Deciphering the landscape of post-translational modifications in the centrosome upon T cell activation.

Intrahepatic Murine CD8 T-Cell Activation Associates With a Distinct Phenotype Leading to Bim-Dependent Death

Diverse post-translational modifications (PTMs) regulate protein function and interaction to fine-tune biological processes. Reversible phosphorylation, cysteines (Cys) modifications, and N-linked glycosylation are all essentially involved in cellular signaling pathways, such as those initiated by the action of pro-inflammatory cytokines, which can induce pancreatic β-cell death and diabetes. Here we have developed a novel strategy for the simultaneous and comprehensive characterization of the proteome and three PTMs including reversibly modified Cysteines (rmCys), phosphorylation, and sialylated N-linked glycosylation from low amount of sample material. This strategy, termed TiCPG, is based on a combination of chemical labeling and titanium dioxide (TiO2) chromatography. We applied the TiCPG strategy to study the proteome and the three PTMs changes in β-cells subject to pro-inflammatory cytokines stimulation. It enabled quantitative analysis of 8346 rmCys sites, 10,321 phosphosites and 962 sialylated N-glycosites from 5496 proteins. Significant regulation was found on 100 proteins at the expression level, while 3020 PTM peptide isoforms from 1468 proteins were significantly regulated. The three PTMs were involved in cytokine mediated β-cell apoptosis, such as the NFκB and the inducible NO synthase signaling pathways. Overall, the TiCPG strategy is a cheap, straightforward, and powerful tool for studies targeting the three PTMs described above. SignificanceThe present study presents a fast and easy method for quantitative assessment of the proteome and three PTMs from minimal amount of sample material. This simple method provides comprehensive and significant knowledge on biological systems and cellular signaling with relatively low analysis time, suitable for younger researchers and researchers that do not have direct access to LC-MSMS in their laboratories. From sub-milligram amount of material, we were able to map known cellular signaling events of proinflammatory cytokine effect on beta-cells and to discover novel PTMs involved in several known signaling pathways.

Purpose/Objectives: NFATc1 (nuclear factor of activated T cells 1) belongs to the NFAT family of transcription factors and regulates T cell activation as well as effector and cytotoxic T cell functions in many tissues. In most of the established tumors, inhibitory receptors like programmed cell death protein 1 (PD-1) contribute to the functional impairment of T cell activation by persistent tumour antigen challenge, a process that is called T cell exhaustion. Cancer immunotherapies are aimed to reawake exhausted T cells by blocking inhibitory checkpoint receptors or immunosuppressive cells. As NFATc1 is important for T cell activation we asked whether this factor could be important for the reactivation of exhausted T cells in non-small cell lung cancer (NSCLC). Materials/Methods: We analyzed NFATc1 and its interplay with PD-1 in a human NSCLC patient cohort using qPCR, IHC and FACS. Furthermore, in a murine model of lung adenocarcinoma, we determined lung tumour growth in mice with a conditional inactivation of NFATc1 in T cells (NFATc1ΔCD4) and analyzed the influence of anti-PD-1 antibody treatment on NFATc1 protein levels in T cells of lung-tumour bearing wild-type mice. Results and Conclusion: In this study, we report a progressive decrease of NFATc1 in lung tumor tissue and in tumor-infiltrating lymphocytes (TIL) of patients suffering from advancedstage NSCLC. NFATc1ΔCD4 mice showed increased lung tumor growth associated with impaired T-cell activation and function. Furthermore, in the absence of NFATc1, reduced IL2 influenced the development of memory CD8+ T cells. Specifically, we discovered a reduction in effector memory and CD103+ tissue-resident memory (TRM) CD8+ T cell numbers in the lung of tumor-bearing NFATc1ΔCD4, underlining an impaired cytotoxic T-cell response and a reduced TRM tissue-homing capacity. Targeting PD-1 resulted in NFATc1 induction in CD4+and CD8+ T cells in tumor-bearing mice and was associated with increased antitumor cytotoxic functions. Thus this study reveals a role of NFATc1 in the activation and cytotoxic functions of T cells, in the development of memory CD8+ T-cell subsets, and in the regulation of T-cell exhaustion. These data underline the indispensability of NFATc1 for successful antitumor immune responses in patients with NSCLC.

The Age of Omics-Driven Precision Medicine.

HLA-DR+ CD38high Expression in T Cells Is Excellent in Quantifying the Amplitude of T-Cell Activation in a Spectrum of Hyperinflammatory Disorders Including HLH

The diverse proteome of an organism arises from such events as single nucleotide substitutions at the DNA level, different RNA processing, and dynamic enzymatic post-translational modifications. This minireview focuses on the measurement of intact proteins to describe the diversity found in proteomes. The field of biological mass spectrometry has steadily advanced, enabling improvements in the characterization of single proteins to proteins derived from cells or tissues. In this minireview, we discuss the basic technology for "top-down" intact protein analysis. Furthermore, examples of studies involved with the qualitative and quantitative analysis of full-length polypeptides are provided.

Citrullination of arginine residues is a post-translational modification (PTM) found on myelin basic protein (MBP), which neutralizes MBPs positive charge, and is implicated in myelin damage and multiple sclerosis (MS). Here we identify lysine acetylation as another neutralizing PTM to MBP that may be involved in myelin damage. We quantify changes in lysine and arginine PTMs on MBP derived from mice induced with an experimental autoimmune encephalomyelitis (EAE) model of MS using liquid chromatography tandem mass spectrometry. The changes in PTMs are correlated to changes in neurological disability scoring (NDS), as a marker of myelin damage. We found that lysine acetylation increased by 2-fold on MBP during peak NDS post-EAE induction. We also found that mono- and dimethyl-lysine, as well as asymmetric dimethyl-arginine residues on MBP were elevated at peak EAE disability. These findings suggest that the acetylation and methylation of lysine on MBP are PTMs associated with the neurological disability produced by EAE. Since histone deacetylase (HDAC) inhibitors have been previously shown to improve neurological disability, we also show that treatment with trichostatin A (a HDAC inhibitor) improves the NDS of EAE mice but does not change MBP acetylation.

HiACT - HIV immune activation and signals through ITAM

133P Novel fully human agonist antibodies against the T-cell costimulatory receptor CD27 shape adaptive anti-tumor immunity

Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.

Dependence of T Cell Antigen Recognition on T Cell Receptor-Peptide MHC Confinement Time

Cirrhosis, an advanced stage of liver disease, induces cirrhosis-associated immune dysfunction syndrome (CAIDS), characterized by both innate and adaptive immune dysfunction. Inflammation triggered by factors such as alcohol, viruses, toxins, and cholesterol induces metabolic reprogramming of both innate and adaptive immune cells. Our study specifically sought to investigate the compromised adaptive immune response in cirrhosis by focusing on assessing T-cell exhaustion and activation markers on helper and cytotoxic T cells. A prospective observational study involving 19 liver cirrhosis patients and 36 healthy controls was conducted. The hepatic decompensation degree was assessed using various parameters, including serum bilirubin, albumin, international normalized ratio, ascites, and hepatic encephalopathy. T cell activation (CD38, CD44, CD69, HLADR) and exhaustion markers (CTLA-4, PD-1, TIM-3, LAG-3) were assessed on helper and cytotoxic T cells by flow cytometry. Cirrhosis patients showed reduced T cells with no alteration in the CD4:CD8 T cell ratio. Among activation markers, HLADR showed increased expression on CD8+ T cells (p = 0.031). Regarding exhaustion markers, LAG-3 and TIM-3 exhibited increased expression in cirrhotic patients compared to controls in both CD4 and CD8 T cells (p = 0.004, p = 0.016, p = 0.001, p = 0.004, respectively). Neither cirrhotic nor healthy controls showed CTLA expression. PD-1 did not differ significantly between the two groups. Co-expression of PD-1/TIM-3 on CD8+ T cells was notably higher in cirrhotic patients (p < 0.002). The observation of impaired adaptive immunity with notable T-cell exhaustion and activation in cirrhosis underscores the potential relevance of immunotherapy.

BackgroundImmune activation markers associate with morbidity and mortality in HIV and hepatitis C virus (HCV) infection. We investigated how T-cell and monocyte activation are related over the course of HCV direct-acting antiviral (DAA) therapy during HCV/HIV coinfection.MethodsPeripheral blood mononuclear cells from AIDS Clinical Trials Group (ACTG) A5329 participants and a single-site separate cohort treated with DAAs were analyzed for central memory (CM)/effector memory (EM) T-cell subsets, monocyte subsets, and cell activation (CD38 and HLA-DR expression) before, during, and after therapy.ResultsBefore therapy, classical and inflammatory monocyte subset HLA-DR expression positively correlated with absolute counts and frequencies of CD38+HLA-DR+-expressing CD4+ and CD8 T cells and corresponding CM and EM subsets. After therapy initiation, CD38+HLA-DR+ co-expression on CD4+ and CD8+ memory T cells decreased by 12 weeks and 36 weeks, and plasma sCD14 positively correlated with CD38+HLA-DR+ CD4+ and CD4+CM T-cell frequencies. Monocyte subset activation remained similar over time.ConclusionsDuring HCV/HIV coinfection, memory T-cell activation is associated with monocyte subset activation, consistent with related underlying mechanisms. Following therapy initiation, memory T-cell, but not monocyte, activation decreased. Residual CD4+ T-cell activation after therapy completion is associated with sCD14, potentially linking the remaining CD4+ T-cell activation to residual factors driving activation in antiretroviral therapy–controlled HIV.

Correlative biomarker analyses of etentamig in patients with Relapsed/Refractory multiple myeloma (RRMM) at optimal dose support its potential to maximize T-cell activity with rapid and transient proinflammatory cytokine responses

Post-translational modification (PTM) is a highly dynamic yet precisely controlled process by which most eukaryotic proteins are chemically diversified. Many critical cellular responses are mediated through PTMs, which lead to modulation of enzyme activity, protein conformation, protein–protein interaction, and cellular localization. Analysis of these modifications at the proteome level could provide invaluable biological insight but remains a technically challenging undertaking. Traditionally, PTMs have been studied by standard molecular biology techniques involving tedious isolation of individual proteins and subsequent direct detection and analysis of amino acids bearing the modification. Recent advances in mass spectrometry, when combined with stable-isotope or metabolic labeling approaches, have enabled several large-scale studies of PTMs and their dynamics. These methods, however, analyze PTM changes of all proteins (old and new) present in the cell at the time of sampling and thus are only able to evaluate PTM dynamics at the ensemble level. With unnatural metabolic building blocks and in vivo compatible conjugation chemistries becoming increasingly available (Scheme 1), we sought to develop a proteomic strategy for the detection and identification of newly synthesized proteomes and their PTMs (Figure 1). We envisioned several advantages of studying the PTM dynamics of newly synthesized proteomes: 1) this method decreases the complexity of the proteome and enables the identification of PTM changes that occur in a predefined protein synthesis window; 2) it gives an accurate estimate of the time scale of different PTM events in transforming newly synthesized, modification-free proteins into mature functional entities; 3) it permits PTM analysis of primary protein synthesis responses to internal and external cues. To isolate a newly synthesized proteome, wemade use of BONCAT (bio-orthogonal noncanonical amino acid tagging), which uses the known methionine surrogates azidohomoalanine (AHA) and homopropargylglycine (HPG) for metabolic incorporation into newly synthesized proteins (Scheme 1, blue) and the corresponding alkyneor azide-modified biotin reporter (Scheme 1, bottom) for subsequent proteome isolation. To monitor dynamic changes of an PTM event, we fed growing cells with an azideor alkynecontaining sugar, fatty acid, or lipid building block (Scheme 1, orange). It should be noted that while our work was in progress, Hang and co-workers reported a tandem labeling and detection method to monitor the dynamic acylation of LCK (a tyrosine kinase) and its turnover. Their work focused on the study of single PTM events (i.e. protein palmitoylation) of a specific protein (i.e., LCK) in a proteome. The work herein, while conceptually similar, greatly expands the scope of this double metabolic incorporation strategy by successfully demonstrating, for the first Scheme 1. Methionine surrogates (blue) and unnatural metabolite PTM probes (orange) form bio-orthogonal pairs for compatible double metabolic incorporation. Those forming pairs are boxed in the same group. Azideand alkyne-containing fluorophore and biotin reporters are also shown (bottom).