T cell differentiation

The Wolkers lab studies how T cell responses against tumors and infections are generated and maintained, and what makes T cells lose their proper function in disease. We study these processes in human blood cells, and in T cells from Non-Small Cell Lung Cancer (NSCLC) and pediatric neuroblastoma. Appropriate T cell function is defined by the proteins they express. To decipher the rules that define protein expression, we use proteomics and transcriptomics analyses, molecular biology assays, high dimensional flow cytometry and functional T cell read outs. Importantly, it has become apparent that transcript levels do not predict well the actual protein content of a cells. We therefore specifically focus on the contribution of post-transcriptional events and the role of RNA binding proteins in defining protein expression. Unraveling these regulatory mechanisms is key for our understanding of T cell functionality in health, and for identifying methods to restore appropriate T cell function in disease.

Deciphering the molecular mechanisms that drive T cell effector function

T cells release effector molecules to kill their target cells, including granzymes and cytokines. Because these molecules are highly toxic, their release must thus be tightly regulated to guarantee a protective yet balanced immune response. In the absence of infection, the constitutively expressed mRNA must be silenced to avoid immunopathology. We found that that T cells use pre-formed mRNA for the rapid cytokine production (Salerno, J. Immunol. 2016; PNAS 2017; Nature Immunol 2018). Importantly, the post-transcriptional events driving cytokine production are impeded in tumor-infiltrating T cells (Salerno, Oncoimmunology 2019). We found which RNA binding proteins contribute to the highly orchestrated production of cytokines, and how they contribute to the dysregulated responses in tumors (Salerno, Nature Immunol 2018; Popovic, Cell Reports 2023; Zandhuis. Eur J Immunol 2024+2025). 
We recently also uncovered how mTOR signaling contributes to this regulation (Jurgens, Mol Cell 2025, Lattanzio+Jurgens, BioRxiv 2026). 

RNA binding proteins contributing to T cell function

RNA binding proteins (RBPs) are fundamental for defining T cell differentiation and T cell responses (Salerno, Trends Immunol 2020; Nicolet, Immunol Rev 2021). RBPs determine the fate of mRNA, including RNA splicing, subcellular localization, stability and the process of translation into protein. These features combined define the actual protein output from a given transcript. Therefore, RBPs contribute to the observed disconcordance of mRNA and protein abundance (Nicolet, PlosOne 2022). Importantly, RBPs display cell-type and cell-type specific expression profiles (Zandhuis, Front Immunol 2021). Indeed, we recently showed how T cell activation alters the RBP binding landscape, and we used this information to study how individual RBPs define T cell activation and differentiation (Lattanzio, BioRxiv 2025).

Technical advances to study gene regulation in T cells

To decipher the regulatory mechanisms of cytokine production in human T cells, we implemented flow cytometry- based fluorescence in situ hybridization (FLOW-FISH) (Nicolet, J Immunol 2017) and single molecule (sm-FISH) (Lattanzio, EMBO J 2025). Using 3D analyses and RBP deletion in primary T cells we were able to observe regulatory mechanisms with unprecedented depth. We now also optimized proximity labeling for human T cells and report how the interactions with the RBP ZFP36L1 are subject to changes upon T cell activation (Jurgens, BioRxiv 2025). Furthermore, we optimized mRNA and protein isolation from FACS-sorted fixed T cells for RNA-seq and Mass spectrometry analysis to determine the molecular features of specific cytokine-producing T cells (Nicolet, PNAS 2020). 
To study the rules that drive protein expression in immune cells we recently presented SONAR, a machine learning tool that can decipher the cell-type and cell-state dependent sequence features that predict protein abundance, reaching > 50% of accuracy (Nicolet, Science Advances, 2025). Importantly, because this tool reports the sequences contributing to the models, the output can be used for improving the design of therapeutics.

TIL therapy against adult and pediatric solid tumors

T cells are critical mediators to clear tumor cells and are thus critical for the efficacy of immunotherapy. Adoptive cell transfer (ACT) showed astounding results for solid tumors, in particular for melanoma. We found that ACT products containing tumor-reactive TILs can be also generated for most non-small-cell lung cancers (NSCLC), but not renal cell carcinoma’s (de Groot, Oncoimmunol 2019; van Asten, Oncoimmunol 2021; Castenmiller, IO-Tech 2022, Oncoimmunology 2024). Based on our findings, we are preparing a clinical trial for TIL therapy in NSCLC.

Recently we shifted our attention towards pediatric neuroblastoma. Importantly, even though neuroblastoma is considered non-immunogenic, we find evidence for tumor-reactive T cells (Castenmiller, Life Science Alliance 2025). However, possibly also owing to the age of the children suffering from this tumor, the immune landscape substantially differs. Together with the Princes Maxima Center, we are currently investigating how to harness T cells for therapeutic purposes.