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: ObjectiveThe aim of this study was to isolate and expand donor-derived human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs) for adoptive transfer of 107 cells per m2 of body surface area to restore protective immunity after stem cell transplantation.: Materials and MethodsA new strategy to generate HCMV-specific CTLs using the interferon-γ (IFN-γ) secretion assay, followed by expansion to numbers sufficient for clinical application with interleukin-2 and feeder cell stimulation, is described.: ResultsFrom 1 to 5 × 104 HCMV peptide-specific T lymphocytes (greater than 90% CD3+CD8+) were isolated from 1 to 2 × 108 peripheral blood mononuclear cells comparable to 50 to 100 mL of blood from HLA-A*0201 HCMV seropositive blood donors (n = 14) and expanded ex vivo after a median of 16 days (range 8–28 days; n = 13) to greater than 107/m2 HCMV peptide-specific CTLs using autologous (n = 2) or allogeneic (n = 11) feeder cell stimulation. In three experiments, expansion to 6 weeks was performed, achieving a median of 1.6 × 109 cells (range 6.1 × 108–3.3 × 109). Characterization of these HCMV-specific CTL lines revealed an average purity of 89.2% (range 66.2–99.3%) using HCMV pp65 peptide HLA-A*0201 tetramer staining (n = 14) and 89.4% (range 64.4–99.5%) by peptide-specific IFN-γ secretion (n = 7). A median of 82.6% (range 76.0–88.0%) showed perforin secretion (n = 3) and 57.5% (range 22.2–80.7%) specific lysis of peptide-pulsed T2 cells (n = 5). A median of 52.2% (range 35.2–7.3%) revealed specific killing of HCMV-infected autologous, but not allogeneic, fibroblasts (n = 6).: ConclusionIFN-γ secretion assay allows development of a simple and rapid protocol with short expansion times for generation of greater than 107/m2 HCMV-specific CTLs for adoptive immunotherapy. [Copyright &y& Elsevier]

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process. [ABSTRACT FROM AUTHOR]

Background aims: Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorders (PTLD) belong to the most dreaded complications of immunosuppression. The efficacy of EBV-specific T-cell transfer for PTLD has been previously shown, yet the optimal choice of EBV-derived antigens inducing polyclonal CD4' and CD8' T cells that cover a wide range of human leukocyte antigen types and efficiently control PTLD remains unclear. Methods: A pool of 125 T-cell epitopes from seven latent and nine lytic EBV-derived proteins (EBVmjx) and peptide pools of EBNA1, EBNA3c, LMP2a and BZLF1 were used to determine T-cell frequencies and to isolate T cells through the use of the interferon (IFN)-y cytokine capture system. We further evaluated the phenotype and functionality of the generated T-cell lines in vitro. Results: EBVmix induced significantly higher T-cell frequencies and allowed selecting more CD4+ IFN-y+ and CD4+ IFN-γ+ cells than single peptide pools. T cells of all specificities expanded similarly in vitro, recognized cognate antigen, and, to a lower extent, EBV-infected cells, exerted moderate cytotoxicity and showed reduced alloreactivity. However, EBVmix-specific cells most efficiently controlled EBV-infected lymphoblastoid cell lines (LCLs). This control was mainly mediated by EBVspecific CD8+ cells with an oligoclonal epitope signature covering both latent and lytic viral proteins. Notably, EBVspecific CD4+ cells unable to control LCLs produced significantly less perforin and granzymc B, probably because of limited LCL epitope presentation. Conclusions: EBVmjx induces a broader T-cell response, probably because of its coverage of latent and lytic EBV-derived proteins that may be important to control EBV-transformed B cells and might offer an improvement of T-cell therapies. [ABSTRACT FROM AUTHOR]