Ex vivo generation of human cytomegalovirus‐specific cytotoxic T cells by peptide‐pulsed dendritic cells

Abstract

Summary. Adoptive transfer of donor-derived human cytomegalovirus (HCMV)-specific T-cell clones can restore protective immunity after stem cell transplantation. Ex vivo induction of HCMV-specific T cells using HCMV-infected fibroblasts as stimulator cells confines this approach to HCMV-seropositive donors and requires the presence of infectious virus during the stimulation procedure. In this study, we describe a potential alternative strategy to generate HCMV-specific T cells ex vivo for adoptive immunotherapy. Generation of HCMV-specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using peptide-pulsed dendritic cells as antigen-presenting cells. HCMV-specific T cells were generated and sufficiently expanded for adoptive immunotherapy in 6 out of 14 HCMV-seropositive and 2 out of 11 HCMV-seronegative donors. The CTLs recognized HCMV-infected autologous fibroblasts. No lysis was observed with either non-infected autologous or HLA-mismatched infected fibroblasts. Staining with tetrameric HLA/peptide complexes revealed significant enrichment for peptidespecific T cells of up to 28% and . 90% of CD8 1 T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of . 1 Â 10 9 HCMV-specific T cells was possible after 6±7 weeks when cultures were initiated with 1± 5 Â 10 6 responder cells. Thus, the approach with peptidepulsed DCs to generate HCMV-specific CTLs is feasible for clinical application after allogeneic stem cell transplantation.

Ex vivo stimulation of cytomegalovirus (CMV)‐specific T cells using CMV pp65‐modified dendritic cells as stimulators

“…Peptide pulsing of DC proved highly efficient for the selective stimulation and expansion of peptide-specific CTL in eight out of nine donors with pre-existing CD8 + cells against the peptide. This is in contrast to the findings of Kleihauer et al (2001), who reported a variable success of expanding CMV-directed CD8 + T cells. The expanded CTL specifically lysed T2 target cells pulsed with the cognate peptide, as also shown by others (Solache et al, 1999;Kleihauer et al, 2001).…”

“…This is in contrast to the findings of Kleihauer et al (2001), who reported a variable success of expanding CMV-directed CD8 + T cells. The expanded CTL specifically lysed T2 target cells pulsed with the cognate peptide, as also shown by others (Solache et al, 1999;Kleihauer et al, 2001). MHC class I tetramers can be used for the direct isolation of CMV-specific T cells from leukapheresis products.…”

Efficient Generation of Antigen-Specific Cytotoxic T Cells Using Retrovirally Transduced CD40-Activated B Cells

“…For example, infusion of CMVspecific CD8 ϩ T cell clones to hemopoietic stem cell recipients resulted in the restoration of CMV-specific immunity (12,19). Various combinations of APC and CMV Ags have been developed for the generation of CMV-specific T cell responses including CMV-infected fibroblasts (20); CMV-infected MoDC (21,22); MoDC pulsed with pp65 peptides (23,24); MoDC pulsed with recombinant pp65 protein (25); adenovirus vector-infected MoDC (26); vaccinia virus vector-infected MoDC (27); LCL transfected with pp65 gene (28); and retrovirus vector-infected LCL (29). All of the methods described to date have limitations.…”

“…The results, however, do not necessarily preclude the possibility of priming naive T cells with CD40-B/pp65 cells. Kleihauer et al (23) showed that cytotoxic T cell lines were generated from CMV-seronegative donors only in 2 of 11 donors starting with 3 ϫ 10 6 PBMC, even after stimulating with pp65 peptidepulsed MoDC. Szmania et al (24) also reported that only 2 of 10 CTL lines were pp65 specific, when 2 ϫ 10 6 PBMC were stimulated with MoDC.…”

CMV-Specific TCR-Transgenic T Cells for Immunotherapy

“…Remarkably, with a small proportion of CMV-seronegative donors it has been possible to generate CMV-specific T cells by in vitro priming (45)(46)(47). However, we expect it to be very difficult to translate these observations into feasible clinical procedures, because the precursor frequency of naive CMV-specific CD8 ϩ T cells in CMV-seronegative individuals, estimated from general considerations on TCR diversity (48), is unlikely to be above 1 in 10 6 naive T cells.…”