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).

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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><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%&amp;nbsp;</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 &amp;mu;m&amp;nbsp;</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>&lt;strong&gt;Flow Cytometry Materials&lt;/strong&gt;</td><td>&lt;strong&gt;Manufacturer&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</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-&amp;gamma;-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 &amp;micro;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>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Manufacturer&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td></td></tr><tr><td>CliniMACS Prodigy Device&amp;nbsp;</td><td>Miltenyi Biotec GmbH</td><td>200-075-301</td><td></td></tr><tr><td>Software V1.0.0.RC</td><td></td><td></td><td></td></tr><tr><td>MACSQuant Analyzer 10</td><td>Miltenyi Biotec GmbH</td><td>130-096-343</td><td></td></tr><tr><td>Software 2.4</td><td></td><td></td><td></td></tr><tr><td>Centrifuge 5415R&amp;nbsp;</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></tr></tbody></table>

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

12.4K 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.

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

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 ...

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 ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Cancer Research

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Medicine

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...

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

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