Aberrant thymic architecture and enhanced interferon signaling in down syndrome

Abstract Disrupting the mechanisms of thymocyte development and central tolerance can severely affect the immune system and lead to autoimmune disease. It is well established that individuals with Down syndrome (DS) have an elevated risk of autoimmune disease, particularly celiac disease, type 1 diabetes, and autoimmune hypothyroidism. DS thymi exhibit significant cellular and structural perturbations, including alterations to thymic epithelial cells (TECs), thymocytes, and functional immune cell subsets resulting in diminution of the cortex, expansion of the medulla, and concurrent enlargement of Hassall's corpuscles. To better understand how the distinct characteristics of the DS thymus may contribute to immune dysregulation and autoimmunity, we performed single cell RNA-sequencing (scRNA-seq), spatial transcriptomics, and histological analyses on age-matched, discarded thymic tissue from infants (0-1 years old) with and without DS undergoing corrective heart surgery. Consistent with previous observations, histological analysis of DS thymi showed expansion of medullary regions by cytokeratin 5 (CK5) staining and increased numbers of atypical, enlarged Hassall's corpuscles, stained by involucrin (IVL), indicative of accelerated thymic involution. To determine how the DS thymic structure affects the composition and transcriptional programs of T cells, we performed scRNA-seq on sorted CD34+ and CD34- thymocytes from 9 non-DS and 6 DS individuals. Interestingly, analysis of the proportions of CD34+ and CD34- clusters revealed no consistent differences between non-DS and DS thymi, suggesting that trisomy 21 does not significantly alter thymocyte diversity. Yet, several transcriptional programs were perturbed. For instance, genes associated with T cell commitment/differentiation such as TCF7 and BCL11B were significantly upregulated in DS CD34+ progenitors. In the most immature DS CD34+ populations, CXCR4—an important chemokine receptor for thymic migration of early progenitors—was significantly downregulated. Also, DS CD4+ single positive (SP) and T regulatory cell (Treg) clusters showed an enrichment of a CD24hi vs CD24low Treg signature, suggesting a bias toward more immature Treg populations in DS thymus. The most striking gene signature enriched across nearly all DS thymocyte clusters was interferon signaling and interferon response. DS has been considered an interferonopathy based on the amplification of four interferon receptors and the interferon-induced GTP-binding protein MX1, all of which reside on chromosome 21. Indeed, we saw significant upregulation of MX1 and INFAR1. However, interferon response enrichment was not solely due to chromosome 21 genes, as other interferon response genes were upregulated, such as ISG1 and STAT1. Importantly, phospho-STAT1 was also increased in the DS thymic medulla, where clusters of activated cells can be readily observed by immunohistochemistry (IHC). Therefore, DS thymocytes appear to be in a chronic interferon-responsive state. While the DS thymus displays unique morphology, the proportion of DS thymocyte clusters by scRNA-seq were relatively unchanged. To determine if thymic cells are improperly organized in DS, we performed GeoMX spatial transcriptomic profiling of 7 pairs of non-DS and DS samples and used our scRNA-seq data to deconvolute cell types in regions of interests (ROI) from cortex, cortical medullary junction, and the medulla. Signatures for early double negative (DN) thymic progenitors and proliferating double positive (DP) cells were significantly enriched in the cortex and medulla of DS thymus, suggesting impaired spatiotemporal kinetics of thymocyte development, as DN and DP cells are typically confined to the cortex. IHC for TdT—a marker of immature thymic progenitors—revealed a variable increase in TdT+ cells in the medulla of DS thymus. Signatures for many antigen presenting cells in DS medullae were also significantly increased, including dendritic cells (DCs), B cells, and medullary TECs (mTECs). Similarly, we saw an increase in staining for CD11c+ DCs and CD123+ plasmacytoid DCs in the DS medulla, consistent with increased interferon signaling. Together, these findings show that the compartmentalization of differentiating thymocytes and support cells is significantly disorganized in the infant DS thymus, and that the consequences of hyperactive interferon response in DS extends to thymocyte production.