Quantification of T-Cell Receptor Excision Circles (TRECs).

Aging is a natural process that compromises the immune system's functionality increasing the risk of infectious, tumors, and autoimmune diseases. The thymus involution is an age-dependent process characterized by decreased cellularity, peripheral lymphocyte infiltration into the perivascular space, and expansion of adipose tissue. All those modifications hamper the functionality of the organ and lead to a decline of naïve T-cell production with a shrinking of the T-cell repertoire. Thymus atrophy is described in several disorders including autoimmune diseases. The quantification of T-cell receptor excision circles (TRECs) in recent thymus emigrants is a standard procedure to investigate the thymic function. In this chapter, we discuss the methodology used to quantify this molecule in peripheral blood mononuclear cells and isolated CD4+ and CD8+ T cells.

Selenium deficiency exacerbates ROS/ER stress mediated pyroptosis and ferroptosis induced by bisphenol A in chickens thymus.

Bisphenol A (BPA) is an industrial pollutant that can cause immune impairment. Selenium acts as an antioxidant, as selenium deficiency often accompanies oxidative stress, resulting in organ damage. This study is the first to demonstrate that BPA and/or selenium deficiency induce pyroptosis and ferroptosis-mediated thymic injury in chicken and chicken lymphoma cell (MDCC-MSB-1) via oxidative stress-induced endoplasmic reticulum (ER) stress. We established a broiler chicken model of BPA and/or selenium deficiency exposure and collected thymus samples as research subjects after 42 days. The results demonstrated that BPA or selenium deficiency led to a decrease in antioxidant enzyme activities (T-AOC, CAT, and GSH-Px), accumulation of peroxides (H2O2 and MDA), significant upregulation of ER stress-related markers (GRP78, IER 1, PERK, EIF-2α, ATF4, and CHOP), a significant increase in iron ion levels, significant upregulation of pyroptosis-related gene (NLRP3, ASC, Caspase1, GSDMD, IL-18 and IL-1ß), significantly increase ferroptosis-related genes (TFRC, COX2) and downregulate GPX4, HO-1, FTH, NADPH. In vitro experiments conducted in MDCC-MSB-1 cells confirmed the results, demonstrating that the addition of antioxidant (NAC), ER stress inhibitor (TUDCA) and pyroptosis inhibitor (Vx765) alleviated oxidative stress, endoplasmic reticulum stress, pyroptosis, and ferroptosis. Overall, this study concludes that the combined effects of oxidative stress and ER stress mediate pyroptosis and ferroptosis in chicken thymus induced by BPA exposure and selenium deficiency.

S1PR1 mediates Th17 cell migration from the thymus to the skin in health and disease.

Th17 cells play crucial roles in host defense and the pathogenesis of autoimmune diseases in the skin. While their differentiation mechanisms have been extensively studied, the origin of skin Th17 cells remains unclear. In this study, we analyzed single-cell RNA-sequencing data and identify the presence of Th17 cells in the human thymus. Thymic Th17 cells were characterized by high expression levels of Sphingosine-1-Phosphate Receptor 1 (S1PR1), a receptor crucial for T cell egress from lymphoid tissues. In mice, Th17 cell-specific knockout of S1pr1 resulted in the accumulation of Th17 cells in the thymus and a corresponding decrease in their numbers in the skin. Th17 cells that accumulated in the thymus exhibited a lower IL-17A production capacity compared to those in the skin, indicating that the local environment in the skin is important for maintaining the Th17 cell phenotype. Additionally, using a murine psoriasis model, we demonstrated that Th17 cell-specific knockout of S1pr1 reduced their migration to the inflamed skin, thereby ameliorating disease progression. Collectively, our data suggest that S1PR1 mediates Th17 cell migration from the thymus to the skin, thereby modulating their functional engagement in both homeostatic and inflammatory conditions.

Case report: Artificial thymic organoids facilitate clinical decisions for a patient with a TP63 variant and severe persistent T cell lymphopenia.

Pathogenic variants in the transcription factor TP63 are associated with clinically overlapping syndromes including ectrodactyly-ectodermal dysplasia clefting (EEC) and ankyloblepharon-ectodermal defects-cleft lip/palate (AEC). T cell lymphopenia has rarely been described in individuals with TP63 variants and the cause of the T cell defect is unclear. Here, we present a case of a female infant born with TP63-related syndrome and profound T cell lymphopenia, first uncovered through newborn screening. Flow cytometry analysis revealed low CD4+ naïve T cells and nearly absent CD8+ T cells with intact B and NK cell compartments. A de novo heterozygous pathogenic variant c.1040 G>A (C347Y) in exon 8 of TP63 was identified. An artificial thymic organoid system, to assess the intrinsic ability of the patient's hematopoietic cells to develop into T cells, was performed twice using separate peripheral blood samples. Ex vivo T cell differentiation was evident with the artificial organoid system, suggesting that a thymic stromal cell defect may be the cause of the T cell lymphopenia. Consistent with this, interrogation of publicly available data indicated that TP63 expression in the human thymus is restricted to thymic epithelial cells. Based on these data, congenital athymia was suspected and the patient received an allogenic cultured thymus tissue implant (CTTI). This is the first report of suspected congenital athymia and attempted treatment with CTTI associated with TP63 variant. At 9 months post-implant, peripheral lymphocyte analysis revealed measurable T cell receptor excision circles and presence of CD4+ recent thymic emigrants suggestive of early thymopoiesis. She will continue regular monitoring to ensure restoration of T cell immunity.

In ovo betaine and heat acclimation affect hatching results, growth performance and immunity of the broilers under cyclic heat stress.

This study investigated the effects of in ovo betaine and thermal manipulation during incubation on growth performance, and some immune parameters of broilers under cyclic heat stress (CHS). Eggs were divided into 5 groups and incubated at 1) 37.8 °C and 60% relative humidity (Control incubation and not-injected, CI); 2) eggs were incubated at CI and in ovo betaine injected into yolk sac on d 11 (E11) (CI + In ovo); 3) eggs were exposed to 38.8 °C for 8 h between 10 and 18d of incubation (heat acclimation, HA); 4) eggs were incubated at HA and in ovo betaine applied (HA + In ovo); 5) positive control: eggs were incubated at CI and injected with saline. Hatched chicks were raised under standard management conditions until 21 d, between 21 and 42 d half of the chickens in each incubation treatment were kept either at optimum (OPT) or at CHS. In ovo and HA did not affect hatching performance. In ovo increased thymus and spleen weights of chicks. Serum IgG was higher in HA + In ovo chicks. From d 7 to 21, in ovo chicks were heavier body weights, consumed more feed, and better feed conversion than those from CI. The body weights of HA chickens were similar in OPT and CHS on d 28 and 35. CHS reduced the body weight of CI chickens which was compatible with their feed consumption. Moreover, feed intake of HA + In ovo chickens exposed to CHS was higher than those not injected indicating that HA + In ovo enhanced thermoregulation of chickens under CHS.

Incontinentia pigmenti underlies thymic dysplasia, autoantibodies to type I IFNs, and viral diseases.

Human inborn errors of thymic T cell tolerance underlie the production of autoantibodies (auto-Abs) neutralizing type I IFNs, which predispose to severe viral diseases. We analyze 131 female patients with X-linked dominant incontinentia pigmenti (IP), heterozygous for loss-of-function (LOF) NEMO variants, from 99 kindreds in 10 countries. Forty-seven of these patients (36%) have auto-Abs neutralizing IFN-α and/or IFN-ω, a proportion 23 times higher than that for age-matched female controls. This proportion remains stable from the age of 6 years onward. On imaging, female patients with IP have a small, abnormally structured thymus. Auto-Abs against type I IFNs confer a predisposition to life-threatening viral diseases. By contrast, patients with IP lacking auto-Abs against type I IFNs are at no particular risk of viral disease. These results suggest that IP accelerates thymic involution, thereby underlying the production of auto-Abs neutralizing type I IFNs in at least a third of female patients with IP, predisposing them to life-threatening viral diseases.

Neuroendocrine neoplasms of the thymus.

Neuroendocrine neoplasms of the thymus (tNENs), including typical carcinoid, atypical carcinoid, large cell neuroendocrine carcinoma, and small cell carcinoma, are rare tumors with scarce clinical and pathological data available in the literature. They share many common features with neuroendocrine neoplasms in other organs, such as those in the lungs, while demonstrating some distinct clinical and pathological features. This review aims to give an updated overview of each category of tNENs, focusing primarily on the pathologic diagnosis and differential diagnosis of these tumors.

Thymic development of human natural killer T cells: recent advances and implications for immunotherapy.

Invariant natural killer T (iNKT) cells are a subset of lipid-reactive, unconventional T cells that have anti-tumor properties that make them a promising target for cancer immunotherapy. Recent studies have deciphered the developmental pathway of human MAIT and Vγ9Vδ2 γδ-T cells as well as murine iNKT cells, yet our understanding of human NKT cell development is limited. Here, we provide an update in our understanding of how NKT cells develop in the human body and how knowledge regarding their development could enhance human treatments by targeting these cells.

Unraveling the spatial organization and development of human thymocytes through integration of spatial transcriptomics and single-cell multi-omics profiling.

The structural components of the thymus are essential for guiding T cell development, but a thorough spatial view is still absent. Here we develop the TSO-his tool, designed to integrate multimodal data from single-cell and spatial transcriptomics to decipher the intricate structure of human thymus. Specifically, we characterize dynamic changes in cell types and critical markers, identifying ELOVL4 as a mediator of CD4+ T cell positive selection in the cortex. Utilizing the mapping function of TSO-his, we reconstruct thymic spatial architecture at single-cell resolution and recapitulates classical cell types and their essential co-localization for T cell development; additionally, previously unknown co-localization relationships such as that of CD8αα with memory B cells and monocytes are identified. Incorporating VDJ sequencing data, we also delineate distinct intermediate thymocyte states during αß T cell development. Overall, these insights enhance our understanding of thymic biology and may inform therapeutic interventions targeting T cell-mediated immune responses.

The thymus road to a T cell: migration, selection, and atrophy.

The thymus plays a pivotal role in generating a highly-diverse repertoire of T lymphocytes while preventing autoimmunity. Thymus seeding progenitors (TSPs) are a heterogeneous group of multipotent progenitors that migrate to the thymus via CCR7 and CCR9 receptors. While NOTCH guides thymus progenitors toward T cell fate, the absence or disruption of NOTCH signaling renders the thymus microenvironment permissive to other cell fates. Following T cell commitment, developing T cells undergo multiple selection checkpoints by engaging with the extracellular matrix, and interacting with thymic epithelial cells (TECs) and other immune subsets across the different compartments of the thymus. The different selection checkpoints assess the T cell receptor (TCR) performance, with failure resulting in either repurposing (agonist selection), or cell death. Additionally, environmental cues such as inflammation and endocrine signaling induce acute thymus atrophy, contributing to the demise of most developing T cells during thymic selection. We discuss the occurrence of acute thymus atrophy in response to systemic inflammation. The thymus demonstrates high plasticity, shaping inflammation by abrogating T cell development and undergoing profound structural changes, and facilitating regeneration and restoration of T cell development once inflammation is resolved. Despite the challenges, thymic selection ensures a highly diverse T cell repertoire capable of discerning between self and non-self antigens, ultimately egressing to secondary lymphoid organs where they complete their maturation and exert their functions.

Multidimensional profiling of human T cells reveals high CD38 expression, marking recent thymic emigrants and age-related naive T cell remodeling.

Thymic involution is a key factor in human immune aging, leading to reduced thymic output and a decline in recent thymic emigrant (RTE) naive T cells in circulation. Currently, the precise definition of human RTEs and their corresponding cell surface markers lacks clarity. Analysis of single-cell RNA-seq/ATAC-seq data distinguished RTEs by the expression of SOX4, IKZF2, and TOX and CD38 protein, whereby surface CD38hi expression universally identified CD8+ and CD4+ RTEs. We further determined the dynamics of RTEs and mature cells in a cohort of 158 individuals, including age-associated transcriptional reprogramming and shifts in cytokine production. Spectral cytometry profiling revealed two axes of aging common to naive CD8+ and CD4+ T cells: (1) a decrease in CD38++ cells (RTEs) and (2) an increase in CXCR3hi cells. Identification of RTEs enables direct assessment of thymic health. Furthermore, resolving the dynamics of naive T cell remodeling yields insight into vaccination and infection responsiveness throughout aging.

Genetically-determined defects of T cell development.

Genetically determined defects of T-cell development comprise a heterogeneous group of conditions characterized by peripheral T-cell lymphopenia due to impaired intrathymic differentiation of T-cell progenitors. Collectively, these conditions are typically referred to as severe combined immune deficiency (SCID). In some cases (leaky SCID), residual function of the defective gene allows partial T-cell development. The vast majority of SCID disorders are due to genetic defects that affect the T-cell differentiation potential of hematopoietic stem cells, through a variety of mechanisms. However, some forms of SCID reflect impaired development or function of thymic stromal cells. A lack of peripheral T cells leads to increased susceptibility to severe infections since early in life. SCID is inevitably fatal unless immune reconstitution is achieved, most often through hematopoietic cell transplantation. Enzyme replacement therapy, gene therapy, and thymus implantation represent other forms of treatment in selected cases. The availability of newborn screening has greatly facilitated prompt recognition of SCID, which allows statistically significant improvement in survival after hematopoietic cell transplantation.

The single-cell transcriptome of mTECs and CD4+ thymocytes under adhesion revealed heterogeneity of mTECs and a network controlled by Aire and lncRNAs.

To further understand the impact of deficiency of the autoimmune regulator (Aire) gene during the adhesion of medullary thymic epithelial cells (mTECs) to thymocytes, we sequenced single-cell libraries (scRNA-seq) obtained from Aire wild-type (WT) (Airewt/wt ) or Aire-deficient (Airewt/mut ) mTECs cocultured with WT single-positive (SP) CD4+ thymocytes. Although the libraries differed in their mRNA and long noncoding RNA (lncRNA) profiles, indicating that mTECs were heterogeneous in terms of their transcriptome, UMAP clustering revealed that both mTEC lines expressed their specific markers, i.e., Epcam, Itgb4, Itga6, and Casp3 in resting mTECs and Ccna2, Pbk, and Birc5 in proliferative mTECs. Both cocultured SP CD4+ thymocytes remained in a homogeneous cluster expressing the Il7r and Ccr7 markers. Comparisons of the two types of cocultures revealed the differential expression of mRNAs that encode transcription factors (Zfpm2, Satb1, and Lef1), cell adhesion genes (Itgb1) in mTECs, and Themis in thymocytes, which is associated with the regulation of positive and negative selection. At the single-cell sequencing resolution, we observed that Aire acts on both Aire WT and Aire-deficient mTECs as an upstream controller of mRNAs, which encode transcription factors or adhesion proteins that, in turn, are posttranscriptionally controlled by lncRNAs, for example, Neat1, Malat1, Pvt1, and Dancr among others. Under Aire deficiency, mTECs dysregulate the expression of MHC-II, CD80, and CD326 (EPCAM) protein markers as well as metabolism and cell cycle-related mRNAs, which delay the cell cycle progression. Moreover, when adhered to mTECs, WT SP CD4+ or CD8+ thymocytes modulate the expression of cell activation proteins, including CD28 and CD152/CTLA4, and the expression of cellular metabolism mRNAs. These findings indicate a complex mechanism through which an imbalance in Aire expression can affect mTECs and thymocytes during adhesion.

Chemokine receptors in primary and secondary lymphoid tissues.

Chemokine receptors are a complex superfamily of surface G protein-coupled receptors present mostly in leukocytes. In this chapter, we review the presence and functions of chemokine receptors in the immune cells of the primary and secondary lymphoid organs. Those include bone marrow, thymus, spleen, lymph nodes, and Peyer's patches as the main components of the gut-associated lymphoid tissue. There are general groups of chemokine receptors: conventional and atypical. We will mostly cover the role of conventional chemokine receptors, which are divided into four classes (CC, CXC, CX3C, and XC). Some relevant members are CXCR4, CXCR5, CCR4 and CCR7. For example, CXCR4 is a key chemokine receptor in the bone marrow controlling from the homing of progenitor cells into the bone marrow, the development of B cells, to the homing of long-lived plasma cells to this primary lymphoid organ. CCR7 and CCR4 are two of the main players in the thymus. CCR7 along with CCR9 control the traffic of thymic seed progenitors into the thymus, while CCR4 and CCR7 are critical for the entry of thymocytes into the medulla and as controllers of the central tolerance in the thymus. CXCR4 and CXCR5 have major roles in the spleen, guiding the maturation and class-switching of B cells in the different areas of the germinal center. In the T-cell zone, CCR7 guides the differentiation of naïve T cells. CCR7 also controls and directs the entry of T cells, B cells, and dendritic cells into secondary lymphoid tissues, including the spleen and lymph nodes. As new technologies emerge, techniques such as high dimensional spectral flow cytometry or single-cell sequencing allow a more comprehensive knowledge of the chemokine receptor network and their ligands, as well as the discovery of new interactions mediating unknown and overlooked mechanisms in health and disease.

Unraveling the phenotypic states of human innate-like T cells: Comparative insights with conventional T cells and mouse models.

The "innate-like" T cell compartment, known as Tinn, represents a diverse group of T cells that straddle the boundary between innate and adaptive immunity. We explore the transcriptional landscape of Tinn compared to conventional T cells (Tconv) in the human thymus and blood using single-cell RNA sequencing (scRNA-seq) and flow cytometry. In human blood, the majority of Tinn cells share an effector program driven by specific transcription factors, distinct from those governing Tconv cells. Conversely, only a fraction of thymic Tinn cells displays an effector phenotype, while others share transcriptional features with developing Tconv cells, indicating potential divergent developmental pathways. Unlike the mouse, human Tinn cells do not differentiate into multiple effector subsets but develop a mixed type 1/type 17 effector potential. Cross-species analysis uncovers species-specific distinctions, including the absence of type 2 Tinn cells in humans, which implies distinct immune regulatory mechanisms across species.

Maternal Undernutrition Affects Fetal Thymus DNA Methylation, Gene Expression, and, Thereby, Metabolism and Immunopoiesis in Wagyu (Japanese Black) Cattle.

We aimed to determine the effects of maternal nutrient restriction (MNR) on the DNA methylation and gene expression patterns associated with metabolism and immunopoiesis in the thymuses of fetal Wagyu cattle. Pregnant cows were allocated to two groups: a low-nutrition (LN; 60% nutritional requirement; n = 5) and a high-nutrition (HN; 120% nutritional requirement, n = 6) group, until 8.5 months of gestation. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing were used to analyze DNA methylation and gene expression, while capillary electrophoresis-Fourier transform mass spectrometry assessed the metabolome. WGBS identified 4566 hypomethylated and 4303 hypermethylated genes in the LN group, with the intergenic regions most frequently being methylated. Pathway analysis linked hypoDMGs to Ras signaling, while hyperDMGs were associated with Hippo signaling. RNA sequencing found 94 differentially expressed genes (66 upregulated, 28 downregulated) in the LN group. The upregulated genes were tied to metabolic pathways and oxidative phosphorylation; the downregulated genes were linked to natural killer cell cytotoxicity. Key overlapping genes (GRIA1, CACNA1D, SCL25A4) were involved in cAMP signaling. The metabolomic analysis indicated an altered amino acid metabolism in the MNR fetuses. These findings suggest that MNR affects DNA methylation, gene expression, and the amino acid metabolism, impacting immune system regulation during fetal thymus development in Wagyu cattle.

Systemic immunostimulation induces glucocorticoid-mediated thymic involution succeeded by rebound hyperplasia which is impaired in aged recipients.

The thymus is the central organ involved with T-cell development and the production of naïve T cells. During normal aging, the thymus undergoes marked involution, reducing naïve T-cell output and resulting in a predominance of long-lived memory T cells in the periphery. Outside of aging, systemic stress responses that induce corticosteroids (CS), or other insults such as radiation exposure, induce thymocyte apoptosis, resulting in a transient acute thymic involution with subsequent recovery occurring after cessation of the stimulus. Despite the increasing utilization of immunostimulatory regimens in cancer, effects on the thymus and naïve T cell output have not been well characterized. Using both mouse and human systems, the thymic effects of systemic immunostimulatory regimens, such as high dose IL-2 (HD IL-2) with or without agonistic anti-CD40 mAbs and acute primary viral infection, were investigated. These regimens produced a marked acute thymic involution in mice, which correlated with elevated serum glucocorticoid levels and a diminishment of naïve T cells in the periphery. This effect was transient and followed with a rapid thymic "rebound" effect, in which an even greater quantity of thymocytes was observed compared to controls. Similar results were observed in humans, as patients receiving HD IL-2 treatment for cancer demonstrated significantly increased cortisol levels, accompanied by decreased peripheral blood naïve T cells and reduced T-cell receptor excision circles (TRECs), a marker indicative of recent thymic emigrants. Mice adrenalectomized prior to receiving immunotherapy or viral infection demonstrated protection from this glucocorticoid-mediated thymic involution, despite experiencing a substantially higher inflammatory cytokine response and increased immunopathology. Investigation into the effects of immunostimulation on middle aged (7-12 months) and advance aged (22-24 months) mice, which had already undergone significant thymic involution and had a diminished naïve T cell population in the periphery at baseline, revealed that even further involution was incurred. Thymic rebound hyperplasia, however, only occurred in young and middle-aged recipients, while advance aged not only lacked this rebound hyperplasia, but were entirely absent of any indication of thymic restoration. This coincided with prolonged deficits in naïve T cell numbers in advanced aged recipients, further skewing the already memory dominant T cell pool. These results demonstrate that, in both mice and humans, systemic immunostimulatory cancer therapies, as well as immune challenges like subacute viral infections, have the potential to induce profound, but transient, glucocorticoid-mediated thymic involution and substantially reduced thymic output, resulting in the reduction of peripheral naive T cells. This can then be followed by a marked rebound effect with naïve T cell restoration, events that were shown not to occur in advanced-aged mice.

Skewed epithelial cell differentiation and premature aging of the thymus in the absence of vitamin D signaling.

Central tolerance of thymocytes to self-antigen depends on the medullary thymic epithelial cell (mTEC) transcription factor autoimmune regulator (Aire), which drives tissue-restricted antigen (TRA) gene expression. Vitamin D signaling regulates Aire and TRA expression in mTECs, providing a basis for links between vitamin D deficiency and autoimmunity. We find that mice lacking Cyp27b1, which cannot produce hormonally active vitamin D, display profoundly reduced thymic cellularity, with a reduced proportion of Aire+ mTECs, attenuated TRA expression, and poorly defined cortical-medullary boundaries. Markers of T cell negative selection are diminished, and organ-specific autoantibodies are present in knockout (KO) mice. Single-cell RNA sequencing revealed that loss of Cyp27b1 skews mTEC differentiation toward Ccl21+ intertypical TECs and generates a gene expression profile consistent with premature aging. KO thymi display accelerated involution and reduced expression of thymic longevity factors. Thus, loss of thymic vitamin D signaling disrupts normal mTEC differentiation and function and accelerates thymic aging.

Comprehensive Postnatal Anatomical, Histological and Geometric Morphometric Analysis of Thymus Development in Dromedary Camels (Camelus dromedarius).

The thymus, a primary lymphoid organ, plays a critical role in T lymphocyte development and adaptive immunity. This study focuses on the anatomical, histological and geometric morphometric characteristics of the thymus in dromedary camels (Camelus dromedarius) during postnatal development. Thymus samples were collected from camels aged approximately 4, 8, 12 and 16 months. Using photogrammetry and 3D modelling, the samples were analysed to generate landmarks and conduct geometric morphometry with the 3D Slicer and ALPACA algorithm. Principal component analysis (PCA) was then performed to evaluate shape variations. Histologically, the samples underwent Haematoxylin and Eosin and Masson's trichrome staining. Image analysis using QuPath software quantified trabeculae, adipose tissue and Hassall's corpuscles. The results revealed significant anatomical and histological changes in the thymus across the different age groups. Notable variations in tissue composition and structural integrity were observed, with the PCA highlighting distinct morphometric patterns associated with age-related development. These findings provide a deeper understanding of thymus maturation in dromedaries and offer valuable data for comparative anatomy and veterinary medicine. This comprehensive analysis enhances our knowledge of species-specific immune development, with important implications for the health and resilience of these animals in arid environments.