Thymic epithelial cells (TECs) are critical for T cell development and self-tolerance but are gradually lost with age. The existence of thymic epithelial progenitors (TEPCs) in the postnatal thymus has been inferred, but their identity has remained enigmatic. Here, we assessed the entire adult TEC compartment in order to reveal progenitor capacity is retained exclusively within a subset of immature thymic epithelium displaying several hallmark features of stem/progenitor function. These adult TEPCs generate mature cortical and medullary lineages in a stepwise fashion, including Aire+ TEC, within fetal thymus reaggregate grafts. Although relatively quiescent in vivo, adult TEPCs demonstrate significant in vitro colony formation and self-renewal. Importantly, 3D-cultured TEPCs retain their capacity to differentiate into cortical and medullary TEC lineages when returned to an in vivo thymic microenvironment. No other postnatal TEC subset exhibits this combination of properties. The characterization of adult TEPC will enable progress in understanding TEC biology in aging and regeneration.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co‐segregation, family cancer history profile, co‐occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case‐control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene‐specific calibration of evidence types used for variant classification.

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin ␣ IIb ␤ 3 , necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin ␣ IIb ␤ 3 activation (affinity modulation) primarily occurs downstream of G i -coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin ␣ IIb ␤ 3 bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110␤ isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin ␣ IIb ␤ 3 activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin ␣ IIb ␤ 3 association with the contractile cytoskeleton. Analysis of integrin ␣ IIb ␤ 3 adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin ␣ IIb ␤ 3 bonds. These studies demonstrate an important role for PI3K p110␤ in regulating the avidity of high affinity integrin ␣ IIb ␤ 3 receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110␤ inhibitors.Platelet adhesion and aggregation at sites of vascular injury are essential for the formation of the primary hemostatic plug and for the development of arterial thrombi (1, 2). Platelet thrombus growth is critically dependent on the adhesive function of the platelet integrin, ␣ IIb ␤ 3 . This receptor engages multiple adhesive ligands, including von Willebrand factor, fibrinogen, and fibronectin, and is indispensable for the formation of stable platelet-platelet adhesion contacts during thrombus development. Qualitative or quantitative defects in integrin ␣ IIb ␤ 3 are associated with a profound defect in platelet aggregation, leading to a severe bleeding disorder. Inhibitors of integrin ␣ IIb ␤ 3 have been developed therapeutically, and these agents are effective at preventing arterial thrombus development in patients with the acute coronary syndromes (3).The ligand binding function of integrin ␣ IIb ␤ 3 is subject to tight regulation by intracellular signaling processes that modulate the conformation of the receptor complex (affinity regulation) (4, 5). Once engaged by adhesive ligand, integrins transduce signals into the cell (outside-in signaling) necessary for cytoskeletal remodeling and clustering of integrin receptors. These latter ch...

BackgroundGenetic testing for BRCA1 and BRCA2 is offered typically to selected women based on age of onset and family history of cancer. However, current internationally accepted genetic testing referral guidelines are built mostly on data from cancer genetics clinics in women of European descent. To evaluate the appropriateness of such guidelines in Asians, we have determined the prevalence of germ line variants in an unselected cohort of Asian patients with breast cancer and healthy controls.MethodsGerm line DNA from a hospital-based study of 2575 unselected patients with breast cancer and 2809 healthy controls were subjected to amplicon-based targeted sequencing of exonic and proximal splice site junction regions of BRCA1 and BRCA2 using the Fluidigm Access Array system, with sequencing conducted on a Illumina HiSeq2500 platform. Variant calling was performed with GATK UnifiedGenotyper and were validated by Sanger sequencing.ResultsFifty-five (2.1%) BRCA1 and 66 (2.6%) BRCA2 deleterious mutations were identified among patients with breast cancer and five (0.18%) BRCA1 and six (0.21%) BRCA2 mutations among controls. One thousand one hundred and eighty-six (46%) patients and 97 (80%) carriers fulfilled the National Comprehensive Cancer Network guidelines for genetic testing.ConclusionFive per cent of unselected Asian patients with breast cancer carry deleterious variants in BRCA1 or BRCA2. While current referral guidelines identified the majority of carriers, one in two patients would be referred for genetic services. Given that such services are largely unavailable in majority of low-resource settings in Asia, our study highlights the need for more efficient guidelines to identify at-risk individuals in Asia.

Although forkhead-box n1 (Foxn1) is a critical thymic epithelial cell regulator in thymus organogenesis, its association with epithelial differentiation and homeostasis in the postnatal and aged thymic microenvironment remains conflicting. Consequently, we have generated a Foxn1 knock-in mouse model that allows for refined investigation of the aging thymic epithelium. This reporter line differs from those previously published in that concomitant expression of enhanced green fluorescent protein enables live cell sorting of Foxn1 cell populations. Our heterozygotes did not exhibit haploinsufficiency, with Foxn1 expression resembling that of wild-type mice. Comparative analysis between Foxn1 and enhanced green fluorescent protein at both the transcriptional and translational levels revealed co-localization, with progressive down-regulation observed predominantly in the aging cortical epithelium. Supplementation with bone morphogenetic protein (Bmp)-4 enhanced Foxn1 expression and colony forming efficiency in both embryonic and adult progenitor 3D cultures. Strikingly, selective maintenance of immature cortical and medullary epithelial cells was observed which is consistent with the higher Bmp receptor 2 expression levels seen in these progenitor populations. This study demonstrates the significance of our mouse model in unraveling the role of this master regulator in thymus development, homeostasis and aging, providing a faithful reporter system for phenotypic and functional investigations.