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

Résumé

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.