Medullary Cells Research Articles

1462-P: ASCL1—A Potentially Novel Type 1 Diabetes Gene

Background: New insights into type 1 diabetes (T1D) have recently come from studies of rare monogenic forms of the disease. Uncovering the genes that drive these monogenic forms can reveal mechanisms and druggable pathways in affected families that may be extended to the general T1D population. We have identified one such candidate gene: Achaete-scute complex 1 (ASCL1). ASCL1 encodes a basic helix-loop-helix (bHLH) transcription factor and is well-known for its role in neurogenesis, however it is less understood how this gene may impact immune tolerance. Recent studies have demonstrated that ASCL1 and its targets are expressed in medullary thymic epithelial cells (mTECs), which are key players in the removal of autoreactive T cells from the thymus. Methods: Whole exome sequencing (WES) was performed on individuals from our monogenic T1D registry and functional studies were performed in a human cell line using lentiviral vectors and bulk RNAseq. Results: We identified a novel coding mutation in the bHLH transcription factor ASCL1 in siblings with suspected monogenic T1D. WES revealed a heterozygous coding variant in ASCL1 that was predicted to be both deleterious and rare. This caused an amino acid substitution within the transcriptional activation domain of the ASCL1 protein. RNAseq analysis showed a broad reduction in the activation of gene targets in cells expressing the ASCL1 patient mutation. Conclusion: ASCL1 may play a role in central tolerance and be a new candidate gene for T1D. Disclosure K.C.Lavelle: None. K.Yang: None. J.Wang: None. C.Chamberlain: None. M.German: Advisory Panel; Encellin, Inc., Stock/Shareholder; ViaCyte, Inc. Funding The Leona M. and Harry B. Helmsley Charitable Trust; Larry L. Hillblom Foundation; Nora Eccles Treadwell Foundation; National Institute of Diabetes and Digestive and Kidney Diseases

Gonadal morphogenesis in the South American toad Rhinella Arenarum (Anura, Bufonidae) unveils an extremely delayed rate of sex differentiation.

Among anurans, Bufonids are recognized for their retarded sex differentiation. However, few studies have addressed gonadal morphogenesis in this family. Here, we analyzed the early gonadogenesis in laboratory-reared Rhinella arenarum. Few germ cells were identified in the genital ridge at Gosner stage 26. At metamorphosis, somatic cells and germ cells were observed in the outer region of the undifferentiated gonad, whereas the central region was occupied by stromal tissue. A cortico-medullary organization was first recognized on Day 7 postmetamorphosis. The cortex was composed of germ cells and encompassing epithelial cells, whereas the medulla contained cells presumptively derived from the coelomic epithelium. Medullary somatic cells formed metameric knots along the length of the undifferentiated gonad. Consequently, a series of 12-14 gonomeres became recognizable externally. The first sign of ovarian differentiation was observed on Day 15 postmetamorphosis, when a cavity was formed within each gonomere. In contrast, testes were recognized by a uniform distribution of germ cells and intermingled somatic cells, as the division into cortex and medulla was lost. By Day 50 postmetamorphosis, the gonadal metameric organization was still apparent both in the ovaries and testes. Follicles containing diplotene oocytes were observed within the ovary. In the testis, an incipient lobular architecture was recognized without initiation of meiosis within the seminiferous cords. These observations reveal an extremely delayed gonadal development in R. arenarum, not reported previously for other anuran species. In addition, the late differentiation of the gonads contrasted with the early appearance of follicles in the Bidder's organ. Lastly, we observed that delayed metamorphs exhibited an undifferentiated gonad, demonstrating that gonadogenesis in this species is more dependent on somatic development than on age.

Canine thyroid carcinomas: A review with emphasis on comparing the compact subtype of follicular thyroid carcinomas and medullary thyroid carcinomas.

Canine thyroid carcinomas are relatively common malignant endocrine neoplasms in dogs derived from either thyroid follicular cells (forming follicular thyroid carcinomas) or medullary cells (parafollicular, C-cells; forming medullary thyroid carcinomas). Older and recent clinical studies often fail to discriminate between compact cellular (solid) follicular thyroid carcinomas and medullary thyroid carcinomas, which may skew conclusions. The compact subtype of follicular thyroid carcinomas appears to be the least differentiated subtype of follicular thyroid carcinomas and needs to be differentiated from medullary thyroid carcinomas. This review includes information on the signalment, presentation, etiopathogenesis, classification, histologic and immunohistochemical diagnosis, clinical management, and biochemical and genetic derangements of canine follicular and medullary carcinomas, and their correlates with human medicine.

Acetate-Mediated Odorant Receptor OR51E2 Activation Results in Calcitonin Secretion in Parafollicular C-Cells: A Novel Diagnostic Target of Human Medullary Thyroid Cancer.

Medullary thyroid cancer originates from parafollicular C-cells in the thyroid. Despite successful thyroidectomy, localizing remnant cancer cells in patients with elevated calcitonin and carcinoembryonic antigen levels remains a challenge. Extranasal odorant receptors are expressed in cells from non-olfactory tissues, including C-cells. This study evaluates the odorant receptor signals from parafollicular C-cells, specifically, the presence of olfactory marker protein, and further assesses the ability of the protein in localizing and treating medullary thyroid cancer. We used immunohistochemistry, immunofluorescent staining, Western blot, RNA sequencing, and real time-PCR to analyze the expression of odorant receptors in mice thyroids, thyroid cancer cell lines, and patient specimens. We used in vivo assays to analyze acetate binding, calcitonin secretion, and cAMP pathway. We also used positron emission tomography (PET) to assess C11-acetate uptake in medullary thyroid cancer patients. We investigated olfactory marker protein expression in C-cells in patients and found that it co-localizes with calcitonin in C-cells from both normal and cancer cell lines. Specifically, we found that OR51E2 and OR51E1 were expressed in thyroid cancer cell lines and human medullary thyroid cancer cells. Furthermore, we found that in the C-cells, the binding of acetate to OR51E2 activates its migration into the nucleus, subsequently resulting in calcitonin secretion via the cAMP pathway. Finally, we found that C11-acetate, a positron emission tomography radiotracer analog for acetate, binds competitively to OR51E2. We confirmed C11-acetate uptake in cancer cells and in human patients using PET. We demonstrated that acetate binds to OR51E2 in C-cells. Using C11-acetate PET, we identified recurrence sites in post-operative medullary thyroid cancer patients. Therefore, OR51E2 may be a novel diagnostic and therapeutic target for medullary thyroid cancer.

Cutting Edge: Aire Is a Coactivator of the Vitamin D Receptor.

Vitamin D deficiency is associated with the development of autoimmunity, which arises from defects in T cell tolerance to self-antigens. Interactions of developing T cells with medullary thymic epithelial cells, which express tissue-restricted Ags, are essential for the establishment of central tolerance. However, vitamin D signaling in the thymus is poorly characterized. We find that stromal and hematopoietic cells in the mouse thymus express the vitamin D receptor (Vdr) and Cyp27b1, the enzyme that produces hormonal 1,25-dihydroxyvitamin D (1,25D). Treatment of cultured thymic slices with 1,25D enhances expression of the critical medullary thymic epithelial cell transcription factor autoimmune regulator (Aire), its colocalization with the Vdr, and enhances tissue-restricted Ag gene expression. Moreover, the Vdr interacts with Aire in a 1,25D-dependent manner and recruits Aire to DNA at vitamin D response elements, where it acts as a Vdr coactivator. These data link vitamin D signaling directly to critical transcriptional events necessary for central tolerance.

Crucial Roles of SATB1 in Regulation of Thymocyte Migration after Positive Selection.

Double-positive thymocytes that have passed positive selection migrate from the cortex to the medulla, where negative selection and the development of thymic regulatory T cells (tTregs) take place. Medullary thymic epithelial cells (mTECs) play important roles in these selections, and their differentiation and maintenance depend on interaction with positively selected CD4+ single-positive cells. Therefore, migration and differentiation after positive selection must be coordinated to establish immune tolerance. However, the regulatory mechanisms of these processes are not fully understood. SATB1 is a genome organizer highly expressed in double-positive thymocytes, and SATB1 deletion causes various defects in T-cell development, including impaired positive and negative selection and tTreg differentiation. Here, we show that SATB1 is critical for temporally coordinated thymocyte trafficking after positive selection in mice. Satb1 knockout (ΔSatb1) led to precocious thymic egress caused by augmented S1pr1 upregulation in positively selected thymocytes, accompanied by lower induction of Ccr7, Tnfsf11, and Cd40lg. Altered thymocyte trafficking and functionality affected the differentiation of mTECs and, in turn, tTreg differentiation. Thus, SATB1 is required to establish immune tolerance, at least in part, by ensuring timely thymic egress and mTEC differentiation.

Post-hatching developmental changes in the adrenal gland of the Japanese quail (Coturnix coturnix japonica): Histological, immunohistochemical, and electron microscopic studies.

The adrenal glands are paired abdominal endocrine organs vital to the bird's health. The present research aimed to provide a comprehensive examination of the histological, ultrastructural, and immunohistochemical investigations of the adrenal gland in Japanese quail during the post hatching period. The current study was performed on 21 healthy Japanese quail chicks at different post hatching periods. Our results showed the adrenal gland is surrounded by a connective tissue capsule, which consists of dense collagen fibers containing large blood vessels, chromaffin cells, autonomic ganglia, fibroblasts, and migrating Schwann cells. The zonation of the adrenal gland is composed of a subcapsular layer, a peripheral zone, and a central zone, which gets more pronounced with age. At the ultrastructural level, the interrenal cells take the steroid-secreting cells characters; they have varying amounts of lipid droplets and abundant mitochondria. Adrenal medullary chromaffin cells showed positive immunoreactivity to the NSE. With increasing age, the chromaffin tissue's Sox10 immunoreactivity increased. β-catenin is expressed within the plasmalemma and the cytoplasm of the interrenal and chromaffin cells and its reactivity increased with age, especially in the chromaffin cells. Our results indicate the adrenal gland undergoes significant morphological changes during the postnatal life. Overall, the postnatal period is an important time for the development and maturation of the adrenal glands.

Voltage-Gated Sodium Channel NaV1.7 Inhibitors with Potent Anticancer Activities in Medullary Thyroid Cancer Cells.

Our results from quantitative RT-PCR, Western blotting, immunohistochemistry, and the tissue microarray of medullary thyroid cancer (MTC) cell lines and patient specimens confirm that VGSC subtype NaV1.7 is uniquely expressed in aggressive MTC and not expressed in normal thyroid cells and tissues. We establish the druggability of NaV1.7 in MTC by identifying a novel inhibitor (SV188) and investigate its mode of binding and ability to inhibit INa current in NaV1.7. The whole-cell patch-clamp studies of the SV188 in the NaV1.7 channels expressed in HEK-293 cells show that SV188 inhibited the INa current in NaV1.7 with an IC50 value of 3.6 µM by a voltage- and use-dependent blockade mechanism, and the maximum inhibitory effect is observed when the channel is open. SV188 inhibited the viability of MTC cell lines, MZ-CRC-1 and TT, with IC50 values of 8.47 μM and 9.32 μM, respectively, and significantly inhibited the invasion of MZ-CRC-1 cells by 35% and 52% at 3 μM and 6 μM, respectively. In contrast, SV188 had no effect on the invasion of TT cells derived from primary tumor, which have lower basal expression of NaV1.7. In addition, SV188 at 3 μM significantly inhibited the migration of MZ-CRC-1 and TT cells by 27% and 57%, respectively.

Revisiting Aire and tissue-restricted antigens at single-cell resolution.

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.

CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance

Abstract Central tolerance ensures autoreactive thymocytes are either eliminated via clonal deletion or are diverted to the regulatory T-cell lineage, thus minimizing T cell autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their TCRs for autoreactivity against the diverse self-antigens displayed by medullary antigen presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection against some self-antigens, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, we use synchronized positive selection assays and 2-photon timelapse microscopy to demonstrate that CCR4 and CCR7 are expressed sequentially after positive selection and promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and promotes medullary entry and negative selection of mature thymocytes. Unlike CCR7 ligands, which are expressed by medullary thymic epithelial cells, CCR4 ligands are expressed by hematopoietic APCs, and we find that CCR4 enforces thymocyte tolerance to self-antigens presented by activated thymic APCs. Our findings demonstrate that CCR7 expression is neither necessary nor sufficient for thymocyte medullary entry and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of central tolerance, respectively, via interactions with distinct APC subsets. Supported by a grant from the NIH/NIAID (R01AI104870)

Identification of neuromuscular medullary thymic epithelial cells in thymoma with myasthenia gravis

Clinical and Experimental NeuroimmunologyAccepted Articles HIGHLIGHT Identification of neuromuscular mTEC (nmTEC) in thymoma with MG Tatsusada Okuno, Corresponding Author Tatsusada Okuno [email protected] Department of Neurology, Graduate School of Medicine, Osaka University Suita, Osaka, Japan Correspondence Tatsusada Okuno, 2-2 Yamadaoka, Suita 565-0871 TEL +81-6-6879-3571, Email: [email protected]Search for more papers by this authorYoshiaki Yasumizu, Yoshiaki Yasumizu Department of Neurology, Graduate School of Medicine, Osaka University Suita, Osaka, JapanSearch for more papers by this authorHideki Mochizuki, Hideki Mochizuki Department of Neurology, Graduate School of Medicine, Osaka University Suita, Osaka, JapanSearch for more papers by this author Tatsusada Okuno, Corresponding Author Tatsusada Okuno [email protected] Department of Neurology, Graduate School of Medicine, Osaka University Suita, Osaka, Japan Correspondence Tatsusada Okuno, 2-2 Yamadaoka, Suita 565-0871 TEL +81-6-6879-3571, Email: [email protected]Search for more papers by this authorYoshiaki Yasumizu, Yoshiaki Yasumizu Department of Neurology, Graduate School of Medicine, Osaka University Suita, Osaka, JapanSearch for more papers by this authorHideki Mochizuki, Hideki Mochizuki Department of Neurology, Graduate School of Medicine, Osaka University Suita, Osaka, JapanSearch for more papers by this author First published: 30 January 2023 https://doi.org/10.1111/cen3.12743AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Accepted ArticlesAccepted, unedited articles published online and citable. The final edited and typeset version of record will appear in the future. RelatedInformation

Discovery of RANKL and the current and future impact of its research

Discovery of RANKL (receptor activator of NF-κB ligand) had an impact on identification of the mechanisms regulating osteoclast differentiation and function, resulting in establishment of research field bridging bone biology and immunology (osteoimmunology), and development of a human anti-RANKL monoclonal antibody (denosumab). Denosumab has been clinically available for treatment of osteoporosis and cancer-induced bone diseases in many countries. Denosumab is a so-called blockbuster of which sales amount was 5.3 billion US dollars in 2021. I will discuss the intense competition between Amgen Inc. and us regarding discovery of RANKL. One of the recent topics is the identification of RANKL reverse signaling with a RANKL-binding peptide, W9 known as a RANKL antagonist. The RANKL reverse signaling stimulates differentiation of osteoblasts and bone formation. The findings revealed the RANKL-RANK (a receptor of RANKL) dual signaling in coupling between bone resorption and bone formation. Interestingly, W9 also stimulates differentiation of chondrocytes and repairs defect of articular cartilage regardless of RANKL. Identification of the mechanisms will be useful for development of pharmaceuticals treating osteoarthritis. I also suggest possible applications of anti-RANKL antibody (anti-RANKL) to the treatment of cancer patients. RANKL has an important role in development of medullary thymic epithelial cells (mTECs) establishing self-tolerance. Anti-RANKL potentiates anticancer immune responses thorough regeneration of tumor-reactive T-cells by inhibiting mTEC development. I expect the synergy of anti-RANKL and immune checkpoint inhibitors such as anti-CTLA-4 antibody and anti-PD-1 antibody for immuno-oncology. Several clinical trials are currently in progress. It is likely that cancers will not be incurable diseases in the near future.

Impaired thymic AIRE expression underlies autoantibodies against type I IFNs in humans with inborn errors of the alternative NF-κB pathway

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency display autoantibodies (auto-Abs) neutralizing type I IFNs, conferring a predisposition to life-threatening COVID-19 pneumonia. We report that patients with autosomal recessive NIK or RelB deficiency, or a specific type of autosomal dominant (AD) NF-κB2 deficiency also display neutralizing auto-Abs against type I IFNs. They are prone to severe viral disease, including life-threatening COVID-19 pneumonia, influenza pneumonia, and severe form of varicella. Among patients with AD NF-κB2 deficiency, these auto-Abs are found only in heterozygotes with variants that are both transcriptionally loss-of-function (p52 activity), due to impaired p100 processing into p52, and regulatory gain-of-function (IκBδ activity), due to accumulation of unprocessed p100, thus increasing the inhibitory IκBδ activity (p52LOF/IκBδGOF). Conversely, neutralizing auto-Abs against type I IFNs are not found in individuals heterozygous for NFKB2 variants causing either p100 and p52 haploinsufficiency (p52LOF/IκBδLOF), or p52 gain-of-function (p52GOF/IκBδLOF). Unlike patients with APS-1, patients with disorders of NIK, RelB, or NF-κB2 harbor very few other auto-Abs. Their thymuses are however abnormally structured, and their medullary thymic epithelial cells (mTECs) have defective AIRE expression. Human inborn errors of the alternative NF-κB pathway impair thymic AIRE expression in mTECs, thereby underlying the production of auto-Ab against type I IFNs and predisposition to viral diseases.

Thymic innate lymphoid cells promote thymocyte differentiation of transplanted hematopoietic progenitors in immunodeficient mice.

Abstract Patients with combined immunodeficiencies can be cured by intravenous transfer of allogeneic hematopoietic stem and progenitor cells (HSPC). However, complications such as an impaired and delayed T cell differentiation are unfortunately common, notably due to the defective thymic architecture in these patients and the requirement of HSPC homing to the thymus. To circumvent these problems, we have developed an innovative approach based on the direct targeting of HSPC into the thymus by intrathymic (IT) injection. Using ZAP70-deficient mice as a paradigm, we previously showed that IT injection of HSPC can support a long-term thymus-autonomous differentiation. Here, we show that the IT injection of WT HSPC results in a rapid engraftment and differentiation. Donor-derived thymocytes expand >100-fold, accounting for >13% of thymus cellularity by 3 weeks post IT transfer and importantly, engraftment occurs in the absence of cytoreductive conditioning. While mature CD4 thymocyte differentiation occurs in 3 weeks, the differentiation of mature Treg thymocytes requires 4 weeks. The kinetics of thymic Treg development parallels the generation of a thymic medulla, with the appearance of mature medullary thymic epithelial cells at 3–4 weeks post transplantation. Notably, the generation of the thymic medulla is associated with the differentiation of donor-derived RORγT+ innate lymphoid cells (ILC). While ILC are not required for physiological thymopoiesis, we find that they are critical for T cell differentiation in ZAP70-deficient mice presenting with an absence of the thymic medulla. These results may open new therapeutic avenues, promoting the differentiation of ILC via IT-mediated HSPC transfer, enhancing T cell differentiation. Supported by grants from ANR (CHIC) and AFM-Telethon

Chemotherapy-Induced Thymic Involution: An Ultrastructural Study

Abstract Endogenous thymic repair is quite complex, demanding coordinated thymocyte and thymic stromal responses for T cell development and establishment of central tolerance. While the importance of thymic stroma, notably cortical (cTEC) and medullary (mTEC) thymic epithelial cells, has been shown in gain- and loss-of-function studies, endogenous thymic epithelial repair has never been resolved ultrastructurally. Using Transmission Electron Microscopy (TEM), we analyzed morphometrically thymi in mice receiving Cyclophosphamide (CTX). In vehicle-treated mice, the cortex was populous with thymocytes neighboring cTEC, which extended long cytoplasmic processes into a dendritic meshwork, called “cytoreticulum”. In CTX-treated mice though, thymocytes were scarcely met in the cortex and TEC appeared more circular/ellipsoid. The cytoreticulum was evidently collapsed, increasing total contact surface area among cTEC/mTEC subsets. Despite that thymic macrophages primarily mediate clearance of thymocytes failing positive/negative selection, those in CTX-treated thymi were rich in secondary lysosomes and many were found phagocytosing TEC. The surviving TEC in CTX-treated mice had increased autophagolysosomes per surface area unit of TEC cytoplasm compared to vehicle-treated ones. Analysis via intensity thresholding revealed higher electron density of enclosed particles, consistent with the presence of partly-digested amorphous material, indicating membranous organelle self-digestion (i.e., stress macroautophagy). These data propose an emerging hypothesis that survival of thymic epithelium following cytotoxic insult is mediated via a collective stress response involving macroautophagy activation in TEC. Funded by: 1)AAI careers in immunology Fellowship - trainee 2)new investigator's start up funds (PI)

Cooperative but Distinct Role of Medullary Thymic Epithelial Cells and Dendritic Cells in the Production of Regulatory T Cells in the Thymus.

Regulatory T cells (Tregs) are produced in the thymus to establish self-tolerance, and agonistic stimuli by self-Ags play a pivotal role in this process. Although two types of APCs, medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), are responsible for presenting self-Ags together with costimulatory/cytokine signals, the distinct role of each APC in producing Tregs remains enigmatic. We have approached this issue by depleting the mTECs and DCs using mice expressing diphtheria toxin receptors driven by Aire and CD11c promoters, respectively. Depletion of mTECs showed an effect on Treg production quantitatively and qualitatively more profound than that of DCs followed by the development of distinct organ-specific autoimmune lesions in the hosts. Because self-Ags produced by mTECs are transferable to DCs through a process known as Ag transfer, we monitored the process of Ag transfer using mice expressing GFP from TECs. Although GFP expressed from total TECs was effectively transferred to DCs, GFP expressed from cortical TECs was not, suggesting that mTECs are the predominant source of self-Ags. We also found that GFP expressed not only from mature mTECs but also from immature mTECs was transferred to DCs, suggesting that a broad spectrum of molecules were subjected to Ag transfer during mTEC development. Interestingly, the numbers of recirculating non-Tregs producing IL-2, an important source for Treg expansion in the thymus, were reduced only in the mTEC-depleted mice. These results suggested the cooperative but distinct role of mTECs and DCs in the production of Tregs to avoid autoimmunity.

Embryonic keratin19+ progenitors generate multiple functionally distinct progeny to maintain epithelial diversity in the adult thymus medulla

The thymus medulla is a key site for immunoregulation and tolerance, and its functional specialisation is achieved through the complexity of medullary thymic epithelial cells (mTEC). While the importance of the medulla for thymus function is clear, the production and maintenance of mTEC diversity remains poorly understood. Here, using ontogenetic and inducible fate-mapping approaches, we identify mTEC-restricted progenitors as a cytokeratin19+ (K19+) TEC subset that emerges in the embryonic thymus. Importantly, labelling of a single cohort of K19+ TEC during embryogenesis sustains the production of multiple mTEC subsets into adulthood, including CCL21+ mTEClo, Aire+ mTEChi and thymic tuft cells. We show K19+ progenitors arise prior to the acquisition of multiple mTEC-defining features including RANK and CCL21 and are generated independently of the key mTEC regulator, Relb. In conclusion, we identify and define a multipotent mTEC progenitor that emerges during embryogenesis to support mTEC diversity into adult life.

Abstract 6662: Studying immune responses against human endogenous retroviruses in immune checkpoint blockade-treated lung adenocarcinoma

Abstract Background: Clinical response to immune checkpoint blockade (ICB) is predicated upon the existence of tumor-reactive T cells, but their precise targets remain poorly defined. Some patients with low tumor mutational burden (TMB) lung adenocarcinoma (LUAD) derive clinical benefit from ICB, suggesting that non-mutated antigens may serve as T cell targets. Human endogenous retroviruses (hERVs) can be differentially expressed in malignant cells due to epigenetic dysregulation and can be targeted by T cells. We hypothesize that the detection of T cell responses against hERVs correlates with a favorable clinical response to ICB in LUAD. Methods: Tumor tissues from patients with Stage I-III LUAD who underwent upfront surgical resection were enzymatically digested. Viable CD45-EPCAM+ cells were sorted. Total RNA was extracted and RNA sequencing was performed. The hervQUANT workflow was used to bioinformatically identify differentially-expressed, open reading frame (ORF)-encoding hERV transcripts. Publicly available RNAseq datasets derived from human medullary thymic epithelial cells (mTECs) and normal tissues were queried to exclude hERV transcripts expressed in these tissues. Overlapping peptide libraries (OPLs) spanning the shared hERV ORFs were synthesized and used in Functional Expansion of Specific T Cells (FEST) assays to detect hERV-specific T cell clones in the peripheral blood of a cohort of patients who met these criteria: (1) had advanced/metastatic LUAD, (2) were treated with ICB, (3) had peripheral blood mononuclear cells isolated at 4-8 weeks post-initiation of ICB and (4) had formal radiographic assessment of response to ICB. Results: We identified 41 differentially-expressed, ORF-encoding hERV transcripts that were shared by ≥4 (out of 11) CD45-EPCAM+ LUAD samples. OPLs covering hERV ORFs with mean expression of ≥ 3 CPM in CD45-EPCAM+ LUAD cells and <3 CPM in mTECs were synthesized. Low expression in normal tissues was also confirmed (<3 CPM). A total of 318 patients were screened and 30 patients (22 radiographic responders and 8 non-responders) met the inclusion criteria. The median age was 66.5 years for responders and 58 years for non-responders. The median PD-L1 TPS was 72.5% and 70%, respectively. The median TMB was 8 and 2.55, respectively. FEST assays utilizing hERV OPLs revealed 4 potential hERV-reactive T cell clonotypes in the peripheral blood of 2 patients with LUAD treated with ICB. Studies are ongoing to validate and characterize these clonotypes. Testing of the remainder of the selected patient cohort is also ongoing. Conclusions: Potential hERV-reactive T cell clonotypes are detectable in ICB-treated patients with LUAD. Correlation with clinical response to ICB will be evaluated upon validation of these clonotypes, but these results are promising findings in the ongoing efforts to identify immunogenic sources of non-mutated antigens in cancer. Citation Format: Khaled Sanber, Sydney Connor, Steven Vensko, Christopher Cherry, Zhen Zeng, Drew Pardoll, Patrick Forde, Benjamin Vincent, Kellie Smith. Studying immune responses against human endogenous retroviruses in immune checkpoint blockade-treated lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6662.

Adrenal Gland of Poultry: Anatomy, Microscopy, Morphometry, and Histochemistry

The adrenal gland plays a crucial role in poultry’s body. Its hormones affect growth, tissue differentiation, and metabolism regulation, as well as the bird body’s resistance to infections, intoxication, stress, and low temperature. For poultry farming, veterinary medicine, and ornithology, it is of scientific interest to study the morphological features of the adrenal gland of birds. This review aimed to assess poultry adrenal anatomy, microscopy, morphometry, and histochemistry by summarizing research data from various published articles. The structure of the adrenal gland has been morphologically investigated in clinically healthy chickens, ducks, geese, and quails. Data from the anatomical level of the adrenal gland have indicated that the shape of this organ in poultry of different species is not the same. In most cases, the shape of the adrenal gland of poultry is close to an oval, triangle, or pyramid. The color of the adrenal gland of poultry varies from gray to brown, which depends on the tissue saturation of this organ with carotenoids. The mass of the adrenal glands of poultry correlates with their age. The left adrenal gland has higher mass, volume, and length indicators than the right gland. The microscopic structure of the adrenal gland corresponds to the general laws of the structure and function of endocrine organs. However, the adrenal glands of poultry are characterized by class features of its histoarchitectonics. The adrenal capsule contains ganglia of the autonomic nervous system, the cell strands of cortical and medullary tissues are intertwined, and the configuration of these cell strands determines the formation of two or three zones of the adrenal gland. Studies of the adrenal glands of poultry at the cellular level have indicated that cortical tissue is represented by acidophilic cells and medullary tissue by basophilic cells. Depending on the shape and electron density of secretory granules, medullary tissue cells are divided into epinephrine and norepinephrine. Data on morphometric parameters (capsule thickness, area of cortical and medullary tissues, cortical-medullary ratio) of the adrenal gland are not the same and depend on the type, age, gender, and sexual activity of poultry. In conclusion, morphologists have paid great attention to studying the features of the anatomy, microscopy, morphometry, and histochemistry of the adrenal gland in clinically healthy poultry. Therefore, the presented data can be used to assess deviations in the morphofunctional state of the adrenal gland in poultry under the influence of various factors and pathology. Keywords: Adrenal gland, Anatomy, Morphological features, Histological and cellular levels, Poultry

Loss- and gain-of-function analyses reveal the essential role of Cyp19a1 in ovarian determination of the red-eared slider turtle.

Estrogen signaling exerts a decisive role in female sex determination and differentiation in chicken and fish. Aromatase encoded by Cyp19a1 is the key enzyme that catalyzes the conversion of androgen to estrogen. Correlative analyses implicate the potential involvement of aromatase in reptilian sexual development, however, the direct genetic evidence is lacking. Herein, we found that Cyp19a1 exhibited temperature-dependent sexually dimorphic expression, and located in the medullary somatic cells in early female embryos of the red-eared slider turtle (Trachemys scripta elegans), before the gonad is distinct. To determine the functional role of Cyp19a1 in turtle ovarian determination, we established loss- and gain-of-function models through in ovo lentivirus-mediated genetic manipulation. At female-producing temperature, inhibition of aromatase or knockdown of Cyp19a1 in turtle embryos resulted in female-to-male sex reversal, with the formation of a testis-like structure and a male distribution pattern of germ cells, as well as ectopic expression of male-specific markers (SOX9 and AMH) and disappearance of ovarian regulator FOXL2. On the contrary, overexpression of Cyp19a1 at male-producing temperature led to male-to-female sex reversal. In conclusion, our results suggest that Cyp19a1 is both necessary and sufficient for ovarian determination in the red-eared slider turtle, establishing causality and a direct genetic link between aromatase and reptilian sex determination and differentiation.