Cellular and Molecular Aspects Of Naïve T cell Priming for Adoptive Immunotherapy

Abstract

Over the past decade immunotherapy has emerged as a powerful tool in the treatment of advanced cancer and recurrent viral diseases. In particular, clinical success with Immune Checkpoint Blockade has led to the rapid approval of immunomodulating drugs for the treatment of metastatic cancers. However, despite recent success, a large proportion of patients do not respond or only experience a partial response. Now, it is becoming increasingly evident that the effectiveness of immunomodulating treatments are reliant on a pre-existing immune response to the tumour. Thus increasing immune recognition of the tumour is a key goal in the advancement of immunotherapy. Adoptive cell therapy (ACT) is a modality that aims to generate a robust tumour-directed response by the infusion of tumour specific, ex vivo expanded, autologous cells. In this thesis we examine aspects of naïve T cell culture with the aim of improving polyclonal and antigen specific expansions for use in adoptive immunotherapy. We show that while traditional monocyte-derived dendritic cells are variable in phenotype, ex vivo monocytes stimulated with a TLR agonist are able to generate a robust antigen-specific response to the model antigen MART1/Melan A as well as the germ-line antigen NY-ESO-1. Building on previous work from the host laboratory, we demonstrate that naïve expansion in high dose IL-15 improves expansion numbers but leads to a loss of ‘naïve-like’ phenotype, in particular lymphoid homing receptors CD62L and CCR7 as well as the important costimulation protein CD28. However, low dose IL-15 during primary and secondary expansion results in improved cell yield while preserving a population of central memory phenotype. To further develop our ex vivo protocol we investigated the effect of independent administration of the costimulation molecule, CD28. We make use of an artificial APC system, and demonstrate that beadbound αCD28 is able to achieve appropriate costimulation when delivered in trans to a TCR stimulus or prior to TCR. We also demonstrate that CD28, when ligated with a bead-bound antibody, is able to generate a phosphorylation signal that is unique from TCR, which may provide a mechanism for the ‘storage’ of costimulation in trans. Finally, we investigate T cell signal transduction and demonstrate differential MAP kinase activation between naïve and memory cells. We also explore phosphoprotein recognition by immunohistochemistry as an application for detection of recent TCR activation in situ.