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

Summary

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.

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.^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.

Protocol

1. Mononuclear Cell Isolation (day 0)

1. 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.
2. Centrifuge cells for 10 minutes at 400 x g. Aspirate supernatant.
3. 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.
4. Harvest the interface containing the mononuclear cells and wash in 20 mL of RPMI. Centrifuge for 10 minutes @ ....

Discussion

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640	Invitrogen	21870-076
DC media	CellGenix	20801-0500
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
IL-15	CellGenix	1013-050
IL-7	R&D Systems	AFL207
IL-1beta	R&D Systems	AFL201
IL-6	CellGenix	1004-050
GM-CSF	TCH Pharmacy
IL-4	R&D Systems	AFL204
TNF-alpha	R&D Systems	AFL210
Ad5f35pp65	BCM CAGT Vector Production Facility
Plasma transfer set with female luer adapter	Charter Medical	89-550-66j
Lymphoprep	Nycomed	1114550