1Center for Cell and Gene Therapy, Baylor College of Medicine, 2Pathology and Immunology, Baylor College of Medicine, 3Department of Stem Cell Transplantation and Cellular Therapy, University of Texas M.D. Anderson Cancer Center, 4Medicine, Baylor College of Medicine, 5Department of Pediatrics, Baylor College of Medicine
This article is a part ofJoVE Immunology and Infection. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Hanley, P. J., Lam, S., Shpall, E. J., Bollard, C. M. Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus. J. Vis. Exp. (63), e3627, doi:10.3791/3627 (2012).
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 naïve 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.
1. Mononuclear Cell Isolation (day 0)
2. Dendritic Cell Generation (starting on day 0)
3. Generation of EBV-LCL (starting on day 0)
4. CTL Initiation – 7 days After Initiation of DCs
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).
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.
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-.
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.
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.
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 Here, we show that this approach can also be extended to cord blood.
It is imperative to use aseptic technique when culturing human primary cells. Because of the naïve nature of the T cells, it is also important that human serum is used in all media that require serum. In our experience, FBS-containing products will result in a massive expansion of T cells targeting proteins derived from FBS.
When isolating the mononuclear cells using the Ficoll gradient, it is often difficult to exclude red blood cells because many are nucleated and not lysed by red cell lysis buffer. If the number of red blood cells is excessive, the product can be re-ficolled.
While the method here is limited to the expansion of virus-specific T cells –namely CMV, EBV, and adenovirus–it is applicable to the generation of other antigen-specific T cells from cord blood as well. The use of dendritic cells and the above mentioned cytokine cocktail is a potent stimulator of naïve T cells and should drive the expansion of antigen-specific CTL.
The authors have nothing to disclose.
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).
|EHAA (Click’s Medium)||Irvine Scientific||9195|
|Human Serum||Gemini Bio Products||100-110|
|Gas Permeable Cultureware18||Wilson Wolf Manufacturing||80040S|
|IL-2||Chiron (TCH Pharmacy)|
|IL-12||National Cancer Institute|
|Ad5f35pp65||BCM CAGT Vector Production Facility|
|Plasma transfer set with female luer adapter||Charter Medical||89-550-66j|