This work was supported by a Dan L. Duncan collaborative research grant (C.M.B and E.J.S), the National Heart, Lung, and Blood Institute (US4HL081007), a Leukemia and Lymphoma Society Clinical Research Scholar award (C.M.B), and the National Cancer Institute (RO1 CA06150816; E.J.S).

1. Mononuclear Cell Isolation (day 0)
 <ol>
 <li>Insert spike of female luer adapter into outlet port of the 20% fraction of the cord blood unit, attach syringe and remove blood. Transfer thawed blood to 20 mL of warmed RPMI in 50 mL centrifuge tube. Rinse cord blood bag with 5 mL of RPMI and transfer to the same centrifuge tube.</li>
 <li>Centrifuge cells for 10 minutes at 400 x g. Aspirate supernatant.</li>
 <li>Resuspend cells in 20 mL of warm RPMI. Layer cells onto 15 mL of Lymphoprep in 50 mL centrifuge tube. Centrifuge 40 minutes @ 400 x g.</li>
 <li>Harvest the interface containing the mononuclear cells and wash in 20 mL of RPMI. 
Centrifuge for 10 minutes @ 450 x g.
</li>
 <li>Aspirate supernatant and wash in 20 mL of RPMI. Count cells and spin for 5 minutes @ 400 x g.</li>
 <li>Aspirate supernatant and resuspend cells at 5 x 106 mononuclear cells/mL of Cellgenix DC media (serum free). Remove 1 mL for EBV-LCL generation and put into 15 mL centrifuge tube.</li>
 </ol>
2. Dendritic Cell Generation (starting on day 0)
 <ol>
 <li>Plate 2 mL of cells (are already at 5 x 106 cells/mL of DC media) per well into a 6-well plate. Leave in incubator for 1-2 hours. </li>
 <li>After 1-2 hours, wash off the non-adherent cells by washing 3-4 times with PBS. Collect the non-adherent cells and freeze. Add 2 mL/well of DC media containing 1000 U/mL of IL-4 and 800 U/mL GM-CSF. </li>
 <li>3-4 days after DC initiation, replenish the IL-4 and GM-CSF by adding 100 &mu;L/well of media containing a final concentration of 1000U/mL IL-4 and 800 U/mL GM-CSF.</li>
 <li>On day 5-6, harvest the DC by scraping the well with a transfer pipette. Count the big cells and centrifuge for 5 minutes @ 400 x g. Aspirate media and resuspend DC at 2x106 cells/mL of DC media. Add 0.5 mL/well of a 24-well plate.</li>
 <li>Transduce the DC using the Ad5f35pp65 vector at an MOI per cell of 10 infectious units. Add vector to each well in a 24-well plate. Incubate cells for 1.5 hours. After 1.5 hours, add 1.5 mL/well of DC media containing: 1000 U/mL IL-4, 800 U/mL GM-CSF, 10 ng/mL TNF-alpha, 1 &mu;g/mL PGE-1, 100 ng/mL IL-6, and 10 ng/mL IL-1beta. These cytokines mature the DCs.</li>
 </ol>
3. Generation of EBV-LCL (starting on day 0)
 <ol>
 <li>Centrifuge the tube containing the 5 x 106 mononuclear cells. Add 200 &mu;L of concentrated EBV B95.8 supernatant and 1.8 mL of complete media (RPMI + 10% FBS + 2mM L-glutamine) containing cyclosporine (1 &mu;g/mL).</li>
 <li>Aliquot 100 &mu;L of cells into 10 wells of a 96-well plate and 200 &mu;L into 5 wells. Add 100 &mu;L of complete media + cyclosporine to the wells containing only 100 &mu;L of cells. Fill remaining wells with sterile water.</li>
 <li>Feed the LCL weekly and expand from a 96-well plate to a 24-well plate after ~2 weeks. After another week, transfer to a T25 flask and in the subsequent week transfer to a T75 flask. At this stage it is recommended that you freeze aliquots of LCL. It typically takes 1 month to generate sufficient numbers of LCL.</li>
 </ol>
4. CTL Initiation &ndash; 7 days After Initiation of DCs
 <ol>
 <li>Harvest DCs and irradiate at 30 Gy. Wash 4 x and count. Resuspend DC in T cell media containing human serum (45% RPMI, 45% CLICK's, 10% human serum, 2mM L-glutamine) @ 1 x 105 DCs/mL.</li>
 <li>Thaw non-adherent cells from step 2.2. Wash cells, and count. Resuspend at 2 x 106 non-adherent cells/mL in T cell media containing human serum. Add 10 ng/mL IL-7 and IL-12, and 5 ng/mL IL-15. Add 1 mL of cells per well in a 24-well plate. Add 1 mL of DC per well. Fill empty wells of 24-well plate with sterile water. 
</li>
 <li>7 days after T cell initiation, feed and/or split the cells with T cell media containing human serum.</li>
 <li>9-12 days after T cell initiation, freeze the T cells until the LCL are ready. </li>
 <li>Once LCL have expanded and are passaged in T75 flasks, transduce the LCL by centrifuging for 5 minutes @ 400 x g. Add the vector to the cell pellet at an MOI of 100 infectious units per cell. Incubate for 1.5 hours. After 1.5 hours, resuspend in complete media at 5 x 105 LCL/mL and add 2 mL per well in a 24-well plate.12 </li>
 <li>After 2 days, harvest the LCL and irradiate at 40 Gy. Wash 4 x and count. Resuspend the LCL in T cell media containing human serum at 2.5 x 105 cells/mL. Add 1 mL of LCL per well of a 24-well plate. Also, add 5 x 106 LCL to a Grex40 culture device as well as 5 ng/mL IL-15.13</li>
 <li>Harvest the T cells. Centrifuge for 5 minutes @ 400 x g , aspirate the supernatant, and resuspend in T cell media containing human serum at 1 x 106 T cells/mL. Add 5 ng/mL IL-15 and add 1 mL/well of T cells (total of 1 x 106 T cells) to the LCL that are already in the 24-well plate. In the Grex40, bring the total volume of media up to 30 mL.</li>
 <li>On day 3-4 post-stimulation, if the cells are confluent then split them (in the plate) 1:1 and add fresh media containing 50 U/mL IL-2. If they are not confluent then simply aspirate &frac12; the media and replace with media containing 50 U/mL IL-2. In the Grex40, aspirate half the media, replace with fresh media, and add 50 U/mL IL-2.</li>
 <li>On day 7, repeat as in step 4.6 but use IL-2 on the day of the stimulation, not IL-15. </li>
 </ol>
5. Representative Results
A schematic of the GMP-compliant FDA-approved manufacturing protocol is depicted in Figure 1. The process takes about 50 days. Typical methods of generating virus-specific T cells expand pre-existing memory T cells; however, cord blood lacks virus-experienced T cells and therefore we need to prime na&iuml;ve T cells ex vivo. To do so, we use dendritic cells as well as the cytokines IL-7, IL-12, and IL-15, necessary for generating viral specificity.
After 3 stimulations, the yield should be over 100x106 cells. If sufficient cells are not available, additional stimulations can be performed until the desired number of CTLs are available. The majority of these cells should be CD3+ with a mixture of CD4+ and CD8+ T cells. There should be less than 15% NK cells (CD3-/CD56+) and less than 1% CD19+ B cells (Figure 2).
The expanded CTL should recognize the antigen pp65 from CMV, hexon and penton from adenovirus, as well as numerous EBV antigens that are expressed on EBV-LCL. When tested in an ELISPOT assay, CTL should secrete more IFN-? in response to these antigens than irrelevant antigens (Figure 3).
The CTL should also lyse viral peptide-pulsed targets such PHA blasts. In a 51Cr release assay, CTL should lyse LCL, CMVpp65-pulsed and Adenovirus hexon- and/or penton-pulsed targets but not targets pulsed with irrelevant peptides (Figure 4).
<img src="/files/ftp_upload/3627/3627fig1.jpg" alt="Figure 1."/> Figure 1. Generation of multi-virus-specific CTL from cord blood. Schematic showing the entire process of CTL manufacture from cord blood. First the cord blood mononuclear cells are isolated from the 20% fraction of the fractionated umbilical cord blood unit. With the exception of 5x106 cells that are saved for LCL generation, all the cells are then plated in dendritic cell media for 1-2 hours, at which point the non-adherent cells are harvested and frozen. The DC are then fed DC media containing IL-4 and GM-CSF. After 5 days of culture, the DC are matured and transduced with an adenoviral vector containing the immunodominant CMV antigen pp65. At initiation, these DC are combined with the non-adherent cells as well as the cytokines IL-7, IL-12, and IL-15. At subsequent stimulations, the same adenoviral vector is used to transduce EBV-LCL, which are used as the antigen presenting cells. IL-15 is used at the second stimulation and IL-2 thereafter.
<img src="/files/ftp_upload/3627/3627fig2.jpg" alt="Figure 2."/> Figure 2. Phenotype of resulting T cells. Shown is the percentage of live cells included in the lymphocyte gate. CTL are CD3+ and CD4+ or CD8+ but largely CD3-/CD56- and CD19-.
<img src="/files/ftp_upload/3627/3627fig3.jpg" alt="Figure 3."/> Figure 3. T cell functionality. T cell specificity was tested by IFN- &gamma;ELISPOT. T cells were pulsed with overlapping peptides spanning the entire protein of hexon, penton, pp65, and the irrelevant antigen IE-1. CTL alone indicates media alone. Autologous LCL were irradiated and added at 1x105/well. Shown is the mean spot forming cell count of triplicate wells.
<img src="/files/ftp_upload/3627/3627fig4.jpg" alt="Figure 4."/> Figure 4. Cytolytic activity of CTL. The ability of the resulting CTL to lyse targets expressing viral antigens was tested in a 51Cr assay. 51Cr-labelled Autologous PHA blasts were pulsed with overlapping peptides spanning the entire antigen or 51Cr-labelled Autologous LCL were cocultured with CTL. After 4 hours, gamma release was counted on a gamma counter.

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The authors have nothing to disclose.

Current strategies aimed at controlling viral infections after CBT can be effective, but they are associated with significant toxicities, are expensive, and do not confer long term protection against later infection. In fact, the use of some antiviral drugs may limit the expansion of virus-specific T cells that would otherwise be protective.14 Another option is the infusion of donor-derived virus-specific T cells. We and others have shown that such T cells are safe, efficacious, and cost-effective.15-17</...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Invitrogen</td>
 <td>21870-076 </td>
 </tr>
 <tr>
 <td>DC media</td>
 <td>CellGenix</td>
 <td>20801-0500 </td>
 </tr>
 <tr>
 <td>EHAA (Click&rsquo;s Medium)</td>
 <td>Irvine Scientific</td>
 <td>9195 </td>
 </tr>
 <tr>
 <td>Human Serum</td>
 <td>Gemini Bio Products</td>
 <td>100-110 </td>
 </tr>
 <tr>
 <td>Gas Permeable Cultureware18 </td>
 <td>Wilson-Wolf</td>
 <td>80040S</td>
 </tr>
 <tr>
 <td>IL-2</td>
 <td>Chiron (TCH Pharmacy)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>IL-12</td>
 <td>NCI/CTEP</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>IL-15</td>
 <td>CellGenix</td>
 <td>1013-050 </td>
 </tr>
 <tr>
 <td>IL-7</td>
 <td>R&amp;D</td>
 <td>AFL207 </td>
 </tr>
 <tr>
 <td>IL-1beta</td>
 <td>R&amp;D</td>
 <td>AFL201 </td>
 </tr>
 <tr>
 <td>IL-6</td>
 <td>CellGenix</td>
 <td>1004-050 </td>
 </tr>
 <tr>
 <td>GM-CSF</td>
 <td>TCH Pharmacy</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>IL-4</td>
 <td>R&amp;D</td>
 <td><a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a></td>
 </tr>
 <tr>
 <td>TNF-alpha</td>
 <td>R&amp;D</td>
 <td>AFL210</td>
 </tr>
 <tr>
 <td>Ad5f35pp65</td>
 <td>BCM CAGT Vector Production Facility</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Plasma transfer set with female luer adapter</td>
 <td>Charter Medical</td>
 <td>89-550-66j</td>
 </tr>
 <tr>
 <td>Lymphoprep</td>
 <td>Nycomed</td>
 <td>1114550</td>
 </tr>
 </tbody>
 </table>

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.

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

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

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 ...

expanding-cytotoxic-t-lymphocytes-from-umbilical-cord-blood-that

Research

JoVE Journal

Immunology and Infection

12.7K 조회수.  Baylor College of Medicine. 여기가 첫 번째 좋은 생산 관행 (GMP)을 준수하는 탯줄 혈액, 주로 나이브 T 세포의 소스에서 바이러스 특정 세포 독성 T 림프구 (CTL)를 생산 방법을 설명합니다.

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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;

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

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, ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...