Flexible vectors for immunotherapy against cancer and pathogens
Application deadline: 23 November 2018
Start date: October 2019
Research theme: Infection, immunity and repair
This study aims to generate new understanding/reagents that will enable the expansion of highly functional NK and T-cells for immunotherapy. CD57 is a carbohydrate antigen, that is found on NK and T-cell subsets, and large granular leukaemias (NK and T-LGL).
Neither the function of CD57, nor the proteins that carry it are known, but it is a signature for highly cytotoxic effector cells that could be used for immunotherapy. Human cytomegalovirus (HCMV) is a herpesvirus that induces large NK and CD8+ T-cell expansions expressing CD57 in vivo. Wang/Stanton have worked on HCMV for 18 years, generating unique reagents/technologies to dissect NK and T-cell responses to the virus. Screening with a new assay shows that stimulating blood-derived cells with customised HCMV vectors enables the in vitro expansion of different T-cell subsets: deletion of the viral genes US2-11 or US18-22 expand CD57- cells, whereas deletion of RL10-UL1 expands CD57+ cells.
Thus, manipulation of HCMV genes in these regions enables the expansion of bespoke T-cell types. Further, Heurich-Sevcenco has developed biochemical methods for analysing CD57 expressing proteins and Wooldridge grows CD8+ T-LGL clones and has access to NK-LGL.
Project aims and methods
We intend to:
- Identify individual HCMV genes within US18-22 and RL10-UL1 that expand CD8+57- and CD8+57+ T cells respectively, and define mechanism of action.
- Perform the same analysis for NK cells targeting expansion of CD57+ NK cells (powerful mediators of antibody-dependent cellular cytotoxicity).
- Compare proteins that carry CD57 on NK-LGL, T-LGL, HCMV-specific T-cells, CD57+ NK cells and define function.
In this way, we will define the pathways that drive the growth of different NK and T-cell subsets, which can then be exploited to expand optimised effector cells for multiple different therapeutic settings, as well as determine why CD57 is a marker for such effective cells.
The new expansion assay will be used to compare HCMV knock-outs (KO) vs wildtype infected cells. Individual HCMV KOs within US18-22 have already been made and analysed by proteomics. Responding cells will be defined by cytometry; CD3, CD8, tetramer, CD45RA, CD45RO, CCR7 (memory); CD57, PD1, Tim3, LAG3 (exhaustion/senescence), CD27, CD95 (stem cell memory), CD56, NKG2C (NK cells). Further expts will use NK and CD8+ T-cell lines in established proliferation/functional assays.
Proteomics (established in Cardiff, with collaborators in Cambridge) comparing wildtype and HCMV KO infected cells will be used to identify host proteins targeted by particular genes that thus may orchestrate effector cell expansion. Hits will be validated in the above assays using inhibitory reagents and mechanism defined via established techniques. In parallel, proteomics will be used to identify proteins carrying CD57, comparing HCMV-specific T-cells, NK cells, NK-LGL and T-LGL. Hits will be validated biochemically and functionally.
Prof Linda Wooldridge, University of Bristol.