· Web viewLim SP, Costantini B, Mian SA, Perez Abellan P, Gandhi SA, Martinez Llordella M, et al. Treg sensitivity to FasL and relative IL-2 deprivation drive Idiopathic Aplastic

2020 CRUK KHP Centre PhD Project Catalogue

Contents

1. Multi-omic data integration to study cancer spatial and temporal heterogeneity in response to immunotherapy at single cell resolution ……………………………………………………………………………………………………………………………………p2

2. How to die well. Manipulating cell death-dependent immune programmes as an adjuvant for chimeric antigen receptor (CAR) T cell therapy……………………………………………………………………………………………………………………………p3

3. Understanding how tumour hypoxia is linked to chemo-resistance and immune evasion of cancers……………….p4

4. Investigation of cancer microenvironment-induced transcriptomic and epigenetic changes in a bioengineered extracellular matrix model of colon cancer liver metastasis……………………………………………………………………………..p5

5. Evaluating immunological responses in lymph nodes as a predictive biomarker for immune checkpoint inhibitor response…………………………………………………………………………………………………………………………………………………………..p6

6. Assessing the interaction of radiation with anti-PD1/PD-L1 antibody and/or cisplatin chemotherapy in patient-derived organoids and mouse models in head and neck squamous cell carcinoma (HNSCC)…………………………….p7

7. Developing inflammatory resistant regulatory T cells for controlling graft versus host disease (GvHD)…………….p8

8. Identifying unconventional neoepitopes and immune phenotypes in Myeloproliferative Neoplasia to facilitate personalised combined immunotherapies………………………………………………………………………………………………………..p9

1. Multi-omic data integration to study cancer spatial and temporal heterogeneity in response to immunotherapy at single cell resolution

Co-supervisor 1: Francesca Ciccarelli

Research Division/Department: Comprehensive Cancer Centre, Cancer and Pharmaceutical Sciences

Email: [email protected]

Co-supervisor 2: Christopher Yau

Research Division/Department: Alan Turing Institute


Co-supervisor 3: Jo Spencer

Research Division/Department: Peter Gorer Department of Immunobiology, Immunology and Microbial Sciences


Background: Despite the overall promising results of cancer immunotherapy, still many cancer patients fail to benefit. This likely reflects the complex nature of the immune response to cancer, which relies on a highly regulated and often organ-specific cascade of events. Furthermore, cancer itself is a complex system that constantly evolves in space and time to adapt to and survive in the surrounding tumour microenvironment (TME). Cancer cells progressively acquire genetic alterations that drive and sustain the oncogenic process, including those that actively favour immune escape and/or reduce immune infiltration.

A deep understanding of the dynamic interplay between cancer and the immune system in space and time is at the forefront of cancer research to gain mechanistic insights, modulate treatment choices, and obtain optimal and durable response to immunotherapy.

Scientific hypothesis: The scientific hypothesis of this project is that a detailed understanding of the spatial and temporal interactions between cancer cells and the immune system is crucial to enhance our knowledge of the molecular and cellular determinants of response to cancer immunotherapy.

We will therefore develop new computational approaches that will enable the integrated analysis of single cell data at spatial resolution of tumour and associated TME to enable modelling of molecular and cellular interactions between cancer and the immune system.

Experimental plan: The student will develop computational tools to integrate high dimensional data of the TME from imaging mass cytometry1 and CODEX2 with spatial transcriptomics of the cancer cells based on 10x Genomics technology in fixed tissues3. This will allow detection of expression changes in key cancer genes and their interactions with immune cells in specific tumour and TME compartments before and after immunotherapy.

The student will be embedded in the team of computational and experimental cancer biologists of the Ciccarelli lab based at the Francis Crick Institute, with full access to top-class High-Performance Computing facility of the CRUK City of London Cancer Centre. The co-supervision of Professors Yau and Spencer will expose the student to the scientific environments of the Alan Turing Institute, devoted to big data analysis, and the Department of Immunobiology at KCL where they will learn how to identify immune cell populations for the validation of computational predictions.

References:

1. Giesen, C. et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nature Methods 11, 417 (2014)

2. Schürch, C.M. et al. Coordinated Cellular Neighborhoods Orchestrate Antitumoral Immunity at the Colorectal Cancer Invasive Front. Cell S0092-8674(20)30870-9 (2020)

3. Villacampa, E.G. et al. Genome-wide Spatial Expression Profiling in FFPE Tissues. bioRxiv, 2020.07.24.219758 (2020)

4. Märtens K, Yau C Neural Decomposition: Functional ANOVA with Variational Autoencoders. Proceedings of the Twenty Third International Conference on Artificial Intelligence and Statistics, PMLR 108:2917-2927 (2020)

5. Bortolomeazzi M, et al… Spencer J, Ciccarelli FD Immunogenomic profile of colorectal cancer response to anti-PD1 immunotherapy. (under review; manuscript available upon request: [email protected])

2. How to die well. Manipulating cell death-dependent immune programmes as an adjuvant for chimeric antigen receptor (CAR) T cell therapy

Co-Supervisor 1: Francesco Dazzi

Research Division/Department: Comprehensive Cancer Centre, School of Cancer & Pharmaceutical Sciences


Co-Supervisor 2: John Maher

Research Division/Department: Comprehensive Cancer Centre, School of Cancer & Pharmaceutical Sciences


Background: Chimeric antigen receptor-engineered T cells (CAR-T cells) have revolutionised the field of cancer immunotherapy. However, therapeutic activity is far from optimal. Probably one of the most important obstacles hindering their efficacy is the immunosuppressive activity exerted by the tumour microenvironment (TME). The modalities and molecular mechanisms underlying TME immunosuppression are incompletely understood. In particular, the consequences of CAR-T cell mediated killing of the tumour target cells have never been investigated. The elicitation of different programmed cell death pathways can in fact differently affect immune responses. Whilst apoptosis tends to be immunologically silent or tolerogenic, necroptosis has been linked to the induction of a pro-inflammatory microenvironment that promotes T cell priming. Furthermore, stromal cells, which constitute a prominent part of the TME, are selectively sensitive to undergo tolerogenic apoptosis when in physical contact with activated cytotoxic T or natural killer cells and this is sufficient to initiate local immunosuppression

Scientific hypothesis: We surmise that CAR-T cells elicit two different sets of programmed cell death in the TME with opposing effects on the recipient’s immune system, in one case with immunogenic properties (tumour cell death) and in the other with immunosuppressive activities (stromal cell death). Dissecting the molecular mechanisms by which the different cell death pathways in tumour cells and MSC modulate the immune system will offer novel opportunities to selectively manipulate cell death modalities therapeutically with the goal of restoring local immunocompetence and achieving long-term remissions.

Experimental plan:

Aim 1.To identify the molecular cell death pathways elicited by CAR-T cells in the tumour-specific target and in stromal cells by using a combination of transcriptomics and proteomics

Aim 2.To characterise the impact of the molecular pathways involved in dying tumour cells (DTC) on cross-priming the recipient against tumour-specific immune responses. The candidate molecules will be assessed for their ability to promote or inhibit the cross-priming of recipient immune system against novel antigens in mouse model.

Aim 3.To characterise the impact of the molecular pathways involved in dying stromal cells (DSC) on suppressing DTC mediated cross-priming

Aim 4.To validate the molecular signature identified in DTC and DSC in biopsies and peripheral blood samples obtained from patients undergoing a clinical trial for CAR-T cells in head and neck cancer.

The project will deliver a molecular classifier of the cell death pathways that regulate immune responses in the context of CAR-T cell therapies.

References

1 Whilding LM, Maher J. CAR T-cell immunotherapy: The path from the by-road to the freeway? Mol. Oncol. 2015;9(10):1994–2018.

2 Galleu A, Riffo-Vasquez Y, Trento C, et al. Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation. Sci. Transl. Med. 2017;9(416)

3 Cheung TS, Dazzi F. Mesenchymal-myeloid interaction in the regulation of immunity. Semin. Immunol. 2018;35:

4 Whilding LM, Halim L, Draper B, Parente-Pereira AC, Zabinski T, Davies DM et al. CAR T-cells targeting the integrin αvβ6 and co-expressing the chemokine receptor CXCR2 demonstrate enhanced homing and efficacy against several solid malignancies. Cancers. 2019 May 14;11(5):1-17. 674

5 Yatim N, Jusforgues-Saklani H, Orozco S, et al. RIPK1 and NF- B signaling in dying cells determines cross-priming of CD8+ T cells. Science (80-. ). 2015;350(6258):328–334

3. Understanding how tumour hypoxia is linked to chemo-resistance and immune evasion of cancers.

Co-supervisor 1: Gilbert Fruhwirth

Research Division/Department: Cancer and Pharmaceutical Sciences


Co-supervisor 2: Timothy Witney

Research Division/Department: Biomedical Engineering and Imaging Sciences


Background: Tumour growth is reliant on oxygen supply, supported within a tumour by angiogenesis and vasculogenesis. Insufficiency in these processes results in tumour hypoxia, which increases tumour aggressiveness and facilitates evasion of the immune system. Immune evasion in hypoxic tumours has been linked to upregulation of immune checkpoint inhibitors such as CTLA-4 and PD-L1, secretion of immunosuppressive cytokines, and the recruitment of immunosuppressive cell types (e.g. regulatory T-cells, myeloid-derived suppressor cells, tumour associated macrophages). Most notably, tumour hypoxia renders cancers resistant to treatment [Eckert-2019]. Many mechanistic aspects of the interplay between hypoxia, chemo-resistance and immune evasion, however, remain elusive.

Scientific hypothesis: In this project, we aim to define how tumour hypoxia changes the susceptibility to chemotherapy, how persistent these effects are, and what consequences they have for the immune compartment of lung tumours. We will exploit a new in vivo fate-mapping approach [Godet-2019] that we modified to identify cancer cells that had experienced hypoxia at any point in their life-courses using radionuclide imaging (Fig.A). Our approach is based on established fluorescence-radionuclide reporters [Fruhwirth-2014, Volpe-2020] exclusively controlled by the hypoxia regulator HIF1alpha. This enables us to track these cells in vivo non-invasively and then isolate them to ex vivo analyse their phenotypes.

Experimental plan: The generation of hypoxia reporter lung cancer cell lines (Year 1, Fruhwirth lab) will enable us to identify, spatiotemporally monitor, and subsequently analyse cells that have previously experienced a hypoxic microenvironment. We will apply this methodology to study the impact of hypoxia on treatment outcomes in lung cancer using normal and chemo-resistant tumours, including the characterisation of their immune cell infiltrates (Year 2, Fruhwirth lab). Single-cell transcriptomics of re-isolated cancer cells will identify phenotypes of chemoresistance in post-hypoxic cancer cells. (Year2, Fruhwirth lab in collaboration with Ciccarelli at Crick). Re-establishment of syngeneic tumour models will further reveal how stable such phenotypes are in vivo and validate molecular imaging of the antioxidant response network (xCT (Fig.B), Nrf2; [Greenwood-2019] as markers for chemo-resistance in this setting (Year 3, Fruhwirth/tumour models and Witney/molecular imaging).

Figure: (A) Scheme illustrating the hypoxia-driven genetic switch that renders normoxic (red fluorescent) cells permanently detectable by non-invasive in vivo imaging (by PET/SPECT-CT) when cells experience hypoxia (cells turn green fluorescent and receptive to import of a probing radiotracer). The switch is driven by a Cre recombinase under hypoxia control (B) Molecular imaging of the cystine/glutamate antiporter (xCT) activity using the positron emission tomography (PET) probe [18F]FSPG (hue). Anatomical context is provided by co-registration with x-ray computed tomography (grayscale). Coronal section of a mouse bearing lung cancer; signals outside of the lung depict other tissues with significant xCT activity or the radiotracer excretion route (bladder).

References

1. Eckert F et al. Front Immunol (2019) 10: 407. doi: 10.3389/fimmu.2019.00407.

2. Fruhwirth GO et al J Nucl Med (2014) 55/4:686. doi: 10.2967/jnumed.113.127480.

3. Godet I et al. Nat Commun (2019) 24;10/1:4862. doi: 10.1038/s41467-019-12412-1.

4. Greenwood HE,…, Witney TH. Clin Cancer Res (2019) 25/8:2471. doi:10.1158/1078-0432.CCR-18-3423.

5. Volpe A,…, Fruhwirth GO. Mol Ther (2020) in press. doi: 10.1016/j.ymthe.2020.06.028.

4. Investigation of cancer microenvironment-induced transcriptomic and epigenetic changes in a bioengineered extracellular matrix model of colon cancer liver metastasis.

Co-supervisor 1: Michelle Holland

Research Division/Department: Department of Medical and Molecular Biology, Faculty of Life Sciences and Medicine


Co-supervisor 2: Luca Urbani

Research Division/Department: Institute of Hepatology, School of immunology & Microbial Sciences


Background: Colorectal cancer (CRC) is the 3rd most common cause of cancer-related deaths worldwide. The liver is the most common site of CRC metastasis and in the majority of cases is unresectable, making it the leading cause of morbidity. Therefore, preventing CRC metastasis formation is an important target for improving the outcome for patients with CRC. As a component of the local microenvironment, the extracellular matrix (ECM) is a critical element in cancer progression, which is characterised by ECM dysregulation and remodeling. Our previous work has shown that isolated ECM from CRC patient liver metastases encourages increased proliferation of CRC cells in 3D cultures and is accompanied by changes in gene expression in the cells, including a number of genes involved in regulating the epigenome1. The epigenome is widely altered in cancer and therefore, this project aims to investigate how ECM interactions influence the transcriptome, epigenome and ultimately cell behaviour.

Scientific Hypothesis: The aim of this study is to investigate the unexplored issue of how the ECM facilitates cancer progression by altering the cellular epigenome and transcriptome of the tumour cell using a novel patient-derived bio-engineered 3D culture system developed with decellularised ECM-scaffolds from CRC, CRC liver metastases and matched cancer-free tissue. This will provide the first direct study of how the ECM induces tumour cell molecular responses. Signatures identified will be validated using data derived from primary tumours to demonstrate clinical relevance. We hypothesise this will identify novel pathways as potential therapeutic targets for further investigation.

Experimental plan: Characterisation of ECM-directed changes in cell behaviour. Archived same-patient tissues (healthy colon, CRC, healthy liver, CRC liver metastasis) will be decellularised using established techniques1,2. All ECM-scaffolds will then be re-cellularised with common CRC cell lines and maintained in custom-made bioreactors for perfusion culture (Figure). Cell proliferation and invasiveness will be analysed using multiple orthogonal approaches.

Profiling transcriptional and DNA methylation changes directed by the ECM. Material will be extracted from the cells grown on scaffolds and genome-wide profiles for RNA expression and DNA methylation generated using state-of-the-art techniques.

Comparison to patient-derived data to demonstrate clinical relevance. Established bioinformatic pipelines will be used to identify differential DNA methylation and transcriptional changes induced by the ECM of healthy-vs-cancerous tissues, colon-vs-liver and primary-vs-metastatic tumours. These will be compared to publicly available data from primary tissues. Identified signature pathways will be disrupted with blocking agents in cells grown on scaffolds for validation.

References

1. E. D'Angelo et al., Patient-Derived Scaffolds of Colorectal Cancer Metastases as an Organotypic 3D Model of the Liver Metastatic Microenvironment. Cancers (Basel) 12, (2020).

2. M. Piccoli et al., Decellularized colorectal cancer matrix as bioactive microenvironment for in vitro 3D cancer research. J Cell Physiol 233, 5937-5948 (2018).

3. Law PP, Holland ML. DNA methylation at the crossroads of gene-environment interactions. Essays Biochem 63, 717-726 (2019).

5. Evaluating immunological responses in lymph nodes as a predictive biomarker for immune checkpoint inhibitor response.

Co-Supervisor 1: Sheeba Irshad

Research Division/Department: School of Cancer and Pharmaceutical Sciences. Email: [email protected]

Co-Supervisor 2: Anita Grigoriadis

Research Division/Department: School of Cancer and Pharmaceutical Sciences, Email: [email protected]

Co-Supervisor 3: Debra Josephs

Research Division/Department: Guy’s & St Thomas’ NHS Foundation Trust, [email protected]

Background: The need for predictive biomarkers that can accurately select patients who will respond to immune checkpoint inhibitor (ICI) immunotherapies remains a clinically unmet need. The majority of research efforts have focused on expression of immune-related markers on the tumour and its associated tumour microenvironment (TME). However, the immune response to tumor neoantigens starts at the regional lymph nodes, where antigen presentation takes place and is regulated by multiple cell types and mechanisms. Knowledge of the immunological responses in bystander lymphoid organs following ICI therapies and their association with changes in the TME, could prove to be a valuable component in understanding the treatment response to these agents. Furthermore, exploration of the spectrum of immunological responses to immune checkpoint inhibitor therapies in regional lymph nodes may allow discovery of new mechanisms for immune potentiation of draining lymph nodes through local delivery of immune modulatory drugs.

Scientific hypothesis: We hypothesize that differences in the immune cell populations and their activation status in regional bystander (involved or non-involved) lymph nodes across responders and non-responders to ICI immunotherapies, will provide new insights into predicting outcomes to these therapies. Additionally, this work may also identify novel targets for immune potentiation by local immune modulatory drugs

Experimental plan: Tissue samples will be obtained at post-mortem within the PEACE (Posthumous Evaluation of Advanced Cancer Environment) study (REC Reference: 13/LO/0972) (D. Josephs). Included patients will be those with any cancer who have received ICI therapy as their final treatment prior to death. Tissue samples will be obtained from the tumour, regional/distant lymph nodes and spleen. Clinical and radiological data collected through the PEACE study will be used to correlate clinical factors with immunological findings, allowing comparison of lymph node profiling in responders compared to non-responders to ICI therapy. We will investigate the differences in gene expression profiles established by RNA sequencing of bio-samples with patient's clinical responses to immunotherapies (A. Grigoriadis).

9

We will develop and sub-study with the aim of profiling the regional axillary lymph nodes of breast cancer patients on immunotherapies before and after treatment (K. Tasoulis, A. Khan, A. Kothari). Although the combination of immunotherapy with chemotherapy appears to be very promising based on the results of the KEYNOTE-522 study, further investigation is needed to identify which patients will benefit most from immunotherapy and which patients will do as well with chemotherapy alone and can be spared from the added toxicities of immunotherapy. We will use time-of-flight mass cytometry (CyTOF) of immunophenotyping of lymph nodes (Figure 1).

References

1. Irshad. International Immuno-Oncology Biomarker Working Group, 1 Dec 2020, In : npj Breast Cancer. 6, 1, 15.

2. Grigoriadis, A., Gazinska, P., Pai, T., Irshad, S., et al., 24 Jan 2018, In : The Journal of Pathology: Clinical Research. DOI: 10.1002/cjp2.87.

3. Grigoriadis, A., Gazinska, P., Pinder, S., Pai, T., Irshad, S., Wu, Y., Gillett, C. E., Tutt, A. N. & Coolen, A. C. C., 1 Oct 2017, In : Annals of oncology : official journal of the European Society for Medical Oncology. 28, p. vii27-vii28

6. Assessing the interaction of radiation with anti-PD1/PD-L1 antibody and/or cisplatin chemotherapy in patient-derived organoids and mouse models in head and neck squamous cell carcinoma (HNSCC)

Co-Supervisor 1: Anthony Kong

Research Division/Department: School of Cancer & Pharmaceutical Sciences

E-mail: [email protected]

Co-Supervisor 2: Mahvash Tavassoli

Research Division/Department: Oral Medicine and Pathology

E-mail: [email protected]

Background: Radiotherapy +/- cisplatin chemotherapy is a standard primary or adjuvant treatment in HNSCC. Irradiation has been shown to upregulate PD-L1 expression in the tumour and its microenvironment, specifically on dendritic cells and macrophages (1). The combination of anti-PD-L1 antibody with radiotherapy enhances radiotherapy response by increasing CD8+ T cell response (1). Similarly, chemoradiation has been shown to affect the immunologic landscape and immune checkpoints, supporting the combination of chemoradiation with immune checkpoint blockade (2). Cancer-associated fibroblasts (CAFs) have also been implicated in immune evasion and poor responses to radiotherapy+/-chemotherapy and immunotherapy (3-4). Understanding how radiation +/- cisplatin chemotherapy affects immune response and CAFs, which in turn determine the treatment response would be crucial. Further preclinical experiments are required to elucidate the best way to combine radiotherapy or chemoradiation with anti-PD-L1 antibody, either sequentially or concurrently, +/- targeting CSFs (5), which would result in maximal anti-tumour response. This would have implication on how to best to combine these treatments in clinical trial design and eventually in the clinics.

Scientific Hypothesis:

1) Radiotherapy alone or the combination of radiotherapy with concurrent cisplatin chemotherapy affects tumour PDL1 expression as well as immune cells and CAFs in a different manner

2) The sequence (either sequentially or concurrently) of adding anti-PD1 antibody to radiotherapy alone or chemoradiation is important in determining immune evasion and responses to these treatments

3) Targeting CAFs (e.g. bortezomib and panobinostat through repurposing of drugs)(4) in relation to radiotherapy or chemoradiation or the combination of anti-PD1 with radiation +/- cisplatin may enhance tumour response

Experimental Plan:

1) Assessing the effect of radiation and/or cisplatin on the peripheral and tumour immune responses and CAFs as well as tumour growth delay in mouse tongue SCC syngeneic MOCL1-4 allograft (5) (now available in Kong’s lab) in C57/BL6 mice

2) Repeat the above experiments but assessing the effect of sequential and/or concurrent combination of anti-PD1 with radiation alone in the same mouse models:

· sequentially: to give anti-PD1 before radiation and/or after radiation

· concurrently: to give anti-PD1 with radiation concurrently

· sequentially and concurrently: to give anti-PD1 before, during and after radiation

3) To repeat the above experiments adding concurrent cisplatin chemotherapy with radiation +/- sequential and/or concurrent combination of anti-PDL1 in the same mouse models above

4) To repeat some of the experiments above with the addition of drugs that target CAFs, e.g. bortezomib (4)

5) Assessing the co-culturing of patient-derived organoids with immune cells and/or CAFs treated with radiation +/- concurrent cisplatin +/- anti-PD1 +/- CAF inhibitor:

· TNBC organoids as proof of principle before proceeding to HNSCC organoids (to start soon in Kong’s lab)

References:

1. Deng L et al. Irradiation and anti-PD-L1 treatment synergistically promote antitumour immunity in mice. J CIin Invest. 2014;124(2): 687-695.

2. Sridharan V et al. Definitive chemoradiation alters the immunologic landscape and

immune checkpoints in head and neck cancer. Br J Cancer. 2016; 115(2): 252–260.

3. Wang Z et al. Cancer-associated fibroblasts in radiotherapy: challenges and new opportunities. Cell Commun Signal. 2019; 17: 47.

4. Lee HM et al. Drug repurposing screening identifies bortezomib and panobinostat as drugs targeting cancer associated fibroblasts (CAFs) by synergistic induction of apoptosis. Invest New Drugs. 2018;36(4):545-560.

5. Chen YF et al. Establishing of mouse oral carcinoma cell lines derived from transgenic mice and their use as syngeneic tumorigenesis models. BMC Cancer. 2019; 19: 281.

7. Developing inflammatory resistant regulatory T cells for controlling graft versus host disease (GvHD)

Co-supervisor 1: Shahram Kordasti



Co-supervisor 2: Sophie Papa



Background: Haematopoietic Stem Cell Transplantation (HSCT) is the treatment of choice in several haematological malignancies and could lead to cure. One of the main side effects of HSCT is an immunological response against self, known as graft versus host disease (GVHD). It has been shown that patients with active GVHD have reduced numbers of Regulatory T cells (Tregs) and strategies to increase Tregs such as cell therapy with ex-vivo expanded Tregs could alleviate GVHD.

Tregs are essential for the prevention of autoimmune diseases in humans. Chronic inflammation could affect the number or function of Tregs. For instance, we have described a specific Tregs’ subpopulation which are more sensitive to FAS mediated cell death and their reduction contribute in further inflammation (1-2). The aims of this project are to further investigate the effect of GVHD on Tregs and expand Tregs which are more resistant to inflammation.

Scientific Hypothesis: We hypothesis that expansion of FAS resistant Tregs could increase the longevity of ex-vivo expanded Tregs and create more efficient cell therapy method of the treatment of GVHD and make the HSCT accessible to a larger group of patients with Haematological malignancies.

Experimental Plan: This project consists of two main parts: a) Investigating the dominant phenotype of Tregs following HSCT b) Genetically modifying Tregs to make Tregs resistant to FAS mediated cell death following ex-vivo expansion.

A. Deep phenotyping of Tregs in patient with GVHD using CyTOF and single-Cell RNA-sequencing to identify the dominant Treg subpopulation, their function and sensitivity to FAS mediated cell death. We would also identify pathway(s) which can be modified to prevent cell death in the most functional Tregs’ subpopulation, known as Treg

B. Based on identified pathways in aim A, we will genetically modify and expand Treg B and test their function and longevity both in-vitro and in-vivo (xerograph mouse model) as we have done before (1). Building on our expertise in CAR T cell engineering we will develop synthetic receptors designed to target defined Treg subsets to the site of inflammation and to maximise tolerance in models of GvHD.

References:

1. Lim SP, Costantini B, Mian SA, Perez Abellan P, Gandhi SA, Martinez Llordella M, et al. Treg sensitivity to FasL and relative IL-2 deprivation drive Idiopathic Aplastic Anemia immune dysfunction. Blood. 2020.

2. Jessica Ann Timms, Susann Winter, Steven Hargreaves, Donal P. McLornan, Uwe Platzbecker, Shahram Kordasti; Novel Simultaneous Single Cell mRNA and Protein Expression Profiling Identifies Distinct Treg and T Effector Signatures in the Bone Marrow of MDS Patients. Blood 2019; 134

3. Povoleri GAM, Nova-Lamperti E, Scottà C, et al. Human retinoic acid-regulated CD161+ regulatory T cells support wound repair in intestinal mucosa. Nat Immunol. 2018;19(12):1403-1414. doi:10.1038/s41590-018-0230-z

4. Olivier Martinez, Jane Sosabowski, John Maher, Sophie Papa. New developments in imaging cell-based therapy. J Nucl Med June 1, 2019 vol. 60 no. 6 730-735

8. Identifying unconventional neoepitopes and immune phenotypes in Myeloproliferative Neoplasia to facilitate personalised combined immunotherapies

Co-supervisor 1: Michele Mishto

Research Division/Department: Peter Gorer Department of Immunobiology


Co-supervisor 2: Shahram Kordasti

Research Division/Department: Comprehensive Cancer Centre, School of Cancer and Pharmaceutical Sciences


Background: Myeloproliferative neoplasms (MPN) are stem cell neoplasms. A cure is still missing. MPN are characterised by the presence of myeloid derived inflammation and known driver mutations such as JAK2 V617F, which is present in 50—95% of MPNs. The impact of neoantigens carrying this predominant mutation in shaping the overall immune response and its potential to keep mutated clones under check have been barely explored. Immunogenic neoantigens are presented to CD8+ T cells through neoepitopes mainly produced by proteasomes. These proteases cut antigens and either release peptide fragments or ligate them thereby generating spliced peptides. Mishto lab is leading expert of these unconventional epitopes1-3. Kordasti lab has extensive experience in immune-profiling, using cutting edge technologies such as CyTOF or scRNAseq4,5. Therefore, the joint expertise of these two labs represents a cutting-edge platform to lay the foundation of novel immunotherapy strategies against MPN.

Scientific Hypothesis: Based on preliminary results, we hypothesise that a combination of conventional and unconventional neoepitopes carrying JAK2 V617F mutation and characteristic cytokines and cellular immune phenotypes (so-called immunome) could define a subpopulation of patients with neoantigen-specific response and better clinical outcome.

The overall objective is to identify these immunome patterns, the neoepitopes, their specific CD8+ T cells and studying the synergic interaction of these immunological players. This can pave the way for novel immunotherapies in MPN by targeting tumour neoantigens and modifying the immunome to avoid immune-escape mechanisms of cancer as well as off-target effects.

Experimental Plan:

To test our hypothesis the PhD student will use a combination of:

(i) discovery of proteasome-generated spliced and non-spliced neoepitopes, by adopting in house pipelines based on bioinformatics and in vitro experiments measured by mass spectrometry.

(ii) Isolation of CD8+ T cell clones and their TCRs specific for neoepitopes. The latter could be use for a future development of adoptive T cell therapy against MPN.

(iii) multidimensional cytometry (CyTOF and tetramers) and assessment of inflammatory mediators.

(iv) clinical data of MPN patients.

Thereby, the PhD student could identify immune-signatures, which can favour immune responses against MPN, in the well-annotated cohorts of MPN patients from the trial cohorts as well as prospective patients.

The project outcome may lay foundation to a synergic immunotherapy strategy through the combination of immune response modulation and targeted CD8+ T cell response against neoepitopes.

The project will be carried out at KCL Guy’s campus and Francis Crick Institute.

References

1. Liepe, J. et al. A large fraction of HLA class I ligands are proteasome-generated spliced peptides. Science 354, 354-358 (2016).

2. Liepe, J., Sidney, J., Lorenz, F. K. M., Sette, A. & Mishto, M. Mapping the MHC Class I-Spliced Immunopeptidome of Cancer Cells. Cancer Immunol Res 7, 62-76, doi:10.1158/2326-6066.CIR-18-0424 (2019).

3. Mishto, M. et al. An in silico-in vitro Pipeline Identifying an HLA-A(*)02:01(+) KRAS G12V(+) Spliced Epitope Candidate for a Broad Tumor-Immune Response in Cancer Patients. Front Immunol 10, 2572, doi:10.3389/fimmu.2019.02572 (2019).

4. Kordasti, S. et al. Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment. Blood 128, 1193-1205, doi:10.1182/blood-2016-03-703702 (2016).

5. Timms, J. A. et al. Novel Simultaneous Single Cell mRNA and Protein Expression Profiling Identifies Distinct Treg and T Effector Signatures in the Bone Marrow of MDS Patients. Blood 134, 2980-2980, doi:10.1182/blood-2019-130355 (2019).

Documents

· Web viewLim SP, Costantini B, Mian SA, Perez Abellan P, Gandhi SA, Martinez Llordella M, et al. Treg sensitivity to FasL and relative IL-2 deprivation drive Idiopathic Aplastic