Canonical and Noncanonical Epitopes of Cancer Antigens presented by HLA-Class I complexes in the Human Thymus: Implications for Cancer Immunotherapy

Primary supervisor: Paola Bonfanti, UCL

Secondary supervisor: Michele Mishto, King’s College London

Project

Recent research has shown that noncanonical epitopes – such as proteasome-generated spliced, cryptic or mutated peptides – represent a sizeable portion of the full set of antigenic peptides presented to CD8+ T cells in the periphery. The CD8+ T cell response against noncanonical epitopes is at the cutting edge of the development of novel immunotherapies against cancer. Whether and how these noncanonical epitopes are presented by thymic epithelial cells (TECs) in human thymi and how the central tolerance occurs for noncanonical epitopes is still unknown.

The overall objective of this project is to investigate the mechanisms of HLA class I processing and presentation (APP) of cancer-associated canonical and noncanonical antigenic peptide by cortical and medullary (cTECs and mTECs, respectively) in an ex vivo reconstituted human thymus model. The outcome will help to understand the extension of the natural response of CD8 T cells against cancer-associated antigens, with strong implications on the understanding of cancer immunosurveillance and developing of immunotherapies against cancer.

This project builds on the expertise of the Bonfanti Lab, a leading expert in human thymic cell biology and in reconstitution of functional thymus ex vivo from multipotent thymic epithelial stem cells, and of the Mishto lab, an expert in HLA-I APP of noncanonical epitopes.

The PhD student will:

• Characterize cTECs and mTECs features in both human thymi and ex vivo reconstituted human thymi from cultured epithelial and stromal cells, and their capability of selecting a functional T cell repertoire from thymocyte progenitors.
• Develop and refine strategies for identifying cancer-associated canonical and noncanonical epitope candidates and integrating them with in-house tools predicting HLA-I APP and recognition by TCRs.
• Characterise HLA-I APP molecules in human thymi and ex vivo reconstituted human thymi.
• Characterise the expression of cancer-associated antigens and cryptic antigens by cTECs and mTECs, both inside the cells and at their surface onto HLA-I molecules.
• Verify the impact of specific HLA-I APP molecules such as thymoproteasomes and immunoproteasomes in the cTECs and mTECs mediated selecting a functional T cell repertoire. These assays will be carried out by applying CRISPR-Cas9 gene editing and siRNA modulation in ex vivo reconstituted human thymi focusing on the presentation of specific cancer-associated canonical and noncanonical epitopes and the selection of CD8 T cell clones able to recognise these epitopes.

Candidate background

The project is highly multi-disciplinary, therefore the student should be motivated in carry out both dry lab and wet lab experiments. The student should have a background and / or skills in both biology and bioinformatics. Mishto and Bonfanti labs have all expertise to supervise the students and help them to acquire all required skills to complete the project. Furthermore, the project is well embedded in the research of the two labs, therefore other members of the labs will be involved in the project to carry out some specific tasks, which will be coordinated by the student.

Potential Research Placements

1. Adrian Hayday, Francis Crick Institute/ Centre for Inflammation Biology & Cancer Immunology, King’s College London
2. Michele Mishto, Francis Crick Institute/ Centre for Inflammation Biology & Cancer Immunology, King’s College London
3. Andrea Ditadi, SR-Telethon Institute of Gene Therapy, Milan (IT)

References

1. Mishto M, Takala I, Bonfanti P, Liepe J. Proteasome isoforms in human thymi and mouse models. Immunological Letters 2024 doi.org/10.1016/j.imlet.2024.106899 Review
2. Soh WT, Roetschke HP, Cormican JA, Teo BF, Chiam NC, Raabe M, Pflanz R, Henneberg F, Becker S, Chari A, Liu H, Urlaub H, Liepe J*, Mishto M*. Degradation of proteins by human 20S proteasomes sheds light on the interplay between peptide hydrolysis and peptide splicing. Nature Comms. 2024 Feb 7;15(1):1147. doi: 10.1038/s41467-024-45339-3.
3. Ragazzini R, Boeing S, Zanieri L, Green M, D’Agostino G, Bartolovic K, Agua-Doce A, Watson SA, Batsivari A, Ariza-McNaughton L, Gjinovci A, Greco M, Scoville D, Han A, Hayday AC, Bonnet D, and Bonfanti P. Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human postnatal thymus. Developmental Cell 2023 doi: org/10.1016/j.devcel.2023.08.017
4. Campinoti S, Gjinovci A, Ragazzini R, Zanieri L, Ariza-McNaughton L, Catucci M, Boeing S, Park JE, Hutchinson JC, Munoz-Ruiz M, Manti PG, Vozza G, Villa CE, Phylactopoulos DE, Maurer C, Testa G, Stauss HJ, Teichmann SA, Sebire NJ, Hayday AC, Bonnet D, and Bonfanti P. Reconstitution of a functional human thymus by postnatal progenitor cells and natural whole organ scaffolds. Nature Comms. 2020, Dec 11:632 doi.org/10.1038/s41467-020-20082-7 – PMID: 33311516.
5. Liepe J, Marino F, Sidney J, Jeko A, Bunting DE, Sette A, Kloetzel PM, Stumpf MP, Heck AJ, Mishto M. A large fraction of HLA class I ligands are proteasome-generated spliced peptides. Science 2016 Oct; 354(6310): 354-358. DOI: 10.1126/science.aaf4384.