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

Gerdemann U, Vera JF, Rooney CM, Leen AM.
Source: J Vis Exp
Publication Date: (2011)
Issue: 51: 2736
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Dendritic cell (NHDC), human
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit
1-2 million cells in 100 ul conductive polymer Nucleocuvettes. Nucleofector Solution P3 and program CB-150 Summary (Lonza): The authors present a method for activating human T cells to fight viruses which can be deadly to immune-compromised patients such as those undergoing stem cell transplantation. Nucleofection™ is used to transfect human dendritic cells which are subsequently used to activate cytotoxic T lymphocytes against multiple viruses. These cytotoxic T lymphocytes could then potentially be used to protect against or fight off viral infection in patients.
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 2x10(5)/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 - 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 10(6) cells that is reduced by >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.