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Isolation of Human Immunodeficiency Virus Type 1 From Peripheral Blood Monocytes — Микроскопическая техника в биологии и медицине

Isolation of Human Immunodeficiency Virus Type 1 From Peripheral Blood Monocytes

Paul R. Gorry, Secondo Sonza, Katherine Kedzierska, and Suzanne M. Crowe


Human immunodeficiency virus type 1 (HIV-1) can infect circulating peripheral blood monocytes and resting CD4+ T lymphocytes despite sustained suppression of plasma viremia to undetectable levels. These persistently infected cell populations pose a barrier for virus eradication by highly active antiretroviral therapy (HAART), and are a significant reservoir of HIV-1 contributing to viral rebound following cessation or failure of HAART. This chapter provides a protocol for isolating replication-competent HIV-1 from peripheral blood monocytes of HIV-1-infected individuals, including those with sustained plasma HIV-1 RNA levels below 50 copies/mL, by co-culture with CD8-depleted, phytohemagglutinin-activated donor peripheral blood mononuclear cells. In our laboratory, this protocol has the sensitivity to achieve a success rate of positive HIV-1 isolation in approx 70% of cases. The study of HIV-1 strains harbored by peripheral blood monocytes of patients undergoing HAART will contribute to the understanding of viral persistence in cellular reservoirs that impede effective HAART.

Key Words: Monocyte; macrophage; human immunodeficiency virus; HIV-1; monocyte isolation; HAART; CD8-depleted PBMC; co-culture.

  1. Introduction

The ability to isolate HIV-1 strains from different cell types assists in elucidating the phenotypic and/or genetic properties of particular viral strains that enable them to persist in various cellular compartments. However, success rates in isolating virus vary greatly depending on the source of tissue and method employed (1). Because fewer monocytes are thought to harbor HIV-1 in vivo compared with T-lymphocytes and tissue macrophages, and because monocytes are less permissive to productive virus replication compared with the

latter cell types, the isolation of HIV-1 from peripheral blood monocytes requires techniques with a greater level of sensitivity. However, our laboratory and other investigators have shown that isolation of HIV-1 from peripheral blood monocytes is readily achievable even from patients undergoing highly active antiretroviral therapy (HAART) for periods of up to 80 wk, with undetectable plasma viral loads (2-4).

Although patterns of virus isolation vary from patient to patient, HIV-1 can generally be detected in culture supernatants by 14 to 21 d of co-culture of monocytes with CD8-depleted, phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC). The levels of HIV-1 recovered from monocytes are typically very low compared with levels normally isolated from other cell types such as PBMC, and tissues such as lymph node, spleen, and brain (1). Subsequently, the detection of virus in culture supernatants is often achievable only by measurement of HIV-1 RNA by reverse transcriptase (RT)-polymerase chain reaction (PCR), especially when isolating virus from monocytes of patients undergoing HAART with suppressed plasma viremia.

There is no apparent correlation between the period of viral suppression by HAART and the ability to recover infectious virus from patients’ monocytes. Although it is well established that resting-memory CD4+ (CD45RO+) T lymphocytes constitute a significant latent reservoir of HIV-1 in individuals receiving HAART (5-10), it is unlikely that monocytes represent a latently infected cellular reservoir, because monocytes have a relatively short period of circulation within blood before differentiation into specific tissue macrophages. In fact, our studies and those of other investigators have demonstrated the presence of circular viral DNA and spliced RNA in HIV-1-infected monocytes, albeit at very low levels (2,11), suggesting that their infection is recent and transcriptionally active rather than latent. Futhermore, findings of genetic evolution of HIV-1 in peripheral blood monocytes indicates HIV-1 replicates in monocytes in vivo in patients with (11) and without HAART (12).

The protocol for isolating HIV-1 from peripheral blood monocytes described in this chapter involves three major procedures: (1) isolation of peripheral blood monocytes from PBMC of HIV-1-infected individuals; (2) preparation of CD8-depleted, PHA-activated donor PBMC; and (3) co-culture of patient monocytes with donor CD8-depleted, PHA-activated PBMC.


2.1.Materials Required for Isolation of Peripheral Blood Monocytes From PBMC of HIV-1-Infected Individuals

  1. 30 mL of anticoagulated patient blood (see Notes 1 and 2).
  2. Supplemented Iscove’s medium (Iscove’s medium containing penicillin [100 U/mL], streptomycin [100 pg/mL], L-glutamine [25 pg/mL], and 10-20% [v/v] heat-inactivated AB+ human serum) (see Note 3).
  3. Phosphate-buffered saline (PBS).
  4. PBS, calcium- and magnesium-free (PBS-CMF).
  5. Heat-inactivated fetal calf serum (FCS).
  6. Ficoll-Hypaque (Amersham-Pharmacia, cat. no. 17-1440-03).
  7. Anti-CD14 mouse monoclonal antibody (Mab) conjugated to phycoerythrin (CD14-PE; Becton Dickinson, cat. no. 347497).
  8. Anti-CD3 mouse Mab conjugated to fluorescein isothiocyanate (CD3-FITC; Becton Dickinson, cat. no. 349201).
  9. 150-cm2 plastic tissue culture plates (e.g., Nunc, cat. no. 168381).
  10. 50-mL conical centrifuge tubes (e.g., Corning, cat. no. 430828).
  11. 5-mL polypropylene round-bottom tubes (Becton Dickinson, cat. no. 35-2063).
  12. Cell scrapers (e.g., Nunc, cat. no. 179707).
  13. 3% (v/v) ultrapure formaldehyde.

2.2.        Materials Required for Preparation of CD8-Depleted, PHA-Activated Donor PBMC

  1. Buffy coat packs from healthy HIV-1-seronegative individuals (obtained from the blood bank).
  2. Supplemented Iscove’s medium (see Subheading 2.1.).
  3. Supplemented RPMI medium (RPMI-1640 medium containing penicillin [100 U/mL], streptomycin [100 pg/mL], L-glutamine [25 pg/mL], and 10% [v/v] heat-inactivated FCS).
  4. Ficoll-Hypaque (Amersham-Pharmacia, cat. no. 17-1440-03).
  5. PBS.
  6. Interleukin (IL)-2 (e.g., Roche, cat. no. 799 068).
  7. PHA (e.g., Murex Diagnostics, cat. no. HA15).
  8. 50-mL conical centrifuge tubes (e.g., Corning, cat. no. 430828).
  9. 15-mL conical tubes (e.g., Corning, cat. no. 430790).
  10. 75-cm2 tissue-culture flasks (e.g., Nunc, cat. no. 156472).
  11. Anti-CD8 magnetic beads (Dynal, cat. no. 111.08).
  12. Magnetic particle concentrator (Dynal, cat. no. 120.21).
  13. Sample mixer (e.g., Dynal, cat. no. 159-03).

2.3. Materials Required for Co-Culture of Patient Monocytes With Donor CD8-Depleted, PHA-Activated PBMC

  1. Supplemented Iscove’s medium (see Subheading 2.1.).
  2. Freshly purified peripheral blood monocytes from HIV-1-infected individuals (from Subheading 3.1.).
  3. CD8-depleted, PHA-activated donor PBMC (from Subheading 3.2.).
  4. IL-2 (e.g., Roche, cat. no. 799 068).
  5. 24-well tissue culture plates (e.g., Costar, cat. no. 3524).
  6. 1.0-mL cryovials (e.g., Nunc, cat. no. 377224).
  7. HIV-1 p24 antigen detection kit (e.g., Perkin Elmer, cat. no. NEK050B001KT).
  8. Amplicor HIV-1 Monitor v1.5 kit (Roche Diagnostics, cat. no. 1118390) (see Note 4).
  1. Methods

3.1.        Isolation of Peripheral Blood Monocytes From PBMC of HIV-1-Infected Individuals

The isolation of peripheral blood monocytes requires (1) the preparation of the PBMC fraction from peripheral blood, (2) the isolation of monocytes from the total PBMC, and (3) testing the purity of the isolated monocytes by flow cytometry.

3.1.1.     Preparation of PBMC Fraction From Peripheral Blood

Peripheral blood mononuclear cells are isolated from the patient blood by first diluting the blood 1:2 with PBS-CMF and gently overlaying 30 mL of the diluted blood onto 15 mL of Ficoll-Hypaque in two 50-mL conical centrifuge tubes. The tubes are then centrifuged at 700g for 20 min at room temperature with the centrifuge brake turned off. Keeping the brake turned off during this step is necessary to ensure that efficient separation of the red and white cell fractions occurs. The buffy coat fraction is then collected into a fresh 50-mL tube and washed once by resuspension in 50 mL of PBS-CMF followed by centrifugation at 500g for 7 min at room temperature. The cells are then washed a further two times by resuspension in 50 mL of PBS-CMF and centrifugation at low speed (300g) for 7 min at room temperature to remove platelets. The resulting PBMC are counted with a hemocytometer, and resuspended at 2 x 107 cells/mL in prewarmed (37°C) supplemented Iscove’s medium.

3.1.2.     Isolation of Monocytes From Total PBMC

The resuspended cells are added to 150-cm2 tissue culture plates at a density of no more than 3 x 108 cells per plate (usually 10 to 15 mL per plate), and incubated for 1 to 2 h in a humidified CO2 incubator at 37°C. The adherence properties of monocytes allows them to remain adherent to the plastic during this step, whereas nonmonocytes will remain nonadherent. After this incubation, plates are examined with an inverted phase contrast microscope to check that they contain adherent cells. The nonadherent cells are removed by repeated gentle washing and aspiration using warm (37°C) PBS. PBS is aspirated using a plastic pipet attached to a suction system. This process is repeated six to eight times, during which the culture plates are repeatedly examined for floating cells using an inverted phase contrast microscope. After a quick wash with cold (4°C) PBS-CMF to remove any remaining PBS containing magnesium and calcium, the adherent monocytes are detached by incubating the cells with 20 mL of cold (4°C) PBS-CMF for 15 min on ice. The cells are gently lifted with a cell scraper and isolated monocytes are collected into 50-mL centrifuge tubes containing 2.5 mL of FCS. The plates are washed twice with cold (4°C) PBS-CMF, and the remaining cells gently lifted and collected into the same 50-mL tubes. A final check under the inverted phase contrast microscope will ensure that plates do not still contain adherent monocytes. The cells within the tube are centrifuged at 500g for 7 min at 4°C. Monocytes are resuspended in 10 mL of supplemented Iscove’s medium and a cell count performed using a hemocytometer.

3.1.3.     Testing the Purity of the Isolated Monocytes by Flow Cytometry

To test the purity of the freshly isolated monocytes, 2 x 105 cells are placed into 5-mL polypropylene round-bottom tubes (one tube for specific Mab, and one tube for control Mab), washed once with cold PBS-CMF, centrifuged at 500g to pellet the cells, and PBS-CMF aspirated, leaving approx 100 q,L. Following this, the cells are incubated with 1 q,g of anti-CD14-PE and anti-CD3-FITC for 30 min on ice. An isotype-matched control Mab conjugated to FITC or PE must be included in each assay. The cells are then washed with cold (4°C) PBS-CMF, fixed with 3% (v/v) ultrapure formaldehyde, and analyzed by flow cytometry.

3.2.        Preparation of CD8-Depleted, PHA-Activated Donor PBMC

The next procedure in the protocol to isolate HIV-1 from peripheral blood monocytes is the preparation of CD8-depleted, PHA-activated donor PBMC that are to be used to co-culture with the patient monocytes. This procedure requires (1) the preparation and PHA-activation of PBMC from buffy coat packs obtained from healthy, HIV-1-seronegative donors, and (2) the depletion of CD8+ cells from the PBMC.

3.2.1.     Preparation of PBMC From Buffy Coat Packs

The buffy coat is diluted 1:2 with PBS, and 30 mL gently overlaid onto 15 mL of Ficoll-Hypaque in each of several (usually four) 50-mL conical centrifuge tubes. Following this step, the method for isolating PBMC from the buffy coat is the same as for isolating PBMC from patient’s peripheral blood, which is outlined in Subheading 3.1.1. The only difference is that after the low-speed centrifugation steps to remove platelets, the cells are counted and resuspended in supplemented RPMI medium at a concentration of 2 x 106 cells/mL, and then added to 75-cm2 tissue culture flasks. The PBMC are then stimulated with PHA (5 pg/mL) and incubated for 3 d in a humidified CO2 incubator at 37 °C (see Note 5).

3.2.2.     Depletion of CD8+ T Cells From PBMC

After activation of the PBMC with PHA, a cell count should be performed using a hemocytometer and PBMCs pooled from different donors at a ratio of 1:1. The cells are centrifuged at 500g for 7 min and resuspended in 5 mL supplemented RPMI medium. The CD8+ T-cells (approx 10% of total PBMC) are depleted using anti-CD8 beads according to the manufacturer’s instructions. First, the anti-CD8 beads are washed by transferring the required number of beads (100 pL of beads per 1 x 107 PBMC) into a 15-mL tube and resuspending them in 1 mL of supplemented RPMI medium. The tube is placed on a magnetic particle concentrator for 3 min and then the medium is gently removed using a sterile transfer pipet. One mL of supplemented RPMI medium is added and the process of washing the beads is repeated. The anti-CD8 beads are mixed with the PBMC suspension in a 15-mL tube and incubated on a rotary sample mixer for 30 min at 4°C. The tube is placed on a magnetic particle concentrator for 3 min and the CD8-depleted PBMCs collected in a new tube. This step should remove >95% of the CD8+ T cells. If desired, a second round of CD8 depletion to remove any remaining CD8+ T cells can be performed but is generally not required. This second time, the PBMCs are incubated with only 10 pL of prewashed beads per 1 x 107 cells, and then the method proceeds as for the first round of depletion. The CD8-depleted PBMCs are transferred into a 15-mL tube, centrifuged at 500g for 7 min, the medium aspirated, and cells resuspended at a concentration of 1 x 106/mL in supplemented Iscove’s medium containing 10 U/mL IL-2.

3.3. Co-Culture of Patient Monocytes With Donor CD8-Depleted, PHA-Activated PBMC

The final procedure in this protocol utilizes the purified cell populations outlined in Subheadings 3.1. and 3.2. in co-culture experiments to isolate replication-competent HIV-1 from the patient monocytes.

The freshly isolated monocytes (described in Subheading 3.1.) are resuspended in supplemented Iscove’s medium at a density of 5 x 105 cells/mL. One mL of this suspension (i.e., 5 x 105 monocytes) is placed into wells of 24-well plates and the cells are allowed to adhere for 1 h at 37°C in a humidified CO2 incubator. This is followed by a thorough wash to remove any residual contaminating nonadherent cells (see Note 6). One mL of the CD8-depleted, PHA-activated PBMCs from HIV-1-negative donors at a concentration of 1 x 106 cells/mL (prepared in Subheading 3.2.) is added to each well containing 5 x 105 monocytes (i.e., at a PBMC to monocyte ratio of 2:1) and the cultures

incubated at 37°C in a humidified CO2 incubator for 3 wk (see Notes 7 and 8). The culture medium is replaced twice weekly. This is done by transferring 1 mL of the culture supernatant into 15-mL conical tubes, centrifuging at 700g for 10 min (to clarify viral supernatant) and resuspending the cell pellet in 1 mL of fresh supplemented Iscove’s medium containing 10 U/mL IL-2 before adding back to the co-cultures. The viral supernatant is filtered through 0.22-q,m filters, and stored in 1-mL cryovials at -70°C. The virus stocks are tested by quantifying HIV-1 p24 antigen (using a commercial p24 kit) or, if levels of HIV-1 are low, by HIV-1 RNA (using the Amplicor HIV-1 Monitor v1.5 kit) according to the manufacturer’s instructions (see Note 4).

  1. Notes
  2. Ethylenediamine tetraacetic acid (EDTA) or acid citrate dextrose (ACD) are suitable anticoagulants that can be used in patient blood samples. The use of heparin is avoided because of the inhibitory effect that heparin has on RT-PCR reactions. It is perhaps unlikely that the detection of HIV-1, produced by cells that have been cultured for several weeks, by RT-PCR will be impeded by the presence of heparin in the initial blood sample. However, its use as an anticoagulant in these studies should nonetheless be avoided if possible.
  3. Procedures described in this chapter involve the use of human blood, cells obtained from HIV-1-seropositive individuals and HIV-1 virus stocks. Appropriate biosafety procedures must be observed while working with human blood, whereas BSL-3 biosafety practices must be performed at all times while handling virus stocks and HIV-1-infected cells.
  4. As an alternative to Iscove’s medium, supplemented RPMI medium may also be used (see Subheading 2.2.). All incubations are performed in a humidified 5% CO2 incubator at 37°C unless otherwise noted. Media should be heated to 37°C, except where specified. All solutions must be endotoxin-free and sterile, and proper sterile techniques are required. Culture supernatants, reagents, and media need to be tested to exclude the presence of endotoxin, as lipopolysaccharide (LPS) contamination has been shown to affect HIV-1 replication in monocyte/ macrophages (13). Each new batch of AB+ human serum should be tested for toxicity and sterility and the ability to support monocyte culture.
  5. In some isolation attempts, very low concentrations of HIV-1 may be recovered; in these cases, the concentration of p24 antigen in culture supernatants may be below the limit of detection of commercial kits. The occurrence of undetectable p24 antigen in culture supernatants is more frequent in isolations from monocytes of patients undergoing HAART with plasma HIV-1 RNA levels below detection, compared to untreated viremic patients or patients failing HAART. In these cases it is advisable to quantify HIV-1 RNA in culture supernatants using the Amplicor HIV-1 Monitor v1.5 kit because of the ultrasensitivity of this assay. However, because of the higher cost associated with measuring HIV-1 RNA, it may be prudent to first test for the presence of HIV-1 by measurement of p24 antigen, and then quantify HIV-1 RNA if necessary.
  6. PBMCs from two different HIV-1-negative donors are required for the most successful amplification of primary HIV-1 isolates. PBMC from different donors are cultured separately for the first 3 d after isolation and then pooled prior to CD8 depletion and incubation with monocytes isolated from HIV-1-infected individuals. It is recommended that at least 1 x 107 PBMCs from each donor are stimulated on the day of isolation.
  7. The purity of the isolated monocytes is very important for these studies, because HIV-1 harbored by any contaminating T-cells may be amplified in the co-culture. Thorough washing to remove nonadherent cells is usually sufficient to minimize T-cell contamination. The use of AB+ human serum instead of FCS enhances the adherence of monocytes to plastic, and adherence may be enhanced further if necessary by increasing the concentration of AB+ human serum from 10% (v/v) to 20% (v/v). The additional adherence step described in Subheading 3.3. increases the purity of the monocyte population to approx 99% (2). The level of T-cell contamination in this purified monocyte population can be determined down to a sensitivity of 1 contaminating T cell in 104 monocytes by analysis of TcR mRNA using RT-PCR (2).
  8. In this protocol care is taken to avoid the presence of LPS in all solutions, media, and media supplements because of the ability of LPS to modulate HIV-1 replication in monocytes/macrophages. However, it should be noted that methods to isolate HIV-1 from patient monocytes employed by other investigators utilize LPS stimulation of the monocytes to activate virus replication (4). We have also found that LPS stimulation of patient monocytes, or stimulation with other agents including M-CSF or tumor necrosis factor (TNF)-a prior to co-culture with CD8-depleted PHA-activated PBMC, may increase the amount of virus isolated, or decrease the number of monocytes required for successful virus isolation. However, successful virus isolation can be achieved in approx 70% of cases without any prior stimulation step (2).
  9.  A common concern during the monocyte-PBMC co-culture is the relatively high density at which the cells are cultured. The co-culture starts at an initial density of 1.5 x 106 cells/mL (ie., 5 x 105 monocytes with 1 x 106 PBMC), but the presence of IL-2 in the culture medium promotes the proliferation of the PBMC during the course of the 3 wk that the cells are cultured. This results in the cell density increasing significantly, and commonly causes the culture medium to turn a yellow shade. In practice, however, this does not appear to have any adverse effect on cell viability nor the ability to successfully isolate HIV-1 from monocytes.


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