Post-transcriptional regulation of cytotoxic CD4⁺ T cell function in tumours

Primary Supervisor: Richard Jenner, UCL

Secondary supervisor: Faraz Mardakheh, Queen Mary University of London

Project

Cancer immunotherapy approaches are primarily focused on boosting cytotoxic CD8⁺ T cell function (1). However, the proportion of patients that do not respond to therapy remains high and some tumours lack MHC I expression. Although primarily considered to provide helper function, there is growing recognition that CD4⁺ T cells can also acquire cytotoxic activity (CD4⁺ T_CTX), and that these cells form a key component of effective anti-tumour responses.

We have shown that CD4⁺ effector T cells (T_EFF) acquire cytotoxic function (marked by GzmB) in mouse MCA205 sarcomas and cause tumour regression in response to depletion of regulatory T cells (T_REG) from the tumour microenvironment (2). Using this model, we have found that although the Gzmb gene is transcribed by CD4⁺ T_EFF, GzmB protein production is inhibited unless T_REG are depleted (Figure 1). Furthermore, scRNA-seq demonstrates that only a handful of genes exhibit changes in mRNA abundance in CD4⁺ T_EFF upon T_REG depletion, suggesting that the acquisition of cytotoxic activity is primarily regulated at the post-transcriptional level.

The aim of the PhD project is to identify the set of genes that are under post-transcriptional control in tumour-infiltrating CD4⁺ T cells and determine the mechanisms responsible. The objectives are to:

1. Identify the set of proteins that exhibit increased abundance upon acquisition of cytotoxic function

The student will purify CD4⁺ T_EFF from tumours by FACS before and after T_REG depletion and analyse them by tandem mass tagging (TMT)-proteomics (3). Proteins exhibiting significant changes in abundance will be examined for functional categories and RNA motifs that could suggest the mechanism of post-transcriptional regulation (e.g. ZFP36 binding to AU-rich elements (ARE) or targeting by specific miRNAs).

2. Determine the contribution of ZFP36 RNA binding proteins to regulation of CD4⁺ T_CTX function

The ZFP36 family of RNA binding proteins bind AREs in target mRNAs, either promoting their decay or inhibiting translation. Knockout of Zfp36 and Zfp36l1 in CD8⁺ T cells increases GzmB protein production (4) and ZFP36 binds Gzmb mRNA (5), suggesting this system may also regulate GzmB protein production in CD4⁺ T_CTX. To test this, the student will establish the MCA205 model in Zfp36 knockout mice and determine whether this leads to increased GzmB production and tumour killing.

3. Identification of other post-transcriptional mechanisms in operation in CD4⁺ T_CTX

If the ZFP36 system is found to be dispensable for the regulation of cytotoxic function in tumour-infiltrating CD4⁺ T_CTX, the student will use the motifs from Aim 1 to identify other candidate RNA binding proteins or miRNAs that could be involved. In parallel, the student will perform OOPS (3) that enables unbiased identification of RNA binding proteins that show changes in their RNA association upon T_REG depletion. The student will then determine the contribution of these candidate factors by transferring CRISPR-edited CD4⁺ T cells into irradiated mice harbouring MCA205 tumours.

In summary, this work will identify the post-transcriptional regulatory mechanisms that restrain acquisition of cytotoxic CD4⁺ T cell function in tumours. This will provide new therapeutic opportunities to enhance these responses in cancer patients.

Candidate background

This project would especially suit candidates with a background in tumour immunology and an interest in molecular mechanisms or candidates with expertise in post-transcriptional regulation who have an interest in applying this to tumour immunology.

Potential Research Placements

1. Faraz Mardakheh, Barts Cancer Institute, Queen Mary University of London
2. Sergio Quezada, UCL Cancer Institute
3. Javier Herrero, UCL Cancer Institute

References

1. Topalian SL, Drake CG, and Pardoll DM. Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell 2015. 27:450-61.
2. Sledzinska A, Vila de Mucha M, Bergerhoff K, Hotblack A, Demane DF, Ghorani E, Akarca AU, Marzolini MAV, Solomon I, Vargas FA, Pule M, Ono M, Seddon B, Kassiotis G, Marafioti T, Ariyan CE, Korn T, Lord GM, Stauss H, Jenner RG, Peggs KS, Quezada SA. Regulatory T Cells Restrain Interleukin-2- and Blimp-1-Dependent Acquisition of Cytotoxic Function by CD4(+) T Cells. Immunity 2020. 52:151-166.
3. Azman MA, Dodel M, Capraro F, Faraway R, Dermit M, Fan W, Ule J, Mardakheh FK. An RNA-binding switch drives ribosome biogenesis and tumorigenesis downstream of RAS oncogene. bioRxiv 2021. doi: https://doi.org/10.1101/2021.12.16.472890.
4. Petkau G, Mitchell TJ, Chakraborty K, Bell SE, D?Angeli V, Matheson L, Turner DJ, Gizlenci O, Salerno F, Katsikis PD, Turner M. The timing of differentiation and potency of CD8 effector function is set by RNA binding proteins. bioRxiv 2021. doi: https://doi.org/10.1101/2021.06.03.446738
5. Moore MJ, Blachere NE, Fak JJ, Park CY, Sawicka K, Parveen S, Zucker-Scharff I, Moltedo B, Rudensky AY, Darnell RB. ZFP36 RNA-binding proteins restrain T cell activation and anti-viral immunity. eLife 2018 7:e330575.