Work

The generation of donor-derived virus-specific cytotoxic T lymphocytes reactive against cytomegalovirus and adenovirus antigens for clinical use

Year: 2005

Type: article

Abstract: Cytomegalovirus (CMV) and adenovirus are major viral pathogens causing morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. Previous studies have shown that prophylactic adoptive immunotherapy with donor-derived cytotoxic T lymphocytes (CTLs) directed against Epstein-Barr virus (EBV) and CMV can effectively prevent the clinical manifestations of these viruses. We have extended these studies by generating CTLs from normal donor PBMCs that should restore cellular immunity to both CMV and adenovirus simultaneously. We have generated a clinical grade recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber (Ad5f35pp65) carrying a transgene for the immunodominant CMV antigen, pp65. We have developed a protocol using an initial round of stimulation with autologous mononuclear cells transduced with Ad5f35pp65, followed by 2 rounds of weekly stimulation with autologous EBV-transformed lymphoblastoid cell lines (LCLs) transduced with the same vector using MOIs of 10 and 100, respectively. After 3 rounds of stimulation, 3 CTL cultures contained a mean of 86% (range, 82%-95%) CD8+ve and a mean of 9.5% (range, 5%-14%) CD4+ve cells. Evaluable CTL lines showed significant cytotoxicity in chromium release assays against autologous fibroblasts and LCL infected with a CMV vector and LCL infected with the Ad5GFP vector. The observed cytotoxicity was specific because nontransduced LCLs, transduced and nontransduced MHC-mismatched LCLs, and autologous PHA blasts were not killed. ELISPOT assays on CTL demonstrated a mean of 459 (range, 284–634) and 497.5 (range, 402–593) numbers of cells secreting interferon-gamma after stimulation with autologous PBMC stimulated with CMV and adenovirus peptides, respectively. Using MHC-peptide tetramers in a HLA-A2/B7+ve donor we have been able to demonstrate the simultaneous presence of CD8+ve cells recognizing peptide epitopes from CMV pp65 (range, 2.41%-9.08%) and adenovirus hexon (1.69%) in the CTL culture. In summary, we have developed a protocol for the efficient generation ex vivo of CTL with virus specificity for CMV and adenovirus. We are currently growing these CTL lines for clinical use in a FDA-approved protocol to restore cellular immunity to these viruses after allogeneic stem cell transplantation.

Source: Transplantation and Cellular Therapy

DOI: 10.1016/j.bbmt.2004.12.043