Altered galectin-3 distribution and migratory function in the pre-diabetic non-obese diabetic mouse thymus.

Galectin-3 is an endogenous lectin which binds mainly to ß-galactosides on the cell surface and extracellular matrix (ECM) glycoproteins. In the thymus, this lectin is constitutively expressed, being involved in thymocyte adhesion, migration, and death. Galectin-3 has been related to type 1 diabetes, an autoimmune disease characterized by pancreatic ß-cell destruction mediated by autoreactive T lymphocytes. Non-obese diabetic (NOD) mice represent a suitable model to study type 1 diabetes, as they develop the disease like humans. We previously described important thymic alterations in these animals such as the development of giant perivascular spaces (PVS), characterized by the retention of T and B cells, intermingled with an ECM network, and associated with a defect in the expression of the fibronectin receptor VLA-5 and reduced sphingosine-1-phosphate receptor expression on developing thymocytes. In order to investigate galectin-3 expression in thymic microenvironmental cells and verify its interaction with cells and ECM molecules in PVS, we performed immunofluorescence following colocalization analysis in the thymic parenchyma of pre-diabetic NOD mice by confocal microscopy. In addition, thymocyte migration assays were performed to evaluate the effect of galectin-3 on NOD thymocyte migration. Herein, we showed a significant enhancement of colocalization with cortical and medullary thymic epithelial cells in NOD mice, as compared to controls. In the giant PVS of these animals, we observed a heterogeneous distribution of galectin-3, predominantly found in clusters of B lymphocytes and dendritic cells. Functionally, NOD thymocyte migratory response towards galectin-3 was impaired and a similar decrease was seen in transendothelial thymocyte migration. Taken together, our data provide the histological and functional background for a potential defective thymocyte migration involving galectin-3, thus placing this molecule as a further player in the intrathymic disturbances observed in pre-diabetic NOD mice.

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.

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.

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.

Age-related epithelial defects limit thymic function and regeneration.

The thymus is essential for establishing adaptive immunity yet undergoes age-related involution that leads to compromised immune responsiveness. The thymus is also extremely sensitive to acute insult and although capable of regeneration, this capacity declines with age for unknown reasons. We applied single-cell and spatial transcriptomics, lineage-tracing and advanced imaging to define age-related changes in nonhematopoietic stromal cells and discovered the emergence of two atypical thymic epithelial cell (TEC) states. These age-associated TECs (aaTECs) formed high-density peri-medullary epithelial clusters that were devoid of thymocytes; an accretion of nonproductive thymic tissue that worsened with age, exhibited features of epithelial-to-mesenchymal transition and was associated with downregulation of FOXN1. Interaction analysis revealed that the emergence of aaTECs drew tonic signals from other functional TEC populations at baseline acting as a sink for TEC growth factors. Following acute injury, aaTECs expanded substantially, further perturbing trophic regeneration pathways and correlating with defective repair of the involuted thymus. These findings therefore define a unique feature of thymic involution linked to immune aging and could have implications for developing immune-boosting therapies in older individuals.

The partitioning of TCR repertoires by thymic selection.

αß T cells are critical components of the adaptive immune system; they maintain tissue and immune homeostasis during health, provide sterilizing immunity after pathogen infection, and are capable of eliminating transformed tumor cells. Fundamental to these distinct functions is the ligand specificity of the unique antigen receptor expressed on each mature T cell (TCR), which endows lymphocytes with the ability to behave in a cell-autonomous, disease context-specific manner. Clone-specific behavioral properties are initially established during T cell development when thymocytes use TCR recognition of major histocompatibility complex (MHC) and MHC-like ligands to instruct survival versus death and to differentiate into a plethora of inflammatory and regulatory T cell lineages. Here, we review the ligand specificity of the preselection thymocyte repertoire and argue that developmental stage-specific alterations in TCR signaling control cross-reactivity and foreign versus self-specificity of T cell sublineages.

Single-cell sequencing has revealed a more complex array of thymic epithelial cells.

Thymic epithelial cells participate in the maturation and selection of T lymphocytes. This review explores recent insights from single-cell sequencing regarding classifying thymic epithelial cells in both normal and neoplastic thymus. Cortical thymic epithelial cells facilitate thymocyte differentiation and contribute to positive selection. Medullary epithelial cells are distinguished by their expression of AIRE. Cells progress from a pre-AIRE state, containing precursors with cortical and medullary characteristics, termed junctional cells. Mature medullary epithelial cells exhibit promiscuous gene expression and after that downregulate AIRE mRNA. Post-AIRE cells can adopt a Hassall corpuscle-like phenotype or exhibit distinctive differentiation characteristics including tuft cells, ionocytes, neuroendocrine cells, and myoid cells.

Induction of Thymus Atrophy and Disruption of Thymocyte Development by Fipronil through Dysregulation of IL-7-Associated Genes.

The susceptibility of the immune system to immunotoxic chemicals is evident, particularly in the thymus, a vital primary immune organ prone to atrophy due to exposure to toxicants. Fipronil (FPN), a widely used insecticide, is of concern due to its potential neurotoxicity, hepatotoxicity, and immunotoxicity. Our previous study showed that FPN disturbed the antigen-specific T-cell functionality in vivo. As T-cell lineage commitment and thymopoiesis are closely interconnected with the normal function of the T-cell-mediated immune responses, this study aims to further examine the toxic effects of FPN on thymocyte development. In this study, 4-week-old BALB/c mice received seven doses of FPN (1, 5, 10 mg/kg) by gavage. Thymus size, medulla/cortex ratio, total thymocyte counts, double-positive thymocyte population, and IL-7-positive cells decreased dose-dependently. IL-7 aids the differentiation of early T-cell precursors into mature T cells, and several essential genes contribute to the maturation of T cells in the thymus. Foxn1 ensures that the thymic microenvironment is suitable for the maturation of T-cell precursors. Lyl1 is involved in specifying lymphoid cells and maintaining T-cell development in the thymus. The c-Kit/SCF collaboration fosters a supportive thymic milieu to promote the formation of functional T cells. The expression of IL-7, IL-7R, c-Kit, SCF, Foxn1, and Lyl1 genes in the thymus was significantly diminished in FPN-treated groups with the concordance with the reduction of IL-7 signaling proteins (IL-7, IL-7R, c-KIT, SCF, LYL1, FOXO3A, and GABPA), suggesting that the dysregulation of T-cell lineage-related genes may contribute to the thymic atrophy induced by FPN. In addition, FPN disturbed the functionality of thymocytes with an increase of IL-4 and IFN-γ production and a decrease of IL-2 secretion after T-cell mitogen stimulation ex vivo. Collectively, FPN significantly deregulated genes related to T-cell progenitor differentiation, survival, and expansion, potentially leading to impaired thymopoiesis.

Fam49b dampens TCR signal strength to regulate survival of positively selected thymocytes and peripheral T cells.

The fate of developing T cells is determined by the strength of T cell receptor (TCR) signal they receive in the thymus. This process is finely regulated through the tuning of positive and negative regulators in thymocytes. The Family with sequence similarity 49 member B (Fam49b) protein is a newly discovered negative regulator of TCR signaling that has been shown to suppress Rac-1 activity in vitro in cultured T cell lines. However, the contribution of Fam49b to the thymic development of T cells is unknown. To investigate this important issue, we generated a novel mouse line deficient in Fam49b (Fam49b-KO). We observed that Fam49b-KO double positive (DP) thymocytes underwent excessive negative selection, whereas the positive selection stage was unaffected. Fam49b deficiency impaired the survival of single positive thymocytes and peripheral T cells. This altered development process resulted in significant reductions in CD4 and CD8 single-positive thymocytes as well as peripheral T cells. Interestingly, a large proportion of the TCRγδ+ and CD8αα+TCRαß+ gut intraepithelial T lymphocytes were absent in Fam49b-KO mice. Our results demonstrate that Fam49b dampens thymocytes TCR signaling in order to escape negative selection during development, uncovering the function of Fam49b as a critical regulator of the selection process to ensure normal thymocyte development and peripheral T cells survival.

Glucocorticoids impair T lymphopoiesis after myocardial infarction.

The thymus, where T lymphocytes develop and mature, is sensitive to insults such as tissue ischemia or injury. The insults can cause thymic atrophy and compromise T-cell development, potentially impairing adaptive immunity. The objective of this study was to investigate whether myocardial infarction (MI) induces thymic injury to impair T lymphopoiesis and to uncover the underlying mechanisms. When compared with sham controls, MI mice at day 7 post-MI exhibited smaller thymus, lower cellularity, as well as less thymocytes at different developmental stages, indicative of T-lymphopoiesis impairment following MI. Accordingly, the spleen of MI mice has less T cells and recent thymic emigrants (RTEs), implying that the thymus of MI mice releases fewer mature thymocytes than sham controls. Interestingly, the secretory function of splenic T cells was not affected by MI. Further experiments showed that the reduction of thymocytes in MI mice was due to increased thymocyte apoptosis. Removal of adrenal glands by adrenalectomy (ADX) prevented MI-induced thymic injury and dysfunction, whereas corticosterone supplementation in ADX + MI mice reinduced thymic injury and dysfunction, indicating that glucocorticoids mediate thymic damage triggered by MI. Eosinophils play essential roles in thymic regeneration postirradiation, and eosinophil-deficient mice exhibit impaired thymic recovery after sublethal irradiation. Interestingly, the thymus was fully regenerated in both wild-type and eosinophil-deficient mice at day 14 post-MI, suggesting that eosinophils are not critical for thymus regeneration post-MI. In conclusion, our study demonstrates that MI-induced glucocorticoids trigger thymocyte apoptosis and impair T lymphopoiesis, resulting in less mature thymocyte release to the spleen.NEW & NOTEWORTHY The thymus is essential for maintaining whole body T-cell output. Thymic injury can adversely affect T lymphopoiesis and T-cell immune response. This study demonstrates that MI induces thymocyte apoptosis and compromises T lymphopoiesis, resulting in fewer releases of mature thymocytes to the spleen. This process is mediated by glucocorticoids secreted by adrenal glands. Therefore, targeting glucocorticoids represents a novel approach to attenuate post-MI thymic injury.

GRP75-dependent mitochondria-ER contacts ensure cell survival during early mouse thymocyte development.

Mitochondria and endoplasmic reticulum contacts (MERCs) control multiple cellular processes, including cell survival and differentiation. Based on the observations that MERCs were specifically enriched in the CD4-CD8- double-negative (DN) stage, we studied their role in early mouse thymocyte development. We found that T cell-specific knockout of Hspa9, which encodes GRP75, a protein that mediates MERC formation by assembling the IP3R-GRP75-VDAC complex, impaired DN3 thymocyte viability and resulted in thymocyte developmental arrest at the DN3-DN4 transition. Mechanistically, GRP75 deficiency induced mitochondrial stress, releasing mitochondrial DNA (mtDNA) into the cytosol and triggering the type I interferon (IFN-I) response. The IFN-I pathway contributed to both the impairment of cell survival and DN3-DN4 transition blockage, while increased lipid peroxidation (LPO) played a major role downstream of IFN-I. Thus, our study identifies the essential role of GRP75-dependent MERCs in early thymocyte development and the governing facts of cell survival and differentiation in the DN stage.

Generation and repair of thymic epithelial cells.

In the vertebrate immune system, thymus stromal microenvironments support the generation of αßT cells from immature thymocytes. Thymic epithelial cells are of particular importance, and the generation of cortical and medullary epithelial lineages from progenitor stages controls the initiation and maintenance of thymus function. Here, we discuss the developmental pathways that regulate thymic epithelial cell diversity during both the embryonic and postnatal periods. We also examine how thymus microenvironments respond to injury, with particular focus on mechanisms that ensure regeneration of thymic epithelial cells for the restoration of thymus function.

Stabilization of ß-Catenin Directs HEB to Limit Thymic Selection.

Activation of ß-catenin in CD4+CD8+ double-positive (DP) thymocytes halts development before the thymic selection stage and predisposes to transformation. Leukemogenesis, but not the developmental block, depends on TCF-1, ß-catenin's DNA-binding partner. In this study, we show that ß-catenin activation directs the DNA-binding protein HEB to block DP thymocyte development. Conditional loss of HEB in DP thymocytes with stabilized ß-catenin restores the frequencies of postselection TCRßhi/CCR7+ and TCRßhi/CD69+ DPs and their cell-cycle profile. This recovery is associated with significant reversal of ß-catenin-induced expression changes, particularly those related to the CD69+ DP cell signature and to cell-cycle pathways. Stabilizing ß-catenin in DP thymocytes directs HEB binding to ≈11,000 novel DNA sites throughout the genome. Novel HEB sites mark most CD69+ DP cell signature genes that change expression upon activation of ß-catenin and then revert after loss of HEB. Moreover, many of the novel HEB sites occupy promoter regions of genes enriched in mitotic cell cycle pathways. HEB binding to those regions correlates with downregulation of the associated genes, and HEB inactivation restores expression to physiologic levels. These findings highlight a molecular interplay between HEB and ß-catenin that can impair thymic development.

Adjusting to self in the thymus: CD4 versus CD8 lineage commitment and regulatory T cell development.

During thymic development, thymocytes adjust their TCR response based on the strength of their reactivity to self-peptide MHC complexes. This tuning process allows thymocytes with a range of self-reactivities to survive positive selection and contribute to a diverse T cell pool. In this review, we will discuss recent advances in our understanding of how thymocytes tune their responsiveness during positive selection, and we present a "sequential selection" model to explain how MHC specificity influences lineage choice. We also discuss recent evidence for cell type diversity in the medulla and discuss how this heterogeneity may contribute to medullary niches for negative selection and regulatory T cell development.

Direct presentation of inflammation-associated self-antigens by thymic innate-like T cells induces elimination of autoreactive CD8+ thymocytes.

Upregulation of diverse self-antigens that constitute components of the inflammatory response overlaps spatially and temporally with the emergence of pathogen-derived foreign antigens. Therefore, discrimination between these inflammation-associated self-antigens and pathogen-derived molecules represents a unique challenge for the adaptive immune system. Here, we demonstrate that CD8+ T cell tolerance to T cell-derived inflammation-associated self-antigens is efficiently induced in the thymus and supported by redundancy in cell types expressing these molecules. In addition to thymic epithelial cells, this included thymic eosinophils and innate-like T cells, a population that expressed molecules characteristic for all major activated T cell subsets. We show that direct T cell-to-T cell antigen presentation by minute numbers of innate-like T cells was sufficient to eliminate autoreactive CD8+ thymocytes. Tolerance to such effector molecules was of critical importance, as its breach caused by decreased thymic abundance of a single model inflammation-associated self-antigen resulted in autoimmune elimination of an entire class of effector T cells.

Notch3-regulated microRNAs impair CXCR4-dependent maturation of thymocytes allowing maintenance and progression of T-ALL.

Malignant transformation of T-cell progenitors causes T-cell acute lymphoblastic leukemia (T-ALL), an aggressive childhood lymphoproliferative disorder. Activating mutations of Notch, Notch1 and Notch3, have been detected in T-ALL patients. In this study, we aimed to deeply characterize hyperactive Notch3-related pathways involved in T-cell dynamics within the thymus and bone marrow to propose these processes as an important step in facilitating the progression of T-ALL. We previously generated a transgenic T-ALL mouse model (N3-ICtg) demonstrating that aberrant Notch3 signaling affects early thymocyte maturation programs and leads to bone marrow infiltration by CD4+CD8+ (DP) T cells that are notably, Notch3highCXCR4high. Newly, our in vivo results suggest that an anomalous immature thymocyte subpopulation, such as CD4-CD8- (DN) over-expressing CD3ɛ, but with low CXCR4 expression, dominates N3-ICtg thymus-resident DN subset in T-ALL progression. MicroRNAs might be of significance in T-ALL pathobiology, however, whether required for leukemia maintenance is not fully understood. The selection of specific DN subsets demonstrates the inverse correlation between CXCR4 expression and a panel of Notch3-deregulated miRNAs. Interestingly, we found that within DN thymocyte subset hyperactive Notch3 inhibits CXCR4 expression through the cooperative effects of miR-139-5p and miR-150-5p, thus impinging on thymocyte differentiation with accumulation of DNCD3ɛ+CXCR4- cells. These data point out that deregulation of Notch3 in T-ALL, besides its role in sustaining dissemination of abnormal DP T cells, as we previously demonstrated, could play a role in selecting specific DN immature T cells within the thymus, thus impeding T cell development, to facilitate T-ALL progression inside the bone marrow.

Restoration of thymic T-cell development by bone marrow transplantation in mouse radiation lymphomagenesis.

Fractionated total body irradiation (TBI) with X-rays induces thymic lymphoma/leukemia (TL) in C57BL/6 mice. Radiation-induced mouse TL (RITL) can be prevented by bone marrow transplantation (BMT) of unirradiated BM cells. However, the mechanisms underlying the prevention of RITL with BMT remain unclear. Here, we show that BMT restores thymic T-cell differentiation in mice subjected to TBI. TBI (four times of 1.8 Gy X-rays weekly) was conducted with C57BL/6 mice. BMT was performed immediately after the last irradiation of TBI in mice by transplantation of BM cells isolated from enhanced green fluorescence protein (eGFP) transgenic mice. Thymic cell numbers were drastically decreased in TBI and TBI + BMT mice compared to those in non-irradiated mice. Flow cytometry showed a dramatic decrease in double negative (DN, CD4-CD8-) thymocytes, especially DN2 (CD25+CD44+) and DN3 (CD25+CD44-) subpopulations, in the TBI mice on Day 10 after the last irradiation. In contrast, the DN2 and DN3 populations were recovered in TBI + BMT mice. Interestingly, these restored DN2 and DN3 cells mainly differentiated from eGFP-negative recipient cells but not from eGFP-positive donor cells, suggesting that transplanted BM cells may interact with recipient cells to restore thymic T-cell development in the RITL model. Taken together, our findings highlight the significance of restoring thymic T-cell differentiation by BMT in RITL prevention.

Testosterone regulates thymic remodeling by activating glucocorticoid receptor signaling pathway to accelerate thymocyte apoptosis in male rats.

Thymic atrophy affects T cell generation and migration to the periphery, thereby affecting T cell pool diversity. However, the mechanisms underlying thymic atrophy have not been fully elucidated. Here, gonadotropin-releasing hormone (GnRH) immunization and surgical castration did not affect thymocyte proliferation, but significantly reduced the apoptosis and increased the survival rate of CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+ thymocytes. Following testosterone supplementation in rats subjected to GnRH immunization and surgical castration, thymocyte proliferation remained unchange, but the apoptosis of CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+ thymocytes significantly increased. Transcriptome analyses of the thymus after GnRH immunization and surgical castration showed a significant reduction in the thymus's response to corticosterone. Cholesterol metabolism and the synthesis and secretion of corticosterone were significantly reduced. Analysis of the enzyme levels involved in the corticosterone synthesis pathway revealed that corticosterone synthesis in thymocytes was significantly reduced after GnRH immunization and surgical castration, whereas exogenous testosterone supplementation relieved this process. Testosterone promoted thymocyte apoptosis in a concentration-dependent manner, and induced corticosterone secretion in vitro. Blocking the intracellular androgen receptor (AR) signaling pathway did not significantly affect thymocyte apoptosis, but blocking the glucocorticoid receptor (GR) signaling pathway significantly reduced it. Our findings indicate that testosterone regulates thymus remodeling by affecting corticosterone synthesis in thymocytes, which activates GR signal transduction and promotes thymocyte apoptosis.

T cell-intrinsic PKD3 fine-tunes differentiation into CD8+ central memory T cells and CD8 single positive thymocyte development.

Members of the Protein kinases D (PKD) family are described as regulators of T cell responses. From the two T cell-expressed isoforms PKD2 and PKD3, so far mainly the former was thoroughly investigated and is well understood. Recently, we have investigated also PKD3 using conventional as well as conditional T cell-specific knockout models. These studies suggested PKD3 to be a T cell-extrinsic regulator of the cells' fate. However, these former model systems did not take into account possible redundancies with the highly homologous PKD2. To overcome this issue and thus properly unravel PKD3's T cell-intrinsic functions, here we additionally used a mouse model overexpressing a constitutively active isoform of PKD3 specifically in the T cell compartment. These transgenic mice showed a slightly higher proportion of central memory T cells in secondary lymphoid organs and blood. This effect could not be explained via differences upon polyclonal stimulation in vitro, however, may be connected to the observed developmental aberrances in the CD8 single positive compartment during thymic development. Lastly, the observed alterations in the CD8+ T cell compartment did not impact proper immune response upon immunization with ovalbumin or in a subcutaneous tumour model suggesting only a small to absent biological relevance. Taking together the knowledge of all our published studies on PKD3 in the T cell compartment, we now conclude that T cell-intrinsic PKD3 is a fine-tuner of central memory T cell as well as CD8 single positive thymocyte 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.