mTEC damage risks immune recovery

Whether autologous hematopoietic stem cell transplantation is free from graft-versus-host disease is controversial. Alawam et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20211239) now demonstrate that prolonged damage in thymic medullary epithelial cells causes the failure in self-tolerance in newly generated T cells and provokes post-transplant autoimmunity.

Aire-dependent genes undergo Clp1-mediated 3’UTR shortening associated with higher transcript stability in the thymus

The ability of the immune system to avoid autoimmune disease relies on tolerization of thymocytes to self-antigens whose expression and presentation by thymic medullary epithelial cells (mTECs) is controlled predominantly by Aire at the transcriptional level and possibly regulated at other unrecognized levels. Aire-sensitive gene expression is influenced by several molecular factors, some of which belong to the 3’end processing complex, suggesting they might impact transcript stability and levels through an effect on 3’UTR shortening. We discovered that Aire-sensitive genes display a pronounced preference for short-3’UTR transcript isoforms in mTECs, a feature preceding Aire’s expression and correlated with the preferential selection of proximal polyA sites by the 3’end processing complex. Through an RNAi screen and generation of a lentigenic mouse, we found that one factor, Clp1, promotes 3’UTR shortening associated with higher transcript stability and expression of Aire-sensitive genes, revealing a post-transcriptional level of control of Aire-activated expression in mTECs.

Aire controls the expression of Prss29 in the thymus.

Aire, the autoimmune regulator, promotes promiscuous transcription of tissue-restricted antigen in the thymus for negative selection (1, 2). By studying the transcriptomes of the thymuses of Aire knock-out mice using two independent published datasets (3, 4), we found that Prss29 was among the genes whose expression most changes between wild-type and Aire knock-out mice in the medullary epithelial cells of the thymus. The expression of Prss29 was significantly decreased in the absence of Aire. Prss29 is an implantation-specific protease that is important for the processes of embryonic invasion and implantation (5). We conclude that Aire controls the expression of Prss29 in the thymus and suggest that this may function in immunological privilege of the fetus.

Interleukin 33 is differentially expressed in the thymus by developmental stage and cell type.

The thymus is the site of positive and negative selection (1-5). Comparing transcriptome data from cortical and medullary epithelial cells of the murine thymus (cTEC and mTEC) at 1, 3, and 6 months of age using a published dataset (6) revealed that the IL-1 family cytokine Interleukin 33 (IL-33) (7) was among the genes whose expression varied most significantly through mouse development in mTEC expressing low levels of the class II major histocompatibility complex MHC-II. At 6 months, the IL-33 was among the genes whose expression was most different between cell types. The alarmin IL-33 is differentially expressed between cells of the epithelium of the thymus and its expression is developmentally-patterned in the medulla of the thymus.

The transcriptional landscape of cTEC and mTEChi.

The thymus is the site of positive and negative selection, the processes by which lymphocytes are selected for that are capable, in their T-cell receptors, of adequately recognizing foreign antigen, and selected against in the event that, by their T-cell receptors, they recognize self antigen (1-16). It is anatomically segregated into a medulla and cortex, and epithelial cells in both the medulla and cortex function in the processes of negative and positive selection but there are limited studies assessing in an unbiased, systematic manner their relative basic transcriptional natures (17-26). In this study we compared the transcriptomes of cortical epithelial cells (cTEC) and medullary epithelial cells expressing high levels of the class II major histocompatibility complex (MHC-II) (mTEChi) (27). We previously described the major transcription factors and epigenetic machinery that uniquely describe cTEC and mTEChi (28) . Here, using a published dataset, we report a series of modules consisting of cell surface receptors, Fbox proteins, proteasomal components, cytochrome P450 components, ATP-related machinery of the lysosome and mitochondria, and matrix metalloproteinases that, by their differential expression, uniquely describe cTEC and mTEChi. These data will serve as a resource for future efforts towards molecular engineering of the thymus for transplant medicine and targeted treatment of autoimmunity.

Stage-selective differential expression of kinesin (Kif) genes in cortical and medullary epithelial cells of the murine thymus.

The thymus is an organ with immunologic functions conserved from mouse to man (1). Both fetal and mammalian thymuses exist (1). The processes of negative and positive selection (2) both occur in the thymus and are thought to be conducted in an anatomically distinct fashion, across development. We performed global differential gene expression profiling using a microarray dataset (6) of the murine cortical epithelial cell (cTEC) and medullary epithelial cell (mTEC) transcriptome at months 1, 3, and 6. This analysis revealed that multiple members of the kinesin (Kif) gene family were differentially expressed by cTECs and by mTEC that express high levels of the class II major histocompatibility complex, in a stage-selective manner, but not by mTEC that express low levels of the class II major histocompatibility complex. The products of Kif genes may serve some important motor or transport function in the cortical or medullary epithelium of the mammalian thymus.

Aire-dependent genes undergo Clp1-mediated 3’UTR shortening associated with higher transcript stability in the thymus

The ability of the immune system to avoid autoimmune disease relies on tolerization of thymocytes to self-antigens whose expression and presentation by thymic medullary epithelial cells (mTECs) is controlled predominantly by Aire at the transcriptional level and possibly regulated at other unrecognized levels. Aire-sensitive gene expression is influenced by several molecular factors, some of which belong to the 3’end processing complex, suggesting they might impact transcript stability and levels through an effect on 3’UTR shortening. We discovered that Aire-sensitive genes display a pronounced preference for short-3’UTR transcript isoforms in mTECs, a feature preceding Aire’s expression and correlated with the preferential selection of proximal polyA sites by the 3’end processing complex. Through an RNAi screen and generation of a lentigenic mouse, we found that one factor, Clp1, promotes 3’UTR shortening associated with higher transcript stability and expression of Aire-sensitive genes, revealing a post-transcriptional level of control of Aire-activated expression in mTECs.

Restriction of non-coding elements in the thymus by Aire

Aire is a unique transcriptional molecule that functions in immunological tolerance by promoting promiscuous expression of tissue-restricted genes. The expression of these otherwise tissue-restricted genes in the medullary epithelial cells of the thymus (mTECs) allows the presentation of self antigens to “teach” cells of the immune system what it means to be “self”. To understand the role of Aire in shaping the mTEC transcriptome, I performed global differential gene expression analysis of wild-type mTECs and mTECs deficient in Aire. I identified nineteen non-coding molecules whose expression were significantly modulated by the absence of Aire. These included pseudogenes, antisense RNA, small nucleolar and small nuclear RNA, and long non-coding RNA. This finding reveals that Aire controls the expression of both coding and non-coding genes, and further suggests that non-coding RNA might play a role in Aire’s instruction of immunological tolerance.