Overexpression of Lmo2 initiates T-lymphoblastic leukemia via impaired thymocyte competition.

Cell competition has recently emerged as an important tumor suppressor mechanism in the thymus that inhibits autonomous thymic maintenance. Here, we show that the oncogenic transcription factor Lmo2 causes autonomous thymic maintenance in transgenic mice by inhibiting early T cell differentiation. This autonomous thymic maintenance results in the development of self-renewing preleukemic stem cells (pre-LSCs) and subsequent leukemogenesis, both of which are profoundly inhibited by restoration of thymic competition or expression of the antiapoptotic factor BCL2. Genomic analyses revealed the presence of Notch1 mutations in pre-LSCs before subsequent loss of tumor suppressors promotes the transition to overt leukemogenesis. These studies demonstrate a critical role for impaired cell competition in the development of pre-LSCs in a transgenic mouse model of T cell acute lymphoblastic leukemia (T-ALL), implying that this process plays a role in the ontogeny of human T-ALL.

Evaluation of thymopoiesis in healthy Turkish children aged 0-6 years.

BACKGROUND: Early diagnosis and effective treatment serve as life-saving procedures for primary immunodeficiencies (PIDs) which are very common and a major public health problem in Turkey. Severe combined immunodeficiency (SCID) is constitutively a T-cell defect in which naïve T-cell development is defective due to the mutations in genes responsible for the T cell differentiation and insufficient thymopoiesis. So, assessment of thymopoiesis is very important in the diagnosis of SCID and several combined immune deficiencies (CIDs). METHODS: The purpose of this study is to examine thymopoiesis in healthy children via measurement of recent thymic emigrants (RTE); T lymphocytes that express CD4, CD45RA and CD31 to establish the RTE reference values in Turkish children. RTE were measured in the peripheral blood (PB) of 120 healthy infants and children between 0-6 years including cord blood samples, by flow cytometry. RESULTS: The absolute count of RTE cells and their relative ratios were found to be higher during the first year of life, being highest at the 6th month and tending to decrease significantly by age following birth (p=0.001). In the cord blood group, both values were lower than those in the 6-month-old group. The absolute lymphocyte count (ALC) varying by age, was found to reduce to 1850/mm³ in 4-years and after. CONCLUSIONS: Here we evaluated normal thymopoiesis and established the normal reference levels of RTE cells in the peripheral blood of healthy children aged between 0-6 years. We believe that the collected data will contribute to early diagnosis and monitoring of immune reconstitution; serving as an additional fast and reliable marker for many PID patients especially for SCID including many other CIDs, especially in nations where newborn screening (NBS) via T cell receptor excision circles (TREC) has not yet become available.

Germline T cell receptor exchange results in physiological T cell development and function.

T cell receptor (TCR) transgenic mice represent an invaluable tool to study antigen-specific immune responses. In the pre-existing models, a monoclonal TCR is driven by a non-physiologic promoter and randomly integrated into the genome. Here, we create a highly efficient methodology to develop T cell receptor exchange (TRex) mice, in which TCRs, specific to the self/tumor antigen mesothelin (Msln), are integrated into the Trac locus, with concomitant Msln disruption to circumvent T cell tolerance. We show that high affinity TRex thymocytes undergo all sequential stages of maturation, express the exogenous TCR at DN4, require MHC class I for positive selection and undergo negative selection only when both Msln alleles are present. By comparison of TCRs with the same specificity but varying affinity, we show that Trac targeting improves functional sensitivity of a lower affinity TCR and confers resistance to T cell functional loss. By generating P14 TRex mice with the same specificity as the widely used LCMV-P14 TCR transgenic mouse, we demonstrate increased avidity of Trac-targeted TCRs over transgenic TCRs, while preserving physiologic T cell development. Together, our results support that the TRex methodology is an advanced tool to study physiological antigen-specific T cell behavior.

Type III interferon drives thymic B cell activation and regulatory T cell generation.

The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate T cell central tolerance. The processes leading to licensing are still not fully understood. By comparing thymic B cells to activated Peyer's patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by TCR/CD40-dependent activation, followed by immunoglobulin class switch recombination (CSR) without forming germinal centers. Transcriptional analysis also demonstrated a strong interferon signature, which was not apparent in the periphery. Thymic B cell activation and CSR were primarily dependent on type III IFN signaling, and loss of type III IFN receptor in thymic B cells resulted in reduced thymocyte regulatory T cell (Treg) development. Finally, from TCR deep sequencing, we estimate that licensed B cells induce development of a substantial fraction of the Treg cell repertoire. Together, these findings reveal the importance of steady-state type III IFN in generating licensed thymic B cells that induce T cell tolerance to activated B cells.

An innovative immunotherapeutic strategy for rheumatoid arthritis: Selectively suppressing angiogenesis and osteoclast differentiation by fully human antibody targeting thymocyte antigen-1.

Thymocyte antigen-1 (THY-1)is a potential target for rheumatoid arthritis (RA) treatment, and THY-1 positive fibroblast-like synoviocytes (FLS) are enriched in the synovium of RA patients and participate in angiogenesis to accelerate RA progression. In this study, we screened an antibody targeting THY-1 (THY-1 Ab) and explored its mechanism in alleviating RA progression. THY-1 Ab was screened from ScFv phage antibody library by phage display technology (PDT). THY-1 Ab-treated collagen induced arthritis (CIA) mice had lower degree of arthritis scores. We explore the mechanism of THY-1 Ab in alleviating RA progression. THY-1 Ab can remarkably inhibit the secretion of pro-inflammatory factors and promote the secretion of anti-inflammatory factors. Further experiments showed that THY1 Ab downregulated the expression of JUNB by the hsa_circ_0094342/miRNA-155-5P/SPI1 axis, inhibited RA angiogenesis and osteoclast differentiation, and relieved RA progression. These findings support that THY-1 Ab is a promising therapeutic antibody for RA treatment.

Characterisation of APS-1 Experimental Models Is Crucial for Development of Novel Therapies.

Autoimmune polyglandular syndrome type 1 (APS-1) is an inherited autosomal disorder. The most common clinical features of the disease include adrenocortical failure, hypoparathyroidism (HP), and chronic mucocutaneous candidiasis (CMC). APS-1 is caused by mutations in the autoimmune regulator (AIRE) gene. AIRE is a transcriptional factor involved in the regulation of thousands of genes in the thymus. It facilitates central tolerance by promoting the ectopic expression of tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs), leading to the deletion of self-reactive thymocytes. Several Aire-deficient mice were developed separately, on different backgrounds; seven published Aire knockout mice show a variety of phenotypes depending on the strain used to generate the experimental model. The first Aire-deficient mice were generated on a "black 6" background almost 20 years ago. The model showed mild phenotype with relatively modest penetrance compared to models generated on BALBc or NOD backgrounds. The generation of all these experimental models is crucial for development and testing new therapeutics as well as reading the response to treatments.

Ca2+ Homeostasis by Plasma Membrane Ca2+ ATPase (PMCA) 1 Is Essential for the Development of DP Thymocytes.

The strength of Ca2+ signaling is a hallmark of T cell activation, yet the role of Ca2+ homeostasis in developing T cells before expressing a mature T cell receptor is poorly understood. We aimed to unveil specific functions of the two plasma membrane Ca2+ ATPases expressed in T cells, PMCA1 and PMCA4. On a transcriptional and protein level we found that PMCA4 was expressed at low levels in CD4-CD8- double negative (DN) thymocytes and was even downregulated in subsequent stages while PMCA1 was present throughout development and upregulated in CD4+CD8+ double positive (DP) thymocytes. Mice with a targeted deletion of Pmca1 in DN3 thymocytes had an almost complete block of DP thymocyte development with an accumulation of DN4 thymocytes but severely reduced numbers of CD8+ immature single positive (ISP) thymocytes. The DN4 thymocytes of these mice showed strongly elevated basal cytosolic Ca2+ levels and a pre-mature CD5 expression, but in contrast to the DP thymocytes they were only mildly prone to apoptosis. Surprisingly, mice with a germline deletion of Pmca4 did not show any signs of altered progression through the developmental thymocyte stages, nor altered Ca2+ homeostasis throughout this process. PMCA1 is, therefore, non-redundant in keeping cellular Ca2+ levels low in the early thymocyte development required for the DN to DP transition.

In Vitro Analysis of Thymocyte Signaling.

When a developing thymocyte expresses a TCR, it is subjected to numerous interactions with self-peptide/MHC complexes that determine its fate. These include death by neglect, negative selection (apoptosis and lineage deviation), positive selection, and lineage commitment. Identifying signals that govern these unique cell fates requires the ability to assess the activity, level of expression, subcellular location, and molecular associations between numerous proteins within the developing T cell. Given the unique, temporal, and developmental changes that occur during development, isolating and analyzing small populations of thymocytes are necessary to get a complete picture of the development process. Thus, this chapter describes methods designed to analyze thymocyte signaling under various types of peptide-based stimulation in vitro.

Generation of CD34+CD43+ Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells.

Immunotherapy using primary T cells has revolutionized medical care in some pathologies in recent years, but limitations associated to challenging cell genome edition, insufficient cell number production, the use of only autologous cells, and the lack of product standardization have limited its clinical use. The alternative use of T cells generated in vitro from human pluripotent stem cells (hPSCs) offers great advantages by providing a self-renewing source of T cells that can be readily genetically modified and facilitate the use of standardized universal off-the-shelf allogeneic cell products and rapid clinical access. However, despite their potential, a better understanding of the feasibility and functionality of T cells differentiated from hPSCs is necessary before moving into clinical settings. In this study, we generated human-induced pluripotent stem cells from T cells (T-iPSCs), allowing for the preservation of already recombined TCR, with the same properties as human embryonic stem cells (hESCs). Based on these cells, we differentiated, with high efficiency, hematopoietic progenitor stem cells (HPSCs) capable of self-renewal and differentiation into any cell blood type, in addition to DN3a thymic progenitors from several T-iPSC lines. In order to better comprehend the differentiation, we analyzed the transcriptomic profiles of the different cell types and demonstrated that HPSCs differentiated from hiPSCs had very similar profiles to cord blood hematopoietic stem cells (HSCs). Furthermore, differentiated T-cell progenitors had a similar profile to thymocytes at the DN3a stage of thymic lymphopoiesis. Therefore, utilizing this approach, we were able to regenerate precursors of therapeutic human T cells in order to potentially treat a wide range of diseases.

Phosphorylation of RIPK1 serine 25 mediates IKK dependent control of extrinsic cell death in T cells.

The Inhibitor of Kappa B Kinase (IKK) complex is a critical regulator of NF-κB activation. More recently, IKK has also been shown to repress RIPK1 dependent extrinsic cell death pathways by directly phosphorylating RIPK1 at serine 25. In T cells, IKK expression is essential for normal development in the thymus, by promoting survival of thymocytes independently of NF-κB activation. RIPK1 undergoes extensive phosphorylation following TNF stimulation in T cells, though which targets are required to repress RIPK1 has not been defined. Here, we show that TNF induced phosphorylation of RIPK1 at S25 is IKK dependent. We test the relevance of this phosphorylation event in T cells using mice with a RIPK1S25D phosphomimetic point mutation to endogenous RIPK1. We find that this mutation protects T cells from TNF induced cell death when IKK activity is inhibited in vitro, and can rescues development of IKK deficient thymocytes in vivo to a degree comparable with kinase dead RIPK1D138N. Together, these data show that phosphorylation of RIPK1S25 by IKK represents a key regulatory event promoting survival of T cells by IKK.

Altered thymocyte development observed in EphA4-deficient mice courses with changes in both thymic epithelial and extracellular matrix organization.

Eph receptors and their ligands, Ephrins, are involved in the thymocyte-thymic epithelial cell (TEC) interactions, key for the functional maturation of both thymocytes and thymic epithelium. Several years ago, we reported that the lack of EphA4, a Eph of the subfamily A, coursed with reduced proportions of double positive (DP) thymocytes apparently due to an altered thymic epithelial stroma [Munoz et al. in J Immunol 177:804-813, 2006]. In the present study, we reevaluate the lymphoid, epithelial, and extracellular matrix (ECM) phenotype of EphA4-/- mice grouped into three categories with respect to their proportions of DP thymocytes. Our results demonstrate a profound hypocellularity, specific alterations of T cell differentiation that affected not only DP thymocytes, but also double negative and single positive T cell subsets, as well as the proportions of positively and negatively selected thymocytes. In correlation, thymic histological organization changed markedly, especially in the cortex, as well as the proportions of both Ly51+UEA-1- cortical TECs and Ly51-UEA-1+ medullary TECs. The alterations observed in the expression of ECM components (Fibronectin, Laminin, Collagen IV), integrin receptors (VLA-4, VLA-6), chemokines (CXCL12, CCL25, CCL21) and their receptors (CXCR4, CCR7, CCR9) and in vitro transwell assays on the capacity of migration of WT and mutant thymocytes suggest that the lack of EphA4 alters T-cell differentiation by presumably affecting cell adhesion between TECs and T-TEC interactions rather than by thymocyte migration.

A double-negative thymocyte-specific enhancer augments Notch1 signaling to direct early T cell progenitor expansion, lineage restriction and ß-selection.

T cell differentiation requires Notch1 signaling. In the present study, we show that an enhancer upstream of Notch1 active in double-negative (DN) mouse thymocytes is responsible for raising Notch1 signaling intrathymically. This enhancer is required to expand multipotent progenitors intrathymically while delaying early differentiation until lineage restrictions have been established. Early thymic progenitors lacking the enhancer show accelerated differentiation through the DN stages and increased frequency of B, innate lymphoid (IL) and natural killer (NK) cell differentiation. Transcription regulators for T cell lineage restriction and commitment are expressed normally, but IL and NK cell gene expression persists after T cell lineage commitment and T cell receptor ß VDJ recombination, Cd3 expression and ß-selection have been impaired. This Notch1 enhancer is inactive in double-positive (DP) thymocytes. Its aberrant reactivation at this stage in Ikaros mutants is required for leukemogenesis. Thus, the DN-specific Notch1 enhancer harnesses the regulatory architecture of DN and DP thymocytes to achieve carefully orchestrated changes in Notch1 signaling required for early lineage restrictions and normal T cell differentiation.

CD11c regulates late-stage T cell development in the thymus.

CD11c, also named integrin αX, has been deemed solely as a dendritic cell marker for decades while the delineation of its biological function was limited. In the current study, we observed in mice that CD11c deficiency led to a defect in T cell development, demonstrated by the loss of CD4+CD8+ double positive (DP) T cells, CD4+CD8-, and CD4-CD8+ single positive (SP) T cells in the thymus and less mature T cells in the periphery. By using bone marrow chimera, we confirmed that CD11c regulated T cell development in the thymus. We further showed that CD11c deficiency led to an accelerated apoptosis of CD3 positive thymocytes, but not CD4-CD8- double negative (DN) T cells. Overall, this study added one more layer of knowledge on the regulatory mechanism of late-stage T cell development that the presence of CD11c in the thymus is critical for maintaining T cell survival.

The TCR Cα Domain Regulates Responses to Self-pMHC Class II.

T cells play a central role in adaptive immunity by recognizing peptide Ags presented by MHC molecules (pMHC) via their clonotypic TCRs. αßTCRs are heterodimers, consisting of TCRα and TCRß subunits that are composed of variable (Vα, Vß) and constant (Cα, Cß) domains. Whereas the Vα, Vß, and Cß domains adopt typical Ig folds in the extracellular space, the Cα domain lacks a top ß sheet and instead has two loosely associated top strands (C- and F-strands) on its surface. Previous results suggest that this unique Ig-like fold mediates homotypic TCR interactions and influences signaling in vitro. To better understand why evolution has selected this unique structure, we asked, what is the fitness cost for development and function of mouse CD4+ T cells bearing a mutation in the Cα C-strand? In both TCR retrogenic and transgenic mice we observed increased single-positive thymocytes bearing mutant TCRs compared with those expressing wild-type TCRs. Furthermore, our analysis of mutant TCR transgenic mice revealed an increase in naive CD4+ T cells experiencing strong tonic TCR signals, increased homeostatic survival, and increased recruitment of responders to cognate pMHC class II upon immunization compared with the wild-type. The mutation did not, however, overtly impact CD4+ T cell proliferation or differentiation after immunization. We interpret these data as evidence that the unique Cα domain has evolved to fine-tune TCR signaling, particularly in response to weak interactions with self-pMHC class II.

IL-7 Receptor Drives Early T Lineage Progenitor Expansion.

IL-7 and IL-7R are essential for T lymphocyte differentiation by driving proliferation and survival of specific developmental stages. Although early T lineage progenitors (ETPs), the most immature thymocyte population known, have a history of IL-7R expression, it is unclear whether IL-7R is required at this stage. In this study, we show that mice lacking IL-7 or IL-7R have a marked loss of ETPs that results mostly from a cell-autonomous defect in proliferation and survival, although no changes were detected in Bcl2 protein levels. Furthermore, a fraction of ETPs responded to IL-7 stimulation ex vivo by phosphorylating Stat5, and IL-7R was enriched in the most immature Flt3+Ccr9+ ETPs. Consistently, IL-7 promoted the expansion of Flt3+ but not Flt3- ETPs on OP9-DLL4 cocultures, without affecting differentiation at either stage. Taken together, our data show that IL-7/IL-7R is necessary following thymus seeding by promoting proliferation and survival of the most immature thymocytes.

Thymosin Beta 15 Alters the Spatial Development of Thymic Epithelial Cells.

The thymus is the most sensitive organ under various pathophysiological conditions, such as aging, starvation, and infection. As a key stromal cell for T cell development, it is well-known that thymic epithelial cells (TECs) play an important role in the thymus response to the external environment. Thymosin beta 15 (Tß15) is a G-actin binding protein secreted by TECs, it plays an important role in maintaining the dynamic balance of actin, angiogenesis, axonal formation, and wound healing, but the relationship between Tß15 and TECs is not clear yet. Here, we show the impact of Tß15 on the TEC's spatial development, as well as the T-cell differentiation and thymic output. As a result, TEC is the main effector cell of Tß15 in the thymus. Tß15 OX inhibits the chemotaxis of TECs to the medulla and subsequently blocks the positive selection of thymocytes from CD3+TCRß+CD4+CD8+ double positive cells to CD3+TCRß+CD4+CD8- single-positive (CD4SP) cells. Tß15-knockdown accelerates the reticular differentiation of astral TECs and medullary TECs. Importantly, mice implanted with Tß15-knockdown iTECs show high thymic output but low peripheral T cell maturity and activity. In a word, our results explain the role of Tß15 on the differentiation and function of TECs and provide a new perspective for understanding the process of thymus development and degeneration.

Higher thymocyte selection-associated high mobility group box (TOX) expression predicts poor prognosis in patients with ovarian cancer.

BACKGROUND: Ovarian cancer is one of the most lethal gynecologic malignancies with a dismal prognosis that poses a serious threat to human health, highlighting the need for more knowledge about what is required for identifying some biomarkers for early diagnosis, prediction of prognosis and disease monitoring. TOX, a critical transcription factor related to the development of malignancies that contributing to lymphocytes not just T cells, had been proved prognostic value in some spectrum of cancers. Here, we aimed to study the prognostic role of TOX in ovarian cancer. RESULTS: We found that TOX was not only expressed in CD8 T cells but also tumor cells. TOX expression score was higher in ovarian cancer tissues and correlated with survival status. Survival analysis revealed that ovarian cancer patients with high TOX expression score generally shorter overall survival and disease-free survival times. Univariate and Multivariate Cox demonstrated that TOX expression score could be used as an independent prognostic factor for patients with ovarian cancer. CONCLUSION: TOX expression in ovarian cancer could be a promising tool for predict overall survival of ovarian cancer patients.

Laminin-2 immobilized on a 3D fibrous structure impacts cortical thymic epithelial cells behaviour and their interaction with thymocytes.

The tri-dimensionality of the thymic extracellular matrix (ECM) supports the crosstalk between thymocytes and thymic epithelial cells (TECs). The thymic ECM component laminin-2 is involved in the regulation of thymocytes and their interaction with cortical TECs (cTECs). Most in vitro studies use planar surfaces to study the interaction between ECM components and thymic cells. Herein, we developed a novel biofunctionalized culture system by immobilizing laminin-2 at the surface of porous and fibrous electrospun meshes. We aimed to study the interaction of cTECs with thymocytes in the presence of laminin-2 presented through this system. The results indicated that the presence of laminin-2, not its density, has a positive effect on the cell viability and proliferation of cTECs. qPCR results demonstrated that laminin-2 density influenced the expression of cTECs genes. An increased percentage of adherent CD4-CD8- thymocytes and a decreased percentage of CD4+CD8+ thymocytes were evident in higher laminin-2 concentrations. Higher concentrations decreased the expression of Il7 and Ccl25 in cTECs after thymocyte adhesion. Altogether, these results indicate that the interaction of thymocytes with the thymic cortical compartment is affected by laminin-2 density and supports the need for immobilized ECM proteins in porous and fibrous substrates for the study of thymus biology.

Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-ß selection thymocytes.

Production of a functional peripheral T cell compartment typically involves massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment of double-negative (DN) 2 cells and after successful rearrangement and selection for functional TCRß chains in DN3 thymocytes, which promotes the transition of DN4 cells to the DP stage. The signals driving the expansion of DN2 thymocytes are well studied. However, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-ß-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2, which suppresses cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor regulating the survival of proliferating post-ß-selection thymocytes and demonstrates a key role for the cGAS/STING pathway in driving apoptosis of developing T cells.