TITLE: Ex vivo differentiation of resting CD4+ T cells coupled with the QVOA (dQVOA)
AUTHORS: Elizabeth Wonderlich, Krupa Subramanian, Carol Lackman-Smith , Roger Ptak, Deanna Kulpa

[DESCRIPTION]
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[STEPS]
?. [Day -8]
Thaw and prepare cryopreserved PBMC from cohort or clinical trial participants.Typically, 50-100x106 cryopreserved PBMC are used to yield sufficient resting CD4+ T cells for each dQVOA. For the referenced publication, a typical dQVOA used 8x106 rCD4+ T cells.
?. [Day -7]
Perform resting enrichment of CD4+ T cells per StemCell protocol with some noted adaptations for optimization.
?. [Day -3]
Replenish media and cytokines by replacing half of the volume of each well of the dQVOA.Any culture supernatant being removed from the dQVOA may be saved for analysis.
?. [Day -1]
(Optional: To assess memory T cell subset distribution and any other parameters after ex vivo differentiation, sacrifice one B well as a "post-differentiation" sample and perform standard surface staining for flow cytometry.)
?. [Day 0]
Set up stimulations and longterm outgrowth cultures in the dQVOA.Small scale differentiation cultures are transferred to larger culture dishes without splitting or cross-contaminating.This step is done to allow enough room during the addition of γ-irradiated allogeneic PBMC in a 10:1 stimulation ratio. A table describing steps 7.1 through 7.3 is found in step 7.4.
?. [Day -8]
Thaw cryovials of PBMC  in a  water bath.  When the cells are almost thawed (check every 10 seconds after one minute) transfer the vial contents to the 50 mL conical tubes containing the cold FBS. For a perfect thawing process, a pea-size frozen sphere should be present. Collect any residual PBMCs by rinsing the cryovial with 1 mL of RPMI-1640 using a P1000 pipet and combine. Q/S tube to 50mL with RPMI-1640.
37 °C
?. [Day -8]
Centrifuge at for 10 minutes at   and discard supernatant.
4 °C
?. [Day -8]
Resuspend PBMC in 5mL cold FBS and Q/S tube to 50mL with RPMI-1640.
?. [Day -8]
Count PBMC using trypan blue.
?. [Day -8]
Centrifuge at for 10 minutes at   and discard supernatant.
4 °C
?. [Day -8]
Resuspend PBMC at 3x106 cells/mL in R10 media and place in appropriate tissue culture treated flask(s) for culture. (R10: RPMI-1640 w/ glutamax, 1% NEAA, 1% PenStrep, 1% 1M HEPES, 10% heat-inactivated FBS)
?. [Day -8]
Incubate PBMC at  and 5% CO2 overnight (between 15-19 hours).
37 °C
?. [Day -7]
Immediately, plate rCD4+ T cells in limiting dilution prior to any differentiation incubationsResting CD4+ T cells must be plated in limiting dilution before differentiation culturing or cellular proliferation may alter the ratio of infected cells. (DO NOT PERFORM DIFFERENTIATION CULTURING IN BULK.)Typically, dQVOA is performed utilizing 8x106 rCD4+ cells.  This generates 12 wells containing 5x105 rCD4+ cells (A wells), 6 wells containing 2x105 rCD4+ cells (B wells, one will be utilized for flow cytometric assessment of memory T cell differentiation), 2 wells containing 4x104 rCD4+ cells (C wells), and  2 wells containing 8x103 rCD4+ cells (D wells).  A total of 22 wells are set up on Day -7.  Two wells that do not contain patient-derived CD4+ cells will be generated on Day 0 (G wells).  The numbers and cellular concentration of each well must be determined and plated at this point.
?. [Day -7]
Acquire PBMC from . Thoroughly resuspend PBMC in each flask and remove a sample to perform cell count.
?. [Day -7]
Count PBMC with trypan blue.
?. [Day -7]
Evenly distribute cells into 50 mL conicals and centrifuge at  for 10 minutes at  .
4 °C
?. [Day -7]
Carefully remove and discard supernatant and resuspend PBMC at 5x107 cells/mL in R10 media.
?. [Day -7]
Add  of CD4 isolation antibody cocktail and  of CD25 antibody cocktail per  of cells.  Mix well by pipetting up and down.
50 µl
50 µl
1 ml
?. [Day -7]
Incubate at  for 10 minutes.
0 Room temperature
?. [Day -7]
Vortex magnetic particles well (>30 seconds). Add  of magnetic particles per  of cells to each enrichment sample.  Mix well by pipetting up and down.
75 µl
1 ml
?. [Day -7]
Incubate at  for 5 minutes.
0 Room temperature
?. [Day -7]
Add R10 to Q/S tube to max volume per StemCell protocol.
?. [Day -7]
Insert tube into appropriate magnet for 10 minutes at  . Through negative selection, the resting enriched CD4+ T cells will remain in the supernatant, unbound by the magnet.
0 Room temperature
?. [Day -7]
Carefully, without removing the tube from the magnet or disturbing the magnetic beads, use a pipet to transfer the resting enriched cell suspension to a new conical tube and take an aliquot for counting.
?. [Day -7]
Count resting-enriched CD4+ T (rCD4+) cells using trypan blue.
?. [Day -7]
Centrifuge the rCD4+ cells at for 10 minutes at  .
4 °C
?. [Day -7]
Carefully remove and discard supernatant.  Resuspend rCD4+ cells at 1x106 cells/mL in freshly prepared differentiation media. (Differentiation media: R10 containing 25ng/mL recombinant human IL-6, IL-7, IL-10, IL-15, and TNF-α, which are solubilized and stored per manufacturer recommendation.)
?. [Day -7]
rCD4+ T cells are already resuspended at 1x106 cells/mL of differentiation media. To generate A wells: Transfer  (5x105) of rCD4+ cell suspension to each well of a 24-well plate. Be sure to leave empty wells between samples to minimize cross contamination. Q/S each well up to 1mL with  of differentiation media.
500 µl
500 µl
?. [Day -7]
rCD4+ T cells are already resuspended at 1x106 cells/mL of differentiation media.To generate B wells: Transfer  (2x105) of rCD4+ cell suspension to each well of a 96-well round-bottom plate. Be sure to leave empty wells between samples to minimize cross contamination.  ( is the final volume, no need to Q/S B wells)
200 µl
200 µl
?. [Day -7]
rCD4+ T cells are already resuspended at 1x106 cells/mL of differentiation media.To generate C wells: Transfer  (4x104) of rCD4+ cell suspension to each well of a 96-well round-bottom plate. Be sure to leave empty wells between samples to minimize cross contamination. Q/S each well up to 200uL with  of differentiation media.
40 µl
160 µl
?. [Day -7]
rCD4+ T cells are already resuspended at 1x106 cells/mL of differentiation media.To generate D wells: Transfer  (8x103) of rCD4+ cell suspension to each well of a 96-well round-bottom plate. Be sure to leave empty wells between samples to minimize cross contamination. Q/S each well up to 200uL with  of differentiation media.
8 µl
192 µl
?. [Day -7]
Add sterile liquid (i.e. PBS or water) to any empty wells in the 24- and 96-well plates to minimize evaporation from assay wells.
?. [Day -7]
Differentiate rCD4+ cells by incubating at  and 5% CO2 for 4 days. If the media turns yellow, prepare fresh differentiation media and replace half of the volume in each well as appropriate.
37 °C
?. [Day -3]
To replenish A wells: Without disturbing cells, remove  of media from each well in the 24-well plate.  Add  of freshly prepared differentiation media to each well.  Take care to add slowly, without splashing.
500 µl
500 µl
?. [Day -3]
To replenish B/C/D wells: Without disturbing cells, remove  of media from each well in the 96-well plate. Add  of freshly prepared differentiation media to each well.  Take care to add slowly, without splashing.
100 µl
100 µl
?. [Day -3]
Continue to differentiate CD4+ cells by incubating at  and 5% CO2 for an additional 3 days. If the media turns yellow, prepare fresh differentiation media and replace half of the volume in each well as appropriate.
37 °C
?. [Day -7]
(Optional: To assess resting CD4+ T cell enrichment purity and memory T cell subset distribution, take 2x105 cells as a "post-enrichment/pre-differentiation" sample and perform standard surface staining for flow cytometry.)
?. [Day 0]
Generate γ-irradiated allogeneic PBMC as a part of the stimulation method for dQVOA.
?. [Day 0]
Irradiate HIV-naive buffy coats from two donors by exposing to 5000R in a Cs6 source irradiator. Immediately place irradiated buffy coats on ice.
?. [Day 0]
Process each buffy coat into PBMC through standard density gradient centrifugation (i.e. ficoll, histopaque, LSM, etc).
?. [Day 0]
After final wash step of PBMC processing, centrifuge γ-irradiated allogeneic PBMC at   for 10 minutes at .
4 °C
?. [Day 0]
Carefully remove and discard supernatant and resuspend γ-irradiated allogeneic PBMC at 2.5x106 cells/mL in freshly prepared stimulation media. (Stimulation media: R10 containing 0.5μg/mL REMEL PHA and 100 IU/mL recombinant human IL-2)  Place γ-irradiated allogeneic PBMC on ice immediately to prevent cell adhesion and store on ice until ready for addition to dQVOA.
?. [Day 0]
To generate full-sized A wells: Carefully without splitting or cross-contaminating resuspend each A well in the 24-well plate and transfer the whole volume from each well () of differentiated CD4+ cell culture to its own fresh well in a 6-well plate. Be sure to skip wells to minimize cross contamination. Add  (5x106) of γ-irradiated allogeneic PBMC to each A well. Q/S each A well up to 8mL with  of stimulation media.
1 ml
2 ml
5 ml
?. [Day 0]
To generate full-sized B/C/D wells: Carefully without splitting or cross-contaminating resuspend each B/C/D well in the 96-well round bottom plate and transfer the whole volume from each well () of differentiated CD4+ cell culture to its own fresh well in a 24-well plate. Be sure to skip wells to minimize cross contamination. Add  (2.5x106) of γ-irradiated allogeneic PBMC to each B/C/D well. Q/S each B/C/D well up to 2mL with  of stimulation media.
0.2 ml
1 ml
0.8 ml
?. [Day 0]
To generate G wells:  Add  (2.5x106) of γ-irradiated allogeneic PBMC to each G well. Q/S each G well up to 2mL with  of stimulation media. G wells serve as a negative control for potential HIV-contamination or cross-contamination sources. Be sure to skip wells to minimize cross contamination.
1 ml
1 ml
?. [Day 0]
Table explicitly outlining the volumes of each reagent entering each well of the stimulation portion of dQVOA:
?. [Day 1]
Remove PHA to minimize cytotoxicity and replace with feeding media (Feeding media: R10 containing 100 IU/mL recombinant human IL-2. Note: TCGF is not included as a reagent in dQVOA.)Any culture supernatant being removed from the dQVOA may be saved for analysis.
?. [Day 0]
Incubate dQVOA 6- and 24-well plates at  and 5% CO2 overnight (>17 hours of stimulation).
37 °C
?. [Day 1]
For A wells: Without disturbing cell layer, remove  of media from each well in the 6-well plates.  Add  of feeding media to each well.  Take care to add slowly, without splashing.
6 ml
6 ml
?. [Day 1]
For B/C/D/G wells: Without disturbing cell layer, remove  of media from each well in the 24-well plate.  Add  of feeding media to each well.  Take care to add slowly, without splashing.
1.5 ml
1.5 ml
?. [Day 0]
Add sterile liquid (i.e. PBS or water) to any empty wells in the 6- and 24-well plates to minimize evaporation from assay wells.
?. [Day 1]
Incubate dQVOA 6- and 24-well plates at  and 5% CO2 for 4 days. If the media turns yellow, replace half of the volume of feeding media in each well as appropriate.
37 °C
?. [Day 5]
Without disturbing or removing any cells, replenish feeding media by replacing half of the volume of each well of the dQVOA.For A wells remove and replace  of feeding media. For B/C/D/G wells remove and replace  of feeding media.Any culture supernatant being removed from the dQVOA may be saved for analysis.
4 ml
1 ml
?. [Day 8]
Without disturbing or removing any cells, replenish feeding media by replacing half of the volume of each well of the dQVOA.For A wells remove and replace  of feeding media. For B/C/D/G wells remove and replace  of feeding media.Any culture supernatant being removed from the dQVOA may be saved for analysis.
4 ml
1 ml
?. [Day 11]
Harvest supernatants for endpoint testing and analysis.  Freeze all vials at  until ready for endpoint analysis.  Harvesting supernatants prior to day 11 of outgrowth may yield a lower reservoir measurement.(Optional: At the end of the assay, cell pellets may be harvested, washed in PBS, and stored as a pellet for future analysis.)
-80 °C
?. [Endpoint Analysis]
PerkinElmer Alliance p24 antigen ELISA kits are used per manufacturer specifications.  Each supernatant is assessed in duplicate ELISA wells.  3.25 pg/mL is used as the cutoff value to determine whether a well is considered positive or negative for HIV outgrowth. Typical wells from dQVOA have p24 concentrations that are either negative or beyond the high end of the standard curve, thus binary (+/-) scoring is used.
?. [IUPM Calculation]
The maximum-likelihood method is used to calculated the infectious units per million rCD4+ T cells (IUPM) using IUPMStats v1.0 (found here: http://silicianolab.johnshopkins.edu/ and published here: https://doi.org/10.1101/018911)
?. [Day -8]
Identify 50 mL conical tubes with Experiment ID and add 10 mL cold FBS to each tube to thaw up to four vials that contain 1mL of DMSO-containing freeze-media.  If needed, prepare additional 50 mL conical tubes with 10 mL cold FBS. The volume of DMSO-containing freeze-media being transferred to each 50 mL conical tube should not exceed .
4 ml
?. [Day -7]
(Optional: To assess resting CD4+ T cell enrichment purity and memory T cell subset distribution, take 2x105 cells as an "ex vivo pre-enrichment" sample and perform standard surface staining for flow cytometry.)