Thymic Medulla Research Articles

Spatial transcriptomics elucidates medulla niche supporting germinal center response in myasthenia gravis-associated thymoma

Myasthenia gravis (MG) is etiologically associated with thymus abnormalities, but its pathology in the thymus remains unclear. In this study, we attempt to narrow down the features associated with MG using spatial transcriptome analysis of thymoma and thymic hyperplasia samples. We find that the majority of thymomas are constituted by the cortical region. However, the small medullary region is enlarged in seropositive thymomas and contains polygenic enrichment and MG-specific germinal center structures. Neuromuscular medullary thymic epithelial cells, previously identified as MG-specific autoantigen-producing cells, are enriched in the cortico-medullary junction. The medulla is characterized by a specific chemokine pattern and immune cell composition, including migratory dendritic cells and effector regulatory Tcells. Similar germinal center structures and immune microenvironments are also observed in the thymic hyperplasia medulla. This study shows that the medulla and junction areas are linked to MG pathology and provides insights into future MG research.

Conventional dendritic cells are more activated in the hyperplastic Thymus of myasthenia gravis patients

IntroductionDendritic cells (DCs) are crucial to form ectopic germinal centers (GCs) in the hyperplastic thymus (HT), which are typically found in anti-acetylcholine receptor autoantibody-positive myasthenia gravis (MG) patients. However, the characteristics of such DCs in the HT and their roles in thymic hyperplasia formation remain unclear. MethodsWe collected thymic tissue from MG patients and patients who underwent cardiac surgery. The tissues were cut into sections for immunohistochemistry and immunofluorescence or digested into a single cell suspension for flow cytometry. ResultsIn addition to formation of ectopic GCs, we found that the proportion of the medulla in the thymic parenchyma was higher than that in the cortex (areacortex/areamedulla, 1.279 vs. 0.6576) in the HT of MG patients. The density of conventional dendritic cells (cDCs) in the HT was 131 ± 64.36 per mm2, whereas in normal thymic tissue, the density was 59.17 ± 22.54 per mm2. The more abundant cDCs expressed co-stimulatory molecules (CD80 and CD86) strongly. Moreover, the more abundant subset was mainly CD141+ DCs (cDC1s), accounting for an increase from 15% to 29%. However, these increased cDC1s appeared to be unrelated to Hassall's corpuscles and ectopic GCs. ConclusionThymic hyperplasia in MG patients is manifested as an increase in the proportion of the thymic medulla accompanied by increases in the density and functional activation as well as changes in the subset composition of cDCs.

Preparation of Single-cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis.

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the thymic cortex induce early T cell development and positive selection of cortical thymocytes. In contrast, medullary TECs (mTECs) in the thymic medulla attract positively selected thymocytes from the cortex and establish self-tolerance in T cells. A variety of molecules, including DLL4 and beta5t expressed in cTECs, as well as Aire and CCL21 expressed in mTECs, contribute to thymus function supporting T cell development and selection. Flow cytometric analysis of functionally relevant molecules in cTECs and mTECs is useful to improve our understanding of the biology of TECs, even though current methods for the preparation of single-cell suspensions of TECs can retrieve only a small fraction of TECs (approximately 1% for cTECs and approximately 10% for mTECs) from young adult mouse thymus. Because many of these functionally relevant molecules in TECs are localized within the cells, we describe our protocols for the preparation of single-cell suspension of mouse TECs and the staining of intracellular molecules for flow cytometric analysis.

Characterization of thymic architecture and lymphocyte populations in X-MAID due to an underlying pathogenic moesin mutation

Background: X-linked Moesin Associated Immunodeficiency (X-MAID) is a combined immunodeficiency caused by deficiency in the moesin protein. Moesin, which is encoded by the MSN gene, is part of the ezrin-radixin-moesin (ERM) family of transmembrane proteins that interact with the actin cytoskeleton and regulate the shape and migration of cells. Deficiency of moesin is associated with aberrant T cell migration and inadequate immune synapse formation, leading to significant immunodeficiency and recurrent infections. While the clinical presentation of X-MAID is diverse, to date, no thymus histopathology findings have been reported. Aim: Describe the thymus histopathology of a patient with X-MAID. Results: Our patient is a 10-year-old male who presented early in life with recurrent infections, dysmorphic features, and severe pulmonary venous stenosis which required a double lung transplant at the age of 4 years. Prior to transplant, he was referred to Immunology for assessment and was subsequently found to harbour a hemizygous variant in the MSN gene (c.278dupT; p.L93FfsX21). Thymus histopathology findings showed significant cortical atrophy and dysplasia and was accompanied by reduction in CD3+ cells in the cortex. Abnormally low numbers of suppressor T cells and T helper cells in the thymic cortex and medulla were noted. Conclusion: Thymic findings in X-MAID can include cortical atrophy, dysplasia, and decreased cellularity. This provides further evidence for the importance of moesin on T cell development and migration in the thymus. Statement of novelty: Description of thymus histopathology in a patient with X-MAID.

Stabilization of β-Catenin directs HEB to limit Thymic Selection

Abstract Activation of β-catenin in CD4+CD8+ (DP) thymocytes stalls progression through thymic selection and predisposes to transformation. Leukemogenesis but not the developmental block depends on Tcf-1, β-catenin’s DNA binding partner. Here we show that β-catenin deregulation directs the DNA-binding protein HEB to stall thymocyte development at the thymic selection stage. Conditional loss of HEB along with β-catenin activation restores the frequency of TCRβ+/CCR7+ DP thymocytes, with reduced CXCR4 expression indicating their potential to migrate to the thymic medulla. It also restores the emergence of TCRβ+/CD69+ DPs and enhances the overall CD69+DP expression profile that were diminished by activating β-catenin, indicating a functional response to TCR stimulation. β-Catenin stabilization redirects the binding of HEB to ~11,000 novel DNA sites throughout the genome. A major fraction of these sites occupy promoter regions of genes associated with cell cycle regulation and mitotic processes. HEB binding to those regions corelates with downregulation of the associated genes, and HEB inactivation restores their expression to physiologic levels. These findings highlight the complex molecular interplay between HEB and β-catenin in impairing thymic development.

Thymocyte migration and emigration

Hematopoietic precursors (HPCs) entering into the thymus undergo a sequential process leading to the generation of a variety of T cell subsets. This developmental odyssey unfolds in distinct stages within the thymic cortex and medulla, shaping the landscape of T cell receptor (TCR) expression and guiding thymocytes through positive and negative selection. Initially, early thymic progenitors (ETPs) take residence in the thymic cortex, where thymocytes begin to express their TCR and undergo positive selection. Subsequently, thymocytes transition to the thymic medulla, where they undergo negative selection. Both murine and human thymocyte development can be broadly classified into distinct stages based on the expression of CD4 and CD8 coreceptors, resulting in categorizations as double negative (DN), double positive (DP) or single positive (SP) cells. Thymocyte migration to the appropriate thymic microenvironment at the right differentiation stage is pivotal for the development and the proper functioning of T cells, which is critical for adaptive immune responses. The journey of lymphoid progenitor cells into the T cell developmental pathway hinges on an ongoing dialogue between the differentiating cell and the signals emanating from the thymus niche. Herein, we review the contribution of the key factors mentioned above for the localization, migration and emigration of thymocytes.

Cerebral and Peripheral Immune Cell Changes following Rodent Juvenile Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability. TBI is associated with neuroinflammation, but temporal changes in immune and inflammatory signaling following TBI have not been fully elucidated. Furthermore, there have been no previous studies on changes in immune cell populations following TBI via the Closed Head Injury Model of Engineered Rotational Acceleration (CHIMERA). The current study aimed to determine the time course changes to inflammatory marker mRNA expression in the acute period following TBI in juvenile rats and to determine acute changes to brain and circulating immune cell populations. For this study, post-natal day (PND)-30 male Long Evans rats sustained a TBI or Sham TBI and were euthanized at 0, 3, 6, 12, 24, or 96 h post-injury. Prefrontal cortex and hippocampus samples were used to determine mRNA expression changes of inflammatory factors. The mRNA expression of the pro-inflammatory cytokine TNF-α was significantly elevated at 6 h post-injury in both regions evaluated. To evaluate immune cell populations, male Long Evans rats were euthanized at 48 h post-injury, and brain and blood samples were used for cell sorting by marker-specific antibodies. In the peripheral blood, there was an elevation in CD3+ total T cells, CD45R+ total B cells, and CD3+CD4+ helper T cells in the TBI subjects. However, there were no changes to natural killer cells or CD3+CD8+ cytotoxic T cell populations. In the brain, there was a reduction in CD11b/c+ monocytes/macrophages, but no changes in other immune cell populations. At 48 h post-injury, the TBI subjects also demonstrated expansion of the thymic medulla. These changes in the cerebral and blood immune cell populations and thymic medulla expansion may implicate the subacute recovery timeframe as a vulnerable window for the immune system in the pediatric population.

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium.

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

H2-O deficiency promotes regulatory T cell differentiation and CD4 T cell hyperactivity.

Regulatory T cells (Treg) are crucial immune modulators, yet the exact mechanism of thymic Treg development remains controversial. Here, we present the first direct evidence for H2-O, an MHC class II peptide editing molecular chaperon, on selection of thymic Tregs. We identified that lack of H2-O in the thymic medulla promotes thymic Treg development and leads to an increased peripheral Treg frequency. Single-cell RNA-sequencing (scRNA-seq) analysis of splenic CD4 T cells revealed not only an enrichment of effector-like Tregs, but also activated CD4 T cells in the absence of H2-O. Our data support two concepts; a) lack of H2-O expression in the thymic medulla creates an environment permissive to Treg development and, b) that loss of H2-O drives increased basal auto-stimulation of CD4 T cells. These findings can help in better understanding of predispositions to autoimmunity and design of therapeutics for treatment of autoimmune diseases.

Assembling the thymus medulla: Development and function of epithelial cell heterogeneity.

The thymus is a unique primary lymphoid organ that supports the production of self-tolerant T-cells essential for adaptive immunity. Intrathymic microenvironments are microanatomically compartmentalised, forming defined cortical, and medullary regions each differentially supporting critical aspects of thymus-dependent T-cell maturation. Importantly, the specific functional properties of thymic cortical and medullary compartments are defined by highly specialised thymic epithelial cells (TEC). For example, in the medulla heterogenous medullary TEC (mTEC) contribute to the enforcement of central tolerance by supporting deletion of autoreactive T-cell clones, thereby counterbalancing the potential for random T-cell receptor generation to contribute to autoimmune disease. Recent advances have further shed light on the pathways and mechanisms that control heterogeneous mTEC development and how differential mTEC functionality contributes to control self-tolerant T-cell development. Here we discuss recent findings in relation to mTEC development and highlight examples of how mTEC diversity contribute to thymus medulla function.

Preclinical Study in Mouse Thymus and Thymocytes: Effects of Treatment with a Combination of Sodium Dichloroacetate and Sodium Valproate on Infectious Inflammation Pathways.

The research presents data from a preclinical study on the anti-inflammatory effects of a sodium dichloroacetate and sodium valproate combination (DCA-VPA). The 2-week treatment with a DCA 100 mg/kg/day and VPA 150 mg/kg/day combination solution in drinking water's effects on the thymus weight, its cortex/medulla ratio, Hassall's corpuscles (HCs) number in the thymus medulla, and the expression of inflammatory and immune-response-related genes in thymocytes of male Balb/c mice were studied. Two groups of mice aged 6-7 weeks were investigated: a control (n = 12) and a DCA-VPA-treated group (n = 12). The treatment did not affect the body weight gain (p > 0.05), the thymus weight (p > 0.05), the cortical/medulla ratio (p > 0.05), or the number of HCs (p > 0.05). Treatment significantly increased the Slc5a8 gene expression by 2.1-fold (p < 0.05). Gene sequence analysis revealed a significant effect on the expression of inflammation-related genes in thymocytes by significantly altering the expression of several genes related to the cytokine activity pathway, the inflammatory response pathway, and the Il17 signaling pathway in thymocytes. Data suggest that DCA-VPA exerts an anti-inflammatory effect by inhibiting the inflammatory mechanisms in the mouse thymocytes.

Morphofunctional features in mice treated by low and high Hsp70 doses

Aim. We sought to assess the effects of exogenous Hsp70 (single subcutaneous low- and high-dose injections) on organ structure and functions in adult mice.Materials and methods. We randomized CD1 90-day-old male mice (n = 30) to three groups (10 mice per group). We injected the animals with single subcutaneous saline solution for Group 1 (control), low dose (500 μg/kg) of recombinant human Hsp70 (HspA1A) for Group 2, and high dose (5000 μg/kg) of the Hsp70 for Group 3. We examined the behavior of the mice on Day 3 after the injections (distance traveled, velocity, and bowel movement number). We lethalized the mice on Day 5 with further histological study and morphometrics of cerebral cortex, thymus, spleen, and liver. The statistics included one-factor ANOVA test with post hoc Tukey test.Results. All study groups exhibited no significant difference of behavioral parameters. Some liver sinusoids were wider in control group and Hsp70 500 μg/kg group comparing to Hsp70 5000 μg/kg group. We obtained also data for morphometrics: no difference was found for the number of neurons in ganglionic cerebral cortex, the lymphocytic cellularity difference between thymic cortex and medulla, the number of lymphocytes in white splenic pulp, and the number of hepatocyte nuclei in the liver. Red splenic pulp exhibited 1774,5 ± 24,8, 1623,0 ± 26,7, 1553,6 ± 47,0 macrophages for control, low-dose and high-dose groups, respectively (р &lt; 0,0001). Tukey test showed a significant difference between control group and each of Hsp70 groups 500 μg/kg (р = 0,012) and 5000 μg/kg (р &lt; 0,0001).Conclusion. Our study revealed no negative impact of subcutaneous Hsp70 administration at low and high doses on organ structure and functions in mice.

Applications of EMbedding and backscattered scanning electron microscopy for morphological and functional assessment of the thymus forcedly removed in children with congenital heart defects during the first weeks of life

Thymectomy in a number of clinical situations is an unavoidable step of cardiac surgical treatment of congenital heart defects, but the issue of its effects on development of immunity in early ontogenesis remains open. There is still debates among scientists about the risk of immune deficiency states in children who underwent thymectomy at an early age. One may see completely different opinions, both in domestic and foreign publications. A tool for resolving this issue may be provided by morpho-functional studies of the removed thymic tissue by means of modern scanning electron microscopy techniques. The objective of our study was to assess the possibilities of EMbedding and backscattered scanning electron microscopy for morphological and functional evaluation of the thymus, which was forcedly removed in the infant patients with congenital heart defects over the first weeks of life. The thymus of a newborn infant (postnatal day 27) with congenital heart disease: ventricular septal defect was examined using EMbedding and backscattered scanning electron microscopy imaging after embedding in epoxy resin. The mass of thymus gland was 15.7 g, the dimensions of thymus were: transverse, 3.4 cm; longitudinal, 4.1 cm; thickness, 1.7 cm; volume, 12.4 cm3. The study showed ability of EMbedding and backscattered scanning electron microscopy in morphological and functional assessment of thymus gland considered the central organ of the immune system. Step-bystep visualization from low to high magnifications, from tissues to cells and intracellular structures, as well as layer-by-layer examination of thymic cortex, medulla, interlobular septa and vessels, allows you to effectively assess the functionality of the thymus. This research method is sufficient for scientific research of the forcedly removed thymus since it enables us to visualize its microanatomy, allowing cell phenotyping at different layers of the thymus, studying intercellular interactions of thymocytes with reticulo-epithelial cells, subtle features of Hassall’s bodies and, finally, the process of T lymphocytes’ release from thymus gland.

Testing the Limits of Endogenous Thymic Regeneration after HCT Conditioning

T cell reconstitution following allogeneic hematopoietic cell transplantation (allo-HCT), recently emerging as a major predictor of clinical outcomes, is critically dependent upon thymic function. Previous clinical studies have implicated a link between thymic function pre-transplant to patient outcomes post-bone marrow transplantation. Thymic function is primarily driven by two functionally distinct groups of thymic epithelial cells (TECs) located in the cortex (cTECs) and the medulla (mTECs). These two stromal compartments contribute to the various stages of T cell development, culminating in the generation of a broad repertoire of self-tolerant T cells leaving the thymus and emigrating into the periphery. However, despite the thymus' crucial role in generating a broad array of T cells, the organ is highly sensitive to damage. Condition regiments required for successful HCT, such as ionizing radiation, have a profound damaging effect on the thymus. Although the thymus is sensitive to damaging agents, it also has a remarkable self-repair capacity through mechanisms that are still incompletely understood; and the organ's regenerative capacity also declines with age. Moreover, the specific thymic epithelial subpopulation that contributes to thymus regeneration remains undefined. Therefore, understanding endogenous cellular mechanisms of thymus regeneration, and limitations to its repair, will be crucial for creating therapies to boost thymic function. Although thymic function was restored to pre-conditioning levels after one round of total body irradiation (TBI), and multiple rounds of corticosteroid (Dexamethasone) damage saw no decline in its restorative ability, thymic regeneration was severely reduced upon subsequent rounds of ionizing radiation damage (Fig. 1A). A phenomenon that one may expect to see in the small subset of patients that need to undergo a second HCT. Using single cell RNA sequencing of highly purified TECs at multiple consecutive days after TBI, we identified that shortly after damage the TEC compartment contracts into distinct TEC subpopulations, including a previously reported CD104 expressing mTEC1 subpopulation implicated to provide pre-cursor support to the thymic medulla (Borstein 2018 Nature). To further understand the fundamental differences in regeneration from these different damage modalities, we compared the regeneration of thymic epithelial cells after either dexamethasone (Dex) or TBI damage. Consistent with its progenitor role, we found a significant decrease in the mTEC1 subpopulation in mice damaged with TBI compared to those damaged with Dex. The striking distinction in the mTEC1 compartments between the damage models can potentially explain a key functional difference in how these two damage modalities differ in their regenerative kinetics (Fig 1B). Overall, the results of these studies implicate a potential contribution of a specific mTEC subset (mTEC1s) to modulating thymic regeneration kinetics, and that the thymus cannot withstand multiple rounds of irradiation. The identification of mTEC1s as crucial mediators of thymic regeneration can inform future work in designing thymic boosting therapies that induce this stromal population to accelerate thymus regeneration after HCT.

MSCs-IL33-ILC2s-AREG-Tecs Axis Promotes the Remodeling of Thymic Function in Acute Gvhd

Background: Previous studies suggested that mesenchymal stromal cells (MSCs) administrated at the acute graft versus host disease (aGVHD) phase effectively decreased the incidence and severity of chronic GVHD (cGVHD) by boosting thymic regeneration. Our research has found that MSCs expressing interleukin 33 (IL33) may participate in thymus regeneration from aGVHD damage through type 2 innate lymphoid cells (ILC2s)-mediated tissue recovery. However, the mechanism for ILC2s repairing the damaged thymus caused by aGVHD is unclear. Here, we further explored the mechanisms on how ILC2s promote thymus recovery post-BMT. Methods: We carried out studies in a aGVHD mice model of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). IL33 knocked-down (MSC/IL33 -) and empty-load MSCs (MSC/Control) were generated by lentivirus transfection, and then administrated intravenously at day 7 post-BMT to intervene in aGVHD and assess the effect of IL33 from MSCs on ILC2s (Lin-CD45+iCD3-CD127+GATA3+) in the thymus by flow cytometery at day 14 post-BMT. Clinical scores and survival were recorded once every days. Furthermore, we isolated, purified and amplified ILC2s from C57BL/6 mouse thymus by immunomagnetic bead sorting and flow cytometry for aGVHD intervention and studied whether ILC2s can home to thymus by fluorescence tracing and the effects of ILC2s on TECs by immunofluorescence. To study the potential mechanism of ILC2s affecting TECs by using transcriptome sequencing (RNA-seq) and related molecular biology experiments. Finally, the potential target amphiregulin (AREG) was found from RNA-seq analyses and the murine recombinant AREG was used as an intervention to evaluate its effect on aGVHD mouse. Results: In this study, we found that knocking down the expression of IL33 in MSCs partially blocked the repair effect of MSCs on thymus and decreased the expansion of thymic ILC2s in aGVHD mice compared with the MSC/Control group. Then, we found that exogenous infusion of thymic ILC2s is very effective in treating thymic tissue injury, and can home to the recipient thymus and further migrate to the thymic medulla region. Furthermore, we found that ILC2s may home to the thymus and migrate to the thymic medulla region through CCR7 and MSCs promote the high expression of AREG in ILC2s through RNA-seq. Importantly, we found that the expression level of intrathymic AREG in No-GVHD, MSCs-GVHD and ILC2s-GVHD group was significantly increased than aGVHD group on day 14 post-BMT and exogenous administration of AREG after transplantation can significantly increase the proliferation of thymic epithelial cells, especially the regeneration of thymic medulla and the generation of new thymus-derived CD4/CD8 double-positive T cells. Conclusion: In conclusion, we demonstrate that MSCs infusion may rely on the MSCs-IL33-ILC2s-AREG-TECs axis mediated by ILC2s to remodeling the thymic microenvironment homeostasis, thus promoting the reconstruction of T cell immune homeostasis. Importantly, the results presented in this report identify a previously unrecognized role for the epidermal growth factor family member AREG in promoting the remodeling of thymus tissue. and providing new insights for the mechanism of MSCs treatment for GVHD.

FRI054 Partial Lipodystrophy As A Manifestation Of Autoimmune Polyglandular Syndrome 1

Abstract Disclosure: S. Agarwal: None. C. Xing: None. M.S. Anderson: Stock Owner; Self; Medtronic Diabetes, Merck. A. Garg: None. Background: Autoimmune polyglandular syndrome 1 (APS1) is a rare autosomal recessive disorder due to mutations in AIRE gene that manifests mainly with chronic mucocutaneous candidiasis, primary hypoparathyroidism and adrenal insufficiency. We report an extremely rare APS1 patient who developed partial lipodystrophy during adulthood. Clinical Case: This 39-year-old white female developed frequent diaper rashes, oral thrush and tetany at the age of one year due to candidiasis and hypoparathyroidism. At 6 years, she developed hypothyroidism. At 11 years, she developed fatigue, excessive thirst, and anorexia due to primary adrenal insufficiency. At 13 years of age, she had thelarche and adrenarche but never attained menarche. At 14 years, she underwent a sibling matched allogenic bone marrow transplant due to multiple antibiotic-refractory fungal infections. At 35 years, her serum triglycerides level was 904 mg/dL and loss of subcutaneous fat from the upper and lower extremities and hips was noted but she had increased subcutaneous fat on the chest and abdomen as well as buffalo hump. Thigh skinfold thickness was 8 mm (&amp;lt;10th percentile of normal). A whole-body dual-energy X-ray absorptiometry revealed a total body fat of 38% with lower extremity fat of 25%-27% (&amp;lt;1st percentile of normal). Whole exome sequencing on DNA extracted from saliva revealed two pathogenic variants in AIRE gene; c.83T&amp;gt;C; p.Leu28Pro and c.769C&amp;gt;T; p.Arg257* confirming the diagnosis of APS1. No other pathogenic variants were noted in the known lipodystrophy genes. Serum was negative for anti-perilipin 1 autoantibody. Discussion: Biallelic mutations in the AIRE hinder the process of selective apoptosis of T-cells in the thymic medulla that have a strong affinity to self-antigens leading to autoimmunity manifesting as APS1. Previously, a 5-year-old boy with APS1 was reported to develop generalized lipodystrophy and his serum was positive for anti-perilipin 1 autoantibody. Recently, serum anti-perilipin-1 autoantibodies have also been reported in the Aire gene knock out mice. In contrast, our patient has partial lipodystrophy and was negative for anti-perilipin 1 autoantibody. This suggests that patients with APS1 may harbor many different autoantibodies against adipocyte expressed proteins and develop different patterns of autoimmune lipodystrophies. Presentation: Friday, June 16, 2023

Immunohistochemical developmental comparative study between thymus and spleen of Peacock(Pavo Cristatus)

In thymus, CD8 showed -ve expression at one week age. At one and two months old, strongly +ve were observed within the thymic parenchyma and around Hassall's corpuscles. At seven months old, CD8 showed variable degrees of +ve expression especially around blood vessels but less frequency than that of the younger ages due to the retarded activity of the thymus gland. CD268 cells showed –ve expressions in cortex and medulla of thymus at the 1st week of age . At the age of one month old Peacock the CD268 exhibited high degree of +ve appearance for B cells. At the age of two months old, CD268 displayed +ve expression in thymic cortex particularly around the septae. At age of seven months old Peacocks, the adult cellular population were formed the whole parenchyma of thymus gland therefore CD268 give slightly +ve expression in the cortex mainly around the septae. While in the spleen, CD8 were showed –ve expression at the age of one week Peacock poult . At the age of (1,2 and 7) months old spleen have great role in the immunity. CD268 indicated –ve expressions at one week of age, because of most of the cells at this age immatured. Few numbers of B lymphocytes were showing +ve expression at one month of age .At age of two months old Peacock B cells showed highly +ve expression. At age of seven months old Peacocks showed +ve expression, and highly increase in the number of mature B lymphocytes compared with other previous younger ages

Nitric oxide-dependent immunosuppressive function of thymus-derived mesenchymal stromal/stem cells

BackgroundThe thymus is required for T cell development and the formation of the adaptive immunity. Stromal cells, which include thymic epithelial cells (TECs) and mesenchymal stromal cells (MSCs), are essential for thymic function. However, the immunomodulatory function of thymus-derived MSCs (T-MSCs) has not been fully explored.MethodsMSCs were isolated from mouse thymus and their general characteristics including surface markers and multi-differentiation potential were characterized. The immunomodulatory function of T-MSCs stimulated by IFN-γ and TNF-α was evaluated in vitro and in vivo. Furthermore, the spatial distribution of MSCs in the thymus was interrogated by using tdTomato-flox mice corssed to various MSC lineage Cre recombinase lines.ResultsA subset of T-MSCs express Nestin, and are mainly distributed in the thymic medulla region and cortical-medulla junction, but not in the capsule. The Nestin-positive T-MSCs exhibit typical immunophenotypic characteristics and differentiation potential. Additionally, when stimulated with IFN-γ and TNF-α, they can inhibit activated T lymphocytes as efficiently as BM-MSCs, and this function is dependent on the production of nitric oxide (NO). Additionally, the T-MSCs exhibit a remarkable therapeutic efficacy in acute liver injury and inflammatory bowel disease (IBD).ConclusionsNestin-positive MSCs are mainly distributed in medulla and cortical-medulla junction in thymus and possess immunosuppressive ability upon stimulation by inflammatory cytokines. The findings have implications in understanding the physiological function of MSCs in thymus.

Endogenous CCL21-Ser deficiency reduces B16–F10 melanoma growth by enhanced antitumor immunity

The chemokine CCL21 regulates immune and cancer cell migration through its receptor CCR7. The Ccl21a gene encodes the isoform CCL21-Ser, predominantly expressed in the thymic medulla and the secondary lymphoid tissues. This study examined the roles of CCL21-Ser in the antitumor immune response in Ccl21a-knockout (KO) mice. The Ccl21a-KO mice showed significantly decreased growth of B16–F10 and YUMM1.7 melanomas and increased growth of MC38 colon cancer, despite no significant difference in LLC lung cancer and EO771 breast cancer. The B16–F10 tumor in Ccl21a-KO mice showed melanoma-specific activated CD8+ T cell and NK cell infiltration and higher Treg counts than wild-type mice. B16–F10 tumors in Ccl21a-KO mice showed a reduction in the positive correlation between the ratio of regulatory T cells (Tregs) to activated CD8+ T cells and tumor weight. In Ccl21a-KO tumor, the intratumoral Tregs showed lower co-inhibitory receptors TIM-3 and TIGIT. Taken together, these results suggest that endogenous CCL21-Ser supports melanoma growth in vivo by maintaining Treg function and suppressing antitumor immunity by CD8+ T cells.