Pathogenic implications for autoimmune mechanisms derived by comparative eQTL analysis of CD4+ versus CD8+ T cells.

Inappropriate activation or inadequate regulation of CD4+ and CD8+ T cells may contribute to the initiation and progression of multiple autoimmune and inflammatory diseases. Studies on disease-associated genetic polymorphisms have highlighted the importance of biological context for many regulatory variants, which is particularly relevant in understanding the genetic regulation of the immune system and its cellular phenotypes. Here we show cell type-specific regulation of transcript levels of genes associated with several autoimmune diseases in CD4+ and CD8+ T cells including a trans-acting regulatory locus at chr12q13.2 containing the rs1131017 SNP in the RPS26 gene. Most remarkably, we identify a common missense variant in IL27, associated with type 1 diabetes that results in decreased functional activity of the protein and reduced expression levels of downstream IRF1 and STAT1 in CD4+ T cells only. Altogether, our results indicate that eQTL mapping in purified T cells provides novel functional insights into polymorphisms and pathways associated with autoimmune diseases.

Cell Specific eQTL Analysis without Sorting Cells.

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn's disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.

Epigenetic profiling in CD4+ and CD8+ T cells from Graves' disease patients reveals changes in genes associated with T cell receptor signaling.

In Graves' disease (GD), a combination of genetic, epigenetic and environmental factors causes an autoimmune response to the thyroid gland, characterized by lymphocytic infiltrations and autoantibodies targeting the thyroid stimulating hormone receptor (TSHR) and other thyroid antigens. To identify the epigenetic changes involved in GD, we performed a genome-wide analysis of DNA methylation and enrichment of H3K4me3 and H3K27ac histone marks in sorted CD4+ and CD8+ T cells. We found 365 and 3322 differentially methylated CpG sites in CD4+ and CD8+ T cells, respectively. Among the hypermethylated CpG sites, we specifically found enrichment of genes involved in T cell signaling (CD247, LCK, ZAP70, CD3D, CD3E, CD3G, CTLA4 and CD8A) and decreased expression of CD3 gene family members. The hypermethylation was accompanied with decreased levels of H3K4me3 and H3K27ac marks at several T cell signaling genes in ChIP-seq analysis. In addition, we found hypermethylation of the TSHR gene first intron, where several GD-associated polymorphisms are located. Our results demonstrate an involvement of dysregulated DNA methylation and histone modifications at T cell signaling genes in GD patients.

CpG sites associated with NRP1, NRXN2 and miR-29b-2 are hypomethylated in monocytes during ageing.

BACKGROUND: Ageing affects many components of the immune system, including innate immune cells like monocytes. They are important in the early response to pathogens and for their role to differentiate into macrophages and dendritic cells. Recent studies have revealed significant age-related changes in genomic DNA methylation in peripheral blood mononuclear cells, however information on epigenetic changes in specific leukocyte subsets is still lacking. Here, we aimed to analyse DNA methylation in purified monocyte populations from young and elderly individuals. FINDINGS: We analysed the methylation changes in monocytes purified from young and elderly individuals using the HumanMethylation450 BeadChip array. Interestingly, we found that among 26 differentially methylated CpG sites, the majority of sites were hypomethylated in elderly individuals. The most hypomethylated CpG sites were located in neuropilin 1 (NRP1; cg24892069) and neurexin 2 (NRXN2; cg27209729) genes, and upstream of miR-29b-2 gene (cg10501210). The age-related hypomethylation of these three sites was confirmed in a separate group of young and elderly individuals. CONCLUSIONS: We identified significant age-related hypomethylation in human purified monocytes at CpG sites within the regions of NRP1, NRXN2 and miR-29b-2 genes.

Cytotoxic CD8+ Temra cells show loss of chromatin accessibility at genes associated with T cell activation.

As humans age, their memory T cell compartment expands due to the lifelong exposure to antigens. This expansion is characterized by terminally differentiated CD8+ T cells (Temra), which possess NK cell-like phenotype and are associated with chronic inflammatory conditions. Temra cells are predominantly driven by the sporadic reactivation of cytomegalovirus (CMV), yet their epigenomic patterns and cellular heterogeneity remain understudied. To address this gap, we correlated their gene expression profiles with chromatin openness and conducted single-cell transcriptome analysis, comparing them to other CD8+ subsets and CMV-responses. We confirmed that Temra cells exhibit high expression of genes associated with cytotoxicity and lower expression of costimulatory and chemokine genes. The data revealed that CMV-responsive CD8+ T cells (Tcmv) were predominantly derived from a mixed population of Temra and memory cells (Tcm/em) and shared their transcriptomic profiles. Using ATAC-seq analysis, we identified 1449 differentially accessible chromatin regions between CD8+ Temra and Tcm/em cells, of which only 127 sites gained chromatin accessibility in Temra cells. We further identified 51 gene loci, including costimulatory CD27, CD28, and ICOS genes, whose chromatin accessibility correlated with their gene expression. The differential chromatin regions Tcm/em cells were enriched in motifs that bind multiple transcriptional activators, such as Jun/Fos, NFkappaB, and STAT, whereas the open regions in Temra cells mainly contained binding sites of T-box transcription factors. Our single-cell analysis of CD8+CCR7loCD45RAhi sorted Temra population showed several subsets of Temra and NKT-like cells and CMC1+ Temra populations in older individuals that were shifted towards decreased cytotoxicity. Among CD8+CCR7loCD45RAhi sorted cells, we found a decreased proportion of IL7R+ Tcm/em-like and MAIT cells in individuals with high levels of CMV antibodies (CMVhi). These results shed new light on the molecular and cellular heterogeneity of CD8+ Temra cells and their relationship to aging and CMV infection.

Single cell characterization of blood and expanded regulatory T cells in autoimmune polyendocrine syndrome type 1.

Immune tolerance fails in autoimmune polyendocrine syndrome type 1 (APS-1) because of AIRE mutations. We have used single cell transcriptomics to characterize regulatory T cells (Tregs) sorted directly from blood and from in vitro expanded Tregs in APS-1 patients compared to healthy controls. We revealed only CD52 and LTB (down) and TXNIP (up) as consistently differentially expressed genes in the datasets. There were furthermore no large differences of the TCR-repertoire of expanded Tregs between the cohorts, but unique patients showed a more restricted use of specific clonotypes. We also found that in vitro expanded Tregs from APS-1 patients had similar suppressive capacity as controls in co-culture assays, despite expanding faster and having more exhausted cells. Our results suggest that APS-1 patients do not have intrinsic defects in their Treg functionality, and that their Tregs can be expanded ex vivo for potential therapeutic applications.

MicroRNA expression profiles of human blood monocyte-derived dendritic cells and macrophages reveal miR-511 as putative positive regulator of Toll-like receptor 4.

Dendritic cells (DCs) and macrophages (MFs) are important multifunctional immune cells. Like other cell types, they express hundreds of different microRNAs (miRNAs) that are recently discovered post-transcriptional regulators of gene expression. Here we present updated miRNA expression profiles of monocytes, DCs and MFs. Compared with monocytes, ∼50 miRNAs were found to be differentially expressed in immature and mature DCs or MFs, with major expression changes occurring during the differentiation. Knockdown of DICER1, a protein needed for miRNA biosynthesis, led to lower DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and enhanced CD14 protein levels, confirming the importance of miRNAs in DC differentiation in general. Inhibition of the two most highly up-regulated miRNAs, miR-511 and miR-99b, also resulted in reduced DC-SIGN level. Prediction of miRNA-511 targets revealed a number of genes with known immune functions, of which TLR4 and CD80 were validated using inhibition of miR-511 in DCs and luciferase assays in HEK293 cells. Interestingly, under the cell cycle arrest conditions, miR-511 seems to function as a positive regulator of TLR4. In conclusion, we have identified miR-511 as a novel potent modulator of human immune response. In addition, our data highlight that miRNA influence on gene expression is dependent on the cellular environment.

Epigenetic quantification of immunosenescent CD8+ TEMRA cells in human blood.

Age-related changes in human T-cell populations are important contributors to immunosenescence. In particular, terminally differentiated CD8+ effector memory CD45RA+ TEMRA cells and their subsets have characteristics of cellular senescence, accumulate in older individuals, and are increased in age-related chronic inflammatory diseases. In a detailed T-cell profiling among individuals over 65 years of age, we found a high interindividual variation among CD8+ TEMRA populations. CD8+ TEMRA proportions correlated positively with cytomegalovirus (CMV) antibody levels, however, not with the chronological age. In the analysis of over 90 inflammation proteins, we identified plasma TRANCE/RANKL levels to associate with several differentiated T-cell populations, including CD8+ TEMRA and its CD28- subsets. Given the strong potential of CD8+ TEMRA cells as a biomarker for immunosenescence, we used deep-amplicon bisulfite sequencing to match their frequencies in flow cytometry with CpG site methylation levels and developed a computational model to predict CD8+ TEMRA cell proportions from whole blood genomic DNA. Our findings confirm the association of CD8+ TEMRA and its subsets with CMV infection and provide a novel tool for their high throughput epigenetic quantification as a biomarker of immunosenescence.

Protective antibodies and T cell responses to Omicron variant after the booster dose of BNT162b2 vaccine.

The high number of mutations in the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes its immune escape. We report a longitudinal analysis of 111 vaccinated individuals for their antibody levels up to 6 months after the third dose of the BNT162b2 vaccine. After the third dose, the antibody levels decline but less than after the second dose. The booster dose remarkably increases the serum ability to block wild-type or Omicron variant spike protein's receptor-binding domain (RBD) interaction with the angiotensin-converting enzyme 2 (ACE2) receptor, and these protective antibodies persist 3 months later. Three months after the booster dose, memory CD4+ and CD8+ T cells to the wild-type and Omicron variant are detectable in the majority of vaccinated individuals. Our data show that the third dose restores the high levels of blocking antibodies and enhances T cell responses to Omicron.

Autoimmune regulator deficiency results in decreased expression of CCR4 and CCR7 ligands and in delayed migration of CD4+ thymocytes.

Autoimmune regulator (Aire) has been viewed as a central player in the induction of tolerance. This study examines whether Aire can modulate the production of the thymic chemokines involved in corticomedullary migration and thus play a role in intrathymic thymocyte migration and maturation. Aire deficiency resulted in reduced gene expression and protein levels of the CCR4 and CCR7 ligands in whole thymi of mice, as determined by quantitative PCR analysis and ELISA. The expression of the CCR4 ligands coincided with Aire expression in the CD80(high) medullary thymic epithelial cells, whereas the expression of the CCR7 ligands was detected in other cell populations. Also, the expression pattern of the CCR4 and CCR7 ligands follows that of Aire during postnatal but not during embryonic development. In vitro, overexpression of Aire resulted in an up-regulation of selected CCR4 and CCR7 ligands, which induced selective migration of double-positive and single-positive CD4(+) cells. In vivo, Aire deficiency resulted in a diminished emigration of mature CD4(+) T cells from the thymi of 5-day-old mice. In conclusion, Aire regulates the production of CCR4 and CCR7 ligands in medullary thymic epithelial cells and alters the coordinated maturation and migration of thymocytes. These results suggest a novel mechanism behind the Aire-dependent induction of central tolerance.

Long-Term Elevated Inflammatory Protein Levels in Asymptomatic SARS-CoV-2 Infected Individuals.

The clinical features of SARS-CoV-2 infection range from asymptomatic to severe disease with life-threatening complications. Understanding the persistence of immune responses in asymptomatic individuals merit special attention because of their importance in controlling the spread of the infections. We here studied the antibody and T cell responses, and a wide range of inflammation markers, in 56 SARS-CoV-2 antibody-positive individuals, identified by a population screen after the first wave of SARS-CoV-2 infection. These, mostly asymptomatic individuals, were reanalyzed 7-8 months after their infection together with 115 age-matched seronegative controls. We found that 7-8 months after the infection their antibodies to SARS-CoV-2 Nucleocapsid (N) protein declined whereas we found no decrease in the antibodies to Spike receptor-binding domain (S-RBD) when compared to the findings at seropositivity identification. In contrast to antibodies to N protein, the antibodies to S-RBD correlated with the viral neutralization capacity and with CD4+ T cell responses as measured by antigen-specific upregulation of CD137 and CD69 markers. Unexpectedly we found the asymptomatic antibody-positive individuals to have increased serum levels of S100A12, TGF-alpha, IL18, and OSM, the markers of activated macrophages-monocytes, suggesting long-term persistent inflammatory effect associated with the viral infection in asymptomatic individuals. Our results support the evidence for the long-term persistence of the inflammation process and the need for post-infection clinical monitoring of SARS-CoV-2 infected asymptomatic individuals.

Dynamics of antibody response to BNT162b2 vaccine after six months: a longitudinal prospective study.

BACKGROUND: SARS-CoV-2 mRNA vaccines have proven high efficacy, however, limited data exists on the duration of immune responses and their relation to age and side effects. METHODS: We studied the antibody and memory T cell responses after the two-dose BNT162b2 vaccine in 122 volunteers up to 6 months and correlated the findings with age and side effects. FINDINGS: We found a robust antibody response to Spike protein after the second dose. However, the antibody levels declined at 12 weeks and 6 months post-vaccination, indicating a waning of the immune response over time. At 6 months after the second dose, the Spike antibody levels were similar to the levels in persons vaccinated with one dose or in COVID-19 convalescent individuals. The antibodies efficiently blocked ACE2 receptor binding to SARS-CoV-2 Spike protein of five variants of concern at one week but this was decreased at three months. 87% of individuals developed Spike-specific memory T cell responses, which were lower in individuals with increased proportions of immunosenescent CD8+ TEMRA cells. We found antibody response to correlate negatively with age and positively with the total score of vaccination side effects. INTERPRETATION: The mRNA vaccine induces a strong antibody response to SARS-CoV-2 and five VOCs at 1 week post-vaccination that decreases thereafter. T cell responses, although detectable in the majority, were lower in individuals with higher T cell immunosenescence. The deterioration of vaccine response suggests the need to monitor for the potential booster vaccination.

Genome-wide promoter analysis of histone modifications in human monocyte-derived antigen presenting cells.

BACKGROUND: Monocyte-derived macrophages and dendritic cells (DCs) are important in inflammatory processes and are often used for immunotherapeutic approaches. Blood monocytes can be differentiated into macrophages and DCs, which is accompanied with transcriptional changes in many genes, including chemokines and cell surface markers. RESULTS: To study the chromatin modifications associated with this differentiation, we performed a genome wide analysis of histone H3 trimethylation on lysine 4 (H3K4me3) and 27 (H3K27me3) as well as acetylation of H3 lysines (AcH3) in promoter regions. We report that both H3K4me3 and AcH3 marks significantly correlate with transcriptionally active genes whereas H3K27me3 mark is associated with inactive gene promoters. During differentiation, the H3K4me3 levels decreased on monocyte-specific CD14, CCR2 and CX3CR1 but increased on DC-specific TM7SF4/DC-STAMP, TREM2 and CD209/DC-SIGN genes. Genes associated with phagocytosis and antigen presentation were marked by H3K4me3 modifications. We also report that H3K4me3 levels on clustered chemokine and surface marker genes often correlate with transcriptional activity. CONCLUSION: Our results provide a basis for further functional correlations between gene expression and histone modifications in monocyte-derived macrophages and DCs.

Interferon autoantibodies associated with AIRE deficiency decrease the expression of IFN-stimulated genes.

Neutralizing autoantibodies to type I, but not type II, interferons (IFNs) are found at high titers in almost every patient with autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), a disease caused by AIRE gene mutations that lead to defects in thymic T-cell selection. Combining genome-wide expression array with real time RT-PCR assays, we here demonstrate that antibodies against IFN-alpha cause highly significant down-regulation of interferon-stimulated gene expression in cells from APECED patients' blood by blocking their highly dilute endogenous IFNs. This down-regulation was lost progressively as these APECED cells matured in cultures without neutralizing autoantibodies. Most interestingly, a rare APECED patient with autoantibodies to IFN-omega but not IFN-alpha showed a marked increase in expression of the same interferon-stimulated genes. We also report unexpected increases in serum CXCL10 levels in APECED. Our results argue that the breakdown of tolerance to IFNs in AIRE deficiency is associated with impaired responses to them in thymus, and highlight APECED as another autoimmune disease with associated dysregulation of IFN activity.

NickFect type of cell-penetrating peptides present enhanced efficiency for microRNA-146a delivery into dendritic cells and during skin inflammation.

MicroRNAs (miRNAs) are post-transcriptional gene expression regulators with potential therapeutic applications. miR-146a is a negative regulator of inflammatory processes in both tissue-resident and specialized immune cells and may therefore have therapeutic effect in inflammatory skin diseases. PepFect (PF) and NickFect (NF) type of cell-penetrating peptides (CPPs) have previously been shown to deliver miRNA mimics and/or siRNAs into cell cultures and in vivo. Here, we first demonstrate that selected PF- and NF-type of CPPs support delivery of fluorescent labelled miRNA mimics into keratinocytes (KCs) and dendritic cells (DCs). Second, we show that both PF- and NF-miR-146a nanocomplexes were equally effective in KCs, while NFs were more efficient in DCs as assessed by downregulation of miR-146a-influenced genes. None of miRNA nanocomplexes with the tested CPPs influenced the viability of KCs and DCs nor caused activation of DCs according to CD86 and CD83 markers. Transmission electron microscopy analysis with Nanogold-labelled miR-146a mimics and assessment of endocytic trafficking pathways revealed endocytosis as an active route of delivery in both KCs and DCs for all tested CPPs. However, consistent with the higher efficiency, NF-delivered miR-146a was detected more often outside endosomes in DCs. Finally, pre-injection of NF71:miR-146a nanocomplexes was confirmed to suppress inflammatory responses in a mouse model of irritant contact dermatitis as shown by reduced ear swelling response and downregulation of pro-inflammatory cytokines, including IL-6, IL-1ß, IL-33 and TNF-α. In conclusion, NF71 efficiently delivers miRNA mimics into KCs as well as DCs, and therefore may have advantage in therapeutic delivery of miRNAs in case of inflammatory skin diseases.

Monocytes present age-related changes in phospholipid concentration and decreased energy metabolism.

Age-related changes at the cellular level include the dysregulation of metabolic and signaling pathways. Analyses of blood leukocytes have revealed a set of alterations that collectively lower their ability to fight infections and resolve inflammation later in life. We studied the transcriptomic, epigenetic, and metabolomic profiles of monocytes extracted from younger adults and individuals over the age of 65 years to map major age-dependent changes in their cellular physiology. We found that the monocytes from older persons displayed a decrease in the expression of ribosomal and mitochondrial protein genes and exhibited hypomethylation at the HLA class I locus. Additionally, we found elevated gene expression associated with cell motility, including the CX3CR1 and ARID5B genes, which have been associated with the development of atherosclerosis. Furthermore, the downregulation of two genes, PLA2G4B and ALOX15B, which belong to the arachidonic acid metabolism pathway involved in phosphatidylcholine conversion to anti-inflammatory lipoxins, correlated with increased phosphatidylcholine content in monocytes from older individuals. We found age-related changes in monocyte metabolic fitness, including reduced mitochondrial function and increased glycose consumption without the capacity to upregulate it during increased metabolic needs, and signs of increased oxidative stress and DNA damage. In conclusion, our results complement existing findings and elucidate the metabolic alterations that occur in monocytes during aging.

IL-22 Paucity in APECED Is Associated With Mucosal and Microbial Alterations in Oral Cavity.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by recessive mutations in the AIRE gene. The hallmark of the disease is the production of highly neutralizing autoantibodies against type I interferons and IL-22. Considering the importance of IL-22 in maintaining mucosal barrier integrity and shaping its microbial community, we sought to study potential changes in the oral cavity in this model of human IL-22 paucity. We found that besides known Th22 cell deficiency, APECED patients have significantly fewer circulating MAIT cells with potential IL-22 secreting capacity. Saliva samples from APECED patients revealed local inflammation, the presence of autoantibodies against IFN-α and IL-22, and alterations in the oral microbiota. Moreover, gene expression data of buccal biopsy samples suggested impaired antimicrobial response and cell proliferation, both of which are processes regulated by IL-22. Our data complement the knowledge gained from mouse models and support the concept of IL-22 being a critical homeostatic cytokine in human mucosal sites.

Age-related profiling of DNA methylation in CD8+ T cells reveals changes in immune response and transcriptional regulator genes.

Human ageing affects the immune system resulting in an overall decline in immunocompetence. Although all immune cells are affected during aging, the functional capacity of T cells is most influenced and is linked to decreased responsiveness to infections and impaired differentiation. We studied age-related changes in DNA methylation and gene expression in CD4+ and CD8+ T cells from younger and older individuals. We observed marked difference between T cell subsets, with increased number of methylation changes and higher methylome variation in CD8+ T cells with age. The majority of age-related hypermethylated sites were located at CpG islands of silent genes and enriched for repressive histone marks. Specifically, in CD8+ T cell subset we identified strong inverse correlation between methylation and expression levels in genes associated with T cell mediated immune response (LGALS1, IFNG, CCL5, GZMH, CCR7, CD27 and CD248) and differentiation (SATB1, TCF7, BCL11B and RUNX3). Our results thus suggest the link between age-related epigenetic changes and impaired T cell function.

The transcriptional landscape of age in human peripheral blood.

Disease incidences increase with age, but the molecular characteristics of ageing that lead to increased disease susceptibility remain inadequately understood. Here we perform a whole-blood gene expression meta-analysis in 14,983 individuals of European ancestry (including replication) and identify 1,497 genes that are differentially expressed with chronological age. The age-associated genes do not harbor more age-associated CpG-methylation sites than other genes, but are instead enriched for the presence of potentially functional CpG-methylation sites in enhancer and insulator regions that associate with both chronological age and gene expression levels. We further used the gene expression profiles to calculate the 'transcriptomic age' of an individual, and show that differences between transcriptomic age and chronological age are associated with biological features linked to ageing, such as blood pressure, cholesterol levels, fasting glucose, and body mass index. The transcriptomic prediction model adds biological relevance and complements existing epigenetic prediction models, and can be used by others to calculate transcriptomic age in external cohorts.

Cytomegalovirus (CMV)-dependent and -independent changes in the aging of the human immune system: a transcriptomic analysis.

Aging is associated with a profound reduction of the immune capacity (i.e., immunosenescence), which is manifested as increased morbidity and mortality due to infectious diseases in the elderly. The association of cytomegalovirus (CMV) with several aging-associated phenomena has been extensively characterized, e.g., the accumulation of CD8+ nonproliferative, apoptosis-resistant memory cells that have lost the expression of the costimulatory molecule CD28. However, as the CMV seroprevalence is notably high in elderly individuals, the role of CMV-independent changes has been difficult to analyze. To address this question, we performed a transcriptomic analysis (Illumina Human HT12 microarray) of the peripheral blood mononuclear cells (PBMCs) in a cohort of 90-year-old individuals (CMV seronegative, n=6; CMV seropositive, n=140) using the PBMCs of young CMV-seronegative individuals (n=11) as the controls. The cell type distribution (CD3, CD4, CD8, CD28 and CD14) was analyzed using FACS. The data showed that the gene expression profiles of the CMV+ and CMV- nonagenarians were different compared to the CMV- controls. Compared to the CMV- controls, 667 genes showed altered expression in the CMV- nonagenarians, and 559 genes were altered in the CMV+ nonagenarians. Of these, 337 genes were common. An analysis of the canonical pathways revealed that the number of affected pathways was also different (42 in CMV-, 13 in CMV+; of these, 9 were common). Taken together, these results indicate that the CMV-dependent and CMV-independent changes in the aging of the immune system are fundamentally different.