A critical epithelial survival axis regulated by MCL-1 maintains thymic function in mice.

T-cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function and tolerance. To uncover new strategies to restore thymic function and adaptive immunity in immunodeficiency, we sought to determine the molecular mechanisms that control life and death decisions in TECs. Guided by gene expression profiling, we created mouse models that specifically deleted prosurvival genes in TECs. We found that although BCL-2 and BCL-XL were dispensable for TEC homeostasis, MCL-1 deficiency impacted on TECs as early as embryonic day 15.5, resulting in early thymic atrophy and T-cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was not necessary for TEC differentiation but was continually required for the survival of mature cortical and medullary TECs and the maintenance of thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. This signaling axis governing TEC survival and thymic function represents a new target for strategies for thymic protection and regeneration.

Dual roles for LUBAC signaling in thymic epithelial cell development and survival.

Thymic epithelial cells (TECs) form a unique microenvironment that orchestrates T cell differentiation and immunological tolerance. Despite the importance of TECs for adaptive immunity, there is an incomplete understanding of the signalling networks that support their differentiation and survival. We report that the linear ubiquitin chain assembly complex (LUBAC) is essential for medullary TEC (mTEC) differentiation, cortical TEC survival and prevention of premature thymic atrophy. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, severely impaired expansion of the thymic medulla and AIRE-expressing cells. Furthermore, HOIL-1-deficiency caused early thymic atrophy due to Caspase-8/MLKL-dependent apoptosis/necroptosis of cortical TECs. By contrast, deficiency in the LUBAC component, SHARPIN, caused relatively mild defects only in mTECs. These distinct roles for LUBAC components in TECs correlate with their function in linear ubiquitination, NFκB activation and cell survival. Thus, our findings reveal dual roles for LUBAC signaling in TEC differentiation and survival.

Thymospheres Are Formed by Mesenchymal Cells with the Potential to Generate Adipocytes, but Not Epithelial Cells.

Evidence suggests that a stem-cell-driven differentiation hierarchy maintains the dynamic thymic epithelial cell (TEC) network that governs T lymphocyte development. The identification of TEC stem/progenitor cells has been a major focus in the field, and several candidates with contrasting phenotypes have been described. We sought to determine the provenance and function of the only population reported to exhibit TEC stem cell properties in the adult, a Foxn1- EpCAM- cell that generates so-called thymospheres. We provide evidence that the thymosphere-forming cell (TSFC) is not a TEC stem cell but can incorporate bystander TECs into thymospheres, providing an explanation for the epithelial activity ascribed to these structures. TSFCs were found to share a phenotype, transcriptional profile, and developmental origin with thymic fibroblasts and can generate adipocytes. In summary, this study redefines the nature of bipotent TEC stem/progenitor cells in the adult thymus and highlights a potentially important mesenchymal progenitor population.

Ubiquitin ligase MARCH 8 cooperates with CD83 to control surface MHC II expression in thymic epithelium and CD4 T cell selection.

Major histocompatibility complex class II (MHC II) expression is tightly regulated, being subjected to cell type-specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHC II expression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHC II in thymic epithelial cells (TECs). March8(-/-) mice have elevated MHC II at the surface of cortical TECs and autoimmune regulator (AIRE)(-) medullary TECs (mTECs), but not AIRE(+) mTECs. Despite this, thymic and splenic CD4(+) T cell numbers and repertoires remained unaltered in March8(-/-) mice. Notably, the ubiquitination of MHC II by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83(anu/anu) mice) have impaired CD4(+) T cell selection, but deleting March8 in Cd83(anu/anu) mice restored CD4(+) T cell selection to normal levels. Therefore, orchestrated regulation of MHC II surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4(+) T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation.

Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis.

The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3+ regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation.

Simple sequence repeat markers for the endangered species Clianthus puniceus and C. maximus (Fabaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Clianthus puniceus using a shotgun sequencing library and tested for cross amplification in the closely related C. maximus to inform population management of these two endangered species. • METHODS AND RESULTS: We constructed a shotgun sequencing library using a Roche 454 sequencer and searched the resulting data set for putative microsatellite regions. We optimized 12 of these regions to produce polymorphic markers for Clianthus. We tested these markers on four populations of C. maximus and on four C. puniceus individuals of known provenance. Alleles per locus ranged from two to nine, while observed and expected heterozygosities per locus ranged from 0.000 to 1.000 and 0.178 to 0.600, respectively. • CONCLUSIONS: These markers will be valuable for ongoing monitoring of the genetic variation in naturally occurring populations of Clianthus and for the selection of individuals for revegetation projects in the species' former range.

Isolation of thymic epithelial cells and analysis by flow cytometry.

The epithelial cells of the thymus govern the differentiation of hematopoietic precursors into T cells, which are critical for acquired immunity. Thymic epithelial cells (TEC) provide molecular cues that direct precursor recruitment, commitment to the T cell lineage, thymocyte proliferation, and the processes of positive and negative selection. Despite the importance of TEC to the immune system, fundamental questions regarding their differentiation, turnover, and function throughout life remain unanswered. This knowledge gap is largely due to technical difficulties in isolating, quantifying, and purifying this rare cell type. Here, we describe methods for the enzymatic digestion of the thymus to obtain single-cell suspensions of TEC, their analysis by flow cytometry, enrichment using magnetic beads, and purification for a variety of downstream applications.

Association of vitamin D receptor gene polymorphisms with insulin resistance and response to vitamin D.

The objectives of the study were to determine associations between single nucleotide polymorphisms (SNPs) of the vitamin D receptor (VDR) gene and insulin resistance and the effects of these SNPs on changes in insulin sensitivity in response to vitamin D supplementation. The research described here was an extension of the Surya study. Genotyping of the Cdx-2, FokI, BsmI, ApaI, and TaqI SNPs was carried out on 239 South Asian women in New Zealand using polymerase chain reaction-based techniques. Associations of these genotypes and 3' end haplotypes with insulin resistance were determined using multiple regression analysis. Associations between SNP genotypes and responses in insulin sensitivity to vitamin D supplementation (4000 IU vitamin D(3) per day) were also determined for a subset (81) of these women. BsmI BB, ApaI AA, and TaqI tt genotypes were significantly associated with lower insulin resistance compared with BsmI bb, ApaI aa, and TaqI TT, respectively, in the cohort of 239 women. Furthermore, homozygosity of the haplotypes baT and BAt was associated with higher and lower insulin resistance, respectively, compared with no copies of their respective alleles. Of the 81 subjects who were supplemented with vitamin D, women with the FokI Ff genotype showed a significantly greater improvement in insulin sensitivity (increase of 29.4 [2.9, 38.1]) compared with women with the FokI FF genotype (increase of 2.3 [-11.5, 10.1]). This study has highlighted the association of vitamin D responsiveness and insulin resistance with VDR gene polymorphisms. This is the first study to determine associations between all three. Genotyping of the VDR gene may provide a predictive measure for insulin resistance in response to vitamin D intervention.