TZA, a Sensitive Reporter Cell-based Assay to Accurately and Rapidly Quantify Inducible, Replication-competent Latent HIV-1 from Resting CD4+ T Cells
TZA, 一种基于敏感报告因子细胞学检测方法, 可用于准确快速量化静息CD4+ T细胞中可诱导的且复制能力强的潜伏HV-1    

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Nature Medicine
Jul 2017

 

Abstract

The latent HIV-1 viral reservoir in resting CD4+ (rCD4+) T cells represents a major barrier to an HIV-1 cure. There is an ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this reservoir. However, clinical investigators lack an assay to determine whether or not a decrease in the latent reservoir has been achieved. Therefore, it is critical to develop assays that can reproducibly quantify the reservoir size and changes therein, in participant’s blood during a therapeutic trial. Quantification of the latent HIV viral reservoir requires a highly sensitive, cost-effective assay capable of measuring the low frequency of rCD4+ T cells carrying functional provirus. Preferably, such an assay should be such that it can be adopted for high throughput and could be adopted under conditions for use in large-scale clinical trials. While PCR-based assays are commonly used to quantify pro-viral DNA or intracellular RNA transcript, they cannot distinguish between replication-competent and defective proviruses. We have recently published a study where a reporter cell-based assay (termed TZA or TZM-bl based quantitative assay) was used to quantify inducible replication-competent latent HIV-1 in blood. This assay is more sensitive, cost-efficient, and faster than available technology, including the quantitative viral outgrowth assay or the Q-VOA. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in virally suppressed participants on ART is approximately 70-fold larger than previous estimates. We describe here in detail an optimized method to quantitate latently infected cells using the TZA.

Keywords: TZM-bl cells (TZM-bl细胞), TZA assay (TZA 检测), Latent reservoir (潜伏库), Latent HIV-1 (潜伏 HIV-1 ), Quantification of latent reservoir (潜伏库量化), Inducible virus (可诱导的病毒), Replication competent virus (复制能力强病毒)

Background

The ability to quantitate the latent HIV-1 viral reservoir in a combination ART suppressed individual requires a highly sensitive assay with the ability to measure low frequency of rCD4+ cells carrying functional provirus. This assay should also be cost-effective and adaptable to be high throughput under different conditions and large-scale clinical applications. Currently, the frequency of the infected cells in latent condition is estimated using Poisson Statistics or by maximum likelihood analysis (Cillo et al., 2014; Rosenbloom et al., 2015). Most of these methods including the original Q-VOA method and its adaptations use limited dilution based techniques of PHA simulated CD4+ T cells which either measures HIV-1 protein or RNA via ELISA or quantitative PCR. These are all labor-intensive, time-consuming, expensive, and requires the frequent addition of activated CD4+ T cells as feeders (Chun et al., 1997; Finzi et al., 1999; Siliciano et al., 2003; Siliciano and Siliciano, 2005). There has also been a modified version of Q-VOA assay reported which implements MOLT-4/CCR5 (Laird et al., 2013) cell line for viral expansion instead of activated CD4+ T cells followed by RNA measurement but this is still more time consuming and labor intensive.

We recently reported the development of a sensitive TZM-bl cell-based assay (termed TZA) (Sanyal et al., 2017) to quantify the latent HIV-1 reservoir in blood. The present protocol consists of these main parts (i) isolation of resting T cells from the blood from HIV-1 positive and negative (control) donors (Steps A1-A4); (ii) activation of these resting CD4+ T cells using a strong LRA like Anti-CD3/CD28 (Siliciano and Siliciano, 2004) antibodies and (iii) washing, counting and plating these activated cells on a TZM-bl reporter cells to quantitate replication-competent HIV-1 by measuring β-gal expression to quantify virus induced. This assay utilizes the TZM-bl cell line, which stably expresses CD4, CCR5, and CXCR4, and carries an integrated copy of the β-galactosidase (β-gal) gene under the control of an HIV-1 long terminal repeat (HIV-1 LTR) promoter that allows for the detection of inducible replication-competent HIV-1. By using TZM-bl cells, quantification of replication-competent HIV-1 can be achieved with high sensitivity (Ananworanich and Mellors, 2015). TZA has been used to calculate fraction of inducible latent virus (fPVE) (Cillo et al., 2014; Sanyal et al., 2017). The level of fPVE can be used to screen various LRAs in-vitro as well as help monitor their in-vivo efficacy especially during clinical trials. This assay reveals that the viral reservoir is likely much larger than previously predicted and estimated which can impact the ongoing therapeutic approaches to eradicate HIV-1.

This assay can be adapted and automated to a 384-well format further down the line to enable an efficient screening platform, which will be capable of handling multiple samples simultaneously. This will make it a high-throughput assay system. In addition, because of the low cell requirement in this system, the TZA may also be useful for quantification of replication-competent HIV-1 in the pediatric population as well as estimation of the reservoir in tissues. We are presently developing a protocol for determining the reservoir size in tissues using this assay.

Materials and Reagents

  1. Pipette tips
  2. 15 ml centrifuge tubes (Fisherbrand, catalog number: 07-200-886)
  3. 50 ml centrifuge tubes (Fisherbrand, catalog number: 0553913)
  4. Tissue culture plates, 24-well transparent (Falcon, catalog number: 353047)
  5. White, 96-well plates, sterile with white lids (PerkinElmer, catalog number: 6005181)
  6. EDTA tubes purple tops for blood collection: Vacutainer brand sterile (BD, catalog number: 366643) 
  7. CryoTubeTM vials for freezing cells (Thermo Fischer Scientific, catalog numbers: 153779, 202209, 363401)
  8. Tissue culture plates, 48-well transparent (Falcon, catalog number: 353078)
  9. 5 ml round-bottom polystyrene tubes (Corning, Falcon®, catalog number: 352054)
  10. TZM-bl cells (NIH AIDS Reagent program, catalog number: 8129)
  11. Animal serum complex (Gemini Bio Products, Fetal PlexTM, catalog number: 100-602), storage in a -18 °C freezer before opening bottle and then store at 4 °C for subsequent use
  12. DMSO (Life Technologies Corp, catalog number: 20688)
  13. FBS (HyClone, catalog number: SH3011803)
  14. Beta-Glo Assay System (Promega, catalog number: E4780), storage in a -18 °C freezer
  15. Custom ordered resting CD4+ T cells negative selection kit (Stemcell Technologies, catalog number: 19309VK) with EasySep D Magnetic Particles (Stemcell Technologies, catalog number: 19250), storage at 4 °C
  16. DynabeadsTM Human T-Activator CD3/CD28 (Thermo Fischer Scientific, GibcoTM, catalog number: 11131D)
  17. Clinical grade Human Recombinant Interleukin-2: IL-2 Powder 22 MU VIAL (65483011607), storage in a -18 °C freezer where MU stands for medical units
    Note: Alternatively, human recombinant IL-2 can be purchased from Life Technologies Corp, catalog number: PHC-0023.
  18. Human Recombinant Interleukin-7: IL-7 Premium grade (Miltenyi Biotec, catalog number: 130-095-361), storage in a -18 °C freezer
  19. Efaviranz (EFV) (Cayman Chemical, catalog number: 14412), need to be re-suspended in IMDM media and diluted according to need of the experimental procedure
  20. Flow Monoclonal Antibodies

    Note: You will use 5 μl of each antibody for staining. Use simple Flow staining protocol for staining the cells. This is just done to check for the purity of the resting T cells. Resting T cells are CD25-/CD69-/HLADR- cells.
    1. Mouse Anti-human CD4-AF700 (BD Biosciences, catalog number: 557922)
    2. Mouse Anti-human CD3-V450 (BD Biosciences, catalog number: 560365)
    3. Mouse Anti-human CD25-APC (BD Biosciences, catalog number: 340939)
    4. Mouse Anti-human CD69-FITC (BD Biosciences, catalog number: 555530)
    5. Mouse Anti-human HLADR-PE (BD Biosciences, catalog number: 347367)
    6. Mouse Anti-human CD279-PeCy7 (BD Biosciences, catalog number: 561272) 

    Note: Isotype controls are used at the same concentration as the specific antibody. So, dilutions have to be determined according to the concentration of the matched antibody.
    1. PE-CyTM 7 Mouse IgG1, κ Isotype Control (BD Biosciences, catalog number: 557646)
    2. FITC Mouse IgG1, κ Isotype Control (BD Biosciences, catalog number: 556649)
    3. V450 Mouse IgG1, κ Isotype Control (BD Biosciences, catalog number: 561504)
    4. Alexa Fluor® 700 Mouse IgG1, κ Isotype Control (BD Biosciences, catalog number: 557882)
    5. APC-H7 Mouse IgG1, κ Isotype Control (BD Biosciences, catalog number: 561427)
    6. PE Mouse IgG2a, κ Isotype Control (BD Biosciences, catalog number: 558595) 
  21. Dye-eFluor 506 (Invitrogen, catalog number: 65-0866-14)
  22. DPBS (1x), Dulbecco’s Phosphate-Buffered Saline (Corning Cellgro, catalog number: 21-031-CV), store at 4 °C for use after opening bottle
  23. Dry Ice
  24. HBSS (1x), Hanks Balanced Salt Solution (Thermo Fischer Scientific, GibcoTM, catalog number: 141775-095), store at 4 °C for use after opening bottle
  25. IMDM (1x) (Thermo Fischer Scientific, GibcoTM, catalog number: 12440-053), store at 4 °C for use after opening bottle
  26. Lymphocyte separation medium (Corning, catalog number: 25-072-CV), store at 4 °C for use after opening bottle
  27. Penicillin/Streptomycin (Thermo Fischer Scientific, GibcoTM, catalog number: 15140-122), store at -18 °C freezer before opening bottle and then store at 4 °C for subsequent use
  28. RPMI 1640 (1x) with L-glutamine and 25 mM HEPES (Corning Cellgro, catalog number: 10-041-CV)
  29. 0.05% Trypsin EDTA (1x) (Thermo Fischer Scientific, GibcoTM, catalog number: 25300-120), store at -20 °C freezer before opening bottle and then store at 4 °C for subsequent use
  30. RoboSepTM Buffer (Stemcell Technologies, catalog number: 20104)
  31. Formalin
  32. 10% RPMI (see Recipes)
  33. 10% IMDM (see Recipes)

Note: This procedure can be done in a BSL2+ lab following safety procedures involving wearing a closed lab coat and double gloves. The blood and processing should all be done in a bio-safety hood with proper airflow.

Equipment

  1. Tissue culture flask vented caps, 70 ml (Falcon, catalog number: 353109)
  2. Tissue culture flask vented caps, 250 ml (Falcon, catalog number: 353110)
  3. Pipettes
  4. Rocker
  5. Incubator (Thermo Electron Corporation, NAPCO SERIES 8000 WJ, CO2 Incubator) 
  6. Centrifuge: Sorvall Legend RT+ Centrifuge (Thermo Scientific)
  7. Freezer
  8. Luminometer (Promega GloMax Navigator, GM2000)
  9. LN2 freezer
  10. Magnets (Stem Cell Technologies, catalog number: 96290)
  11. Light Microscope
  12. 4 °C refrigerator

  13. Flow cytometer (BD, model: LSR II)
  14. Water Bath (Fisher Scientific, model: 2223)
  15. Haemocytometer 0.100 mm deep (Hausser Scientific)

Software

  1. FlowJo software (Tree Star)
  2. BD FACSDivaTM software (BD Biosciences)
  3. IUPMStats v1.0 Infection Frequency Calculator (Online) http://silicianolab.johnshopkins.edu/
  4. Microsoft Excel

Procedure

Notes:

  1. In order to get enough cells to keep the control for sets of experiment same, we leukophoresied control HIV-1 negative donors and also participants of the study, and froze those cells down at -140 °C. 
  2. The TZA protocol may be adapted for fresh as well as frozen PBMCs.
  3. This procedure can be done in a BSL2+ lab following safety procedures involving wearing a closed lab coat and double gloves. The blood and processing should all be done in a bio-safety hood with proper airflow.

  1. Isolation of rCD4+ T cells from patients and controls
    1. Collect blood from participants in EDTA tubes. This is usually done in the hospital by nurses and then sent over for processing. 
    2. Dilute the blood in DPBS (1:2). The total volume in a 40 ml tube should be 10 ml blood, 20 ml DPBS. 
    3. Then use 10 ml of lymphocyte separation medium or ficoll to separate the layers of cells in the blood. This is done by putting the pipette with ficoll at the bottom of the 50 ml tubes and slowly pushing the liquid in and a layer of clear ficoll will be at the bottom of the tube. The total volume of liquid will not become 50 ml in the tube.
    4. Every reagent should be at room temperature before use. 
    5. Spin this in a centrifuge at 2,000 rpm (448 x g) for 20 min to get distinct PBMC layer which will appear in between the ficoll layer and the DPBS and serum mixture. RBC will appear at the bottom of the tube. 
    6. Collect the PBMCs layer and wash it again with DPBS spinning in a centrifuge at 1,000 rpm (112 x g) for 10 min.
    7. Discard the supernatant and collect the pellet (PBMCs). At this point you may either carry on with the rest of the procedure or freeze your cells in a freezing media (10% DMSO in FBS) and store them at -140 °C for long term storage. 
    8. Count the cells recovered and then use resting CD4+ T cell Isolation kit to separate out the rCD4+ T cells from the PBMCs. It is always a good idea to not run less than 50 million cells at once since the yield in this kit is not high. It is a high purity but a low recovery kit. Also do not do two 5 min separation for less than 100 million cells, rather do one-time separation through the magnet. The details of the separation are listed in the protocol that is sent with the separation kit.
    9. Count the cells one more time before proceeding to the next step.
    Notes: 
    1. You will have to take some cells out and stain for resting T cells. This is important since it helps you determine whether the cells selected are purely resting T cells. 
    2. If we are working with rCD4+ T cells, you can expect about 5 to 15 million rCD4+T cells from thawing about 100 million PBMCs for patients and about 20-25 million rCD4+ T cells from 100 million normal control PBMCs. This will not change irrespective of fresh or frozen cells. 
    3. For thawing frozen cells, put the frozen cells dropwise slowly while moving the tube in circular motion in a 15 ml tube containing 10 ml of 10% IMDM. Then spin the cells down at 1,000 rpm (112 x g) for 10 min to wash it off from the freezing media.
    4. Follow the same Steps A1-A9 for cells from negative control participants. For every experiment you will need to do a control PBMCs with which the cells will be compared.

  2. Assess the purity of resting T cells using Flow cytometry staining. (Figure 1)
    1. First, transfer cells (2 x 105 to 2.5 x 105) into a tube and centrifuge at 400 x g at 4 °C for 5 min and remove the supernatant. 
    2. Perform the viability staining: resuspend the cell pellets with 250 μl of DPBS and 1 μl of Viability Dye-eFluor506. Incubate for 30 min at 4 °C.
    3. Wash the cells with DPBS and centrifuge the plate at 400 x g at 4 °C for 5 min and remove the supernatant. 

    4. Resuspend the cell pellets first in 100 μl of DPBS and then put this in a mixture of the antibodies in 5 ml round-bottom polystyrene tubes with 5 μl of each antibody listed in Materials and Reagents and incubate for 30 min at 4 °C. 
    5. Wash the cells at 400 x g at 4 °C for 10 min and remove the supernatant. Do this two times.
    6. Fix the cells in 1% Formalin to run your flow. You can run your flow in plates or tubes, but we prefer tubes. 
    7. First gate on singlet population and them on lymphocytes. Follow this up by gating on the live population. In the live population of cells look specifically at the CD4+/CD3+ cells as these are the T cells.
    8. Then the rest of the gating is from the CD4+/CD3+ cells. Gate them for CD69, HLADR and CD25, which are all activation markers. PD1 is used for gating by us for determining cell death signal but it is not important to use if your follow up experiments do not require it.
    Note: You can choose antibodies of your choice for activation marker but we used CD-25, HLA-DR, CD-69 as activation markers. We also used PD1 as a secondary marker since activated cells have lower PD1. You will be looking for cells which are negative for CD-25, HLA-DR, CD-69. We gated on CD4 and CD3 positive cells since we were looking for resting T cells. 95 % or more purity if good for the resting T cells.


    Figure 1. Purity of resting T cells: the resting T cells should be CD25/HLADR/CD69-CD3+/CD4+ cells. A. Gating on singlet population that is live and then to CD4+/CD3+ cells. B. Gating on CD3+/CD4+ cells to determine their activation status (CD25+/CD69+/HLADR+).

  3. Activation of resting T cells
    1. For the next step you will need to first stimulate the cells for activation. Use Anti-CD3/CD28 Dynabeads in a concentration of 12.5 μl of the beads per million cells you put in the culture and they will stay in the culture together for 6 days till you do the next phase of the experiment. 
    2. Wash the beads before using them for activation. Put beads in a 5 ml polystyrene tube, put RoboSepTM/EasySepTM buffer (1 ml) and put in magnet for 5 min. Then after decanting the liquid, resuspend the beads in 10% IMDM culture media exactly the amount you took out. For example, if you took out 100 μl of beads for 8 million cells, you wash the beads, then resuspend it back in 100 μl of 10% IMDM media and then out it in the final activation cocktail. (Figure 2).
    3. The culture for stimulation is usually placed in 1.0 million cells/ml of media. Each well in a 24-well plate will usually have 2 million resting T cells, 2 ml of 10% IMDM media and 25 μl of resuspended AntiCD3/CD28 Dynabead. After re-suspending the cells in media containing beads, put 300 nm of EFV per ml of the media to prevent cell-cell infection. Always prepare this fresh according to the amount of cell yield. 
    4. For best stimulation, the cells can be concentrated in smaller wells (48-well plates with 1 million/ml cells in one well) and then transferred to a 24-well plate next day. Plate 2 million/2 ml of cells per well in a 24-well plate (Figure 2).
    5. After 24 h post activation, add 10 μl of 105 units/ml IL-2 and 10 μl of 105 units of IL-7per million cells as the secondary signaling cytokines to maintain the cells and help them proliferate. Do not remove the Anti-CD3/CD28 Dynabeads.
    6. The cultures need to be monitored every day and if there is overcrowding which is usually when the media becomes very yellow in color and the cell in each well is more than 4 million cells. Put more base media in the wells and split it into two wells in such cases. 
    7. The base media during splitting should always contain IL-2, IL-7 (on Day 2) and EFV along with 10% IMDM. No beads are required during addition of media to the cells in culture (Figure 2).
    8. These cells stay in culture for a total of 6 days (Figure 2).


      Figure 2. Processing latent cells for activation

  4. Harvesting, plating and reading Beta-gal activity of TZM-bl cells
    1. On Day 5 of the culture (one day before the T-cells are taken out of culture), seed 96-Well white PerkinElmer plates with 5 x 104 cells/well and rest of the plate with 3 x 104 cells/well of TZM-bl cells totaling 64-wells. Use 10% RPMI for media since TZM-bl cells grow in 10% RPMI. The total volume of media in each well should be 200 μl. Keep this overnight so that cells adhere to the bottom of wells by next day.
      Note: Don’t maintain TZM-bl cells in culture for more than 3 weeks and split the cells every three days. When splitting, do not use more than 1 million/10 ml of 10% RPMI. TZM-bl cells grow in 10% RPMI in a 250 ml vented Falcon flask and need to be trypsinized when ready to split. When a frozen cell culture is started, make sure it is done in a 70 ml Falcon vented flask and then expanded over time.
    2. On Day 6 from the start of culture, pull the activated T cells from the 24-well plates in 15 ml conical tubes and centrifuge at 1,000 rpm (112 x g) for 10 min to pellet the cells.
      Note: You can save the supernatants for RNA if you want to compare the RNA yield of these activated latently infected cells. This can be frozen in -80 °C freezer. You will have to count the cells and wash them two more times with DPBS. You can also save 0.5 million cells for DNA quantification from each patient. For this, just count the cells, put them in Eppendorf tubes, spin them down at 1,000 rpm (112 x g) for 10 min and pellet the cells. Then directly just freeze these pellets in -80 °C freezer. This is used if you want to calculate the fraction of provirus induced for the particular participant.
    3. With the rest of the activated T-cells, start to make a dilution series. Make a 4 fold dilution (1:3) starting from 1.25 x 105 cells/well in the highest dilution downward. (You can start as low as 6 x 104 cells/well on the top for this).
      Note: If you are starting from 1.25 x 105 cells/well in the first row, each well in the top row (8 wells) should have 1.25 x 105 cells/well in 200 μl of 10% RPMI. The second row will have 1.25 x 105/4 cells in all eight wells and so on downwards for five more dilutions. This should give you a total of 6 dilutions (Figure 3).
    4. After making the dilution series in 15 ml tubes, the media from the TZM-bl cell plates will have to be removed by aspiration before the patient cells are plated on it. Do not do it before you start to plate as it dries out the cells in the plate. The seventh row is usually not used and the eighth row is fresh media on the TZM-bl cells. (Figure 3).
    5. All the dilutions will have to be in 10% RPMI.


      Figure 3. Plating cells for reading in 96 wells using TZM-bl cells

    6. Keep this plate in a 37 °C incubator for 48 h after which it has to be read. This reading can be taken anywhere between 48 and 72 h.
    7. Take the plates out and aspirate the media out without disturbing the cells. 
    8. Put back DPBS in the wells with multi-channel pipettes and lightly wash and take the washout and discard. Do this two times. Do not be very rigorous while washing as this could take the cells off from the plates.
    9. Put 100 μl of Promega Beta-Glo reagent on each well and incubate in the dark for 45 min-1 h. Try to do all these steps in very little light. This reagent is extremely sensitive to light.
    10. Then read in a Promega Glomax machine to get the light units following software prompt. This is an automated machine where you will have to select any of the cell-titer protocols, which will be able to read your plate. Then you can name and save the plate reads as excel files.

  5. Analysis of readings and IUPM calculation
    1. After the reading is taken, transport the Excel file out to analyze these results. First, calculate a mean (average) of the bottom media control row and deduct this value from all the other individual readings in the plate. This will be your blank. This should be done for both the control HIV-1 negative plate as well as the HIV-1 positive patient plate. This will also assure that you ideally have similar conditions for both the test and control cells. 
    2. In the control plate, which has the control HIV-1 negative T cells, calculate the average of each row with all the eight wells for each dilution. Do this for each dilution. Also, calculate the standard deviation for each dilution.
    3. Add the average + 2 times the standard deviation (avg. + 2 SD) for each dilutions. This will give you a value for each dilution in the control well based on which you will calculate the positive wells in the test plate.
    4. After you have the value of (avg. + 2 SD) for each dilution in the control plate, subtract it from each individual reading of wells for that particular dilution in the participant plate.
    5. This will give you some wells with positive and some with negative values. The positive values can vary from 0/8 to 8/8.
    6. Put these readings in the algorithm developed by Silicianos lab http://silicianolab.johnshopkins.edu/ (Rosenbloom et al., 2005) and calculate the Infectious unit per million cells or IUPM. 
    7. For DNA copies/million cells, use a standard DNA q-PCR (Cillo et al., 2014) for integrated provirus in the cells and calculate the fraction of provirus that can be induced by the stimulation to form replication competent virus. This is calculated with the formula: fPVE = IUPM/DNA per million cells x 100.

Data analysis

  1. For the purity estimation of the resting T cells, samples are acquired on an LSR II flow cytometer using FACSDiVaTM software before putting the cells in culture for activation. Flow cytometry data are analyzed using FlowJo to gate, quantify, and analyze the resting T cell population. 
  2. For the IUPM calculations, the maximum likelihood estimate was applied to determine the infectious unit per million (IUPM) cells for the TZA assay using online software, available at http://silicianolab.johnshopkins.edu/, developed by Rosenbloom et al. (2015).

Notes

During the entire protocol, you have to make sure that you are doing exactly the same things for another set of cells which are HIV-1 negative or control cells. If you do not have your controls, your experiments will not work. You will have to make sure even the control cells are stimulated the same way as the patient cells so that ideally you have similar conditions for both the test and control cells.

Recipes

  1. 10% RPMI
    RPMI 1640
    1x medium with 10% Animal Serum Complex and 1% Penicillin-streptomycin
    Keep sterile and at room temperature during experiment and store at 4 °C
  2. 10% IMDM
    Mix 450 ml of IMDM w/L-glutamine with 50 ml FBS and 1% Penicillin-streptomycin (5 ml) from storage at -20 °C
    This will make a final volume of 10% FBS in IMDM
    This should always be stored at 4 °C for long term storage

Acknowledgments

This protocol was adapted from our publication (Sanyal et al., 2017). We thank the ex-members of the lab, Lori Caruso, Deena Ratner and Ming Ding (University of Pittsburgh, Pittsburgh) for their technical assistance; N. Sluis-Cremer for the DNA measurement and study design inputs; P. Tarwater (University of Texas, El Paso) and C. Shen (University of Pittsburgh, Pittsburgh) for statistical consultation and development of the assay statistics; W. Buchanan (University of Pittsburgh, Pittsburgh) for recruitment of the Multicenter AIDS Cohort Study participants for the study; and all the participants of the Pittsburgh portion of the MACS for donating blood for this study.
  This work was supported by NIH grants R21AI138716 (Phalguni Gupta), U01-AI35041 (Charles R. Rinaldo.), R21-AI119117 (Nicolas P. Sluis-Cremer and NIH Fogarty training grant fellowship D43TW010039 (Phalguni Gupta).

Competing interests

The authors declare no conflict of interest.

Ethics

Blood was collected and handled according to protocols approved by the University of Pittsburgh institutional review board and was pulled from the patient in the hospital by nurses. The participants both HIV-1 negative and positive were recruited from the Multicenter AIDS Cohort Study.
  Written consent was obtained from all the participants and the study was explained to them in simple terms before proceeding with it.

References

  1. Ananworanich, J. and Mellors, J. W. (2015). How much HIV is alive? The challenge of measuring replication competent HIV for HIV cure research. EBioMedicine 2(8): 788-789.
  2. Chun, T. W., Carruth, L., Finzi, D., Shen, X., DiGiuseppe, J. A., Taylor, H., Hermankova, M., Chadwick, K., Margolick, J., Quinn, T. C., Kuo, Y. H., Brookmeyer, R., Zeiger, M. A., Barditch-Crovo, P. and Siliciano, R. F. (1997). Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387(6629): 183-188. 
  3. Cillo, A. R., Sobolewski, M. D., Bosch, R. J., Fyne, E., Piatak, M., Jr., Coffin, J. M. and Mellors, J. W. (2014). Quantification of HIV-1 latency reversal in resting CD4+ T cells from patients on suppressive antiretroviral therapy. Proc Natl Acad Sci U S A 111(19): 7078-7083. 
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  5. Laird, G. M., Eisele, E. E., Rabi, S. A., Lai, J., Chioma, S., Blankson, J. N., Siliciano, J. D. and Siliciano, R. F. (2013). Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay. PLoS Pathog 9(5): e1003398. 
  6. Rosenbloom, D. I., Elliott, O., Hill, A. L., Henrich, T. J., Siliciano, J. M. and Siliciano, R. F. (2015). Designing and interpreting limiting dilution assays: General principles and applications to the latent reservoir for human immunodeficiency virus-1. Open Forum Infect Dis 2(4): ofv123. 
  7. Sanyal, A., Mailliard, R. B., Rinaldo, C. R., Ratner, D., Ding, M., Chen, Y., Zerbato, J. M., Giacobbi, N. S., Venkatachari, N. J., Patterson, B. K., Chargin, A., Sluis-Cremer, N. and Gupta, P. (2017). Novel assay reveals a large, inducible, replication-competent HIV-1 reservoir in resting CD4+ T cells. Nat Med 23(7): 885-889. 
  8. Siliciano, J. D., Kajdas, J., Finzi, D., Quinn, T. C., Chadwick, K., Margolick, J. B., Kovacs, C., Gange, S. J. and Siliciano, R. F. (2003). Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med 9(6): 727-728. 
  9. Siliciano, J. D. and Siliciano, R. F. (2005). Enhanced culture assay for detection and quantitation of latently infected, resting CD4+ T-cells carrying replication-competent virus in HIV-1-infected individuals. Methods Mol Biol 304: 3-15. 
  10. Siliciano, J. D. and Siliciano, R. F. (2004). A long-term latent reservoir for HIV-1: discovery and clinical implications. J Antimicrob Chemother 54(1): 6-9.

简介

摘要:静息CD4 + (rCD4 + )T细胞中潜伏的HIV-1病毒储库代表了HIV-1治愈的主要障碍。目前正在努力确定消除或减少该储层尺寸的治疗方法。然而,临床研究人员缺乏一种测定方法来确定是否已经实现了潜在储层的减少。因此,在治疗试验期间,在参与者的血液中开发可重复量化储库大小和其中的变化的测定是至关重要的。潜在HIV病毒储库的定量需要高灵敏度,成本有效的测定,能够测量携带功能性原病毒的rCD4 + T细胞的低频率。优选地,这样的测定应该使得它可以用于高通量并且可以在用于大规模临床试验的条件下采用。虽然基于PCR的测定通常用于定量前病毒DNA或细胞内RNA转录物,但它们不能区分复制能力和有缺陷的原病毒。我们最近发表了一项研究,其中使用基于报告基因细胞的测定(称为基于TZA或TZM-bl的定量测定)来定量血液中诱导型复制能力的潜伏HIV-1。该测定比现有技术更灵敏,更具成本效益且更快,包括定量病毒生长测定或Q-VOA。使用该测定,我们显示在病毒抑制的ART参与者中诱导型潜伏HIV-1储库的大小比先前估计大约70倍。我们在此详细描述了使用TZA定量潜伏感染细胞的优化方法。


背景:在组合ART抑制个体中定量潜伏HIV-1病毒储库的能力需要高度灵敏的测定,其具有测量携带功能性原病毒的rCD4 + 细胞的低频率的能力。该测定还应该是成本有效的并且适合于在不同条件和大规模临床应用下的高通量。目前,使用泊松统计或最大似然分析估计潜伏状态下感染细胞的频率(Cillo et al。,2014; Rosenbloom et al。,2015) 。这些方法中的大多数包括最初的Q-VOA方法及其适应性使用PHA模拟的CD4 + T细胞的有限稀释技术,其通过ELISA或定量PCR测量HIV-1蛋白或RNA。这些都是劳动密集型,耗时,昂贵,并且需要频繁添加活化的CD4 + T细胞作为饲养者(Chun et al。,1997; Finzi < em> et al。,1999; Siliciano et al。,2003; Siliciano and Siliciano,2005)。还报道了Q-VOA测定的修改版本,其实施了用于病毒扩增的MOLT-4 / CCR5(Laird 等人,2013)细胞系而不是活化的CD4 + T细胞,然后进行RNA测量,但这仍然是更耗时和劳动密集的。

我们最近报道了开发一种敏感的TZM-bl细胞分析(称为TZA)(Sanyal et al。,2017)来量化血液中潜伏的HIV-1储库。本方案由以下主要部分组成:(i)从HIV-1阳性和阴性(对照)供体血液中分离静息T细胞(步骤A1-A4); (ii)使用抗CD3 / CD28(Siliciano和Siliciano,2004)抗体等强LRA激活这些静息CD4 + T细胞,以及(iii)在这些抗体上洗涤,计数和接种这些活化细胞。通过测量β-gal表达来定量病毒诱导的TZM-bl报告细胞以定量具有复制能力的HIV-1。该试验利用TZM-bl细胞系,该细胞系稳定表达CD4,CCR5和CXCR4,并携带在HIV-1长末端重复(HIV-)控制下的β-半乳糖苷酶(β-gal)基因的整合拷贝。 1 LTR)启动子,允许检测诱导型复制能力的HIV-1。通过使用TZM-bl细胞,可以高灵敏度地实现具有复制能力的HIV-1的定量(Ananworanich和Mellors,2015)。 TZA已被用于计算诱导型潜伏病毒(fPVE)的分数(Cillo et al。,2014; Sanyal et al。,2017)。 fPVE的水平可用于筛选体外的各种LCA ,以及帮助监测其体内效力,尤其是在临床试验期间。该分析显示,病毒库可能比先前预测的大得多,并且估计可能影响正在进行的根除HIV-1的治疗方法。

该测定可以进一步调整并自动化为384孔格式,以实现有效的筛选平台,该平台能够同时处理多个样品。这将使其成为高通量测定系统。此外,由于该系统中细胞需求量低,TZA也可用于量化儿科人群中具有复制能力的HIV-1以及估计组织中的储库。我们目前正在开发一种使用该测定法确定组织中储库大小的方案。

关键字:TZM-bl细胞, TZA 检测, 潜伏库, 潜伏 HIV-1 , 潜伏库量化, 可诱导的病毒, 复制能力强病毒

材料和试剂

  1. 移液器吸头
  2. 15毫升离心管(Fisherbrand,目录号:07-200-886)
  3. 50毫升离心管(Fisherbrand,目录号:0553913)
  4. 组织培养板,24孔透明(Falcon,目录号:353047)
  5. 白色,96孔板,无菌白色盖子(PerkinElmer,目录号:6005181)
  6. EDTA管紫色上衣用于采血:Vacutainer牌无菌(BD,目录号:366643)&nbsp;
  7. 用于冷冻细胞的CryoTube TM 小瓶(Thermo Fischer Scientific,目录号:153779,202209,363401)
  8. 组织培养板,48孔透明(Falcon,目录号:353078)
  9. 5毫升圆底聚苯乙烯管(Corning,Falcon ®,目录号:352054)
  10. TZM-bl细胞(NIH AIDS Reagent program,目录号:8129)
  11. 动物血清复合物(Gemini Bio Products,Fetal Plex TM ,目录号:100-602),在开瓶前储存于-18°C冰箱中,然后储存在4°C以备后续使用
  12. DMSO(Life Technologies Corp,目录号:20688)
  13. FBS(HyClone,目录号:SH3011803)
  14. Beta-Glo分析系统(Promega,目录号:E4780),储存在-18°C冰箱中
  15. 定制有序的静息CD4 + T细胞阴性选择试剂盒(Stemcell Technologies,目录号:19309VK),带EasySep D磁性颗粒(Stemcell Technologies,目录号:19250),储存于4°C
  16. Dynabeads TM 人类T-Activator CD3 / CD28(Thermo Fischer Scientific,Gibco TM ,目录号:11131D)
  17. 临床级人类重组白细胞介素-2:IL-2粉末22 MU VIAL(65483011607),储存在-18°C冰箱中,其中MU代表医疗单位
    注意:或者,人重组IL-2可从Life Technologies Corp购买,目录号:PHC-0023。
  18. 人重组白细胞介素-7:IL-7 Premium级(Miltenyi Biotec,目录号:130-095-361),储存于-18°C冰箱中
  19. Efaviranz(EFV)(Cayman Chemical,目录号:14412)需要重新悬浮在IMDM介质中并根据实验程序的需要进行稀释
  20. 流式单克隆抗体
    注意:您将使用5μl的每种抗体进行染色。使用简单的Flow染色方法染色细胞。这样做是为了检查静息T细胞的纯度。静息T细胞是CD25- / CD69- / HLADR-细胞。
    1. 小鼠抗人CD4-AF700(BD Biosciences,目录号:557922)
    2. 小鼠抗人CD3-V450(BD Biosciences,目录号:560365)
    3. 小鼠抗人CD25-APC(BD Biosciences,目录号:340939)
    4. 小鼠抗人CD69-FITC(BD Biosciences,目录号:555530)
    5. 小鼠抗人HLADR-PE(BD Biosciences,目录号:347367)
    6. 小鼠抗人CD279-PeCy7(BD Biosciences,目录号:561272)&nbsp;

    注意:同种型对照的使用浓度与特异性抗体相同。因此,必须根据匹配抗体的浓度确定稀释度。
    1. PE-Cy TM 7小鼠IgG 1 ,κ同型对照(BD Biosciences,目录号:557646)
    2. FITC小鼠IgG 1 ,κ同型对照(BD Biosciences,目录号:556649)
    3. V450小鼠IgG 1 ,κ同型对照(BD Biosciences,目录号:561504)
    4. Alexa Fluor ® 700小鼠IgG 1 ,κ同型对照(BD Biosciences,目录号:557882)
    5. APC-H7小鼠IgG 1 ,κ同型对照(BD Biosciences,目录号:561427)
    6. PE小鼠IgG 2a ,κ同型对照(BD Biosciences,目录号:558595)&nbsp;
  21. Dye-eFluor 506(Invitrogen,目录号:65-0866-14)
  22. DPBS(1x),Dulbecco磷酸盐缓冲盐水(Corning Cellgro,目录号:21-031-CV),储存于4°C,开瓶后使用
  23. 干冰
  24. HBSS(1x),Hanks平衡盐溶液(Thermo Fischer Scientific,Gibco TM ,目录号:141775-095),储存于4°C,用于开瓶后
  25. IMDM(1x)(Thermo Fischer Scientific,Gibco TM ,目录号:12440-053),储存于4°C,开瓶后使用
  26. 淋巴细胞分离培养基(Corning,目录号:25-072-CV),储存于4℃,开瓶后使用
  27. 青霉素/链霉素(Thermo Fischer Scientific,Gibco TM ,目录号:15140-122),在开瓶前储存于-18°C冰箱,然后储存在4°C以备后续使用
  28. 含有L-谷氨酰胺和25 mM HEPES的RPMI 1640(1x)(Corning Cellgro,目录号:10-041-CV)
  29. 0.05%胰蛋白酶EDTA(1x)(Thermo Fischer Scientific,Gibco TM ,目录号:25300-120),在开瓶前储存在-20°C冰箱中,然后储存在4°C以备后续使用
  30. RoboSep TM 缓冲液(Stemcell Technologies,目录号:20104)
  31. 福尔马林
  32. 10%RPMI(见食谱)
  33. 10%IMDM(见食谱)

注意:此程序可在BSL2 +实验室中按照安全程序进行,包括穿着封闭的实验室外套和双层手套。血液和处理都应该在生物安全罩中进行,并保持适当的气流。

设备

  1. 组织培养瓶通气帽,70 ml(Falcon,目录号:353109)
  2. 组织培养瓶通气帽,250 ml(Falcon,目录号:353110)
  3. 移液器
  4. 摇臂
  5. 培养箱(Thermo Electron Corporation,NAPCO SERIES 8000 WJ,CO 2 Incubator)&nbsp;
  6. 离心机:Sorvall Legend RT +离心机(Thermo Scientific)
  7. 冰箱
  8. 发光计(Promega GloMax Navigator,GM2000)
  9. LN2冷冻柜
  10. 磁铁(干细胞技术,目录号:96290)
  11. 光学显微镜
  12. 4°C冰箱
  13. 流式细胞仪(BD,型号:LSR II)
  14. 水浴(Fisher Scientific,型号:2223)
  15. 血细胞计数器深度为0.100 mm(Hausser Scientific)

软件

  1. FlowJo软件(Tree Star)
  2. BD FACSDiva TM 软件(BD Biosciences)
  3. IUPMStats v1.0感染频率计算器(在线) http://silicianolab.johnshopkins.edu/
  4. Microsoft Excel

程序

注意:

  1. 为了获得足够的细胞以保持对实验组的控制相同,我们对HIV-1阴性供体以及该研究的参与者进行白细胞介导,并在-140°C下将这些细胞冷冻。
  2. TZA协议可适用于新鲜和冷冻PBMC。

  1. 从患者和对照中分离rCD4 + T细胞
    1. 从EDTA管的参与者收集血液。这通常由护士在医院完成,然后送去处理。&nbsp;
    2. 用DPBS稀释血液(1:2)。 40毫升管中的总体积应为10毫升血液,20毫升DPBS。&nbsp;
    3. 然后使用10ml淋巴细胞分离培养基或ficoll分离血液中的细胞层。这是通过将带有ficoll的移液管放在50ml管的底部并缓慢推入液体并在管的底部放置一层透明的ficoll来完成的。管中的液体总量不会达到50毫升。
    4. 每种试剂在使用前都应处于室温下。&nbsp;
    5. 将其在离心机中以2,000rpm(448 x g )旋转20分钟,以得到不同的PBMC层,其将出现在ficoll层与DPBS和血清混合物之间。 RBC将出现在试管的底部。&nbsp;
    6. 收集PBMC层并再次用离心机中的DPBS旋转以1,000rpm(112 x g )洗涤10分钟。
    7. 弃去上清液并收集沉淀(PBMC)。此时,您可以继续进行剩余的程序,或将细胞冷冻在冷冻培养基(FBS中的10%DMSO)中,并将其储存在-140°C下长期储存。&nbsp;
    8. 计数回收的细胞,然后使用静息CD4 + T细胞分离试剂盒从PBMC中分离出rCD4 + T细胞。由于此套件的产量不高,因此不要一次运行少于5000万个电池,这总是一个好主意。它是一种高纯度但低回收率的试剂盒。对于少于1亿个电池,也不要进行两次5分钟的分离,而是通过磁铁进行一次性分离。分离的详细信息列在与分离试剂盒一起发送的协议中。
    9. 在继续下一步之前,再次计算细胞数。
    注意:&NBSP;
    1. 你必须取出一些细胞并染色静息T细胞。这很重要,因为它可以帮助您确定所选细胞是否是纯静息T细胞。&nbsp;
    2. 如果我们正在使用rCD4 + T细胞,您可以预期大约500万到1500万rCD4 + 为患者解冻约1亿个PBMC的T细胞和约20-25百万个rCD4 + 来自1亿个正常对照PBMC的T细胞。无论新鲜细胞还是冷冻细胞,这都不会改变。
    3. 为了解冻冷冻细胞,将冷冻细胞缓慢滴入,同时在含有10ml 10%IMDM的15ml管中以圆周运动移动管。然后以1,000 rpm(112 x g )旋转细胞10分钟,将其从冷冻培养基中洗掉。
    4. 对于来自阴性对照参与者的细胞,遵循相同的步骤A1-A9。对于每个实验,您都需要进行对照PBMC,并与其进行比较。

  2. 使用流式细胞术染色评估静息T细胞的纯度。 (图1)
    1. 首先,将细胞(2 x 10 5 至2.5 x 10 5 )转移到试管中,并在4℃下以400 xg 离心5分钟分钟并去除上清液。&nbsp;
    2. 进行活力染色:用250μlDPBS和1μlViabilityDye-eFluor506重悬细胞沉淀。在4°C孵育30分钟。
    3. 用DPBS洗涤细胞,并在4℃下以400μL离心平板5分钟并除去上清液。
    4. 首先将细胞沉淀重悬于100μlDPBS中,然后将其置于5 ml圆底聚苯乙烯管中的抗体混合物中,材料和试剂中列出5μl每种抗体,并在4°C孵育30分钟。 ;
    5. 将细胞在400℃下洗涤,在4℃下洗涤10分钟并除去上清液。这样做两次。
    6. 将细胞固定在1%福尔马林中以运行流动。您可以在板材或管材中运行流动,但我们更喜欢管材。&nbsp;
    7. 关于单重群体和淋巴细胞的第一道门。通过对现场人口进行控制来实现这一目标。在活细胞群中,特异性地观察CD4 + / CD3 + 细胞,因为它们是T细胞。
    8. 然后其余的门控来自CD4 + / CD3 + 细胞。为CD69,HLADR和CD25控制它们,它们都是激活标记。 PD1用于我们用于确定细胞死亡信号的门控,但如果您的后续实验不需要它,则使用它并不重要。
    注意:您可以选择您选择的抗体作为激活标记,但我们使用CD-25,HLA-DR,CD-69作为激活标记。我们还使用PD1作为次要标记物,因为活化的细胞具有较低的PD1。您将寻找CD-25,HLA-DR,CD-69阴性的细胞。我们对CD4和CD3阳性细胞进行门控,因为我们正在寻找静息T细胞。如果对静息T细胞有益,则纯度为95%或更高。


    图1.静息T细胞的纯度:静息T细胞应为CD25 / HLADR / CD69 - CD3 + / CD4 + 细胞。 A.对有效的单重群体进行门控,然后进入CD4 + / CD3 + 细胞。 B.对CD3 + / CD4 + 细胞进行门控以确定其活化状态(CD25 + / CD69 + / HLADR + )。

  3. 静息T细胞的激活
    1. 对于下一步,您需要首先刺激细胞进行激活。使用抗CD3 / CD28 Dynabeads,浓度为每百万个细胞中12.5μl珠子放入培养物中,它们将一起在培养物中培养6天,直到您进行下一个实验阶段。&nbsp;
    2. 在使用它们进行活化之前清洗珠子。将珠子放入5ml聚苯乙烯管中,放入RoboSep TM / EasySep TM 缓冲液(1ml)并放入磁铁中5分钟。然后在倾析液体后,将珠子重新悬浮在10%IMDM培养基中,与您取出的量完全重复。例如,如果您为800万个细胞取出100μl珠子,则清洗珠子,然后将其重悬于100μl10%IMDM培养基中,然后在最终激活混合物中将其重新吸出。 (图2)。
    3. 刺激培养物通常置于1.0百万细胞/ ml培养基中。 24孔板中的每个孔通常具有200万个静息T细胞,2ml 10%IMDM培养基和25μl重悬的抗CD3 / CD28 Dynabead。将细胞重新悬浮在含有珠子的培养基中后,每毫升培养基中加入300nm的EFV以防止细胞 - 细胞感染。始终根据细胞产量准备新鲜的。&nbsp;
    4. 为了获得最佳刺激,可以将细胞浓缩在较小的孔中(48孔板,在一个孔中具有1百万/ ml细胞),然后第二天转移到24孔板中。在24孔板中每孔培养2百万/ 2ml细胞(图2)。
    5. 活化后24小时,加入10μl10 5 单位/ ml IL-2和10μl10 5 单位的IL-7百万细胞作为第二信号细胞因子维持细胞并帮助它们增殖。请勿卸下Anti-CD3 / CD28 Dynabeads。
    6. 需要每天监测培养物,如果存在过度拥挤,通常当培养基变成非常黄的颜色并且每个孔中的细胞超过4百万个细胞时。在这种情况下,将更多的基础培养基放入孔中并将其分成两个孔。&nbsp;
    7. 分裂期间的基础培养基应始终含有IL-2,IL-7(第2天)和EFV以及10%IMDM。在培养基中添加培养基期间不需要珠子(图2)。
    8. 这些细胞共培养6天(图2)。


      图2.处理潜在的细胞以进行激活

  4. 收获,接种和读取TZM-bl细胞的β-gal活性
    1. 在培养的第5天(T细胞从培养物中取出前一天),种子96孔白色PerkinElmer平板,每孔5×10 5个细胞/孔,剩余培养皿3个总共64孔的TZM-bl细胞的x 10 4 细胞/孔。使用10%RPMI用于培养基,因为TZM-bl细胞在10%RPMI中生长。每个孔中的培养基总体积应为200μl。保持过夜,使细胞在第二天粘附在孔的底部。
      注意:不要将培养物中的TZM-bl细胞维持超过3周,并且每隔三天将细胞分开。分裂时,不要使用超过100万/ 10毫升的10%RPMI。 TZM-bl细胞在250ml通气的Falcon烧瓶中在10%RPMI中生长,并且在准备分裂时需要进行胰蛋白酶消化。当冷冻细胞培养开始时,确保在70毫升Falcon排气瓶中进行,然后随着时间的推移进行扩增。
    2. 在培养开始的第6天,将活化的T细胞从24孔板中取出15ml锥形管,并以1,000rpm(112 xg )离心10分钟以沉淀细胞。
      注意:如果您想比较这些激活的潜伏感染细胞的RNA产量,您可以保存RNA的上清液。这可以在-80°C冰箱中冷冻。您将不得不计算细胞并用DPBS洗涤两次。您还可以为每位患者节省50万个细胞用于DNA定量。为此,只需计数细胞,将它们放入Eppendorf管中,以1,000 rpm(112 x g )旋转10分钟并沉淀细胞。然后直接将这些颗粒冷冻在-80°C冰箱中。如果您想计算针对特定参与者诱导的原病毒的比例,则使用此方法。
    3. 使用其余的活化T细胞,开始进行稀释系列。从最高稀释度向下的1.25×10 5个细胞/孔开始进行4倍稀释(1:3)。 (你可以从顶部开始低至6 x 10 4 细胞/孔)。
      注意:如果您从第一行中的1.25 x 10 5 单元格/井开始,则每行都在顶行( 8孔)在200μl10%RPMI中应具有1.25×10 5 5 细胞/孔。第二行将在所有八个孔中具有1.25×10 5 / 4个细胞,依此类推,再进行五次稀释。这应该给你总共6个稀释液(图3)。
    4. 在15ml管中制备稀释系列后,必须通过抽吸除去TZM-bl细胞板中的培养基,然后将患者细胞接种在其上。在开始研磨之前不要这样做,因为它会使盘中的细胞变干。通常不使用第七行,第八行是TZM-bl细胞上的新鲜培养基。 (图3)。
    5. 所有稀释液必须在10%RPMI中。


      图3.使用TZM-bl细胞在96孔中读取细胞

    6. 将该板置于37°C培养箱中48小时,之后必须将其读取。这种读数可以在48到72小时之间的任何时间进行。
    7. 取出平板,吸出培养基,不要打扰细胞。&nbsp;
    8. 用多通道移液器将DPBS放回孔中并轻轻洗涤并取出冲洗液并丢弃。这样做两次。洗涤时不要太严格,因为这可能会从板上取下细胞。
    9. 每孔加入100μlPromegaBeta-Glo试剂,在黑暗中孵育45分钟-1小时。尝试在很少的光线下完成所有这些步骤。该试剂对光非常敏感。
    10. 然后在Promega Glomax机器中读取以按照软件提示获取灯具。这是一台自动化机器,您必须选择任何细胞滴度协议,这些协议将能够读取您的平板。然后,您可以将板读取命名并保存为excel文件。

  5. 分析读数和IUPM计算
    1. 读取后,将Excel文件传输出去以分析这些结果。首先,计算底部介质控制行的平均值(平均值),并从板中所有其他单个读数中减去该值。这将是你的空白。对于对照HIV-1阴性平板以及HIV-1阳性患者平板,应该这样做。这也可以确保您在测试和控制单元格中具有相似的条件。&nbsp;
    2. 在具有对照HIV-1阴性T细胞的对照板中,计算每行的平均值,每个稀释度的所有8个孔。每次稀释都要这样做。另外,计算每种稀释液的标准偏差。
    3. 为每个稀释液添加标准偏差(平均值+ 2 SD)的平均值+ 2倍。这将为您提供对照孔中每种稀释度的值,您可以根据该值计算测试板中的阳性孔。
    4. 在对照板中每个稀释度的值(平均值+ 2 SD)后,从参与者平板中特定稀释度的每个孔读数中减去它。
    5. 这将为您提供一些具有正值且一些具有负值的井。正值可以在0/8到8/8之间变化。
    6. 将这些读数放入Silicianos实验室开发的算法 http://silicianolab.johnshopkins.edu/ (Rosenbloom et al。,2005)并计算每百万细胞或IUPM的感染单位。&nbsp;
    7. 对于DNA拷贝/百万个细胞,使用标准DNA q-PCR(Cillo et al。,2014)在细胞中整合原病毒并计算可由刺激诱导形成的原病毒部分复制型病毒。用公式计算:fPVE = IUPM /每百万个细胞DNA×100。

数据分析

  1. 对于静息T细胞的纯度估计,使用FACSDiVa TM 软件在LSR II流式细胞仪上获得样品,然后将细胞置于培养物中用于活化。使用FlowJo分析流式细胞仪数据以对静息T细胞群进行门控,定量和分析。&nbsp;
  2. 对于IUPM计算,应用最大似然估计来确定TZA测定的感染单位百万分率(IUPM)细胞,使用在线软件,可在 http://silicianolab.johnshopkins.edu/ ,由Rosenbloom 等人开发(2015)。

笔记

在整个方案中,您必须确保对另一组HIV-1阴性或对照细胞的细胞做同样的事情。如果您没有控件,那么您的实验将无效。您必须确保控制细胞甚至与患者细胞一样受到刺激,因此理想情况下您对测试细胞和对照细胞具有相似的条件。

食谱

  1. 10%RPMI
    RPMI 1640
    1x培养基,含10%动物血清复合物和1%青霉素 - 链霉素
    在实验过程中保持无菌和室温,并在4°C下储存
  2. 10%IMDM
    在-20°C下储存,混合450毫升IMDM w / L-谷氨酰胺与50毫升FBS和1%青霉素 - 链霉素(5毫升)
    这将使IMDM的最终成交量达到10%FBS 应始终将其储存在4°C以便长期储存

致谢

该协议改编自我们的出版物(Sanyal et al。,2017)。我们感谢实验室前成员Lori Caruso,Deena Ratner和Ming Ding(匹兹堡匹兹堡大学)的技术援助; N. Sluis-Cremer用于DNA测量和研究设计输入; P. Tarwater(德克萨斯大学埃尔帕索分校)和C. Shen(匹兹堡匹兹堡大学)进行统计咨询和开发分析统计数据; W. Buchanan(匹兹堡匹兹堡大学)招募多中心艾滋病队列研究参与者进行研究;以及匹兹堡匹兹堡部分参与者为本研究献血的所有参与者。
&NBSP;这项工作得到NIH资助R21AI138716(Phalguni Gupta),U01-AI35041(Charles R. Rinaldo。),R21-AI119117(Nicolas P. Sluis-Cremer和NIH Fogarty培训资助奖学金D43TW010039(Phalguni Gupta))的支持。

利益争夺

作者宣称没有利益冲突。

伦理

根据匹兹堡大学机构审查委员会批准的方案收集和处理血液,并由护士从医院中取出血液。 HIV-1阴性和阳性的参与者是从多中心艾滋病队列研究中招募的。
&NBSP;所有参与者都获得了书面同意,并在继续进行之前以简单的方式向他们解释了研究。

参考

  1. Ananworanich,J。和Mellors,J。W.(2015)。 艾滋病病毒存活多少?测量具有HIV复制能力的HIV用于HIV治疗研究的挑战。 EBioMedicine 2(8):788-789。
  2. Chun,TW,Carruth,L.,Finzi,D.,Shen,X.,DiGiuseppe,JA,Taylor,H.,Hermankova,M.,Chadwick,K.,Margolick,J.,Quinn,TC,Kuo,YH ,Brookmeyer,R.,Zeiger,MA,Barditch-Crovo,P。和Siliciano,RF(1997)。 HIV-1感染中潜伏组织储库和全身病毒载量的定量。 Nature 387(6629):183-188。&nbsp;
  3. Cillo,A.R.,Sobolewski,M.D.,Bosch,R.J.,Fyne,E.,Piatak,M.,Jr.,Coffin,J.M。和Mellors,J.W。(2014)。 量化静息CD4 + T细胞中HIV-1潜伏期逆转抑制性抗逆转录病毒治疗的患者。 Proc Natl Acad Sci USA 111(19):7078-7083。&nbsp;
  4. Finzi,D.,Blankson,J.,Siliciano,JD,Margolick,JB,Chadwick,K.,Pierson,T.,Smith,K.,Lisziewicz,J.,Lori,F.,Flexner,C.,Quinn, TC,Chaisson,RE,Rosenberg,E.,Walker,B.,Gange,S.,Gallant,J。和Siliciano,RF(1999)。 潜伏感染CD4 + T细胞提供终身持久性的机制HIV-1,即使是有效联合治疗的患者。 Nat Med 5(5):512-517。&nbsp;
  5. Laird,G.M.,Eisele,E.E.,Rabi,S.A.,Lai,J.,Chioma,S.,Blankson,J.N.,Siliciano,J.D。和Siliciano,R.F。(2013)。 使用病毒生长试验快速定量HIV-1潜伏库。 PLoS Pathog 9(5):e1003398。&nbsp;
  6. Rosenbloom,D.I.,Elliott,O.,Hill,A.L.,Henrich,T.J。,Siliciano,J.M。和Siliciano,R.F。(2015)。 设计和解释有限稀释分析:人类免疫缺陷病毒潜伏储库的一般原则和应用-1 。 Open Forum Infect Dis 2(4):ofv123。&nbsp;
  7. Sanyal,A.,Mailliard,RB,Rinaldo,CR,Ratner,D.,Ding,M.,Chen,Y.,Zerbato,JM,Giacobbi,NS,Venkatachari,NJ,Patterson,BK,Chargin,A.,Sluis -Cremer,N。和Gupta,P。(2017)。 新的检测结果显示静息CD4中有一个大的,可诱导的,具有复制能力的HIV-1水库 + T细胞。 Nat Med 23(7):885-889。&nbsp;
  8. Siliciano,J.D.,Kajdas,J.,Finzi,D.,Quinn,T.C.,Chadwick,K.,Margolick,J.B.,Kovacs,C.,Gange,S.J。和Siliciano,R.F。(2003)。 长期随访研究证实静息CD4中潜伏性HIV-1水库的稳定性 + T细胞。 Nat Med 9(6):727-728。&nbsp;
  9. Siliciano,J。D.和Siliciano,R。F.(2005)。 用于检测和定量潜伏感染,静息CD4 + 的增强型培养检测在感染HIV-1的个体中携带有复制能力的病毒的T细胞。 Methods Mol Biol 304:3-15。&nbsp;
  10. Siliciano,J。D.和Siliciano,R。F.(2004)。 HIV-1的长期潜在储存库:发现和临床意义。 J Antimicrob Chemother 54(1):6-9。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sanyal, A., Rangachar, V. S. and Gupta, P. (2019). TZA, a Sensitive Reporter Cell-based Assay to Accurately and Rapidly Quantify Inducible, Replication-competent Latent HIV-1 from Resting CD4+ T Cells. Bio-protocol 9(10): e3232. DOI: 10.21769/BioProtoc.3232.
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