Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers
S. Szmania et al., Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers, BLOOD, 98(3), 2001, pp. 505-512
Cytomegalovirus (CMV) reactivation in immunocompromised recipients of allog
eneic stem cell transplantation is a cause of morbidity and mortality from
viral pneumonitis. Antiviral drugs given to reactivating patients have redu
ced the mortality from CMV but have toxic side effects and do not always pr
event late CMV disease. Cellular immunotherapy to prevent CMV disease Is le
ss toxic and could provide prolonged protection. However, a practical appro
ach to generating sufficient quantities of CMV-specific cytotoxic T cells (
CTLs) Is required. This study describes a system for generating sufficient
CMV-specific CTLs for adoptive immunotherapy of HLA-A*0201 bone marrow tran
splant recipients from 200 mL donor blood. Donor monocytes are used to gene
rate dendritic cells (DCs) in medium with autologous plasma, interleukin 4,
granulocyte-macrophage colony-stimulating factor, and CD40 ligand, The DCs
are pulsed with the immunodominant HLA-A*0201-restricted CMV peptide pp65(
495-503), and incubated with donor T cells. These cultures are restimulated
twice with peptide-pulsed lymphoblastoid cell lines (LCLs) or CD40-ligated
B cells and purified with phycoerythrin (PE)-labeled pp65(495-503)/ HLA-A*
0201 tetramers by flow sorting, or with anti-PE paramagnetic beads. The pur
e tetramer-positive population is then rapidly expanded to obtain sufficien
t cells for clinical immunotherapy. The expanded CTLs are more than 80% pur
e, of memory phenotype, with a Tc1 cytokine profile. They efficiently kill
CMV-infected fibroblasts and express the integrin VLA-4, suggesting that th
e CTLs could cross endothelial barriers. This technique is reproducible and
could be used for generating CMV-specific CTLs to prevent CMV disease afte
r allogeneic blood and marrow transplantation.