Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8+ T cell tolerance.

Naturally occurring cases of monogenic type 1 diabetes (T1D) help establish direct mechanisms driving this complex autoimmune disease. A recently identified de novo germline gain-of-function (GOF) mutation in the transcriptional regulator STAT3 was found to cause neonatal T1D. We engineered a novel knock-in mouse incorporating this highly diabetogenic human STAT3 mutation (K392R) and found that these mice recapitulated the human autoimmune diabetes phenotype. Paired single-cell TCR and RNA sequencing revealed that STAT3-GOF drives proliferation and clonal expansion of effector CD8+ cells that resist terminal exhaustion. Single-cell ATAC-seq showed that these effector T cells are epigenetically distinct and have differential chromatin architecture induced by STAT3-GOF. Analysis of islet TCR clonotypes revealed a CD8+ cell reacting against known antigen IGRP, and STAT3-GOF in an IGRP-reactive TCR transgenic model demonstrated that STAT3-GOF intrinsic to CD8+ cells is sufficient to accelerate diabetes onset. Altogether, these findings reveal a diabetogenic CD8+ T cell response that is restrained in the presence of normal STAT3 activity and drives diabetes pathogenesis.

Combined transient ablation and single-cell RNA-sequencing reveals the development of medullary thymic epithelial cells.

Medullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene-expression patterns within the mTEC compartment are heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single-cell RNA-sequencing in Mus musculus. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. We propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

Specialized cells in the immune system known as T cells protect the body from infection by destroying disease-causing microbes, such as bacteria or viruses. T cells use proteins on their surface called receptors to stick to infectious microbes and remove them from the body. Some newly developed T-cells, however, contain receptors that recognize and bind to cells that belong in the body. If these faulty T cells are released, they can attack healthy tissues and cause an autoimmune disease. After a new T cell is developed, it gets carried to a gland in the chest known as the thymus. Cells in the thymus called mTECs screen T cells for receptors that may bind to the body's tissues. mTECs do this by presenting T cells with proteins that are commonly found on the surface of healthy cells in the body. If a T cell recognizes any of these 'tissue specific proteins', it is destroyed or given a new role in the body. Some faulty T cells, however, still manage to evade detection. One way to uncover why this might happen is to investigate how mTECs develop. Previous work showed that mTECs transition through various stages before reaching their final form. However, the order in which these events occur remained unclear. To gain a better understanding of these developmental steps, Wells, Miller et al. extracted mTECs from the thymus of mice and analyzed the genetic make-up of individual cells. This uncovered a missing link in mTEC development: a new type of cell that is the immediate predecessor of the final mTEC. These 'predecessor' cells were actively growing, highlighting that mTECs can be constantly generated in the body. By probing the genes that generate tissue-specific proteins in mTECs, Wells, Miller et al. revealed that these proteins were only produced for short periods and in the late stages of mTEC development. These findings contribute to our understanding of how mTECs develop to screen T cells. Mapping these developmental stages will make it easier to identify when faulty T cells are able to evade mTECs. This will lead to earlier detection of autoimmune diseases which could result in better treatments.