Epigenetic immune cell quantification for diagnostic evaluation and monitoring of patients with inborn errors of immunity and secondary immune deficiencies.

BACKGROUND: Early detection and monitoring of primary immunodeficiencies (PID) in humans require quantitative determination of immune cells from fresh blood analyzed by flow cytometry. However, epigenetic immune cell quantification allows analysis from fresh, frozen, or dried blood samples. We demonstrate the utility of epigenetic immune cell quantification for patients with PID. METHODS: Epigenetic quantification of basic lymphocyte subpopulations of 259 samples from PID patients were compared to flow cytometric data. Epigenetic analysis was extended to T-cell subsets (Treg, Th17, Tfh, PD-1+, CCR6+) and memory B-cells and compared between venous EDTA and dried blood. RESULTS: A high correlation of >0.9 was observed for basic T- and B-cell subsets. Extended epigenetic analysis showed quantitative trends within PID subgroups, but individually these varied substantially within these groups. Epigenetic analysis of dried blood samples was equivalent to EDTA blood. CONCLUSION: Epigenetic immune cell quantification is suitable for immune cell profiling in PID patients.

Epigenetic and immunological indicators of IPEX disease in subjects with FOXP3 gene mutation.

BACKGROUND: Forkhead box protein 3 (FOXP3) is the master transcription factor in CD4+CD25hiCD127lo regulatory T (Treg) cells. Mutations in FOXP3 result in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome. Clinical presentation of IPEX syndrome is broader than initially described, challenging the understanding of the disease, its evolution, and treatment choice. OBJECTIVE: We sought to study the type and extent of immunologic abnormalities that remain ill-defined in IPEX, across genetic and clinical heterogeneity. METHODS: We performed Treg-cell-specific epigenetic quantification and immunologic characterization of severe "typical" (n = 6) and "atypical" or asymptomatic (n = 9) patients with IPEX. RESULTS: Increased number of cells with Treg-cell-Specific Demethylated Region demethylation in FOXP3 is a consistent feature in patients with IPEX, with (1) highest values in those with typical IPEX, (2) increased values in subjects with pathogenic FOXP3 but still no symptoms, and (3) gradual increase over the course of disease progression. Large-scale profiling using Luminex identified plasma inflammatory signature of macrophage activation and TH2 polarization, with cytokines previously not associated with IPEX pathology, including CCL22, CCL17, CCL15, and IL-13, and the inflammatory markers TNF-α, IL-1A, IL-8, sFasL, and CXCL9. Similarly, both Treg-cell and Teff compartments, studied by Mass Cytometry by Time-Of-Flight, were skewed toward the TH2 compartment, especially in typical IPEX. CONCLUSIONS: Elevated TSDR-demethylated cells, combined with elevation of plasmatic and cellular markers of a polarized type 2 inflammatory immune response, extends our understanding of IPEX diagnosis and heterogeneity.

Epigenomics AG.

Earlier diagnosis and a more individualized choice of treatment options has the potential to greatly improve the outcome of life-threatening diseases. DNA methylation has proven to be a rich source of biomarkers for diagnosis, prognosis and drug response prediction in cancer and other diseases. Epigenomics AG makes use of DNA methylation biomarkers to develop in vitro diagnostic test products. The product pipeline comprises screening tests for the early detection of cancer in body fluids and molecular pathology tests on routinely available tissue samples for cancer prognosis. In collaborations with pharmaceutical and biotechnology companies, Epigenomics provides access to its broad range of technologies for the support of drug development and commercialization by patient stratification and drug response prediction.

Integrated technology platform protein kinases for drug development in oncology.

Protein kinases are among the most promising targets for drug discovery and development, mostly in oncology but also in other fields such as inflammation, Alzheimer's, and infectious diseases. The Integrated Technology Platform Protein Kinases was designed as a comprehensive tool for drug discovery in thefield of oncology. It combines modules for the identification and validation of novel target protein kinases, a unique panel of active recombinant protein kinases, high-throughput screening, selectivity profiling, cellular testing, and in vivo tumor models. Here we give an overview of the Integrated Technology Platform Protein Kinases as well as data that validate each module.

SRC catalytic but not scaffolding function is needed for integrin-regulated tyrosine phosphorylation, cell migration, and cell spreading.

Src family kinases (SFKs) are crucial for signaling through a variety of cell surface receptors, including integrins. There is evidence that integrin activation induces focal adhesion kinase (FAK) autophosphorylation at Y397 and that Src binds to and is activated by FAK to carry out subsequent phosphorylation events. However, it has also been suggested that Src functions as a scaffolding molecule through its SH2 and SH3 domains and that its kinase activity is not necessary. To examine the role of SFKs in integrin signaling, we have expressed various Src molecules in fibroblasts lacking other SFKs. In cells plated on fibronectin, FAK could indeed autophosphorylate at Y397 independently of Src but with lower efficiency than when Src was present. This step was promoted by kinase-inactive Src, but Src kinase activity was required for full rescue. Src kinase activity was also required for phosphorylation of additional sites on FAK and for other integrin-directed functions, including cell migration and spreading on fibronectin. In contrast, Src mutations in the SH2 or SH3 domain greatly reduced binding to FAK, Cas, and paxillin but had little effect on tyrosine phosphorylation or biological assays. Furthermore, our indirect evidence indicates that Src kinase activity does not need to be regulated to promote cell migration and FAK phosphorylation. Although Src clearly plays important roles in integrin signaling, it was not concentrated in focal adhesions. These results indicate that the primary role of Src in integrin signaling is as a kinase. Indirect models for Src function are proposed.