The Wolkers lab studies how T cell responses against tumors and infections are generated and maintained. We specifically study the role of post-transcriptional gene regulation herein. To tackle this, we use two model systems. Mouse models allow us to study the fundamental aspects of T cell responses against ongoing infections and tumor challenges. In addition, with our study on tumor-reactive human T cells, we can now translate our findings from model systems to clinically relevant models, and we investigate how T cell effector function can be maintained against tumors.

 

Deciphering the molecular mechanisms that drive T cell effector function

Memory T cells protect us from recurring infections by rapidly releasing cytokines. Cytokines, however, are highly toxic. Their release must thus be tight regulated to guarantee a protective yet balanced immune response. Here, we show that post-transcriptional regulation is critical to modulate the cytokine production of T cells. We show that memory T cells use pre-formed mRNA for the immediate cytokine production in response to recall infections (Salerno et al. PNAS 2017). In the absence of infection, however, the constitutively expressed mRNA must be silenced to avoid immunopathology. We found that the RNA-binding protein ZFP36L2 blocks translation of cytokine mRNA, which is rapidly released upon T cell activation (Salerno et al. Nature Immunology 2018). In effector T cells, post-transcriptional events dictate the duration and magnitude of cytokine production depending on the cues a T cell receives (Salerno et al. J. Immunol. 2016, PNAS 2017), and these post-transcriptional events driving cytokine production are impeded in tumor-infiltrating T cells (Salerno et al. Oncoimmunology 2019). We are currently investigating the expression of RBPs in T cells and their role in driving effective T cell responses.

 

Circular RNA in hematopoietic cells and T cells

Circular RNA expression is found in many cell types but our current understanding of these highly stable RNA variants is very limited. We showed that circRNA expression in hematopoietic cells is cell-type specific (Nicolet et al. NAR 2018). We are currently investigating the expression and the function of circRNAs in hematopoiesis and in human T cells.

 

Technical advances to study gene regulation in T cells

To decipher the regulatory mechanisms of cytokine production, we developed a multiparameter flow-cytometry-based fluorescence in situ hybridization (Flow-FISH) essay for human and mouse T cells to simultaneously measure specific mRNAs and proteins in heterogeneous T cell populations (Nicolet et al. J. Immunol. 2017, Salerno&Freen-van Heeren et al. J.Immunol. 2019). 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 et al. PNAS 2020). This analysis revealed that only a part of human CD8⁺ T cells are highly cytotoxic, a feature that is maintained over time. Because we identified surface markers to select for these cytotoxic T cells, we are currently investigating whether this finding can be exploited to improve TIL products.

 

TIL therapy against human solid tumors.

T cells are critical mediators to clear tumor cells, and can thus be used for immunotherapy. Adoptive cell transfer (ACT) showed astounding results for solid tumors, in particular for melanoma. We questioned whether TIL therapy could also be employed for the treatment of non-small-cell lung cancers (NSCLC). In close collaboration with the Netherlands Cancer Institute and Sanquin Laboratory of Cell therapy, we found that 13/17 (76%) TIL products from NSCLC are tumor-reactive, as defined by IFN-g production in response to tumor digest. Importantly, high responders also produced TNF-α and/or IL-2, demonstrating that expanded NSCLC-derived TILs can be polyfunctional, a hallmark of effective T cell responses (de Groot et al. Oncoimmunology 2019). We have now extended and validated these findings to stage IV patients, and we are preparing a clinical trial for TIL therapy in NSCLC.

 

 

Key Publications (aub vervangen met deze lijst):

 

1. Nicolet BP, Guislain A, van Alphen F, van den Biggelaar M, Gomez-Eerland R, Schumacher TN, Wolkers MC. CD29 identifies IFN-g producing human CD8⁺ T cells with an increased cytotoxic potential. Proceedings of the National Academy of Science. 2020. 117: 6686-6696
2. De Groot R*, van Loenen MM*, Guislain A, van den Heuvel MM, de Jong J, Patrick Burger P, Spaapen RM, Amsen D, Haanen JBAG, Monkhorst K, Hartemink KJ, Wolkers MC. Polyfunctional tumor-reactive T cells are effectively expanded from non-small cell lung cancers, and correlate with an immune-engaged T cell profile.  Oncoimmunology 2019. Doi:10.1080/2162402X.2019.1648170.
3. Nicolet BP, Engels S, Aglialoro F, van den Akker E, von Lindern M, Wolkers MC. Circular RNA expression in human hematopoietic cells is widespread and cell-type specific. Nucleic Acid Research, 2018. 46: 8168-8180.
4. Salerno F, Engels S, van den Biggelaar M, van Alphen F, Guislain A, Zhao W, Hodge D, Bell SE, Medema JP, von Lindern M, Turner M, Young HA, and Wolkers MC. Translation repression through AU-rich elements prevents aberrant cytokine production in memory T cells. Nature Immunology, 2018. 19:828-837.
5. Salerno F, Pasolini NA, Stark R, von Lindern M, Wolkers MC. Distinct PKC-mediated post-transcriptional events set cytokine production kinetics in CD8+ T cells. Proceedings of the National Academy of Science, 2017. 114:9677-9682.