Leucine Zipper-based Cell Sorting for Purification of Dual-vector-transduced Cells

Summary

Here, we describe a protocol for selectively purifying dual-transduced T cells from a mixture of co-transduced T cells. This approach combines two retroviral vectors that each encode one component of a paired leucine zipper-based cell sorting methodology that enables selective immunomagnetic purification of dual-vector-transduced cells.

Abstract

Adoptive T cell therapies, including chimeric antigen receptor (CAR) T cells, have demonstrated impressive clinical activity against hematologic malignancies and are showing promise in treating solid tumor malignancies. Despite these successes, multiple mechanisms of resistance to T cell immunotherapy have been identified that limit therapeutic success, including loss of or weak expression of target antigens, development of T cell exhaustion, and the presence of an immunosuppressive tumor microenvironment. Multiple cell engineering strategies have been developed to overcome these mechanisms of resistance. However, multiple mechanisms of resistance can occur simultaneously, necessitating a combination of multiple engineering strategies to optimize anti-tumor activity. Vector packaging constraints limit the delivery of large amounts of genetic information to T cells and present a challenge in co-expressing multiple engineered constructs. Here, we describe a protocol for co-transducing T cells with two vectors to encode multiple transgenes, thereby increasing the number of engineered functions. By co-expressing a leucine zipper-based cell sorting methodology, termed Zip-sort, we direct selective immunomagnetic purification of dual-transduced cells that have incorporated two distinct vectors.

Introduction

CAR T cell therapy has revolutionized the treatment of B cell leukemia, lymphoma, and multiple myeloma^1,2. CAR T cells are also being investigated for solid tumor malignancies³. However, recurrent mechanisms for failure of CAR T cell therapy have been identified, including poor expression or loss of the CAR target antigen molecule, development of T cell dysfunction (exhaustion), active immune suppression by the tumor microenvironment, and limited T cell trafficking to and persistence in the tumor microenvironment^3,4,5,6. While individual engineering solutions have been devised to attempt to overcome each of these CAR T cell resistance mechanisms³, introducing multiple engineered functions into a cell therapy product to simultaneously overcome multiple resistance mechanisms is challenging due to the limited genetic packaging capacity of retroviral and lentiviral vector systems⁷.

To overcome limitations in vector packaging capacity, co-transduction of T cells with multiple vectors has been used increasingly. Vector co-transduction has been used to study logic-gated and drug-regulated T cell engineering strategies in pre-clinical studies^8,9,10 and in a recent clinical trial evaluating CD19/CD22 dual-CAR T cells to overcome vector packaging limitations¹¹. However, co-transduction generates mixed cell products containing combinations of single- and dual-transduced T cells, which may impair functionality of the product if it depends on components encoded by both vectors and may also lead to competition between single-transduced T cells¹².

To overcome these challenges, we have developed a methodology called Zip-sort, which uses heterodimerizing leucine zippers - each expressed from one of two co-transduced vectors - to direct single-step immunomagnetic purification of dual-transduced cells¹³. In this protocol, we describe methods for constructing retroviral vectors incorporating our Zip-sort methodology, retroviral supernatant production and concentration, T cell activation and co-transduction, and immunomagnetic purification of dual-transduced cells (Zip-sort).

Protocol

Animal studies were conducted in the City of Hope (COH) Parvin Animal Research Laboratory under protocol #23145 approved by the COH Institutional Animal Care and Use Committee and in the Memorial Sloan Kettering Cancer Center (MSKCC) vivarium under protocol #99-07-025 as approved by the MSKCC Institutional Animal Care and Use Committee. Human T cells were isolated from de-identified Peripheral Blood Mononuclear Cells (PBMCs) that were Ficoll-Paque purified from leukocyte reduction filters obtained from the City of Hope Blood Donor Center and used under protocol #24427 as approved by the City of Hope Institutional Review Board. All procedures were carried out in accordance with institutional biosafety standards.

1. Zip-sort vector design

1. Select the expression vector of choice for genetically modifying cells of interest.
   NOTE: The zip-sort methodology comprises secreted zippers and capture zippers, both of which are expressed from different vectors, and which pair intracellularly and traffic to the cell surface to enable selective purification of cells incorporating both vectors (Figure 1A-D)¹³. Secreted zippers are expressed from Vector 1 and contain an affinity tag. Capture zippers are expressed from Vector 2 and retain the secreted zipper on the surface of the cell. The blocking zipper modification is comprised of a covalently-linked second zipper, which blocks non-specific extracellular zipper pairing of the secreted zipper. The blocking zipper modification is required to prevent extracellular pairing when adding a capture zipper to proteins with elongated extracellular domains (typically extracellular domains larger than a short-hinge domain).
2. Select a set of secreted and capture zippers from Supplementary Table 1 and Supplementary File 1 to test and build vectors.
3. Clone zipper sequences into paired expression vectors using standard molecular biology techniques¹³- each of two vectors will encode either the affinity tag secreted zipper or capture zipper (Figure 1D).
   1. See Supplementary Table 1 and Supplementary File 1 for zipper construct sequences. The zipper sequences have been placed at the 5' (N terminus) of a polycistronic message to enhance their expression and thereby boost sort yield. Place subsequent transgenes downstream, separated by self-cleaving 2A peptides (Figure 1D). However, prior experiments have had success placing the zippers in a 3' configuration if high expression is maintained.

2. Retrovirus production

1. Prepare Phoenix-Eco cells (ATCC CRL-3214, mouse-tropic virus) or Phoenix-Eco ɑ-V β-3 cells¹³ (addition of ɑ-V β-3 designed to enhance adhesion of Phoenix-Eco) or 293Vec-RD114¹⁴ (human-tropic virus) or 293Vec-RD114 ɑ-V β-3 cells (addition of ɑ-V β-3 designed to enhance adhesion of 293Vec-RD114) 1 day prior to transfection.
   NOTE: This protocol does not require modification of packaging lines with ɑ-V β-3 integrins, but this modification improves adhesion of this cell line to tissue culture flasks and facilitates retroviral supernatant collection.
   1. Trypsinize cells in T175 flask containing Phoenix or 293Vec-RD114 cells at ~80% confluence with 0.25% trypsin-EDTA.
      1. Remove media, wash adherent cells with 10 mL of PBS, keeping cells non-disturbed to remove serum components that inhibit trypsin, add 5 mL of 0.25% trypsin-EDTA, and incubate cells at 37 °C for 5 min.
      2. Dissociate and collect the cells in 5 mL of DMEM supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, and 1% GlutaMAX (referred to as complete DMEM).
      3. Pellet the cells in a 15 mL conical tube by centrifuging at 340 x g for 5 min at 4 °C. Decant media.
   2. Resuspend centrifuge-pelleted cells in 10 mL of complete DMEM and plate on a 10 cm tissue culture dish approximately 1.5 x 10⁶ Phoenix-Eco cells, Phoenix-Eco ɑ-V β-3, 293Vec-RD114 cells, or 293Vec-RD114 ɑ-V β-3 cells counted with a hemacytometer.
   3. Gently shake or swirl the plate to distribute cells evenly and incubate overnight at 37 °C to allow the cells to adhere to the plate.
2. Approximately 18-24 h later, verify that cells are 60%-70% confluent, with healthy, adherent cells exhibiting dendritic projections.
3. Stably transfect Phoenix-Eco, Phoenix-Eco ɑ-V β-3 cells, 293Vec-RD114, or 293Vec-RD114 ɑ-V β-3 cells with retroviral LZRS-vector constructs (Addgene Plasmid #31601, EBNA-based episomal stable transfection) or PB-vector constructs¹³ (piggyBac transposon-based genomic stable integration, Supplementary File 1) using the transfection kit (Table of Materials) as described below.
   1. Combine 2 µg LZRS-vector DNA or PB-vector DNA and 16 µL of enhancer reagent in 300 µL of EC buffer in a microcentrifuge tube for each transfection reaction.
   2. For stable integration of PB-vector constructs, also add 1 µg of the hyperactive piggyBac transposase vector pCMV-hyPBase¹⁵ (Sanger Institute).
   3. Gently vortex for 5 s and incubate at room temperature for 5 min. Add 20 µL of transfection reagent, vortex for 10 s, and incubate at room temperature for 10 min to generate DNA micelle complexes.
   4. Add 300 µL of complete DMEM to the tube, pipette several times to thoroughly mix, and add micelle suspension dropwise to the plated packaging line cells using a 1 mL pipette.
   5. Swirl gently to ensure uniform deposition of micelles. After 24 h, trypsinize transfected cells and assess initial retroviral encoded construct expression by flow cytometry.
   6. Transfer trypsinized cells to a T75 flask with 20 mL of complete DMEM and add 2 µg/mL puromycin or 10 µg/mL blasticidin, depending on the antibiotic resistance genes, to select the transfected cells. Once cells reach 70%-75% confluency, trypsinize and transfer to a T175 flask with 40 mL of complete DMEM. Maintain selection of antibiotics in the media during expansion of the packaging line.
   7. When the cells are 80%-85% confluent, trypsinize again and confirm construct expression by flow cytometry. Freeze down an aliquot of cells using freezing media (50% complete DMEM, 40% FBS, 10% DMSO) and passage cells into two T175 flasks (1:10-1:12 split ratio) with 40 mL of complete DMEM.
4. Collect and concentrate retroviral supernatant from fully selected stable packaging lines grown in T175 flasks when cells reach 80%-85% confluency.
   1. Remove antibiotics from the media before the first harvest by replacing the media in T175 with 40 mL of fresh, complete DMEM 24 h before the first intended collection.
   2. Collect and filter supernatants 24 h after media replacement by passing collected supernatant through 50 mL syringes fitted with syringe filters (0.45 µm, polyvinylidene fluoride). Collect in a 50 mL conical tube.
   3. Add 5x polyethylene glycol (PEG) solution concentrate (40% (wt/vol) PEG 8,000 MW containing 2.4% (wt/vol) NaCl) filtered supernatant and invert the tube several times to thoroughly mix (PEG precipitation step).
   4. Add 40 mL of complete DMEM back to the original harvested T175 flask, incubate for another 24 h, and collect viral supernatant daily for another 2 consecutive days in total.
   5. Precipitate the virus for 1-2 days at 4 °C. The precipitated virus will appear as flocculent, floating, or sedimented material.
   6. Centrifuge PEG-precipitated virus suspension at 2,100 x g for 15 min at 4 °C. Discard non-precipitated supernatant.
   7. Resuspend each PEG-precipitated viral pellet in 500 µL of RPMI supplemented with 10% FBS, 1% Penicillin/Streptomycin, 1% GlutaMAX, 1% Sodium Pyruvate, 1% Non-Essential Amino Acids, and 0.1% β-mercaptoethanol (referred to as complete RPMI).
   8. Use the resuspended virus immediately for transduction or transfer to a microcentrifuge tube and store at −80 °C.

3. Day 0: Mouse T cell enrichment and activation

1. Coat a tissue culture-treated 6-well plate with 1.5 mL of PBS per well containing 2 µg/mL anti-CD3 (clone 145-2C11) and 2 µg/mL anti-CD28 (clone 37.51). Incubate for 2 h at 37 °C.
2. Harvest spleen from donor mouse, dissociate cells through a 40 µm filter, and collect splenocytes in complete RPMI. Pellet splenocytes by centrifuging at 340 x g for 5 min at 4 °C.
   NOTE: Female BALB/cJ mice between the ages of 6-8 weeks were used as donor mice in this study.
3. Discard supernatant, gently vortex pellet, add 1 mL of RBC lysis buffer, pipette 3x-5x to mix, incubate at room temperature for 1 min, quench with 10 mL of complete RPMI, and spin down cells by centrifuging at 340 x g for 5 min at 4 °C.
4. Discard supernatant, add 800 µL of PBS with 0.5% BSA and 2 mM EDTA (referred to as MACS buffer), and add 200 µL of mouse-CD19 microbeads. Pipette to mix and incubate for 15 min at 4 °C.
5. Prepare 1 LD column per mouse spleen. Fix the LD column to a MACS separator. Place a 15 mL conical tube underneath to collect CD19-depleted splenocytes. Wet the column with 2 mL of MACS buffer.
6. After incubation with CD19 microbeads, wash cells with 5 mL of cold MACS buffer, pellet cells by centrifuging at 340 x g for 5 min at 4 °C, and discard the supernatant. Resuspend in 1 mL of MACS buffer and add cells to the LD column. Wait for the suspension to pass through before washing with 1 mL and then 2 mL of MACS buffer.
7. Collect flow-through of the CD19-depleted splenocytes to use downstream. Discard LD column, spin down CD19-depleted splenocytes by centrifuging at 340 x g for 5 min at 4 °C. Discard supernatant and resuspend in 18 mL of complete RPMI. Add recombinant human IL-2 (rhIL-2) to a final concentration of 50 IU/mL.
8. Discard the antibody solution from the antibody-coated 6-well plate. Add 3 mL of resuspended B cell-depleted splenocytes to each well of the antibody-coated 6-well plate.

4. Day 1: Retroviral transduction of mouse T cells

1. Coat a non-tissue-culture-treated 6-well plate with 1.5 mL of PBS per well containing 2 µg/mL anti-CD3 (clone 145-2C11), 2 µg/mL anti-CD28 (clone 37.51), and 20 µg/mL RetroNectin.
   NOTE: Non-tissue-culture plates are used during RetroNectin coating according to the manufacturer's recommendations.
2. Incubate for 2 h at 37 °C. Discard coating solution or save for re-use (may reuse once). Block plate with 1 mL of PBS with 2% BSA per well, incubate for 10 min at 37 °C, and discard the blocking solution.
3. In the meantime, thaw PEG-precipitated viral supernatant in a 37 °C water bath.
   NOTE: 500 µL of each co-transduced PEG-concentrated retrovirus generated from a T175 flask should be sufficient to dual-transduce up to 2 x 10⁶ activated T cells per well with > 20% dual-transduction efficiency.
4. Add co-transduction viral supernatants to the 6-well plate well according to desired conditions, bring the final volume up to at least 2 mL with complete RPMI and 50 IU/mL rhIL-2, and spin for 1 h at 2,100 x g and 32 °C.
5. Add 0.5-2 x 10⁶ activated T cells per well and spin plate at 340 x g for 10 min at 32 °C. If cells are not adherent, repeat spin at 340 x g for another 10-20 min at 32 °C. Incubate at 37 °C overnight.

5. Day 2: Vector expression assessment and optional re-transduction

1. Disrupt 100-200 µL of adherent cells per condition from the transduction plate for flow cytometry. The user should identify a flow cytometry-detectable marker for each individual vector and affinity tag and use them to assess vector single-vector transduction efficiency, double-vector transduction efficiency, and affinity tag presentation using flow cytometry.
2. Perform flow cytometry on co-transduced cells as described below.
   1. Set up acquisition parameter axes to display vector reporters for Vector 1 and Vector 2 (Figure 1C).
   2. Plot histograms for affinity tag zipper staining (e.g., FLAG or CD34) for each of the four Vector 1 and Vector 2 expression quadrants to ensure that the affinity tag staining is limited to cells co-expressing Vector 1 and Vector 2 transduction reporters.
3. If cells are sufficiently dual-transduced to ensure sufficient cells for downstream experiments (generally 20% or greater dual-transduction), perform the following steps.
   1. Coat double the number of the initial wells used to transduce each T cell line with PBS containing 2 µg/mL anti-CD3 and anti-CD28 antibodies to maintain T cell stimulation. Incubate for 2 h at 37 °C.
   2. Transfer cells into new wells with complete RPMI with 50 IU/mL rhIL-2 (maintains T cell stimulation).
4. If cells are insufficiently dual-transduced to ensure sufficient cells for downstream experiments (generally < 20% dual-transduction), perform the following steps.
   1. Re-transduce using the same protocol as Day 1, but increasing to double the number of wells initially used to account for T cell expansion.
   2. Incubate at 37 °C overnight. Repeat flow cytometry 24 h later.
      NOTE: Vector-encoded constructs, including the affinity tag zipper, show maximum expression 2 days after initial transduction.

6. Day 3: T cell immunomagnetic selection (Zip-sort)

1. Pool wells of the cells generated using the same co-transduction conditions into a 15 mL conical tube.
2. Centrifuge cells at 340 x g for 5 min at 4 °C. Discard supernatant and incubate cells in 500 µL of MACS buffer containing 30 µL of microbeads (DYKDDDDK (FLAG) or human CD34) per 1 x 10⁷ cells for 30 min at 4 °C.
3. After incubation, wash cells with 5 mL of cold MACS buffer. Centrifuge cells at 340 x g for 5 min at 4 °C. Discard supernatant and resuspend pellet in 1 mL of MACS buffer.
4. Positively select transduced T cells using an LS column as described below.
   1. Wet the column with 2 mL of MACS buffer. Apply cell suspension to the LS column in the magnetic stand.
   2. Wash sequentially with MACS buffer (1 mL, 2 mL, 3 mL). Elute retained cells from the column by removing the column from the magnetic stand, adding 5 mL of complete RPMI to the column, and inserting the cell plunger.
   3. Capture cells in a 15 mL conical tube or directly in a T25 flask. Equilibrate cells at 0.5 x 10⁶ cells/mL in complete RPMI with 50 IU/mL rhIL-2.
   4. If starting from < 20% dual-transduction, optionally elute cells onto a second LS column for sequential positive selection to increase post-sort purity.
5. Culture CAR T cells in T25 or T75 flasks at approximately 0.5 x 10⁶ cells/mL of complete RPMI and 50 IU/mL rhIL-2.
6. Optional: If T cells will be used for in vivo experiments, consider culturing in 1 µM dasatinib to attenuate CAR T cell tonic signaling-induced dysfunction encountered during the initial culture period^13,16.
7. Perform flow cytometry as per steps 2.1 and 2.2 above for pre-sorted and post-sorted T cells to assess post-Zip-sort yield and purity (% dual-transduced).
   Yield = 100 x total dual-transduced cells recovered / total dual-transduced cells present in pre-sort population)

7. Day 4: Continued T cell culture

1. Add fresh complete RPMI and 50 IU/mL rhIL-2 for the entire culture volume to maintain T cell concentration at 0.5-1 x 10⁶ cells/mL.
2. Optional: If T cells will be used for in vivo experiments, add fresh 1 µM dasatinib.

8. Day 5: Characterization of Zip-sorted T cells

1. Optional: Prepare 100-200 µL of T cell suspension per condition stained with antibodies to detect CARs or other protein products encoded by the Zip-sort vectors using flow cytometry.
2. Perform in vitro or in vivo experiments with CAR T cells.
3. Optional: If T cells will be used for in vivo experiments, wash cells to remove dasatinib. Resuspend cells at the concentration required for retro-orbital or tail-vein injection according to institutional guidelines.

9. Day 0: Purification and stimulation of human T cells

1. Quickly thaw a vial of cryopreserved human peripheral blood mononuclear cells (PBMCs) in a water bath (or use freshly isolated PBMCs). Add thawed PBMCs to a 15 mL conical tube containing 5 mL of MACS buffer, prepare an aliquot for counting, and pellet cells by centrifuging at 340 x g for 5 min at 4 °C.
2. Discard supernatant and resuspend cells in MACS buffer (use 40 µL of buffer per 1 x 10⁷ total cells). Proceed with T cell isolation using the Pan T cell isolation Kit, human, as per the manufacturer's instructions.
   1. Briefly, add 10 µL of Pan T Cell Biotin-Antibody Cocktail per 1 x 10⁷ total cells. Incubate for 5 min at 4 °C.
   2. Add 30 µL of MACS buffer per 1 x 10⁷ total cells. Add 20 µL of Pan T Cell MicroBead Cocktail per 1 x 10⁷ total cells.
   3. Incubate for 10 min at 4 °C. Fill up the volume of cell suspension using MACS buffer to at least 500 µL.
   4. Proceed to magnetic cell separation using an LS column and collect the flow through containing enriched (untouched) T cells.
3. Count the T cell yield and pellet cells by centrifuging at 340 x g for 5 min at 4 °C. Discard supernatant. Resuspend human T cells in complete RPMI at 1 x 10⁶ cells/mL with rhIL-2 at 50 IU/mL.
4. To a 15 mL conical tube containing 2 mL of PBS, add required volume of Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (25 µL beads per 1 x 10⁶ cells, using a 1:1 bead to T cell ratio) and spin down at 340 x g for 5 min at 4 °C to wash the beads. Discard supernatant.
5. Mix resuspended T cells with washed beads and transfer to a T25 flask for culture.

10. Day 3: Retroviral transduction of human T cells

1. Coat a non-tissue-culture 6-well plate with 1.5 mL of PBS per well containing 20 µg/mL RetroNectin.
2. Incubate for 2 h at 37 °C. Discard coating solution or save for re-use (may reuse once).
3. In the meantime, thaw PEG-precipitated viral supernatants for co-transduction in a 37 °C water bath.
4. Add co-transduction viral supernatant to wells of RetroNectin-coated 6-well plate according to desired transduction conditions, count and add desired amount of T cells (generally 0.5 - 2 x 10⁶ T cells/well), bring volume up to 3 mL with complete RPMI containing rhIL-2 and spin for 45 min at 340 x g at 32 °C.
5. After 24 h, maintain T cells at 1 x 10⁶ cells/mL in complete RPMI with 50 IU/mL rhIL-2.
6. Transfer cells from 6-well plate to T25 flask if volume needed to maintain the desired concentration of T cells exceeds the volume capacity of the 6-well plate.

11. Day 6: Zip-sorting human CAR T cells

1. Remove Dynabeads from T cells prior to affinity tag-based immunomagnetic purification (Zip-sort) to avoid possible Dynabead-associated retention in the magnetic column and prior to flow cytometry to avoid potential interference with flow cytometric analysis.
2. Vortex briefly to dissociate Dynabeads and transfer T cells to a 15 mL conical tube and insert into Dynabead Magnet. Invert the tube a few times and wait for 5 min until the Dynabeads are bound to the magnet.
3. Carefully transfer cell suspension without Dynabeads into a new 15 mL conical tube by aspirating with a pipette. Count recovered cell concentration and use volume to calculate total cell yield.
4. Evaluate CAR expression using flow cytometry as per the mouse transduction protocol above.
5. Spin down cells by centrifuging at 340 x g for 5 min at 4 °C and incubate in 500 µL MACS buffer containing 30 µL of microbeads (DYKDDDDK (FLAG) or human CD34) per 1 x 10⁷ cells.
6. Follow steps 6.3-6.5 to obtain Zip-sorted human CAR T cells.
7. Evaluate post-sort viability by hemacytometer viability check using trypan blue exclusion. Pre-sort viability is expected to be > 80%. This may drop below 80% following Zip-sorting but recovers over 4-5 days.
8. Expand human T cells in complete RPMI with 50 IU/mL rhIL-2 and maintain T cell concentration at approximately 1 x 10⁶ cells/mL. Post-sort T cell expansion and maintenance can be adapted according to the user's standard expansion and usage protocols.
9. Carry out downstream experimental testing such as killing assays, cytokine production, in vivo analysis using T cells generally after day 8.

Results

In this protocol, we demonstrate co-transduction and single-step immunomagnetic selection of T cells dual-transduced with two distinct retroviral vectors (termed Zip-sort, Figure 1)¹³. Each vector encodes one part of a heterodimerizing leucine zipper pair that is selectively displayed on the surface of dual-transduced T cells (Figure 1A). An affinity tag secreted zipper is expressed from Vector 1 and pairs intracellularly with a membrane-...

Discussion

Here, we have described a protocol for generating Zip-sort vector sets, producing retrovirus, co-transducing mouse and human T cells, and immunomagnetically purifying dual-transduced cells (Zip-sort). The above protocol was optimized for retroviral vectors and for mouse and human T cells. While the Zip-sort methodology was not evaluated with other vector systems, in principle, other vector systems are likely to be compatible, including lentivirus¹⁷, transposons¹⁸, or site-s...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tumor cell line related
C1498	ATCC	TIB-49
BM185-CD19	Ref. 13
BM185-CD20	Ref. 13
4D-Nucleofector Core Unit	Lonza	AAF-1003B
4D-Nucleofector X Unit	Lonza	AAF-1003X
Lonza SF buffer kit
Virus Production
10 cm Tissue culture (TC)-treated Cell Culture Dish	Corning	353003
50 mL conical tube	Thermo Scientific	339652
Effectene	Qiagen	301425
pENTR1a plasmid	Addgene	17398
LZRS-Rfa	Addgene	31601
PB MMLV puro (viral vector for mouse T cells)	Ref. 13
PB SFG blastR (viral vector for human T cells)	Ref. 13
pCMV-hyPBase	Sanger Institute
Phoenix-Eco	ATCC	CRL-3214
Phoenix-Eco alpha V beta 3	Ref. 13		Phoenix-Eco modified to adhere to culture flasks with higher avidity
PB-EF1a intron WPRE mCD4 P2A MCS integrin beta 3	Ref. 13		optional to enhance adhesion of packaging line cells to culture flasks
PB-EF1a intron WPRE hCD8a 2A integrin alpha V	Ref. 13		optional to enhance adhesion of packaging line cells to culture flasks
Biovec-RD113	Biovec Pharma
Biovec-RD113 alpha V beta 3	Biovec Pharma	This study, transfected with alpha V beta 3 vectors
Millex-HV syringe filters (0.45 μm)	Millipore	SLHVR33RRS
Polyethylene glycol 8000 (PEG)	Fisher Scientific	BP233-1
Sodium Chloride	Fisher Scientific	S271-500
Puromycin	Santa Cruz Biotech	sc-108071B
Blasticidin	Santa Cruz Biotech	sc-204655
T175 TC-treated flasks	Corning	353112
Trypsin-EDTA (0.25%), phenol red	Gibco	25200056
Cell media
2-mercaptoethanol	Gibco	21985023
RPMI1640	Gibco	11-875-119
DMEM	Gibco	11-965-118
FBS	Sigma	F2442
Glutamax	Gibco	35050061
MEM non-essential amino acids	Gibco	11140050
Penicillin-streptomycin	Gibco	15140-163
Sodium pyruvate	Gibco	11360070
T cell stimulation/transduction
15 mL conical tube	Thermo Scientific	339650
6-well non-TC plate	Gibco	351146
6-well TC plate	Gibco	353046
anti-CD19 microbeads	Miltenyi Biotec	130-121-301
InVivoMab anti-mouse CD28 (Clone 37.51)	BioXCell	BE0015-1
InVivoPlus anti-mouse CD3ε (Clone 145-2C11)	BioXCell	BE0001-1
Bovine Serum Albumin	Sigma Aldrich	A4737-1G
Dasatinib	Cayman Chemical	11498
Dynabeads Human T-Activator CD3/CD28	Gibco	11131D
EDTA (0.5 M), pH 8.0, RNase-free	Invitrogen	AM9260G
HybriMax RBC lysis buffer	Sigma	R7757-100ML
LD column	Miltenyi Biotec	130-042-901
Pan T Cell Isolation Kit, human	Miltenyi Biotec	130-096-535
Dynamag-15 Magnet	Invitrogen	12301D
Phosphate Buffered Saline (1X)	Corning	21040CV
Retronectin	Takara	T100B
rhIL-2	Proleukin (aldesleukin)
T25 cell culture flask	Corning	353109
Zip-sort
µMACS DYKDDDDK Isolation Kit	Miltenyi Biotec	130-101-591
CD34 MicroBead Kit, human	Miltenyi Biotec	130-046-702
QuadroMACS	Miltenyi Biotec	130-090-976
MACS MultiStand	Miltenyi Biotec	130-042-303
LS column	Miltenyi Biotec	130-042-401
Flow Cytometry and cell monitoring
Anti-CD90.1 Thy1.1 APC (Clone OX-7)	BD Biosciences	Cat# 561409; RRID:AB_10683163
Anti-V5 tag Antibody, (Clone 4C12E11) iFluor 488	GenScript	Cat# A01803, RRID:AB_2925231
Myc PE	Cell Signaling Technology	Cat# 2233; RRID:AB_823474
Myc Alexa 647	Cell Signaling Technology	Cat# 2233; RRID:AB_823474
APC anti-DYKDDDK Tag (Clone L5)	BioLegend	Cat# 637307; RRID:AB_2561496
Mouse Anti CD34 Antibody, Alexa Fluor 647, (Clone QBEnd/10)	Novus Biologicals	Cat# NBP2-34713AF647; RRID:AB_2925234
Streptag FITC	GenScript	Cat# A01736; RRID:AB_2622221
Incucyte SX5	Sartorius
Penteon Flow Cytometer	Agilent