Thymic gene expression analysis reveals a potential link between HIF-1A and Th17/Treg imbalance in thymoma associated myasthenia gravis.

Myasthenia gravis (MG) is an immune-mediated disease frequently associated with thymic changes. Increased T helper 17 (Th17) cell activity and dysfunctional regulatory T (Treg) cells have been demonstrated in subgroups of MG. On the other hand, hypoxia-inducible factor 1 (HIF-1) has been shown to regulate the Th17/Treg balance by inducing Th17 differentiation while attenuating Treg development. To identify the underlying mechanisms of different thymic pathologies in MG development, we evaluated thymic samples from thymoma-associated myasthenia gravis (TAMG), MG with hyperplasia (TFH-MG) and thymoma without MG (TOMA) patients. Differential gene expression analysis revealed that TAMG and TFH-MG cells are associated with different functional pathways. A higher RORC/FOXP3 ratio provided evidence for Th17/Treg imbalance in TAMG potentially related to increased HIF1A. The hypoxic microenvironment in thymoma may be a driver of TAMG by increasing HIF1A. These findings may lead to new therapeutic approaches targeting HIF1A in the development of TAMG.

Congenital cystic neck mass of thymic origin.

Neck masses are frequently seen in children. The differential diagnosis includes infectious, congenital and neoplastic lesions. We report a case of rare thymic neck mass in a boy in his middle childhood presented with a history of a left anterolateral neck mass not associated with fever, dysphagia or shortness of breath. The radiographic evaluation showed a picture of a thymopharyngeal duct cyst. Thymic remnant and thymopharyngeal duct cyst are caused by the failure of obliteration and might appear as a lateral neck mass in children. The most effective treatment for a thymopharyngeal duct cyst is total surgical excision. This particular case highlights the importance for clinicians to have a high index of suspicion for a broad differential diagnosis when evaluating paediatric patients who present with neck mass. Additionally, we emphasise the importance of consistently considering thymopharyngeal cyst as differential diagnosis.

The role of thymic epithelium in thymus development and age-related thymic involution.

The establishment of an adaptive immune system is critical for protecting our bodies from neoplastic cancers and invading pathogens such as viruses and bacteria. As a primary lymphoid organ, the thymus generates lymphoid T cells that play a major role in the adaptive immune system. T cell generation in the thymus is controlled by interactions between thymocytes and other thymic cells, primarily thymic epithelial cells. Thus, the normal development and function of thymic epithelial cells are important for the generation of immunocompetent and self-tolerant T cells. On the other hand, the degeneration of the thymic epithelium due to thymic aging causes thymic involution, which is associated with the decline of adaptive immune function. Herein we summarize basic and current knowledge of the development and function of thymic epithelial cells and the mechanism of thymic involution. J. Med. Invest. 71 : 29-39, February, 2024.

Generating an organ-deficient animal model using a multi-targeted CRISPR-Cas9 system.

Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNAms system that induces cell death with a single-guide RNA (sgRNAms) targeting multiple sites in the genome. The Cas9-sgRNAms system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNAms at the Rosa26 locus (Foxn1Cas9; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1Cas9; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNAms system because Rosa26_ms could be maintained as homozygous.

Does Surgical Removal of the Thymus Have Deleterious Consequences?

The role of immunosenescence, particularly the natural process of thymic involution during aging, is increasingly acknowledged as a factor contributing to the development of autoimmune diseases and cancer. Recently, a concern has been raised about deleterious consequences of the surgical removal of thymic tissue, including for patients who undergo thymectomy for myasthenia gravis (MG) or resection of a thymoma. This review adopts a multidisciplinary approach to scrutinize the evidence concerning the long-term risks of cancer and autoimmunity postthymectomy. We conclude that for patients with acetylcholine receptor antibody-positive MG and those diagnosed with thymoma, the removal of the thymus offers prominent benefits that well outweigh the potential risks. However, incidental removal of thymic tissue during other thoracic surgeries should be minimized whenever feasible.

Effect of Flavonols of Aronia melanocarpa Fruits on Morphofunctional State of Immunocompetent Organs of Rats under Cyclophosphamide-Induced Immunosuppression.

Aronia melanocarpa berries contain many compounds with potential benefits for human health. The food flavonoids quercetin and rutin, found in significant amounts in the fruits of A. melanocarpa, are known to have favourable effects on animal and human organisms. However, data on the effect of flavonols isolated from black chokeberry on immune functions during immunosuppression are not available in the literature. Thus, the aim of this study was to evaluate the effect of flavonol fraction isolated from A. melanocarpa fruits, in comparison with pure quercetin and rutin substances, on the dysfunctional state of rat thymus and spleen in immunodeficiency. The study was performed on Wistar rats. The animals were orally administered solutions of the investigated substances for 7 days: water, a mixture of quercetin and rutin and flavonol fraction of A. melanocarpa. For induction of immunosuppression, the animals were injected once intraperitoneally with cyclophosphamide. Substance administration was then continued for another 7 days. The results showed that under the influence of flavonols, there was a decrease in cyclophosphamide-mediated reaction of lipid peroxidation enhancement and stimulation of proliferation of lymphocytes of thymus and spleen in rats. At that, the effect of the flavonol fraction of aronia was more pronounced.

Invariant γδTCR natural killer-like effector T cells in the naked mole-rat.

The naked mole-rat (Heterocephalus glaber) is a long-lived rodent species showing resistance to the development of cancer. Although naked mole-rats have been reported to lack natural killer (NK) cells, γδ T cell-based immunity has been suggested in this species, which could represent an important arm of the immune system for antitumor responses. Here, we investigate the biology of these unconventional T cells in peripheral tissues (blood, spleen) and thymus of the naked mole-rat at different ages by TCR repertoire profiling and single-cell gene expression analysis. Using our own TCR annotation in the naked mole-rat genome, we report that the γδ TCR repertoire is dominated by a public invariant Vγ4-2/Vδ1-4 TCR, containing the complementary-determining-region-3 (CDR3)γ CTYWDSNYAKKLF / CDR3δ CALWELRTGGITAQLVF that are likely generated by short-homology-repeat-driven DNA rearrangements. This invariant TCR is specifically found in γδ T cells expressing genes associated with NK cytotoxicity and is generated in both the thoracic and cervical thymus of the naked mole-rat until adult life. Our results indicate that invariant Vγ4-2/Vδ1-4 NK-like effector T cells in the naked mole-rat can contribute to tumor immunosurveillance by γδ TCR-mediated recognition of a common molecular signal.

Dynamics of somatostatin 4 receptor expression during chronic-stress loading and its potential as a chronic-stress marker.

Chronic stress has been implicated in mental illnesses and depressive behaviors. Somatostatin 4 receptor (SSTR4) has been shown to mediate anxiolytic and depression-like effects. Here, we aimed to explore the potential of SSTR4 as a diagnostic marker for chronic stress in mice. The mice were divided into single stress, chronic restraint stress, and control groups, and Sstr4 mRNA expression in the pituitary, lungs, and thymus, its protein expression in the thymus, were analyzed. Compared to controls, Sstr4 mRNA expression decreased significantly in the pituitary gland of the chronic and single-stress groups (P = 0.0181 and 0.0022, respectively) and lungs of the single-stress group (P = 0.0124), whereas it significantly increased in the thymus of the chronic-stress group (P = 0.0313). Thymic SSTR4 expression did not decrease significantly in stress groups compared to that in the control group (P = 0.0963). These results suggest that SSTR4 expression fluctuates in response to stress. Furthermore, Sstr4 mRNA expression dynamics in each organ differed based on single or chronic restraint stress-loading periods. In conclusion, this study suggests that investigating SSTR4 expression in each organ could allow for its use as a stress marker to estimate the stress-loading period and aid in diagnosing chronic stress.

Aire mediates tolerance to insulin through thymic trimming of high-affinity T cell clones.

Insulin is a central autoantigen in the pathogenesis of T1D, and thymic epithelial cell expression of insulin under the control of the Autoimmune Regulator (Aire) is thought to be a key component of maintaining tolerance to insulin. In spite of this general working model, direct detection of this thymic selection on insulin-specific T cells has been somewhat elusive. Here, we used a combination of highly sensitive T cell receptor transgenic models for detecting thymic selection and sorting and sequencing of Insulin-specific CD4+ T cells from Aire-deficient mice as a strategy to further define their selection. This analysis revealed a number of unique t cell receptor (TCR) clones in Aire-deficient hosts with high affinity for insulin/major histocompatibility complex (MHC) ligands. We then modeled the thymic selection of one of these clones in Aire-deficient versus wild-type hosts and found that this model clone could escape thymic negative selection in the absence of thymic Aire. Together, these results suggest that thymic expression of insulin plays a key role in trimming and removing high-affinity insulin-specific T cells from the repertoire to help promote tolerance.

Our experience with robotic-assisted thymic surgery.

Thoracic surgery is increasingly influenced by the development of minimally invasive approaches which have also influenced surgery in the area of the anterior mediastinum. The previously standard approach to the thymus via partial sternotomy was gradually replaced by the videothoracoscopic approach in most cases. In recent years, robotically assisted surgery has been gaining ground worldwide in this area, as well. The aim of our paper is to provide a comprehensive overview of procedures in the field of the thymus, including their indications, and to share our first experience with robot-assisted thymus surgery. At the 3rd Department of Surgery, since the start of the robot-assisted thymus surgery program, 23 thymectomies have been performed using this approach, of which 17 were performed for thymoma, 3 for myasthenia gravis, and 3 for parathyroid adenoma localized in thymus tissue. From our experience and the available data, it follows that the length of hospitalization, the rate of complications and the resulting effect of robot-assisted procedures is comparable to VTS procedures; however, the robot-assisted surgery also allows for mini-invasive treatment even in significantly obese patients and in patients with advanced thymic tumors who would otherwise be indicated for open thymectomy.

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.

Structure characterization and intestinal immune promotion effect of polysaccharide purified from Alhagi camelorum Fisch.

This study investigated the structure of acid Alhagi camelorum Fischa polysaccharide (aAP) and its impact on intestinal activity in mice. The results showed that aAP comprised of the fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid with the molar ratio of 0.81:14.97:10.84:11.14:3.26:0.80:0.80:54.92:2.47 with the molecular weight (Mw) of 22.734 kDa. Additionally, the composition of aAP was assessed via FT-IR, methylation, and NMR analyses, indicating that the backbone of the aAP was consisted of →4)-α-D-GalpA-6-OMe-(1 â†’ 4)-α-GalpA-(1 â†’ and →4)-α-D-GalpA-6-OMe-(1 â†’ 2)-α-L-Rhap-(1→, as well as →4)-ß-D-Galp- and →5)-α-L-Araf- for the branched chain. Furthermore, ICR mice underwent intragastric administration of different concentrations of aAP for 7 consecutive days. The results showed that aAP enhanced the murine spleen and thymus indices, promoted the secretion of serum lgG antibody, intestinal lgA antibody and intestinal cytokines, improved the morphology of intestinal villi and crypts, enhanced quantity of intestinal IELs and IgA+ cells, and activated T lymphocytes and DC cells in MLNs. In summary, these findings suggest that the utilization of aAP could enhance the immune response of the murine intestinal mucosa.

Foxo3 regulates cortical and medullary thymic epithelial cell homeostasis with implications in T cell development.

Within the thymus, thymic epithelial cells (TECs) create dedicated microenvironments for T cell development and selection. Considering that TECs are sensitive to distinct pathophysiological conditions, uncovering the molecular elements that coordinate their thymopoietic role has important fundamental and clinical implications. Particularly, medullary thymic epithelial cells (mTECs) play a crucial role in central tolerance. Our previous studies, along with others, suggest that mTECs depend on molecular factors linked to genome-protecting pathways, but the precise mechanisms underlying their function remain unknown. These observations led us to examine the role of Foxo3, as it is expressed in TECs and involved in DNA damage response. Our findings show that mice with TEC-specific deletion of Foxo3 (Foxo3cKO) displayed a disrupted mTEC compartment, with a more profound impact on the numbers of CCL21+ and thymic tuft mTEClo subsets. At the molecular level, Foxo3 controls distinct functional modules in the transcriptome of cTECs and mTECs under normal conditions, which includes the regulation of ribosomal biogenesis and DNA damage response, respectively. These changes in the TEC compartment resulted in a reduced total thymocyte cellularity and specific changes in regulatory T cell and iNKT cell development in the Foxo3cKO thymus. Lastly, the thymic defects observed in adulthood correlated with mild signs of altered peripheral immunotolerance in aged Foxo3cKO mice. Moreover, the deficiency in Foxo3 moderately aggravated the autoimmune predisposition observed in Aire-deficient mice. Our findings highlight the importance of Foxo3 in preserving the homeostasis of TECs and in supporting their role in T cell development and tolerance.

[The Effect of SIRT5 Deletion on Recovery of Hematopoietic Stem Cells after Injury in Mouse].

OBJECTIVE: To investigate the effect of deacylase Sirtuin 5 in the recovery of hematopoietic stem cells (HSCs) after treated by 5-FU in mouse. METHODS: Flow cytometry was used to analyze the effect of SIRT5 deletion on the proportion of hematopoietic stem/progenitor cells (HSPCs) in bone marrow (BM), the proportion of T cells, B cells and myeloid cells (TBM) in peripheral blood (PB) and spleen, and the development of T cells in thymus. Mouse were treated with 5-FU to study the effect of SIRT5 deletion on the cell cycle, apoptosis and the proportion of HSPCs in BM. The effect of SIRT5 deletion on the proliferation of HSCs was analyzed by flow sorting in vitro. RESULTS: SIRT5 deletion did not affect the development of T cells in thymus and the proportion of TBM cells in PB and spleen compared with wild type mice. SIRT5 deletion increased proportion of HSPCs in BM. After 5-FU treatment, the proportion of HSCs in SIRT5 deletion mice was significant decreased (P < 0.05), the HSPC in SIRT5 deletion mice was activated from G0 to G1 phase (P < 0.05), and the proportion of early apoptosis increased (P < 0.05). By monoclonal culture in vitro, the ability of HSCs to form clones in SIRT5 deletion mice was decreased significantly (P < 0.05). CONCLUSION: SIRT5 deletion lead to a decreased the ability of HSCs to clone in vitro. SIRT5 deletion is not conducive to the recovery of HSPCs injury in mice under hematopoietic stress.

Combined effects of radiation and simulated microgravity on intestinal tumorigenesis in C3B6F1 ApcMin/+ mice.

Explorations of the Moon and Mars are planned as future manned space missions, during which humans will be exposed to both radiation and microgravity. We do not, however, know the health effects for such combined exposures. In a ground-based experiment, we evaluated the combined effects of radiation and simulated microgravity on tumorigenesis by performing X-irradiation and tail suspension in C3B6F1 ApcMin/+ mice, a well-established model for intestinal tumorigenesis. Mice were irradiated at 2 weeks of age and underwent tail suspension for 3 or 11 weeks using a special device that avoids damage to the tail. The tail suspension treatment significantly reduced the thymus weight after 3 weeks but not 11 weeks, suggesting a transient stress response. The combination of irradiation and tail suspension significantly increased the number of small intestinal tumors less than 2 mm in diameter as compared with either treatment alone. The combined treatment also increased the fraction of malignant tumors among all small intestinal tumors as compared with the radiation-only treatment. Thus, the C3B6F1 ApcMin/+ mouse is a useful model for assessing cancer risk in a simulated space environment, in which simulated microgravity accelerates tumor progression when combined with radiation exposure.

[Salivary carcinoma showing thymus-like differentiation: clinicopathological analysis of 7 cases].

Objective: To analyze the clinicopathological features of salivary carcinoma showing thymus-like differentiation(CASTLE). Methods: Cases diagnosed with salivary CASTLE from January 2020 to December 2023 were collected and selected from the Department of Oral Pathology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. A total of 7 cases of salivary CASTLE were identified. All the cases originated from parotid. There were 3 males and 4 females. The patients' age range was 11-70 years.The clinical, microscopic, immunohistochemical and prognostic features of these cases were analyzed. Results: The duration of disease ranged from 1 month to 1 year, and 1 patient had facial numbness and 1 with swelling sensation occasionally. Radiographically, 4 cases showed malignant signs. Microscopically, 4 cases involved in parotid gland, and all the tumors had different degrees of lymphoid tissue background. The tumor cells arranged in nests, 5 cases with lymphoepithelial carcinoma-like and 2 cases with squamous cell carcinoma morphology. The tumor cells expressed CD5 and CD117 proteins diffusely in lymphoepithelial carcinoma-like cases. However, the tumor cells expressed CD5 diffusely and CD117 focally in cases with squamous cell carcinoma morphology. All the cases had no Epstein-Barr virus infection. Among the 6 patients with follow-up information, all of them underwent postoperative radiotherapy, and none of them had local recurrence and lymph node metastasis. Conclusions: Salivary CASTLE is a rare tumor, it should be distinguished from lymphoepithelial carcinoma and squamous cell carcinoma. The patients often have better prognosis and CD5 protein expression has a valuable role in the differential diagnosis.

Whole-transcriptome profiling reveals potential biomarkers for the reversal of thymic epithelial cell senescence by umbilical cord mesenchymal stem cells.

BACKGROUND: Reduced numbers and dysfunction of thymic epithelial cells (TECs) are important factors of thymic degeneration. Previous studies have found that umbilical cord mesenchymal stem cells (UCMSCs) reverse the structure and function of the senescent thymus in vivo. However, the transcriptomic regulation mechanism is unclear. METHODS: TECs were cultured with H2O2 for 72 hours to induce senescence. UCMSCs were cocultured with senescent TECs for 48 hours to detect SA-ß-gal, P16 and Ki67. The cocultured TECs were collected for lncRNA, mRNA and miRNA sequencing to establish a competitive endogenous regulatory network (ceRNA). And RT-qPCR, immunofluorescence staining, and western blot were used to identified key genes. RESULTS: Our results showed that H2O2 induced TEC aging and that UCMSCs reversed these changes. Compared with those in aged TECs, 2260 DE mRNAs, 1033 DE lncRNAs and 67 DE miRNAs were differentially expressed, and these changes were reversed by coculturing the cells with UCMSCs. Differential mRNA enrichment analysis of ceRNA regulation revealed that the PI3K-AKT pathway was a significant signaling pathway. UCMSC coculture upregulated VEGFA, which is the upstream factor of the PI3K-AKT signaling pathway, and the expression of the key proteins PI3K and AKT. Thus, the expression of the cell cycle suppressor P27, which is downstream of the PI3K-AKT signaling pathway, was downregulated, while the expression of the cell cycle regulators CDK2 and CCNE was upregulated. CONCLUSION: UCMSC coculture upregulated the expression of VEGFA, activated the PI3K-AKT signaling pathway, increased the expression of CDK2 and CCNE, decreased the expression of P27, and promoted the proliferation of TECs.

Is the exquisite specificity of lymphocytes generated by thymic selection or due to evolution?

We have previously argued that the antigen receptors of T and B lymphocytes evolved to be sufficiently specific to avoid massive deletion of clonotypes by negative selection. Their optimal 'specificity' level, i.e., probability of binding any particular epitope, was shown to be inversely related to the number of self-antigens that the cells have to be tolerant to. Experiments have demonstrated that T lymphocytes also become more specific during negative selection in the thymus, because cells expressing the most crossreactive receptors have the highest likelihood of binding a self-antigen, and hence to be tolerized (i.e., deleted, anergized, or diverted into a regulatory T cell phenotype). Thus, there are two -not mutually exclusive- explanations for the exquisite specificity of T cells, one involving evolution and the other thymic selection. To better understand the impact of both, we extend a previously developed mathematical model by allowing for T cells with very different binding probabilities in the pre-selection repertoire. We confirm that negative selection tends to tolerize the most crossreactive clonotypes. As a result, the average level of specificity in the functional post-selection repertoire depends on the number of self-antigens, even if there is no evolutionary optimization of binding probabilities. However, the evolutionary optimal range of binding probabilities in the pre-selection repertoire also depends on the number of self-antigens. Species with more self antigens need more specific pre-selection repertoires to avoid excessive loss of T cells during thymic selection, and hence mount protective immune responses. We conclude that both evolution and negative selection are responsible for the high level of specificity of lymphocytes.

Shorter telomere length increases the risk of lymphocyte immunodeficiency: A Mendelian randomization study.

BACKGROUND: For a long time, the prevailing viewpoint suggests that shorter telomere contribute to chromosomal instability, which is a shared characteristic of both aging and cancer. The newest research presented that T cell immune deficiency rather than chromosome instability predisposes patients with short telomere syndromes to some cancers. However, the relationship between genetically determined telomere length (TL) and immune cells remains unclear. METHODS: The two-sample Mendelian randomization analysis was conducted to elucidate the potential causal relationship. The genetic data of TL and immune cells were obtained from the Genome-Wide Association Study. The inverse variance weighted (IVW) method was used to estimate the effects primarily and another four methods were as a supplement. Sensitivity analysis was used to test the results. RESULTS: The IVW method showed a significant correlation between TL and the percentage of T cells in lymphocytes (odds ratio [OR]: 1.222, 95% confidence interval [CI]: 1.014-1.472, p = .035), indicating that shorter TL significantly increases the risk of low T cell percentage. Further analysis of T cell subsets indicated that shorter TL may primarily lead to a lower percentage of Natural Killer T cells (OR: 1.574, 95% CI: 1.281-1.935, p < .001). Analysis of B cell subsets revealed that shorter TL may be associated with a higher percentage of Naive-mature B cells, and a lower percentage of Memory B cells. And the sensitivity analysis indicated the validity and robustness of our findings. CONCLUSION: In summary, our findings suggest that shorter TL may be associated with a decline in the percentage of T cell, as well as impediments in the differentiation of B cell, consequently leading to the onset of immunosenescence and immunodeficiency. The relevant mechanisms and potential therapeutic avenues still need further investigation.