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. Author manuscript; available in PMC: 2025 Sep 5.
Published in final edited form as: Methods Mol Biol. 2022;2407:57–68. doi: 10.1007/978-1-0716-1871-4_5

A reliable primary cell model for HIV latency: The QUECEL (Quiescent Effector Cell Latency) method

Meenakshi Shukla 1, Fredrick Kizito 1, Uri Mbonye 1, Kien Nguyen 1, Curtis Dobrowolski 1,1, Jonathan Karn 1
PMCID: PMC12410993  NIHMSID: NIHMS2105521  PMID: 34985657

Abstract

One of the main methods to generate the HIV reservoir is during the transition of infected activated effector CD4 T cells to a memory phenotype. The QUECEL (Quiescent Effector Cell Latency) protocol mimics this process efficiently and allows production of large numbers of latently infected CD4+ T cells. After polarization and expansion, CD4+ T cells are infected with a single round reporter virus which expressed GFP/CD8a. The infected cells were purified and coerced into quiescence using a defined cocktail of cytokines including TGF-β, IL-10, and IL-8, producing a homogeneous population of latently infected cells. Since homogeneous populations of latently infected cells can be recovered, the QUECEL model has an excellent signal to noise ratio, and has been extremely consistent and reproducible in numerous experiments performed during the last 5 years. The ease, efficiency, and accurate mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency.

Keywords: HIV latency, memory T-cells, QUECEL model, cytokines, cellular quiescence

1. Introduction

HIV persists due to a pool of transcriptionally silenced, but replication-competent proviruses, found in a small population of resting memory CD4+ T cells (1 to 100 per 106 cells) accumulating in the peripheral blood [1] and tissues [2]. Since silenced proviruses produce only minimal viral RNA and proteins, they are refractory to antiviral drugs and effectively evade immune surveillance.

Our understanding of HIV latency and persistence has been complicated by the small numbers of latently infected cells found in the circulation, the difficulty of obtaining comprehensive sets of tissue samples from patients, the lack of known phenotypic markers that can distinguish latently infected cells from uninfected ones, and limited information about the behavior of tissue reservoirs in vivo. Contemporary HIV research has therefore been propelled by the development of models of T cell latency that can be implemented in the laboratory setting. Although latency can be established in transformed T cell lines, such as Jurkat T-cells [3–7], this can lead to potentially unrepresentative mechanisms of HIV latency since the host cell is not quiescent [7, 8] [9]. A more physiologically relevant approach is to infect primary cells isolated from healthy donors. In many instances, HIV latency is established during the transition of effector memory cells to a quiescent memory cell phenotype [10–12]. Primary cell models of HIV latency [13] therefore typically involve infection of an activated cell population that is then allowed to enter quiescence [14–16]. Alternatively, resting cells can be infected directly [12, 17, 18].

Unfortunately, most primary cell models for HIV latency correlate poorly with the reactivation behavior of patient cells [7, 19]. The QUECEL model [8, 19–21], which is described in detail in this article, is a refinement of the model of Bosque and Planelles [14, 22]. Briefly Naïve helper T cells are polarized into the four major effector T cell subsets (Th1, Th2, Th17, and Treg). The Th17 polarization phenotype, which we routinely use since these cells tend to retain a higher degree of viability than the other polarization phenotypes, represents the most abundant effector T cell population in the lamina propria of the GI tract [23–25]. QUECEL generates a large and homogenous population of latently infected CD4+ memory cells. By purifying HIV infected cells and inducing cell quiescence with a defined cocktail of cytokines, we have eliminated the largest problems with previous primary cell models of HIV latency: variable infection levels, ill-defined polarization states, and inefficient shut down of cellular transcription. This scalable and highly reproducible model of HIV latency therefore permits a detailed analysis of cellular mechanisms controlling HIV latency and reactivation and we have used it successfully to study the epigenetic, transcriptomic and cell biological underpinnings of HIV latency.

2. Materials

2.1. Cell Growth Media

The primary cells need to be grown in primary cell media (RPMI, 10% FBS, Primocin and 25 mM HEPES) together with their required cytokines (Table 1).

Table 1:

Maintenance Cytokines

Subtype Cytokine Stock Conc. Dilution Source Catalog No.
Th1, Th2, TReg rIL-2 60,000 IU/ml 1:1000 NIH AIDS Reagent Program 136
Th17 rIL-2 60,000 IU/ml 1:1000 NIH AIDS Reagent Program 136
rIL-23 50 μg/ml 1:1000 PeproTech 200–23
IL-7 10 μg/ml 1:1000 200–07
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2.2. Polarization cytokines

Cells are polarized using a cocktail of antibodies and cytokines (Table 2).

Table 2:

Polarization Cytokines

Subtype Cytokine or Antibody Stock Conc. Dilution Source Catalog No.
Th1 Anti-Human IFN-γ 10 μg/ml 1:1000 Preprotech 500-M90
Anti-Human IL-4 500 μg/ml 1:1000 Preprotech 500-M04
Th2 Anti-Human IL-4 500 μg/ml 1:1000 Preprotech 500-M04
Anti-Human IFN-γ 10 μg/ml 1:1000 Preprotech 500-M90
Th17 TGF-β 5 μg/ml 1:1000 Preprotech 100–21C
Anti-Human IL-4 500 μg/ml 1:1000 Preprotech 500-M04
Anti-Human IFN-γ 10 μg/ml 1:1000 Preprotech 500-M90
IL-1β 10 μg/ml 1:1000 Preprotech 200–01B
IL-6 30 μg/ml 1:1000 Preprotech 200–06
IL-23 50 μg/ml 1:1000 Preprotech 200–23
TReg TGF-β 5 μg/ml 1:1000 Preprotech 100–21C
Anti-Human IL-4 500 μg/ml 1:1000 Preprotech 500-M04
Anti-Human IFN-γ 10 μg/ml 1:1000 Preprotech 500-M90
Anti-Human IL-12 500 μg/ml 1:1000 Preprotech 500-M12
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2.3. Quiescence Medium

Cells are polarized using a cocktail of antibodies and cytokines (Table 2).

3. Methods

An outline of the QUECEL method is shown in Figure 1. Naïve T-cells are polarized and after 6 days of expansion the replicating cells are infected using a single round VSV pseudotyped reporter virus that expresses GFP/CD8a (Figure 1A) [3, 4, 26]. The infected cells are then purified by magnetic bead isolation and coerced into quiescence using a defined cocktail of cytokines including TGF-β, IL-10, and IL-8, producing a homogeneous population of latently infected cells (Figure 1B). Flow cytometry using the cell cycle markers demonstrated that the cells maintained the correct polarization phenotype and had withdrawn from the cell cycle. Both GFP and HIV nef expression can be used to monitor the fraction of infected cells and entry of cells into quiescence (Figure 1C). Once the cells have entered quiescence, the level of HIV Nef protein expression is reduced to almost undetectable levels (1%), which is indicative of HIV latency. GFP levels also decline, but because of the high stability of the CD8a-GFP fusion protein, a moderate level of GFP persists in the quiescent cells. Upon stimulation through the TCR (α-CD3/ α-CD28 Dynal magnetic beads), the fraction of cells expressing Nef increases dramatically (>82%) and there is a concomitant increase in GFP levels (Figure 1C). The QUECEL model is extremely robust and shows minimal variation between replicate samples and between donors in assays performed over a period of more than 2 years (Figure 1D). RNA FISH and immunofluorescence can also be used to show that the latently infected cells produce minimal levels of HIV RNA and the HIV Tat transactivator protein, Tat (Figure 1E). We also routinely monitor cells using an antibody to CDK9 pSer175 as a marker for transcriptionally active P-TEFb [27] since there is a strict correlation between cells that have activated P-TEFb and cells that produce HIV proteins.

Figure 1.

Figure 1.

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Ex vivo QUECEL model for HIV latency. (A) Structure of lentiviral reporter virus. (B) Time line for polarization and infection of naïve CD4+ T cells. In this example cells were polarized to a Th17 phenotype. After infection, cells are placed in media containing a limiting amount of cytokines to allow the cells to return to a resting state. (C) Flow cytometric analysis of the expression of the reporter genes GFP (vertical) and Nef (horizontal) after infection, after isolation of infected cells, after entering quiescence and after restimulation through the TCR. (D) Reproducibility of the QUECEL assay. (E) Induction of HIV RNA and 7SK snRNA after reactivation of latent proviruses by T-cell receptor activation of quiescent Th17 cells (Top) and immunofluorescence imaging of HIV Tat. Images were taken at 100X using a high resolution DeltaVision deconvolution microscope. Scale bars represent a length of 10 μm.

An improvement to the original published QUECEL method [19], which is described here, is the addition of IL-7 to improve cell viability. The ease, efficiency, and accurate mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency.

3.1. Day 0: Activation and Initial Polarization

  1. Isolate naïve CD4 T cells using EasySep™ Human Naïve CD4+ T Cell Enrichment Kit (Stem Cell Technologies, Catalog No. 19155F) and the RoboSep™ Fully Automated Cell Separator (Catalog # 21000, 20119, 20155) following the manufacturer’s directions. Typically, 50 × 106 PBMCs yields 5 × 106 naïve CD4 T cells. Resuspend the naïve CD4 T cells at 5 × 105 cells per ml, which is approximately 10 ml of media per 50 × 106 PBMCs used. 5 × 106 of polarized cells yields 100 to 150 × 106 effector cells after the expansion phase of the protocol.

  2. Add polarization cytokines and antibodies (Table 2), together with Concanavalin A (Millipore Sigma, 234567–1GM) at a final concentration of 10 μg/ml.

  3. Distribute cells into an upright T25 flask to allow for cell to cell contact.

  4. Incubate cells for 72 hours at 37 °C in a CO2 incubator.

3.2. Day 3: Continuing Polarization

  1. Measure out the same amount of primary cell media used at day 0, and add polarization cytokines following the dilutions shown in Table 2, together with 10 μg/ml Concanavalin A and 120 IU/ml of IL-2.

  2. Incubate cells for 72 hours at 37 °C in a CO2 incubator.

3.3. Day 6: Infection with pseudotyped reporter virus

Cells are ready for infection after the 6th day of polarization. Since primary cells are notoriously hard to infect the most efficient infections are achieved by spinoculation for a long period of time using a high titer virus stock as follows:

  1. Pellet the polarized cells and discard the supernatant.

  2. Use the pHR’-Nef+-CD8a/GFP virus when using the CD8a protein for purification of the infected cells. Use 1 ml of a high titer concentrated virus stock (MOI of 10 on Jurkat cells) per 10 × 106 polarized cells.

  3. Dilute the virus in sufficient primary cell media and cell-specific cytokines (Table 1) to resuspend the cells at 5 × 106 cells per ml (i.e. for 10 × 106 cells, add 1 ml primary cell media to 1 ml concentrated virus and 2 μl of cytokine stock).

  4. Aliquot 1 ml of cells into each well of a 24 well plate. It is convenient to split the cells evenly between into two plates to provide a balance for the centrifugation.

  5. Spin the plates at 2000 RCF for 90 min 23 °C and 5 acceleration/9 deceleration.

  6. Remove the cells from the centrifuge and place at 37 °C in a CO2 incubator overnight. This will increase the amount of infection.

3.4. Day 7: Dilution of Virus

Cells should have grown in number after the overnight incubation. Pool the cells and replate at 1 × 106 cells per ml. Do not remove the virus; simply dilute the cells with fresh media until cells are at the desired concentration.

3.5. Days 8–13: Cell Husbandry and Expansion

The cells were kept growing by addition of normal cytokines and fresh media once the media turns yellow (or after 3 days). Cell densities were maintained above 1 ×106 million cells per ml until the needed amount of cells was reached. Check the amount of GFP+ cells 48 hrs after infection and use this to determine the number of cells expressing GFP/CD8a (Total number of cells × % GFP+ cells). Typically, approximately half of the GFP+ cells are isolated by magnetic bead purification.

3.6. Day 14: CD8a+ Cell Isolation

CD8a-expressing cells are isolated using the RoboSep™ Fully Automated Cell Separator (Catalog # 21000, 20119, 20155) and the Mouse CD8a Isolation II kit (Stem Cell Technologies Catalog No.18953) as follows:

  1. Resuspend cells at 10 × 106 cells per 100 μl of RoboSep buffer supplemented with IL-2 (1:2000 of IL-2, Table 1) and placed in a 14 ml polystyrene round bottom sterile test tube. Be careful not to get bubbles in cell suspension or on the sides of the tube, since this will make the isolation less efficient.

  2. Turn on the RoboSep and pick the 1st quadrant and select the 1st program labeled Biotin Isolation. When working with more than one type of cell/donor use one quadrant per cell type for a total of 4 different cell types/donors per isolation. When prompted enter the amount of buffer used to resuspend the cells. If there is a low frequency of infection (less than 10%) enter twice the amount of buffer, which will result in a two-fold concentration of antibody and cocktail during the isolation.

  3. Load the RoboSep carousel based on the directions from the machine (it will give a diagram indicating what type of tubes are required and what each tube will have at the end of the procedure). The Positive fraction tube (located in the magnet) will be the CD8a+ cells. The negative fraction tube will be a mixed population of both infected and uninfected cells but the frequency of infected cells will be less than it was before isolation. This mixed population can be used for controls, or for additional CD8a+ cell isolations, if additional cells are required.

3.7. Days 14 to 28: Cell Quiescence

  1. Vortex the positive fraction tube from the RoboSep to remove cells and beads from the side of the tube and then centrifuge and remove the RoboSep buffer.

  2. Resuspend at 1 × 106 cells per ml, in primary cell media with the addition of the maintenance cytokine (Table 1) cytokines depending on cell type. The cells are cultured at normal levels of cytokines for 5 days without changing the media or adding any additional cytokines. During this period the cells slowly use up the cytokines and cell growth slows.

  3. For the cells to enter quiescence the amount of cytokines need to be reduced to slow growth, mimicking what happens in vivo; this is done by using the dilutions of cytokines listed in Table 3.

Table 3.

Cytokines for Quiescence Medium

Subtype Cytokine Stock Conc. Quiescence Media Maintanence Media Source Catalog No.
Th1, rIL-2 15,000 IU/ml 1:4000 1:4000 NIH 136
Th2, TGF-β1 10 μg/ml 1:1000 Preprotech 100–21C
TReg, IL-8 50 μg/ml 1:1000 Preprotech 200–08M
IL-10 10 μg/ml 1:1000 Preprotech 200–10
Th17 rIL-7 10 μg/ml 1:2000 1:2000 Peprotech 200–07
IL-23 50 μg/ml 1:1600 1:1600 Preprotech 200–23
TGF-β 10 μg/ml 1:1000 Preprotech 100–21C
IL-8 50 μg/ml 1:1000 Preprotech 200–08M
IL-10 10 μg/ml 1:1000 Preprotech 200–10
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4. Notes

  1. After polarization, the cells must remain in the growth cytokines at the specified concentration. These cells need to be grown at high density due to autocrine production of cytokines. A concentration of 1 to 3 × 106 per ml is ideal, and the cells will stop growing at a concentration below 1 × 106 per ml. It is preferable not to remove any old media from these cells, and simply add fresh media and cytokines to the cells to bring them up to the ideal concentration once the media begins to turn yellow.

  2. Thy1.2 can be used as a selection marker using our pHR’-Nef+-Thy1.2-T2A-GFP virus. This vector uncouples GFP from the surface protein so that GFP can be used as an HIV activation marker. Thy1.2 is also easier to purify resulting in a higher yield of HIV infected cells during isolation. To use this option, infect using the pHR’-Nef+-Thy1.2-T2A-GFP virus in the same manner as described for the pHR’-Nef+-CD8a/GFP virus. To select for Thy1.2 you can use the previous described protocol except use the EasySep™ Mouse CD90.2 Positive Selection Kit II (Stemcell, 18951RF).

  3. The cells must remain in quiescence media for at least 1 week to allow the cells to fully enter quiescence. The viability of your cells drops to about 60% due to apoptosis during entry into quiescence. Entry into quiescence should be monitored by cell cycle monitoring, typically by flow cytometry for EdU incorporation and CycB1 and CycD3 levels.

  4. Since the cells will continue to expand at the beginning of the cell quiescence protocol (Days 14 to 28), it is possible to start the step down phase in the middle of the expansion phase. For example, if you want the cells to expand for 7 days, place in normal media at 1 × 106 cells per ml, then 2 days later add more cytokine media to bring them to 1 × 106 cells per ml, and culture for 5 more days without changing media or cytokines to allow them to step down.

  5. The addition of IL-7, as described in this protocol, increases cell viability but also partially blocks entry of cells into full quiescence. If needed, cells can be cultured for a further week in quiescence media containing 15 IU/ml IL-2 in place of IL-7.

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