Epigenetic networks driving T cell identity and plasticity during immunosenescence.

The aging process is associated with the accumulation of epigenetic alterations in immune cells, although the origin of these changes is not clear. Understanding this epigenetic drift in the immune system can provide essential information about the progression of the aging process and the immune history of each individual.

Exploring kidney allograft rejection: A proof-of-concept study using spatial transcriptomics.

In this proof-of-concept study, spatial transcriptomics combined with public single-cell ribonucleic acid-sequencing data were used to explore the potential of this technology to study kidney allograft rejection. We aimed to map gene expression patterns within diverse pathologic states by examining biopsies classified across nonrejection, T cell-mediated acute rejection, interstitial fibrosis, and tubular atrophy. Our results revealed distinct immune cell signatures, including those of T and B lymphocytes, monocytes, mast cells, and plasma cells, and their spatial organization within the renal interstitium. We also mapped chemokine receptors and ligands to study immune cell migration and recruitment. Finally, our analysis demonstrated differential spatial enrichment of transcription signatures associated with kidney allograft rejection across various biopsy regions. Interstitium regions displayed higher enrichment scores for rejection-associated gene expression patterns than tubular areas, which had negative scores. This implies that these signatures are primarily driven by processes unfolding in the renal interstitium. Overall, this study highlights the value of spatial transcriptomics for revealing cellular heterogeneity and immune signatures in renal transplant biopsies and demonstrates its potential for studying the molecular and cellular mechanisms associated with rejection. However, certain limitations must be borne in mind regarding the development and future applications of this technology.

Propionate and butyrate counteract renal damage and progression to chronic kidney disease.

BACKGROUND: Short-chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, are produced by gut microbiota through fermentation of complex carbohydrates that cannot be digested by the human host. They affect gut health and can contribute at the distal level to the pathophysiology of several diseases, including renal pathologies. METHODS: SCFA levels were measured in chronic kidney disease (CKD) patients (n = 54) at different stages of the disease and associations with renal function and inflammation parameters were examined. The impact of propionate and butyrate in pathways triggered in tubular cells under inflammatory conditions was analysed using genome-wide expression assays. Finally, a pre-clinical mouse model of folic acid-induced transition from acute kidney injury to CKD was used to analyse the preventive and therapeutic potential of these microbial metabolites in the development of CKD. RESULTS: Faecal levels of propionate and butyrate in CKD patients gradually reduce as the disease progresses, and do so in close association with established clinical parameters for serum creatinine, blood urea nitrogen and the estimated glomerular filtration rate. Propionate and butyrate jointly downregulated the expression of 103 genes related to inflammatory processes and immune system activation triggered by TNF-α in tubular cells. In vivo, the administration of propionate and butyrate, either before or soon after injury, respectively prevented and slowed the progression of damage. This was indicated by a decrease in renal injury markers, the expression of pro-inflammatory and pro-fibrotic markers, and recovery of renal function over the long term. CONCLUSIONS: Propionate and butyrate levels are associated with a progressive loss of renal function in CKD patients. Early administration of these SCFAs prevents disease advancement in a pre-clinical model of acute renal damage, demonstrating their therapeutic potential independently of the gut microbiota.

Gut microbiota and inflammatory mediators differentiate IgE mediated and non-IgE mediated cases of cow's milk protein at diagnosis.

OBJECTIVE: Analyze fecal and blood samples at point of diagnosis in IgE mediated cow's milk protein allergy (CMPA) and non-IgE mediated (NIM)-CMPA patients to look for potential new biomarkers. PATIENTS AND METHODS: Fourteen patients with IgE mediated CMPA and 13 with NIM-CMPA were recruited in three hospitals in the north of Spain, and were compared with 25 infants from a control group of the same age range. To characterize intestinal microbiota, 16S rDNA gene and internal transcribed spacer amplicons of bifidobacteria were sequenced with Illumina technology. Fatty acids were analyzed by gas chromatography, meanwhile intestinal inflammation markers were quantified by enzyme-linked immunosorbent assay and a multiplex system. Immunological analysis of blood was performed by flow cytometry. RESULTS: The fecal results obtained in the NIM-CMPA group stand out. Among them, a significant reduction in the abundance of Bifidobacteriaceae and Bifidobacterium sequences with respect to controls was observed. Bifidobacterial species were also different, highlighting the lower abundance of Bifidobacterium breve sequences. Fecal calprotectin levels were found to be significantly elevated in relation to IgE mediated patients. Also, a higher excretion of IL-10 and a lower excretion of IL-1ra and platelet derived growth factor-BB was found in NIM-CMPA patients. CONCLUSIONS: The differential fecal parameters found in NIM-CMPA patients could be useful in the diagnosis of NIM food allergy to CM proteins.

The transcription factor Fosl1 preserves Klotho expression and protects from acute kidney injury.

Increased expression of AP-1 transcription factor components has been reported in acute kidney injury (AKI). However, the role of specific components, such as Fosl1, in tubular cells or AKI is unknown. Upstream regulator analysis of murine nephrotoxic AKI transcriptomics identified AP-1 as highly upregulated. Among AP-1 canonical components, Fosl1 was found to be upregulated in two transcriptomics datasets from nephrotoxic murine AKI induced by folic acid or cisplatin and from proximal tubular cells exposed to TWEAK, a cytokine mediator of AKI. Fosl1 was minimally expressed in the kidneys of control uninjured mice. Increased Fosl1 protein was localized to proximal tubular cell nuclei in AKI. In human AKI, FOSL1 was found present in proximal tubular cells in kidney sections and in urine along with increased urinary FOSL1 mRNA. Selective Fosl1 deficiency in proximal tubular cells (Fosl1Δtub) increased the severity of murine cisplatin- or folate-induced AKI as characterized by lower kidney function, more severe kidney inflammation and Klotho downregulation. Indeed, elevated AP-1 activity was observed after cisplatin-induced AKI in Fosl1Δtub mice compared to wild-type mice. More severe Klotho downregulation preceded more severe kidney dysfunction. The Klotho promoter was enriched in Fosl1 binding sites and Fosl1 bound to the Klotho promoter in cisplatin-AKI. In cultured proximal tubular cells, Fosl1 targeting increased the proinflammatory response and downregulated Klotho. In vivo, recombinant Klotho administration protected Fosl1Δtub mice from cisplatin-AKI. Thus, increased proximal tubular Fosl1 expression during AKI is an adaptive response, preserves Klotho, and limits the severity of tubular cell injury and AKI.

The CCN2 Polymorphism rs12526196 Is a Risk Factor for Ascending Thoracic Aortic Aneurysm.

Cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a downstream mediator of other profibrotic factors including transforming growth factor (TGF)-ß and angiotensin II. However, recent evidence from our group demonstrated the direct role of CCN2 in maintaining aortic wall homeostasis and acute and lethal aortic aneurysm development induced by angiotensin II in the absence of CCN2 in mice. In order to translate these findings to humans, we evaluated the potential association between three polymorphisms in the CCN2 gene and the presence of a thoracic aortic aneurysm (TAA). Patients with and without TAA retrospectively selected were genotyped for rs6918698, rs9402373 and rs12526196 polymorphisms related to the CCN2 gene. Multivariable logistic regression models were performed. In our population of 366 patients (69 with TAA), no associations were found between rs6918698 and rs9402373 and TAA. However, the presence of one C allele from rs12526196 was associated with TAA comparing with the TT genotype, independently of risk factors such as sex, age, hypertension, type of valvulopathy and the presence of a bicuspid aortic valve (OR = 3.17; 95% CI = 1.30-7.88; p = 0.011). In conclusion, we demonstrated an association between the C allele of rs12526196 in the CCN2 gene and the presence of TAA. This study extrapolates to humans the relevance of CCN2 in aortic aneurysm observed in mice and postulates, for the first time, a potential protective role to CCN2 in aortic aneurysm pathology. Our results encourage future research to explore new variants in the CCN2 gene that could be predisposed to TAA development.

Type IV Collagen and SOX9 Are Molecular Targets of BET Inhibition in Experimental Glomerulosclerosis.

Progressive glomerulonephritis (GN) is characterized by an excessive accumulation of extracellular (ECM) proteins, mainly type IV collagen (COLIV), in the glomerulus leading to glomerulosclerosis. The current therapeutic approach to GN is suboptimal. Epigenetic drugs could be novel therapeutic options for human disease. Among these drugs, bromodomain and extra-terminal domain (BET) inhibitors (iBETs) have shown beneficial effects in experimental kidney disease and fibrotic disorders. Sex-determining region Y-box 9 (SOX9) is a transcription factor involved in regulating proliferation, migration, and regeneration, but its role in kidney fibrosis is still unclear. We investigated whether iBETs could regulate ECM accumulation in experimental GN and evaluated the role of SOX9 in this process. For this purpose, we tested the iBET JQ1 in mice with anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS). In NTS-injected mice, JQ1 treatment reduced glomerular ECM deposition, mainly by inhibiting glomerular COLIV accumulation and Col4a3 gene overexpression. Moreover, chromatin immunoprecipitation assays demonstrated that JQ1 inhibited the recruitment and binding of BRD4 to the Col4a3 promoter and reduced its transcription. Active SOX9 was found in the nuclei of glomerular cells of NTS-injured kidneys, mainly in COLIV-stained regions. JQ1 treatment blocked SOX9 nuclear translocation in injured kidneys. Moreover, in vitro JQ1 blocked TGF-ß1-induced SOX9 activation and ECM production in cultured mesangial cells. Additionally, SOX9 gene silencing inhibited ECM production, including COLIV production. Our results demonstrated that JQ1 inhibited SOX9/COLIV, to reduce experimental glomerulosclerosis, supporting further research of iBET as a potential therapeutic option in progressive glomerulosclerosis.

Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment.

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.

CCN2 Increases TGF-ß Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-ß Pathway Regulation.

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor ß (TGF-ß). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-ß together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-ß receptors (TßR) in the TGF-ß pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TßRI and TßRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TßRI levels, an increase in TßRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TßRI inhibitor did not modify TßRII increment induced by CCN2(VI), demonstrating a TGF-ß-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TßRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TßRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-ß pathway activation by increasing TßRII expression in an EGFR-SMAD dependent manner activation.

Epigenetic Networks Regulate the Transcriptional Program in Memory and Terminally Differentiated CD8+ T Cells.

Epigenetic mechanisms play a critical role during differentiation of T cells by contributing to the formation of stable and heritable transcriptional patterns. To better understand the mechanisms of memory maintenance in CD8+ T cells, we performed genome-wide analysis of DNA methylation, histone marking (acetylated lysine 9 in histone H3 and trimethylated lysine 9 in histone), and gene-expression profiles in naive, effector memory (EM), and terminally differentiated EM (TEMRA) cells. Our results indicate that DNA demethylation and histone acetylation are coordinated to generate the transcriptional program associated with memory cells. Conversely, EM and TEMRA cells share a very similar epigenetic landscape. Nonetheless, the TEMRA transcriptional program predicts an innate immunity phenotype associated with genes never reported in these cells, including several mediators of NK cell activation (VAV3 and LYN) and a large array of NK receptors (e.g., KIR2DL3, KIR2DL4, KIR2DL1, KIR3DL1, KIR2DS5). In addition, we identified up to 161 genes that encode transcriptional regulators, some of unknown function in CD8+ T cells, and that were differentially expressed in the course of differentiation. Overall, these results provide new insights into the regulatory networks involved in memory CD8+ T cell maintenance and T cell terminal differentiation.

The macrophage phenotype and inflammasome component NLRP3 contributes to nephrocalcinosis-related chronic kidney disease independent from IL-1-mediated tissue injury.

Primary/secondary hyperoxalurias involve nephrocalcinosis-related chronic kidney disease (CKD) leading to end-stage kidney disease. Mechanistically, intrarenal calcium oxalate crystal deposition is thought to elicit inflammation, tubular injury and atrophy, involving the NLRP3 inflammasome. Here, we found that mice deficient in NLRP3 and ASC adaptor protein failed to develop nephrocalcinosis, compromising conclusions on nephrocalcinosis-related CKD. In contrast, hyperoxaluric wild-type mice developed profound nephrocalcinosis. NLRP3 inhibition using the ß-hydroxybutyrate precursor 1,3-butanediol protected such mice from nephrocalcinosis-related CKD. Interestingly, the IL-1 inhibitor anakinra had no such effect, suggesting IL-1-independent functions of NLRP3. NLRP3 inhibition using 1,3-butanediol treatment induced a shift of infiltrating renal macrophages from pro-inflammatory (CD45+F4/80+CD11b+CX3CR1+CD206-) and pro-fibrotic (CD45+F4/80+CD11b+CX3CR1+CD206+TGFß+) to an anti-inflammatory (CD45+F4/80+CD11b+CD206+TGFß-) phenotype, and prevented renal fibrosis. Finally, in vitro studies with primary murine fibroblasts confirmed the non-redundant role of NLRP3 in the TGF-ß signaling pathway for fibroblast activation and proliferation independent of the NLRP3 inflammasome complex formation. Thus, nephrocalcinosis-related CKD involves NLRP3 but not necessarily via intrarenal IL-1 release but rather via other biological functions including TGFR signaling and macrophage polarization. Hence, NLRP3 may be a promising therapeutic target in hyperoxaluria and nephrocalcinosis.

Epigenetic Modification Mechanisms Involved in Inflammation and Fibrosis in Renal Pathology.

The growing incidence of obesity, hypertension, and diabetes, coupled with the aging of the population, is increasing the prevalence of renal diseases in our society. Chronic kidney disease (CKD) is characterized by persistent inflammation, fibrosis, and loss of renal function leading to end-stage renal disease. Nowadays, CKD treatment has limited effectiveness underscoring the importance of the development of innovative therapeutic options. Recent studies have identified how epigenetic modifications participate in the susceptibility to CKD and have explained how the environment interacts with the renal cell epigenome to contribute to renal damage. Epigenetic mechanisms regulate critical processes involved in gene regulation and downstream cellular responses. The most relevant epigenetic modifications that play a critical role in renal damage include DNA methylation, histone modifications, and changes in miRNA levels. Importantly, these epigenetic modifications are reversible and, therefore, a source of potential therapeutic targets. Here, we will explain how epigenetic mechanisms may regulate essential processes involved in renal pathology and highlight some possible epigenetic therapeutic strategies for CKD treatment.

Inhibition of Bromodomain and Extraterminal Domain Family Proteins Ameliorates Experimental Renal Damage.

Renal inflammation has a key role in the onset and progression of immune- and nonimmune-mediated renal diseases. Therefore, the search for novel anti-inflammatory pharmacologic targets is of great interest in renal pathology. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) proteins, was previously found to preserve renal function in experimental polycystic kidney disease. We report here that JQ1-induced BET inhibition modulated the in vitro expression of genes involved in several biologic processes, including inflammation and immune responses. Gene silencing of BRD4, an important BET protein, and chromatin immunoprecipitation assays showed that JQ1 alters the direct association of BRD4 with acetylated histone-packaged promoters and reduces the transcription of proinflammatory genes (IL-6, CCL-2, and CCL-5). In vivo, JQ1 abrogated experimental renal inflammation in murine models of unilateral ureteral obstruction, antimembrane basal GN, and infusion of Angiotensin II. Notably, JQ1 downregulated the expression of several genes controlled by the NF-κB pathway, a key inflammatory signaling pathway. The RelA NF-κB subunit is activated by acetylation of lysine 310. In damaged kidneys and cytokine-stimulated renal cells, JQ1 reduced the nuclear levels of RelA NF-κB. Additionally, JQ1 dampened the activation of the Th17 immune response in experimental renal damage. Our results show that inhibition of BET proteins reduces renal inflammation by several mechanisms: chromatin remodeling in promoter regions of specific genes, blockade of NF-κB pathway activation, and modulation of the Th17 immune response. These results suggest that inhibitors of BET proteins could have important therapeutic applications in inflammatory renal diseases.

Regulation of the transcriptional program by DNA methylation during human αß T-cell development.

Thymocyte differentiation is a complex process involving well-defined sequential developmental stages that ultimately result in the generation of mature T-cells. In this study, we analyzed DNA methylation and gene expression profiles at successive human thymus developmental stages. Gain and loss of methylation occurred during thymocyte differentiation, but DNA demethylation was much more frequent than de novo methylation and more strongly correlated with gene expression. These changes took place in CpG-poor regions and were closely associated with T-cell differentiation and TCR function. Up to 88 genes that encode transcriptional regulators, some of whose functions in T-cell development are as yet unknown, were differentially methylated during differentiation. Interestingly, no reversion of accumulated DNA methylation changes was observed as differentiation progressed, except in a very small subset of key genes (RAG1, RAG2, CD8A, PTCRA, etc.), indicating that methylation changes are mostly unique and irreversible events. Our study explores the contribution of DNA methylation to T-cell lymphopoiesis and provides a fine-scale map of differentially methylated regions associated with gene expression changes. These can lay the molecular foundations for a better interpretation of the regulatory networks driving human thymopoiesis.

Links between coagulation, inflammation, regeneration, and fibrosis in kidney pathology.

Acute kidney injury (AKI) involves nephron injury leading to irreversible nephron loss, ie, chronic kidney disease (CKD). Both AKI and CKD are associated with distinct histological patterns of tissue injury, but kidney atrophy in CKD involves tissue remodeling with interstitial inflammation and scarring. No doubt, nephron atrophy, inflammation, fibrosis, and renal dysfunction are associated with each other, but their hierarchical relationships remain speculative. To better understand the pathophysiology, we provide an overview of the fundamental danger response programs that assure host survival upon traumatic injury from as early as the first multicellular organisms, ie, bleeding control by coagulation, infection control by inflammation, epithelial barrier restoration by re-epithelialization, and tissue stabilization by mesenchymal repair. Although these processes assure survival in the majority of the populations, their dysregulation causes kidney disease in a minority. We discuss how, in genetically heterogeneous population, genetic variants shift balances and modulate danger responses toward kidney disease. We further discuss how classic kidney disease entities develop from an insufficient or overshooting activation of these danger response programs. Finally, we discuss molecular pathways linking, for example, inflammation and regeneration or inflammation and fibrosis. Understanding the causative and hierarchical relationships and the molecular links between the danger response programs should help to identify molecular targets to modulate kidney injury and to improve outcomes for kidney disease patients.

The inflammatory cytokine TWEAK decreases PGC-1α expression and mitochondrial function in acute kidney injury.

Studies of mitochondria-targeted nephroprotective agents suggest a key role of mitochondrial injury in AKI. Here we tested whether an improved perception of factors responsible for mitochondrial biogenesis may provide clues to novel therapeutic approaches to AKI. TWEAK is an inflammatory cytokine which is upregulated in AKI. Transcriptomic analysis of TWEAK-stimulated cultured murine tubular epithelial cells and folic acid-induced AKI in mice identified downregulation of peroxisome proliferator- activated receptor-γ coactivador-1α (PGC-1α) and its target genes (mitochondrial proteins Ndufs1, Sdha, and Tfam) as a shared feature. Neutralizing anti-TWEAK antibodies prevented the decrease in kidney PGC-1α and its targets during AKI. TWEAK stimulation decreased kidney PGC-1α expression in healthy mice and decreased expression of PGC-1α and its targets as well as mitochondrial membrane potential in cultured tubular cells. Adenoviral-mediated PGC-1α overexpression prevented TWEAK-induced downregulation of PGC-1α-dependent genes and the decrease in mitochondrial membrane potential. TWEAK promoted histone H3 deacetylation at the murine PGC-1α promoter. TWEAK-induced downregulation of PGC-1α was prevented by histone deacetylase or NF-κB inhibitors. Thus, TWEAK decreases PGC-1α and target gene expression in tubular cells in vivo and in vitro. Approaches that preserve mitochondrial function during kidney injury may be therapeutic for AKI.

DNA methylation: a promising landscape for immune system-related diseases.

During hematopoiesis, a unique hematopoietic stem cell (HSC) from the bone marrow gives rise to a subset of mature blood cells that directs all the immune responses. Recent studies have shown that this well-defined, hierarchical process is regulated in part by epigenetic mechanisms. Changes in the DNA methylation profile have a critical role in the division of these stem cells into the myeloid and lymphoid lineages and in the establishment of a specific phenotype and functionality in each terminally differentiated cell type. In this review, we describe how the DNA methylation patterns are modified during hematopoietic differentiation and what their role is in cell plasticity and immune function. An in-depth knowledge of these epigenetic mechanisms will help clarify how cell type-specific gene programs are established, and how they can be leveraged in the development of novel strategies for treating immune system-related pathologies.

Autoantibodies against MHC class I polypeptide-related sequence A are associated with increased risk of concomitant autoimmune diseases in celiac patients.

BACKGROUND: Overexpression of autologous proteins can lead to the formation of autoantibodies and autoimmune diseases. MHC class I polypeptide-related sequence A (MICA) is highly expressed in the enterocytes of patients with celiac disease, which arises in response to gluten. The aim of this study was to investigate anti-MICA antibody formation in patients with celiac disease and its association with other autoimmune processes. METHODS: We tested serum samples from 383 patients with celiac disease, obtained before they took up a gluten-free diet, 428 patients with diverse autoimmune diseases, and 200 controls for anti-MICA antibodies. All samples were also tested for anti-endomysium and anti-transglutaminase antibodies. RESULTS: Antibodies against MICA were detected in samples from 41.7% of patients with celiac disease but in only 3.5% of those from controls (P <0.0001) and 8.2% from patients with autoimmune disease (P <0.0001). These antibodies disappeared after the instauration of a gluten-free diet. Anti-MICA antibodies were significantly prevalent in younger patients (P <0.01). Fifty-eight patients with celiac disease (15.1%) presented a concomitant autoimmune disease. Anti-MICA-positive patients had a higher risk of autoimmune disease than MICA antibody-negative patients (P <0.0001; odds ratio = 6.11). The risk was even higher when we also controlled for age (odds ratio = 11.69). Finally, we found that the associated risk of developing additional autoimmune diseases was 16 and 10 times as high in pediatric patients and adults with anti-MICA, respectively, as in those without. CONCLUSIONS: The development of anti-MICA antibodies could be related to a gluten-containing diet, and seems to be involved in the development of autoimmune diseases in patients with celiac disease, especially younger ones.

A promoter DNA demethylation landscape of human hematopoietic differentiation.

Global mechanisms defining the gene expression programs specific for hematopoiesis are still not fully understood. Here, we show that promoter DNA demethylation is associated with the activation of hematopoietic-specific genes. Using genome-wide promoter methylation arrays, we identified 694 hematopoietic-specific genes repressed by promoter DNA methylation in human embryonic stem cells and whose loss of methylation in hematopoietic can be associated with gene expression. The association between promoter methylation and gene expression was studied for many hematopoietic-specific genes including CD45, CD34, CD28, CD19, the T cell receptor (TCR), the MHC class II gene HLA-DR, perforin 1 and the phosphoinositide 3-kinase (PI3K) and results indicated that DNA demethylation was not always sufficient for gene activation. Promoter demethylation occurred either early during embryonic development or later on during hematopoietic differentiation. Analysis of the genome-wide promoter methylation status of induced pluripotent stem cells (iPSCs) generated from somatic CD34(+) HSPCs and differentiated derivatives from CD34(+) HSPCs confirmed the role of DNA methylation in regulating the expression of genes of the hemato-immune system, and indicated that promoter methylation of these genes may be associated to stemness. Together, these data suggest that promoter DNA demethylation might play a role in the tissue/cell-specific genome-wide gene regulation within the hematopoietic compartment.

Assessing Predictive Value of SARS-CoV-2 Epitope-Specific CD8+ T-Cell Response in Patients with Severe Symptoms.

Specific T cell responses against SARS-CoV-2 provided an overview of acquired immunity during the pandemic. Anti-SARS-CoV-2 immunity determines the severity of acute illness, but also might be related to the possible persistence of symptoms (long COVID). We retrospectively analyzed ex vivo longitudinal CD8+ T cell responses in 26 COVID-19 patients diagnosed with severe disease, initially (1 month) and long-term (10 months), and in a cohort of 32 vaccinated healthcare workers without previous SARS-CoV-2 infection. We used peptide-human leukocyte antigen (pHLA) dextramers recognizing 26 SARS-CoV-2-derived epitopes of viral and other non-structural proteins. Most patients responded to at least one of the peptides studied, mainly derived from non-structural ORF1ab proteins. After 10 months follow-up, CD8+ T cell responses were maintained at long term and reaction against certain epitopes (A*01:01-ORF1ab1637) was still detected and functional, showing a memory-like phenotype (CD127+ PD-1+). The total number of SARS-CoV-2-specific CD8+ T cells was significantly associated with protection against long COVID in these patients. Compared with vaccination, infected patients showed a less effective immune response to spike protein-derived peptides restricted by HLA. So, the A*01:01-S865 and A*24:02-S1208 dextramers were only recognized in vaccinated individuals. We conclude that initial SARS-CoV-2-specific CD8+ T cell response could be used as a marker to understand the evolution of severe disease and post-acute sequelae after SARS-CoV-2 infection.