Insm1 regulates the development of mTECs and immune tolerance

Background

Developing T cells (thymocytes), derived from a common lymphoid progenitor in the bone marrow, undergo a series of maturation steps in the thymus. Initially, immature thymocytes are positively selected for self-major histocompatibility complex (MHC) recognition via interactions with cortical thymic epithelial cells (cTECs). In turn, medullary thymic epithelial cells (mTECs) are important mediators of subsequent negative selection of T cells because they ectopically express tissue-restricted antigens (TRA) in a mosaic fashion. T cell interaction with mTECs allows them to sample the pool of TRAs and, if they are reactive to such self-antigens, they will be eliminated or converted to Tregs to avoid autoimmunity. TRA expression is thought to be regulated, in part, by a subset of mTECs expressing the transcription factor (TF) Aire (autoimmune regulator) via mechanisms of chromatin looping and activation of gene (super-) enhancers.

Single-cell RNA-sequencing (scRNA-seq) has made it possible for researchers to profile the various cell types found in the thymus. Recent single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) studies revealed that mTECs are highly heterogeneous and are comprised of a number of subsets¹. A couple of these subsets are referred to as “mimetic cells” because their transcriptome strikingly resembles that of distinct peripheral cells. Mimetic cells differ from Aire⁺ mTECs because they express lineage-defining TFs specific to peripheral cell types. For example, the TF FoxA specifically defines neuroendocrine mimetic cells. It seems that the induction of central tolerance is not only due to promiscuous TRA expression by Aire⁺ mTECs, but also by thymic mimetic cells. Aire deficiency is marked by a reduction of several mimetic cell subsets, suggesting that Aire can both directly induce TRAs in mTECs and indirectly drive TRA expression in mimetic cells by modulating their frequency. However, how Aire itself is regulated remains unanswered. In this study, Tao et al. characterized the expression and function of Insulinoma-associated protein 1 (Insm1), a new potential regulator of Aire, in Aire+ mTECs and mimetic cells of the thymus.

 

Key findings

Aire⁺ mTECs and neuroendocrine mimetic cells express Insm1

Tao et al. started off by assessing the expression of Insm1 in the thymus of young and adult mice. Using a β-Gal assay to monitor the expression of Insm1, they detected the Insm1 protein in the nuclei and cytoplasm of thymic cells. Insm1 expression was specifically observed in mTECs, and no expression was detected in lymphocytes, dendritic cells or cTECs. Additionally, 70% of Insm1-positive mTECs co-expressed Aire. Using a publicly available scATAC-seq dataset¹, in which mimetic cells were first characterized, Tao et al. found that neuroendocrine mimetic cells particularly express high levels of Insm1, which the authors validated by immunofluorescent staining in 6-week-old mouse thymi.

 

Insm1 affects thymic cell proportions and the expression of mTEC-related genes

Using flow cytometry, the authors compared the thymic cell populations in wild-type and Insm1-mutant mice at E18.5 and adult stage. They observed similar counts of cTECs and mTECs overall, however, the proportion of Aire+ mTECs was significantly lower in E18.5 Insm1-mutants compared to the controls. In line with that, Insm1 protein and transcript levels were reduced in Insm1-mutant mice as well as animals harboring a thymus-specific Insm1 mutation. These findings indicate that Insm1 plays a role in Aire⁺ mTEC development in the prenatal thymus and that mutation of Insm1 negatively impacts Aire expression.

Differential gene expression (DEG) analysis performed on RNA-seq data of mTECs isolated from Insm1-mutant E18.5 and adult mice with a thymus-specific mutation in Insm1 revealed that several genes were deregulated in both timepoints. Consistent with previous observations, the expression of Aire, but also Insm1-dependent genes in several mimetic cell types, was downregulated in both E18.5 and adult mice. Additionally, the authors found Aire-dependent and -independent TRAs among the deregulated genes. Of the 81 downregulated TRAs, 26 were found to be deregulated in both, Aire and Insm1 mutants, while 55 of them were unique to Insm1 mutants. Interestingly, the downregulated TRAs were not restricted to neuroendocrine mimetic cells. In line with this, Insm1-dependent TRAs were expressed in multiple peripheral tissues, particularly the gastrointestinal tract and neuroendocrine tissues.

 

Insm1 promotes mTEC fate and regulates mTEC gene expression through co-occupancy of super-enhancers with Aire

Overexpression of Insm1 in mTECs resulted in an enlarged mTEC compartment, an increase in Insm1-positive cells and a slightly lower increase in Aire⁺ mTECs. Moreover, the expression levels of Aire, neuroendocrine markers and some TRA genes, which were downregulated in Insm1 mutants, significantly increased in Insm1-overexpressing mTECs. These findings suggest that Insm1 promotes mTEC fate and is associated with the induction of mTEC-specific genes. By performing Cut & Tag in mTECs of E18.5 and adult mice, the authors profiled genome-wide binding sites of Insm1. They found numerous Insm1 peaks within promoter regions of genes, whose expression was rarely affected in Insm1 mutants. Interestingly, the majority of Insm1 binding sites were co-occupied by Aire and enriched in super-enhancer regions. Genes within 500kb of these super-enhancers turned out to be among the dysregulated genes in Insm1 mutants, suggesting that Insm1 regulates mTEC gene expression through binding of super-enhancers.

 

Insm1 mutation in the thymus leads to autoimmune phenotypes

Upon transplantation of Insm1-mutant versus control thymi into nude mice the authors did not report any differences in overall thymic morphology and in the number of T cells. However, mice transplanted with Insm1-knockout thymi exhibited splenomegaly (an enlarged spleen), lymphocyte infiltration at the site of  several peripheral tissues, including the pancreatic islets, lungs, kidney and salivary glands, as well as autoantibody production against some of those tissues. A similar phenotype, suggestive of a mounting autoimmune response, was observed in thymus-specific Insm1-mutant mice. In addition, thymus-specific Insm1-mutants showed reduced numbers of Tregs in the thymus and lymph nodes. Altogether, these findings highlight how thymic expression of Insm1 is crucial for the establishment of central tolerance, while limiting autoimmune reactions in the periphery.

 

Why is this work important

Negative selection is intriguing because mTECs express thousands of TRAs to expose developing T cells to peptide-MHC complexes specific to our different organs and prohibit autoimmune reactions later in life. Most work done so far has focused on Aire-dependent TRA expression. However, mTEC heterogeneity brings new questions: Do mimetic cells express specific TRAs regulated by other TFs? Can other TFs individually or in cooperation with Aire be drivers of TRA expression and what is their role in mTEC fate? Are the quasi-random, Aire-mediated TRA expression in mTECs and antigen presentation by mimetic cells completely independent processes, serially occurring during thymocyte development? How can we avoid overly redundant TRA expression by mTECs and mimetic cells? Or alternatively, are there different outcomes to identical TRA expression by mTECs and mimetic cells? Although many questions remain, Tao and colleagues have highlighted the potential role of the Insm1 TF in mTEC development, Aire regulation and the establishment of immune tolerance, which other researchers may build upon.

 

Questions for the authors

• Do you have a possible explanation for the decrease in Aire⁺ mTECs in Insm1mutants at E18.5 but not in Insm1 adult mutants?
• According to Figure 1F, Insm1 is also highly expressed in transient amplifying mTECs. By flow cytometry do you observe a loss in the transient amplifying mTEC compartment in Isnm1 mutant mice as well?
• Given recent reports² on Aire regulation – do you know whether Insm1 interacts with other key regulators of Aire, such as Tbx21, Tcf7, Irf4 or Irf8?
• Can you speculate why the pancreas and salivary glands are continuously affected in the autoimmune phenotype of Insm1 mutants? Is the abundance of Insm1+ neuroendocrine cells in those tissues higher than in other peripheral sites?
• It has been reported that lineage-specific mTECs produce distinct cytokines (f.e. tuft mTECs express Il10 and Il25) – do you know whether Insm1+ mTECs produce specific cytokines, chemokines, or other molecules? If so, how might those affect T cell education and selection?

 

References

1          Michelson, D. A., Hase, K., Kaisho, T., Benoist, C. & Mathis, D. Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells. Cell 185, 2542-2558.e2518, doi:https://doi.org/10.1016/j.cell.2022.05.018 (2022).

2          Herzig, Y. et al. Transcriptional programs that control expression of the autoimmune regulator gene Aire. Nature Immunology 18, 161-172, doi:10.1038/ni.3638 (2017).

 

 

 

 

 

 

Tags: aire, autoimmunity, immune tolerance, mtecs

doi: https://doi.org/10.1242/prelights.33911

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