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J Vis Exp. 2012 May 7;(63):e3627. doi: 10.3791/3627.

Expanding cytotoxic T lymphocytes from umbilical cord blood that target cytomegalovirus, Epstein-Barr virus, and adenovirus.

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1
Center for Cell and Gene Therapy, Baylor College of Medicine.

Abstract

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. We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective. 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. 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. 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 50x10(6) CB mononuclear cells we are able to generate upwards of 150 x 10(6) virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation. 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.

PMID:
22588077
PMCID:
PMC3466930
DOI:
10.3791/3627
[Indexed for MEDLINE]
Free PMC Article
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