Name: expansion and enrichment of gamma-delta (&#947;&#948;) t cells from apheresed human product
Uploaded: 2021-09-22T14:00:00
Duration: 11 min 51 s
Description: Watch this Scientific Journal Video about Expansion and Enrichment of Gamma-Delta ÃŽÂ³ÃŽÂ´ T Cells from Apheresed Human Product at JoVE.com

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hanks Balanced Salt Solution</td>
			<td>R&#38;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&#34; 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&#945;/&#946; 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&#160; 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 ÃŽÂ³ÃŽÂ´ T Cells from Apheresed Human Product at JoVE.com

Expansion and Enrichment of Gamma-Delta ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ 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 ...

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