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Title: Ex vivo manipulation of CD8 T cells to improve adoptive cell therapy against cancer
Author: Leonard, Henry
ISNI:       0000 0004 8501 8807
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2018
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Adoptive cell transfer (ACT) is a potentially curative cancer therapy in which autologous tumourspecific lymphocytes undergo vast levels of ex vivo expansion before being reinfused into the patient. Despite many promising results, ACT efficacy is limited by both poor persistence and weak anti-tumour responses, mainly due to high levels of terminal differentiation displayed by transferred T cell and immunosuppressive factors associated with the tumour micro-environment. Technical developments in genetic modifications have allowed for the manipulation of T cell longevity and function, improving the potency of ACT and broadening the range of treatable cancers. The investigation of which genetic modifications are best suited for enhancing the function of CD8 T cells in the context of ACT is therefore a growing area of research. Here, I developed a protocol using OT-I T cells for the treatment of mice bearing EG7 tumours, including vaccination and sublethal-irradiation strategies, where the impact of genetic modifications could be assessed. Firstly, I aimed to enhance the formation of memory CD8 T cells, as this phenotype is positively correlated with improved ACT therapy in clinical and preclinical settings. Here, the overexpression of either FOXO1, FOXO3a, or Eomes was sufficient to drive cells towards aspects of the classic memory profile, yet this did not lead to improved responsiveness during in vivo vaccination of immune competent mice. While enhanced engraftment was achieved by T cells expressing constitutively active FOXO1 in a lymphopenic setting, these cells had limited peripheral surveillance and showed reduced anti-tumour immunity in an ACT model. These data highlighted the complexities of interpreting the phenotype of cells in relation to function while overexpressing transcription factors. Following this work, I aimed to improve the sensitivity of CD8 T cell stimulation through the knockdown of phosphatases or a kinase that are known to limit aspects of TCR signalling. With a mostly unaltered phenotype following the IL-2 mediated expansion, the in vitro restimulation of transduced CD8 T cells demonstrated that the knockdown of PTPN2, PTPN22, or CSK enhanced IL-2 expression in response to TCR stimulation. However, neither cytotoxic molecule production nor ability to kill target cells was observed in vitro. In vivo, it was shown that PTPN2 knockdown uniquely improved CD8 T cell accumulation and granzyme B expression in response to vaccination while also mediating a positive bystander effect for nontransduced cells. As such the knockdown of PTPN2 granted improved anti-tumour immunity within a preclinical ACT model, accompanied by substantial increased in the accumulation of T cells in vivo. The work here highlights the benefits of disrupting factors that suppress T cell stimulation and opens the possibility of future work utilising PTPN2 knockdown alone or in combination with other therapeutic strategies to augment the efficacy of ACT.
Supervisor: Al-Shamkhani, Aymen ; Rogel, Anne Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available