This work is supported by a Production Assistance for Cellular Therapies (contract NIH-NHLBI (HB-10-03) HHSN26820100000C) (C.M.R.), a Specialized Centers for Cell-based Therapy Grant NIH-NHLBI 1 U54 HL081007 (C.M.R.), an ASBMT Young Investigator Award (U.G. and J.V.), a Leukemia and Lymphoma Society Special Fellow in Clinical Research Award (U.G.), and an Amy Strelzer Manasevit Scholar Award (A.M.L).

1. DC nucleofection
<ol>
<li>Harvest monocyte-derived DCs, which have been enriched using plastic adherence, cultured for 5 days using Cell Genix Media supplemented with IL4(1000U/ml), GMCSF (800IU/ml) and further matured for 24hrs using the DC maturation cytokines IL4(1000U/ml), GMCSF (800IU/ml), IL6100ng/ml, TNF-&alpha; 10ng/ml, IL1-&beta; 10ng/ml and PGE2 (1&mu;g/ml)1, by gentle resuspension with a 3ml transfer pipette.</li> 
<li>Count viable DCs using trypan blue, transfer into 3x 15ml tubes with no fewer than 0.5x106 and no more than 2x106 cells/tube.</li>
<li>Centrifuge DCs for 10mins @ 200g. During this time pre-warm Cell Genix media supplemented with the DC maturation cytokines (DC maturation media) - 2ml/well in three wells of a 12-well tissue culture treated plate in a 37&deg;C/5% CO2 incubator.</li> 
<li>Once cells have finished spinning, aspirate the supernatant and add the relevant DNA plasmids to each of the tubes in a final concentration of 5&mu;g DNA/tube. In this case add the plasmid encoding IE1-pp65 to tube #1, Hexon-Penton to tube #2, and EBNA1-LMP2-BZLF1 to tube #3.</li>
<li>Resuspend DCs and DNA with 100&mu;l of Amaxa nucloefection solution, mix well and transfer to the nucleofection cuvettes.</li>
<li>Place cuvettes in the 4D nucleofector, choose program CB150 (Amaxa/Lonza), and press start.</li>
<li>Immediately after nucleofection add 500&mu;l of the 2ml pre-warmed Cell Genix DC maturation media to the cuvette, mix gently by pipetting up and down 2-3 times, and transfer nucleofected DCs to the prepared 12-well plate containing the remaining 1.5 ml of prewarmed DC maturation media. Transfer to the 37&deg;C/5% CO2 incubator for a further 12-18hrs.</li>
</ol>
2. T cell stimulation
<ol>
<li>Harvest and count nucleofected DCs, and irradiate at 30Gy. Wash once with 10ml of CTL medium (45% RPMI, 45% Clicks EHAA, 10% FBS, 2mM Glutamax) and resuspend @ 3 x 105 DCs per ml of CTL media.</li> 
<li>Pool a minimum of 7.5x105 (2.5ml) and a maximum of 15x105 (5ml) of DCs containing each of the plasmids and transfer the pooled DCs to the G-Rex device which will then be placed in the incubator.</li>
<li>For the preparation of responder cells use either previously frozen PBMCs or non-adherent mononuclear cells that remain after the DC selection (adherence or CD14 selection). Thaw the cells, transfer to prewarmed culture medium, wash once with CTL media. Resuspend the cells in CTL Media, count the cells and bring them to a concentration of 2x106 cells per ml. Take 15x106 cells or 7.5ml and supplement with 30000U IL4 (1000U/ml &#x2013; final conc.) and 300ng IL7 (10ng/ml &#x2013; final conc.). </li>
<li>Transfer 7.5ml of PBMC (15x106 cells) to the G-Rex and top up the bioreactor with CTL media to a total volume of 30ml.</li> 
<li>Culture the G-Rex for 6-7 days in a 37&deg;C/5% CO2 humidified incubator.</li>
</ol>
3. T cell expansion
<ol>
<li>On day 6-7, aspirate 10ml of media, then mix the cells in the remaining 20ml of media with a 10ml pipette and count viable cells using trypan blue. If there are &lt;50x106 replenish with fresh media + cytokines. If there are &gt;50x106 cells remove 10ml of cell suspension, transfer to a new G-Rex, and then feed both G-Rexs with fresh CTL media + cytokines.</li>
<li>Culture for an additional 4-6 days. Once sufficient cells have been expanded, perform phenotypic and functional characterization of the CTL and cryopreserve excess for future use.</li>
</ol>
4. Representative Results: 
A schematic of our FDA-approved multivirus-specific CTL generation process is shown in Figure 1. In contrast to convention multivirus CTL protocols which use adenovectors and EBV-LCL to stimulate virus-reactive T cells2 we have replaced infectious virus material with DNA plasmids that encode multiple antigens derived from each of the viruses3. To stimulate trivirus CTL we designed three multicistronic plasmids encoding Hexon and Penton of adenovirus, IE1 and pp65 of CMV, and EBNA1, LMP2, and BZLF1 of EBV. These antigens were chosen based on encouraging clinical results of our own and other groups showing that T cells directed against Adv-hexon and penton2,4-6, and to CMV-1E1 and to CMV-pp65 are protective in vivo7. For EBV, EBNA1 is an immunodominant CD4+ T cell target antigen expressed in all EBV-associated malignancies and in normal EBV-infected B cells8,9, LMP2 is immunogenic across multiple HLA types and expressed in most EBV malignancies,10,11 while BZLF1 encodes an immunodominant, immediate early lytic cycle antigen that stimulates both CD4+ and CD8+ T cells from most individuals and is likely important for the control of cells replicating virus12. To further optimize our manufacturing methods we collaborated with Nature Technology who generated minimalized, antibiotic-free (FDA-compliant) plasmids for CTL stimulation13,14. Using this strategy we consistently achieve nucleofection efficiencies of &gt;35% while maintaining high cell viability (data not shown)3. Figure 2 shows that the frequency of virus-specific T cells in response to optimized DNA plasmids as measured by IFN&gamma; ELIspot, was greater than in response to conventional pShuttle-based expression plasmids expressing the same antigens (n=8 Adenovirus, n=4 CMV, and n=2 EBV). The optimal ratio of DC:PBMC was important for potent T cell stimulation as shown in Figure 3 where a ratio of 1:50 produced sub-optimal activation compared to a 1:20 S:R ratio (n=2 donors). Production of sufficient CTL numbers with broad antigen specificity is a pre-requisite for clinical efficacy against all three viruses. This is achieved by CTL culture in the G-Rex, which supports superior T cell expansion compared with conventional 24-well plates (Figure 4A)15, while the addition of IL4 and IL7 to cultures increases the repertoire and specificity as shown in Figure 4B where the frequency of T cells reactive against the CMV-pp65-derived HLA-A2 resticted NLV peptide was assessed in cultures generated in the presence or absence of IL4 and/or IL716,17. To assess the phenotype and functional capacity of the expanded cells we perform flow cytometric analysis, intracellular cytokine staining/IFN&gamma; ELIspot, and Cr51 release assays on the final product for cryopreservation/infusion. Typically the generated cells are polyclonal with a mixed population of CD4+ and CD8+ T cells with antigen-specificity detectable in both T cell compartments. The CTL are able to kill viral antigen-expressing target cells but not virus negative partially-HLA matched targets, indicating that they should not induce graft-versus-host disease (GvHD) in vivo (Figure 5).
<img src="/files/ftp_upload/2736/2736fig1.jpg" alt="Figure 1"/> Figure 1. rCTL generation protocol. First, DCs are nucleofected with the viral antigen-encoding plasmids and then mixed with autologous PBMCs at an R:S of 10 or 20:1. Cells are expanded in the G-Rex for 10-14 days in the presence of IL4 and IL7, then harvested, counted, tested for function, identity and sterility, and then cryopreserved for clinical use.
<img src="/files/ftp_upload/2736/2736fig2.jpg" alt="Figure 2"/> Figure 2. Optimized DNA plasmids induce superior T cell activation in vitro. DCs were nucleofected with optimized, FDA-compliant plasmids encoding Hexon and Penton (Adv), IE1 and pp65 (CMV), and EBNA1, LMP2, and BZLF1 (EBV) or conventional pShuttle plasmids encoding the same antigens. These were used to stimulate T cells and specificity was analyzed by IFN&gamma; ELIspot 10 days post-stimulation.
<img src="/files/ftp_upload/2736/2736fig3.jpg" alt="Figure 3"/> Figure 3. Optimal DC:T cell ratios for CTL activation. DCs from 2 donors were nucleofected with all three optimized plasmids and then used to stimulate autologous PBMCs at 1:20 or 1:50 DC:PBMC ratio. The frequency of reactivated T cells was assessed on day 10 by IFN&gamma; ELIspot. 
<img src="/files/ftp_upload/2736/2736fig4.jpg" alt="Figure 4"/> Figure 4. T cell expansion in the G-Rex using enhancing cytokines. Panel A shows the G-Rex device as well as CTL appearance on the gas permeable membrane, evaluated by microscopy. A comparison between cell output achieved in convention tissue culture treated plates vs G-Rex is also shown. Panel B shows the frequency of CMV pentamer positive CTL achieved in cultures expanded in the presence of no cytokine, IL4 alone, IL7 alone and IL4+IL7.
<img src="/files/ftp_upload/2736/2736fig5.jpg" alt="Figure 5"/> Figure 5. Phenotype and function of expanded CTL. Panel A shows a representative example of the phenotype of the expanded multivirus CTL, which are polyclonal with a mixture of CD4+ (45% - helper) and CD8+ (42% - cytotoxic) T cells, of which the majority (95%) expressed the memory marker CD45RO+/CD62L+. Panel B shows that these cells are specific for all the stimulating antigens and are polyfunctional as assessed by intracellular cytokine staining to detect production of IFN&Gamma; and TNF&alpha; after antigen stimulation. Panel C shows that the expanded CTL are functional as measured by Cr51 assay. Autologous LCL, either alone or transduced with a null vector or an adenoviral vector expressing CMV-pp65 were used as targets. Alloreactivity was assessed using allogeneic PHA blasts as a target.

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Juan F. Vera is a consultant for Wilson Wolf Manufacturing. The other authors have no relevant disclosures.

Viral infections account for substantial morbidity and mortality in patients who are immunocompromised by their disease or its treatment. After HSCT, for example, infections caused by persistent herpesviruses such as EBV and CMV, as well as by respiratory viruses such as Respiratory Syncytial Virus (RSV), are well known, while the importance of infections caused by Adv, BK virus, and human herpesvirus (HHV)-6 have more recently been appreciated. While pharmacological agents are standard therapy for some infections, they have substantial toxicities, generate resistant variants, and are frequently ineffective. In contrast, virus-specific T cells derived from stem cell donors have proven safe and effective for the prevention and treatment of viral infection or disease in the hemopoietic stem cell transplant (HSCT) setting2,5,6,18-21. However, the broader implementation of T cell immunotherapy is ultimately limited by the cost, complexity and time required for CTL production. 
Our novel and rapid approach to generate multivirus CTL, described in the current manuscript, should substantially increase the feasibility of cytotoxic T cell therapy for viral diseases, enabling the strategy to become a standard of care for the immunocompromised host. The use of plasmid nucleofected DCs as APCs enables antigen presentation on both MHC class I and II without competition from viral vectors or indeed from multiple viral antigens being expressed within a single cell since different DC populations are utilized for each plasmid3. The use of IL-4/7 increases T cell survival and proliferation, which correspondingly helps increase the frequency and repertoire of responding antigen-specific T cells16,17. Finally, culture in the G-Rex dramatically reduces T cell apoptosis during culture. Gas exchange (O2 in and CO2 out) occurs across a gas permeable silicon membrane at the base of the flask, preventing hypoxia while allowing a greater depth of medium above the cells, providing more nutrients and diluting waste products. This platform can also be extended to additional viruses as when protective antigens are identified.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 	<td>CellGenix</td>
 <td>CellGenix</td>
 <td>2005</td>
 </tr>
 <tr>
 	<td>IL4</td>
 <td>R+D Systems</td>
 <td>204-IL/CF</td>
 </tr>
 <tr>
 	<td>IL7</td>
 <td>Peprotech</td>
 <td>200-15</td>
 </tr>
 <tr>
 	<td>Hyclone RPMI 1640</td>
 <td>Thermo Scientific</td>
 <td>SH30096.01</td>
 </tr>
 <tr>
 	<td>Human AB serum</td>
 <td>Valley Biomedical Inc.</td>
 <td>HP1022</td>
 </tr>
 <tr>
 	<td>Nucleofector</td>
 <td>Amaxa/Lonza</td>
 <td>AAF-1001B &amp; AAF-1001X</td>
 </tr>
 <tr>
 	<td>Nucleofection Kit</td>
 <td>Amaxa/Lonza</td>
 <td>V4XP-3012</td>
 </tr>
 <tr>
 	<td>Plasmids</td>
 <td>NTC</td>
 <td>n/a</td>
 </tr>
 <tr>
 	<td>GM-CSF</td>
 <td>R&amp;D</td>
 <td>215-GM/CF</td>
 </tr>
 <tr>
 	<td>IL1</td>
 <td>R&amp;D</td>
 <td>201-LB-025</td>
 </tr>
 <tr>
 	<td>IL6</td>
 <td>R&amp;D</td>
 <td>206-IL-CF</td>
 </tr>
 <tr>
 	<td>TNF&alpha;</td>
 <td>R&amp;D</td>
 <td>210-TA-010</td>
 </tr>
 <tr>
 	<td>PGE2</td>
 <td>Sigma</td>
 <td>P6532-1MG</td>
 </tr>
 <tr>
 	<td>G-Rex</td>
 <td>Wilson Wolf Manufacturing</td>
 <td>AY11-00027</td>
 </tr>
 
 </tbody>

</table>

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.

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

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

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 ...

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Research

JoVE Journal

Immunology and Infection

17.1K vistas.  Baylor College of Medicine. Un protocolo de forma rápida, sencilla y rentable para la generación de donantes de origen multivirus CTL específicos (RCTL) para la perfusión del trasplante alogénico de células madre hematopoyéticas de trasplante (HSCT) con riesgo de desarrollar infecciones por CMV, EBV o Adv. Este proceso de fabricación es conforme a las GMP y debe garantizar la aplicación más amplia de células T inmunoterapia más allá de los centros especializados.

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

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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 ...

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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...