The University of Southampton
University of Southampton Institutional Repository

Ex vivo manipulation of CD8 T cells to improve adoptive cell therapy against cancer

Ex vivo manipulation of CD8 T cells to improve adoptive cell therapy against cancer
Ex vivo manipulation of CD8 T cells to improve adoptive cell therapy against cancer
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.
University of Southampton
Leonard, Henry
e536c6af-c363-47fa-8ff8-18587f1ddc48
Leonard, Henry
e536c6af-c363-47fa-8ff8-18587f1ddc48
Al-Shamkhani, Aymen
0a40b3ce-9d71-4d41-9369-7212f0a84504
Rogel, Anne
5a895ba8-c877-484f-a9c1-34a2b1af6414

Leonard, Henry (2018) Ex vivo manipulation of CD8 T cells to improve adoptive cell therapy against cancer. University of Southampton, Doctoral Thesis, 212pp.

Record type: Thesis (Doctoral)

Abstract

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.

Text
HL Thesis - Version of Record
Available under License University of Southampton Thesis Licence.
Download (9MB)

More information

Published date: June 2018

Identifiers

Local EPrints ID: 435542
URI: http://eprints.soton.ac.uk/id/eprint/435542
PURE UUID: 0f8dc809-963d-431c-ad76-76b9ee8a5f44
ORCID for Aymen Al-Shamkhani: ORCID iD orcid.org/0000-0003-0727-4189

Catalogue record

Date deposited: 08 Nov 2019 17:30
Last modified: 27 Jun 2020 04:01

Export record

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×