No abstract

Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore longlasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4 ؉ and CD8 ؉ CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinicalscale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMVspecific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)-restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-␥ (IFN-␥) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 ؋ 10 8 combined CD4 ؉ and CD8 ؉ CMV-specific T cells, on average, were generated, as determined by antigentriggered IFN-␥ production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4 ؉ and CD8 ؉ T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4 ؉ and CD8 ؉ T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4 ؉ and CD8 ؉ CMV-specific T cells under conditions mimicking good manufacturing practice.

Reconstitution of human cytomegalovirus (HCMV)-specific cytotoxic T lymphocytes (CTLs),

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.

SummaryT cell lines with defined cytokine profiles are an invaluable tool for assessing the control of immune responses both in vitro and in vivo. Production of such cell lines can be complex and time-consuming. Here we present a powerful technique to assay the cytokines produced by T cells activated polyclonally or with specific antigens. This paper presents a detailed methodology for the identification and isolation of cytokine-producing T cells activated with the artificial superantigen, CytoStim, or viral and fungal antigens. These cells can be analysed for different cytokines simultaneously, or cultured further to rapidly establish T cell lines making known cytokine types. We highlight the enumeration, isolation and phenotype of interleukin-17-producing T cells, and the rapid generation of virus-specific Th1 T cell lines.

No abstract

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-gamma (IFN-gamma) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01 +/- 0.84 x 10(6) total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56% +/- 20.8%, yield=51% +/- 32.4%) but also CD8+ (mean=42% +/- 27%, yield = 56% +/- 39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-gamma and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

Die Perowskite Ba2BIIISbO6 kristallisieren monoklin (BIII  La, Pr, Nd) bzw. kubisch (BIII  Sm, Eu, Gd, Tb, Dy, Yb, Lu, Y). Die Sr‐Verbindungen, Sr2BIIISbO6, besitzen eine monokline (BIII  Nd, Sm, Eu, Dy), rhombische (BIII  Yb, Lu, Y, Sc) bzw. kubische (BIII  In, Ga) Perowskitstruktur. Mit Intensitätsberechnungen und schwingungsspektroskopischen Methoden ließen sich Abweichungen von einer vollständigen Ordnung der BIII‐ und SbV‐Ionen nachweisen. In den Perowskiten Ba2BIIIMVO6 mit MV  Nb, Ta ist ebenfalls keine vollständige Kationenordnung erkennbar.