Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium.

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium.

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

The thymoproteasome hardwires the TCR repertoire of CD8+ T cells in the cortex independent of negative selection.

The thymoproteasome expressed specifically in thymic cortical epithelium optimizes the generation of CD8+ T cells; however, how the thymoproteasome contributes to CD8+ T cell development is unclear. Here, we show that the thymoproteasome shapes the TCR repertoire directly in cortical thymocytes before migration to the thymic medulla. We further show that the thymoproteasome optimizes CD8+ T cell production independent of the thymic medulla; independent of additional antigen-presenting cells, including medullary thymic epithelial cells and dendritic cells; and independent of apoptosis-mediated negative selection. These results indicate that the thymoproteasome hardwires the TCR repertoire of CD8+ T cells with cortical positive selection independent of negative selection in the thymus.

Short Communication: Ultrasensitive Immunoassay for Assessing Residual Simian-Tropic HIV in Nonhuman Primate Models of AIDS.

Persistence of replication-competent viral reservoirs during infection remains a barrier to HIV cure, despite the ability of combination antiretroviral therapy (cART) to effectively suppress viral replication. Simian-tropic HIV (stHIV) is a minimally chimeric HIV-1 that is comprised of 94% HIV-1 sequence, contains HIV-1 drug and immunologic targets, and is capable of replicating to high levels and causing authentic HIV-like pathogenesis leading to clinical AIDS in pigtail macaques. Suppression of stHIV replication by cART provides a model for study of viral reservoirs and HIV-specific intervention strategies targeting them. Accurate measurement of reservoir size is crucial for evaluating the effect of any such intervention strategies. Although there are a variety of assays that allow for indirect monitoring of viral reservoir size ex vivo, they each quantify a different aspect of viral reservoirs, and are characterized by conceptual and/or technical limitations. Measurement of viral protein in ex vivo cell culture assays captures the immunologically relevant viral-antigen producing component of the reservoir. This study demonstrates the utility of an ultrasensitive digital HIV Gag p24 immunoassay, which enabled earlier, and more sensitive detection of viral protein in culture supernatants from stimulated CD4+ T cells from stHIV-infected pigtail macaques receiving cART compared with conventional enzyme-linked immunosorbent assay. Protein measurements were highly correlated with cell-free stHIV RNA, as measured by quantitative reverse transcription polymerase chain reaction. This ultrasensitive p24 assay can be used to complement other reservoir measurement tools to assess ongoing replication and reactivation of infectious virus from reservoirs in stHIV-infected pigtail macaques.

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.