Single-cell functional genomics reveals determinants of sensitivity and resistance to natural killer cells in blood cancers.

Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.

Early DNA methylation changes in children developing beta cell autoimmunity at a young age.

AIMS/HYPOTHESIS: Type 1 diabetes is a chronic autoimmune disease of complex aetiology, including a potential role for epigenetic regulation. Previous epigenomic studies focused mainly on clinically diagnosed individuals. The aim of the study was to assess early DNA methylation changes associated with type 1 diabetes already before the diagnosis or even before the appearance of autoantibodies. METHODS: Reduced representation bisulphite sequencing (RRBS) was applied to study DNA methylation in purified CD4+ T cell, CD8+ T cell and CD4-CD8- cell fractions of 226 peripheral blood mononuclear cell samples longitudinally collected from seven type 1 diabetes-specific autoantibody-positive individuals and control individuals matched for age, sex, HLA risk and place of birth. We also explored correlations between DNA methylation and gene expression using RNA sequencing data from the same samples. Technical validation of RRBS results was performed using pyrosequencing. RESULTS: We identified 79, 56 and 45 differentially methylated regions in CD4+ T cells, CD8+ T cells and CD4-CD8- cell fractions, respectively, between type 1 diabetes-specific autoantibody-positive individuals and control participants. The analysis of pre-seroconversion samples identified DNA methylation signatures at the very early stage of disease, including differential methylation at the promoter of IRF5 in CD4+ T cells. Further, we validated RRBS results using pyrosequencing at the following CpG sites: chr19:18118304 in the promoter of ARRDC2; chr21:47307815 in the intron of PCBP3; and chr14:81128398 in the intergenic region near TRAF3 in CD4+ T cells. CONCLUSIONS/INTERPRETATION: These preliminary results provide novel insights into cell type-specific differential epigenetic regulation of genes, which may contribute to type 1 diabetes pathogenesis at the very early stage of disease development. Should these findings be validated, they may serve as a potential signature useful for disease prediction and management.

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes.

AIMS/HYPOTHESIS: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. METHODS: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. RESULTS: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate <0.05. CONCLUSIONS/INTERPRETATION: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.

Standard of hygiene and immune adaptation in newborn infants.

The prevalence of immune-mediated diseases, such as allergies and type 1 diabetes, is on the rise in the developed world. In order to explore differences in the gene expression patterns induced in utero in infants born in contrasting standards of living and hygiene, we collected umbilical cord blood RNA samples from infants born in Finland (modern society), Estonia (rapidly developing society) and the Republic of Karelia, Russia (poor economic conditions). The whole blood transcriptome of Finnish and Estonian neonates differed from their Karelian counterparts, suggesting exposure to toll-like receptor (TLR) ligands and a more matured immune response in infants born in Karelia. These results further support the concept of a conspicuous plasticity in the developing immune system: the environmental factors that play a role in the susceptibility/protection towards immune-mediated diseases begin to shape the neonatal immunity already in utero and direct the maturation in accordance with the surrounding microbial milieu.

Single-cell analysis of immune recognition in chronic myeloid leukemia patients following tyrosine kinase inhibitor discontinuation.

Immunological control of residual leukemia cells is thought to occur in patients with chronic myeloid leukemia (CML) that maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. To study this, we analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced samples (scRNA+TCRαß-seq) from patients with CML (n = 13, N = 25), other cancers (n = 28), and healthy (n = 7). The high number and active phenotype of natural killer (NK) cells in CML separated them from healthy and other cancers. Most NK cells in CML belonged to the active CD56dim cluster with high expression of GZMA/B, PRF1, CCL3/4, and IFNG, with interactions with leukemic cells via inhibitory LGALS9-TIM3 and PVR-TIGIT interactions. Accordingly, upregulation of LGALS9 was observed in CML target cells and TIM3 in NK cells when co-cultured together. Additionally, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 and quantified anti-PR1 T cells in 90 CML and 786 healthy TCRß-sequenced samples. Anti-PR1 T cells were more prevalent in CML, enriched in bone marrow samples, and enriched in the mature, cytotoxic CD8 + TEMRA cluster, especially in a patient maintaining TFR. Our results highlight the role of NK cells and anti-PR1 T cells in anti-leukemic immune responses in CML.

The tumor and plasma cytokine profiles of renal cell carcinoma patients.

Renal cell carcinoma (RCC) accounts for 90% of all renal cancers and is considered highly immunogenic. Although many studies have reported the circulating peripheral cytokine profiles, the signatures between the tumor tissue and matching healthy adjacent renal tissue counterparts have not been explored. We aimed to comprehensively investigate the cytokine landscape of RCC tumors and its correlation between the amount and phenotype of the tumor infiltrating lymphocytes (TILs). We analyzed the secretion of 42 cytokines from the tumor (n = 46), adjacent healthy kidney tissues (n = 23) and matching plasma samples (n = 33) with a Luminex-based assay. We further explored the differences between the tissue types, as well as correlated the findings with clinical data and detailed immunophenotyping of the TILs. Using an unsupervised clustering approach, we observed distinct differences in the cytokine profiles between the tumor and adjacent renal tissue samples. The tumor samples clustered into three distinct profiles based on the cytokine expressions: high (52.2% of the tumors), intermediate (26.1%), and low (21.7%). Most of the tumor cytokines positively correlated with each other, except for IL-8 that showed no correlation with any of the measured cytokine expressions. Furthermore, the quantity of lymphocytes in the tumor samples analyzed with flow cytometry positively correlated with the chemokine-family of cytokines, CXCL10 (IP-10) and CXCL9 (MIG). No significant correlations were found between the tumor and matching plasma cytokines, suggesting that circulating cytokines poorly mirror the tumor cytokine environment. Our study highlights distinct cytokine profiles in the RCC tumor microenvironment and provides insights to potential biomarkers for the treatment of RCC.

Permutation-based significance analysis reduces the type 1 error rate in bisulphite sequencing data analysis of human umbilical cord blood samples.

DNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2% of human CpG sites. To detect such associations outside these regions, we chose the bisulphite sequencing approach. We collected and curated clinical data on 200 newborn infants; whose umbilical cord blood samples were analysed with the reduced representation bisulphite sequencing (RRBS) method. A generalized linear mixed-effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables, such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis. We discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. The inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.

Characterization and non-parametric modeling of the developing serum proteome during infancy and early childhood.

Children develop rapidly during the first years of life, and understanding the sources and associated levels of variation in the serum proteome is important when using serum proteins as markers for childhood diseases. The aim of this study was to establish a reference model for the evolution of a healthy serum proteome during early childhood. Label-free quantitative proteomics analyses were performed for 103 longitudinal serum samples collected from 15 children at birth and between the ages of 3-36 months. A flexible Gaussian process-based probabilistic modelling framework was developed to evaluate the effects of different variables, including age, living environment and individual variation, on the longitudinal expression profiles of 266 reliably identified and quantified serum proteins. Age was the most dominant factor influencing approximately half of the studied proteins, and the most prominent age-associated changes were observed already during the first year of life. High inter-individual variability was also observed for multiple proteins. These data provide important details on the maturing serum proteome during early life, and evaluate how patterns detected in cord blood are conserved in the first years of life. Additionally, our novel modelling approach provides a statistical framework to detect associations between covariates and non-linear time series data.

Genome-wide Analysis of STAT3-Mediated Transcription during Early Human Th17 Cell Differentiation.

The development of therapeutic strategies to combat immune-associated diseases requires the molecular mechanisms of human Th17 cell differentiation to be fully identified and understood. To investigate transcriptional control of Th17 cell differentiation, we used primary human CD4+ T cells in small interfering RNA (siRNA)-mediated gene silencing and chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) to identify both the early direct and indirect targets of STAT3. The integrated dataset presented in this study confirms that STAT3 is critical for transcriptional regulation of early human Th17 cell differentiation. Additionally, we found that a number of SNPs from loci associated with immune-mediated disorders were located at sites where STAT3 binds to induce Th17 cell specification. Importantly, introduction of such SNPs alters STAT3 binding in DNA affinity precipitation assays. Overall, our study provides important insights for modulating Th17-mediated pathogenic immune responses in humans.

Serum proteomes distinguish children developing type 1 diabetes in a cohort with HLA-conferred susceptibility.

We determined longitudinal serum proteomics profiles from children with HLA-conferred diabetes susceptibility to identify changes that could be detected before seroconversion and positivity for disease-associated autoantibodies. Comparisons were made between children who seroconverted and progressed to type 1 diabetes (progressors) and those who remained autoantibody negative, matched by age, sex, sample periodicity, and risk group. The samples represented the prediabetic period and ranged from the age of 3 months to 12 years. After immunoaffinity depletion of the most abundant serum proteins, isobaric tags for relative and absolute quantification were used for sample labeling. Quantitative proteomic profiles were then measured for 13 case-control pairs by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, a label-free LC-MS/MS approach was used to analyze depleted sera from six case-control pairs. Importantly, differences in abundance of a set of proteins were consistently detected before the appearance of autoantibodies in the progressors. Based on top-scoring pairs analysis, classification of such progressors was observed with a high success rate. Overall, the data provide a reference of temporal changes in the serum proteome in healthy children and children progressing to type 1 diabetes, including new protein candidates, the levels of which change before clinical diagnosis.

Reactive oxygen species deficiency induces autoimmunity with type 1 interferon signature.

AIMS: Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the phagocyte reactive oxygen species (ROS)-producing NOX2 enzyme complex and characterized by recurrent infections associated with hyperinflammatory and autoimmune manifestations. A translational, comparative analysis of CGD patients and the corresponding ROS-deficient Ncf1(m1J) mutated mouse model was performed to reveal the molecular pathways operating in NOX2 complex deficient inflammation. RESULTS: A prominent type I interferon (IFN) response signature that was accompanied by elevated autoantibody levels was identified in both mice and humans lacking functional NOX2 complex. To further underline the systemic lupus erythematosus (SLE)-related autoimmune process, we show that naïve Ncf1(m1J) mutated mice, similar to SLE patients, suffer from inflammatory kidney disease with IgG and C3 deposits in the glomeruli. Expression analysis of germ-free Ncf1(m1J) mutated mice reproduced the type I IFN signature, enabling us to conclude that the upregulated signaling pathway is of endogenous origin. INNOVATION: Our findings link the previously unexplained connection between ROS deficiency and increased susceptibility to autoimmunity by the discovery that activation of IFN signaling is a major pathway downstream of a deficient NOX2 complex in both mice and humans. CONCLUSION: We conclude that the lack of phagocyte-derived oxidative burst is associated with spontaneous autoimmunity and linked with type I IFN signature in both mice and humans.

Innate immune activity is detected prior to seroconversion in children with HLA-conferred type 1 diabetes susceptibility.

The insult leading to autoantibody development in children who will progress to develop type 1 diabetes (T1D) has remained elusive. To investigate the genes and molecular pathways in the pathogenesis of this disease, we performed genome-wide transcriptomics analysis on a unique series of prospective whole-blood RNA samples from at-risk children collected in the Finnish Type 1 Diabetes Prediction and Prevention study. We studied 28 autoantibody-positive children, out of which 22 progressed to clinical disease. Collectively, the samples covered the time span from before the development of autoantibodies (seroconversion) through the diagnosis of diabetes. Healthy control subjects matched for date and place of birth, sex, and HLA-DQB1 susceptibility were selected for each case. Additionally, we genotyped the study subjects with Immunochip to identify potential genetic variants associated with the observed transcriptional signatures. Genes and pathways related to innate immunity functions, such as the type 1 interferon (IFN) response, were active, and IFN response factors were identified as central mediators of the IFN-related transcriptional changes. Importantly, this signature was detected already before the T1D-associated autoantibodies were detected. Together, these data provide a unique resource for new hypotheses explaining T1D biology.

Probe-level estimation improves the detection of differential splicing in Affymetrix exon array studies.

The recent advent of exon microarrays has made it possible to reveal differences in alternative splicing events on a global scale. We introduce a novel statistical procedure that takes full advantage of the probe-level information on Affymetrix exon arrays when detecting differential splicing between sample groups. In comparison to existing ranking methods, the procedure shows superior reproducibility and accuracy in distinguishing true biological findings from background noise in high agreement with experimental validations.