Normal and Aberrant TCRγδ+ T Cells and T Cell Large Granular Lymphocyte Leukemia

Throughout this thesis different aspects of T cells and T-LGL leukemias – with special emphasis on TCRγδ-variants – are studied

Proteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualisation, identification and quantification of N-myristoylation, N- and S-acylation, Ocholesterylation, S-farnesylation and S-geranylgeranylation

Protein lipidation is one of the most widespread post-translational modifications (PTMs) found in nature, regulating protein function, structure and subcellular localization. Lipid transferases and their substrate proteins are also attracting increasing interest as drug targets because of their dysregulation in many disease states. However, the inherent hydrophobicity and potential dynamic nature of lipid modifications makes them notoriously challenging to detect by many analytical methods. Chemical proteomics provides a powerful approach to identify and quantify these diverse protein modifications by combining bespoke chemical tools for lipidated protein enrichment with quantitative mass spectrometry–based proteomics. Here, we report a robust and proteome-wide approach for the exploration of five major classes of protein lipidation in living cells, through the use of specific chemical probes for each lipid PTM. In-cell labeling of lipidated proteins is achieved by the metabolic incorporation of a lipid probe that mimics the specific natural lipid, concomitantly wielding an alkyne as a bio-orthogonal labeling tag. After incorporation, the chemically tagged proteins can be coupled to multifunctional ‘capture reagents’ by using click chemistry, allowing in-gel fluorescence visualization or enrichment via affinity handles for quantitative chemical proteomics based on label-free quantification (LFQ) or tandem mass-tag (TMT) approaches. In this protocol, we describe the application of lipid probes for N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation in multiple cell lines to illustrate both the workflow and data obtained in these experiments. We provide detailed workflows for method optimization, sample preparation for chemical proteomics and data processing. A properly trained researcher (e.g., technician, graduate student or postdoc) can complete all steps from optimizing metabolic labeling to data processing within 3 weeks. This protocol enables sensitive and quantitative analysis of lipidated proteins at a proteome-wide scale at native expression levels, which is critical to understanding the role of lipid PTMs in health and disease

Inhibition of vaccinia virus L1 N-myristoylation by the host N-myristoyltransferase inhibitor IMP-1088 generates non-infectious virions defective in cell entry

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors

Dysregulated signaling, proliferation and apoptosis impact on the pathogenesis of TCRγδ+ T cell large granular lymphocyte leukemia

TCRγδ+ T-LGL leukemia is a rare form of chronic mature T cell disorders in elderly, which is generally characterized by a persisten

Next-generation sequencing analysis of the human TCRγδ+ T-cell repertoire reveals shifts in Vγ- and Vδ-usage in memory populations upon aging

Immunological aging remodels the immune system at several levels. This has been documented in particular for the T-cell receptor (TCR)αβ+ T-cell compartment, showing reduced naive T-cell outputs and an accumulation of terminally differentiated clonally expanding effector T-cells, leading to increased proneness to autoimmunity and cancer development at older age. Even though TCRαβ+ and TCRγδ+ T-cells follow similar paths of development involving V(D)J-recombination of TCR genes in the thymus, TCRγδ+ T-cells tend to be more subjected to peripheral rather than central selection. However, the impact of aging in shaping of the peripheral TRG/TRD repertoire remains largely elusive. Next-generation sequencing analysis methods were optimized based on a spike-in method using plasmid vector DNA-samples for accurate TRG/TRD receptor diversity quantification, resulting in optimally defined primer concentrations, annealing temperatures and cycle numbers. Next, TRG/TRD repertoire diversity was evaluated during TCRγδ+ T-cell ontogeny, showing a broad, diverse repertoire in thymic and cord blood samples with Gaussian CDR3-length distributions, in contrast to the more skewed repertoire in mature circulating TCRγδ+ T-cells in adult peripheral blood. During aging the naive repertoire maintained its diversity with Gaussian CDR3-length distributions, while in the central and effector memory populations a clear shift from young (Vγ9/Vδ2 dominance) to elderly (Vγ2/Vδ1 dominance) was observed. Together with less clear Gaussian CDR3-length distributions, this would be highly suggestive of differentially heavily selected repertoires. Despite the apparent age-related shift from Vγ9/Vδ2 to Vγ2/Vδ1, no clear aging effect was observed on the Vδ2 invariant T nucleotide and canonical Vγ9-Jγ1.2 selection determinants. A more detailed look into the healthy TRG/TRD repertoire revealed known cytomegalovirus-specific TRG/TRD clonotypes in a few donors, albeit without a significant aging-effect

Ageing and latent CMV infection impact on maturation, differentiation and exhaustion profiles of T-cell receptor gammadelta T-cells

Ageing is a broad cellular process, largely affecting the immune system, especially T-lymphocytes. Additionally to immunosenescence alone, cytomegalovirus (CMV) infection is thought to have major impacts on T-cell subset composition and exhaustion. These impacts have been studied extensively in TCRαβ+ T-cells, with reduction in naive, increase in effector (memory) subsets and shifts in CD4/CD8-ratios, in conjunction with morbidity and mortality in elderly. Effects of both ageing and CMV on the TCRγδ+ T-cell compartment remain largely elusive. In the current study we investigated Vγ- and Vδ-usage, maturation, differentiation and exhaustion marker profiles of both CD4 and CD8 double-negative (DN) and CD8+TCRγδ+ T-cells in 157 individuals, age range 20–95. We observed a progressive decrease in absolute numbers of total TCRγδ+ T-cells in blood, affecting the predominant Vγ9/Vδ2 population. Aged TCRγδ+ T-cells appeared to shift from naive to more (late-stage) effector phenotypes, which appeared more prominent in case of persistent CMV infections. In addition, we found effects of both ageing and CMV on the absolute counts of exhausted TCRγδ+ T-cells. Collectively, our data show a clear impact of ageing and CMV persistence on DN and CD8+TCRγδ+ T-cells, similar to what has been reported in CD8+TCRαβ+ T-cells, indicating that they undergo similar ageing processes