Hold your breath and tighten your belt – engineering γδT cells to survive in hostile tumour environments

Primary supervisor: Jonathan Fisher, UCL

Secondary supervisor: James Arnold, King’s College London

Project

Whilst cellular therapy for haematological cancers has been highly successful, results against solid tumours have been disappointing. Several bottlenecks exist, including a challenging tumour microenvironment and a dependence on autologous starting material for the majority of platforms.

γδT cells are an immune-subset which is not allo-reactive, has innate anti-tumour activity and, if present in tumours confers the strongest indicator of good clinical outcome. In normoxia, Vδ1 γδT cells have potent activity against childhood tumours including neuroblastoma [1]. Key metabolic responses to nutrient and oxygen-poor environments blunt their anti-cancer activity however [2, 3] – this project aims to overcome this.

Aim 1: Using established techniques which facilitate efficient γδT engineering, Vδ1 will be expanded from healthy donor blood and engineered to secrete IL-15, a strategy conferring enhanced persistence [4]. Engineered cells will be exposed to neuroblastoma cell lines in the presence of hypoxic and/or glucose-poor conditions, and key transcriptomic changes determined by bulk RNA sequencing. Phenotypic markers including memory, exhaustion and innate immune phenotype will also be determined, as these are frequently used as indicators of immune competence, but typically in normoxia. Controls will include target-free and normoxic/glucose replete conditions.

Aim 2: Using the gene signatures obtained in Aim 1 in conjunction with established hypoxic gene signatures from both γδT cells and αβT cells, a CRISPR guide library will be prepared and introduced to expanded γδT cells using established nucleofection techniques. Nucelofected cells will be exposed to hypoxic and/or glucose-poor conditions in the presence or absence of neuroblastoma targets, and after co-culture the surviving γδT will be isolated and the relative abundance of each gRNA determined and compared to baseline. This work will be supported by a placement in the laboratory of Dr Franziska Blaeschke (KiTZ-Heidelberg) who is an expert on the use of CRISPR screens to guide T-cell engineering.

Aim 3: Lead targets identified in the CRISPR screen will be validated for both function and mechanism – the leads will be chosen based on statistical significance across 5 donors and also the pathways they engage. Whilst the specifics of mechanistic evaluation will be guided by the nature of the gene target, the functional validation will remain consistent. Targeted knockdown of the lead genes will precede evaluation of engineered cells’ ability to serially kill neuroblastoma targets in hypoxic and glucose poor conditions. We will also determine how generalisable the findings are by using an alternative cancer model. For this we will use the patient-derived colorectal cancer organoid systems developed in Dr Chris Tape’s laboratory, using established high-throughput CyTOF analysis to probe not only cytotoxicity but also key markers of γδT cell state and immunophenotype. Importantly, this excludes the potential of confounding factors which would be introduced by using a murine model, where the oxygen and glucose tension would be uncontrollable.

Candidate background

This project would suit candidates with background in immunology and molecular biology, who are familiar with basic bioinformatic techniques.

Potential Research Placements

1. James Arnold, Comprehensive Cancer Centre, King’s College London
2. Franziska Blaeschke, Hopp-Kindertumorzentrum Heidelberg (KiTZ), German Cancer Research Center (DKFZ) – Heidelberg (D)
3. Chris Tape, Cancer Institute, UCL

References

1. J. P. H. Fisher, M. Yan, J. Heuijerjans, L. Carter, A. Abolhassani, J. Frosch, R. Wallace, B. Flutter, A. Capsomidis, M. Hubank, N. Klein, R. Callard, K. Gustafsson, J. Anderson, Neuroblastoma killing properties of Vδ2 and Vδ2-negative γδT cells following expansion by artificial antigen-presenting cells. Clinical Cancer Research 20 (2014), doi:10.1158/1078-0432.CCR-13-3464.
2. M. Barisa, D. Fowler, J. Fisher, Interplay between γδT-Cell Metabolism and Tumour Microenvironment Offers Opportunities for Therapeutic Intervention. (2021), doi:10.20900/immunometab20210026.
3. J. H. Park, H.-J. Kim, C. W. Kim, H. C. Kim, Y. Jung, H.-S. Lee, Y. Lee, Y. S. Ju, J. E. Oh, S.-H. Park, J. H. Lee, S. K. Lee, H. K. Lee, Tumor hypoxia represses γδT cell-mediated antitumor immunity against brain tumors. Nat Immunol 22, 336-346 (2021).
4. D. Fowler, M. Barisa, A. Southern, C. Nattress, E. Hawkins, E. Vassalou, A. Kanouta, J. Counsell, E. Rota, P. Vlckova, B. Draper, T. De Mooij, A. Farkas, H. Brezovjakova, A. T. Baker, K. Scotlandi, M. C. Manara, C. Tape, K. Chester, J. Anderson, J. Fisher, Payload-delivering engineered γδT cells display enhanced cytotoxicity, persistence, and efficacy in preclinical models of osteosarcoma. Sci Transl Med 16, eadg9814 (2024).
5. Kosti, P., et al (2021) Hypoxia-sensing CAR T-cells provide safety and efficacy in treating solid tumors. Cell Reports Medicine, 12:658315. * co-senior authors