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Ess2, also known as Dgcr14, is a transcriptional co-regulator of CD4+ T cells. Ess2 is located in a chromosomal region, the loss of which has been associated with 22q11.2 deletion syndrome (22q11DS), which causes heart defects, skeletal abnormalities, and immunodeficiency. However, the specific association of Ess2 with 22q11DS remains unclear. To elucidate the role of Ess2 in T-cell development, we generated Ess2 floxed (Ess2fl/fl) and CD4+ T cell-specific Ess2 KO (Ess2ΔCD4/ΔCD4) mice using the Cre/loxP system. Interestingly, Ess2ΔCD4/ΔCD4 mice exhibited reduced naïve T-cell numbers in the spleen, while the number of thymocytes (CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+) in the thymus remained unchanged. Furthermore, Ess2ΔCD4/ΔCD4 mice had decreased NKT cells and increased γδT cells in the thymus and spleen. A genome-wide expression analysis using RNA-seq revealed that Ess2 deletion alters the expression of many genes in CD4 single-positive thymocytes, including genes related to the immune system and Myc target genes. In addition, Ess2 enhanced the transcriptional activity of c-Myc. Some genes identified as Ess2 targets in mice show expressional correlation with ESS2 in human immune cells. Moreover, Ess2ΔCD4/ΔCD4 naïve CD4+ T cells did not maintain survival in response to IL-7. Our results suggest that Ess2 plays a critical role in post-thymic T-cell survival through the Myc and IL-7 signaling pathways. [ABSTRACT FROM AUTHOR]

Objective: Congenital heart diseases (CHD) are often associated with thymus development disorders and immune dysfunctions. However, the features of thymocyte differentiation in cyanotic and acyanotic CHD remain unknown. Materials and Methods: We have analyzed the main thymocyte subsets depending on CD4 and CD8 co-expression, and the number of natural regulatory T-cells (nTreg) and invariant natural killer T-cells (iNKT) precursors in the co-cultures of thymocytes with thymic plasmacytoid (p) dendritic cells (DCs) in vitro, isolated from the thymus of children with acyanotic (without hypoxia) and cyanotic (with severe hypoxia) CHD. Results: In the thymocyte co-cultures with pDCs in cyanotic CHD, compared to acyanotic CHD, a decreased number of thymocytes expressing αβ chains of the T-cell receptor with CD4 and CD8 lower levels (CD4loCD8loαβTCR+ ), but the increased numbers of CD4hiCD8-/loαβTCR+ cells were detected. The numbers of CD4- CD8- αβTCR+, CD4hiCD8hiαβTCR+, CD4-/loCD8hiαβTCR+ cells did not differ between cyanotic or acyanotic CHD. In cyanotic CHD in the thymocyte co-cultures with pDCs, the decreased number of CD4+ CD25+ FOXP3+ cells, nTreg precursors, was detected in comparison with acyanotic CHD. In cyanotic CHD, the number of CD3hiVα24Jα18+ cells, iNKT precursors, in the thymocyte co-cultures with pDCs did not differ in comparison with acyanotic CHD. Hypoxia in cyanotic CHD increased the resistance to apoptosis of thymocytes in co-cultures with pDCs in comparison with acyanotic CHD. Conclusion: Thus, hypoxia affected the main CD4+ and CD8+ αβTCR T-cell subsets and the number of CD4+ CD25+ FOXP3+ cells in the thymocyte co-cultures with pDCs isolated from thymus of children with CHD [ABSTRACT FROM AUTHOR]

The main function of the thymus is to support the establishment of a wide repertoire of T lymphocytes capable of eliminating foreign pathogens, yet tolerant to self‐antigens. Thymocyte development in the thymus is dependent on the interaction with thymic stromal cells, a complex mixture of cells comprising thymic epithelial cells (TEC), mesenchymal and endothelial cells. The exchange of signals between stromal cells and thymocytes is referred to as "thymic cross‐talk". Genetic defects affecting either side of this interaction result in defects in thymic development that ultimately lead to a decreased output of T lymphocytes to the periphery. In the present review, we aim at providing a summary of inborn errors of immunity (IEI) characterized by T‐cell lymphopenia due to defects of the thymic stroma, or to hematopoietic‐intrinsic defects of T‐cell development, with a special focus on recently discovered disorders. Additionally, we review the novel diagnostic tools developed to discover and study new genetic causes of IEI due to defects in thymic development. Finally, we discuss therapeutic approaches to correct thymic defects that are currently available, in addition to potential novel therapies that could be applied in the future. [ABSTRACT FROM AUTHOR]

ICAP‐1 regulates β1‐integrin activation and cell adhesion. Here, we used ICAP‐1‐null mice to study ICAP‐1 potential involvement during immune cell development and function. Integrin α4β1‐dependent adhesion was comparable between ICAP‐1‐null and control thymocytes, but lack of ICAP‐1 caused a defective single‐positive (SP) CD8+ cell generation, thus, unveiling an ICAP‐1 involvement in SP thymocyte development. ICAP‐1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP‐1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP‐1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP‐1–/– spleen T and B cells displayed upregulation of α4β1‐mediated adhesion, indicating that ICAP‐1 negatively controls their attachment. Furthermore, CD3+‐ and CD19+‐selected spleen cells from ICAP‐1‐null mice showed reduced proliferation in response to T‐ and B‐cell stimuli, respectively. Finally, loss of ICAP‐1 caused a remarkable decrease in marginal zone B‐ cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP‐1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B‐cell numbers. [ABSTRACT FROM AUTHOR]

Introduction: The thymus is active mainly during the neonatal and pre-adolescent periods. Objective: To test naïve thymocytes proliferation and monocytes stimulation. Materials and methods: We collected fresh thymus tissue from neonate mice after surgery. Suspension cells were coated onto Ficoll-Hypaque support. The obtained cells (thymocytes) were cultured measuring the proliferation of naïve T cells stimulated by Crotalus durissus cumanensis (Cdc) venom at sub-lethal doses (20 ng). Then, we supplemented the wells with AlamarBlue™ and incubated them for 5 h to test their proliferation. Mononuclear cells from mice peripheral blood were collected and layered onto the support of the Ficoll-Hypaque solution. We added the thymocytes actively dividing (25 x 105 cells) from cultures stimulated with Cdc venom at 20 ng/well to cultured monocytes freshly obtained from the Ficoll-Hypaque separation. Both cell populations were incubated for 36 h until monocytes matured to macrophages. Results: The naïve thymocytes rapidly proliferated after stimulation with the Cdc venom (NTCdc) and these successively induced the maturation and function of monocytes progenitor cells to mature macrophages, which ingested Chinese ink. Conclusions: The naïve thymocytes proliferated by stimulation with the Cdc venom and subsequently the NT/Cdc induced the rapid maturation and function of monocytes progenitor cells becoming mature macrophages with their phenotypic characteristics.

T cells are generated from hematopoietic stem cells through a highly organized developmental process, in which stage-specific molecular events drive maturation towards ab and gd T cells. Although many of the mechanisms that control ab- and gd-lineage differentiation are shared between human and mouse, important differences have also been observed. Here, we studied the regulatory dynamics of the E and ID protein encoding genes during pediatric human T cell development by evaluating changes in chromatin accessibility, histone modifications and bulk and single cell gene expression. We profiled patterns of ID/E protein activity and identified up- and downstream regulators and targets, respectively. In addition, we compared transcription of E and ID protein encoding genes in human versus mouse to predict both shared and unique activities in these species, and in prenatal versus pediatric human T cell differentiation to identify regulatory changes during development. This analysis showed a putative involvement of TCF3/E2A in the development of gd T cells. In contrast, in ab T cell precursors a pivotal pre-TCR-driven population with high ID gene expression and low predicted E protein activity was identified. Finally, in prenatal but not postnatal thymocytes, high HEB/TCF12 levels were found to counteract high ID levels to sustain thymic development. In summary, we uncovered novel insights in the regulation of E and ID proteins on a crossspecies and cross-developmental level. [ABSTRACT FROM AUTHOR]

T cell development in the thymus is tightly controlled by complex regulatory mechanisms at multiple checkpoints. Currently, many studies have focused on the transcriptional and posttranslational control of the intrathymic journey of T-cell precursors. However, over the last few years, compelling evidence has highlighted cell metabolism as a critical regulator in this process. Different thymocyte subsets are directed by distinct metabolic pathways and signaling networks to match the specific functional requirements of the stage. Here, we epitomize these metabolic alterations during the development of a T cell and review several recent works that provide insights into equilibrating metabolic quiescence and activation programs. Ultimately, understanding the interplay between cellular metabolism and T cell developmental programs may offer an opportunity to selectively regulate T cell subset functions and to provide potential novel therapeutic approaches to modulate autoimmunity. [ABSTRACT FROM AUTHOR]

Thymic Epithelial Tumors (TETs) represent a rare tumor family, originating from the epithelial component of the thymus gland. Clinicopathologically, they are segregated into six major subtypes, associated with distinct histological features and clinical outcomes. Their emergence and evolution are accompanied by the generation of a complex tumor microenvironment (TME), dominated by phenotypically and functionally divergent immune cellular subsets, in different maturation states and in analogies that vary significantly among different subtypes. These heterogenous leukocyte populations exert either immune-permissive and tumor-suppressive functions or vice versa, and the dynamic equilibrium established among them either dictates the tumor immune milieu towards an immune-tolerance state or enables the development of a productive spontaneous tumoricidal response. The immunologically "hot" microenvironment, defining a significant proportion of TETs, makes them a promising candidate for the implementation of immune checkpoint inhibitors (ICIs). A number of phase I and II clinical trials have already demonstrated significant, type-specific clinical efficacy of PD-L1 inhibitors, even though substantial limitations in their utilization derive from their immune-mediated adverse effects. Moreover, the completed clinical studies involved relatively restricted patient samples and an expansion in the enrolled cohorts is required, so that more trustworthy conclusions regarding the benefit from ICIs in TETs can be extracted. [ABSTRACT FROM AUTHOR]

The β chain rearrangement in T cells is a two-step process where first Dβ and Jβ bind, and only then Vβ is joined to the complex. We here show that the frequency of human and mouse Vβ Jβ combinations deviates from the one expected based on each gene usage frequency. This bias is observed mainly in functional (F) rearrangements, but also slightly in non-functional (NF) rearrangements. Preferred Vβ Jβ combinations in F clones are shared between donors and samples, suggesting a common structural mechanism for these biases in addition to any host-specific antigen-induced peripheral selection. The sharing holds even in clones with Jβ1 that share the same Dβ 1 gene. Vβ Jβ usage is correlated with the Molecular Weight and Isoelectric Point in F clones. The pairing is also observed in the Double Positive cells in mice thymocytes, suggesting that the selection leading to such a pairing occurs before thymic selection. These results suggest an additional structural checkpoint in the beta chain development prior to thymic selection during the T cell receptor expression. Understanding this structural selection is important for the distinction between normal and aberrant T cell development, and crucial for the design of engineered TCRs. [ABSTRACT FROM AUTHOR]

Sanyal I et al i .12 reported the differential expression pattern of the CD20 homolog MS4A4A on the cell surface of human monocyte, dendritic cell (DC) and plasma cell populations in the peripheral blood and bone marrow. Integration of T-cell receptor, notch and cytokine signals programs mouse T-cell effector differentiation. To investigate how the therapeutic application of Toll-like receptor 2 (TLR2) activators can mobilize innate effector cells to inflammatory sites, Haidl I et al i .10 examined the mechanisms of TLR2-induced natural killer (NK) cell migration into the peritoneal cavity. On behalf of the Canadian Society for Immunology (CSI), it is our great pleasure to highlight a few of the many outstanding articles in I Immunology & Cell Biology i ( I ICB i ) authored by Canadian immunologists. [Extracted from the article]

Signaling via the TCR, which is initiated by the Src-family tyrosine kinase Lck, is crucial for the determination of cell fates in the thymus. Because of its pivotal role, ablation of Lck results in a profound block of T-cell development. Here, we show that, in addition to its well-known function in the initiation of TCR signaling, Lck also acts at a more downstream level. This novel function of Lck is determined by the tyrosine residue (Y192) located in its SH2 domain. Thymocytes from knock-in mice expressing a phosphomimetic Y192E mutant of Lck initiate TCR signaling upon CD3 cross-linking up to the level of PLC-γ1 phosphorylation. However, the activation of downstream pathways including Ca2+ influx and phosphorylation of Erk1/2 are impaired. Accordingly, positive and negative selections are blocked in LckY192E knock-in mice. Collectively, our data indicate that Lck has a novel function downstream of PLCγ-1 in the regulation of thymocyte differentiation and selection. [ABSTRACT FROM AUTHOR]

The thymus is a primary lymphoid organ essential for the induction of central immune tolerance. Maturing T cells undergo several steps of expansion and selection mediated by thymic epithelial cells (TECs). In APECED and other congenital pathologies, a deficiency in genes that regulate TEC development or their ability to select non auto-reactive thymocytes results in a defective immune balance, and consequently in a general autoimmune syndrome. Restoration of thymic function is thus crucial for the emergence of curative treatments. The last decade has seen remarkable progress in both gene editing and pluripotent stem cell differentiation, with the emergence of CRISPR-based gene correction, the trivialization of reprogramming of somatic cells to induced pluripotent stem cells (iPSc) and their subsequent differentiation into multiple cellular fates. The combination of these two approaches has paved the way to the generation of genetically corrected thymic organoids and their use to control thymic genetic pathologies affecting self-tolerance. Here we review the recent advances in differentiation of iPSc into TECs and the ability of the latter to support a proper and efficient maturation of thymocytes into functional and non-autoreactive T cells. A special focus is given on thymus organogenesis and pathway modulation during iPSc differentiation, on the impact of the 2/3D structure on the generated TECs, and on perspectives for therapeutic strategies in APECED based on patient-derived iPSc corrected for AIRE gene mutations. [ABSTRACT FROM AUTHOR]

γδT cells mature in the human thymus, and mainly produce IL-17A or IFN-γ, but can also produce IL-22 and modulate a variety of immune responses. Here, we aimed to evaluate whether IgG from AD patients (AD IgG) can functionally modulate thymic nonatopic γδT cells. Thymic tissues were obtained from 12 infants who had not had an atopic history. Thymocytes were cultured in mock condition, or in the presence of either AD IgG or therapeutic intravenous IgG (IVIg). Following these treatments, intracellular cytokine production, phenotype, and microRNA expression profiles were investigated. AD IgG could downregulate α4β7, upregulate CLA, and induce the production of IFN-γ, IL-17, and IL-22 in γδT cells. Although both AD IgG and IVIg could directly interact with γδT cell membranes, AD IgG could reduce γδT cell apoptosis. AD IgG could upregulate nine miRNAs compared to IVIg, and six when compared to the mock condition. In parallel, some miRNAs were downregulated. Target gene prediction and functional analysis indicated that some target genes were enriched in the negative regulation of cellular transcription. This study shows that AD IgG influences the production of IL-17 and IL-22 by intrathymic nonatopic γδT cells, and demonstrates epigenetic implications mediated by miRNAs. [ABSTRACT FROM AUTHOR]

One of the earliest hallmarks of immune aging is thymus involution, which not only reduces the number of newly generated and exported T cells, but also alters the composition and organization of the thymus microenvironment. Thymic T‐cell export continues into adulthood, yet the impact of thymus involution on the quality of newly generated T‐cell clones is not well established. Notably, the number and proportion of medullary thymic epithelial cells (mTECs) and expression of tissue‐restricted antigens (TRAs) decline with age, suggesting the involuting thymus may not promote efficient central tolerance. Here, we demonstrate that the middle‐aged thymic environment does not support rapid motility of medullary thymocytes, potentially diminishing their ability to scan antigen presenting cells (APCs) that display the diverse self‐antigens that induce central tolerance. Consistent with this possibility, thymic slice assays reveal that the middle‐aged thymic environment does not support efficient negative selection or regulatory T‐cell (Treg) induction of thymocytes responsive to either TRAs or ubiquitous self‐antigens. This decline in central tolerance is not universal, but instead impacts lower‐avidity self‐antigens that are either less abundant or bind to TCRs with moderate affinities. Additionally, the decline in thymic tolerance by middle age is accompanied by both a reduction in mTECs and hematopoietic APC subsets that cooperate to drive central tolerance. Thus, age‐associated changes in the thymic environment result in impaired central tolerance against moderate‐avidity self‐antigens, potentially resulting in export of increasingly autoreactive naive T cells, with a deficit of Treg counterparts by middle age. [ABSTRACT FROM AUTHOR]

CD4+ T cell responses to self-antigens are pivotal for immunological self-tolerance. Activation of Foxp3– T-conventional (T-conv) cells can precipitate autoimmune disease, whereas activation of Foxp3+ T-regulatory (T-reg) cells is essential to prevent autoimmune disease. This distinction indicates the importance of the thymus in controlling the differentiation of self-reactive CD4+ T cells. Thymocytes and thymic antigen-presenting cells (APC) depend on each other for normal maturation and differentiation. In this Hypothesis and Theory article, we propose this mutual dependence dictates which self-antigens induce T-reg cell development in the thymic medulla. We postulate self-reactive CD4+ CD8– thymocytes deliver signals that stabilize and amplify the presentation of their cognate self-antigen by APC in the thymic medulla, thereby seeding a niche for the development of T-reg cells specific for the same self-antigen. By limiting the number of antigen-specific CD4+ thymocytes in the medulla, thymocyte deletion in the cortex may impede the formation of medullary T-reg niches containing certain self-antigens. Susceptibility to autoimmune disease may arise from cortical deletion creating a "hole" in the self-antigen repertoire recognized by T-reg cells. [ABSTRACT FROM AUTHOR]

Thymic homing of hematopoietic progenitor cells (HPCs) is tightly regulated for proper T cell development. Previously we have identified a subset of specialized thymic portal endothelial cells (TPECs), which is important for thymic HPC homing. However, the underlying molecular mechanism still remains unknown. Here, we found that signal regulatory protein alpha (SIRPα) is preferentially expressed on TPECs. Disruption of CD47-SIRPα signaling in mice resulted in reduced number of thymic early T cell progenitors (ETPs), impaired thymic HPC homing, and altered early development of thymocytes. Mechanistically, Sirpa-deficient ECs and Cd47-deficient bone marrow progenitor cells or T lymphocytes demonstrated impaired transendothelial migration (TEM). Specifically, SIRPα intracellular ITIM motif-initiated downstream signaling in ECs was found to be required for TEM in an SHP2- and Src-dependent manner. Furthermore, CD47 signaling from migrating cells and SIRPα intracellular signaling were found to be required for VE-cadherin endocytosis in ECs. Thus, our study reveals a novel role of endothelial SIRPα signaling for thymic HPC homing for T cell development. [ABSTRACT FROM AUTHOR]

The transcription factors of the nuclear factor of activated T cell (NFAT) family play a crucial role in multiple aspects of T cell function. It has recently been reported that NFATs play an important role in the suppressive function of CD4+CD25+Foxp3+ regulatory T (Treg) cells. In this study, we have investigated the role of NFATs in the thymic development of Treg cells in mice. We show that NFAT factors are dispensable for the development of Foxp3+ Treg cells in the thymus but are critical for the maintenance of both the phenotype and survival of Treg cells in the thymus as well as in peripheral lymphoid organs. Specifically, the homeostasis of CD4+CD25+Foxp3+ but not the CD4+CD25−Foxp3+ fraction is severely perturbed when NFAT signaling is blocked, leading to a strongly reduced Treg population. We underscored this intriguing effect of NFAT on CD4+CD25+Foxp3+ Treg cells to the disruption of survival signals provided by interleukin 2 (IL-2). Accordingly, blocking Treg cell death by abolishing the activity of pro-apoptotic Bcl-2 family member Bim, compensated for the survival defects induced due to a lack of NFAT-IL-2-IL-2R signaling. Inhibition of NFAT activity led to a strong reduction in the number of Foxp3+ Treg cells; however, it did not influence the level of Foxp3 expression on an individual cell basis. In addition, we show a differential effect of IL-2 and IL-7 signaling on Foxp3+ Treg versus CD4+CD25− T cell development, again underlining the dispensability of NFAT signaling in the development, but not in the maintenance of Foxp3+ Treg cells. [ABSTRACT FROM AUTHOR]

Establishing both central and peripheral tolerance requires the appropriate TCR signaling strength to discriminate self‐ from agonist‐peptide bound to self MHC molecules. ZAP70, a cytoplasmic tyrosine kinase, directly interacts with the TCR complex and plays a central and requisite role in TCR signaling in both thymocytes and peripheral T cells. By studying ZAP70 hypomorphic mutations in mice and humans with a spectrum of hypoactive or hyperactive activities, we have gained insights into mechanisms of central and peripheral tolerance. Interestingly, both hypoactive and hyperactive ZAP70 can lead to the development of autoimmune diseases, albeit through distinct mechanisms. Immature thymocytes and mature T cells rely on normal ZAP70 function to complete their development in the thymus and to modulate T cell responses in the periphery. Hypoactive ZAP70 function compromises key developmental checkpoints required to establish central tolerance, allowing thymocytes with potentially self‐reactive TCRs a greater chance to escape negative selection. Such 'forbidden clones' may escape into the periphery and may pose a greater risk for autoimmune disease development since they may not engage negative regulatory mechanisms as effectively. Hyperactive ZAP70 enhances thymic negative selection but some thymocytes will, nonetheless, escape negative selection and have greater sensitivity to weak and self‐ligands. Such cells must be controlled by mechanisms involved in anergy, expansion of Tregs, and upregulation of inhibitory receptors or signaling molecules. However, such potentially autoreactive cells may still be able to escape control by peripheral negative regulatory constraints. Consistent with findings in Zap70 mutants, the signaling defects in at least one ZAP70 substrate, LAT, can also lead to autoimmune disease. By dissecting the similarities and differences among mouse models of patient disease or mutations in ZAP70 that affect TCR signaling strength, we have gained insights into how perturbed ZAP70 function can lead to autoimmunity. Because of our work and that of others on ZAP70, it is likely that perturbations in other molecules affecting TCR signaling strength will be identified that also overcome tolerance mechanisms and cause autoimmunity. Delineating these molecular pathways could lead to the development of much needed new therapeutic targets in these complex diseases. [ABSTRACT FROM AUTHOR]

We have previously demonstrated lactational transfer of T cell-based immunity from dam to foster pup. In the short term, a significant part of transferred immunity is passive cellular immunity. However, as time progresses, this is replaced by what we have described as maternal educational immunity such that by young adulthood, all immune cells responding to a foster dam immunogen are the product of the foster pup's thymus. To reduce confounding factors, this original demonstration used congenic/syngeneic dam and foster pup pairs. In this study, we investigated lactational transfer of immunity to Mycobacterium tuberculosis in MHC class I-mismatched animals, as well as from Th1-biased dams to Th2-biased foster pups. Using immunized C57BL/6J dams, lactational transfer to nonimmunized BALB/cJ foster pups resulted in much greater immunity than direct immunization in 5-wk-old pups (ex vivo assay of pup splenocytes). At this age, 82% of immunogen-responding cells in the pup spleen were produced through maternal educational immunity. FVB/NJ nonimmunized foster recipients had a greater number of maternal cells in the spleen and thymus but a much larger percentage was Foxp3+, resulting in equivalent immunity to direct immunization. Depletion of maternal Foxp3+ cells from pup splenocytes illustrated a substantial role for lactationally transferred dam regulatory T cells in suppression of the ex vivo response in FVB/NJ, but not BALB/cJ, recipients. We conclude that lactational transfer of immunity can cross MHC class I barriers and that Th1 immunity can be imparted to Th2-biased offspring; in some instances, it can be greater than that achieved by direct immunization.

The Src Family kinase Lck sets a critical threshold for T cell activation because it phosphorylates the TCR complex and the Zap70 kinase. How a T cell controls the abundance of active Lck molecules remains poorly understood. We have identified an unappreciated role for a phosphosite, Y192, within the Lck SH2 domain that profoundly affects the amount of active Lck in cells. Notably, mutation of Y192 blocks critical TCR-proximal signaling events and impairs thymocyte development in retrogenic mice. We determined that these defects are caused by hyperphosphorylation of the inhibitory C-terminal tail of Lck. Our findings reveal that modification of Y192 inhibits the ability of CD45 to associate with Lck in cells and dephosphorylate the C-terminal tail of Lck, which prevents its adoption of an active open conformation. These results suggest a negative feedback loop that responds to signaling events that tune active Lck amounts and TCR sensitivity.

Post-thymic naïve T cells constitute a key cellular arm of adaptive immunity, with a well-known characteristic of the specificity and robustness of responses to cognate foreign antigens which is presented as a form of antigen-derived peptides bound to major histocompatibility complex (MHC) molecules by antigen-presenting cells (APCs). In a steady state, however, these cells are resting, quiescent in their activity, but must keep full ranges of functional integrity to mount rapid and robust immunity to cope with various infectious pathogens at any time and space. Such unique property of resting naïve T cells is not acquired in a default manner but rather requires an active mechanism. Although our understanding of exactly how this process occurs and what factors are involved remains incomplete, a particular role of self-recognition by T cells has grown greatly in recent years. In this brief review, we discuss recent data on how the interaction of T cells with self-peptide MHC ligands regulates their functional responsiveness and propose that variable strength of self-reactivity imposes distinctly different levels of functional competence and heterogeneity.

T cells are generated from hematopoietic stem cells through a highly organized developmental process, in which stage-specific molecular events drive maturation towards αβ and γδ T cells. Although many of the mechanisms that control αβ- and γδ-lineage differentiation are shared between human and mouse, important differences have also been observed. Here, we studied the regulatory dynamics of the E and ID protein encoding genes during pediatric human T cell development by evaluating changes in chromatin accessibility, histone modifications and bulk and single cell gene expression. We profiled patterns of ID/E protein activity and identified up- and downstream regulators and targets, respectively. In addition, we compared transcription of E and ID protein encoding genes in human versus mouse to predict both shared and unique activities in these species, and in prenatal versus pediatric human T cell differentiation to identify regulatory changes during development. This analysis showed a putative involvement of TCF3/E2A in the development of γδ T cells. In contrast, in αβ T cell precursors a pivotal pre-TCR-driven population with high ID gene expression and low predicted E protein activity was identified. Finally, in prenatal but not postnatal thymocytes, high HEB/TCF12 levels were found to counteract high ID levels to sustain thymic development. In summary, we uncovered novel insights in the regulation of E and ID proteins on a cross-species and cross-developmental level.

Premature lymphocytes develop into non-autoreactive, mature naïve CD4+ or CD8+ T cells in the thymus before entering the circulation. However, in-depth characterization of human thymocyte development remains challenging due to limited availability of human thymus samples and the fragile nature of thymocyte populations. Thymocytes often do not survive cryopreservation and thawing procedures, especially the fragile CD4+CD8+ double positive population. It is generally recommended to use fresh human thymus tissue on the day of excision to avoid any biases in thymocyte composition. This hampers the possibility to perform multiple experiments on the same thymus sample. To establish how the thymocyte viability and composition can be maintained, we compared two thymocyte isolation methods used for human and/or mice thymi, three cryopreservation methods in combination with our most gentle thawing technique. Based on our findings we established that fresh human thymi remain viable in cold storage for up to two days post-surgery without compromising thymocyte composition. Thymocytes can be cryopreserved if required, although the CD4+CD8+ double positive populations may be reduced. Our study provides thoroughly optimized methods to study human thymocyte development over a considerable time-frame post-surgery. [ABSTRACT FROM AUTHOR]

The thymus is a crucial organ for the development of T cells. T cell progenitors first migrate from the bone marrow into the thymus. During the journey to become a mature T cell, progenitors require interactions with many different cell types within the thymic microenvironment, such as stromal cells, which include epithelial, mesenchymal and other non-T-lineage immune cells. There are two crucial decision steps that are required for generating mature T cells: positive and negative selection. Each of these two processes needs to be performed efficiently to produce functional MHC-restricted T cells, while simultaneously restricting the production of auto-reactive T cells. In each step, there are various cell types that are required for the process to be carried out suitably, such as scavengers to clean up apoptotic thymocytes that fail positive or negative selection, and antigen presenting cells to display self-antigens during positive and negative selection. In this review, we will focus on thymic non-T-lineage immune cells, particularly dendritic cells and macrophages, and the role they play in positive and negative selection. We will also examine recent advances in the understanding of their participation in thymus homeostasis and T cell development. This review will provide a perspective on how the thymic microenvironment contributes to thymocyte differentiation and T cell maturation. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are suggested to play a discriminative role between some stages of thymocyte differentiation. However, differential aspects of the stage of mature single-positive thymocytes remain to be explored. The purpose of this study is to investigate the differential expression pattern of circRNAs in three different development stages of human thymocytes, including mature single-positive cells, and perform predictions in silico regarding the ability of specific circRNAs when controlling the expression of genes involved in thymocyte differentiation. We isolate human thymocytes at three different stages of intrathymic differentiation and determine the expression of circRNAs and mRNA by RNASeq. We show that the differential expression pattern of 50 specific circRNAs serves to discriminate between the three human thymocyte populations. Interestingly, the downregulation of RAG2, a gene involved in T-cell differentiation in the thymus, could be simultaneously controlled by the downregulation of two circRNASs (hsa_circ_0031584 and hsa_circ_0019079) through the hypothetical liberation of hsa-miR-609. Our study provides, for the first time, significant insights into the usefulness of circRNAs in discriminating between different stages of thymocyte differentiation and provides new potential circRNA–miRNA–mRNA networks capable of controlling the expression of genes involved in T-cell differentiation in the thymus. [ABSTRACT FROM AUTHOR]

A basic study on the development of Hybridoma was undertaken, while using buffalo Prolactin as the immunogen. The fusion protocol was standardized with respect to the ratio of splenocytes vs myeloma cells, percentage of polyethylene glycol (PEG), volume of PEG, the nutrient selection of hybrids with HAT medium, the day of HAT shock and the propagation of hybrid secreting desired antibodies. The effectiveness of primary immunization at intradermal and subcutaneous site/sites was also studied. The use of splenocytes or thymocytes from the non-immunised mice as feeder layers and the day of plating them in 96 well plates was standardized too. [ABSTRACT FROM AUTHOR]

Background: Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown that AIRE knockout (KO) mice have a small and disorganized thymic medulla; however, whether AIRE influences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed an in vitro model system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. Results: The comparisons between AIRE wild type (AIREWT) and AIRE KO (AIRE−/−) 3D mTEC spheroid formation showed that the absence of AIRE: i) disorganizes the 3D structure of mTEC spheroids, ii) increases the proportion of cells at the G0/G1 phase of the cell cycle, iii) increases the rate of mTEC apoptosis, iv) decreases the strength of mTEC-mTEC adhesion, v) promotes a differential regulation of mTEC classical surface markers, and vi) modulates genes encoding adhesion and other molecules. Conclusions: Overall, the results show that AIRE influences the 3D structuring of mTECs when these cells begin the spheroid formation through controlling cell adhesion genes. [ABSTRACT FROM AUTHOR]

EZH2 plays an essential role at the β-selection checkpoint of T lymphopoiesis by regulating histone H3 lysine 27 trimethylation (H3K27me3) via its canonical mode of action. Increasing data suggest that EZH2 could also regulate other cellular functions, such as cytoskeletal reorganization, via its noncanonical pathway. Consequently, we investigated whether the EZH2 noncanonical pathway could be involved in early T-cell maturation, which requires cell polarization. We observed that EZH2 localization is tightly regulated during the early stages of T-cell development and that EZH2 relocalizes in the nucleus of double-negative thymocytes enduring TCRβ recombination and β-selection processes. Furthermore, we observed that EZH2 and EED, but not Suz12, colocalize with the microtubule organization center (MTOC), which might prevent its inappropriate polarization in double negative cells. In accordance with these results, we evidenced the existence of direct or indirect interaction between EED and α-tubulin. Taken together, these results suggest that the EZH2 noncanonical pathway, in association with EED, is involved in the early stages of T-cell maturation. [ABSTRACT FROM AUTHOR]

Simple Summary: Because children have a long life expectancy relative to adults and their tissues and organs are growing and developing rapidly, the risk of radiation carcinogenesis for children is considered higher than that for adults. However, the underlying mechanism(s) is unclear. To uncover the mechanism, we previously revealed that principal causative genes in mouse thymic lymphomas arising in irradiated infants or adults as Pten or Ikzf1, respectively, suggesting that cells with mutation in these genes might be the origin of lymphomas arising after irradiation depending on age at exposure. Here, we clarified the age-dependent differences in thymus-cell dynamics in mice during the initial post-irradiation period. Our results demonstrate that the dynamics of thymocytes during the post-irradiation period depends on the age at exposure. For irradiated infants in particular, the number of proliferating cells increase dramatically, and this correlate with activation of the PI3K-AKT-mTOR pathway. Thus, we conclude that the PI3K-AKT-mTOR pathway in infants contributed, at least in part, to thymus-cell dynamics through the modification of cell proliferation and survival after irradiation, which may be associated with the risk of Pten mutation-associated thymic lymphoma. The risk of radiation-induced carcinogenesis depends on age at exposure. We previously reported principal causative genes in lymphomas arising after infant or adult exposure to 4-fractionated irradiation as Pten or Ikzf1, respectively, suggesting that cells with mutation in these genes might be the origin of lymphomas arising after irradiation depending on age at exposure. Here, we clarified the age-dependent differences in thymus-cell dynamics in mice during the initial post-irradiation period. The thymocyte number initially decreased, followed by two regeneration phases. During the first regeneration, the proportion of phosphorylated-AKT-positive (p-AKT+) cells in cell-cycle phases S+G2/M of immature CD4−CD8− and CD4+CD8+ thymocytes and in phases G0/G1 of mature CD4+CD8− and CD4−CD8+ thymocytes was significantly greater in irradiated infants than in irradiated adults. During the second regeneration, the proportion of p-AKT+ thymocytes in phases G0/G1 increased in each of the three populations other than CD4−CD8− thymocytes more so than during the first regeneration. Finally, PI3K-AKT-mTOR signaling in infants contributed, at least in part, to biphasic thymic regeneration through the modification of cell proliferation and survival after irradiation, which may be associated with the risk of Pten mutation-associated thymic lymphoma. [ABSTRACT FROM AUTHOR]

Negative selection of developing T cells plays a significant role in T‐cell tolerance to self‐antigen. This process relies on thymic antigen‐presenting cells which express both self‐antigens and cosignaling molecules. Inducible T‐cell costimulator (ICOS) belongs to the CD28 family of cosignaling molecules and binds to ICOS ligand (ICOSL). The ICOS signaling pathway plays important roles in shaping the immune response to infections, but its role in central tolerance is less well understood. Here we show that ICOSL is expressed by subsets of thymic dendritic cells and medullary thymic epithelial cells as well as thymic B cells. ICOS expression is upregulated as T cells mature in the thymus and correlates with T‐cell receptor signal strength during thymic selection. We also provide evidence of a role for ICOS signaling in mediating negative selection. Our findings suggest that ICOS may fine‐tune T‐cell receptor signals during thymic selection contributing to the generation of a tolerant T‐cell population. [ABSTRACT FROM AUTHOR]

In the quest for more effective radiation treatment options that can improve both cell killing and healthy tissue recovery, combined radiation therapies are lately in the spotlight. The molecular response to a combined radiation regime where exposure to an initial low dose (priming dose) of ionizing radiation is administered prior to a subsequent higher radiation dose (challenging dose) after a given latency period have not been thoroughly explored. In this study we report on the differential response to either a combined radiation regime or a single challenging dose both in mouse in vivo and in human ex vivo thymocytes. A differential cell cycle response including an increase in the subG1 fraction on cells exposed to the combined regime was found. Together with this, a differential protein expression profiling in several pathways including cell cycle control (ATM, TP53, p21CDKN1A), damage response (γH2AX) and cell death pathways such as apoptosis (Cleaved Caspase-3, PARP1, PKCδ and H3T45ph) and ferroptosis (xCT/GPX4) was demonstrated. This study also shows the epigenetic regulation following a combined regime that alters the expression of chromatin modifiers such as DNMTs (DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L) and glycosylases (MBD4 and TDG). Furthermore, a study of the underlying cellular status six hours after the priming dose alone showed evidence of retained modifications on the molecular and epigenetic pathways suggesting that the priming dose infers a "radiation awareness phenotype" to the thymocytes, a sensitization key to the differential response seen after the second hit with the challenging dose. These data suggest that combined-dose radiation regimes could be more efficient at making cells respond to radiation and it would be interesting to further investigate how can these schemes be of use to potential new radiation therapies. [ABSTRACT FROM AUTHOR]

T-cell development in the thymus undergoes the process of differentiation, selective proliferation, and survival from CD4-CD8-double negative (DN) stage to CD4+CD8+ double positive (DP) stage prior to the formation of CD4+ helper and CD8+ cytolytic T cells ready for circulation. Each developmental stage is tightly regulated by sequentially operating molecular networks, of which only limited numbers of transcription regulators have been deciphered. Here, we identified Zfp335 transcription factor as a new player in the regulatory network controlling thymocyte development in mice. We demonstrate that Zfp335 intrinsically controls DN to DP transition, as T-cell-specific deficiency in Zfp335 leads to a substantial accumulation of DN3 along with reduction of DP, CD4+, and CD8+ thymocytes. This developmental blockade at DN stage results from the impaired intracellular TCRβ (iTCRβ) expression as well as increased susceptibility to apoptosis in thymocytes. Transcriptomic and ChIP-seq analyses revealed a direct regulation of transcription factors Bcl6 and Rorc by Zfp335. Importantly, enhanced expression of TCRβ and Bcl6/Rorc restores the developmental defect during DN3 to DN4 transition and improves thymocytes survival, respectively. These findings identify a critical role of Zfp335 in controlling T-cell development by maintaining iTCRβ expression-mediated β-selection and independently activating cell survival signaling. [ABSTRACT FROM AUTHOR]

Type-I interferon (IFN-I) signals exert a critical role in disease progression during viral infections. However, the immunomodulatory mechanisms by which IFN-I dictates disease outcomes remain to be fully defined. Here we report that IFN-I signals mediate thymic atrophy in viral infections, with more severe and prolonged loss of thymic output and unique kinetics and subtypes of IFN-α/β expression in chronic infection compared to acute infection. Loss of thymic output was linked to inhibition of early stages of thymopoiesis (DN1-DN2 transition, and DN3 proliferation) and pronounced apoptosis during the late DP stage. Notably, infection-associated thymic defects were largely abrogated upon ablation of IFNαβR and partially mitigated in the absence of CD8 T cells, thus implicating direct as well as indirect effects of IFN-I on thymocytes. These findings provide mechanistic underpinnings for immunotherapeutic strategies targeting IFN-1 signals to manipulate disease outcomes during chronic infections and cancers. • Critical role of IFN-I signals in mediating thymic atrophy in chronic viral infection through IFN-α/βR. • Unique IFN-Iα program associated with aberrant thymopoiesis and thymic atrophy in chronic viral infection. • IFN-I induces thymic atrophy through both CD8 T cell dependent and independent mechanisms. [ABSTRACT FROM AUTHOR]

PTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor involved in numerous types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). In human, loss-of-function mutations of PTEN are correlated to mature T-ALL expressing a T-cell receptor (TCR) at their cell surface. In accordance with human T-ALL, inactivation of Pten gene in mouse thymocytes induces TCRαβ+ T-ALL development. Herein, we explored the functional interaction between TCRαβ signaling and PTEN. First, we performed single-cell RNA sequencing (scRNAseq) of PTEN-deficient and PTEN-proficient thymocytes. Bioinformatic analysis of our scRNAseq data showed that pathological Ptendel thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Ptendel thymocytes display a lower calcium pathway activity score compared to their physiological counterparts. We confirmed this result using ex vivo calcium flux assay and showed that upon TCR activation tumor Ptendel blasts were unable to release calcium ions (Ca2+) from the endoplasmic reticulum to the cytosol. In order to understand such phenomena, we constructed a mathematical model centered on the mechanisms controlling the calcium flux, integrating TCR signal strength and PTEN interactions. This qualitative model displays a dynamical behavior coherent with the dynamics reported in the literature, it also predicts that PTEN affects positively IP3 (inositol 1,4,5-trisphosphate) receptors (ITPR). Hence, we analyzed Itpr expression and unraveled that ITPR proteins levels are reduced in PTEN-deficient tumor cells compared to physiological and leukemic PTEN-proficient cells. However, calcium flux and ITPR proteins expression are not defective in non-leukemic PTEN-deficient T cells indicating that beyond PTEN loss an additional alteration is required. Altogether, our study shows that ITPR/Calcium flux is a part of the oncogenic landscape shaped by PTEN loss and pinpoints a putative role of PTEN in the regulation of ITPR proteins in thymocytes, which remains to be characterized. [ABSTRACT FROM AUTHOR]

PTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor involved in numerous types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). In human, loss-of-function mutations of PTEN are correlated to mature T-ALL expressing a T-cell receptor (TCR) at their cell surface. In accordance with human T-ALL, inactivation of Pten gene in mouse thymocytes induces TCRαβ+ T-ALL development. Herein, we explored the functional interaction between TCRαβ signaling and PTEN. First, we performed single-cell RNA sequencing (scRNAseq) of PTEN-deficient and PTEN-proficient thymocytes. Bioinformatic analysis of our scRNAseq data showed that pathological Ptendel thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Ptendel thymocytes display a lower calcium pathway activity score compared to their physiological counterparts. We confirmed this result using ex vivo calcium flux assay and showed that upon TCR activation tumor Ptendel blasts were unable to release calcium ions (Ca2+) from the endoplasmic reticulum to the cytosol. In order to understand such phenomena, we constructed a mathematical model centered on the mechanisms controlling the calcium flux, integrating TCR signal strength and PTEN interactions. This qualitative model displays a dynamical behavior coherent with the dynamics reported in the literature, it also predicts that PTEN affects positively IP3 (inositol 1,4,5-trisphosphate) receptors (ITPR). Hence, we analyzed Itpr expression and unraveled that ITPR proteins levels are reduced in PTEN-deficient tumor cells compared to physiological and leukemic PTEN-proficient cells. However, calcium flux and ITPR proteins expression are not defective in non-leukemic PTEN-deficient T cells indicating that beyond PTEN loss an additional alteration is required. Altogether, our study shows that ITPR/Calcium flux is a part of the oncogenic landscape shaped by PTEN loss and pinpoints a putative role of PTEN in the regulation of ITPR proteins in thymocytes, which remains to be characterized.

The ability of T cells to respond to a wide array of foreign antigens while avoiding reactivity to self is largely determined by cellular selection of developing T cells in the thymus. While a great deal is known about the cell types and molecules involved in T-cell selection in the thymus, our understanding of the spatial and temporal aspects of this process remain relatively poorly understood. Thymocytes are highly motile within the thymus and travel between specialized microenvironments at different phases of their development while interacting with distinct sets of self-peptides and peptide presenting cells. A knowledge of when, where, and how thymocytes encounter self-peptide MHC ligands at different stages of thymic development is key to understanding T-cell selection. In the past several years, our laboratory has investigated this topic using two-photon time-lapse microscopy to directly visualize thymocyte migration and signaling events, together with a living thymic slice preparation to provide a synchronized experimental model of T-cell selection in situ. Here, we discuss recent advances in our understanding of the temporal and spatial aspects of T-cell selection, highlighting our own work, and placing them in the context of work from other groups.

The receptor-like tyrosine phosphatase CD45 regulates antigen receptor signaling by dephosphorylating the C-terminal inhibitory tyrosine of the src family kinases. However, despite its abundance, the function of the large, alternatively spliced extracellular domain of CD45 has remained elusive. We used normally spliced CD45 transgenes either incorporating a phosphatase-inactivating point mutation or lacking the cytoplasmic domain to uncouple the enzymatic and noncatalytic functions of CD45 in lymphocytes. Although these transgenes did not alter T-cell signaling or development irrespective of endogenous CD45 expression, both partially rescued the phenotype of CD45-deficient B cells. We identify a noncatalytic role for CD45 in regulating tonic, but not antigen-mediated, B-cell antigen receptor (BCR) signaling through modulation of the function of the inhibitory coreceptor CD22. This finding has important implications for understanding how naïve B cells maintain tonic BCR signaling while restraining inappropriate antigen-dependent activation to preserve clonal ignorance.

How multipotential cells initiate distinct gene expression programs in response to external cues to instruct cell fate choice remains a fundamental question in biology. Establishment of CD4 and CD8 T cell fates during thymocyte development is critically regulated by T cell receptor (TCR) signals, which in turn control expression of the CD4-determining transcription factor ThPOK. However, the mechanism whereby differential TCR signals are molecularly interpreted to promote or antagonize ThPOK expression, and thereby CD4 versus CD8 lineage fates remains unknown. Here we show, using reverse genetic and molecular approaches that an autonomous, position-independent TCR-sensing switch is embedded within the ThPOK locus. Further, using an in vivo mutagenesis approach, we demonstrate that differential TCR signals are interpreted during lineage commitment by relative binding of EGR, NFAT and Ebox factors to this bistable switch. Collectively our study reveals the central molecular mechanism whereby TCR signaling influences differential lineage choice. Ultimately, these findings may provide an important new tool for skewing T cell fate to treat cancer and autoimmune diseases. Basu Jayati et al. examine how T-cell receptor (TCR) engagement with MHC ligands can contribute toward adoption of CD4 and CD8 cell fates during development. By using an in vivo mutagenesis approach in mice, their results suggest potential mechanisms linking TCR signaling to ThPOK expression and differential immune lineage choice. [ABSTRACT FROM AUTHOR]

In the thymus, the thymic epithelium provides a microenvironment essential for the development of functionally competent and self-tolerant T cells. Previous findings showed that modulation of Wnt/β-catenin signaling in mouse thymic epithelial cells (TECs) disrupts embryonic thymus organogenesis. However, the role of β-catenin in TECs for postnatal T-cell development remains to be elucidated. Here, we analyzed gain-of-function (GOF) and loss-of-function (LOF) of β-catenin highly specific in mouse TECs. We found that GOF of β-catenin in TECs results in severe thymic dysplasia and T-cell deficiency beginning from the embryonic period. By contrast, LOF of β-catenin in TECs reduces the number of cortical TECs and thymocytes modestly and only postnatally. These results indicate that fine-tuning of β-catenin expression within a permissive range is required for TECs to generate an optimal microenvironment to support postnatal T-cell development. [ABSTRACT FROM AUTHOR]

Fucoxanthin is a natural carotenoid obtained from seaweed which exhibits antioxidant properties. This research aimed to assess whether fucoxanthin, immobilized on aluminum-silicon carrier particles, has a toxic effect on immune cells. The viability, proliferation, nitric oxide production and myeloperoxidase activity of thymocytes and splenocytes of mice in vitro were studied. It was shown that fucoxanthin, immobilized on aluminum-silicon carrier particles, increased the survival rate and proliferation of mature immunocytes (splenocytes) after 24 hours exposure and increased the survival rate of naïve immunocytes (thymocytes) when exposed for 120 hours. In terms of myeloperoxidase, the activity of the immune cells was not affected by fucoxanthin immobilized on the carrier particles. The obtained results indicated that fucoxanthin, immobilized on particles of an aluminum-silicon carrier, did not have a toxic effect on mouse immunocytes. [ABSTRACT FROM AUTHOR]

X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesinR171W). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesinR171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesinR171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesinR171W. Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesinR171W, and will provide new insights into how the actin cortex regulates lymphocyte function. [ABSTRACT FROM AUTHOR]

Objectives: Flow cytometric detection of T-cell clonality is challenging, particularly in differential diagnosis of immature T-cell proliferations. Studies have shown utility of TRBC1, in conjunction with other T-cell markers, as reliable means to identify T-cell clonality by flow cytometry. One limitation of surface TRBC1 (sTRBC1) evaluation is it cannot be detected in surface CD3 (sCD3)-negative T cells, such as normal or abnormal immature T-cell precursors. Here, we assess surface and cytoplasmic TRBC1 expression patterns in the differential diagnosis of T-lymphoblastic leukemia/lymphoma (T-ALL) vs normal thymocyte expansions.Methods: Forty-three samples containing T-ALL, thymoma, normal thymus, and/or indolent T-lymphoblastic proliferation (i-TLBP), were evaluated.Results: All 24 cases with normal thymocytes or i-TLBPs revealed a characteristic and reproducible sCD3/sTRBC1 expression pattern indicative of polytypic T-cell maturation. In contrast, all 19 T-ALLs lacked this polytypic maturation pattern and were either completely negative for sCD3/sTRBC1 or showed a minor sCD3-positive subset with a monotypic TRBC1 expression pattern. Cytoplasmic TRBC1 evaluation in 9 T-ALLs demonstrated a monotypic intracellular TRBC1-positive (n = 4) or TRBC1-negative (n = 5) expression, indicative of clonality.Conclusions: Our findings demonstrate flow cytometric evaluation of surface and cytoplasmic TRBC1 expression can aid detection of T-cell clonality and differential diagnosis of immature T-cell proliferations. [ABSTRACT FROM AUTHOR]

Central tolerance is achieved through positive and negative selection of thymocytes mediated by T cell receptor (TCR) signaling strength. Thus, dysregulation of the thymic selection process often leads to autoimmunity. Here, we show that Capicua (CIC), a transcriptional repressor that suppresses autoimmunity, controls the thymic selection process. Loss of CIC prior to T-cell lineage commitment impairs both positive and negative selection of thymocytes. CIC deficiency attenuated TCR signaling in CD4+CD8+ double-positive (DP) cells, as evidenced by a decrease in CD5 and phospho-ERK levels and calcium flux. We identified Spry4, Dusp4, Dusp6, and Spred1 as CIC target genes that could inhibit TCR signaling in DP cells. Furthermore, impaired positive selection and TCR signaling were partially rescued in Cic and Spry4 double mutant mice. Our findings indicate that CIC is a transcription factor required for thymic T cell development and suggests that CIC acts at multiple stages of T cell development and differentiation to prevent autoimmunity. [ABSTRACT FROM AUTHOR]

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme intrinsic to the purine salvage pathway, leads to severe combined immunodeficiency (SCID) both in humans and mice. Lack of ADA results in the intracellular accumulation of toxic metabolites which have effects on T cell development and function. While untreated ADA-SCID is a fatal disorder, there are different therapeutic options available to restore ADA activity and reconstitute a functioning immune system, including enzyme replacement therapy (ERT). Administration of ERT in the form of pegylated bovine ADA (PEG-ADA) has proved a life-saving though non-curative treatment for ADA-SCID patients. However, in many patients treated with PEG-ADA, there is suboptimal immune recovery with low T and B cell numbers. Here, we show reduced thymus cellularity in ADA-SCID mice despite weekly PEG-ADA treatment. This was associated with lack of effective adenosine (Ado) detoxification in the thymus. We also show that thymocyte development in ADA-deficient thymi is arrested at the DN3-to-DN4 stage transition with thymocytes undergoing dATP-induced apoptosis rather than defective TCRβ rearrangement or β-selection. Our studies demonstrate at a detailed level that exogenous once-a-week enzyme replacement does not fully correct intra-thymic metabolic or immunological abnormalities associated with ADA deficiency. [ABSTRACT FROM AUTHOR]

iNKT cells play a critical role in controlling the strength and character of adaptive and innate immune responses. Their unique functional characteristics are induced by a transcriptional program initiated by positive selection mediated by CD1d expressed by CD4+CD8+ (double positive, DP) thymocytes. Here, using a novel Vα14 TCR transgenic strain bearing greatly expanded numbers of CD24hiCD44loNKT cells, we examined transcriptional events in four immature thymic iNKT cell subsets. A transcriptional regulatory network approach identified transcriptional changes in proximal components of the TCR signalling cascade in DP NKT cells. Subsequently, positive and negative selection, and lineage commitment, occurred at the transition from DP NKT to CD4 NKT. Thus, this study introduces previously unrecognised steps in early NKT cell development, and separates the events associated with modulation of the T cell signalling cascade prior to changes associated with positive selection and lineage commitment. [ABSTRACT FROM AUTHOR]

• WAire KO mTECs were compared to Aire WT mTECs for lncRNA transcriptional profiling both in vitro and in vivo model systems. •In conjunction with Aire, mTEC-thymocyte adhesion modulates the expression of lncRNAs in mTECs in vitro. • The mTECs isolated from Aire KO mice differentially express a set of lncRNAs. • lncRNAs that are unique or shared between mTECs cultured in vitro and mTECs from fresh thymus (in vivo) were identified. • We show a synergistic mechanism involving a link between thymocyte adhesion, Aire, and lncRNAs in mTECs. The autoimmune regulator (Aire) gene in medullary thymic epithelial cells (mTECs) encodes the AIRE protein, which interacts with its partners within the nucleus. This "Aire complex" induces stalled RNA Pol II on chromatin to proceed with transcription elongation of a large set of messenger RNAs and microRNAs. Considering that RNA Pol II also transcribes long noncoding RNAs (lncRNAs), we hypothesized that Aire might be implicated in the upstream control of this RNA species. To test this, we employed a loss-of-function approach in which Aire knockout mTECs were compared to Aire wild-type mTECs for lncRNA transcriptional profiling both in vitro and in vivo model systems. RNA sequencing enables the differential expression profiling of lncRNAs when these cells adhere in vitro to thymocytes or do not adhere to them as a way to test the effect of cell adhesion. Sets of lncRNAs that are unique and that are shared in vitro and in vivo were identified. Among these, we found the Aire-dependent lncRNAs as for example, Platr28, Ifi30, Morrbid, Malat1, and Xist. This finding represents the first evidence that Aire mediates the transcription of lncRNAs in mTECs. Microarray hybridizations enabled us to observe that temporal thymocyte adhesion modulates the expression levels of such lncRNAs as Morrbid, Xist, and Fbxl12o after 36 h of adhesion. This finding shows the existence of a synergistic mechanism involving a link between thymocyte adhesion, Aire, and lncRNAs in mTECs that might be important for immune self-representation. [ABSTRACT FROM AUTHOR]

The prolactin hormone (PRL), in addition to its known effects on breast development and lactation, exerts effects on the immune system, including pleiotropic effects on the thymus. The aim of this study was to evaluate the influence of PRL on the epithelial compartment of the thymus. Thymic epithelial cells (TECs) (2BH4 cells) and fresh thymocytes were used. Immunofluorescence assay revealed that PRL treatment (10 ng/mL) increases the deposition of laminin and expression of the chemokine CXCL12 in 2BH4 cells. However, no change was observed in the deposition of fibronectin. Moreover, PRL altered F-actin polymerisation, allowing the formation of focal adhesion complexes in treated cells. When 2BH4 cells were pre-treated with PRL, thymocyte adhesion was not altered. However, in the cell migration assay, pre-treatment with PRL potentiated the chemotactic effect of CXCL12 on the migration of total, double-positive, CD4-positive, and CD8-positive thymocytes. Together, the results of this study demonstrate the effect of PRL on thymic epithelial cells, particularly on CXCL12-driven thymocyte migration, confirming that this hormone is a regulator of thymic physiology. [ABSTRACT FROM AUTHOR]

Ataxia Telangiectasia (A-T) and Ataxia with Ocular Apraxia Type 1 (AOA1) are devastating neurological disorders caused by null mutations in the genome stability genes, A-T mutated (ATM) and Aprataxin (APTX), respectively. Our mechanistic understanding and therapeutic repertoire for treating these disorders are severely lacking, in large part due to the failure of prior animal models with similar null mutations to recapitulate the characteristic loss of motor coordination (i.e., ataxia) and associated cerebellar defects. By increasing genotoxic stress through the insertion of null mutations in both the Atm (nonsense) and Aptx (knockout) genes in the same animal, we have generated a novel mouse model that for the first time develops a progressively severe ataxic phenotype associated with atrophy of the cerebellar molecular layer. We find biophysical properties of cerebellar Purkinje neurons (PNs) are significantly perturbed (e.g., reduced membrane capacitance, lower action potential [AP] thresholds, etc.), while properties of synaptic inputs remain largely unchanged. These perturbations significantly alter PN neural activity, including a progressive reduction in spontaneous AP firing frequency that correlates with both cerebellar atrophy and ataxia over the animal's first year of life. Double mutant mice also exhibit a high predisposition to developing cancer (thymomas) and immune abnormalities (impaired early thymocyte development and T-cell maturation), symptoms characteristic of A-T. Finally, by inserting a clinically relevant nonsense-type null mutation in Atm, we demonstrate that Small Molecule Read-Through (SMRT) compounds can restore ATM production, indicating their potential as a future A-T therapeutic. [ABSTRACT FROM AUTHOR]