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DNA methylation (DNAm) has pervasive effects on gene expression and associations with ageing-related traits. Here we describe monocyte DNAm responses to inflammatory stimuli across 192 individuals. We find that, unlike the similarly widespread changes in gene expression elicited by LPS and IFNγ, DNAm is markedly more sensitive to LPS. Exposure to LPS caused differential methylation at 20,858 immune-modulated CpGs (imCpGs) which display distinct genomic localisation and transcription factor usage, dependent upon whether methylation is lost or gained. Demethylated imCpGs are profoundly enriched for enhancers, and are over-represented by genes implicated in human diseases, most notably cancer. We find LPS-induced demethylation follows hydroxymethylation and for most sites the degree of demethylation correlates with baseline signal. Notably, we find LPS exposure triggers gain in epigenetic age by approximately 6 months, identifying a potential cause of accelerated epigentic aging which has diverse negative health associations. Finally, we explore the effect of genetic variation on LPS-induced changes in DNAm, identifying 209 imCpGs under genetic control. Exploring shared causal loci between LPS-induced DNAm responses and human disease traits highlights examples of human disease associated loci that also modulate imCpG formation.In summary, our findings suggest innate immune activity continually remodels DNAm in a highly punctate, enhancerenriched fashion that is under tight genetic control and predominantly involves genes commonly mutated in cancer.

Immune checkpoint blockade (ICB) has markedly improved outcomes across a range of tumours, including metastatic melanoma (MM). However, peripheral biomarkers of response remain lacking and underlying mechanisms of action incompletely described. A number of studies have demonstrated the value of T cell receptor (TCR) repertoire analysis in determining associations with response, however identifying key groups of T cells based on their TCR usage has remained elusive. Here we performed deep sequencing of the TCR of CD8+T cells isolated from peripheral blood of patients receiving ICB for MM (n=91) at multiple time points, along with healthy control samples (n=42) and resected tumour specimens (from n=7 patients). Using the GLIPH2 algorithm to cluster TCR based on putative shared antigen specificity, we describe groups of TCR which expand post-ICB in responding patients which we term ‘Emergent Responder’ (ER) clones. We find that these ER clones are typically large and of a memory phenotype, with increased expression of genes encoding cytotoxic proteins. Analysis of tumours resected in advance of ICB demonstrates ER clones are enriched and expanded within the tumour compared to the periphery at pre-treatment. Significantly, we note the proportion of the peripheral repertoire occupied by ER clones strongly correlates with long-term clinical response. Clinical outcome further associated with HLA type and, crucially, can be validated across replication and independent datasets. This work provides the first-in-kind description of TCR-defined CD8+T cells that mediate the response to ICB in MM, demonstrating the prognostic utility of the peripheral immune repertoire with potential widespread therapeutic and prognostic applications.

Background Immune checkpoint blockers (ICBs) activate CD8+ T cells, eliciting both anti-cancer activity and immune-related adverse events (irAEs). The relationship of irAEs with baseline parameters and clinical outcome is unclear. Methods Retrospective evaluation of irAEs on survival was performed across primary (N = 144) and secondary (N = 211) independent cohorts of patients with metastatic melanoma receiving single agent (pembrolizumab/nivolumab—sICB) or combination (nivolumab and ipilimumab—cICB) checkpoint blockade. RNA from pre-treatment and post-treatment CD8+ T cells was sequenced and differential gene expression according to irAE development assessed. Results 58.3% of patients developed early irAEs and this was associated with longer progression-free (PFS) and overall survival (OS) across both cohorts (log-rank test, OS: P P = 2.8 × 10−6). Pre-treatment monocyte and neutrophil counts, but not BMI, were additional predictors of clinical outcome. Differential expression of numerous gene pathway members was observed in CD8+ T cells according to irAE development, and patients not developing irAEs demonstrating upregulated CXCR1 pre- and post-treatment. Conclusions Early irAE development post-ICB is associated with favourable survival in MM. Development of irAEs is coupled to expression of numerous gene pathways, suggesting irAE development in-part reflects baseline immune activation.

Contains fulltext : 286832.pdf (Publisher’s version ) (Open Access) BACKGROUND: Direct evaluation of vascular inflammation in patients with COVID-19 would facilitate more efficient trials of new treatments and identify patients at risk of long-term complications who might respond to treatment. We aimed to develop a novel artificial intelligence (AI)-assisted image analysis platform that quantifies cytokine-driven vascular inflammation from routine CT angiograms, and sought to validate its prognostic value in COVID-19. METHODS: For this prospective outcomes validation study, we developed a radiotranscriptomic platform that uses RNA sequencing data from human internal mammary artery biopsies to develop novel radiomic signatures of vascular inflammation from CT angiography images. We then used this platform to train a radiotranscriptomic signature (C19-RS), derived from the perivascular space around the aorta and the internal mammary artery, to best describe cytokine-driven vascular inflammation. The prognostic value of C19-RS was validated externally in 435 patients (331 from study arm 3 and 104 from study arm 4) admitted to hospital with or without COVID-19, undergoing clinically indicated pulmonary CT angiography, in three UK National Health Service (NHS) trusts (Oxford, Leicester, and Bath). We evaluated the diagnostic and prognostic value of C19-RS for death in hospital due to COVID-19, did sensitivity analyses based on dexamethasone treatment, and investigated the correlation of C19-RS with systemic transcriptomic changes. FINDINGS: Patients with COVID-19 had higher C19-RS than those without (adjusted odds ratio [OR] 2·97 [95% CI 1·43-6·27], p=0·0038), and those infected with the B.1.1.7 (alpha) SARS-CoV-2 variant had higher C19-RS values than those infected with the wild-type SARS-CoV-2 variant (adjusted OR 1·89 [95% CI 1·17-3·20] per SD, p=0·012). C19-RS had prognostic value for in-hospital mortality in COVID-19 in two testing cohorts (high [≥6·99] vs low [

Although treatment with immune checkpoint blockade (ICB) has transformed outcomes for patients with melanoma (1, 2), it frequently triggers immune related adverse events (irAEs), causing serious morbidity and presenting a major hurdle to immuno-oncology (3, 4). The degree to which germline genetic variation predisposes to irAEs is unknown. Here, studying a cohort of 214 patients receiving ICB for melanoma, we observe increased risk of severe irAEs in carriers of the minor allele of rs16906115, intronic to IL7. We find rs16906115 forms a B cell specific expression quantitative trait locus to IL7 in patients only, with risk allele carriers demonstrating increased B cell IL7 expression, immunoglobulin class switching and somatic hypermutation. Notably, increased B cell IL7 expression is independently associated with risk of irAEs. Consistent with the role of IL-7 in T cell development, risk allele carriers have distinct ICB induced CD8+ T cell subset responses, with allelic effects on gene expression and clonality. Risk allele carriers display significantly reduced CD8+ T cell mitotic responses to ICB, with skewing of T cell clonality and increased counts of large clones previously associated with disease response. These observations highlight key roles for both B cells and IL-7 in ICB response and toxicity, and demonstrate the power of agnostic human genetic studies to reveal novel determinants of response to cancer immunotherapy.

Treatment with immune checkpoint blockade (ICB) frequently triggers immune-related adverse events (irAEs), causing considerable morbidity. In 214 patients receiving ICB for melanoma, we observed increased severe irAE risk in minor allele carriers of rs16906115, intronic to IL7. We found that rs16906115 forms a B cell-specific expression quantitative trait locus (eQTL) to IL7 in patients. Patients carrying the risk allele demonstrate increased pre-treatment B cell IL7 expression, which independently associates with irAE risk, divergent immunoglobulin expression and more B cell receptor mutations. Consistent with the role of IL-7 in T cell development, risk allele carriers have distinct ICB-induced CD8+ T cell subset responses, skewing of T cell clonality and greater proportional repertoire occupancy by large clones. Finally, analysis of TCGA data suggests that risk allele carriers independently have improved melanoma survival. These observations highlight key roles for B cells and IL-7 in both ICB response and toxicity and clinical outcomes in melanoma.

The antitumor action of immune checkpoint blockade (ICB) is primarily mediated by CD8(+) T cells. How sensitivity to ICB varies across CD8(+) T cell subsets and clonotypes and the relationship of these with clinical outcome is unclear. To explore this, we used single-cell V(D)J and RNA-sequencing to track gene expression changes elicited by ICB across individual peripheral CD8(+) T cell clones, identify baseline markers of CD8(+) T cell clonal sensitivity, and chart how CD8(+) T cell transcriptional changes vary according to phenotypic subset and clonal size. We identified seven subsets of CD8(+) T cells with divergent reactivity to ICB and found that the cytotoxic effector subset showed the greatest number of differentially expressed genes while remaining stable in clonal size after ICB. At the level of CD8(+) T cell clonotypes, we found a relationship between transcriptional changes and clone size, with large clones showing a greater number of differentially regulated genes enriched for pathways including T cell receptor (TCR) signaling. Cytotoxic CD8(+) effector clones were more likely to persist following ICB and were more likely to correspond with public tumor-infiltrating lymphocyte clonotypes. Last, we demonstrated that individuals whose CD8(+) T cell pretreatment showed low cytotoxicity and had fewer expanded clones typically had worse outcomes after ICB treatment. This work further advances understanding of the molecular determinants of ICB response, assisting in the search for peripheral prognostic biomarkers and highlighting the importance of the baseline CD8(+) immune landscape in determining ICB response in metastatic melanoma.

Immune checkpoint blockade (ICB) of PD-1 and CTLA-4 to treat metastatic melanoma (MM) has variable therapeutic benefit. To explore this in peripheral samples, we characterized CD8+ T cell gene expression across a cohort of patients with MM receiving anti-PD-1 alone (sICB) or in combination with anti-CTLA-4 (cICB). Whereas CD8+ transcriptional responses to sICB and cICB involve a shared gene set, the magnitude of cICB response is over fourfold greater, with preferential induction of mitosis- and interferon-related genes. Early samples from patients with durable clinical benefit demonstrated overexpression of T cell receptor–encoding genes. By mapping T cell receptor clonality, we find that responding patients have more large clones (those occupying >0.5% of repertoire) post-treatment than non-responding patients or controls, and this correlates with effector memory T cell percentage. Single-cell RNA-sequencing of eight post-treatment samples demonstrates that large clones overexpress genes implicated in cytotoxicity and characteristic of effector memory T cells, including CCL4, GNLY and NKG7. The 6-month clinical response to ICB in patients with MM is associated with the large CD8+ T cell clone count 21 d after treatment and agnostic to clonal specificity, suggesting that post-ICB peripheral CD8+ clonality can provide information regarding long-term treatment response and, potentially, facilitate treatment stratification. Transcriptomic analysis of peripheral CD8+ T cells in a cohort of patients with metastatic melanoma receiving checkpoint inhibitors shows that the number of large clones early post-treatment is strongly associated with six-month clinical outcome.

IL-7 is a key factor in T cell immunity and common variants at IL7R, encoding its receptor, are associated with autoimmune disease susceptibility. IL7R mRNA is induced in stimulated monocytes, yet a function for IL7R in monocyte biology remains unexplored. Here we characterize genetic regulation of IL7R at the protein level in healthy individuals, and find that monocyte surface and soluble IL7R (sIL7R) are markedly induced by lipopolysaccharide. In monocytes, both surface IL7R and sIL7R expression strongly associate with allelic carriage of rs6897932, a disease-associated IL7R polymorphism. Monocytes produce more sIL7R than CD4 + T cells, and the amount is additionally correlated with the expression of DDX39A, encoding a splicing factor. Synovial fluid-derived monocytes from patients with spondyloarthritis are enriched for IL7R+ cells with a unique transcriptional profile that overlaps with IL-7-induced gene sets. Our data thus suggest a previously unappreciated function for monocytes in IL-7 biology and IL7R-associated diseases., Interleukin-7 (IL-7) is a central cytokine in T cell homeostasis. Here the authors show that allelic variation at rs6897932, an autoimmune GWAS risk allele at IL7R, regulates surface and soluble IL-7R in stimulated monocytes, indicating a function of monocytes in IL-7-related autoimmunity.

NP105-113-B*07:02 specific CD8+ T-cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02 specific T-cell clones and single cell sequencing were performed concurrently, with functional avidity and anti-viral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with TCR usage, transcriptome signature, and disease severity (acute N=77, convalescent N=52). We demonstrated a beneficial association of NP105-113-B*07:02 specific T-cells in COVID-19 disease progression, linked with expansion of T-cell precursors, high functional avidity and anti-viral effector function. Broad immune memory pools were narrowed post-infection but NP105-113-B*07:02 specific T-cells were maintained 6 months after infection with preserved anti-viral efficacy to the SARS-CoV-2 Victoria strain, as well as new Alpha, Beta and Gamma variants. Our data shows that NP105-113-B*07:02 specific T-cell responses associate with mild disease and high anti-viral efficacy, pointing to inclusion for future vaccine design.

NP

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design and personalized medicine approaches for COVID-19., Graphical Abstract, A multi-omic analysis of patient blood samples reveals both similarities and specific features of COVID-19 when compared with samples obtained from sepsis or influenza patients which could yield better targetted therapies for severe COVID-19.

NP105–113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105–113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105–113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105–113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105–113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design. Peng et al. find that immunodominant cytotoxic T lymphocytes (CTLs) specific for NP105–113-B*07:02 are associated with reduced COVID-19 severity. Mechanistically, NP105–113-B*07:02-specific CTLs show potent antiviral functionality and may represent rational T cell vaccine targets.

The pathogenesis of immune checkpoint inhibitor (ICI)-colitis remains incompletely understood. We sought to identify key cellular drivers of ICI-colitis and their similarities to idiopathic ulcerative colitis, and to determine potential novel therapeutic targets.We used a cross-sectional approach to study patients with ICI-colitis, those receiving ICI without the development of colitis, idiopathic ulcerative colitis, and healthy controls. A subset of patients with ICI-colitis were studied longitudinally. We applied a range of methods, including multiparameter and spectral flow cytometry, spectral immunofluorescence microscopy, targeted gene panels, and bulk and single-cell RNA sequencing.We demonstrate CD8Interferon gamma-producing CD8

Immune checkpoint blockers (ICB) exert their anti-cancer effects via CD8+ T cells, although how responses vary over sub-populations and across clones is incompletely understood. We performed single-cell RNA-sequencing of CD8+ T cells and their receptors pre- and post-ICB across eight patients, integrating results with bulk-sequencing data (n=209). We identify seven subsets with divergent responses to ICB, finding the effector cluster demonstrates the most pronounced changes. Likewise, transcriptomic response to ICB relates to clone size, with large clones demonstrating increased numbers of regulated genes of higher immunological pertinence. Cytotoxic effector clones were more likely to persist long-term following ICB and overlapped with public tumour-infiltrating lymphocyte clonotypes. Notably, pre-treatment CD8+ cytotoxicity associated with progression-free survival, highlighting the importance of the baseline CD8+ immune landscape in long-term response. This work further advances understanding of the molecular determinants of ICB response and assists in the search for peripheral prognostic biomarkers.ONE SENTENCE SUMMARYUsing single-cell and bulk RNA sequencing we explore checkpoint immunotherapy activity on peripheral CD8+ T cells in metastatic melanoma; demonstrating that cell subset and clone size determine gene expression responses to treatment, and that pre-treatment cytotoxicity and clonality of peripheral CD8+ T cells is clinically prognostic.

1Abstract1.1BackgroundImmune checkpoint blockers (ICBs) activate CD8+ T cells to elicit anti-cancer activity but frequently lead to immune-related adverse events (irAEs). The relationship of irAE with baseline parameters and clinical outcome is unclear. We investigated associations between irAE development, CD8+ T cell receptor diversity and expression and clinical outcome in a non-trial setting.1.2MethodsPatients ≥18 years old with metastatic melanoma (MM) receiving combination ICB (ipilimumab plus nivolumab – cICB, n=60) or single-agent ICB (nivolumab/pembrolizumab – sICB, n=78) were prospectively recruited. We retrospectively evaluated the impact of irAEs on survival. This analysis was repeated in an independent cohort of MM patients treated at a separate institution (n=210, cICB:74, sICB:136). We performed RNA sequencing of CD8+ T cells isolated from patients prior to treatment, analysing T cell receptor clonality differential transcript expression according to irAE development.1.3Results48.6% of patients experienced treatment-related irAEs within the first 5 cycles of treatment. Development of irAE prior to the 5th cycle of ICB was associated with longer progression-free and overall survival (PFS, OS) in the primary cohort (log-rank test, PFS: P=0.00034; OS: PP=0.00064). Across cohorts median survival for those patients not experiencing irAE was 14.4 (95% CI:9.6-19.5) months vs not-reached (95% CI:28.9 - Inf), P=3.0×10−7. Pre-treatment performance status and neutrophil count, but not BMI, were additional predictors of clinical outcome. Analysis of CD8+ T cells from 128 patients demonstrated irAE development was associated with increased T cell receptor diversity post-treatment (P=4.3×10−5). Development of irAE in sICB recipients was additionally associated with baseline differential expression of 224 transcripts (FDRCYP4F3 and PTGS2.1.4ConclusionsEarly irAE development post-ICB is strongly associated with favourable survival in MM and increased diversity of peripheral CD8+ T cell receptors after treatment. irAE post-sICB is associated with pre-treatment upregulation of inflammatory pathways, indicating irAE development may reflect baseline immune activation states.Key messageImmune-related adverse events (irAEs) commonly occur in patients with metastatic melanoma treated with immune checkpoint blockade (ICB) therapy. In real world setting we find development of early irAEs post-ICB treatment is associated with survival benefit, indicative of a shared mechanism with anti-tumour efficacy. CD8+ T cells from patients who develop irAE show increased receptor diversity, and pre-treatment samples from patients who develop irAE post single-agent anti-PD1 show over-expression of inflammatory pathways, indicating baseline immune state can determine irAE development.

Background & Aims The pathogenesis of immune checkpoint inhibitor (ICI)–colitis remains incompletely understood. We sought to identify key cellular drivers of ICI-colitis and their similarities to idiopathic ulcerative colitis, and to determine potential novel therapeutic targets. Methods We used a cross-sectional approach to study patients with ICI-colitis, those receiving ICI without the development of colitis, idiopathic ulcerative colitis, and healthy controls. A subset of patients with ICI-colitis were studied longitudinally. We applied a range of methods, including multiparameter and spectral flow cytometry, spectral immunofluorescence microscopy, targeted gene panels, and bulk and single-cell RNA sequencing. Results We demonstrate CD8+ tissue resident memory T (TRM) cells are the dominant activated T cell subset in ICI-colitis. The pattern of gastrointestinal immunopathology is distinct from ulcerative colitis at both the immune and epithelial-signaling levels. CD8+ TRM cell activation correlates with clinical and endoscopic ICI-colitis severity. Single-cell RNA sequencing analysis confirms activated CD8+ TRM cells express high levels of transcripts for checkpoint inhibitors and interferon-gamma in ICI-colitis. We demonstrate similar findings in both anti–CTLA-4/PD-1 combination therapy and in anti–PD-1 inhibitor-associated colitis. On the basis of our data, we successfully targeted this pathway in a patient with refractory ICI-colitis, using the JAK inhibitor tofacitinib. Conclusions Interferon gamma–producing CD8+ TRM cells are a pathological hallmark of ICI-colitis and a novel target for therapy.

We present a new model of ESR1 network regulation based on analysis of Doxorubicin, Estradiol, and TNFα combination treatment in MCF-7. We used Doxorubicin as a therapeutic agent, TNFα as marker and mediator of an inflammatory microenvironment and 17β-Estradiol (E2) as an agonist of Estrogen Receptors, known predisposing factor for hormone-driven breast cancer, whose pharmacological inhibition reduces the risk of breast cancer recurrence. Based on the results of transcriptomics analysis, we found 71 differentially expressed genes that are specific for the combination treatment with Doxorubicin + Estradiol + TNFα in comparison with single or double treatments. The responsiveness to the triple treatment was examined for seven genes by qPCR, of which six were validated, and then extended to four additional cell lines differing for p53 and/or ER status. The results of differential regulation enrichment analysis highlight the role of the ESR1 network that included 36 of 71 specific differentially expressed genes. We propose that the combined activation of p53 and NF-kB transcription factors significantly influences ligand-dependent, ER-driven transcriptional responses, also of the ESR1 gene itself. These results provide a model of coordinated interaction of TFs to explain the Doxorubicin, E2 and TNFα induced repression mechanisms.

Background Immune Checkpoint blockade for the treatment of metastatic melanoma is associated with highly variable clinical outcomes between individuals in terms of both oncological benefit and immune-related adverse events. Early markers of response are urgently sought. Whereas numerous intra-tumoural determinants of sensitivity to immunotherapy are known, the identification of peripheral predictors of response is limited. We aimed to characterize the CD8+ T-cell transcriptomic and clonal changes to treatment across a large cohort of patients in an effort to gain further insight into the peripheral markers of response. Methods We performed paired-end 75bp read RNA-sequencing to assay the peripheral CD8+ response at baseline and across multiple cycles of treatment (n = 105 patients, 69 controls, 315 separate transcriptomes). We validated identified clonal subsets indicative of response using 10X Chromium single cell sequencing (16 samples, 8 patients), flow-cytometry and targeted PCR. Results After adjusting for multiple testing, we identify >5,800 transcripts induced by treatment. These fall into discrete gene modules, with several markedly diverging between combination immunotherapy and anti-PD1 alone. Patients demonstrating a durable radiological response to checkpoint immunotherapy (absence of progression at 6 months) have significantly increased numbers of large peripheral CD8+ circulating clones by day 21 after treatment, compared to non-responders and controls. We replicate this observation in a separately recruited cohort. Large peripheral clones have a distinct gene expression profile, characterized by high expression of CCL5, BCL2L1and NKG7 amongst other genes. Conclusions We identify robust and reproducible predictors of 6 month clinical and radiological responses to immune checkpoint blockade in the transcriptomes of peripheral circulating CD8+ T-cells from metastic melanoma patients after 21 days of treatment. These observations can be used to further our understanding of determinants of patient response, and may provide a mechanism for early treatment stratification of patients with a non-favourable peripheral profile. Legal entity responsible for the study Oxford Radcliffe Biobank. Funding Wellcome Trust, Oxford NIHR Biomedical Research Centre, Cancer Research UK. Disclosure All authors have declared no conflicts of interest.

IL-7 is a key factor in T-cell immunity and IL7R polymorphisms are implicated in autoimmune pathogenesis. IL7R mRNA is induced in stimulated monocytes in a genetically determined manner, yet a role for IL7R in monocyte biology remains unexplored. Here we characterize genetic regulation of IL7R at the protein level across multiple cell subsets and conditions in healthy individuals. We find monocyte surface and soluble IL7R (sIL7R) protein are markedly expressed in response to lipopolysaccharide (LPS). We further demonstrate alleles of rs6897932, a non-synonymous IL7R polymorphism associated with susceptibility to Multiple Sclerosis, Ankylosing Spondylitis and Primary Biliary Cirrhosis, form the key determinant of both surface IL7R and sIL7R in the context of inflammation. No effect of this allele was observed in unstimulated monocytes or across lymphoid subsets. Production of sIL7R by monocytes greatly exceeded that of CD4+ T-cells, and was strongly associated with both rs6897932 genotype and expression of the splicing factor gene DDX39A. Stimulated monocytes were sensitive to exogenous IL-7, which elicits a defined transcriptional signature. Flow cytometry and single-cell sequencing of synovial fluid derived monocytes from patients with spondyloarthritis showed an enlarged subset of IL7R+ monocytes with a unique transcriptional profile that markedly overlaps that induced by IL-7 in-vitro and shows similarity to the previously described ‘Mono4’ subset. These data demonstrate disease-associated genetic variants at IL7R specifically impact monocyte surface IL7R and sIL7R following innate immune stimulation, suggesting a previously unappreciated key role for monocytes in IL-7 pathway biology and IL7R-associated diseases.

A signaling pathway is a sequence of proteins and passenger molecules that transmits information from the cell surface to target molecules. Understanding signal transduction process requires detailed description of the involved pathways. Several methods and tools resolved this problem by incorporating genomic and proteomic data. However, the difficulty of obtaining prior knowledge of complex signaling networks limited the applicability of these tools. In this study, based on the simulation of signal flow in signaling network, we introduce a method for determining dominant pathways and signal response to stimulations. The model uses topology-weighted transit compartment approach and comprises four main steps which include weighting the edges, simulating signal transduction in the network (weighting the nodes), finding paths between initial and target nodes, and assigning a significance score to each path. We applied the proposed model to eighty-three signaling networks by using biologically derived source and sink molecules. The recovered dominant paths matched many known signaling pathways and suggesting a promising index to analyze the phenotype essentiality of molecule encoding paths. We also modeled the stimulus-response relations in long and short-term synaptic plasticity based on the dominant signaling pathway concept. We showed that the proposed method not only accurately determines dominant signaling pathways, but also identifies effective points of intervention in signal transduction.

The investigation of the complex processes involved in cellular differentiation must be based on unbiased, high throughput data processing methods to identify relevant biological pathways. A number of bioinformatics tools are available that can generate lists of pathways ranked by statistical significance (i.e. by p-value), while ideally it would be desirable to functionally score the pathways relative to each other or to other interacting parts of the system or process. We describe a new computational method (Network Activity Score Finder - NASFinder) to identify tissue-specific, omics-determined sub-networks and the connections with their upstream regulator receptors to obtain a systems view of the differentiation of human adipocytes. Adipogenesis of human SBGS pre-adipocyte cells in vitro was monitored with a transcriptomic data set comprising six time points (0, 6, 48, 96, 192, 384 hours). To elucidate the mechanisms of adipogenesis, NASFinder was used to perform time-point analysis by comparing each time point against the control (0 h) and time-lapse analysis by comparing each time point with the previous one. NASFinder identified the coordinated activity of seemingly unrelated processes between each comparison, providing the first systems view of adipogenesis in culture. NASFinder has been implemented into a web-based, freely available resource associated with novel, easy to read visualization of omics data sets and network modules.

Cell morphological phenotypes, including shape, size, intensity, and texture of cellular compartments have been shown to change in response to perturbation with small molecule compounds. Image-based cell profiling or cell morphological profiling has been used to associate changes of cell morphological features with alterations in cellular function and to infer molecular mechanisms of action. Recently, the Library of Integrated Network-based Cellular Signatures (LINCS) Project has measured gene expression and performed image-based cell profiling on cell lines treated with 9515 unique compounds. These data provide an opportunity to study the interdependence between transcription and cell morphology. Previous methods to investigate cell phenotypes have focused on targeting candidate genes as components of known pathways, RNAi morphological profiling, and cataloging morphological defects; however, these methods do not provide an explicit model to link transcriptomic changes with corresponding alterations in morphology. To address this, we propose a cell morphology enrichment analysis to assess the association between transcriptomic alterations and changes in cell morphology. Additionally, for a new transcriptomic query, our approach can be used to predict associated changes in cellular morphology. We demonstrate the utility of our method by applying it to cell morphological changes in a human bone osteosarcoma cell line.

Mutability varies significantly along nucleotide sequences, and mutations occur at higher frequencies at certain positions of a genome. The high rate of mutation in some regions is independent from the function and structure of corresponding regions in protein products. This raises the possibility of DNA sequence and cis-elements as causes of mutations. We used computational methods to examine the surrounding region of 20 mutation hotspots related to different human genetic disorders, and combinatorial patterns of gene mutations in cancers. We introduced A(C/G)AA(C/G)(A/T) as an associated sequence motif in fourteen hotspot of mutations in different cancers. These observations are support the correlation between DNA motifs with hotspots of mutation in cancer and promising marker for selection of suitable regions for genes mutation screening.

Cancer is a complex process in which the abnormalities of many genes appear to be involved. The synchronized patterns of gene mutations may reveal the functional relations between genes and pathways in tumor-genesis as well as identify targets for treatment. Co-occurrence database represents a comprehensive core collection of data on published coincidental mutations in nuclear genes underlying human cancers. By August 2013, the database contained 126 different coincidental lesions in 43 different genes in 25 different types of human cancers, and 8 cellular signaling pathways. In next step, this model for knowledge representation about coincidental mutations in human cancers can be extended to other synchronized patterns of genes alterations in human diseases. Although, co-occurrence database originally established for the scientific study of coincidental mutational mechanisms in human cancer genes, it is also applicable for physicians and genetic counselors. The database is freely available from http://lbb.ut.ac.ir/project/Co-Occurrence/home.php

Simulating signal transduction in cellular signaling networks provides predictions of network dynamics by quantifying the changes in concentration and activity-level of the individual proteins. Since numerical values of kinetic parameters might be difficult to obtain, it is imperative to develop non-parametric approaches that combine the connectivity of a network with the response of individual proteins to signals which travel through the network. The activity levels of signaling proteins computed through existing non-parametric modeling tools do not show significant correlations with the observed values in experimental results. In this work we developed a non-parametric computational framework to describe the profile of the evolving process and the time course of the proportion of active form of molecules in the signal transduction networks. The model is also capable of incorporating perturbations. The model was validated on four signaling networks showing that it can effectively uncover the activity levels and trends of response during signal transduction process.

Distinguish harmful missense nucleotide substitutions from single nucleotide polymorphisms is a difficult problem usually to be accomplished through functional in vivo analyses. In this study, instead current biochemical methods, the effects of missense mutations upon protein structure and function were assayed by means of computational methods and information from the databases. For this order, the effects of new missense mutations in exon 5 of PTEN gene upon protein structure and function were examined. The gene coding for PTEN was identified and localized on chromosome region 10q23.3 as the tumor suppressor gene. The utilization of these methods were shown that c.319G>A and c.341T>G missense mutations that were recognized in patients with breast cancer and Cowden disease, could be pathogenic. This method could be use for analysis of missense mutation in others genes.

The single-strand conformation polymorphism (SSCP), accompanied by sequencing, is a useful methods for identifying mutations in a DNA fragment. In this study, we have developed a modified SSCP with the aid of sodium bisulfite treatment. The corresponding PCR products for exon 3 of Hb gene were sequenced and samples with homozygote and heterozygote single nucleotide substitutions were identified. The PCR products were treated with sodium bisulfite, which deaminates all the cytosine residues. The reaction mixture was then analyzed on non-denaturing polyacrylamide gels. The modified method, which is called deaminated SSCP (DSSCP), was applied successfully in analysis of mutations in the beta-globin gene at positions relevant to codon 6. DSSCP is a very effective and reproducible method providing clear results that are easy to interpret without the involvement of radioactivity.

p16 INK4A is a tumor suppressor encoding the Cdk inhibitor protein, which acts to repress Cdk4/6 and pRb phosphorylation. p16 INK4A gene can be inactivated by a variety of events, including promoter hypermethylation. To investigate the methylation status of the p16 INK4A gene in Iranian patients with breast carcinoma, promoter methylation was studied by methylation-specific PCR (MSP) and restriction enzyme-related PCR (REP). In addition, p16 INK4A promoter was analyzed by PCR-SSCP in order to detection of mutation and single nucleotide polymorphisms. Analysis of 70 patients by MPS and REP showed hypermethylation of p16 INK4A promoter in 35.7% (25/70) and 40% (28/70) of samples, respectively. Comparison of the molecular data and pathological information of the samples suggested that p16 INK4A gene might be inactivated at the early stages in breast cancer. Therefore, it could be suggested that hypermethylation of p16 INK4A promoter is one of the epigenetic factors affecting the progress of sporadic breast carcinogenesis in Iranian patients.