Epstein-Barr Virus Specific Cytotoxic T Cells for Clinical Use in Immune Reconstitution Post Haemopoietic Stem Cell Transplant.

Uncontrolled EBV replication can lead to life threatening post-transplant lymphoproliferative disorder (PTLD). The incidence of EBV reactivation and PTLD is proportional to the degree of cellular immunosuppression. Treatment with EBV specific cytotoxic T lymphocytes (CTL) effectively controls EBV replication but the generation of CTL using autologous EBV-transformed B cells as stimulators is cumbersome and not compliant with more strict regulatory requirements.

To develop a rapid and reliable method for production of a clinical grade EBV specific T cell product using an adenoviral vector encoding genes for EBNA-1 and immunogenic LMP peptides.

Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood by Ficoll-paque centrifugation. Monocyte derived dendritic cells (mo-DC) were generated from PBMC by adherence to plastic and exposure to IL-4 and GM-CSF. On day 6 mo-DC were transfected with a clinical grade adenoviral vector encoding EBNA-1 and HLA Class I epitopes of LMP-1 and LMP-2a and matured with the addition of TNF. Transfected PBMC or mo-DC were used to stimulate T cells from the same donor and cultures were continued for 21 days from the first stimulation with a second stimulation on day 7 and increasing doses of IL-2 thereafter. The cellular product was analysed on day 21 for phenotype, antigen specificity and functional capacity by tetramer staining, cytokine production in response to antigen stimulation and cytotoxic activity against antigen coated targets.

Cellular proliferation was seen in all of 11 EBV seropositive normal donors with a mean fold increase in total cell number of 5.41 (SEM 1.3). In all donors, the cellular product was CD3 positive (mean 94.7%, range 81 to 99.4) with both CD4 (mean 65.7% of CD3, range 17.0 to 93.2) and CD8 cells present (mean 29.6%, range 4.4 to 74.4). Cells were of memory phenotype, being CD28+ (mean 91%, SEM 3.1), CD45RO+ (mean 89.4%, SEM 3.0) and a variable proportion were of central memory phenotype (CD62L+ mean 35.9%, range 10.6 to 73.3). EBV specificity was demonstrated by tetramer staining or intracellular cytokine detection in 7 of 10 donors. Tetramer analysis of 6 HLA-A2 positive donors showed up to 2498-fold expansion of LMP-2a tetramer specific CD8 cells compared to baseline (for epitope CLGGLLTMV, mean 469-fold increase, range 5.2 to 2498; for epitope FLYALALLL, mean 186-fold increase, range 2.1 to 601). Cytokine responses to stimulation with EBNA, LMP and adenoviral peptides were present in both CD4 and CD8 cells and showed significant individual variation. Cytotoxic activity was seen with up to 75% specific lysis of EBV antigen coated targets at an effector to target ratio of 20:1.

The use of an adenoviral vector containing genes encoding EBNA-1 and LMP antigens allows the rapid generation of a clinical grade EBV-specific T cell product from the majority of EBV seropositive normal donors. This technique will permit the incorporation of adoptive immunotherapy for EBV into routine haemopoietic stem cell transplantation.

No relevant conflicts of interest to declare.