Isolation of Highly Suppressive CD25+FoxP3+ T Regulatory Cells from G-CSF-Mobilized Donors with Retention of Cytotoxic Anti-Viral CTLs: Application for Multi-Functional Immunotherapy Post Stem Cell Transplantation.

Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells

Guía de recursos para la investigación antropológica.

Sin resume

Evaluation and optimization of immunomagnetic methods for the purification and selection of CMV-specific cytotoxic T lymphocytes from G-CSF primed peripheral blood mononuclear cells for adoptive immunotherapy

Adoptive transfer of cytomegalovirus (CMV)-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or weeks after granulocyte-colony stimulating factor (G-CSF) mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. However, the tolerogenic effects induced by G-CSF on T cells could affect the anti-viral in vivo functionality of CMV-specific T cells obtained from G-CSF mobilized donor samples if they were infused into the patient. In this thesis it has been investigated the use of G-CSF mobilized samples for the manufacture of CMV-specific T cells using two direct selection methods. First, CMV-specific T cells were isolated based on activation-dependent CD137 expression, which demonstrated a high specificity for CMV, the secretion of cytotoxic effector molecules, proliferation in response to the antigen and lysis of CMV-loaded target cells, showing that they shared the same characteristics as CMV-specific T cells obtained from non-mobilized cells. On the other hand, CMV-specific CD8+ T cells were isolated using MHC-multimers and showed that products obtained from G-CSF mobilized samples were able to respond to the antigen by expressing activation markers and secreting pro-inflammatory cytokines and lytic molecules, but the in vitro proliferative capacity was impaired

Evaluation and optimization of immunomagnetic methods for the purification and selection of CMV-specific cytotoxic T lymphocytes from G-CSF primed peripheral blood mononuclear cells for adoptive immunotherapy

Adoptive transfer of cytomegalovirus (CMV)-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or weeks after granulocyte-colony stimulating factor (G-CSF) mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. However, the tolerogenic effects induced by G-CSF on T cells could affect the anti-viral in vivo functionality of CMV-specific T cells obtained from G-CSF mobilized donor samples if they were infused into the patient. In this thesis it has been investigated the use of G-CSF mobilized samples for the manufacture of CMV-specific T cells using two direct selection methods. First, CMV-specific T cells were isolated based on activation-dependent CD137 expression, which demonstrated a high specificity for CMV, the secretion of cytotoxic effector molecules, proliferation in response to the antigen and lysis of CMV-loaded target cells, showing that they shared the same characteristics as CMV-specific T cells obtained from non-mobilized cells. On the other hand, CMV-specific CD8+ T cells were isolated using MHC-multimers and showed that products obtained from G-CSF mobilized samples were able to respond to the antigen by expressing activation markers and secreting pro-inflammatory cytokines and lytic molecules, but the in vitro proliferative capacity was impaired

Sintesi autopropagante ad alta temperatura in condizioni di microgravità

Self-propagating high temperature synthesis (SHS) under microgravity conditions was studied. The green density of the reacting sample also influenced SHS processes since it affected system reactivity and thermal conductivity of the compact. Either for the case of cylindrical pellets or during combustion front quenching (CFQ) experiments, temperature during reaction evolution was measured using thermocouples embedded in the pellet