Sensitive Measurement of Drug-Target Engagement by a Cellular Thermal Shift Assay with Multiplex Proximity Extension Readout.

The ability to monitor target engagement in cellular contexts is a key for successful drug discovery and also valuable in clinical routine. A cellular thermal shift assay (CETSA) provides realistic information about drug binding in cells and tissues, revealing drug-target engagement in clinically relevant samples. The CETSA combined with mass spectrometry (MS) detection can be applied in the early hit identification phase to generate target engagement data for large sets of proteins. However, the analysis is slow, requires substantial amounts of the sample material, and often misses proteins of specific interest. Here, we combined the CETSA and the multiplex proximity extension assay (PEA) for analysis of target engagement of a set of 67 proteins from small amounts of the sample material treated with kinase inhibitors. The results were concordant with the corresponding analyses read out via MS. Our approach allows analyses of large numbers of specific target proteins at high sensitivity in limited sample aliquots. Highly sensitive multiplex CETSA-PEA assays are therefore promising for monitoring drug-target engagement in small sample aliquots in the course of drug development and potentially in clinical settings.

Leukocytes with chromosome Y loss have reduced abundance of the cell surface immunoprotein CD99.

Mosaic loss of chromosome Y (LOY) in immune cells is a male-specific mutation associated with increased risk for morbidity and mortality. The CD99 gene, positioned in the pseudoautosomal regions of chromosomes X and Y, encodes a cell surface protein essential for several key properties of leukocytes and immune system functions. Here we used CITE-seq for simultaneous quantification of CD99 derived mRNA and cell surface CD99 protein abundance in relation to LOY in single cells. The abundance of CD99 molecules was lower on the surfaces of LOY cells compared with cells without this aneuploidy in all six types of leukocytes studied, while the abundance of CD proteins encoded by genes located on autosomal chromosomes were independent from LOY. These results connect LOY in single cells with immune related cellular properties at the protein level, providing mechanistic insight regarding disease vulnerability in men affected with mosaic chromosome Y loss in blood leukocytes.

A combined approach for single-cell mRNA and intracellular protein expression analysis.

Combined measurements of mRNA and protein expression in single cells enable in-depth analysis of cellular states. We present SPARC, an approach that combines single-cell RNA-sequencing with proximity extension essays to simultaneously measure global mRNA and 89 intracellular proteins in individual cells. We show that mRNA expression fails to accurately reflect protein abundance at the time of measurement, although the direction of changes is in agreement during neuronal differentiation. Moreover, protein levels of transcription factors better predict their downstream effects than do their corresponding transcripts. Finally, we highlight that protein expression variation is overall lower than mRNA variation, but relative protein variation does not reflect the mRNA level. Our results demonstrate that mRNA and protein measurements in single cells provide different and complementary information regarding cell states. SPARC presents a state-of-the-art co-profiling method that overcomes current limitations in throughput and protein localization, including removing the need for cell fixation.

Nuclear gene proximity and protein interactions shape transcript covariations in mammalian single cells.

Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.

A compendium on single-cell analysis for the curious.

Single-cell analysis is impacting biology and medicine by changing the scale and resolution at which we investigate multicellular organisms. A particular, overarching aim of this field is to characterize the programmed development of all different cell types in the human body, as well as their individual spatial, molecular, and functional characteristics. This vast research program is generating a much-needed source of fundamental biological insights that will provide a basis for new diagnostic and therapeutic approaches. With this Focus Issue on Single-Cell Analyses, The FEBS Journal offers interested readers an excellent introduction to this exciting research field, including ideas on how a wide community can benefit from the powerful approaches and technologies as well as the biological knowledge generated.

Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assays.

Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA, and KLK6, whereas prostasomes carried NKX31, GSTP1, and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.

Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction.

We present a scalable, integrated strategy for coupled protein and RNA detection from single cells. Our approach leverages the DNA polymerase activity of reverse transcriptase to simultaneously perform proximity extension assays and complementary DNA synthesis in the same reaction. Using the Fluidigm C1™ system, we profile the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation, benchmarking against in situ hybridizations and immunofluorescence staining, as well as recombinant proteins, ERCC Spike-Ins, and population lysate dilutions. Through supervised and unsupervised analyses, we demonstrate synergies enabled by simultaneous measurement of single-cell protein and RNA abundances. Collectively, our generalizable approach highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses.

Identification of a major locus, TNF1, that controls BCG-triggered tumor necrosis factor production by leukocytes in an area hyperendemic for tuberculosis.

BACKGROUND: Tumor necrosis factor (TNF) is a key immune regulator of tuberculosis resistance, as exemplified by the highly increased risk of tuberculosis disease among individuals receiving TNF-blocker therapy. METHODS: We determined the extent of TNF production after stimulation with BCG or BCG plus interferon gamma (IFN-γ) using a whole blood assay in 392 children belonging to 135 nuclear families from an area hyperendemic for tuberculosis in South Africa. We conducted classical univariate and bivariate genome-wide linkage analysis of TNF production using the data from both stimulation protocols by means of an extension of the maximum-likelihood-binomial method for quantitative trait loci to multivariate analysis. RESULTS: Stimulation of whole blood by either BCG or BCG plus IFN-γ resulted in a range of TNF release across subjects. Extent of TNF production following both stimulation protocols was highly correlated (r = 0.81). We failed to identify genetic linkage of TNF release when considering each stimulus separately. However, using a multivariate approach, we detected a major pleiotropic locus (P < 10(-5)) on chromosome region 11p15, termed TNF locus 1 (TNF1), that controlled TNF production after stimulation by both BCG alone and BCG plus IFN-γ. CONCLUSIONS: The TNF1 locus was mapped in the vicinity of the TST1 locus, previously identified in the same family sample, that controls tuberculin skin test (TST) negativity per se, that is, T-cell-independent resistance to Mycobacterium tuberculosis infection. This suggested that there is a connection between TST negativity per se and TNF production.

Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic lupus erythematosus in a large-scale multiracial replication study.

Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 (IRF8; rs11644034; p(meta-Euro) = 2.08 × 10(-10)), transmembrane protein 39A (TMEM39A; rs1132200; p(meta-all) = 8.62 × 10(-9)), and 17q21 (rs1453560; p(meta-all) = 3.48 × 10(-10)) between IKAROS family of zinc finger 3 (AIOLOS; IKZF3) and zona pellucida binding protein 2 (ZPBP2). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 × 10(-8) < p(meta-Euro) < 9.99 × 10(-5)) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4, FAM19A2, and STXBP6. The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.

Evaluation of TRAF6 in a large multiancestral lupus cohort.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with significant immune system aberrations resulting from complex heritable genetics as well as environmental factors. We undertook to study the role of TRAF6 as a candidate gene for SLE, since it plays a major role in several signaling pathways that are important for immunity and organ development. METHODS: Fifteen single-nucleotide polymorphisms (SNPs) across TRAF6 were evaluated in 7,490 SLE patients and 6,780 control subjects from different ancestries. Population-based case-control association analyses and meta-analyses were performed. P values, false discovery rate q values, and odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated. RESULTS: Evidence of associations was detected in multiple SNPs. The best overall P values were obtained for SNPs rs5030437 and rs4755453 (P = 7.85 × 10(-5) and P = 4.73 × 10(-5) , respectively) without significant heterogeneity among populations (P = 0.67 and P = 0.50, respectively, in Q statistic). In addition, SNP rs540386, which was previously reported to be associated with rheumatoid arthritis (RA), was found to be in linkage disequilibrium with these 2 SNPs (r(2) = 0.95) and demonstrated evidence of association with SLE in the same direction (meta-analysis P = 9.15 × 10(-4) , OR 0.89 [95% CI 0.83-0.95]). The presence of thrombocytopenia improved the overall results in different populations (meta-analysis P = 1.99 × 10(-6) , OR 0.57 [95% CI 0.45-0.72], for rs5030470). Finally, evidence of family-based association in 34 African American pedigrees with the presence of thrombocytopenia was detected in 1 available SNP (rs5030437) with a Z score magnitude of 2.28 (P = 0.02) under a dominant model. CONCLUSION: Our data indicate the presence of association of TRAF6 with SLE, consistent with the previous report of association with RA. These data provide further support for the involvement of TRAF6 in the pathogenesis of autoimmunity.

Identification of a systemic lupus erythematosus susceptibility locus at 11p13 between PDHX and CD44 in a multiethnic study.

Systemic lupus erythematosus (SLE) is considered to be the prototypic autoimmune disease, with a complex genetic architecture influenced by environmental factors. We sought to replicate a putative association at 11p13 not yet exceeding genome-wide significance (p < 5 × 10(-8)) identified in a genome-wide association study (GWAS). Our GWA scan identified two intergenic SNPs located between PDHX and CD44 showing suggestive evidence of association with SLE in cases of European descent (rs2732552, p = 0.004, odds ratio [OR] = 0.78; rs387619, p = 0.003, OR = 0.78). The replication cohort consisted of >15,000 subjects, including 3562 SLE cases and 3491 controls of European ancestry, 1527 cases and 1811 controls of African American (AA) descent, and 1265 cases and 1260 controls of Asian origin. We observed robust association at both rs2732552 (p = 9.03 × 10(-8), OR = 0.83) and rs387619 (p = 7.7 × 10(-7), OR = 0.83) in the European samples with p(meta) = 1.82 × 10(-9) for rs2732552. The AA and Asian SLE cases also demonstrated association at rs2732552 (p = 5 × 10(-3), OR = 0.81 and p = 4.3 × 10(-4), OR = 0.80, respectively). A meta-analysis of rs2732552 for all racial and ethnic groups studied produced p(meta) = 2.36 × 10(-13). This locus contains multiple regulatory sites that could potentially affect expression and functions of CD44, a cell-surface glycoprotein influencing immunologic, inflammatory, and oncologic phenotypes, or PDHX, a subunit of the pyruvate dehydrogenase complex.

A 3'-untranslated region variant is associated with impaired expression of CD226 in T and natural killer T cells and is associated with susceptibility to systemic lupus erythematosus.

OBJECTIVE: Costimulatory receptor CD226 plays an important role in T cell activation, differentiation, and cytotoxicity. This study was undertaken to investigate the genetic association of CD226 with susceptibility to systemic lupus erythematosus (SLE) and to assess the functional implications of this association. METHODS: Twelve tag single-nucleotide polymorphisms (SNPs) in CD226 were typed in 1,163 SLE patients and 1,482 healthy control subjects from Europe or of European ancestry. Analyses of association were performed by single-marker Cochran-Mantel-Haenszel meta-analysis, followed by haplotype analysis. Gene expression was analyzed by quantitative real-time polymerase chain reaction analyses of RNA from peripheral blood mononuclear cells, and by fluorescence-activated cell sorter analysis. To study the functional impact of the associated variants, luciferase reporter constructs containing different portions of the 3'-untranslated region (3'-UTR) of the gene were prepared and used in transfection experiments. RESULTS: A 3-variant haplotype, rs763361;rs34794968;rs727088 (ATC), in the last exon of CD226 was associated with SLE (P = 1.3 × 10(-4) , odds ratio 1.24, 95% confidence interval 1.11-1.38). This risk haplotype correlated with low CD226 transcript expression and low CD226 protein levels on the surface of CD4+ and CD8+ T cells and natural killer T (NKT) cells. NK cells expressed high levels of CD226, but this expression was independent of the haplotype. Reporter assays with deletion constructs indicated that only the presence of rs727088 could account for the differences in the levels of luciferase transcripts. CONCLUSION: This study identified an association of CD226 with SLE in individuals of European ancestry. These data support the importance of the 3'-UTR SNP rs727088 in the regulation of CD226 transcription both in T cells and in NKT cells.

High heritability of antimycobacterial immunity in an area of hyperendemicity for tuberculosis disease.

Human antimycobacterial immunity is a critical component of tuberculosis (TB) pathogenesis that is often used to infer the presence of TB infection. We report high heritability (>50%) for in vitro secretion of tumor necrosis factor alpha and interferon gamma (IFN-gamma), and the frequency of antigen-specific IFN-gamma(+)CD4(+) and IFN-gamma(+)CD8(+) cells in the response of whole blood to mycobacterial challenge. In principal component analysis, the first 3 components explain 78% of the overall variance consistent with the effect of pleiotropic regulatory genes of human antimycobacterial immunity. These results directly demonstrate the pivotal role played by host genetics in quantitative measures of antimycobacterial immunity underlying immune diagnosis of TB infection.

Tuberculin skin test and in vitro assays provide complementary measures of antimycobacterial immunity in children and adolescents.

BACKGROUND: Although many studies have compared in vitro TB diagnostic tests with the venerable tuberculin skin test (TST), there is little understanding of the quantitative relationship between critical measures of antimycobacterial immunity used to detect TB infection. We, therefore, decided to determine the degree of redundancy between quantitative read-outs of in vivo and in vitro assays of antimycobacterial immunity. METHODS: We enrolled 475 healthy HIV-negative children and young adults living in a hyperendemic area of TB. We measured in vivo TST responses, and a 1:10 diluted 3- or 7-day whole-blood assay was used to determine the in vitro antigen-specific interferon (IFN)-gamma cytokine release. The frequency of antigen-specific IFN-gamma(+)CD4(+) and IFN-gamma(+)CD8(+) cells was tested using intracellular cytokine staining after 1 day incubation. RESULTS: In vivo TST responses segregated into two well-separated groups with either no measurable response (TST induration < 5 mm; n = 164) or a normally distributed group with TST indurations > or = 5 mm with peak at 15 mm (n = 260). In vitro assays provided a less pronounced separation of responders and nonresponders. Correlation analysis of responses among persons with TST > or = 5 mm demonstrated that extent of TST response was poorly correlated with IFN-gamma release (coefficients of correlation rho = 0.17-0.22) and frequency of IFN-gamma(+)CD4(+)/CD8(+) cells (rho = 0.05-0.17) across three stimulating antigens (Mycobacterium bovis bacillus Calmette-Guérin, purified protein derivative, early-secreted antigenic target-6). CONCLUSION: We conclude that in vivo and in vitro assays are nonredundant, complementary measures of antimycobacterial immunity. Both TST and in vitro assays provided valuable information about antimycobacterial immunity and by inference latent TB in the studied high-incidence TB settings.

Two loci control tuberculin skin test reactivity in an area hyperendemic for tuberculosis.

Approximately 20% of persons living in areas hyperendemic for tuberculosis (TB) display persistent lack of tuberculin skin test (TST) reactivity and appear to be naturally resistant to infection by Mycobacterium tuberculosis. Among those with a positive response, the intensity of TST reactivity varies greatly. The genetic basis of TST reactivity is not known. We report on a genome-wide linkage search for loci that have an impact on TST reactivity, which is defined either as zero versus nonzero (TST-BINa) or as extent of TST in millimeters (TST-quantitative trait locus [QTL]) in a panel of 128 families, including 350 siblings, from an area of South Africa hyperendemic for TB. We detected a major locus (TST1) on chromosomal region 11p14 (P = 1.4 x 10(-5)), which controls TST-BINa, with a lack of responsiveness indicating T cell-independent resistance to M. tuberculosis. We also detected a second major locus (TST2) on chromosomal region 5p15 (P < 10(-5)), which controls TST-QTL or the intensity of T cell-mediated delayed type hypersensitivity (DTH) to tuberculin. Fine mapping of this region identified SLC6A3, encoding the dopamine transporter DAT1, as a promising gene for further studies. Our results pave the way for the understanding of the molecular mechanisms involved in resistance to M. tuberculosis infection in endemic areas (TST1) and for the identification of critical regulators of T cell-dependent DTH to tuberculin (TST2).

Susceptibility to leprosy is associated with PARK2 and PACRG.

Leprosy is caused by Mycobacterium leprae and affects about 700,000 individuals each year. It has long been thought that leprosy has a strong genetic component, and recently we mapped a leprosy susceptibility locus to chromosome 6 region q25-q26 (ref. 3). Here we investigate this region further by using a systematic association scan of the chromosomal interval most likely to harbour this leprosy susceptibility locus. In 197 Vietnamese families we found a significant association between leprosy and 17 markers located in a block of approx. 80 kilobases overlapping the 5' regulatory region shared by the Parkinson's disease gene PARK2 and the co-regulated gene PACRG. Possession of as few as two of the 17 risk alleles was highly predictive of leprosy. This was confirmed in a sample of 975 unrelated leprosy cases and controls from Brazil in whom the same alleles were strongly associated with leprosy. Variants in the regulatory region shared by PARK2 and PACRG therefore act as common risk factors for leprosy.