HDAC1/2 inhibitor therapy improves multiple organ systems in aged mice.

Aging increases the risk of age-related diseases, imposing substantial healthcare and personal costs. Targeting fundamental aging mechanisms pharmacologically can promote healthy aging and reduce this disease susceptibility. In this work, we employed transcriptome-based drug screening to identify compounds emulating transcriptional signatures of long-lived genetic interventions. We discovered compound 60 (Cmpd60), a selective histone deacetylase 1 and 2 (HDAC1/2) inhibitor, mimicking diverse longevity interventions. In extensive molecular, phenotypic, and bioinformatic assessments using various cell and aged mouse models, we found Cmpd60 treatment to improve age-related phenotypes in multiple organs. Cmpd60 reduces renal epithelial-mesenchymal transition and fibrosis in kidney, diminishes dementia-related gene expression in brain, and enhances cardiac contractility and relaxation for the heart. In sum, our two-week HDAC1/2 inhibitor treatment in aged mice establishes a multi-tissue, healthy aging intervention in mammals, holding promise for therapeutic translation to promote healthy aging in humans.

Anti-retroviral treatment with zidovudine alters pyrimidine metabolism, reduces translation, and extends healthy longevity via ATF-4.

The human population is aging, and the need for interventions to slow progression of age-related diseases (geroprotective interventions) is growing. Repurposing compounds already used clinically, usually at modified doses, allows rapid implementation of geroprotective pharmaceuticals. Here we find the anti-retroviral nucleoside reverse transcriptase inhibitor (NRTI) zidovudine robustly extends lifespan and health span in C. elegans, independent of electron transport chain impairment or ROS accumulation. Rather, zidovudine treatment modifies pyrimidine metabolism and transcripts related to proteostasis. Testing regulators of mitochondrial stress and proteostasis shows that lifespan extension is dependent on activating transcription factor 4 (ATF-4). ATF-4 regulates longevity induced by mitochondrial stress, specifically communication between mitochondrial and cytosolic translation. Translation is reduced in zidovudine-treated worms, also dependent on ATF-4. Finally, we show ATF-4-dependent lifespan extension induced by didanosine, another NRTI. Altogether, our work elucidates the geroprotective effects of NRTIs such as zidovudine in vivo, via reduction of translation and ATF-4.

Chronic lymphocytic leukemia presence impairs antigen-specific CD8+ T-cell responses through epigenetic reprogramming towards short-lived effectors.

T-cell dysregulation in chronic lymphocytic leukemia (CLL) associates with low response rates to autologous T cell-based therapies. How CLL affects antigen-specific T-cell responses remains largely unknown. We investigated (epi)genetic and functional consequences of antigen-specific T-cell responses in presence of CLL in vitro and in an adoptive-transfer murine model. Already at steady-state, antigen-experienced patient-derived T cells were skewed towards short-lived effector cells (SLEC) at the expense of memory-precursor effector cells (MPEC). Stimulation of these T cells in vitro showed rapid induction of effector genes and suppression of key memory transcription factors only in presence of CLL cells, indicating epigenetic regulation. This was investigated in vivo by following antigen-specific responses of naïve OT-I CD8+ cells to mCMV-OVA in presence/absence of TCL1 B-cell leukemia. Presence of leukemia resulted in increased SLEC formation, with disturbed inflammatory cytokine production. Chromatin and transcriptome profiling revealed strong epigenetic modifications, leading to activation of an effector and silencing of a memory profile through presence of CLL cells. Secondary challenge in vivo confirmed dysfunctional memory responses by antigen-experienced OT-I cells generated in presence of CLL. Altogether, we show that presence of CLL induces a short-lived effector phenotype and impaired memory responses by epigenetic reprogramming during primary responses.

Sterol-regulated transmembrane protein TMEM86a couples LXR signaling to regulation of lysoplasmalogens in macrophages.

Lysoplasmalogens are a class of vinyl ether bioactive lipids that have a central role in plasmalogen metabolism and membrane fluidity. The liver X receptor (LXR) transcription factors are important determinants of cellular lipid homeostasis owing to their ability to regulate cholesterol and fatty acid metabolism. However, their role in governing the composition of lipid species such as lysoplasmalogens in cellular membranes is less well studied. Here, we mapped the lipidome of bone marrow-derived macrophages (BMDMs) following LXR activation. We found a marked reduction in the levels of lysoplasmalogen species in the absence of changes in the levels of plasmalogens themselves. Transcriptional profiling of LXR-activated macrophages identified the gene encoding transmembrane protein 86a (TMEM86a), an integral endoplasmic reticulum protein, as a previously uncharacterized sterol-regulated gene. We demonstrate that TMEM86a is a direct transcriptional target of LXR in macrophages and microglia and that it is highly expressed in TREM2+/lipid-associated macrophages in human atherosclerotic plaques, where its expression positively correlates with other LXR-regulated genes. We further show that both murine and human TMEM86a display active lysoplasmalogenase activity that can be abrogated by inactivating mutations in the predicted catalytic site. Consequently, we demonstrate that overexpression of Tmem86a in BMDM markedly reduces lysoplasmalogen abundance and membrane fluidity, while reciprocally, silencing of Tmem86a increases basal lysoplasmalogen levels and abrogates the LXR-dependent reduction of this lipid species. Collectively, our findings implicate TMEM86a as a sterol-regulated lysoplasmalogenase in macrophages that contributes to sterol-dependent membrane remodeling.

Lymphoma-associated mutations in autoreactive memory B cells of patients with Sjögren's syndrome.

We recently demonstrated that normal memory B lymphocytes carry a substantial number of de novo mutations in the genome. Here, we performed exome-wide somatic mutation analyses of bona fide autoreactive rheumatoid factor (RF)-expressing memory B cells retrieved from patients with SjÓ§gren's syndrome (SS). The amount and repertoire of the de novo exome mutations of RF B cells were found to be essentially different from those detected in healthy donor memory B cells. In contrast to the mutation spectra of normal B cells, which appeared random and non-selected, the mutations of the RF B cells were greater in number and enriched for mutations in genes also found mutated in B-cell non-Hodgkin lymphomas. During the study, one of the SS patients developed a diffuse large B-cell lymphoma (DLBCL) out of an RF clone that was identified 2 years earlier in an inflamed salivary gland biopsy. The successive oncogenic events in the RF precursor clone and the DLBCL were assessed. In conclusion, our findings of enhanced and selected genomic damage in growth-regulating genes in RF memory B cells of SS patients together with the documented transformation of an RF-precursor clone into DLBCL provide unique novel insight into the earliest stages of B-cell derailment and lymphomagenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

SMG1, a nonsense-mediated mRNA decay (NMD) regulator, as a candidate therapeutic target in multiple myeloma.

Early data suggested that CC-115, a clinical molecule, already known to inhibit the mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK) may have additional targets beyond TORK and DNA-PK. Therefore, we aimed to identify such target(s) and investigate a potential therapeutic applicability. Functional profiling of 141 cancer cell lines revealed inhibition of kinase suppressor of morphogenesis in genitalia 1 (SMG1), a key regulator of the RNA degradation mechanism nonsense-mediated mRNA decay (NMD), as an additional target of CC-115. CC-115 treatment showed a dose-dependent increase of SMG1-mediated NMD transcripts. A subset of cell lines, including multiple myeloma (MM) cell lines sensitive to the endoplasmic reticulum stress-inducing compound thapsigargin, were highly susceptible to SMG1 inhibition. CC-115 caused the induction of UPR transcripts and cell death by mitochondrial apoptosis, requiring the presence of BAX/BAK and caspase activity. Superior antitumor activity of CC-115 over TORK inhibitors in primary human MM cells and three xenograft mouse models appeared to be via inhibition of SMG1. Our data support further development of SMG1 inhibitors as possible therapeutics in MM.

Myeloid CD40 deficiency reduces atherosclerosis by impairing macrophages' transition into a pro-inflammatory state.

AIMS: CD40 and its ligand, CD40L, play a critical role in driving atherosclerotic plaque development. Disrupted CD40-signalling reduces experimental atherosclerosis and induces a favourable stable plaque phenotype. We recently showed that small molecule-based inhibition of CD40-tumour necrosis factor receptor associated factor-6 interactions attenuates atherosclerosis in hyperlipidaemic mice via macrophage-driven mechanisms. The present study aims to detail the function of myeloid CD40 in atherosclerosis using myeloid-specific CD40-deficient mice. METHOD AND RESULTS: Cd40flox/flox and LysM-cre Cd40flox/flox mice on an Apoe-/- background were generated (CD40wt and CD40mac-/-, respectively). Atherosclerotic lesion size, as well as plaque macrophage content, was reduced in CD40mac-/- compared to CD40wt mice, and their plaques displayed a reduction in necrotic core size. Transcriptomics analysis of the CD40mac-/- atherosclerotic aorta revealed downregulated pathways of immune pathways and inflammatory responses. Loss of CD40 in macrophages changed the representation of aortic macrophage subsets. Mass cytometry analysis revealed a higher content of a subset of alternative or resident-like CD206+CD209b- macrophages in the atherosclerotic aorta of CD40mac-/- compared to CD40wt mice. RNA-sequencing of bone marrow-derived macrophages of CD40mac-/- mice demonstrated upregulation of genes associated with alternatively activated macrophages (including Folr2, Thbs1, Sdc1, and Tns1). CONCLUSIONS: We here show that absence of CD40 signalling in myeloid cells reduces atherosclerosis and limits systemic inflammation by preventing a shift in macrophage polarization towards pro-inflammatory states. Our study confirms the merit of macrophage-targeted inhibition of CD40 as a valuable therapeutic strategy to combat atherosclerosis.

Molecular Changes Following Induction of Hepatocellular Carcinoma by Diethylnitrosamine and Thioacetamide, and Subsequent Treatment with Dioscorea membranacea Extract.

Hepatocellular carcinoma (HCC) is a primary liver cancer commonly found in adults. Previously, we showed the anticancer effects of Thai herbal plant extract, Dioscorea membranacea Pierre (DM), in HCC-bearing rats. In the present study, we further examined the proposed mechanism of DM, including apoptosis and antioxidant activity. Moreover, we used RNA sequencing (RNA-seq) to analyze molecular pathways in the rat model in which HCC was induced by diethylnitrosamine (DEN) and thioacetamide (TAA). The HCC-bearing rats were then treated with 40 mg/kg of DM for 8 weeks, after which experimental and control rats were sacrificed and liver tissues were collected. The RNA-seq data of DEN/TAA-treated rats exhibited upregulation of 16 hallmark pathways, including epithelial mesenchymal transition, inflammatory responses, and angiogenesis (p<0.01). DM extract expanded the Bax protein-positive pericentral zone in the tumor areas and decreased hepatic malondialdehyde levels, implying a decrease in lipid peroxidation in liver. However, DM treatment did not ameliorate the molecular pathways induced in DEN/TAA-treated livers. Our findings indicate that DM extract has antioxidant activity and exerts its pro-apoptotic effect on rat HCCs in vivo at the (post-)translational level.

TRIB3 Modulates PPARγ-Mediated Growth Inhibition by Interfering with the MLL Complex in Breast Cancer Cells.

Aberrant expression or activity of proteins are amongst the best understood mechanisms that can drive cancer initiation and progression, as well as therapy resistance. TRIB3, a member of the Tribbles family of pseudokinases, is often dysregulated in cancer and has been associated with breast cancer initiation and metastasis formation. However, the underlying mechanisms by which TRIB3 contributes to these events are unclear. In this study, we demonstrate that TRIB3 regulates the expression of PPARγ, a transcription factor that has gained attention as a potential drug target in breast cancer for its antiproliferative actions. Proteomics and phosphoproteomics analyses together with classical biochemical assays indicate that TRIB3 interferes with the MLL complex and reduces MLL-mediated H3K4 trimethylation of the PPARG locus, thereby reducing PPARγ mRNA expression. Consequently, the overexpression of TRIB3 blunts the antiproliferative effect of PPARγ ligands in breast cancer cells, while reduced TRIB3 expression gives the opposite effect. In conclusion, our data implicate TRIB3 in epigenetic gene regulation and suggest that expression levels of this pseudokinase may serve as a predictor of successful experimental treatments with PPARγ ligands in breast cancer.

In rheumatoid arthritis inflamed joints share dominant patient-specific B-cell clones.

Background: In patients with rheumatoid arthritis (RA) different joints were shown to share the same dominant T-cell clones, suggesting shared characteristics of the inflammatory process and indicating that strategies to selectively target the antigen receptor might be feasible. Since T- and B-lymphocytes closely interact in adaptive responses, we analysed to what extent different joints also share dominant B-cell clones. Methods: In 11 RA patients, quantitative B-cell receptor (BCR) repertoire analysis was performed in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, from synovial fluid (SF) and peripheral blood (PB). Results: ST biopsies from different locations in the same joint showed clear overlap in the top-25 dominant BCR clones (16.7%, SD 12.5), in the same range as the overlap between ST and SF in the same joint (8.0%, SD 8.8) and the overlap between ST-ST between different joints (9.1%, SD 8.2), but clearly higher than the overlap between ST and PB (1.7%, SD 2.4; p<0.05) and SF and PB (2.7%, SD 4.1; p<0.05). Interestingly, these figures were substantially lower than the overlap observed in previous T-cell clonality studies. Conclusions: We conclude that in RA BCR clonal responses may be more localized than TCR clonal responses, pointing to antigen-selective influx, proliferation and/or maturation of B-cells. B lineage cells in the SF may adequately represent the dominant BCR clones of the ST, which is in contrast to T-cells. Collectively, the presence of shared B- and especially T-cells in different joints from the same patient suggests that approaches might be feasible that aim to develop antigen-receptor specific targeting of lymphocyte clones in RA as an alternative to more generalized immunosuppressive strategies.

An animal model for Pierpont syndrome: a mouse bearing the Tbl1xr1Y446C/Y446C mutation.

Pierpont syndrome is a rare disorder characterized mainly by global developmental delay, unusual facial features, altered fat distribution in the limbs and hearing loss. A specific mutation (p.Tyr446Cys) in TBL1XR1, encoding a WD40 repeat-containing protein, which is a component of the SMRT/NCoR (silencing mediator retinoid and thyroid hormone receptors/nuclear receptor corepressors), has been reported as the genetic cause of Pierpont syndrome. Here, we used CRISPR-cas9 technology to generate a mutant mouse with the Y446C mutation in Tbl1xr1, which is also present in Pierpont syndrome. Several aspects of the phenotype were studied in the mutant mice: growth, body composition, hearing, motor behavior, thyroid hormone state and lipid and glucose metabolism. The mutant mice (Tbl1xr1Y446C/Y446C) displayed delayed growth, altered body composition with increased relative lean mass and impaired hearing. Expression of several genes involved in fatty acid metabolism differed in white adipose tissue, but not in liver or muscle of mutant mice compared to wild-type mice (Tbl1xr1+/+). No difference in thyroid hormone plasma concentrations was observed. Tbl1xr1Y446C/Y446C mice can be used as a model for distinct features of Pierpont syndrome, which will enable future studies on the pathogenic mechanisms underlying the various phenotypic characteristics.

Extracellular matrix remodeling precedes atrial fibrillation: Results of the PREDICT-AF trial.

BACKGROUND: To which extent atrial remodeling occurs before atrial fibrillation (AF) is unknown. OBJECTIVE: The PREventive left atrial appenDage resection for the predICtion of fuTure Atrial Fibrillation (PREDICT-AF) study investigated such subclinical remodeling, which may be used for risk stratification and AF prevention. METHODS: Patients (N = 150) without a history of AF with a CHA2DS2-VASc score of ≥2 at an increased risk of developing AF were included. The left atrial appendage was excised and blood samples were collected during elective cardiothoracic surgery for biomarker discovery. Participants were followed for 2 years with Holter monitoring to determine any atrial tachyarrhythmia after a 50-day blanking period. RESULTS: Eighteen patients (12%) developed incident AF, which was associated with increased tissue gene expression of collagen I (COL1A1), collagen III (COL3A1), and collagen VIII (COL8A2), tenascin-C (TNC), thrombospondin-2 (THBS2), and biglycan (BGN). Furthermore, the fibroblast activating endothelin-1 (EDN1) and sodium voltage-gated channel ß subunit 2 (SCN2B) were associated with incident AF whereas the Kir2.1 channel (KCNJ2) tended to downregulate. The plasma levels of COL8A2 and TNC correlated with tissue expression and predicted incident AF. A gene panel including tissue KCNJ2, COL1A1, COL8A2, and EDN1 outperformed clinical prediction models in discriminating incident AF. CONCLUSION: The PREDICT-AF study demonstrates that atrial remodeling occurs long before incident AF and implies future potential for early patient identification and therapies to prevent AF (ClinicalTrials.gov identifier NCT03130985).

Framing the potential of public frameshift peptides as immunotherapy targets in colon cancer.

Approximately 15% of Colon Cancers are Microsatellite Instable (MSI). Frameshift Peptides (FPs) formed in MSI Colon Cancer are potential targets for immunotherapeutic strategies. Here we comprehensively characterize the mutational landscape of 71 MSI Colon Cancer patients from the cancer genome atlas (TCGA). We confirm that the mutations in MSI Colon Cancers are frequently frameshift deletions (23% in MSI; 1% in microsatellite stable), We find that these mutations cluster at specific locations in the genome which are mutated in up to 41% of the patients. We filter these for an adequate variant allele frequency, a sufficient mean mRNA level and the formation of a Super Neo Open Reading Frame (SNORF). Finally, we check the influence of Nonsense Mediated Decay (MMD) by comparing RNA and DNA sequencing results. Thereby we identify a set of 20 NMD-escaping Public FPs (PFPs) that cover over 90% of MSI Colon, 62.2% of MSI Endometrial and 58.8% of MSI Stomach cancer patients and 3 out of 4 Lynch patients in the TCGA-COAD. This underlines the potential for PFP directed immunotherapy, both in a therapeutic and a prophylactic setting in multiple types of MSI cancers.

TBX2, a Novel Regulator of Labour.

Background and Objectives: Therapeutic interventions targeting molecular factors involved in the transition from uterine quiescence to overt labour are not substantially reducing the rate of spontaneous preterm labour. The identification of novel rational therapeutic targets are essential to prevent the most common cause of neonatal mortality. Based on our previous work showing that Tbx2 (T-Box transcription factor 2) is a putative upstream regulator preceding progesterone withdrawal in mouse myometrium, we now investigate the role of TBX2 in human myometrium. Materials and Methods: RNA microarray analysis of (A) preterm human myometrium samples and (B) myometrial cells overexpressing TBX2 in vitro, combined with subsequent analysis of the two publicly available datasets of (C) Chan et al. and (D) Sharp et al. The effect of TBX2 overexpression on cytokines/chemokines secreted to the myometrium cell culture medium were determined by Luminex assay. Results: Analysis shows that overexpression of TBX2 in myometrial cells results in downregulation of TNFα- and interferon signalling. This downregulation is consistent with the decreased expression of cytokines and chemokines of which a subset has been previously associated with the inflammatory pathways relevant for human labour. In contrast, CXCL5 (C-X-C motif chemokine ligand 5), CCL21 and IL-6 (Interleukin 6), previously reported in relation to parturition, do not seem to be under TBX2 control. The combined bioinformatical analysis of the four mRNA datasets identifies a subset of upstream regulators common to both preterm and term labour under control of TBX2. Surprisingly, TBX2 mRNA levels are increased in preterm contractile myometrium. Conclusions: We identified a subset of upstream regulators common to both preterm and term labour that are activated in labour and repressed by TBX2. The increased TBX2 mRNA expression in myometrium collected during a preterm caesarean section while in spontaneous preterm labour compared to tissue harvested during iatrogenic preterm delivery does not fit the bioinformatical model. We can only explain this by speculating that the in vivo activity of TBX2 in human myometrium depends not only on the TBX2 expression levels but also on levels of the accessory proteins necessary for TBX2 activity.

miR-511 Deficiency Protects Mice from Experimental Colitis by Reducing TLR3 and TLR4 Responses via WD Repeat and FYVE-Domain-Containing Protein 1.

Antimicrobial responses play an important role in maintaining intestinal heath. Recently we reported that miR-511 may regulate TLR4 responses leading to enhanced intestinal inflammation. However, the exact mechanism remained unclear. In this study we investigated the effect of miR-511 deficiency on anti-microbial responses and DSS-induced intestinal inflammation. miR-511-deficient mice were protected from DSS-induced colitis as shown by significantly lower disease activity index, weight loss and histology scores in the miR-511-deficient group. Furthermore, reduced inflammatory cytokine responses were observed in colons of miR-511 deficient mice. In vitro studies with bone marrow-derived M2 macrophages showed reduced TLR3 and TLR4 responses in miR-511-deficient macrophages compared to WT macrophages. Subsequent RNA sequencing revealed Wdfy1 as the potential miR-511 target. WDFY1 deficiency is related to impaired TLR3/TLR4 immune responses and the expression was downregulated in miR-511-deficient macrophages and colons. Together, this study shows that miR-511 is involved in the regulation of intestinal inflammation through downstream regulation of TLR3 and TLR4 responses via Wdfy1.

ITGA6+ Human Testicular Cell Populations Acquire a Mesenchymal Rather than Germ Cell Transcriptional Signature during Long-Term Culture.

Autologous spermatogonial stem cell transplantation is an experimental technique aimed at restoring fertility in infertile men. Although effective in animal models, in vitro propagation of human spermatogonia prior to transplantation has proven to be difficult. A major limiting factor is endogenous somatic testicular cell overgrowth during long-term culture. This makes the culture both inefficient and necessitates highly specific cell sorting strategies in order to enrich cultured germ cell fractions prior to transplantation. Here, we employed RNA-Seq to determine cell type composition in sorted integrin alpha-6 (ITGA6+) primary human testicular cells (n = 4 donors) cultured for up to two months, using differential gene expression and cell deconvolution analyses. Our data and analyses reveal that long-term cultured ITGA6+ testicular cells are composed mainly of cells expressing markers of peritubular myoid cells, (progenitor) Leydig cells, fibroblasts and mesenchymal stromal cells and only a limited percentage of spermatogonial cells as compared to their uncultured counterparts. These findings provide valuable insights into the cell type composition of cultured human ITGA6+ testicular cells during in vitro propagation and may serve as a basis for optimizing future cell sorting strategies as well as optimizing the current human testicular cell culture system for clinical use.

Functional Differences Between EBV- and CMV-Specific CD8+ T cells Demonstrate Heterogeneity of T cell Dysfunction in CLL.

Acquired T cell dysfunction is a hallmark of chronic lymphocytic leukemia (CLL), and is linked to an increased risk of infections, but also reduced immune surveillance and disappointing responses to autologous T cell-based immunotherapy. The mechanisms of T cell dysfunction in CLL are not well understood. Studying immunity against chronic viruses allows for detailed analysis of the effect of CLL on T cells chronically exposed to a specific antigen. Cytomegalovirus (CMV) reactivations are rare in CLL, which corroborates with preserved CMV-specific T cell function. Epstein-Barr virus (EBV) is another herpesvirus that results in chronic infection, but unlike CMV, is characterized by subclinical reactivations in CLL patients. Since both herpesviruses induce strong CD8+ T cell responses, but have different clinical outcomes, studying these specific T cells may shed light on the mechanisms of CLL-induced T cell dysfunction. We first analyzed the phenotype of EBV-specific CD8+ T cells in CLL and healthy controls, and found that in CLL EBV-specific CD8+ T cells are in an advanced differentiation state with higher expression of inhibitory receptors. Secondly, CLL-derived EBV-specific CD8+ T cells show reduced cytotoxic potential, in contrast to CMV-specific T cells. Finally, we performed transcriptome analysis to visualize differential modulation by CLL of these T cell subsets. While T cell activation and differentiation genes are unaffected, in EBV-specific T cells expression of genes involved in synapse formation and T cell exhaustion is altered. Our findings on the heterogeneity of antigen specific T cell function in CLL aids in understanding immune-dysregulation in this disease.

Comparing genome-scale DNA methylation and CNV marks between adult human cultured ITGA6+ testicular cells and seminomas to assess in vitro genomic stability.

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture.

Glycine promotes longevity in Caenorhabditis elegans in a methionine cycle-dependent fashion.

The deregulation of metabolism is a hallmark of aging. As such, changes in the expression of metabolic genes and the profiles of amino acid levels are features associated with aging animals. We previously reported that the levels of most amino acids decline with age in Caenorhabditis elegans (C. elegans). Glycine, in contrast, substantially accumulates in aging C. elegans. In this study we show that this is coupled to a decrease in gene expression of enzymes important for glycine catabolism. We further show that supplementation of glycine significantly prolongs C. elegans lifespan, and early adulthood is important for its salutary effects. Moreover, supplementation of glycine ameliorates specific transcriptional changes that are associated with aging. Glycine feeds into the methionine cycle. We find that mutations in components of this cycle, methionine synthase (metr-1) and S-adenosylmethionine synthetase (sams-1), completely abrogate glycine-induced lifespan extension. Strikingly, the beneficial effects of glycine supplementation are conserved when we supplement with serine, which also feeds into the methionine cycle. RNA-sequencing reveals a similar transcriptional landscape in serine- and glycine-supplemented worms both demarked by widespread gene repression. Taken together, these data uncover a novel role of glycine in the deceleration of aging through its function in the methionine cycle.

Improving CLL Vγ9Vδ2-T-cell fitness for cellular therapy by ex vivo activation and ibrutinib.

The efficacy of autologous (αß) T-cell-based treatment strategies in chronic lymphocytic leukemia (CLL) has been modest. The Vγ9Vδ2-T cell subset consists of cytotoxic T lymphocytes with potent antilymphoma activity via a major histocompatibility complex-independent mechanism. We studied whether Vγ9Vδ2-T cells can be exploited as autologous effector lymphocytes in CLL. Healthy control Vγ9Vδ2-T cells were activated by and had potent cytolytic activity against CLL cells. However, CLL-derived Vγ9Vδ2-T cells proved dysfunctional with respect to effector cytokine production and degranulation, despite an increased frequency of the effector-type subset. Consequently, cytotoxicity against malignant B cells was hampered. A comparable dysfunctional phenotype was observed in healthy Vγ9Vδ2-T cells after coculture with CLL cells, indicating a leukemia-induced mechanism. Gene-expression profiling implicated alterations in synapse formation as a conceivable contributor to compromised Vγ9Vδ2-T-cell function in CLL patients. Dysfunction of Vγ9Vδ2-T cells was fully reversible upon activation with autologous monocyte-derived dendritic cells (moDCs). moDC activation resulted in efficient expansion and predominantly yielded Vγ9Vδ2-T cells with a memory phenotype. Furthermore, ibrutinib treatment promoted an antitumor T helper 1 (TH1) phenotype in Vγ9Vδ2-T cells, and we demonstrated binding of ibrutinib to IL-2-inducible kinase (ITK) in Vγ9Vδ2-T cells. Taken together, CLL-mediated dysfunction of autologous Vγ9Vδ2-T cells is fully reversible, resulting in potent cytotoxicity toward CLL cells. Our data support the potential use of Vγ9Vδ2-T cells as effector T cells in CLL immunotherapy and favor further exploration of combining Vγ9Vδ2-T-cell-based therapy with ibrutinib.