Cytomegalovirus-specific cytolytic T-cell lines and clones generated against adenovirus-pp65-infected dendritic cells

Cytomegalovirus (CMV) infection is a serious complication of allogeneic bon e marrow transplantation (BMT). CMV disease can usually be prevented by pas sive immunization with donor-derived CMV-pp65-specific T-cell clones if pro vided early post-EMT. The classic method of generating CMV-specific T-cell clones requires donor-derived fibroblast lines infected with CMV as stimula tors, thus limiting the availability of CMV immunotherapy to those patients for whom a donor skin biopsy can be obtained 6 to 8 weeks pretransplantati on. To overcome this limitation we have used monocyte-derived dendritic cel ls (DCs) to induce donor anti-CMV cytotoxic T lymphocytes (CTLs). Matured, adenopp65-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 compa red with cultures stimulated in an identical fashion with CMV-infected fibr oblasts or with adeno-pp65-infected freshly isolated blood monocytes. Speci fic killing of CMV-infected fibroblasts was detected in all except the cult ure stimulated with PP65-infected monocytes. DCs infected after maturation elicited greater CTL activity than did DCs matured after infection. A serie s of 5 CD8(+) clones from a fibroblast-stimulated culture and 7 CD8(+)-clon es from a mature-DC-stimulated culture derived from a single HLA-A*0201(+) individual were characterized. Ah 12 clones lysed autologous CMV-infected f ibroblasts. Ah except 1 clone from the CMV-infected fibroblast arm (fibrobl ast arm) lysed vaccinia-pp65-infected B-lymphoblastoid cell lines (BLCLs); none lysed vaccinia-pp150-infected or noninfected BLCLs. Ten of 10 CD8(+) c lones tested were restricted by HLA-A*0201. Seven of the 12 clones were V b eta6(+) (2 from the fibroblast arm and 5 from the DC arm) with an identical V beta6.1-J1.4 sequence. Three clones from the fibroblast arm and 5 clones from the DC arm recognized the pp65 peptide NLVPMVATV (amino acids [aa], 4 95-503). These data show that CMV-specific T-cell clones with similar restr iction patterns, T cell-receptor usage, and specificity can be generated us ing monocyte-derived pp65-infected-DC or CMV-infected-fibroblast stimulator s. This approach should broaden the applicability of CMV-specific T-cell im munotherapy to a wider spectrum of patients by reducing the time required t o generate CMV-specific T-cell clones.