Biology subjects

Wolkers, M. C.

Publications and source records attributed to Wolkers, M. C..

17 recordsLinked to original sources

bioRxiv2026

Proteomics of human cancer-associated T cells identifies regulators of T cell functionality

CD8+ T cells in solid cancers progressively lose anti-tumor activity, yet the cell-intrinsic mechanisms driving this loss of function remain incompletely defined. Here, we performed matched proteomic and transcriptomic profiling of dysfunctional and bystander CD8+ tumor-infiltrating T cells isolated from primary tumors of treatment-naive non-small cell lung cancer patients. Proteomic analysis revealed widespread discordance with mRNA expression, with 8% of all quantified proteins displaying differential expression exclusively at the protein level. Genetic perturbation of such differentially expressed proteins identified the chromatin remodeler CHD4 and fatty acid synthase (FASN) as cell-intrinsic regulators of T cell function. CHD4 deletion resulted in altered gene-regulatory networks that promoted effector differentiation and enhanced cytokine production. In contrast, FASN deletion preserved mitochondrial fitness and sustained T cell functionality under chronic T cell receptor stimulation. Together, these findings demonstrate that proteomic profiling uncovers regulators of T cell functionality that are not apparent from transcriptomic analyses alone, highlighting an additional layer of regulatory control.

immunology↗

bioRxiv2026

An early mTOR-dependent window during human T cell activation programs T cell state

T cell activation results in profound proteome remodeling that programs T cells into distinct cellular states. The mechanistic target of rapamycin (mTOR) biases T cell differentiation toward a cytotoxic fate at the expense of memory-precursor formation, making mTOR inhibition an attractive strategy to boost T cell memory during vaccination. Here, we used matched time-resolved mRNA sequencing and quantitative mass spectrometry to define how the human T cell proteome is remodeled during the first 24 hours of activation. We found that human T cells rapidly remodel their proteome in distinct, temporally ordered modules that drive translation and proliferation while promoting a cytotoxic T cell state. Notably, mTOR inhibition during the first 24 hours of T cell activation perturbed these protein modules. Strikingly, transient mTOR inhibition limited to the first 16 hours of T cell priming was sufficient to imprint a memory-like T cell state, while preserving the capacity to produce inflammatory cytokines and mediate target cell killing. Together, these findings indicate that mTOR activity dictates stable functional trajectories during early T cell activation, revealing a therapeutic window to refine vaccination responses.

immunology↗

bioRxiv2025

Mapping the dynamic RNA binding proteome in human effector T 1 cells identifies differentiation and cytotoxicity regulators

RNA-binding proteins (RBPs) are key regulators of T cell function by controlling (m)RNA fate and fine-tuning protein expression dynamics. Dysregulated RBPs can drive immune diseases and malignancies, highlighting their potential as therapeutic targets. To achieve this, a systematic analysis of the dynamic RBP-RNA interactions is required. Here, we mapped the RNA-binding proteome in human T cells and measured its alterations upon T cell activation using orthogonal organic phase separation (OOPS), analysed with PROMOGEB, a Bayesian linear regression model. This approach uncovered the intricate RNA-binding dynamics of the RBProteome. Gene-editing of such dynamic RNA binders revealed that TUT1 (Star-PAP) maintains the integrity of the T cell differentiation program, and that mutating SF3A1 enhanced the cytotoxic molecule expression and thus target cell killing. Our work provides the most comprehensive analysis of the effector T cell RBProteome to date and shows the potential of identifying RBPs and their binding dynamics as therapeutic agents. TeaserOOPS analysed with PROMOGEB maps RBP dynamics in human Teff cells, identifying TUT1 and SF3A1 as regulators of T cell fidelity.

immunology↗

bioRxiv2025

γδ T cells are the prime anti-tumoral T cells in pediatric neuroblastoma

High-risk pediatric neuroblastoma patients have a dismal survival rate despite intensive treatment regimens. New treatment options are thus required. Even though HLA expression in neuroblastoma is low and immune cell infiltrates are limited, the presence of tumor infiltrating lymphocytes (TILs) is indicative for better patient survival. Here, we show that most tumor lesions contain viable immune cell infiltrates after induction chemotherapy, with high percentages of CD3+ T cells. We therefore expanded the TILs and tested their anti-tumoral activity. With sufficient starting material, TIL expansion was as efficient as for adult solid tumors. However, whereas TIL products from adult tumors almost exclusively contained {beta} T cells, in neuroblastoma-derived TILs, {gamma}{delta} T cells expanded with similar efficacy as {beta} T cells. Importantly, the anti-tumor responses in response to autologous tumor digest primarily originated from (V{delta}1- and V{delta}3-expressing) {gamma}{delta} T cells, and not from {beta} T cells. In conclusion, this finding creates a window of opportunity for immunotherapy for neuroblastoma patients, with {gamma}{delta} T cells as potential prime responders.

cancer biology↗

bioRxiv2024

Single molecule imaging of transcription dynamics, RNA localization and fate in T cells

T cells are critical effector cells against infections and malignancies. To achieve this, they produce pro-inflammatory cytokines, including IFN-{gamma} and TNF. Cytokine production is a tightly regulated process. The relative contribution of transcriptional and post-transcriptional regulation to mRNA expression is, however, unknown. We therefore optimized single-molecule FISH for primary human T cells (T-cell smFISH) to simultaneously quantify nascent RNA, mature mRNA levels and its localization with single-cell resolution. T-cell smFISH uncovered heterogeneous cytokine mRNA levels, with high cytokine producers displaying biallelic IFNG/TNF RNA transcription activity. Throughout activation, nuclear cytokine mRNAs accumulated, whereas cytoplasmic cytokine mRNA was degraded through translation-dependent decay. Lastly, T-cell smFISH uncovered cytokine-specific regulation by the RNA-binding protein HuR. Thus, T-cell smFISH provides novel insights in the intricate (post)-transcriptional processes in T cells.

immunology↗

bioRxiv2024

mTOR signaling promotes cytokine production in T cells through 3UTR-mediated translation control

T cells are key contributors to clear our body from infected and malignant cells. When T cells respond to target cells, they undergo profound translational alterations. The evolutionary and highly conserved kinase mammalian target of rapamycin (mTOR) is a central mediator of T cell differentiation, homeostasis, and T cell activation, including the production of the key pro-inflammatory cytokines TNF, IL2, and IFN{gamma}. mTOR was shown to execute its translation activity through TOP motifs located in the 5 Untranslated region (5UTR) of its target genes. Here, we uncovered a distinct mechanism of mTOR signaling on cytokine production in T cells, which is under control of the 3UTR. Even though non-classical TOP motifs are present in cytokine 3UTRs, they do not contribute to mTOR-mediated translation regulation. Rather, AU-rich elements (AREs) are required for mTOR-mediated cytokine production. Furthermore, we discovered that the RNA binding protein DDX21 binds to 3UTR AREs and confers the mTOR-mediated translation control. In conclusion, we here present a previously unappreciated ARE-dependent, 3UTR-mediated mode of action that mTOR employs to regulate cytokine production.

immunology↗

bioRxiv2024

Regulation of IFNgamma production by ZFP36L2 in T cells is context-dependent

CD8+ T cells kill target cells by releasing cytotoxic molecules and pro-inflammatory cytokines, such as TNF and IFN{gamma}. The magnitude and duration of cytokine production is defined by post-transcriptional regulation, and a critical regulator herein are RNA-binding proteins (RBPs). Although the functional importance of RBPs in regulating cytokine production is established, the kinetics and mode of action through which RBPs control cytokine production is not well understood. Previously, we showed that the RBP ZFP36L2 blocks translation of pre-formed cytokine encoding mRNA in quiescent memory T cells. Here, we uncover that ZFP36L2 regulates cytokine production in a context-dependent manner. T cell-specific deletion of ZFP36L2 (CD4-cre) had no effect on T cell development, or on cytokine production during early time points (2-6h) of T cell activation. In contrast, ZFP36L2 specifically dampened the production of IFN{gamma} during prolonged T cell activation (20-48h). ZFP36L2 deficiency also resulted in increased production of IFN{gamma} production in tumour-infiltrating T cells that are chronically exposed to antigen. Mechanistically, ZFP36L2 regulates IFN{gamma} production at late time points of activation by destabilizing Ifng mRNA in an AU-rich element-dependent manner. Together, our results reveal that ZFP36L2 employs different regulatory nodules in effector and memory T cells to regulate cytokine production.

immunology↗

bioRxiv2022

Effective generation of tumor-infiltrating lymphocyte products from metastatic non-small-cell lung cancer (NSCLC) lesions irrespective of location and previous treatments.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Because current treatment regimens show limited success rates, alternative therapeutic approaches are needed. We recently showed that treatment-naive, stage I/II primary NSCLC tumors contain a high percentage of tumor-reactive T cells, and that these tumor-reactive T cells can be effectively expanded and used for the generation of autologous TIL therapy. Whether these promising findings also hold true for metastatic lesions is unknown yet critical for the translation into the clinic. We here studied the lymphocytic composition and the feasibility to generate tumor-responsive TIL products from metastatic NSCLC lesions that were derived from different locations and from patients who had previously received different treatment regimens, or who were treatment naive. The overall numbers and composition of lymphocyte infiltrates from this various metastatic lesions was by and large comparable to that of early-stage primary NSCLC tumors. With the clinically approved TIL expansion protocol, we effectively expanded TILs from metastatic NSCLC lesions to numbers that were compatible with TIL transfusion, irrespective of the location of the metastasis and of the previous treatment. Importantly, 16 of 21 (76%) tested TIL products displayed anti-tumoral activity, as determined by their production of the key pro-inflammatory cytokines IFN-{gamma}, TNF, IL-2 and of CD137 expression upon incubation with autologous tumor digests by CD8+ and CD4+ T cells. In summary, metastatic NSCLC lesions constitute a viable source for the generation of tumor-reactive TIL products for therapeutic purposes irrespective of their location and the pretreatment regimens.

cancer biology↗

bioRxiv2021

Mapping RNA-binding proteins in human B cells and Tcells upon differentiation

B cells and T cells are key players in the defence against infections and malignancies. To exert their function, B cells and T cells differentiate into effector and memory cells. Tight regulation of these differentiation processes is key to prevent their malfunction, which can result in life-threatening disease. Lymphocyte differentiation relies on the appropriate timing and dosage of regulatory molecules, and post-transcriptional gene regulation (PTR) is a key player herein. PTR includes the regulation through RNA-binding proteins (RBPs), which control the fate of RNA and its translation into proteins. To date, a comprehensive RBP expression map throughout lymphocyte differentiation is lacking. Using transcriptome and proteome analyses, we here provide an RBP expression map for human B cells and T cells. We observed that even though the overall RBP expression is conserved, the relative RBP expression is distinct between B cells and T cells. Differentiation into effector and memory cells alters the RBP expression, resulting into preferential expression of different classes of RBPs. For instance, whereas naive T cells express high levels of translation-regulating RBPs, effector T cells preferentially express RBPs that modulate mRNA stability. Lastly, we found that cytotoxic CD8+ and CD4+ T cells express a common RBP repertoire. Combined, our study reveals a cell type-specific and differentiation-dependent RBP expression landscape in human lymphocytes, which will help unravel the role of RBPs in lymphocyte function.

immunology↗

bioRxiv2021

CD29 enriches for cytotoxic human CD4+ T cells

CD4+ T cell are key contributors in the induction of adaptive immune responses against pathogens. Even though CD4+ T cells are primarily classified as non-cytotoxic helper T cells, it has become appreciated that a subset of CD4+ T cells is cytotoxic. However, tools to identify these cytotoxic CD4+ T cells are lacking. We recently showed that CD29 (Integrin Beta 1, ITGB1) expression on human CD8+ T cells enriches for the most potent cytotoxic T cells. Here, we questioned whether CD29 expression also associates with cytotoxic CD4+ T cells. We show that human peripheral blood-derived CD29hiCD4+ T cells display a cytotoxic gene expression profile, which closely resembles that of CD29hi cytotoxic CD8+ T cells. This CD29hi cytotoxic phenotype was observed ex vivo and was maintained in in vitro cultures. CD29 expression enriched for CD4+ T cells, which effectively produced the pro-inflammatory cytokines IFN-{gamma}, IL-2, and TNF-, and cytotoxic molecules. Lastly, CD29-expressing CD4+ T cells transduced with a MART-1 specific TCR showed target cell killing in vitro. In conclusion, we here demonstrate that CD29 can be employed to enrich for cytotoxic human CD4+ T cells.

immunology↗

bioRxiv2020

Abundant circular RNA expression in terminal myeloid differentiation does not correlate with frequent circRNA translation, or with circRNA-mediated translation regulation

Each day, about 1012 erythrocytes and platelets are released into the blood stream. This substantial output from hematopoietic stem cells is tightly regulated by transcriptional and epigenetic factors. Whether and how circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not known. We recently reported that erythrocytes and platelets contain the highest levels and numbers of circRNAs amongst hematopoietic cells. Here, we provide the first detailed analysis of circRNA expression during erythroid and megakaryoid differentiation. CircRNA expression not only significantly increased upon enucleation, but also had limited overlap between progenitor cells and mature cells, suggesting that circRNA expression stems from regulated processes rather than resulting from mere accumulation. To study circRNA function in hematopoiesis, we first compared the expression levels of circRNAs with the translation efficiency of their mRNA-counterpart. We found that only 1 out of 2531 (0.04%) circRNAs associated with mRNA-translation regulation. Furthermore, irrespective of 1000s of identified putative open reading frames, deep ribosome-footprinting sequencing and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs. In conclusion, circRNAs alter their expression profile during terminal hematopoietic differentiation, yet their contribution to regulate cellular processes remains enigmatic.

molecular biology↗

bioRxiv2020

T cells expanded from renal cell carcinoma are tumor-reactive but fail to produce IFN-γ, TNF-α or IL-2

Metastatic renal cell carcinoma (RCC) has a poor prognosis. Recent advances have shown beneficial responses to immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 antibodies. As only a subset of RCC patients respond, alternative strategies should be explored. Patients refractory to anti-PD-1 therapy may benefit from autologous tumor infiltrating lymphocyte (TIL) therapy. Even though efficient TIL expansion was reported from RCC lesions, it is not well established how many RCC TIL products are tumor-reactive, how well they produce pro-inflammatory cytokines in response to autologous tumors, and whether their response correlates with the presence of specific immune cells in the tumor lesions. We here compared the immune infiltrate composition of RCC lesions with that of autologous kidney tissue of 18 RCC patients. T cell infiltrates were increased in the tumor lesions, and CD8+ T cell infiltrates were primarily of effector memory phenotype. Nine out of 16 (56%) tested TIL products we generated were tumor-reactive, as defined by CD137 upregulation after exposure to autologous tumor digest. Tumor reactivity was found in particular in TIL products originating from tumors with a high percentage of infiltrated T cells compared to autologous kidney, and coincided with increased ex vivo CD25 expression on CD8+ T cells. Importantly, although TIL products had the capacity to produce the key effector cytokines IFN-{gamma}, TNF- or IL-2, they failed to do so in response to autologous tumor digests. In conclusion, TIL products from RCC lesions contain tumor-reactive T cells. Their lack of tumor-specific cytokine production requires further investigation of immunosuppressive factors in RCC and subsequent optimization of RCC-derived TIL culture conditions.

immunology↗

bioRxiv2020

Limited but gene-class specific correlation of mRNA and protein expression in human CD8+ T cells

T cells are key players in our defence against infections and malignancies. When T cells differentiate or become activated, they undergo substantial alterations in gene expression. Even though RNA expression levels are now well documented throughout different stages of T cells, it is not well understood how mRNA expression translates into the protein landscape. By combining paired RNA sequencing and massspectrometry data of primary human CD8+ T cells, we report that mRNA expression is a poor proxy for the overall protein output, irrespective of the differentiation or activation status. Yet, gene class stratification revealed a function-specific correlation of mRNA with protein expression. This gene class-specific expression pattern associated with differences in gene characteristics such as sequence conservation and untranslated region (UTR) lengths. In addition, the presence of AU-rich elements in the 3UTR associated with alterations in mRNA and protein abundance T cell activation dependent, gene class-specific manner. In conclusion, our study highlights the role of gene characteristics as a determinant for gene expression in T cells.

immunology↗

bioRxiv2019

Human T cells employ conserved AU-rich elements to fine-tune IFN-γ production

Long-lasting CD8+ T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-{gamma} is a key effector molecule herein. We recently showed that in murine T cells, the production of IFN-{gamma} is tightly regulated through AU-rich elements (AREs) that are located in the 3 Untranslated Region (UTR). Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of sustained IFN-{gamma} protein-producing T cells. Importantly, this was also true for tumor antigen-specific T cells. MART-1 TCR engineered T cells that were gene-edited for ARE-deletion showed increased percentages of IFN-{gamma} producing MART-1-specific ARE-Del T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated post-transcriptional regulation is highly conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be exploited for therapeutical purposes.

immunology↗

bioRxiv2018

Polyfunctional tumor-reactive T cells are effectively expanded from non-small cell lung cancers, and correlate with an immune-engaged T cell profile in situ

Non-small cell lung cancer (NSCLC) is the second most prevalent type of cancer. With the current treatment regimens, the mortality rate remains high. Therefore, better therapeutic approaches are necessary. NSCLCs generally possess many genetic mutations and are well infiltrated by T cells (TIL), making TIL therapy an attractive option. Here we show that T cells from treatment naive, stage I-IVa NSCLC tumors can effectively be isolated and expanded, with similar efficiency as from normal lung tissue. Importantly, 76% (13/17) of tested TIL products isolates from NSCLC lesions exhibited clear reactivity against primary tumor digests, with 0.5%-30% of T cells producing the inflammatory cytokine Interferon (IFN)-{gamma}. Both CD4+ and CD8+ T cells displayed tumor reactivity. The cytokine production correlated well with CD137 and CD40L expression. Furthermore, almost half (7/17) of the TIL products contained polyfunctional T cells that produced Tumor Necrosis Factor (TNF)- and/or IL-2 in addition to IFN-{gamma}, a hallmark of effective immune responses. Tumor-reactivity in the TIL products correlated with high percentages of CD103+CD69+CD8+ T cell infiltrates in the tumor lesions, with PD-1hiCD4+ T cells, and with FoxP3+CD25+CD4+ regulatory T cell infiltrates, suggesting that the composition of T cell infiltrates may predict the level of tumor reactivity. In conclusion, the effective generation of tumor-reactive and polyfunctional TIL products implies that TIL therapy will be a successful treatment regimen for NSCLC patients.

immunology↗

bioRxiv2018

Critical role of post-transcriptional regulation for IFN-γ in tumor-infiltrating T cells

Protective T cell responses against tumors require the production of Interferon gamma (IFN-{gamma}). However, tumor-infiltrating T cells (TILs) gradually lose their capacity to produce IFN-{gamma} and therefore fail to clear malignant cells. Dissecting the underlying mechanisms that block cytokine production is thus key for improving T cell products. Here we show that although TILs express substantial levels of Ifng mRNA, post-transcriptional mechanisms impede the production of IFN-{gamma} protein due to loss of mRNA stability. CD28 triggering, but not PD1 blocking antibodies, effectively restores the stability of Ifng mRNA. Intriguingly, TILs devoid of AU-rich elements within the 3untranslated region maintain stabilized Ifng mRNA and produce more IFN-{gamma} protein than wild-type TILs. This sustained IFN-{gamma} production translates into effective suppression of tumor outgrowth, which is almost exclusively mediated by direct effects on the tumor cells. We therefore conclude that post-transcriptional mechanisms could be modulated to potentiate effective T cell therapies in cancer.

immunology↗

bioRxiv2018

Circular RNA expression in human hematopoietic cells is widespread and cell-type specific

Hematopoietic stem cells differentiate into a broad range of specialized blood cells. This process is tightly regulated and depends on transcription factors, micro-RNAs, and long non-coding RNAs. Recently, also circular RNA (circRNA) were found to regulate cellular processes. Their expression pattern and their identity is however less well defined. Here, we provide the first comprehensive analysis of circRNA expression in human hematopoietic progenitors, and in differentiated lymphoid and myeloid cells. We here show that the expression of circRNA is cell-type specific, and increases upon maturation. circRNA splicing variants can also be cell-type specific. Furthermore, nucleated hematopoietic cells contain circRNA that have higher expression levels than the corresponding linear RNA. Enucleated blood cells, i.e. platelets and erythrocytes, were suggested to use RNA to maintain their function, respond to environmental factors or to transmit signals to other cells via microvesicles. Here we show that platelets and erythrocytes contain the highest number of circRNA of all hematopoietic cells, and that the type and numbers of circRNA changes during maturation. This cell-type specific expression pattern of circRNA in hematopoietic cells suggests a hithero unappreciated role in differentiation and cellular function.

immunology↗