Regulating cytotoxic T lymphocyte Activity through endogenous and synthetic costimulation

Abstract

Cytotoxic T lymphocytes (CTLs) are pivotal effector cells in the immune response against tumour cells. Costimulation, typically provided through CD28, increases activation efficiency of CTLs. Tumour cells frequently modulate expression of costimulatory ligands, reducing CTL activation efficiency and inducing anergy. Immunotherapies are being developed to enhance CTL costimulation and activation by introducing costimulation through genetically engineered synthetic receptors.
Here we investigate the impacts for one synthetic receptor, CoStAR™, designed to provide costimulation to human CTLs upon engagement of its ligand, the cancer-associated folate receptor α (FRα). CoStAR™ contains CD28 and CD40 cytoplasmic costimulatory domains which activate following ligand engagement to provide costimualtion to engineered CTLs. We aimed to determine the impact CoStAR™ expression and engagement of FRα has on human CTL cell coupling with tumour cells and CTL morphology, its localisation within CTLs, and a possible mechanism of action. Our findings demonstrate CoStAR™ engagement increases CTL tight cell coupling with tumour cells, following initial contact, from 18% to 78%. Additionally, CoStAR™ forms clusters at the immunological synapse following engagement of its ligand, FRα, and we show how CoStAR™ promotes the formation of CoStAR™+ filipodia-like structures.
In addition, it was investigated how overexpression of costimulatory ligands, ICAM-1 and CD86, on tumour cells impacts CTL killing and secretion of the immune-stimulatory cytokine interferon gamma (IFN-γ) using two-dimensional (2D) and three-dimensional (3D) murine tumour models. CD86 ligates CD28, while ICAM-1 binds the integrin LFA-1. Current literature present CD86 as an ambiguous prognostic marker in tumours, while little is known about the impact of LFA-1-stimualted costimualtion. Here we show how high levels of CD86 reduces CTL killing efficiency, and ICAM-1 expression retains IFN-γ secretion in CTLs under low peptide presentation conditions. Furthermore, we show how murine renal carcinoma cells overexpressing CD86 and ICAM-1 form 3D tumour spheroids suitable for future 3D cytotoxicity assays.

Date of Award	5 Dec 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Christoph Wuelfing (Supervisor)