Single-cell multiomics defines tolerogenic extrathymic Aire-expressing populations with unique homology to thymic epithelium.

The autoimmune regulator (Aire), a well-defined transcriptional regulator in the thymus, is also found in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells and inducers of immune tolerance, but their precise identity has remained unclear. Here, we use single-cell multiomics, transgenic murine models, and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and an Airehi population coexpressing Aire and retinoic acid receptor­related orphan receptor γt (RORγt) that we term Janus cells (JCs). Both JCs and AmDCs have the highest transcriptional and genomic homology to CCR7+ migratory dendritic cells. eTACs, particularly JCs, have highly accessible chromatin and broad gene expression, including a range of tissue-specific antigens, as well as remarkable homology to medullary thymic epithelium and RANK-dependent Aire expression. Transgenic self-antigen expression by eTACs is sufficient to induce negative selection and prevent autoimmune diabetes. This transcriptional, genomic, and functional symmetry between eTACs (both JCs and AmDCs) and medullary thymic epithelium­the other principal Aire-expressing population and a key regulator of central tolerance­identifies a core program that may influence self-representation and tolerance across the spectrum of immune development.

Single-cell transcriptional profiling of human thymic stroma uncovers novel cellular heterogeneity in the thymic medulla.

The thymus' key function in the immune system is to provide the necessary environment for the development of diverse and self-tolerant T lymphocytes. While recent evidence suggests that the thymic stroma is comprised of more functionally distinct subpopulations than previously appreciated, the extent of this cellular heterogeneity in the human thymus is not well understood. Here we use single-cell RNA sequencing to comprehensively profile the human thymic stroma across multiple stages of life. Mesenchyme, pericytes and endothelial cells are identified as potential key regulators of thymic epithelial cell differentiation and thymocyte migration. In-depth analyses of epithelial cells reveal the presence of ionocytes as a medullary population, while the expression of tissue-specific antigens is mapped to different subsets of epithelial cells. This work thus provides important insight on how the diversity of thymic cells is established, and how this heterogeneity contributes to the induction of immune tolerance in humans.

Elastase 3B mutation links to familial pancreatitis with diabetes and pancreatic adenocarcinoma.

While improvements in genetic analysis have greatly enhanced our understanding of the mechanisms behind pancreatitis, it continues to afflict many families for whom the hereditary factors remain unknown. Recent evaluation of a patient with a strong family history of pancreatitis sparked us to reexamine a large kindred originally reported over 50 years ago with an autosomal dominant inheritance pattern of chronic pancreatitis, diabetes and pancreatic adenocarcinoma. Whole exome sequencing analysis identified a rare missense mutation in the gene encoding pancreas-specific protease Elastase 3B (CELA3B) that cosegregates with disease. Studies of the mutant protein in vitro, in cell lines and in CRISPR-Cas9 engineered mice indicate that this mutation causes translational upregulation of CELA3B, which upon secretion and activation by trypsin leads to uncontrolled proteolysis and recurrent pancreatitis. Although lesions in several other pancreatitic proteases have been previously linked to hereditary pancreatitis, this is the first known instance of a mutation in CELA3B and a defect in translational control contributing to this disease.

A broad range of self-reactivity drives thymic regulatory T cell selection to limit responses to self.

The degree of T cell self-reactivity considered dangerous by the immune system, thereby requiring thymic selection processes to prevent autoimmunity, is unknown. Here, we analyzed a panel of T cell receptors (TCRs) with a broad range of reactivity to ovalbumin (OVA(323-339)) in the rat insulin promoter (RIP)-mOVA self-antigen model for their ability to trigger thymic self-tolerance mechanisms. Thymic regulatory T (Treg) cell generation in vivo was directly correlated with in vitro TCR reactivity to OVA-peptide in a broad ~1,000-fold range. Interestingly, higher TCR affinity was associated with a larger Treg cell developmental "niche" size, even though the amount of antigen should remain constant. The TCR-reactivity threshold to elicit thymic negative selection and peripheral T cell responses was ~100-fold higher than that of Treg cell differentiation. Thus, these data suggest that the broad range of self-reactivity that elicits thymic Treg cell generation is tuned to secure peripheral tolerance to self.

Absence of P-selectin in recipients of allogeneic bone marrow transplantation ameliorates experimental graft-versus-host disease.

Alloreactive T cells are crucial for graft-versus-host disease (GVHD) pathophysiology, and modulating their trafficking patterns has been efficacious in ameliorating experimental disease. We report in this paper that P-selectin, a glycoprotein found on resting and inflamed endothelium, is important for donor alloreactive T cells trafficking into GVHD target organs, such as the intestines and skin. Compared with wild-type (WT) recipients of allogeneic bone marrow transplantation, P-selectin(-/-) recipients exhibit decreased GVHD mortality and decreased GVHD of the skin, liver, and small bowels. This was associated with diminished infiltration of alloactivated T cells into the Peyer's patches and small bowels, coupled with increased numbers of donor T cells in the spleen and secondary lymphoid organs (SLOs). Surprisingly, however, donor T cells deficient for P-selectin glycoprotein ligand 1, the most well described P-selectin ligand, mediated GVHD similar to WT T cells and accumulated in SLO and target organs in similar numbers as WT T cells. This suggests that P-selectin may be required for trafficking into inflamed tissues but not SLO and that donor T cells may use multiple P-selectin ligands apart from P-selectin glycoprotein ligand 1 to interact with P-selectin and traffic into inflamed tissues during GVHD. We conclude that targeting P-selectin may be a viable strategy for GVHD prophylaxis or treatment.

Intraclonal competition limits the fate determination of regulatory T cells in the thymus.

Because the deletion of self-reactive T cells is incomplete, thymic development of natural Foxp3+CD4+ regulatory T cells (Treg cells) is required for preventing autoimmunity. However, the function of T cell antigen receptor (TCR) specificity in thymic Treg cell development remains controversial. To address this issue, we generated a transgenic line expressing a naturally occurring Treg cell-derived TCR. Unexpectedly, we found that efficient thymic Treg cell development occurred only when the antigen-specific Treg cell precursors were present at low clonal frequency (o1%) in a normal thymus. Using retroviral vectors and bone marrow chimeras, we observed similar activity with two other Treg cell-derived TCRs. Our data demonstrate that thymic Treg cell development is a 'TCR-instructive' process involving a niche that can be saturable at much lower clonal frequencies than is the niche for positive selection.