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><tbody><tr><td>RPMI 1640</td><td>Invitrogen</td><td>21870-076</td><td></td></tr><tr><td>DC media</td><td>CellGenix</td><td>20801-0500</td><td></td></tr><tr><td>EHAA (Click&amp;rsquo;s Medium)</td><td>Irvine Scientific</td><td>9195</td><td></td></tr><tr><td>Human Serum</td><td>Gemini Bio Products</td><td>&lt;strong&gt;100-110&lt;/strong&gt;&lt;strong&gt; &lt;/strong&gt;</td><td></td></tr><tr><td>Gas Permeable Cultureware&lt;sup&gt;18&lt;/sup&gt; </td><td>Wilson Wolf Manufacturing</td><td>80040S</td><td></td></tr><tr><td>IL-2</td><td>Chiron (TCH Pharmacy)</td><td></td><td></td></tr><tr><td>IL-12</td><td>National Cancer Institute</td><td></td><td></td></tr><tr><td>IL-15</td><td>CellGenix</td><td>1013-050</td><td></td></tr><tr><td>IL-7</td><td>R&amp;amp;D Systems</td><td>AFL207</td><td></td></tr><tr><td>IL-1beta</td><td>R&amp;amp;D Systems</td><td>AFL201</td><td></td></tr><tr><td>IL-6</td><td>CellGenix</td><td>1004-050</td><td></td></tr><tr><td>GM-CSF</td><td>TCH Pharmacy</td><td></td><td></td></tr><tr><td>IL-4</td><td>R&amp;amp;D Systems</td><td>&lt;a href=&quot;http://www.rndsystems.com/pdf/AFL204.pdf&quot; target=&quot;_blank&quot;&gt;AFL204&lt;/a&gt;</td><td></td></tr><tr><td>TNF-alpha</td><td>R&amp;amp;D Systems</td><td>AFL210</td><td></td></tr><tr><td>Ad5f35pp65</td><td>BCM CAGT Vector Production Facility</td><td></td><td></td></tr><tr><td>Plasma transfer set with female luer adapter</td><td>Charter Medical</td><td>89-550-66j</td><td></td></tr><tr><td>Lymphoprep</td><td>Nycomed</td><td>1114550</td><td></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 ...

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Immunology and Infection

12.7K Views.  Baylor College of Medicine. 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 naîve T cells.

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

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 ...

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

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

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

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

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, ...

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.

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 ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting proteins. For example, chimeric antigen receptor (CAR)-T cells to treat cancers and viral infections involve the transduction of T cells with synthetic genes encoding CAR molecules. The CAR molecules make the T cells specifically recognize and kill cancer or virally infected cells. Cells can also be co-transduced with other genes of interest. For example, cells can be co-transduced with genes encoding proteins that target cells to specific locations. Here, we present a protocol to transduce primary peripheral blood mononuclear cells (PBMCs) with genes encoding a virus-specific CAR and the B cell follicle homing molecule chemokine receptor type 5 (CXCR5). This procedure takes nine days and results in transduced T cell populations that maintain a central memory phenotype. Maintenance of a central memory or less differentiated phenotype has been shown to associate with persistence of cells post-infusion. Furthermore, cells produced with this method show high levels of viability, high levels of co-expression of the two transduced genes, and large enough quantities of cells for immunotherapeutic infusion. This nine-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches. The methods described here are based on studies presented in our previous publications.

We outline a 9-day protocol for the transduction and expansion of rhesus macaque peripheral blood mononuclear cells which yields cells with excellent co-expression of the genes of interest in sufficient number for infusion studies of cell efficacy.

Emerging immunotherapies to treat infectious diseases and cancers often involve transduction of cellular populations with genes encoding disease-targeting ...

Bioengineering

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Medicine

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

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

Summary

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Chapters in this video

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

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

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

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

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

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis