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Despite the clear distinction between cortical (cTECs) and medullary thymic epithelial cells (mTECs) in physiology, the cell of origin of thymic carcinomas (TCs) and other thymic epithelial tumors remained enigmatic. We addressed this issue by focusing on AIRE, an mTEC-specific transcriptional regulator that is required for immunological self-tolerance. We found that a large proportion of TCs expressed AIRE with typical nuclear dot morphology by immunohistochemistry. AIRE expression in TCs was supported by the RNA-seq data in the TCGA-THYM database. Furthermore, our bioinformatics approach to the recent single-cell RNA-seq data on human thymi has revealed that TCs hold molecular characteristics of multiple mTEC subpopulations. In contrast, TCs lacked the gene signatures for cTECs. We propose that TCs are tumors derived from mTECs.

The thymus is a highly specialized organ that plays an indispensable role in the establishment of self-tolerance, a process characterized by the “education” of developing T-cells. To provide competent T-cells tolerant to self-antigens, medullary thymic epithelial cells (mTECs) orchestrate negative selection by ectopically expressing a wide range of genes, including various tissue-restricted antigens (TRAs). Notably, recent advancements in the high-throughput single-cell analysis have revealed remarkable heterogeneity in mTECs, giving us important clues for dissecting the mechanisms underlying TRA expression. We overview how recent single-cell studies have furthered our understanding of mTECs, with a focus on the role of Aire in inducing mTEC heterogeneity to encompass TRAs.

Background: Shellfish allergy is one of the most common food allergies. Recent studies have shown that sensitization to allergens via the skin is involved in the development of food allergies. In this study, a mouse model of shrimp allergy was generated by epicutaneous sensitization and used to identify skin conditions associated with susceptibility to sensitization. Methods: Four-week-old female BALB/c mice were sensitized by repeated application of 0.1 mg of tropomyosin to tape-stripped skin on days 0, 7, and 15, followed by a challenge on days 28 and 35. Results: Epicutaneously sensitized mice exhibited higher serum levels of tropomyosin-specific IgE on day 15 than control mice. After the oral challenge, model mice had higher anaphylaxis scores and lower rectal temperature. After three tape-strip treatments for sensitization, the skin was analyzed by Raman microscopy. The sensitized mice exhibited lower relative intensities of Raman bands at 399, 915, and 1073 cm−1 than control mice, which could be helpful noninvasive markers in screening for potential sensitization via the skin. Conclusions: An epicutaneous sensitization shellfish allergy model was generated. This model will be useful in studies to elucidate the pathogenesis of skin sensitization. Raman microscopy may also be valuable for capturing subtle skin changes leading to sensitization.

Ly6C comprises two homologous components of Ly6C1 and Ly6C2, and the expression of either of the Ly6C molecules defines unique functional subsets of monocytes. Ly6C is also expressed by other immune cell types, including Aire-expressing medullary thymic epithelial cells. Because the role of Ly6C expression in determining the functional subsets remains unclear, we generated mice deficient for both Ly6C1 and Ly6C2 with CRISPR-Cas9–mediated deletion. Mice deficient for Ly6C1/Ly6C2 showed no major alterations in the subsets and function of monocyte and other immune cells, including the cells involved in the dextran sulfate sodium salt–induced colitis model. By generating the mice deficient for Ly6C1 alone, we have also investigated the expression pattern of Ly6C1 and Ly6C2 in immune cells. Except for medullary thymic epithelial cells and CD4 single-positive T cells, immune cells predominantly expressed Ly6C2. Thus, despite the importance as a marker with a unique differential expression pattern, the Ly6C molecules have no major impact on determining the functional subsets and maintaining immune homeostasis.

Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare subtype of renal cell carcinoma. Although usually indolent, high-grade MTSCC has been reported to exhibit an aggressive clinical course. Herein, we report a case of high-grade renal MTSCC. An 86-year-old man visited our hospital with fever and fatigue. Based on contrast-enhanced computed tomography findings, the patient was diagnosed with clinical stage T2aN0M0 right renal cell carcinoma and underwent laparoscopic radical nephrectomy. Histological examination showed tubular to tubulopapillary structures accompanied by mucinous stroma, suggesting high-grade renal MTSCC. He remained recurrence- and metastasis-free 6 months after nephrectomy. Since high-grade renal MTSCC may have an aggressive clinical course, such patients should be observed carefully after radical nephrectomy.

The deficiency of Aire, a transcriptional regulator whose defect results in the development of autoimmunity, is associated with reduced expression of tissue-restricted self-Ags (TRAs) in medullary thymic epithelial cells (mTECs). Although the mechanisms underlying Aire-dependent expression of TRAs need to be explored, the physical identification of the target(s) of Aire has been hampered by the low and promiscuous expression of TRAs. We have tackled this issue by engineering mice with augmented Aire expression. Integration of the transcriptomic data from Aire-augmented and Aire-deficient mTECs revealed that a large proportion of so-called Aire-dependent genes, including those of TRAs, may not be direct transcriptional targets downstream of Aire. Rather, Aire induces TRA expression indirectly through controlling the heterogeneity of mTECs, as revealed by single-cell analyses. In contrast, Ccl25 emerged as a canonical target of Aire, and we verified this both in vitro and in vivo. Our approach has illuminated the Aire’s primary targets while distinguishing them from the secondary targets.

Deficiency for AIRE/Aire in both humans and mice results in the development of organ-specific autoimmune disease. We tested whether augmented and/or dysregulated AIRE/Aire expression might be also prone to the breakdown of self-tolerance. To define the effect of augmented Aire expression on the development of autoimmunity, antigen-specific clonal deletion and production of clonotypic regulatory T cells (Tregs) in the thymus were examined using mice expressing two additional copies of Aire in a heterozygous state (3xAire-knockin mice: 3xAire-KI). We found that both clonal deletion of autoreactive T cells and production of clonotypic Tregs in the thymus from 3xAire-KI were impaired in a T-cell receptor-transgenic system. Furthermore, 3xAire-KI females showed higher scores of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein than wild-type littermates, suggesting that augmented Aire expression exacerbates organ-specific autoimmunity under disease-prone conditions. In humans, we found that one patient with amyopathic dermatomyositis showed CD3–CD19– cells expressing AIRE in the peripheral blood before the treatment but not during the remission phase treated with immunosuppressive drugs. Thus, not only loss of function of AIRE/Aire but also augmented and/or dysregulated expression of AIRE/Aire should be considered for the pathogenesis of organ-specific autoimmunity. We suggest that further analyses should be pursued to establish a novel link between organ-specific autoimmune disease and dysregulated AIRE expression in clinical settings.

Various cells such as macrophages and hepatic stellate cells interact in the generation of fibrosis in nonalcoholic steatohepatitis (NASH), but the mechanism remains unclear. We employed a high fat/cholesterol/cholate (HFCC) diet to generate a model of NASH-related fibrosis and investigate the pathogenesis of fibrosis. Two mouse strains differing in susceptibility to obesity, the susceptible strain C57BL/6J (B6) and the relatively resistant strain A/J, developed hepatic histological features of NASH including fat deposition, intralobular inflammation, hepatocyte ballooning, and fibrosis after 9 weeks of HFCC diet feeding. The severity of hepatic inflammation and fibrosis was greater in A/J mice than in B6 mice. A/J mice fed the HFCC diet exhibited characteristic CD204-positive lipid-laden macrophage aggregation in hepatic parenchyma. Polarized light visualized the Maltese cross, namely cholesterol crystals within the aggregated macrophages. Moreover, fibrosis developed in a ring-shape from the periphery of the aggregated macrophages, i.e., the starting point of fibrosis could be visualized histologically. Furthermore, matrix assisted laser desorption/ionization mass spectrometry imaging analysis detected a molecule at m/z 772.462, which corresponds to the protonated ion of phosphatidylcholine (P-18:1 (11Z)/18:0) and phosphatidylethanolamine (18:0/20:2 (11Z, 14Z)), in aggregated macrophages in adjacent to the fibrotic lesions. In conclusion, the present HFCC diet-fed A/J model provides an ideal tool to study fibrogenesis and enables novel insights into the pathophysiology of NASH-related fibrosis.

Metabolic syndrome (MS) is a risk factor for type 2 diabetes mellitus, vascular inflammation, atherosclerosis, and renal, liver, and heart diseases. Non-alcoholic steatohepatitis (NASH) is a progressive representative liver disease and may lead to the irreversible calamities of cirrhosis and hepatocellular carcinoma. Metabolic disorders such as hyperglycemia have been broadly reported to be related to hepatocarcinogenesis in NASH; however, direct evidence of a link between hyperglycemia and carcinogenesis is still lacking. Tsumura Suzuki Obese Diabetic (TSOD) mice spontaneously develop metabolic syndrome, including obesity, insulin resistance, and NASH-like liver phenotype, and eventually develop hepatocellular carcinomas. TSOD mice provide a spontaneous human MS-like model, even with significant individual variations. In this study, we monitored mice in terms of their changes in blood glucose levels, body weights, and pancreatic and liver lesions over time. As a result, liver carcinogenesis was delayed in non-hyperglycemic TSOD mice compared to hyperglycemic mice. Moreover, at the termination point of 40 weeks, liver tumors appeared in 18 of 24 (75%) hyperglycemic TSOD mice; in contrast, they only appeared in 5 of 24 (20.8%) non-hyperglycemic mice. Next, we investigated three kinds of oligosaccharide that could lower blood glucose levels in hyperglycemic TSOD mice. We monitored the levels of blood and urinary glucose and assessed pancreatic lesions among the experimental groups. As expected, significantly lower levels of blood and urinary glucose and smaller deletions of Langerhans cells were found in TSOD mice fed with milk-derived oligosaccharides (galactooligosaccharides and lactosucrose). At the age of 24 weeks, mild steatohepatitis was found in the liver but there was no evidence of liver carcinogenesis. Steatosis in the liver was alleviated in the milk-derived oligosaccharide-administered group. Taken together, suppressing the increase in blood glucose level