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

Abstract

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.

Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers

“…32 Alternatives to the use of tetramer include the transfection of pp65 into LCLs or DCs and the generation of EBVand CMV-bispecific T-cells lines. [33][34][35] This approach is attractive because pp65 is naturally processed and there is no requirement for matching HLA allele with peptide. Furthermore, these polyclonal lines will contain T-helper cells, which will increase the longevity of the transfused cells.…”

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

“…CMV-infected fibroblasts as APC [6] have generally been replaced either by unseparated PBMC [5] or by defined professional APC populations such as dendritic cells (DC) [9][10][11][12] or activated B cells [13,14]. The CMV antigenic stimulus has been provided by infection of APC with recombinant viral vectors introducing relevant CMV antigens [9,10,13,14], loading with epitope peptides [12] or CMV protein lysates [5,11].…”

Langerhans-Type Dendritic Cells Genetically Modified to Express Full-Length Antigen Optimally Stimulate CTLs in a CD4-Dependent Manner

“…Even the peptide-pulsed LCs stimulated a background CD4 ϩ T cell response (ϳ5%), presumably due to a bystander effect from cytokines secreted during the initial stimulatory culture. Importantly, the CD4 ϩ T cell responses that we detected were not confounded by the introduction of exogenous cytokines that are often used to expand T cells for adoptive immunotherapy (11)(12)(13)(14)21).…”

“…Our intention was not to expand large numbers of T cells ex vivo for adoptive immunotherapy, where the role of CD4 ϩ T cells in such expansion has been difficult to distinguish from the role of exogenous cytokines (11)(12)(13)(14). Furthermore, even though most tumor Ags are either self or self-differentiation Ags, we considered recall responses against an immunogenic viral Ag useful for establishing the principal parameters of Ag presentation by gene-transduced LCs.…”