We thank Miltenyi Biotec, Germany for providing reagents and CliniMACS Prodigy equipment for evaluation studies. We thank George T. McNamara (Pediatric department, MD Anderson Cancer Center) for proof reading the manuscript. Grant support: Cancer Center Core Grant (CA16672); RO1 (CA124782, CA120956, CA141303; CA141303); R33 (CA116127); P01 (CA148600); Burroughs Wellcome Fund; Cancer Prevention and Research Institute of Texas; CLL Global Research Foundation; Estate of Noelan L. Bibler; Gillson Longenbaugh Foundation; Harry T. Mangurian, Jr., Fund for Leukemia Immunotherapy; Institute of Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lymphoma Research Foundation; MDACC&#8217;s Sister Institution Network Fund; Miller Foundation; Mr. Herb Simons; Mr. and Mrs. Joe H. Scales; Mr. Thomas Scott; National Foundation for Cancer Research; Pediatric Cancer Research Foundation; William Lawrence and Blanche Hughes Children's Foundation.

1. Preparation of Materials under Sterile Conditions (See Materials and Equipment Table)
<ol>
	<li>Prepare 3 L of PBS/EDTA buffer supplemented with human serum albumin (HSA) to a final concentration of 0.5% (w/v).</li>
	<li>Prepare 1 L bag of clinical grade 0.9 % sodium chloride (NaCl) solution and 2 L of GMP grade cell culture medium.</li>
	<li>Prepare 60 nmol of CMV-specific peptide antigen cocktail by reconstituting one vial of CMV pp65 with 8 ml of sterile water.</li>
	<li>Transfer CMV pp65 peptide cocktail into a 50 ml volume freezing bag using a Luer/Spike interconnector and clamp with locking forceps to avoid subsequent distribution of cocktail into the tubing set. Open cell enrichment tubing set (TS 500) under sterile conditions.</li>
	<li>Using sterile tubing welder, connect peptide cocktail freezing bag into tube connection for valve 2 of tubing set TS 500. Do not open the peptide cocktail bag clamp at this time.</li>
	<li>Remove 1 x 109 TNC from starting cellular product and suspend in PBS/EDTA buffer containing 2.5% HSA to a total volume of 50 ml. Inject the cellular product into a 150 ml transfer bag.</li>
</ol>
2. Preparation and Use of Automated Cell Enrichment System (See Materials and Equipment Table)
<ol>
	<li>Switch on the cell enrichment system (Figure 1B) and select the program &#8220; CCS_IFN-&#947; Enrichment&#8221;. Observe a user interface showing screens with instructions and pictures guiding the operator through the procedure.</li>
	<li>Enter the parameter &#8220;Operator&#8221; and &#8220;Tubing Set P/N No&#8221;. Next, install the Tubing Set 500 to automated cell enrichment device as per instructions displayed on the interactive monitor screen.</li>
	<li>Follow the step by step instructions displayed on the screen to connect the medium and buffers to the device. Record catalogue number and lot number of the reagents before connecting to the instrument.</li>
	<li>After the final check of the tubing set, open the clamp of the peptide cocktail bag. Open the medium bag and initiate automatic priming of the tubing set.</li>
	<li>After the priming step is completed, supplement HSA (2.5%) into NaCl buffer in the reservoir bag (200 ml) with the help of the sterile tubing welder. Transfer the starting cellular product into the &#8220;Application bag&#8221; using the sterile tubing welder.</li>
	<li>Connect CCS (IFN&#947;) reagents into respective tubing via adapters. Enter preferred time to collect a fraction of cellular material before enrichment process. Review and verify the accuracy of all data/parameters entered. Start the process.</li>
	<li>Before the start of automated cell enrichment process, remove the Quality Control Bag (QCB, original fraction (ori) contains approximately 1.3 ml out of 100 ml chamber content diluted with PBS/EDTA buffer). Seal the QCB, weigh, and store at 4 &#176;C.</li>
	<li>Start the enrichment process. At the end of the process, target cells will be eluted with an approximate volume of elution buffer from the reservoir bag.</li>
	<li>Seal the Non Target Cell Bag (NTCB, negative fraction = neg) and Target Cell Bag (TCB, positive fraction = pos) and weigh each bag. The weights will be used later for calculation of the cell numbers.</li>
	<li>Immediately after the enrichment procedure collect two aliquots per fraction for flow cytometry analysis, and store the rest of the samples at 4 &#176;C. Use one sample aliquot for cell count determination and the other sample aliquot for the enrichment performance analysis (Table 1).</li>
	<li>Remove the tubing set from the cell enrichment instrument. Transfer the log file to a USB drive for future use. 
	NOTE:&#160; All reagents should be prepared under sterile conditions. The use of a Biosafety type II hood is highly recommended. Use steady-state apheresis cellular product (non-mobilized) isolated from a healthy CMV-seropositive donor to enrich CMV antigen specific T cells. Only FDA licensed HSA should be used. The buffer for cell preparation should be kept at&#160; +19 &#176;C to +25 &#176;C as lower or higher ambient temperatures will result in reduced purity and a reduced yield of the target cells.</li>
</ol>
3. Cell Count Determination
<ol>
	<li>Take the aliquots of QCB , NTCB and TCB for cell counts as shown in Table 1. Add CD45-VoBlue to each aliquot (titer 1:11) and incubate in the dark for 10 min at 4 &#176;C.</li>
	<li>Add 1.5 ml freshly prepared red blood cell lysis solution (1x) to the original fraction and negative fraction, 450 &#181;l freshly prepared red blood cell lysis solution to the positive fraction, and incubate all fractions for 15 min at RT.</li>
	<li>Just prior to analysis, add propidium iodide to a final concentration of 1 &#181;g/ml (1:100 dilution of 100 &#181;g/ml). Use automatic cell counter to determine cell count and viability. Use cell counter device recommended software for flow cytometry analysis. Determine the absolute counts of leukocytes for original, negative and positive fractions. 
	&#160;NOTE: The cell count of viable leukocytes per ml of the samples taken for cell count analysis is determined using the cell analyzer recommended software.</li>
	<li>Set the region as shown in Figure 2 (region 5, viable leukocytes). Use the following gating strategy to determine cell count. A viable leukocyte in original fraction is shown in Figure 2.</li>
	<li>The indicated regions (Figure 2, 1-6) are hierarchically as follows: 
	1: Time gate &#8594; 2: Single cells &#8594; 3: CD45+ cells &#8594; 4: Leukocytes (debris excluded) &#8594; 5: Viable leukocytes&#160;&#8594; 6: Viable lymphocytes</li>
	<li>Repeat the same steps to determine cell counts for negative and positive fractions. Calculate the cell count of the whole fraction by considering the diluent factor of the sample and total volume of the fraction (Table 2).</li>
</ol>
4. Examination of the Separation Performance
<ol>
	<li>Wash the aliquots of QCB, NTCB and TCB fraction cells with pre-chilled PBS/EDTA buffer/0.5% AB serum. Centrifuge the cells at 300 x g for 5 min at 4 &#176;C and aspirate the supernatant.</li>
	<li>Resuspend cells in 100 &#181;l antibody-fluorochrome staining mixture containing: CD3-FITC, CD4-APC, CD8-APC-Vio770, CD14-PerCP, CD20-PerCP, CD45-VioBlue and anti-IFN&#947;-PE (titer 1:11) and incubate in the dark for 10 min at 4 &#176;C.</li>
	<li>Add 1 ml freshly prepared red blood cell lysis solution (1x) and incubate for 15 min at RT. Centrifuge at 300 x g for 5 min at 4 &#176;C and aspirate the supernatant. Resuspend the cells in an adequate volume of PBS/EDTA Buffer/ 0.5% AB Serum.</li>
	<li>Add propidium iodide to a final concentration of 1 &#181;g/ml just prior to analysis (1:100 dilution of 100 &#181;g/ml). Perform flow cytometry analysis to evaluate the purity of the sample.</li>
	<li>Use the following gating strategy to calculate the CD3+ T cells in viable leukocyte Gating strategy for determining the CD3+ T cells is shown in positive fraction after CCS enrichment process. The indicated regions (Figure 3A and 3B, 1-6) are hierarchically linked as follows: 
	1: Time gate &#8594; 2: Single cells &#8594; 3: CD45+ cells &#8594; 4: Cells (debris excluded) &#8594; 5: Viable leukocytes &#8594; 6: Viable CD3+ cells population</li>
	<li>Determine the frequencies of CD4+, CD8+, CD4+ IFN-&#947;+ and CD8+ IFN-&#947;+ T cells after CCS enrichment process (Table 2).</li>
	<li>Use the gating strategy to determine the frequencies of CD4+, CD8+, CD4+ IFN-&#947;+ and CD8+ IFN-&#947;+ T cells shown below for an original and enriched (captured) positive fraction after CCS process. The indicated regions are hierarchically linked and named as follows: 
	1: Time gate &#8594; 2: Single cells&#160;&#8594; 3: CD45+ cells&#160;&#8594; 4: Cells (debris excluded) &#8594; 5: Viable Leukocytes &#8594; 6: Viable CD3+ cells &#8594; 7: CD4+ cells &#8594; 7a: CD4+ IFN-&#947;+ cells (box) &#8594; 8: CD8+ cells &#8594; 8a: CD8+ IFN-&#947;+ cells (box) &#160;</li>
</ol>
NOTE: The first 6 indicated regions of the hierarchy links are the same as Figure 3, (1-6) and last 2 regions are shown in Figure 4 (6-8a).

<ol>
	<li>Syed, B. A., Evans, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+Analyst's+Couch+Stem+Cell+Therapy+Market.">From the Analyst's Couch Stem Cell Therapy Market.</a> Nat Rev Drug Discov. 12 (3), 185-186 (2013).</li><li>Maus, M. V., 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=Adoptive+Immunotherapy+for+Cancer+or+Viruses.">Adoptive Immunotherapy for Cancer or Viruses.</a> Annu Rev Immunol. 32, 189-225 (2014).</li><li>Kumaresan, P. R., 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=Bioengineering+T+cells+to+target+carbohydrate+to+treat+opportunistic+fungal+infection.">Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.</a> Proc Natl Acad Sci U S A. 111 (29), 10660-10665 (2014).</li><li>Singh, H., 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=Redirecting+specificity+of+T-cell+populations+for+CD19+using+the+Sleeping+Beauty+system.">Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system.</a> Cancer Res. 68 (8), 2961-2971 (2008).</li><li>Kumaresan, P. 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Rise of Cell-Based Immunotherapy : Personalized Medicine Takes Next Step Forward.</a> Genetic Engineering &amp; Biotechnology News. 33 (5), 12-13 (2013).</li><li>Apel, 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=Integrated+Clinical+Scale+Manufacturing+System+for+Cellular+Products+Derived+by+Magnetic+Cell+Separation,+Centrifugation+and+Cell+Culture.">Integrated Clinical Scale Manufacturing System for Cellular Products Derived by Magnetic Cell Separation, Centrifugation and Cell Culture.</a> Chem-Ing-Tech. 85 (1-2), 103-110 (2013).</li><li>Brestrich, G., 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=Adoptive+T-Cell+Therapy+of+a+Lung+Transplanted+Patient+with+Severe+CMV+Disease+and+Resistance+to+Antiviral+Therapy.">Adoptive T-Cell Therapy of a Lung Transplanted Patient with Severe CMV Disease and Resistance to Antiviral Therapy.</a> Am J Transplant. 9 (7), 1679-1684 (2009).</li><li>Feuchtinger, T., 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+grade+generation+of+hexon-specific+T+cells+for+adoptive+T-cell+transfer+as+a+treatment+of+adenovirus+infection+after+allogeneic+stem+cell+transplantation.">Clinical grade generation of hexon-specific T cells for adoptive T-cell transfer as a treatment of adenovirus infection after allogeneic stem cell transplantation.</a> J Immunother. 31 (2), 199-206 (2008).</li><li>Peggs, K. 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M., Remberger, M., Alvin, O., Karlsson, H., Ringden, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Costs+after+Allogeneic+Haematopoietic+Sct+Are+Associated+with+Major+Complications+and+Re-Transplantation.">Increased Costs after Allogeneic Haematopoietic Sct Are Associated with Major Complications and Re-Transplantation.</a> Biol Blood Marrow Transplant. 18 (2), S339-S339 (2012).</li><li>Leen, A. 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=Multicenter+study+of+banked+third-party+virus-specific+T+cells+to+treat+severe+viral+infections+after+hematopoietic+stem+cell+transplantation.">Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation.</a> Blood. 121 (26), 5113-5123 (2013).</li></ol>

Both MD Anderson Cancer Center and Dr. Cooper have a financial interest in ZIOPHARM Oncology, Inc., and Intrexon Corporation. On May 7, 2015, Dr. Cooper was appointed as the Chief Executive Officer at ZIOPHARM Oncology.&#160; Dr. Cooper is now a Visiting Scientist at MD Anderson. Dr. Cooper founded and owns InCellerate, Inc. He has patents with Sangamo BioSciences with artificial nucleases. He consults with Targazyme, Inc. (formerly American Stem cells, Inc.), GE Healthcare, Ferring Pharmaceuticals, Fate Therapeutics, Janssen Pharmaceuticals, and Bristol-Myers Squibb. He is on the Scientific Advisory Board of Cellectis. He receives honoraria from Miltenyi Biotec.

Adoptive T-cell therapy has emerged as a viable option to treat B-cell malignancies4. Its therapeutic potential is dependent on infusing the desired number of target antigen specific T cells that lack replicative senescence2. This can be achieved by sorting out a pure population of antigen specific T cells from expanded T cells in compliance with current good manufacturing practices. Two sorting procedures are widely used, namely, fluorescence-activated cell sorting (FACS) and magnetic activated cel...

Hematopoietic stem-cell transplantation (HSCT) 1 can be combined with adoptive T-cell therapy to improve graft-versus-tumor effect and to provide immunity to opportunistic infections2. Generation of antigen-specific donor-derived T cells for infusion has historically required skilled personnel and use of specialized facilities that are GMP-compliant. The delivery of such T cells has resulted in resolution of opportunistic infections3 as well as treating the underlying malignancy4. Recently, investigators have demonstrated that the adoptive transfer of only few thousand virus-specific T cells (~ 1 x 104 &#8211;&#160; 2.5 x 105 cells/kg recipient body weight) can successfully treat opportunistic CMV infections after allogeneic HSCT5-9. A limited number of GMP facilities with associated skilled manufacturing requirements and the high cost associated with cell production has, however, restricted patient access to promising T-cell therapies10. One approach to isolating antigen-specific T cells is based on the CCS using a bi-specific reagent to recognize CD45 and IFN-&#947;. As is shown, this methodology can be used to generate clinical-grade CMV-specific T cells employing an automated cell enrichment CCS device (Figure 1B).
CMV-specific T cells are generated by incubating overlapping peptides from CMV pp65 antigen with leukapheresis total nuclear cells (TNC) from CMV-seropositive donors. These peptides, displayed in the context of human leukocyte antigen (HLA), activate the CMV pp65-specific T cells within the TNC to secrete IFN-&#947;. These T cells can then be &#8220;captured&#8221; and magnetically separated. The operation of the first-generation cell enrichment device (Figure 1A) required personnel skilled in cell culture under GMP conditions, and coordination of staff to undertake the multiple steps necessary to generate a &#8220;captured&#8221; product.
The procedure typically required 10 to 12 hr of continuous operation, and therefore personnel likely need to work over two shifts in the GMP facility. These constraints are now obviated by the implementation of a second-generation device (shown in Figure 1B). This device undertakes magnetic enrichment, similar to the first generation device, but automates other aspects of the CCS in an unbreached approach. This significantly reduces the burden on the GMP team as most of the steps can be accomplished unattended by staff. Furthermore, since the device operates as a closed system, the antigen-specific T cells can be captured and processed on the benchtop except the steps involved in leukapheresis isolation and preparation of materials before starting the instrument. Details of the complete instrumentation and functionality of this second-generation cell enrichment device have been published11.
Here, we describe the steps to enrich CMV pp65-specific T cells from a steady-state apheresis product using the automated cell enrichment CCS system. Once isolated, these CMV-specific T cells may be immediately infused into a patient.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>CliniMACS PBS/EDTA Buffer 3 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>700-29</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Tubing Set TS 500</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-182</td>
			<td></td>
		</tr>
		<tr>
			<td>5 L waste bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>110-004-067</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Cytokine Capture System (IFN-gamma)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>279-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin (Human) 25%&#160;</td>
			<td>Grifols</td>
			<td>58516-5216-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer/Spike Interconnector</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-701</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9 % NaCl Solution (1 L)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS GMP PepTivator HCMV pp65</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for injections</td>
			<td>Hospira, inc, Lake Forest, IL</td>
			<td>NDC-0409-4887-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLEX GV Filter Unit 0.22 &#956;m&#160;</td>
			<td>Millipore</td>
			<td>SLGV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>TexMACS GMP Medium 2 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-306</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Bag, 150 mL (for cellular starting material)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-301</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoMACS Freezing Bag 50</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>200-074-400</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL Syringes, sterile</td>
			<td>BD, Laagstraat, Temse, Belgium</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>CMV sero positive apheresis product</td>
			<td>Key Biologics, LLC, Memphis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Materials</td>
			<td>Manufacturer</td>
			<td>Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>AB Serum, GemCell</td>
			<td>Gemini Bio-Products, West Sacramento, USA</td>
			<td>100-512</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-FITC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-080-401</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-APC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-033</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8-APC-Vio770</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-065</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-072</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-077</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-VioBlue</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-136</td>
			<td></td>
		</tr>
		<tr>
			<td>aIFN-&#947;-PE, human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-940</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-PE</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-091-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide Solution (100 &#181;g/mL)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-093-233</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Device&#160;</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">200-075-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Software V1.0.0.RC</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQuant Analyzer 10</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">130-096-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Software 2.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5415R&#160;</td>
			<td>Eppendorf AG</td>
			<td>22331</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer K2</td>
			<td>Nexelom Bioscience, Lawrence, MA</td>
			<td>LB-001-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tubing welder SCDIIB</td>
			<td>Terumo Medical Corp., Elkton, MA</td>
			<td>7811</td>
			<td></td>
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automated cell enrichment of cytomegalovirus-specific t cells for clinical applications using the cytokine-capture system

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

In this study, an automated cell enrichment CCS System was used for automated production of CMV pp65-specific T cells. CMV-specific T cells were enriched from three apheresis cell products. The steady-state apheresis product was harvested over 2 hr from a CMV-seropositive donor and generated 1010 total nuclear cells (TNC). 109 TNC were then activated with CMV pp65-derived peptides (60 nmol) for 4 hr and the IFN-&#947; secreting T cells were isolated using the CCS on the automated cell enrichment dev...

Watch this Scientific Journal Video about Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System at JoVE.com

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

automated-cell-enrichment-cytomegalovirus-specific-t-cells-for

Research

JoVE Journal

Immunology and Infection

12.3K Views.  U.T. MD Anderson Cancer Center. The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.