Medullary Epithelial Cells Research Articles

Abnormal Cellular Populations Shape Thymic Epithelial Tumor Heterogeneity and Anti-Tumor by Blocking Metabolic Interactions in Organoids.

A variety of abnormal epithelial cells and immature and mature immune cells in thymic epithelial tumors (TETs) affect histopathological features, the degree of malignancy, and the response to treatment. Here, gene expression, trajectory inference, and T cell antigen receptor (TCR)-based lineage tracking are profiled in TETs at single-cell resolution. An original subpopulation of KRT14+ progenitor cells with a spindle cell phenotype is shown. An abnormal infiltration of immature T cells with a TCR hyper-rearrangement state is revealed, due to the lack of CCL21+ medullary epithelial cells. For thymic carcinoma, the novel biomarkers of MSLN, CCL20, and SLC1A5 are identified and observed an elevated expression of LAG3 and HAVCR2 in malignant tumorn-infiltrating mature T cells. These common features based on the single-cell populations may inform pathological reclassification of TETs. Meanwhile, it is found that macrophages (MACs) attract thymic tumor cells through the LGALS9-SLC1A5 axis, providing them with glutamine to elicit metabolic reprogramming. This MAC-based metabolic pattern can promote malignancy progression. Additionally, an interactive immune environment in TETs is identified that correlates with the infiltration of abnormal FOXI1+ CFTR- ionocytes. Collectively, the data broaden the knowledge of TET cellular ecosystems, providing a basis for tackling histopathological diagnosis and related treatment.

Functionally diverse thymic medullary epithelial cells interplay to direct central tolerance

Medullary thymic epithelial cells (mTECs) are essential for the establishment of self-tolerance in Tcells. Promiscuous gene expression by a subpopulation of mTECs regulated by the nuclear protein Aire contributes to the display of self-genomic products to newly generated Tcells. Recent reports have highlighted additional self-antigen-displaying mTEC subpopulations, namely Fezf2-expressing mTECs and a mosaic of self-mimetic mTECs including thymic tuft cells. In addition, a functionally different subset of mTECs produces chemokine CCL21, which attracts developing thymocytes to the medullary region. Here, we report that CCL21+ mTECs and Aire+ mTECs non-redundantly cooperate to direct self-tolerance to prevent autoimmune pathology by optimizing the deletion of self-reactive Tcells and the generation of regulatory Tcells. We also detect cooperation for self-tolerance between Aire and Fezf2, the latter of which unexpectedly regulates thymic tuft cells. Our results indicate an indispensable interplay among functionally diverse mTECs for the establishment of central self-tolerance.

Clinical Correlation of Function and TCR vβ Diversity of MAGE-C2-Specific CD8+ T Cell Response in Esophageal Cancer.

Melanoma-associated Ag (MAGE)-C2, an immunogenic cancer germline (testis) Ag, is highly expressed by various tumor cells, thymic medullary epithelial cells, and germ cells. In this study, we aimed to explore the immunologic properties of MAGE-C2-specific CD8+ T cells and the relationship of its TCR β-chain V region (TCR vβ) subfamily distribution to prognosis of patients with esophageal cancer. PBMCs and tumor-infiltrating lymphocytes expanded by CD3/CD28 Dynabeads and MAGE-C2 peptides in vitro resulted in the induction of lysosome-associated membrane protein-1 (LAMP-1 or CD107a) on the cell surface and the production of IFN-γ by MAGE-C2-specific CD8+ T cells. We found differential TCR vβ subfamily distribution among flow-sorted CD107a+IFN-γ+ and CD107a-IFN-γ- CD8+ T cells. The proportion of CD107a+ and/or IFN-γ+ tetramer+ CD8+ T cells was lower in patients with lymph node metastasis, late tumor stage, and poorly differentiated state (p < 0.05). T-box transcription factor was positively correlated with CD107a and IFN-γ. Kaplan-Meier analysis showed that patients whose MAGE-C2-specific CD8+ T cells expressed high CD107a and/or IFN-γ had a longer survival time when compared with patients whose MAGE-C2-specific CD8+ T cells expressed low levels of CD107a and/or IFN-γ. Moreover, analysis of TCR vβ subfamily distribution revealed that a higher frequency of TCR vβ16 in MAGE-C2-specific CD8+ T cells was positively correlated with a better prognosis. These results suggest that the presence of functional MAGE-C2-specific CD8+ T cells had an independent prognostic impact on the survival of patients with esophageal cancer.

Immunohistochemical association with histological features of Hassall’s corpuscles in human fetal thymus

Background - Hassall’s corpuscles (HC) are balls of flattened medullary epithelial cells which are characteristic features of the thymus. Thymus is truly an ‘organ of mystery’ which has kept scholars in dark still continues to elucidate researchers till date. Aim- To study themicroscopic features of human fetal thymus along withthe differentiation and maturation process of the Hassall’s Corpuscles (HC) in human fetal thymus by using the immunohistochemical markers. Materials &amp; Methods- Total 20 aborted and still born fetuses ranging from 17-39 weeks of gestation were used for the study. After embalming, meticulous dissection, thymusgland was weighed and fixed with formalin. The various histological and histometric parameters were observed. Another set of microscopic slides were subjected to immunohistochemical studies by staining with primary antibodies (Cytokeratin, S100 and Vimentin) to study the presence of epithelial, histiocytic and mesenchymal components in fetal thymus and to know the nature of differentiation of the Hassall’s corpuscles, epithelial reticular cells and dendritic cells in the fetal thymus. Observations &amp; Results: Microscopic examination of serial section of thymus revealed few patterns of shapes of HC’s. Based on the morphological features, the corpuscles were classified as Solid Hassal Corpuscles (SHC), Cystic I (CHCI) and Cystic II(CHC II) and Degenerating(DHC). Epithelial cells of the HC were positive for cytokeratin which was less intensely positive in Solid Hassall Corpuscles (SHC) and intensely positive in primary and secondary cystic Hassall Corpuscles (CHC I and CHC II). The solid corpuscles (SHC) with homogenous mass were seen to be intensely positive whereas the central core in cystic varieties (CHC 1 &amp; CHC II) seems to be less positive with S 100 (marker for dendritic cells, histocytes and keratinocytes). Vimentin is intermediate cytoplasmic proteins expressed in the cells of mesenchymal differentiation found negative in the complete sections studied. Conclusion:The various types of HC encountered with different structural morphology could represent a stage of maturation in the developmental process. Hence HCs’ were arranged in a sequential order of development stages within the age group of the present study. The presence of Solid Hassall’s Corpuscle at the periphery of the medulla and Degenerating Hassall’s Corpuscle(DHC) at the central core of the medulla confirms the direction of maturation is from the periphery to the center of the medulla. Hence there is gradual increase in the expression of cytokeratin from solid to cystic Hassall’s corpuscles. Bangladesh Journal of Medical Science Vol. 21 No. 03 July’22 Page: 529-539

Gonadotropin-releasing hormone stimulation of annexin A5 expression in the thymus of male rats.

As gonadotropin-releasing hormone (GnRH) is expressed in the thymus, its direct action on thymic cells, including thymic involution, has been suggested. Annexin A5 (ANXA5), a biomarker of GnRH, was used to determine whether GnRH affects the thymus of male rats. Immunohistochemistry showed positive reactions for ANXA5 in large medullary epithelial cells at 30 days of age, and the expression continued until 180 days of age. Organ culture of thymus pieces was performed to examine the direct action of a GnRH agonist (GnRHa) on the expression of Anxa5 and Gnrh mRNA. Thymus tissues obtained from male rats (40–60 days old) were cut into small pieces (2–3 mm3) and incubated for 3 hr with the GnRHa. The expression levels of Anxa5 and Gnrh mRNA were augmented by the GnRHa. Immunohistochemistry of these tissue fragments showed that ANXA5 expression was enhanced, especially in medullary epithelial cells. These results revealed that GnRH synthesis in the thymus could affect thymic epithelial cells after puberty.

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.

CD8+ T Cells Variably Recognize Native Versus Citrullinated GRP78 Epitopes in Type 1 Diabetes.

In type 1 diabetes, autoimmune β-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Retracted: Primary Culture and Characterization of Human Renal Inner Medullary Collecting Duct Epithelial Cells.

Retracted: Primary Culture and Characterization of Human Renal Inner Medullary Collecting Duct Epithelial Cells.

MHCII-mediated antigen presentation by thymic stromal cells is required to enforce central T-cell tolerance

Abstract Central T-cell tolerance relies on elimination of self-reactive CD4+ T-cell clones in the thymus by either deletion or conversion to regulatory T cells (tTregs). Developing thymocytes are selected by exposure to tissue-specific antigens (TSAs) expressed by thymic medullary epithelial cells (mTECs) under the control of Autoimmune Regulator (Aire). TSA-derived peptides-MHCII complexes can be presented either directly by mTECs or indirectly by thymic DCs. Dissecting the contributions of these antigen presenting cell (APC) subsets to thymic tolerance has been hindered by the lack of appropriate genetic tools to selectively target presentation by mTECs. To address this, we created a mouse model in which MHCII can be inducibly ablated in Aire-expressing mTECs (iAire-CreERT2;MHCIIfl/fl). We show that treatment of these mice with tamoxifen leads to a complete loss of MHCII expression by Aire+ and post-Aire mTECs without affecting other thymic APCs or perturbing thymic homeostasis. In a TCR-restricted mouse model of negative selection, ablation of MHCII on mTECs led to a failure to delete self-reactive T cells, allowing escape and activation in the periphery. Surprisingly, we also found a defect in conversion of self-reactive CD4+ T cells to FoxP3+ tTregs. Similarly, in the diverse repertoire, loss of direct antigen presentation by mTECs resulted in tTreg reduction. In contrast, using CD11c-Cre;MHCIIfl/fl mice, in which MHCII is absent on thymic DCs but not mTECs, we found no disruption to negative selection or Treg generation. In sum, our data support a distinct and non-redundant role for direct antigen presentation by mTECs in shaping thymic T cell selection.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Restore Thymic Architecture and T Cell Function Disrupted by Neonatal Hyperoxia.

Treating premature infants with high oxygen is a routine intervention in the context of neonatal intensive care. Unfortunately, the increase in survival rates is associated with various detrimental sequalae of hyperoxia exposure, most notably bronchopulmonary dysplasia (BPD), a disease of disrupted lung development. The effects of high oxygen exposure on other developing organs of the infant, as well as the possible impact such disrupted development may have on later life remain poorly understood. Using a neonatal mouse model to investigate the effects of hyperoxia on the immature immune system we observed a dramatic involution of the thymic medulla, and this lesion was associated with disrupted FoxP3+ regulatory T cell generation and T cell autoreactivity. Significantly, administration of mesenchymal stromal cell-derived extracellular vesicles (MEx) restored thymic medullary architecture and physiological thymocyte profiles. Using single cell transcriptomics, we further demonstrated preferential impact of MEx treatment on the thymic medullary antigen presentation axis, as evidenced by enrichment of antigen presentation and antioxidative-stress related genes in dendritic cells (DCs) and medullary epithelial cells (mTECs). Our study demonstrates that MEx treatment represents a promising restorative therapeutic approach for oxygen-induced thymic injury, thus promoting normal development of both central tolerance and adaptive immunity.

Follistatin-like 1 deficiency impairs T cell development to promote lung metastasis of triple negative breast cancer.

Our study aims to detect the underlying mechanism of the suppressive effect of Follistatin-like 1 (FSTL1) on lung metastasis of triple negative breast cancer (TNBC). We found that FSTL1 had no effect on the proliferation and metastasis of 4T1 cells in vitro, while in the tumor-bearing Fstl1 heterozygous (Fstl1+/-) mice, the number of anti-tumor T lymphocytes in the lung was significantly reduced with the increase in lung metastasis. Impaired development of T cells can cause dysfunction of adaptive immune system, which promotes cancer metastasis. Therefore the effect of FSTL1 on T cell development was further investigated.Lower population of T cells in periphery and decreased proliferation of CD4- CD8- double negative (DN) thymocytes and impairment development of T cells were found in Fstl1+/- mice. Furthermore, high expression of FSTL1 in medullary thymus epithelial (mTEC) cells and decreased mRNA expression of inducible costimulator on activated T-cell ligand (Icosl) in mTECsh Fstl1 were detected. Combining other studies that the generation of ICOSL by mTEC cells promotes CD4+ single positive (SP) thymocytes to produce IL-2, which promotes T cell development. Our results indicate FSTL1 deficiency in mTEC cells impairs T cell development to promote the lung metastasis of TNBC.

Fibroblasts as a source of self-antigens for central immune tolerance.

Fibroblasts are one of the most common but also neglected types of stromal cells, the heterogeneity of which underlies the specific function of tissue microenvironments in development and regeneration. In the thymus, autoreactive T cells are thought to be negatively selected by reference to the self-antigens expressed in medullary epithelial cells, but the contribution of other stromal cells to tolerance induction has been poorly examined. In the present study, we report a PDGFR+ gp38+ DPP4- thymic fibroblast subset that is required for T cell tolerance induction. The deletion of the lymphotoxin β-receptor in thymic fibroblasts caused an autoimmune phenotype with decreased expression of tissue-restricted and fibroblast-specific antigens, offering insight into the long-sought target of lymphotoxin signaling in the context of the regulation of autoimmunity. Thus, thymic medullary fibroblasts play an essential role in the establishment of central tolerance by producing a diverse array of self-antigens.

Thymus aging in mice deficient in either EphB2 or EphB3, two master regulators of thymic epithelium development.

The epithelial microenvironment is involved in thymus aging, but the possible role of EphB receptors that govern the thymic epithelium development has not been investigated. Herein, we study the changes undergone by the thymus of EphB-deficient mice throughout their life. Immune alterations occurring throughout life were more severe in mutant than in wild-type (WT) mice. Mutant thymuses exhibit lower cellularity than WT ones, as well as lower proportions of early thymic progenitors cells and double-positive (CD4+ CD8+ ) thymocytes, but higher of double-negative (CD4- CD8- ) and single-positive (CD4+ CD8- , CD4- CD8+ ) cells. Throughout life, CD4+ naïve cells decreased particularly in mutant mice. In correlation, memory T cells, largely CD8+ cells, increased. Aged thymic epithelium undergoes changes including appearance of big epithelial free areas, decrease of K8+ K5- areas, which, however, contain higher proportions of Ly51+ UEA1- cortical epithelial cells, in correlation with reduced Aire+ medullary epithelial cells. Also, aged thymuses particularly those derived from mutant mice exhibited increased collagen IV, fat-storing cells, and connective cells. The absence of EphB accelerates the alterations undergone throughout life by both thymic epithelium and thymocytes, and the proportions of peripheral naïve and memory T cells, all of which are hallmarks of immune aging.

P0011THE ROLE OF AQUAPORIN-11 (AQP11) IN THE THYMUS DEVELOPMENT

Abstract Background and Aims Some aquaporins transport not only water but also small non-ionic molecules such as glycerol. In fact, aquaporin-9 (AQP9) has been shown to transport glycerol which is critical to support memory T cell survival through metabolic fitness (Cui G et al. Cell. 161:750, 2015). As AQP11 is widely expressed and transport glycerol as well as water, AQP11 may also play a role in immunological system, which will expand the repertoire of the aquaporin research field. Method The AQP11 expression in the thymus was examined by RT-PCR, Western blot and immunohistochemistry in mice. Phenotypic analysis and microarray analysis of the thymus were conducted in wild and AQP11 null mice at 21 day old before the development of uremia due to polycystic kidneys. Results Both mRNA and protein expressions of AQP11 in the thymus were higher than those of the kidney by RT-PCR and Western blot. Interestingly, the size of the thymus from AQP11 null mice was smaller than that of the wild mice: 28mg vs. 56mg, even after the correction by the body weight: 0.59 % vs. 0.82 %, as the body size of AQP11 null mice was smaller. The vacuolated medullary epithelial cells were observed in the thymus of AQP11 null mice with narrowed cortex. The immunohistochemical analysis revealed the AQP11 expression at the stromal-epithelial cells in the thymus. The microarray analysis of the gene expression in the thymus was compared between the wild and AQP11 null thymus by the annotation analysis based on the David Bioinformatics Resources 6.8 (beta). The up-regulated 66 genes more than 1.5 folds in AQP11 null thymus are mainly related to the PI3K/Akt signaling pathway to promote metabolism, proliferation, cell survival, growth and angiogenesis. On the other hand, the down-regulated 55 genes less than half in AQP11 null thymus are related to energy production and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Further RT-qPCR analysis confirmed the enhanced expression of Egfr (Epidermal Growth Factor Receptor), Itgb4 (Integrin beta-4) and Il2ra (interleukin 2 receptor alpha) and the diminished expression of aquaglyceroporin AQP7, Pck1 (Phosphoenolpyruvate carboxy-kinase 1) and Ucp1 (Mitochondrial uncoupling protein 1). The up-regulated genes for growth signaling was unexpected with the smaller thymus but may support the survival of the hypoplastic thymus as a compensation. On the other hand, the down-regulated genes may compromise fat-glucose-energy metabolism in the thymus leading to the smaller thymus, which may be aggravated by the decreased expression of AQP7, another glycerol channel. Conclusion AQP11 may play an important role in the metabolic control of the developing thymus possibly through its glycerol transport. The smaller thymus with narrower cortex in AQP11 null mice may lead to immunological dysfunction, which is currently under study.

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 is induced in oral squamous cell carcinoma and promotes cancer gene expression.

Autoimmune regulator (AIRE) is a transcriptional regulator that is primarily expressed in medullary epithelial cells, where it induces tissue-specific antigen expression. Under pathological conditions, AIRE expression is induced in epidermal cells and promotes skin tumor development. This study aimed to clarify the role of AIRE in the pathogenesis of oral squamous cell carcinoma (OSCC). AIRE expression was evaluated in six OSCC cell lines and in OSCC tissue specimens. Expression of STAT1, ICAM1, CXCL10, CXCL11, and MMP9 was elevated in 293A cells stably expressing AIRE, and conversely, was decreased in AIRE-knockout HSC3 OSCC cells when compared to the respective controls. Upregulation of STAT1, and ICAM in OSCC cells was confirmed in tissue specimens by immunohistochemistry. We provide evidence that AIRE exerts transcriptional control in cooperation with ETS1. Expression of STAT1, ICAM1, CXCL10, CXCL11, and MMP9 was increased in 293A cells upon Ets1 transfection, and coexpression of AIRE further increased the expression of these proteins. AIRE coprecipitated with ETS1 in a modified immunoprecipitation assay using formaldehyde crosslinking. Chromatin immunoprecipitation and quantitative PCR analysis revealed that promoter fragments of STAT1, ICAM1, CXCL10, and MMP9 were enriched in the AIRE precipitates. These results indicate that AIRE is induced in OSCC and supports cancer-related gene expression in cooperation with ETS1. This is a novel function of AIRE in extrathymic tissues under the pathological condition.

Identification of an Intronic Regulatory Element Necessary for Tissue-Specific Expression of Foxn1 in Thymic Epithelial Cells.

The thymus is critical for the establishment of the adaptive immune system and the development of a diverse T cell repertoire. T cell development depends upon cell-cell interactions with epithelial cells in the thymus. The thymus is composed of two different types of epithelial cells: cortical and medullary epithelial cells. Both of these express and critically depend on the transcription factor Foxn1 Foxn1 is also expressed in the hair follicle, and disruption of Foxn1 function in mice results in severe thymic developmental defects and the hairless (nude) phenotype. Despite its importance, little is known about the direct regulation of Foxn1 expression. In this study, we identify a cis-regulatory element (RE) critical for expression of Foxn1 in mouse thymic epithelial cells but dispensable for expression in hair follicles. Analysis of chromatin accessibility, histone modifications, and sequence conservation identified regions within the first intron of Foxn1 that possessed the characteristics of REs. Systematic knockout of candidate regions lead us to identify a 1.6 kb region that, when deleted, results in a near total disruption of thymus development. Interestingly, Foxn1 expression and function in the hair follicle were unaffected. RNA fluorescent in situ hybridization showed a near complete loss of Foxn1 mRNA expression in the embryonic thymic bud. Our studies have identified a genomic RE with thymic-specific control of Foxn1 gene expression.

Some histological studies on thymus gland of mature and senile rabbits

The present study was carried out to investigate some histological changes of thymus gland of mature and senile rabbits. The study was carried out on fifteen apparently healthy mature (six-month-old) and senile (twelve-month-old) rabbits which were humanlysacrificed, eviscerated and subsequently tissue specimens from the thymuswere taken.It was found that the thymus of mature rabbits was lobulated organ consisted of stroma and parenchyma, the stroma consisted of connective tissue capsule and septa. The septal connective tissue rich in collagen fibers and contain low elastic fibers. Within the septal connective tissue found the muscular artery which characterized by duplicated internal elastic lamina. The artery branched to give small arteriole. Also, septa contained medium size vein. Parenchyma consisted of peripheral dark cortex and central light stained medulla. The cortex heavily populated by small lymphocytes which had dark nucleus and also contained epithelial reticular cells which had large vesicular nucleus. Medulla contained large lymphocytes, epithelial reticular cells and Hassall’s corpuscles. The medullary epithelial reticular cells showed positive reaction for PAS stain. The thymus of senile rabbits showed signs ofinvolution, but it not completely disappeared. There were some changes, occurredthinning and irregularity of thymic lobule, progressive cortical thymocyte depletion, thymic cyst formation and lipid infiltrationwithin the capsule and septa. The present study revealed that thymus was present through all the life, after maturity occurred involution but complete absence of thymus not occurred.

Autoimmune Regulator is required in female mice for optimal embryonic development and implantation†.

Autoimmune Regulator (AIRE) regulates central immune tolerance by inducing expression of tissue-restricted antigens in thymic medullary epithelial cells, thereby ensuring elimination of autoreactive T cells. Aire mutations in humans and targeted Aire deletion in mice result in multiorgan autoimmune disease, known in humans as autoimmune polyglandular syndrome type 1 (APS-1). APS-1 is characterized by the presence of adrenal insufficiency, chronic mucosal candidiasis, and/or hypoparathyroidism. Additionally, females often present with gonadal insufficiency and infertility. Aire-deficiency (KO) in mice results in oophoritis and age-dependent depletion of follicular reserves. Here, we found that while the majority of young 6-week-old Aire-KO females had normal follicular reserves, mating behavior, and ovulation rates, 50% of females experienced embryonic loss between gestation day (GD) 5.5 and 7.5 that could not be attributed to insufficient progesterone production or decidualization. The quality of GD0.5 embryos recovered from Aire KO mice was reduced, and when cultured in vitro, embryos displayed limited developmental capacity in comparison to those recovered from wild-type (WT) mice. Further, embryos flushed from Aire KO dams at GD3.5 were developmentally delayed in comparison to WT controls and had reduced trophoblastic outgrowth in vitro. We conclude that AIRE does not play a direct role in uterine decidualization. Rather, reduced fertility of Aire-deficient females is likely due to multiple factors, including oophoritis, delayed preimplantation development, and compromised implantation. These effects may be explained by autoimmune targeting of the ovary, embryo, or both. Alternatively, altered embryonic development could be due to a direct role for AIRE in early embryogenesis.

An Improved Method for Cell Type-Selective Glycomic Analysis of Tissue Sections Assisted by Fluorescence Laser Microdissection.

Lectin microarray (LMA) is a highly sensitive technology used to obtain the global glycomic profiles of endogenous glycoproteins in biological samples including formalin-fixed paraffin-embedded tissue sections. Here, we describe an effective method for cell type-selective glycomic profiling of tissue fragments collected by laser microdissection (LMD) under fluorescent histochemical visualization. We optimized each step of histochemical staining and confirmed the reliability and validity of glycomic profiling. Using the optimized procedure, glycomic profiles were obtained with 0.5 mm2 of stained thymic sections (5-μm-thick) from 8-week-old C57BL/6J male mice. The glycomic profiles of Ulex europaeus agglutinin-I (UEA-I)-stained medullary regions showed higher UEA-I signals than those of the morphologically determined medulla regions, indicating the utility of this method for UEA-I(+) cell-selective analysis. To further evaluate this method, tissue fragments was serially collected from stained and unstained areas of medullary epithelial cell probes (UEA-I and anti-cytokeratin 5 antibody) and a cortex-staining probe (peanut agglutinin). The medullary regions assigned by the three probes showed significantly different glycomic profiles, highlighting the difference in subpopulation recognition among the three probes, which was consistent with previous reports. In conclusion, our fluorescence LMD-LMA method enabled cell type-selective tissue glycomic analysis of pathological specimens and animal models, especially for glyco-biomarker discovery.