Cytomegalovirus-specific cytolytic T-cell lines and clones generated against adenovirus-pp65-infected dendritic cells.
Cytomegalovirus (CMV) infection is a serious complication of allogeneic bone marrow transplantation (BMT). CMV disease can usually be prevented by passive immunization with donor-derived CMV-pp65-specific T-cell clones if provided early post-BMT. The classic method of generating CMV-specific T-cell clones requires donor-derived fibroblast lines infected with CMV as stimulators, thus limiting the availability of CMV immunotherapy to those patients for whom a donor skin biopsy can be obtained 6 to 8 weeks pretransplantation. To overcome this limitation we have used monocyte-derived dendritic cells (DCs) to induce donor anti-CMV cytotoxic T lymphocytes (CTLs). Matured, adeno-pp65-infected DCs were added at day 0 and at day 7 of a 2-week culture of donor peripheral blood mononuclear cells. DC-primed cultures were compared with cultures stimulated in an identical fashion with CMV-infected fibroblasts or with adeno-pp65-infected freshly isolated blood monocytes. Specific killing of CMV-infected fibroblasts was detected in all except the culture stimulated with pp65-infected monocytes. DCs infected after maturation elicited greater CTL activity than did DCs matured after infection. A series of 5 CD8+ clones from a fibroblast-stimulated culture and 7 CD8+ clones from a mature-DC-stimulated culture derived from a single HLA-A*0201+ individual were characterized. All 12 clones lysed autologous CMV-infected fibroblasts. All except 1 clone from the CMV-infected fibroblast arm (fibroblast arm) lysed vaccinia-pp65-infected B-lymphoblastoid cell lines (BLCLs); none lysed vaccinia-pp150-infected or noninfected BLCLs. Ten of 10 CD8+ clones tested were restricted by HLA-A*0201. Seven of the 12 clones were Vbeta6+ (2 from the fibroblast arm and 5 from the DC arm) with an identical Vbeta6.1-J1.4 sequence. Three clones from the fibroblast arm and 5 clones from the DC arm recognized the pp65 peptide NLVPMVATV (amino acids [aa], 495-503). These data show that CMV-specific T-cell clones with similar restriction patterns, T cell-receptor usage, and specificity can be generated using monocyte-derived pp65-infected-DC or CMV-infected-fibroblast stimulators. This approach should broaden the applicability of CMV-specific T-cell immunotherapy to a wider spectrum of patients by reducing the time required to generate CMV-specific T-cell clones.
Keever-Taylor
C A
CA
Department of Medicine, Medical College of Wisconsin, Milwaukee 53226, USA. ckeever@mcw.edu
Margolis
D
D
Konings
S
S
Sandford
G R
GR
Nicolette
C A
CA
Lawendowski
C
C
Burns
W H
WH
D000256:Adenoviridae / Q000235:genetics; D017951:Antigen Presentation / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D001782:Blood Donors; D018929:Cell Culture Techniques / Q000379:methods; D002999:Clone Cells / Q000276:immunology*; D003587:Cytomegalovirus / Q000276:immunology*; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D006680:HLA Antigens / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D009000:Monocytes / Q000166:cytology; D010750:Phosphoproteins / Q000276:immunology*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014161:Transduction, Genetic; D014763:Viral Matrix Proteins / Q000276:immunology*
D000956:Antigens, Viral; D000939:Epitopes; D006680:HLA Antigens; D010750:Phosphoproteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa