Demultiplexing of single-cell RNA-sequencing data using interindividual variation in gene expression.

Motivation: Pooled designs for single-cell RNA sequencing, where many cells from distinct samples are processed jointly, offer increased throughput and reduced batch variation. This study describes expression-aware demultiplexing (EAD), a computational method that employs differential co-expression patterns between individuals to demultiplex pooled samples without any extra experimental steps. Results: We use synthetic sample pools and show that the top interindividual differentially co-expressed genes provide a distinct cluster of cells per individual, significantly enriching the regulation of metabolism. Our application of EAD to samples of six isogenic inbred mice demonstrated that controlling genetic and environmental effects can solve interindividual variations related to metabolic pathways. We utilized 30 samples from both sepsis and healthy individuals in six batches to assess the performance of classification approaches. The results indicate that combining genetic and EAD results can enhance the accuracy of assignments (Min. 0.94, Mean 0.98, Max. 1). The results were enhanced by an average of 1.4% when EAD and barcoding techniques were combined (Min. 1.25%, Median 1.33%, Max. 1.74%). Furthermore, we demonstrate that interindividual differential co-expression analysis within the same cell type can be used to identify cells from the same donor in different activation states. By analysing single-nuclei transcriptome profiles from the brain, we demonstrate that our method can be applied to nonimmune cells. Availability and implementation: EAD workflow is available at https://isarnassiri.github.io/scDIV/ as an R package called scDIV (acronym for single-cell RNA-sequencing data demultiplexing using interindividual variations).

An isoform quantitative trait locus in SBNO2 links genetic susceptibility to Crohn's disease with defective antimicrobial activity.

Despite major advances in linking single genetic variants to single causal genes, the significance of genetic variation on transcript-level regulation of expression, transcript-specific functions, and relevance to human disease has been poorly investigated. Strawberry notch homolog 2 (SBNO2) is a candidate gene in a susceptibility locus with different variants associated with Crohn's disease and bone mineral density. The SBNO2 locus is also differentially methylated in Crohn's disease but the functional mechanisms are unknown. Here we show that the isoforms of SBNO2 are differentially regulated by lipopolysaccharide and IL-10. We identify Crohn's disease associated isoform quantitative trait loci that negatively regulate the expression of the noncanonical isoform 2 corresponding with the methylation signals at the isoform 2 promoter in IBD and CD. The two isoforms of SBNO2 drive differential gene networks with isoform 2 dominantly impacting antimicrobial activity in macrophages. Our data highlight the role of isoform quantitative trait loci to understand disease susceptibility and resolve underlying mechanisms of disease.

Severe acute myositis and myocarditis on initiation of 6-weekly pembrolizumab post-COVID-19 mRNA vaccination.

We describe three cases of critical acute myositis with myocarditis occurring within 22 days of each other at a single institution, all within 1 month of receiving the initial cycle of the anti-PD-1 drug pembrolizumab. Analysis of T cell receptor repertoires from peripheral blood and tissues revealed a high degree of clonal expansion and public clones between cases, with several T cell clones expanded within the skeletal muscle putatively recognizing viral epitopes. All patients had recently received a COVID-19 mRNA booster vaccine prior to treatment and were positive for SARS-CoV2 Spike antibody. In conclusion, we report a series of unusually severe myositis and myocarditis following PD-1 blockade and the COVID-19 mRNA vaccination.

Characterization of the genetic determinants of context-specific DNA methylation in primary monocytes.

To better understand inter-individual variation in sensitivity of DNA methylation (DNAm) to immune activity, we characterized effects of inflammatory stimuli on primary monocyte DNAm (n = 190). We find that monocyte DNAm is site-dependently sensitive to lipopolysaccharide (LPS), with LPS-induced demethylation occurring following hydroxymethylation. We identify 7,359 high-confidence immune-modulated CpGs (imCpGs) that differ in genomic localization and transcription factor usage according to whether they represent a gain or loss in DNAm. Demethylated imCpGs are profoundly enriched for enhancers and colocalize to genes enriched for disease associations, especially cancer. DNAm is age associated, and we find that 24-h LPS exposure triggers approximately 6 months of gain in epigenetic age, directly linking epigenetic aging with innate immune activity. By integrating LPS-induced changes in DNAm with genetic variation, we identify 234 imCpGs under local genetic control. Exploring shared causal loci between LPS-induced DNAm responses and human disease traits highlights examples of disease-associated loci that modulate imCpG formation.

A common NFKB1 variant detected through antibody analysis in UK Biobank predicts risk of infection and allergy.

Infectious agents contribute significantly to the global burden of diseases through both acute infection and their chronic sequelae. We leveraged the UK Biobank to identify genetic loci that influence humoral immune response to multiple infections. From 45 genome-wide association studies in 9,611 participants from UK Biobank, we identified NFKB1 as a locus associated with quantitative antibody responses to multiple pathogens, including those from the herpes, retro-, and polyoma-virus families. An insertion-deletion variant thought to affect NFKB1 expression (rs28362491), was mapped as the likely causal variant and could play a key role in regulation of the immune response. Using 121 infection- and inflammation-related traits in 487,297 UK Biobank participants, we show that the deletion allele was associated with an increased risk of infection from diverse pathogens but had a protective effect against allergic disease. We propose that altered expression of NFKB1, as a result of the deletion, modulates hematopoietic pathways and likely impacts cell survival, antibody production, and inflammation. Taken together, we show that disruptions to the tightly regulated immune processes may tip the balance between exacerbated immune responses and allergy, or increased risk of infection and impaired resolution of inflammation.

A role for SETD2 loss in tumorigenesis through DNA methylation dysregulation.

SETD2-dependent H3 Lysine-36 trimethylation (H3K36me3) has been recently linked to the deposition of de-novo DNA methylation. SETD2 is frequently mutated in cancer, however, the functional impact of SETD2 loss and depletion on DNA methylation across cancer types and tumorigenesis is currently unknown. Here, we perform a pan-cancer analysis and show that both SETD2 mutation and reduced expression are associated with DNA methylation dysregulation across 21 out of the 24 cancer types tested. In renal cancer, these DNA methylation changes are associated with altered gene expression of oncogenes, tumour suppressors, and genes involved in neoplasm invasiveness, including TP53, FOXO1, and CDK4. This suggests a new role for SETD2 loss in tumorigenesis and cancer aggressiveness through DNA methylation dysregulation. Moreover, using a robust machine learning methodology, we develop and validate a 3-CpG methylation signature which is sufficient to predict SETD2 mutation status with high accuracy and correlates with patient prognosis.

scQCEA: a framework for annotation and quality control report of single-cell RNA-sequencing data.

BACKGROUND: Systematic description of library quality and sequencing performance of single-cell RNA sequencing (scRNA-seq) data is imperative for subsequent downstream modules, including re-pooling libraries. While several packages have been developed to visualise quality control (QC) metrics for scRNA-seq data, they do not include expression-based QC to discriminate between true variation and background noise. RESULTS: We present scQCEA (acronym of the single-cell RNA sequencing Quality Control and Enrichment Analysis), an R package to generate reports of process optimisation metrics for comparing sets of samples and visual evaluation of quality scores. scQCEA can import data from 10X or other single-cell platforms and includes functions for generating an interactive report of QC metrics for multi-omics data. In addition, scQCEA provides automated cell type annotation on scRNA-seq data using differential gene expression patterns for expression-based quality control. We provide a repository of reference gene sets, including 2348 marker genes, which are exclusively expressed in 95 human and mouse cell types. Using scRNA-seq data from 56 gene expressions and V(D)J T cell replicates, we show how scQCEA can be applied for the visual evaluation of quality scores for sets of samples. In addition, we use the summary of QC measures from 342 human and mouse shallow-sequenced gene expression profiles to specify optimal sequencing requirements to run a cell-type enrichment analysis function. CONCLUSIONS: The open-source R tool will allow examining biases and outliers over biological and technical measures, and objective selection of optimal cluster numbers before downstream analysis. scQCEA is available at https://isarnassiri.github.io/scQCEA/ as an R package. Full documentation, including an example, is provided on the package website.

IFNγ signaling in cytotoxic T cells restricts anti-tumor responses by inhibiting the maintenance and diversity of intra-tumoral stem-like T cells.

IFNγ is an immune mediator with concomitant pro- and anti-tumor functions. Here, we provide evidence that IFNγ directly acts on intra-tumoral CD8 T cells to restrict anti-tumor responses. We report that expression of the IFNγ receptor ß chain (IFNγR2) in CD8 T cells negatively correlates with clinical responsiveness to checkpoint blockade in metastatic melanoma patients, suggesting that the loss of sensitivity to IFNγ contributes to successful antitumor immunity. Indeed, specific deletion of IFNγR in CD8 T cells promotes tumor control in a mouse model of melanoma. Chronic IFNγ inhibits the maintenance, clonal diversity and proliferation of stem-like T cells. This leads to decreased generation of T cells with intermediate expression of exhaustion markers, previously associated with beneficial anti-tumor responses. This study provides evidence of a negative feedback loop whereby IFNγ depletes stem-like T cells to restrict anti-tumor immunity. Targeting this pathway might represent an alternative strategy to enhance T cell-based therapies.

IL7 genetic variation and toxicity to immune checkpoint blockade in patients with melanoma.

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.

Germline variants associated with toxicity to immune checkpoint blockade.

Immune checkpoint inhibitors (ICIs) have yielded remarkable responses but often lead to immune-related adverse events (irAEs). Although germline causes for irAEs have been hypothesized, no individual variant associated with developing irAEs has been identified. We carried out a genome-wide association study of 1,751 patients on ICIs across 12 cancer types. We investigated two irAE phenotypes: (1) high-grade (3-5) and (2) all-grade events. We identified 3 genome-wide significant associations (P < 5 × 10-8) in the discovery cohort associated with all-grade irAEs: rs16906115 near IL7 (combined P = 3.6 × 10-11; hazard ratio (HR) = 2.1); rs75824728 near IL22RA1 (combined P = 3.5 × 10-8; HR = 1.8); and rs113861051 on 4p15 (combined P = 1.2 × 10-8, HR = 2.0); rs16906115 was replicated in 3 independent studies. The association near IL7 colocalized with the gain of a new cryptic exon for IL7, a critical regulator of lymphocyte homeostasis. Patients carrying the IL7 germline variant exhibited significantly increased lymphocyte stability after ICI initiation, which was itself predictive of downstream irAEs and improved survival.

Genome-wide association study of leprosy in Malawi and Mali.

Leprosy is a chronic infection of the skin and peripheral nerves caused by Mycobacterium leprae. Despite recent improvements in disease control, leprosy remains an important cause of infectious disability globally. Large-scale genetic association studies in Chinese, Vietnamese and Indian populations have identified over 30 susceptibility loci for leprosy. There is a significant burden of leprosy in Africa, however it is uncertain whether the findings of published genetic association studies are generalizable to African populations. To address this, we conducted a genome-wide association study (GWAS) of leprosy in Malawian (327 cases, 436 controls) and Malian (247 cases, 368 controls) individuals. In that analysis, we replicated four risk loci previously reported in China, Vietnam and India; MHC Class I and II, LACC1 and SLC29A3. We further identified a novel leprosy susceptibility locus at 10q24 (rs2015583; combined p = 8.81 × 10-9; OR = 0.51 [95% CI 0.40 - 0.64]). Using publicly-available data we characterise regulatory activity at this locus, identifying ACTR1A as a candidate mediator of leprosy risk. This locus shows evidence of recent positive selection and demonstrates pleiotropy with established risk loci for inflammatory bowel disease and childhood-onset asthma. A shared genetic architecture for leprosy and inflammatory bowel disease has been previously described. We expand on this, strengthening the hypothesis that selection pressure driven by leprosy has shaped the evolution of autoimmune and atopic disease in modern populations. More broadly, our data highlights the importance of defining the genetic architecture of disease across genetically diverse populations, and that disease insights derived from GWAS in one population may not translate to all affected populations.

Natural Killer cells demonstrate distinct eQTL and transcriptome-wide disease associations, highlighting their role in autoimmunity.

Natural Killer cells are innate lymphocytes with central roles in immunosurveillance and are implicated in autoimmune pathogenesis. The degree to which regulatory variants affect Natural Killer cell gene expression is poorly understood. Here we perform expression quantitative trait locus mapping of negatively selected Natural Killer cells from a population of healthy Europeans (n = 245). We find a significant subset of genes demonstrate expression quantitative trait loci specific to Natural Killer cells and these are highly informative of human disease, in particular autoimmunity. A Natural Killer cell transcriptome-wide association study across five common autoimmune diseases identifies further novel associations at 27 genes. In addition to these cis observations, we find novel master-regulatory regions impacting expression of trans gene networks at regions including 19q13.4, the Killer cell Immunoglobulin-like Receptor region, GNLY, MC1R and UVSSA. Our findings provide new insights into the unique biology of Natural Killer cells, demonstrating markedly different expression quantitative trait loci from other immune cells, with implications for disease mechanisms.

Toward a better understanding of T cells in cancer.

T cells mediate anti-tumor immune responses and are the key target of immune checkpoint therapy, but they can also promote immune tolerance. A clear understanding of the specific contributions and biology of different T cell subsets is required to fully harness the curative potential of immunotherapies. Experts discuss the state of the field and key challenges for moving forward.

Immune checkpoint blockade sensitivity and progression-free survival associates with baseline CD8+ T cell clone size and cytotoxicity.

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.

Large-scale cis- and trans-eQTL analyses identify thousands of genetic loci and polygenic scores that regulate blood gene expression.

Trait-associated genetic variants affect complex phenotypes primarily via regulatory mechanisms on the transcriptome. To investigate the genetics of gene expression, we performed cis- and trans-expression quantitative trait locus (eQTL) analyses using blood-derived expression from 31,684 individuals through the eQTLGen Consortium. We detected cis-eQTL for 88% of genes, and these were replicable in numerous tissues. Distal trans-eQTL (detected for 37% of 10,317 trait-associated variants tested) showed lower replication rates, partially due to low replication power and confounding by cell type composition. However, replication analyses in single-cell RNA-seq data prioritized intracellular trans-eQTL. Trans-eQTL exerted their effects via several mechanisms, primarily through regulation by transcription factors. Expression of 13% of the genes correlated with polygenic scores for 1,263 phenotypes, pinpointing potential drivers for those traits. In summary, this work represents a large eQTL resource, and its results serve as a starting point for in-depth interpretation of complex phenotypes.

Interferon-Gamma-Producing CD8+ Tissue Resident Memory T Cells Are a Targetable Hallmark of Immune Checkpoint Inhibitor-Colitis.

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.

EPISPOT: An epigenome-driven approach for detecting and interpreting hotspots in molecular QTL studies.

We present EPISPOT, a fully joint framework which exploits large panels of epigenetic annotations as variant-level information to enhance molecular quantitative trait locus (QTL) mapping. Thanks to a purpose-built Bayesian inferential algorithm, EPISPOT accommodates functional information for both cis and trans actions, including QTL hotspot effects. It effectively couples simultaneous QTL analysis of thousands of genetic variants and molecular traits with hypothesis-free selection of biologically interpretable annotations which directly contribute to the QTL effects. This unified, epigenome-aided learning boosts statistical power and sheds light on the regulatory basis of the uncovered hits; EPISPOT therefore marks an essential step toward improving the challenging detection and functional interpretation of trans-acting genetic variants and hotspots. We illustrate the advantages of EPISPOT in simulations emulating real-data conditions and in a monocyte expression QTL study, which confirms known hotspots and finds other signals, as well as plausible mechanisms of action. In particular, by highlighting the role of monocyte DNase-I sensitivity sites from >150 epigenetic annotations, we clarify the mediation effects and cell-type specificity of major hotspots close to the lysozyme gene. Our approach forgoes the daunting and underpowered task of one-annotation-at-a-time enrichment analyses for prioritizing cis and trans QTL hits and is tailored to any transcriptomic, proteomic, or metabolomic QTL problem. By enabling principled epigenome-driven QTL mapping transcriptome-wide, EPISPOT helps progress toward a better functional understanding of genetic regulation.

Checkpoint-blocker-induced autoimmunity is associated with favourable outcome in metastatic melanoma and distinct T-cell expression profiles.

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 < 0.0001). Median survival for patients without irAEs was 16.6 months (95% CI: 10.9-33.4) versus not-reached (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.

Changes in epigenetic profiles throughout early childhood and their relationship to the response to pneumococcal vaccination.

BACKGROUND: Pneumococcal infections are a major cause of morbidity and mortality in young children and immaturity of the immune system partly underlies poor vaccine responses seen in the young. Emerging evidence suggests a key role for epigenetics in the maturation and regulation of the immune system in health and disease. The study aimed to investigate epigenetic changes in early life and to understand the relationship between the epigenome and antigen-specific antibody responses to pneumococcal vaccination. METHODS: The epigenetic profiles from 24 healthy children were analyzed at 12 months prior to a booster dose of the 13-valent pneumococcal conjugate vaccine (PCV-13), and at 24 months of age, using the Illumina Methylation 450 K assay and assessed for differences over time and between high and low vaccine responders. RESULTS: Our analysis revealed 721 significantly differentially methylated positions between 12 and 24 months (FDR < 0.01), with significant enrichment in pathways involved in the regulation of cell-cell adhesion and T cell activation. Comparing high and low vaccine responders, we identified differentially methylated CpG sites (P value < 0.01) associated with HLA-DPB1 and IL6. CONCLUSION: These data imply that epigenetic changes that occur during early childhood may be associated with antigen-specific antibody responses to pneumococcal vaccines.

Dissecting genetic determinants of variation in human immune responses.

The immune system is paradigmatic for a complex arrangement of heterogenous cells performing distinct, frequently temporally and anatomically dissociated, functions. Immune dysfunction is a common characteristic across most diseases and human genetic approaches have revealed that many disease risk loci are associated with expression profiles and counts of specific immune subsets. Furthermore, genetic regulators of immune function may only demonstrate activity in specific disease-linked contexts. Here we explore steps taken to dissect the genetic determinants of variation in immune response across cell counts and function, and the insights these have provided into human immunity.