (Bottom).

Figure 6.

Reactivation profiles as assessed by P-TEFb immuno-flow following treatment of quiescent, uninfected Th17 cells with latency-reversing stimuli

Quiescent, uninfected Th17 cells were stimulated or not as shown, then immunostained for Cyclin T1, pSer175 CDK9, CD25, and CD69 as described in the text. The 2D plots at the top show the extent of P-TEFb expression as monitored by Cyclin T1 versus pSer175 CDK9, while those in the middle and bottom show the extent of T-cell activation based on the measured expression of CD25 and CD69.

Figure 7.

Coupling P-TEFb immuno-flow with cell viability measurements using a fixable viability dye

Quiescent, uninfected Th17 cells were stimulated or not as shown, then subjected to staining with the fixable viability dye eFluor450 (e450 FVD). Thereafter, the fixed and permeabilized cells were immunostained for Cyclin T1 and pSer175 CDK9. Top, Forward/side scatter plots with events color-coded to distinguish between viable (red) and non-viable e450-positive (blue) cells. Middle, Identification of e450-positive cells and gating of viable cells. Bottom, 2D plots showing the extent of P-TEFb expression in the gated viable subset.

Figure 8.

Proviral reactivation in the QUECEL Th17 model tightly correlates with the extent of inducible P-TEFb expression

(A) Graphed immunofluorescence flow cytometry measurements of the extent latent HIV reactivation (left) and P-TEFb (dual Cyclin T1 and pSer175 CDK9) expression (right) following 24 h treatment with latency-reversing stimuli.

(B) Coupling the detection of the pSer175 CDK9 marker of P-TEFb activation with the assessment of latent HIV reactivation. Latently infected Th17 cells (left graph) were stimulated through the T-cell receptor (TCR; right graph) and then co-stained with fluorophore-conjugated antibodies against pSer175 CDK9 and the viral factor Nef.

Limitations

The Tat/Nef immuno-flow protocol is optimized for latently infected primary T cells generated in vitro. An important extension of the procedure, which we are currently testing, is to overcome the challenge of sensitively monitoring latency reversal in CD4⁺ T cells derived from virally suppressed individuals, like the intracellular p24 HIV-Flow assay developed by Chomont and colleagues.¹⁷ The QUECEL procedure generates millions of viable latently infected Th17 cells. Studies are ongoing to improve the survivability and yield of HIV-infected cells that have been polarized into the Th1, Th2, or Treg phenotypes.

The immuno-flow assays described here emphasize the use of directly conjugated primary antibodies and are incompatible with fluorophore-conjugated secondary antibodies. This enables the utilization of multiple primary antibodies from the same species and avoids the high background signal emanating from the amplification step with the secondary antibody. We have noted that primary antibody conjugates prepared with any of the four labeling kits shown in the key resources table must be stored at 4°C and tend to substantially lose their fluorophore activity after 2 months. Thus, it is strongly recommended that these conjugates be utilized within 1 month of their preparation.

Troubleshooting

Problem 1

During the last week of entry into quiescence, the viability of the latently infected Th17 cells drops drastically below 50% (related to Step 45).

Potential solution

If this problem arises, it may be avoided by opting not to change the cell media at Step 43 for the remaining duration of the QUECEL procedure. Ensure the cell culture is replenished with the quiescence cytokine cocktail (Table 5) as recommended at Days 24 and 26 (Steps 44 and 46).

Problem 2

There is an inadequate yield of expanded polarized Th17 cells (related to Steps 13–15).

Potential solution

A clear indication that the Th17 cells have properly expanded is the visible accumulation of plump settlements of cells at the bottom of the 96 wells at the end of the 7-day polarization phase (Figure 9). If this is not observed, it could be due to a weak response of the polarizing Th17 cells to activation with Con A. Ensure that the Con A stock solution is well prepared and homogeneously mixed before addition to the cells on Days 1 and 4. If the Con A stock is not identified as the issue, try using a different healthy donor source of naive CD4⁺ T cells.

Figure 9.

Appearance of well expanded, polarized Th17 cells at the end of the 7-day polarization phase

From a starting isolation of 6–10 million naïve CD4⁺ T cells, a good yield of expanded and polarized Th17 cells will be between 25 and 40 million cells.

Problem 3

Percent of HIV-infected Th17 cells is low (10% or less; related to Steps 16–35).

Potential solution

Test the concentrated packaged virus for infectivity by examining reporter GFP expression in infected HEK 293T cells. Poorly expanded Th17 cells will also yield a poor infection (refer to the proposed solution to problem 2).

Problem 4

The yield of HIV-infected Th17 cells following enrichment by anti-CD8a positive selection is unusually low despite a good percent infection (related to Steps 24–35).

Potential solution

Perform a second round of anti-CD8a positive selection of the negatively sorted cells 2–3 days after conducting the first round. Ensure that the magnetic beads are removed 48 h after the positive selection.

Problem 5

The recommended infection of the polarized Th17 cells (addition of 1 mL of concentrated virus per 5 million cells) is noted to cause substantial cytotoxicity 2–3 days post-infection (related to Steps 17–21).

Potential solution

Adjust the proportion of concentrated virus to Th17 cells by infecting a larger number of cells; for instance, try infecting 20 million cells with the 1 mL of concentrated virus. Incubate the virus with the cells for at least 72 h prior to performing anti-CD8a positive selection of the infected cells. The same exact procedure described in Steps 24–35 can be used to enrich for HIV-infected cells from this larger mixed population of cells. If this alternative procedure does not lead to a significant reduction in virus-induced cytotoxicity, prepare another batch of packaged virus concentrate to use for infection.

Problem 6

An antibody that is well-validated for its cross-reactivity towards a particular protein target elicits a poor immunofluorescence signal by flow cytometry (related to Steps 50–57).

Potential solution

We had previously encountered this problem with the Tat monoclonal antibody (NT3 2D1.1). We found that it exhibited poor immunoreactivity if labeled with Alexa Fluor 555 or Alexa Fluor 647 before identifying that the antibody works well if conjugated with Pacific Blue. Thus, this problem could be resolved by identifying a fluorophore conjugate that does not seem to interfere with the antibody’s immunoreactivity.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Uri Mbonye (uri.mbonye@case.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contacts, Uri Mbonye (uri.mbonye@case.edu) and Jonathan Karn (jonathan.karn@case.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

There is no dataset/code associated with the paper.

Acknowledgments

We thank the Rustbelt CWRU/Pitt Center for AIDS Research for the provision of flow cytometry services. This work was supported by National Institutes of Health grants R01 AI48083 to J.K. and U.M. and R01 DEO25464 and R61 AI169629 to J.K. and a developmental grant to U.M. from the CWRU/Pitt Center for AIDS Research (P30 AI036219).

Author contributions

Conceptualization, U.M. and J.K.; methodology and optimization of protocols, U.M., A.A., M.Y., and J.K.; writing – original draft, U.M.; writing – reviewing and editing, U.M., A.A., M.Y., and J.K.; funding acquisition, U.M. and J.K.

Declaration of interests

The authors declare no competing interests.