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
. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded
expression. We propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to
- and
-expressing mTEC subsets. We further use experimental techniques to show that within the
expressing developmental branch, TSA expression peaked as
expression decreased, implying
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.
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.
Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (
) 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.
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