We give thanks to the Cellular Immunotherapies-Investigator Initiated Trials Award Intramural Funding Opportunity from Moffitt Cancer Center for providing the funding for this protocol development. We also thank Dr. Claudio Anasetti for his invaluable help and guidance through this project. Finally, we thank Dr. Justin Boucher for his insights and review of the manuscript.

NOTE: IRB approval was obtained, and informed consent was obtained from the donors.
1. Lymphocyte isolation
<ol>
	<li>Transfer the apheresis product to a clean room. 
	NOTE: Process validation was performed using normal donor apheresis from an external commercial vendor compliant with raw cellular material collection regulations.</li>
	<li>Collect samples for sterility testing, cell count, and cell phenotyping.</li>
	<li>Elutriate on the counterflow centrifugation device using a primary medium of Hanks Balanced Salt Solution with 1% human serum albumin (HSA) and a secondary medium of saline solution (0.9% sodium chloride Injection USP) or Dulbecco&#39;s Phosphate-Buffered Saline (DPBS). Set the elutriation centrifugation speed at 900 &#215; g and collect fractions based on flow rate and time.</li>
	<li>Collect samples from fraction 2 and perform the following tests: 2 mL for sterility testing; 0.5 mL for cell count and viability using acridine orange/propidium iodide (AO/PI); 5 &#215; 106 cells for cell phenotyping by flow cytometry.</li>
	<li>Expand a pure lymphocyte fraction (fraction 2) of cells in culture at 10 &#215; 106 cells/cm2 in a 1 L closed-system bioreactor with 5 &#181;mol/L of zoledronic acid and 300 IU/mL of IL-2.</li>
	<li>Incubate for seven days in an incubator set at 37 &#176;C with 5% CO2.</li>
</ol>
2. Alpha-beta (&#945;&#946;) T cell depletion
<ol>
	<li>Harvest cells from the 1 L closed-system bioreactor flask. Sterile-weld a 1 L transfer pack to the red line of the closed-system bioreactor, and use the appropriate pharmaceutical pump to transfer the cells into the transfer pack.</li>
	<li>Take the following samples: 10 mL for spent medium sterility; 0.5 mL of cells for cell count and viability using AO/PI; 5 &#215; 106 cells for flow cytometry</li>
	<li>Resuspend the cells at ~5 &#215; 108 cells/mL in phosphate-buffered saline (PBS) or (PBS/ethylenediaminetetraacetic acid (EDTA)) buffer + 0.5% HSA and biotinylated TCR &#945;&#946;-specific antibody.</li>
	<li>Place the shaker in the refrigerator and incubate the cells at 2-8 &#176;C for approximately 15 min with shaking.</li>
	<li>Wash the cells with a total of 600 mL of PBS/EDTA buffer + 0.5% HSA. Centrifuge to remove the unbound antibody at 200-500 &#215; g for 15 min at 2-8 &#176;C. Resuspend ~5 &#215; 108 cells/mL in PBS/EDTA buffer + 0.5% HSA with anti-biotin-specific microbeads (7.5 mL/1 vial).</li>
	<li>Place the shaker in the refrigerator and incubate the cells at 2-8 &#176;C for approximately 15 min with shaking. After incubation, centrifuge the cells at 200-500 &#215; g for 15 min at 2-8 &#176;C to remove the unbound microbeads. Resuspend ~6 &#215; 107 cells/mL in PBS/EDTA buffer + 0.5% HSA and transfer them to a transfer pack bag.</li>
	<li>Install the tubing set in the clinical grade magnetic cell separation device by following the manufacturer&#39;s instructions, place the packs with the PBS/EDTA buffer and the transfer pack with the cell product in the instrument and spike it when instructed by the instrument.</li>
	<li>Select the Depletion 1.2 protocol for the depletion of the labeled &#945;&#946; T cells.</li>
	<li>Centrifuge the target fraction (enriched &#947;&#948; T cells) and resuspend the cells in medium supplemented with 10% human AB serum.</li>
	<li>Take a 0.5 mL sample and perform a cell count and viability with AO/PI stain. Bring cells to a final concentration of approximately 1 &#215; 106 cells/mL. Take a sample of 5 &#215; 106 cells of the product for flow cytometry phenotyping post-depletion.</li>
</ol>
3. Co-culture with aAPCs
<ol>
	<li>Irradiate 5 &#215; 107 aAPCs/flask at 100 Gy on the X-ray generating instrument.</li>
	<li>Use the aAPCs in co-culture at a 10:1 ratio with the &#947;&#948; T cells. Place the irradiated aAPCs (5 &#215; 107 cells/flask) and &#947;&#948; T cells (5 &#215; 106 cells/flask) in 1 L closed-system bioreactor flasks with 1 L of culture medium supplemented with 10% human AB serum. Seed up to 10 flasks.</li>
	<li>Expand the cells in culture for 10 days in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>Monitor glucose and lactate levels every 3-4 days using strips, glucose, and a lactate meter.</li>
	<li>If glucose drops to 250 mg/dL, reduce the volume in the flask to 200 mL using a pharmaceutical pump by sterile-welding a 1 L transfer pack to the red line of the closed system bioreactor.</li>
	<li>Mix the cells in the remaining 200 mL, and take a 0.5 mL sample for cell counting and viability measurement by AO-PI staining. If the cell count is &#8805;109, split one flask into two flasks and fill each flask up to 1 L with AIM-V supplemented with 10% human AB serum. If the cell count is &#60;109, feed the cells with a fresh liter of culture medium supplemented with 10% human AB serum.</li>
	<li>Repeat step 3.6 for all the flasks and return them to the incubator at 37 &#176;C and 5% CO2. Repeat steps 3.4-3.7 every 3 to 4 days.</li>
</ol>
4. Cell harvest
<ol>
	<li>At the end of 10 days in co-culture, harvest all the bioreactor flasks. Harvest 1 bioreactor flask at a time, and pool all the cells into a transfer pack of appropriate size. Sterile-weld the transfer pack to the red line of the closed-system bioreactor, and use the pharmaceutical pump to transfer the cells into the transfer pack.</li>
	<li>Remove the following quality control samples: 1% of the drug product (DP) for sterility by blood culture and gram staining; 0.5 mL for cell counting and viability using AO/PI; 5-10 &#215; 106 cells for flow cytometry (see Figure 1 for gating strategy); 0.5 mL for endotoxin; 0.5 mL for gram staining; 106 cells spiked into 10 mL of spent medium for Mycoplasma testing.</li>
	<li>Centrifuge the cells at 200-500 &#215; g for 15 min at room temperature and discard the supernatant.</li>
	<li>Wash the cells in a solution of balanced crystalloid solution+ 0.5% HSA at 200-500 &#215; g for 15 min at room temperature. Resuspend them in a target volume of 100-300 mL of balanced crystalloid solution + 0.5% HSA.</li>
</ol>
5. Release testing
<ol>
	<li>Perform quality control testing of the &#947;&#948; T cell DP for the following: purity and Identity by flow cytometry (Live/Dead, CD45, CD3, TCR &#945;&#946;, TCR &#947;&#948;, CD20, CD56, CD16); viability by AO/PI staining; endotoxin; Mycoplasma testing by polymerase chain reaction (PCR); sterility by gram staining and aerobic and anaerobic blood cultures; residual K562 assay by flow cytometry (CD3-CD16-CD56-CD71+).</li>
</ol>

<ol>
	<li>Bejanyan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+patients+with+acute+myeloid+leukemia+relapsing+after+allogeneic+hematopoietic+cell+transplantation:+a+center+for+international+blood+and+marrow+transplant+research+study.">Survival of patients with acute myeloid leukemia relapsing after allogeneic hematopoietic cell transplantation: a center for international blood and marrow transplant research study.</a> Biology of Blood and Marrow Transplantation. 21 (3), 454-459 (2015).</li><li>Bejanyan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcomes+of+AML+patients+relapsing+after+matched-related+donor+and+umbilical+cord+blood+transplantation.">Clinical outcomes of AML patients relapsing after matched-related donor and umbilical cord blood transplantation.</a> Bone Marrow Transplantation. 49 (8), 1029-1035 (2014).</li><li>Schmid, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment,+risk+factors,+and+outcome+of+adults+with+relapsed+AML+after+reduced+intensity+conditioning+for+allogeneic+stem+cell+transplantation.">Treatment, risk factors, and outcome of adults with relapsed AML after reduced intensity conditioning for allogeneic stem cell transplantation.</a> Blood. 119 (6), 1599-1606 (2012).</li><li>Siegers, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-leukemia+activity+of+in+vitro-expanded+human+gamma+delta+T+cells+in+a+xenogeneic+Ph++leukemia+model.">Anti-leukemia activity of in vitro-expanded human gamma delta T cells in a xenogeneic Ph+ leukemia model.</a> PLoS One. 6 (2), 16700 (2011).</li><li>Airoldi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#947;&#948;+T-cell+reconstitution+after+HLA-haploidentical+hematopoietic+transplantation+depleted+of+TCR-&#945;&#946;+/CD19++lymphocytes.">&#947;&#948; T-cell reconstitution after HLA-haploidentical hematopoietic transplantation depleted of TCR-&#945;&#946;+/CD19+ lymphocytes.</a> Blood. 125 (15), 2349-2358 (2015).</li><li>Acuto, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+T+cell+receptor:+appearance+in+ontogeny+and+biochemical+relationship+of+alpha+and+beta+subunits+on+IL-2+dependent+clones+and+T+cell+tumors.">The human T cell receptor: appearance in ontogeny and biochemical relationship of alpha and beta subunits on IL-2 dependent clones and T cell tumors.</a> Cell. 34 (3), 717-726 (1983).</li><li>Xiao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+expansion+of+V&#947;9V&#948;2+T+cells+with+engineered+K562+feeder+cells+in+G-Rex+vessels+and+their+use+as+chimeric+antigen+receptor-modified+effector+cells.">Large-scale expansion of V&#947;9V&#948;2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor-modified effector cells.</a> Cytotherapy. 20 (3), 420-435 (2018).</li><li>Peters, C., Kouakanou, L., Oberg, H. H., Wesch, D., Kabelitz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+V&#947;9V&#948;2+T+cells+for+immunotherapy.">In vitro expansion of V&#947;9V&#948;2 T cells for immunotherapy.</a> Methods in Enzymology. 631, 223-237 (2020).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+V&#947;9V&#948;2+T-cell+immunotherapy+exhibits+promising+clinical+safety+and+prolongs+the+survival+of+patients+with+late-stage+lung+or+liver+cancer.">Allogeneic V&#947;9V&#948;2 T-cell immunotherapy exhibits promising clinical safety and prolongs the survival of patients with late-stage lung or liver cancer.</a> Cellular &amp; Molecular Immunology. 18 (2), 427-439 (2021).</li></ol>

The authors have no conflicts of interest to disclose.

The Moffit Cell Therapy Lab has developed a protocol with a biphasic expansion of highly pure &#947;&#948; T cells for use as a DP in clinical trials. This protocol provides a manufacturing method under cGMP guidelines in a closed system that yields a highly pure &#947;&#948; T cell DP that is successfully activated and expanded by zoledronic acid and the WCB aAPCs. This protocol has been approved by the FDA for the manufacture of an allogeneic &#947;&#948; T cell DP for AML patients. Using healthy donors, we successfully expanded the small population of donor &#947;&#948; T cells in just 7 days from 2.06 &#177; 0.45% to 54.45 &#177; 28.34%. After the 7-day expansion with zoledronic acid, it was observed that donor 2 had an increase in the NK population.
Zoledronic acid inhibits farnesyl diphosphate synthase (FDPS) in monocytes, which, in turn, leads to the accumulation of isopentenyl pyrophosphate (IPP), which has been correlated with a significant increase in the proliferation of T cells and natural NK cells7,8,9. This increased NK population hinders the 2nd phase of expansion with the aAPCs, as the aAPCs will only contribute to the further expansion of NK cells. For this reason, the donor criteria were modified to exclude donors with high NK populations. After depletion of the &#945;&#946; T cells, the &#947;&#948; T cells were further enriched to 76.61 &#177; 27.51%. This unique protocol includes a second expansion utilizing the Moffit-manufactured aAPCs to target the CD8, CD28, and CD127L receptors in the &#947;&#948; T cells. This second expansion phase with the aAPCs yielded a DP with &#8805;65% for CD3+TCR &#947;&#948;+ T cells, &#8804;1% TCR&#945;&#946; T, and &#60;35% CD3-CD16+CD56+ NK cells. Owing to the use of K562-derived aAPCs, it was necessary to demonstrate that these aAPCs comprised &#60;1% of the final product.
The Moffit CTF developed a flow cytometric assay used for the release criteria to measure the percentage of the residual K562 cells in the final DP. This flow cytometric assay mitigates all the issues of utilizing cell surface antigens to identify the K562 cells. As activated T cells can express CD71, we devised a strategy to exclude all T cells and NK cells by gating on CD3- CD56- and CD16- populations and then examining the CD71+ cells, which would be exclusively K562 cells. This protocol demonstrates that the &#947;&#948; T cell DP yields 0.48 &#177; 0.42% of residual K562 cells and meets all the release criteria of &#8805;70% viability, Mycoplasma negativity by PCR, no organisms seen by gram staining, &#8804;2 EU/mL of endotoxin, and no growth final (14 days) blood culture sterility.

Leukemia relapse is the leading cause of mortality after hematopoietic cell transplantation (HCT) in patients with AML1,2,3. Better leukemia-free survival was reported with the increased recovery of blood &#947;&#948; T cells after HCT without increased risk of Graft-versus-Host Disease (GVHD)4. The ability of &#947;&#948; T cells to recognize antigens in an MHC-independent manner and develop strong cytolytic and Th1-like effector functions make this minor subpopulation of T cells ideal for the treatment of AML patients undergoing allogeneic transplantation at risk for relapse5. Given that the V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes ranging from 0.5% to 5% of T cells in the periphery6, we set out to establish a robust system to expand this rare population of blood cells to achieve potentially therapeutic doses for clinical trials.
Although others have successfully expanded &#947;&#948; T cells using zoledronic acid and even aAPCs, we have developed a process that can potentially expand &#947;&#948; T cells by 229,749-fold. The expansion is biphasic: first, lymphocytes are obtained by elutriation using the separation instrument. The equipment provides a closed system that allows the separation of cells based on their size, shape, and density by counterflow centrifugation. After enriching for lymphocytes, selective expansion of V&#947;9V&#948;2 T cells is achieved by treatment with zoledronic acid and IL-2 for 7 days. Immediately following this treatment, TCR-&#945;&#946; T cells are depleted using microbead technology, allowing for subsequent expansion of the &#947;&#948; T cells with K562-derived aAPCs.
For process validation, only 5 &#215; 106 zoledronic acid-expanded &#947;&#948; T cells were used for the phase-2 co-culture expansion with aAPCs. In this second phase of expansion, &#947;&#948; T cells are activated using a current Good Manufacturing Practices (cGMP)-compliant Working Cell Bank (WCB) of genetically engineered K562-derived aAPCs (K562VL6(scFv-CD3-41BBL;scFv-CD28-IL15-RA)) manufactured at Moffitt. The rationale of this biphasic expansion is based on the ability of zoledronic acid to inhibit farnesyl diphosphate synthase (FDPS) in monocytes, leading to the accumulation of isopentenyl pyrophosphate, which directly stimulates the V&#947;2V&#948;2 cells. In the second phase of expansion, the K562-derived aAPCs (K562VL6(scFv-CD3-41BBL;scFv-CD28-IL15-RA)) provide a robust stimulation to all T cells. However, the cell product has already been enriched for the &#947;&#948; T cells, resulting in a robust expansion of the &#947;&#948; T cells.
With the use of specific equipment and flasks, the process is a functionally closed system, thus decreasing the risk of contamination. In addition, the 1 L closed-system bioreactor facilitates maximal growth and expansion of cells in a total volume of 1 L of medium with minimal need for feeding. The advantage of the Moffitt method is that it provides a rapid, reproducible, and highly feasible GMP system to produce a highly pure, donor-derived &#947;&#948; T cell product for allogeneic administration. This method can be applied to any clinical trial that aims at using human &#947;&#948; T cells expressing V&#947;2V&#948;2 T cell receptors as adoptive immunotherapy to mediate immunity against microbes and tumors in cancer patients with partial and complete remission. In addition, it provides a robust platform for the development and production of &#947;&#948; chimeric antigen receptor-positive (CAR+) T cells.

<table><tbody><tr><td>Hanks Balanced Salt Solution</td><td>R&amp;amp;D</td><td>285-GMP</td><td></td></tr><tr><td>Human Albumin 25%</td><td>Grifolis</td><td>65483-16-071</td><td></td></tr><tr><td>Plasmalyte A</td><td>Fisher</td><td>2B2543Q</td><td></td></tr><tr><td>Zoledronic Acid (Zometa)</td><td>Hos pira</td><td>4215-04--8</td><td>FDA approved drug</td></tr><tr><td>DMSO</td><td>WAK-CHEMIE MEDICAL GMBH</td><td>WAK-DMSO-10</td><td></td></tr><tr><td>CS10</td><td>BIOLIFE SOLUTIONS</td><td>210374</td><td></td></tr><tr><td>3 mL syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 mL syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>20 mL syringe</td><td>BD</td><td>302830</td><td></td></tr><tr><td>50 mL syringe</td><td>BD</td><td>309653</td><td></td></tr><tr><td>100 mL syringe</td><td>JMS</td><td>992861</td><td></td></tr><tr><td>18g Needle</td><td>Fisher</td><td>305198</td><td></td></tr><tr><td>Cryovials 1.8 mL</td><td>Fisher</td><td>375418</td><td></td></tr><tr><td>5 mL pipette</td><td>Fisher</td><td>1367811D</td><td></td></tr><tr><td>50 mL pipette</td><td>Fisher</td><td>1367610Q</td><td></td></tr><tr><td>10 mL pipette</td><td>Fisher</td><td>1367811E</td><td></td></tr><tr><td>100 mL pipette</td><td>Fisher</td><td>07-200-620</td><td></td></tr><tr><td>15 mL conical</td><td>Fisher</td><td>05-539-12</td><td></td></tr><tr><td>50 mL conical</td><td>Fisher</td><td>05-539-7</td><td></td></tr><tr><td>250 mL conical</td><td>Fisher</td><td>430776</td><td></td></tr><tr><td>600 mL Transfer Pack</td><td>TERUMO BCT INC</td><td>1BBT060CB71</td><td></td></tr><tr><td>4&quot; Plasma Transfer Set</td><td>INDEPENDENT MEDICSL ASSOCIATES</td><td>03-220-90</td><td></td></tr><tr><td>Elutra Tubing Set</td><td>TerumoBCT</td><td>70800</td><td></td></tr><tr><td>100 MCS GREX</td><td>WILSON WOLF MFG CORP</td><td>81100-CS</td><td></td></tr><tr><td>Ashton Sterile Pumpmatic Liquid dispensing system</td><td>Fisher Scientific</td><td>22-246660</td><td></td></tr><tr><td>Acacia Pump boot</td><td>MPS Medical In</td><td>17789HP3MLL</td><td></td></tr><tr><td>CliniMACS PBS/EDTA Buffer</td><td>Miltenyi Biotec Inc</td><td>130-070-525</td><td></td></tr><tr><td>Dornase Alpha</td><td>Genentech, Inc</td><td>50242-100-40/186-0055</td><td>FDA approved drug</td></tr><tr><td>1000 mL 0.22 um Filter</td><td>Fisher</td><td>157-0020</td><td></td></tr><tr><td>Blood Filter 170um</td><td>B.Braun</td><td>V2500</td><td></td></tr><tr><td>CliniMACs Tubing set</td><td>Miltenyi Biotec Inc</td><td>130-090-719</td><td></td></tr><tr><td>CliniMACS TCR&amp;alpha;/&amp;beta; Kit</td><td>Miltenyi Biotec Inc</td><td>130-021-301</td><td></td></tr><tr><td>Y-Type blood set</td><td>Fenwal</td><td>FWL4C2498H</td><td></td></tr><tr><td>75 mL Flask</td><td>Fisher</td><td>430641U</td><td></td></tr><tr><td>IL-2</td><td>Prometheus</td><td>65483-116-071</td><td>FDA approved drug</td></tr><tr><td>AIM-V</td><td>Fisher</td><td>0870112BK</td><td></td></tr><tr><td>Human AB serum</td><td>Gemini Bio-Product</td><td>100H41T</td><td></td></tr><tr><td>3 Liter Transfer pack</td><td>Independent Medical Associates</td><td>T3109</td><td></td></tr><tr><td>1000&amp;nbsp; pipette tips</td><td>Fisher Scientific</td><td>5991040</td><td></td></tr><tr><td>CF-250</td><td>KOLBio</td><td>CF-250</td><td></td></tr><tr><td>Elutra</td><td>TERUMOBCT</td><td></td><td></td></tr><tr><td>CliniMACS</td><td>Miltenyi Biotec Inc</td><td></td><td></td></tr><tr><td>GatheRex Liquid Handling, Cell Harvest Pump</td><td>WILSON WOLF MFG CORP</td><td></td><td></td></tr><tr><td>HERAcell Vios CO2 Incubator</td><td>Thermo Scientific</td><td></td><td></td></tr></tbody></table>

expansion and enrichment of gamma-delta (&#947;&#948;) t cells from apheresed human product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Watch this Scientific Journal Video about Expansion and Enrichment of Gamma-Delta Î³Î´ T Cells from Apheresed Human Product at JoVE.com

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

expansion-enrichment-gamma-delta-t-cells-from-apheresed-human

Research

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Cancer Research

8.8K Views.  Moffitt Cancer Center and Research Institute. Presented is a protocol for the expansion of Gamma-Delta (γδ) T cell drug product. Lymphocytes are isolated by elutriation and γδ-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The γδ cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded. 

Video: Expansion and Enrichment of Gamma-Delta γδ T Cells from Apheresed Human Product

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells 

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Immunology and Infection

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

Engineering

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Expansion and Enrichment of Gamma-Delta (γδ) T Cells from Apheresed Human Product

Summary

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Chapters in this video

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Expansion of Human Peripheral Blood γδ T Cells using Zoledronate

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

Isolation and Ex Vivo Culture of Vδ1⁺CD4⁺γδ T Cells, an Extrathymic αβT-cell Progenitor

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles