Name: Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus (Video) | JoVE Journal
Uploaded: 2012-05-07T13:00:00.000+00:00
Duration: 11 min 18 s
Description: Learn to generate virus-specific cytotoxic T lymphocytes from umbilical cord blood using a GMP-compliant method. This protocol produces CTL targeting CMV, EBV, and adenovirus for immunotherapy applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 μL/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 μg/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 μL of concentrated EBV B95.8 supernatant and 1.8 mL of complete media (RPMI + 10% FBS + 2mM L-glutamine) containing cyclosporine (1 μg/mL).</li>
 <li>Aliquot 100 μL of cells into 10 wells of a 96-well plate and 200 μL into 5 wells. Add 100 μL of complete media + cyclosporine to the wells containing only 100 μL 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 – 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 ½ 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 naïve 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="DC maturation process diagram showing PBMC isolation, cytokine induction, and LCL establishment." data-altupdatedat="1787630408000" data-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="Flow cytometry dot plots for CD3, CD4, CD8, CD19, CD56 markers, showing cell population percentages." data-altupdatedat="1787630408000" data-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="Bar graph showing IFN-γ spots/10^5 CTL; immune response assay results for CTL, IE-1, Ad Hexon, CMV pp65, EBV LCL." data-altupdatedat="1787630408000" data-alt="Figure 3."> Figure 3. T cell functionality. T cell specificity was tested by IFN- γELISPOT. 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="Effector-to-target ratio effect on cell lysis; line graph; IE-1, Ad Hexon, CMV pp65, EBV LCL comparison." data-altupdatedat="1787630408000" data-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.

<ol><li>Kennedy-Nasser, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparable+outcome+of+alternative+donor+and+matched+sibling+donor+hematopoietic+stem+cell+transplant+for+children+with+acute+lymphoblastic+leukemia+in+first+or+second+remission+using+alemtuzumab+in+a+myeloablative+conditioning+regimen%5BTitle%5D)+AND+%22Biol.+Blood+Marrow+Transplant%22%5BJournal%5D)">Comparable outcome of alternative donor and matched sibling donor hematopoietic stem cell transplant for children with acute lymphoblastic leukemia in first or second remission using alemtuzumab in a myeloablative conditioning regimen.</a> Biol. Blood Marrow Transplant. 14, 1245-1245 (2008).</li>
<li>Hanley, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+clinical+outcomes+using+adoptively+transferred+immune+cells+from+umbilical+cord+blood%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Improving clinical outcomes using adoptively transferred immune cells from umbilical cord blood.</a> Cytotherapy. 12, 713(2010).</li>
<li>Szabolcs, P., Cairo, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unrelated+umbilical+cord+blood+transplantation+and+immune+reconstitution%5BTitle%5D)+AND+%22Semin.+Hematol%22%5BJournal%5D)">Unrelated umbilical cord blood transplantation and immune reconstitution.</a> Semin. Hematol. 47, 22(2010).</li>
<li>Canto, E., Rodriguez-Sanchez, J. L., Vidal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinctive+response+of+naive+lymphocytes+from+cord+blood+to+primary+activation+via+TCR%5BTitle%5D)+AND+%22J.+Leukoc.+Biol%22%5BJournal%5D)">Distinctive response of naive lymphocytes from cord blood to primary activation via TCR.</a> J. Leukoc. Biol. 74, 998-998 (2003).</li>
<li>Leen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monoculture-derived+T+lymphocytes+specific+for+multiple+viruses+expand+and+produce+clinically+relevant+effects+in+immunocompromised+individuals%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals.</a> Nat. Med. 12, 1160-1160 (2006).</li>
<li>Riddell, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Restoration+of+viral+immunity+in+immunodeficient+humans+by+the+adoptive+transfer+of+T+cell+clones%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones.</a> Science. 257, 238(1992).</li>
<li>O'Reilly, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adoptive+transfer+of+antigen-specific+T-cells+of+donor+type+for+immunotherapy+of+viral+infections+following+allogeneic+hematopoietic+cell+transplants%5BTitle%5D)+AND+%22Immunol.+Res%22%5BJournal%5D)">Adoptive transfer of antigen-specific T-cells of donor type for immunotherapy of viral infections following allogeneic hematopoietic cell transplants.</a> Immunol. Res. 38, 237-237 (2007).</li>
<li>Peggs, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adoptive+cellular+therapy+for+early+cytomegalovirus+infection+after+allogeneic+stem-cell+transplantation+with+virus-specific+T-cell+lines%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines.</a> Lancet. 362, 1375-1375 (2003).</li>
<li>Sili, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+expansion+of+dendritic+cell-primed+polyclonal+human+cytotoxic+T-lymphocyte+lines+using+lymphoblastoid+cell+lines+for+adoptive+immunotherapy%5BTitle%5D)+AND+%22J.+Immunother%22%5BJournal%5D)">Large-scale expansion of dendritic cell-primed polyclonal human cytotoxic T-lymphocyte lines using lymphoblastoid cell lines for adoptive immunotherapy.</a> J. Immunother. 26, 241(2003).</li>
<li>Bollard, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Good+manufacturing+practice-grade+cytotoxic+T+lymphocytes+specific+for+latent+membrane+proteins+%28LMP%29-1+and+LMP2+for+patients+with+Epstein-Barr+virus-associated+lymphoma%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Good manufacturing practice-grade cytotoxic T lymphocytes specific for latent membrane proteins (LMP)-1 and LMP2 for patients with Epstein-Barr virus-associated lymphoma.</a> Cytotherapy. 13, 518(2011).</li>
<li>Hanley, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functionally+active+virus-specific+T+cells+that+target+CMV%2C+adenovirus%2C+and+EBV+can+be+expanded+from+naive+T-cell+populations+in+cord+blood+and+will+target+a+range+of+viral+epitopes%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Functionally active virus-specific T cells that target CMV, adenovirus, and EBV can be expanded from naive T-cell populations in cord blood and will target a range of viral epitopes.</a> Blood. 114, 1958(1958).</li>
<li>Hanley, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion+of+T+cells+targeting+multiple+antigens+of+cytomegalovirus%2C+Epstein-Barr+virus+and+adenovirus+to+provide+broad+antiviral+specificity+after+stem+cell+transplantation%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Expansion of T cells targeting multiple antigens of cytomegalovirus, Epstein-Barr virus and adenovirus to provide broad antiviral specificity after stem cell transplantation.</a> Cytotherapy. , (2011).</li>
<li>Gerdemann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+Multivirus-specific+T+Cells+to+Prevent%2Ftreat+Viral+Infections+after+Allogeneic+Hematopoietic+Stem+Cell+Transplant%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant.</a> J. Vis. Exp. (51), e2736(2011).</li>
<li>Mori, T., Kato, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytomegalovirus+infection%2Fdisease+after+hematopoietic+stem+cell+transplantation%5BTitle%5D)+AND+%22Int.+J.+Hematol%22%5BJournal%5D)">Cytomegalovirus infection/disease after hematopoietic stem cell transplantation.</a> Int. J. Hematol. 91, 588(2010).</li>
<li>Einsele, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infusion+of+cytomegalovirus+%28CMV%29-specific+T+cells+for+the+treatment+of+CMV+infection+not+responding+to+antiviral+chemotherapy%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy.</a> Blood. 99, 3916(2002).</li>
<li>Bao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion+of+cytomegalovirus+pp65+and+IE-1+specific+cytotoxic+T+lymphocytes+for+cytomegalovirus-specific+immunotherapy+following+allogeneic+stem+cell+transplantation%5BTitle%5D)+AND+%22Biol.+Blood+Marrow+Transplant%22%5BJournal%5D)">Expansion of cytomegalovirus pp65 and IE-1 specific cytotoxic T lymphocytes for cytomegalovirus-specific immunotherapy following allogeneic stem cell transplantation.</a> Biol. Blood Marrow Transplant. 14, 1156(2008).</li>
<li>Shpall, E. J., Bollard, C. M., Brunstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+cord+blood+transplant+therapies%5BTitle%5D)+AND+%22Biol.+Blood+Marrow+Transplant%22%5BJournal%5D)">Novel cord blood transplant therapies.</a> Biol. Blood Marrow Transplant. 17, Suppl 1. S39-S45 (2011).</li>
<li>Vera, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accelerated+production+of+antigen-specific+T+cells+for+preclinical+and+clinical+applications+using+gas-permeable+rapid+expansion+cultureware+%28G-Rex%5BTitle%5D)+AND+%22J.+Immunother%22%5BJournal%5D)">Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex.</a> J. Immunother. 33, 305(2010).</li>
</ol>

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

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

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

The overall goal of this procedure is to generate cord blood derived cytotoxic T lymphocytes or CTL that recognize antigens from multiple viruses. This is accomplished by first isolating the cord blood mononuclear cells from the 20%fraction of the cord blood unit. The second step is to use the mononuclear cells to generate dendritic cells, Epstein-Barr virus, lymphoblasts cell lines, or E-B-V-L-C-L and the virus specific CTL.The mononuclear cells, which contain the T cells, are stimulated with cells that have been transduced with the A five F 35 PP 65 vector. The final step is to confirm the specificity of the multi virus specific CTL and to freeze the CTL. Ultimately, the specificity of the expansion of the CTL for at least one viral antigen can be evaluated through Ellie SPA analysis and cytotoxicity assays.Generally, individuals new to this method will struggle because the volume of core blood available in the 20%fraction is very small, which means that there are very few cells to work with. The procedure is also quite intricate and involves a generation of at least three different cell types from these limited cell numbers. We first had the idea for this method after we analyzed the data from a clinical trial where we made multi virus specific T cells for patients after bone marrow transplant.In that study we saw that the T cells were not only safe but efficacious in these patients and prevented and treated viral infection. However, the patient population who have the greatest need for such a therapy are recipients of cord blood transplants and these patients, however, we are unable to go back to the donor and to get blood to make T cells. So we had to develop a novel strategy to generate multi virus specific T cells from the cord blood from the 20%fraction of the cord blood that would not be needed for the transplant procedure.To begin warm 20 milliliters of RPMI in a 50 milliliter centrifuge tube and thaw the 20%fraction of cord blood. Then insert the spike of a female lure adapter into the outlet port of the 20%fraction and attach a syringe. Next, remove the blood and transfer it into the prepared centrifuge tube.Rinse the cord blood bag with five milliliters of RPMI and then transfer the wash to the same tube. Now centrifuge the cells for 10 minutes at 400 Gs at room temperature and then after aspirating the re suspend the cells in 20 milliliters of warm RPMI transfer 15 milliliters of lymph prep to a 50 milliliter centrifuge tube. Layer the resuspended cells on top of it and separate the cells for 40 minutes at 400 Gs.Harvest the interface.Then after washing the mononuclear cells in 20 milliliters of RPMI for 10 minutes at 450 GS and aspirating the supernatant, resuspend the pellet in 20 milliliters of RPMI count the cells and then after pelleting the cells again for five minutes at 400 Gs and aspirating the supernatant once more, resus suspend the cells at five times 10 to the six mononuclear cells per milliliter in serum free cellgenic dendritic cell media. Finally transfer one milliliter of the cells into a 15 milliliter conical tube for the E-B-V-L-C-L. Generation incubate two milliliters per well of a six well plate of the just prepared mononuclear cells for one to two hours after the incubation.Wash off the non-adherent cells three to four times with PBS collecting the floating cells into a 50 milliliter centrifuge tube each time. Then freeze the collected cells at a controlled rate of one degree per minute and store in a minus 80 degrees Celsius freezer overnight. The next day transfer the cells to liquid nitrogen storage until the DC are ready.Now add two milliliters per well of dendritic cell media supplemented with IL four and G-M-C-S-F to each well of the six well plate replenish the cytokines three to four days after DC initiation, adding the IL four and G-M-C-S-F in a total volume of 100 microliters of media to each. Well harvest the DC on day five to six by scraping the wells with a transfer pipette count the big cells and then after pelleting the collected cells for five minutes at 400 Gs and aspirating the supernatant resuspend the DC at two times 10 of the six cells per milliliter in DC media add half a milliliter of the cell solution per well into a 24 well plate. Then transduce the DC by adding the add five F 35 PP 65 vector at an MOI of 10 infectious units per cell to each of the wells of the 24 well plate.Finally incubate the plate for 1.5 hours after the incubation at 1.5 milliliters of DC media supplemented with the DC maturation cytokine cocktail. Spin down the reserved mononuclear cells, then add 200 microliters of concentrated EBVB 95.8 supernatant and 1.8 milliliters of complete media supplemented with cyclosporine to the pellet. Next aliquot 100 microliters of the cells into 10 wells of a 96 well plate and 200 microliters five wells.Add 100 microliters of complete media plus cyclosporine to the wells containing only 100 microliters of cells, and then fill the remaining wells with sterile water. Feed the LCL weekly. Eventually splitting the expanding cell cultures into T 75 flasks.After harvesting the seven day old DC irradiate the cells at 30 gras. Then after washing them four times with PBS count, the DC resus suspend the DC in T-cell media containing human serum at one times 10 to the fifth DC per milliliter and then thaw an Eloqua of the previously frozen non-adherent cells after washing in T-cell medium and centrifuging for five minutes at 400 Gs.Count the thought cells and resus suspend them at two times 10 to the six cells per milliliter in T-cell media supplemented with human serum add IL seven IL 12 and IL 15 to the cells and then add one milliliter of cells to each well of a 24 well plate. Next, add one milliliter of the DC to each well containing non-adherent cells.Fill any empty wells of the plate with sterile water and then incubate the T cells. After feeding the T cells on day seven, freeze the T cells nine to 12 days after T-cell initiation until the LCL are ready. Once the LCL have been passaged into the T 75 flasks, add the vector to the cells after they've been spun down at an MOI of 100 infectious units per cell and incubate the cells with the virus for 1.5 hours after the incubation.Re suspend the cells in complete media at five times 10 to the fifth LCL per milliliter and add two milliliters of the transduced cells per well to a new 24 well plate after two days. Harvest the LCL and irradiate them at 40 gras after washing the cells four times with PBS and centrifuging at 400 Gs for five minutes. Count them and then resuspend the LCL in T-cell media containing human serum at 2.5 times 10 to the fifth cells per milliliter.Now either add one milliliter of LCL per well of a new 24 well plate or add five times 10 of the sixth LCL to a GRE 40 culture device. Supplementing the culture with IL 15, harvest the T cells after centrifusion the T-cells for five minutes at 400 Gs and aspirating the supernatant reus. Suspend the cells in T-cell media supplemented with human serum at one times 10 of the six T-cells per milliliter.After adding IL 15, either add one milliliter of the T-cell per well to the LCL in the 24 well plate or one milliliter of T-cell to the LCL in the GRE X 40 culture device. If a GRE X 40 is used, bring the total volume of media up to 30 milliliters. Finally on day seven, harvest and wash the CTL and LCL and irradiate the LCL as before.Then after Resus suspending the LCL and T-cell media supplemented with human serum at 2.5 times 10 of the PIP cells per milliliter add one milliliter of LCL per well of a 24 well plate add T cells and supplement with IL two rather than IL 15 after three stimulations. Typically the majority of the cells are CD three positive with a mixture of CD four positive and CD eight positive T cells. There also are typically less than 15%of CD three negative CD 56 positive natural killer cells and less than 1%of CD 19 positive B cells.The expanded CTL recognized the antigen PP 65 from CMV Heon and Penton from adenovirus, as well as numerous EBV antigens that are expressed on E-B-V-L-C-L. When tested in an Ellie SPA assay, CTL secrete more interferon gamma in response to these antigens than irrelevant antigens. As expected in a chromium release assay, CTL lys L-C-L-C-M-V-P-P 65, pulse and adenovirus, heon and or Penton pulse targets, but not targets pulse with irrelevant peptides.While attempting this procedure, it's important to remember that all cells will be used in the manufacturing process and that cell numbers are extremely limited. It's also important to remember that because you are dealing with naive T cells, that you must always use human serum because the use of fetal calf serum might result in the generation of T-cell specific for fetal calf proteins. By following this procedure, tumor specific T cells or other virus to T cells likely can be generated as well.

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

Research

JoVE Journal

Immunology and Infection

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.

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

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.

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

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.

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

Novel generations of functional assays such as gamma interferon (IFN-γ) 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).

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

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.

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

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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.

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

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

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.

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

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.

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

Presented is a protocol for the expansion of Gamma-Delta (γδ) T cell drug product. Lymphocytes are isolated by elutriation and γδ-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The γδ cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Learn to generate virus-specific cytotoxic T lymphocytes from umbilical cord blood using a GMP-compliant method. This protocol produces CTL targeting CMV, EBV, and adenovirus for immunotherapy applications.

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

Explore More Videos