Adoptive T cell therapies have revolutionised cancer treatment in some haematological tumours. Despite astounding advances, patients often relapse because the transferred T cells have low persistence and they lose their effector function against cancer cells. The prime challenge for improving T cell therapies is to decipher the mechanisms that define T cell effector function and memory formation. The MA6TCELL project aims to disentangle this problem by defining how m6A methylation on mRNA regulates human CD8 T cell differentiation and function. m6A is the most abundant mRNA methylation and determines gene expression by regulating mRNA metabolism, altering as such cell function. Using the miCLIP assay, I previously mapped genome-wide the m6A sites on the mRNAs of human CD8 T cells (unpublished data I will provide the host with). This map revealed that m6A occurs not only at the known DRACH sequences, but also at AU-rich sequences (AREs). Intriguingly, the host has uncovered that AREs are critical regulators of cytokine production in memory CD8 T cells (host expertise). My pilot experiments revealed that chemical inhibition of a m6A demethylase in CD8 T cells decreased cytokine production, further supporting my hypothesis that m6A methylation controls CD8 T cell differentiation and function. To test this hypothesis the M6ATCELL project will: 1) define the regulatory mechanisms of m6A methylation in human CD8 T cell differentiation and effector function and 2) test whether m6A enzyme inhibitors improve the potency of CD8 T cell therapies using chimeric antigen receptor (CAR) T cells. Our findings will yield fundamental insights in T cell biology, which can be relevant for multiple adoptive T cell therapies. My long-term aim is to transfer my scientific findings from bench-to-bedside. Thus, I also secured a placement at the European Medicines Agency and if this proposal is granted, I will be trained on the regulatory guidelines of medicinal T cell products.

Sex differences play a significant role in immune function, with women generally mounting stronger immune responses than men. A key factor behind these differences is the X chromosome, which contains many genes that are important for immune function. In females, who have two X chromosomes, one of the X chromosomes is usually inactivated in each cell to prevent an overdose of X-linked gene expression. This process is known as X-chromosome inactivation (XCI). However, some genes can escape this inactivation and are expressed from both X chromosomes. These “escapee” genes are often expressed at higher levels in females than in males, who only have one X chromosome. The X-CITE (X-Chromosome Inactivation and T cell Escape) project aims to elucidate the molecular mechanisms by which these XCI escapee genes influence T cell responses. Understanding these mechanisms is crucial, as T cells are central to the success of cancer immunotherapy, where sex-based differences in T cell function may impact treatment outcomes. By exploring the sex-biased expression of XCI escapee genes, this project will advance our understanding of sex differences in immune responses and provide new insights into how T cells can be manipulated to improve the efficacy of cancer immunotherapy. The research will be conducted at the Landsteiner Laboratory Amsterdam UMC-Sanquin (The Netherlands), under the supervision of Dr. Monika Wolkers, whose lab specializes in T cell biology and the enhancement of T cell functions in immunotherapy settings.

CD8+ T cells are critical to fight infections and to clear tumor cells through the production of inflammatory cytokines and cytotoxic molecules. These effector molecules must be tightly controlled: too little leads to the inability to control the pathogen, and too much can result in a life-threatening cytokine storm and tissue damage. While transcriptional control of effector genes is well-studied, regulation at the levels of RNA stability and translation efficiency by RNA-binding proteins (RBPs) has remained underappreciated. We recently found that several cytokines are tightly regulated through these processes, and we identified ZFP36L2 as one of the responsible RBPs. However, much is still to be learned about the underlying molecular mechanisms. Moreover, there are >1000 putative RBPs, and a systematic analysis of their regulatory activity in T cells is lacking, particularly with regard to the control of effector proteins. Here, we will use a combination of mouse genetics, and molecular and cellular biology to gain a deep understanding of the control of cytokine production by RBPs, using ZFP36L2 as a paradigm. Next, we will take a novel, highly sensitive proteomics approach to systematically identify the RBP repertoire in resting and activated primary human T cells. Complementary functional screens will identify those RBPs that control specific effectors. Selected RBPs identified in these screens will be studied in-depth to understand their roles in T cell responses to acute infection and in tumor models. Lastly, we will define how RBPs can imprint and/or maintain the killer phenotype of human CD8+ T cells. This research will significantly advance our understanding of post-transcriptional regulation of T cell effector activity, and it should help us to develop novel tools to drive effective T cell responses against pathogens and malignant cells.

Humans are equipped with a sophisticated immune system, especially adaptive immunity, which is characterized by the finest antigen specificity. The evolution of such fine antigen specificity across human lifespan is far from being known, although it is observe that the strength of the adaptive immune response varies throughout human lifetime. Published data suggest that T cells receptors (TCRs) continuously change their characteristics. I aim to provide fundamental knowledge on the evolution of T cell repertoires during immunologically-distinct phases of human lifetime by dissecting the evolution of the influenza virus-specific T cell repertoire in children, adults and elderly at the greatest level of resolution and technological innovation and link this to their TCR characteristics. At the Peter Doherty Institute, Melbourne, Australia, I will use state-of-the-art comprehensive immunological analyses to identify molecular switches, which I will use to establish an immunological model to predict the strength of the CD8+ T cell response. I will bring this cutting-edge multi-disciplinary platform to Europe (Sanquin, Amsterdam, The Netherlands) where I will use this platform to study the underlining mechanisms that drive tissue residency of T cells. I will gain knowledge and skills on studying TCRs, tissue resident T cells, bioinformatics and structural biology, while working in close collaboration with world-renowned experts in the field, namely Profs. Kedzierska, van Lier, Luciani, Gras, Beyer and Schulze. These state-of-the-art comprehensive immunological analyses will inevitably yield important insights into protective vs detrimental human immune responses, which will aid early interventions, treatment and help understand and overcome technical challenges of developing broadly-protective vaccines. The MSCA will strengthen my position as an independent researcher and will greatly increase my chances for a tenure-track/fixed position at an European research institute.

Numerous mucosal vaccines and IgA-based monoclonal antibodies aimed at a robust immunity within the respiratory tract are in development to combat viral endemics and pandemics. However, investigations into humoral immunity have predominantly focused on circulating antibodies, particularly those of the IgG isotype. This has left a significant void in our understanding of the role of mucosal IgA proteoforms in conveying effective immune protection. This crucial knowledge gap deprives vaccine and antibody development of crucial determinants or immune characteristics to replicate. In response to this, we will conduct in-depth investigations of mucosal IgA including in vitro and in vivo functional evaluations. We will capitalize on recent advances in liquid chromatography and mass spectrometry-based approaches for the detailed characterization of mucosal IgA clonal repertoires and glycosylation profiles, a field that has remained completely unexplored until now, B-cell receptor sequencing, monoclonal IgA glycoengineering and in vitro functional assays. We will take advantage of our unparalleled biobank of human nasal secretion samples with clear documentations of infection outcomes on an individual level. What sets our approach apart is thereby the unprecedented molecular-level characterization of IgA, directly linked to their in vitro functionality and pre-defined clinical outcomes, which clearly surpasses current state-of-the-art. By harnessing our in-depth characterization of mucosal IgA coupled to functional traits, we will generate IgA templates with enhanced binding and neutralization capabilities as well as functionally advantageous Fc effector potencies. Our overarching aim is to provide molecular-level blueprints for protective monoclonal IgA-antibodies, enabling the fine-tuning of vaccine formulations and monoclonal antibody generation with a higher degree of accuracy, ultimately enhancing their efficacy and safety.