Human Thymus Research Articles

Advanced three-dimensional X-ray imaging unravels structural development of the human thymus compartments

BackgroundThe thymus, responsible for T cell-mediated adaptive immune system, has a structural and functional complexity that is not yet fully understood. Until now, thymic anatomy has been studied using histological thin sections or confocal microscopy 3D reconstruction, necessarily for limited volumes.MethodsWe used Phase Contrast X-Ray Computed Tomography to address the lack of whole-organ volumetric information on the microarchitecture of its structural components. We scanned 15 human thymi (9 foetal and 6 postnatal) with synchrotron radiation, and repeated scans using a conventional laboratory x-ray system. We used histology, immunofluorescence and flow cytometry to validate the x-ray findings.ResultsApplication to human thymi at pre- and post-natal stages allowed reliable tracking and quantification of the evolution of parameters such as size and distribution of Hassall’s Bodies and medulla-to-cortex ratio, whose changes reflect adaptation of thymic activity. We show that Hassall’s bodies can occupy 25% of the medulla volume, indicating they should be considered a third thymic compartment with possible implications on their role. Moreover, we demonstrate compatible results can be obtained with standard laboratory-based x-ray equipment, making this research tool accessible to a wider community.ConclusionsOur study allows overcoming the resolution and/or volumetric limitations of existing approaches for the study of thymic disfunction in congenital and acquired disorders affecting the adaptive immune system.

Rediscovering the human thymus through cutting-edge technologies.

Recent technological advances have transformed our understanding of the human thymus. Innovations such as high-resolution imaging, single-cell omics, and organoid cultures, including thymic epithelial cell (TEC) differentiation and culture, and improvements in biomaterials, have further elucidated the thymus architecture, cellular dynamics, and molecular mechanisms underlying T cell development, and have unraveled previously unrecognized levels of stromal cell heterogeneity. These advancements offer unprecedented insights into thymic biology and hold promise for the development of novel therapeutic strategies for immune-related disorders.

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels.

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

Sex-biased human thymic architecture guides Tcell development through spatially defined niches.

Within the thymus, regulation of the cellular crosstalk directing Tcell development depends on spatial interactions within specialized niches. To create a spatially defined map of tissue niches guiding humanpostnatal Tcell development, we employed the multidimensional imaging platform co-detection by indexing (CODEX) as well as cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) and assay for transposase accessible chromatin sequencing (ATAC-seq). We generated age-matched 4- to 5-month-old human postnatal thymus datasets for male and female donors, identifying significant sex differences in both Tcell and thymus biology. We demonstrate a possible role for JAG ligands in directing thymic-like dendritic cell development, identify important functions of a population ofextracellular matrix (ECM)- fibroblasts, and characterize the medullary niches surrounding Hassall's corpuscles. Together, these data represent an age-matched spatial multiomic resource to investigate how sex-based differences in thymus regulation and Tcell development arise, providing an essential resource to understand the mechanisms underlying immune function and dysfunction in males and females.

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

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

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

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

Expansion of human γδ T cells in periphery: Lessons learned from development, infections, and compromised thymic function.

γδ T cells predominantly develop in the fetal period. Post birth they respond swiftly to environmental insults, pathogens and tumors, especially when other immune effector cells are less ready to function. Most of our understanding of γδ T-cell development, peripheral adaptation, and function derives from murine studies. The recent advancement of immunological methods allows now to decipher human γδ T-cell biology in patient cohorts and tissue samples, and to manipulate them using in vitro systems. In this review, we summarize γδ T-cell development in the human thymus, their functional adaptation to the microbial environment from birth until old age, and their capacity to expand and fill up the peripheral niche under conditions of perturbations of conventional T-cell development.

Transposable elements regulate thymus development and function

Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN ɑ/β. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.

Transposable elements regulate thymus development and function.

Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN ɑ/β. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.

Abstract 4188: Kyinno PBMC humanized mouse platform: Pioneering service in biomedical research

Abstract Tumor immunotherapy is the use of the human immune system to kill tumors. Compared with traditional chemotherapy and radiotherapy, immunotherapy has significant advantages such as significant efficacy, long-lasting effects, and low toxicity. Wild-type mice are widely used as preclinical models for immunotherapy due to their normal immune system, but they have many shortcomings, such as only being able to inoculate mouse tumor cells, differences between mouse and human immune systems, etc., which cannot accurately simulate the interaction between drugs and tumor cells in the human body. Humanized mouse models include PBMCs humanized mouse models, HSC humanized mouse models, and human embryonic bone, liver, and thymus tissue (BLT) transplantation models. Among them, the PBMCs humanized mouse model is widely used due to its simple preparation, short cycle, and high levels of hCD3+ T cells in the peripheral blood of mice. To assist in the development of immunotherapy, KYINNO has established a PBMC humanized mouse platform that can perform in vivo drug efficacy verification of various immunotherapeutic drugs such as CD3 bispecific antibodies or multi-specific antibodies, immune checkpoint inhibitors, etc. We screen many PBMC donors and select PBMC donors with light GvHD and good hCD3+ T cell reconstruction effects. The drug administration window of the PBMC humanized model has been extended to 6 weeks. Platform verification has been performed on subcutaneous and intravenous injection models of various tumors. Overall, we successfully established the PBMC humanized mouse platform, which is a powerful tool for immunotherapy. Citation Format: Tongtong Liu, Guojin Wu, Feng He, Longtan Luo, Zhengcheng Guo, Shuliang Li, Jinying Ning, Feng Hao. Kyinno PBMC humanized mouse platform: Pioneering service in biomedical research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4188.

Methodological optimisation of thymocyte isolation and cryopreservation of human thymus samples

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.

P014 First-time-in-human visualisation of CCR9 expression in the gut by positron emission tomography

Abstract Background CCR9/CCL25 is the key axis for recruiting lymphocytes to the small intestine and represents a potential treatment target in Crohn’s disease (CD). AZD7798 is a first-in-class monoclonal antibody for CD that depletes CCR9+ lymphocytes. Monitoring CCR9+ cell depletion and repletion in the blood is straightforward, but difficult in the primary tissues of interest (small bowel, mesenteric lymph nodes) which require invasive procedures to access. For AZD7798, an understanding of pharmacodynamics in gut is crucial to guide further clinical development. To address this need, we have developed a CCR9-specific PET tracer based on the small molecule CCR9 antagonist vercirnon. Methods Specific binding of [3H]vercirnon was assessed in-vitro using autoradiography. Specific binding and dosimetry of [11C]vercirnon was assessed in-vivo in cynomolgus monkeys. Subsequently, a first-time-in-human study (NCT05818514) was performed in healthy volunteers to assess the biodistribution, binding to CCR9 in abdominal regions of interest, and safety of [11C]vercirnon, administered intravenously at microdoses (<10μg). Two PET-CT examinations were performed 14 days apart to assess reproducibility. Results In-vitro specific binding of [3H]vercirnon to CCR9 was confirmed in human thymus sections and CCR9-expressing cell pellets. In-vivo specific binding of [11C]vercirnon to CCR9 was putatively demonstrated in the gut of non-human primates (NHPs) in pre-treatment/displacement studies with excess unlabelled vercirnon (20mg/kg). 8 healthy volunteers (5 male) were enrolled in the FTIH study. Detectable intestinal uptake of [11C]vercirnon was demonstrated in all participants (Figure 1) with mean±SD standardised uptake value ratio (SUVR) 1.4±0.6 (reference abdominal aorta), and a proximal to distal gradient (SUVR jejunum 1.8±0.7/ileum 0.9±0.4/colon 0.6±0.2) consistent with CCR9 gene expression data. Test-retest variability of intestinal uptake was 9.6% (intraclass correlation coefficient 0.96), in line with values for other clinically useful PET tracers. Mean±SD injected radioactivity per PET examination was 351±105 MBq, corresponding to a radiation exposure of 1.4 mSv. This low radiation exposure (c.f. conventional FDG PET ~9 mSv) allows multiple PET examinations per participant. Administration of [11C]vercirnon was well-tolerated with no serious adverse events. Conclusion We have developed a CCR9-specific PET tracer which potentially enables repeatable quantification of CCR9 expression in the gut. Ongoing studies will assess the effects of AZD7798 on CCR9+ cell depletion/repletion in the gut, providing critical data to support the clinical development programme.

Transcriptional profile of human thymus reveals IGFBP5 is correlated with age-related thymic involution.

Thymus is the main immune organ which is responsible for the production of self-tolerant and functional T cells, but it shrinks rapidly with age after birth. Although studies have researched thymus development and involution in mouse, the critical regulators that arise with age in human thymus remain unclear. We collected public human single-cell transcriptomic sequencing (scRNA-seq) datasets containing 350,678 cells from 36 samples, integrated them as a cell atlas of human thymus. Clinical samples were collected and experiments were performed for validation. We found early thymocyte-specific signaling and regulons which played roles in thymocyte migration, proliferation, apoptosis and differentiation. Nevertheless, signaling patterns including number, strength and path completely changed during aging, Transcription factors (FOXC1, MXI1, KLF9, NFIL3) and their target gene, IGFBP5, were resolved and up-regulated in aging thymus and involved in promoting epithelial-mesenchymal transition (EMT), responding to steroid and adipogenesis process of thymic epithelial cell (TECs). Furthermore, we validated that IGFBP5 protein increased at TECs and Hassall's corpuscle in both human and mouse aging thymus and knockdown of IGFBP5 significantly increased the expression of proliferation-related genes in thymocytes. Collectively, we systematically explored cell-cell communications and regulons of early thymocytes as well as age-related differences in human thymus by using both bioinformatic and experimental verification, indicating IGFBP5 as a functional marker of thymic involution and providing new insights into the mechanisms of thymus involution.

Thymic Carcinoma: Unraveling Neuroendocrine Differentiation and Epithelial Cell Identity Loss.

The histogenesis of thymic epithelial tumors (TETs) has been a subject of debate. Recent technological advancements have revealed that thymic carcinomas often exhibit a phenotype akin to tuft cells, which is a subset of medullary TECs. Here, we further explored the gene expression signatures of thymic carcinomas in relation to tuft cells and their kinships-ionocytes and neuroendocrine cells (neuroendocrine group). We analyzed a single-cell RNA sequencing dataset from the normal human thymus. Concurrently, we examined publicly available datasets on the mRNA expression and methylation status of TECs and lung cancers. Real-time quantitative PCR was also conducted with our tissue samples. Thymic carcinomas displayed a neuroendocrine phenotype biased toward tuft cells and ionocytes. When exploring the possible regulators of this phenotype, we discovered that HDAC9 and NFATC1 were characteristically expressed in the neuroendocrine group in adult TECs and thymic carcinomas. Additionally, the pan-thymic epithelium markers, exemplified by PAX9 and SIX1, were significantly suppressed in thymic carcinomas. Thymic carcinomas might be characterized by unique neuroendocrine differentiation and loss of identity as thymic epithelial cells. Future studies investigating the role of HDAC9 and NFATC1 in thymic epithelium are warranted to explore their potential as therapeutic targets in TETs.

Recirculating Regulatory T Cells Mediate Thymic Regeneration in a ZFP36L1 and Amphiregulin Dependent Manner

T cell reconstitution correlates with outcomes in patients undergoing stem cell transplantation (HCT). A functional thymus is needed for the maturation of T cells, but unfortunately, the thymus is highly sensitive to acute injury, such as cytoreductive conditioning given to HCT patients. To enhance T cell reconstitution, it is therefore imperative to explore therapies that can boost thymic regeneration and T cell output. Regulatory T cells (Tregs) are known to mediate homeostasis and tissue regeneration in a variety of tissues, but their role in thymic injury is not known. This study aims to shed light on the role of regulatory T cells (Tregs) in thymic regeneration. Using a model of thymic injury and regeneration in mice receiving sublethal total body irradiation (SL-TBI, 550cGy), we observed a significant increase in Tregs ( Fig. 1A), located in both the medullary and cortical areas of the thymus on multiparameter immunohistochemistry. Employing a targeted ablation of Tregs at the time of injury using the FOXP3DTR mouse model, we could observe significant delay in thymic regeneration in mice depleted of Tregs (day 7 post SL-TBI, p=<0.0001, n=10, Fig. 1B). RAG2GFP mice, where GFP is expressed under the RAG2 promoter, have been used to discern recirculating and de-novo produced Tregs. To characterize the nature of the Treg expansion, we treated RAG2GFP mice with SL-TBI and analyzed their thymus at multiple time points after injury. Interestingly, the increase in Tregs was mainly due to recirculating RAG2-GFP- Tregs but not de-novo produced RAG2+GFP+ Tregs after injury. Brdu administration after injury indicated proliferation of these RAG2-GFP- Tregs. Parabiotic experiments linking RAG2GFP mice expressing either the congenic marker CD45.1 or CD45.2 confirmed the recirculating nature of these cells, both at steady state and after injury. Furthermore, when adoptively transferring sorted Thymic Tregs from FOXP3RFP mice to mice receiving SL-TBI, we significantly enhanced thymic cellularity after injury and homing of these Tregs to the thymus. To discern the transcriptional profile of these cells, we used single-cell sequencing of CD45+ RAG2- cells in 2- and 18-month-old RAG2GFP mice before and at day 1, 4, and 7 after sublethal irradiation. This enabled us to discover the age- and injury-associated changes in recirculating T cells. While the aged thymus had a similar surge of Treg numbers after injury, this response was blunted compared to their young counterparts. Factor normalization methods enabled us to find two distinct gene regulatory networks highly specific to the RAG2-GFP-Tregs. One gene network was highly enriched for Zfp36l1, a poised RNA known to mediate cell stability. Selectively ablating Zfp36l1 in Tregs using the Foxp3Cre ZFP36L1 flox/flox model impaired regeneration significantly after SL-TBI. The other distinct RAG2-GFP- Treg specific gene network was enriched for the regenerative factor Amphiregulin ( Areg). Amphiregulin was seen to be increased in thymic supernatants, and its receptor Egfr was seen to be transcriptionally upregulated in thymic epithelial cells after injury. In parallel, the most significantly upregulated pathway using cell-to-cell interaction prediction models on single-cell data indicated a strong likelihood of Treg to thymic epithelial cell interactions through amphiregulin to Egfr. Functional inhibition of amphiregulin with an antibody enabled us to observe delayed thymic regeneration, indicating amphiregulin as a factor in the regenerative capacity of thymic recirculating Tregs. Mapping human gene orthologs of the gene regulatory program provided by factor normalization in murine RAG2GFP- Tregs on single cell data of human pediatric thymic samples from children undergoing open heart surgeries enabled us to distinguish a putatively analogous human population of recirculating thymic Tregs. This population was found to be amphiregulin positive. And human thymic epithelial cell cultures with recombinant amphiregulin enhanced the growth of human thymic epithelial cells, as measured by life imaging, and promoted signalling through MAPK ERK1/2. Taken together, our findings reveal a novel role for injury-resistant recirculating thymic regulatory T cells existing in both the human and murine thymus and may enable the development of new therapeutic targets for thymic regeneration for patients undergoing cytoreductive therapies.

CamoTSS: analysis of alternative transcription start sites for cellular phenotypes and regulatory patterns from 5' scRNA-seq data

Five-prime single-cell RNA-seq (scRNA-seq) has been widely employed to profile cellular transcriptomes, however, its power of analysing transcription start sites (TSS) has not been fully utilised. Here, we present a computational method suite, CamoTSS, to precisely identify TSS and quantify its expression by leveraging the cDNA on read 1, which enables effective detection of alternative TSS usage. With various experimental data sets, we have demonstrated that CamoTSS can accurately identify TSS and the detected alternative TSS usages showed strong specificity in different biological processes, including cell types across human organs, the development of human thymus, and cancer conditions. As evidenced in nasopharyngeal cancer, alternative TSS usage can also reveal regulatory patterns including systematic TSS dysregulations.

Accelerating Immune Reconstitution in HSCT Patients through iPSC-Derived Thymic Epithelial Cells

T cells are central to host defense and tolerance, yet the critical role of the thymus in facilitating T cell development and TCR repertoire selection is often overlooked. Thymic function declines in the second decade of life and is susceptible to a wide range of pathogenic insults. Compromise of thymic function is a frequent complication after HSCT as a result of radiation, anti-thymocyte globulin, CMV infection and acute GVHD. Thymic injury can lead to delayed immune reconstitution, opportunistic infections, autoimmunity and chronic GVHD. Reconstituting thymic function, e.g. through induced pluripotent stem cell (iPSC)-derived thymic epithelial cells (TECs), holds great therapeutic promise. However, the regenerative potential of the thymus remains clinically untapped as the signals that drive thymic ontogeny beyond the anterior foregut are incompletely understood. In this study, we elucidated pathways instructing commitment and specialization of the human thymic epithelial stroma through orthogonal single cell transcriptomic analysis strategies. First, we inferred inductive cues that contribute to the initiation of thymic organogenesis by comparing gene regulatory networks expressed in human fetal epithelial cells of the thymus and other anterior foregut-derived organs. Then we defined lineage trajectories within the thymic epithelium by evaluating human thymus samples sequentially across embryonic, fetal, and early postnatal stages. Our data revealed that responses to types I and II interferon distinguish epithelial cells of the thymus from other anterior foregut-derived organs. We further found that the downstream effects of interferon extend differentially within the cortical and medullary lineages, reflected in characteristic NFκB and IRF signatures (Figure 1A). We have translated the core set of gene regulatory networks identified in our transcriptomic study into a morphogen-based approach to advance the differentiation of iPSCs into TECs. TECs derived from this protocol demonstrate high transcriptional fidelity with primary human fetal intertypic TECs that can give rise to cortical and medullary lineages (Fig. 1B). To test the functional capacity of iPSC-derived TECs to give rise to human T cells, and specifically to execute positive and negative selection, we transplanted iPSC-derived TECs into humanized athymic NSG-nude mice (NSG-Foxn1 Null) (Figure 1C). 12 weeks after transplantation, flowcytometric analysis of dissociated TEC grafts showed robust human T cell development recapitulating the physiologic progression from double-negative to double-positive to TCRab-rearranged single CD4 and CD8 positive T cells observed in primary human thymus. (Figure 1D) These results provide proof of concept that transplantable iPSC-derived TECs can promote T cell development in vivo and have potential as a regenerative thymic replacement therapy for patients suffering from complications of defective T cell immune reconstitution after HSCT.

DHODH Inhibitor Therapy for the Treatment of T-ALL Spares the Developing Thymus

Acute T-cell lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in children and young adults. When compared to B-ALL, T-ALL has a higher rate of induction failure as well as relapse, with very poor overall survival in both scenarios. Furthermore, T-ALL has not benefitted from immune-mediated therapies and continues to pose an ongoing treatment challenge with an unmet therapeutic need. DHODH has been highlighted as a potential therapeutic target in many hematologic and solid tumor malignancies including AML, neuroblastoma, glioblastoma, pancreatic cancer, and breast cancer. DHODH inhibition has a robust anti-leukemia effect in both in vitro and in vivo models of T-ALL. We have confirmed this sensitivity in vitro as well as in vivo in three murine models of T-ALL, including an aggressive NOTCH driven model in syngeneic recipients and two human patient derived xenografts. Given the goal of making DHODHi therapy available to young children with relapsed/refractory T-ALL, we wished to understand the ramifications of nucleotide starvation on normal T-cells and other hematopoietic progenitors. To specifically address this, we turned our attention to the effects of DHODH inhibition on the developing thymus. This compartment would be of particular interest in pediatric patients, whose thymi have not yet finished developing. We studied juvenile mice (3 weeks) whose thymi are just reaching their peak size and growth rate. These mice were treated with the small molecule DHODHi brequinar (BRQ) 50 mg/kg every 72 hours, and were paired with age and gender-matched controls at each time point of analysis. A subset of mice were analyzed following two doses of BRQ, and thymus, bone marrow, spleen, and peripheral blood were analyzed for T-cell populations, both relative and absolute numbers. Another cohort was allowed to “recover” for 2 weeks following treatment before analysis, and a third cohort was allowed a four week recovery period. We discovered that initial treatment with brequinar significantly impacts T-cells across all stages of development ( Figure 1). This is expected, as developing T-cells are rapidly proliferative (often dividing as quickly as every 6 hours) and therefore highly dependent on their supply of nucleotides for DNA replication as well as transcription. However, after the two-week and the four-week recovery periods, these differences are no longer detectable, demonstrating the resilience of the developing thymus, and demonstrating that the effects of nucleotide starvation are temporary and reversible. These data are encouraging, as this may also suggest that the developing human thymus which is similarly rapidly proliferative in nature would also recover once nucleotide starvation was removed. Based on these data, we were interested in understanding the differential sensitivity of normal thymocytes as compared to malignant T-lymphoblasts to DHODHi. The resilience shown by normal thymocytes following treatment with BRQ may be due to their ability to tolerate periods of inhibition of de novo pyrimidine synthesis by preferentially relying on extracellular nucleotide salvage, whereas the sensitivity of malignant T-cells to DHODH may be due to their lack of ability to utilize the salvage pathway. We performed experiments to understand how cells balance de novo synthesis vs extracellular salvage. First, we performed metabolite flux tracing in Jurkat cells to directly measure nucleotide salvage. Here, we followed incorporation of isotopically labeled glutamine ( 15N) and uridine ( 13C) to determine the relative contributions of synthesis vs salvage, as de novo pyrimidine synthesis initiates from glutamine and aspartate while a cell can also use membrane nucleoside transporters (SLC28A and SLC29A family of proteins) to salvage extracellular uridine. These experiments were performed in the presence and absence of BRQ. Once BRQ is added and the cell's ability to synthesize pyrimidines is shut down, the cell must rely more on extracellular salvage as shown in Figure 2. A defective or less efficient process of extracellular salvage could help to explain this cell line's unusual dependence on de novo nucleotide synthesis. Overall, we hope that these findings will pave the way for additional preclinical studies and eventually clinical trials for pediatric patients with T-cell disease.

Human thymic putative CD8αα precursors exhibit a biased TCR repertoire in single cell AIRR-seq

Thymic T cell development comprises T cell receptor (TCR) recombination and assessment of TCR avidity towards self-peptide-MHC complexes presented by antigen-presenting cells. Self-reactivity may lead to negative selection, or to agonist selection and differentiation into unconventional lineages such as regulatory T cells and CD8mathrm{alpha alpha } T cells. To explore the effect of the adaptive immune receptor repertoire on thymocyte developmental decisions, we performed single cell adaptive immune receptor repertoire sequencing (scAIRR-seq) of thymocytes from human young paediatric thymi and blood. Thymic PDCD1+ cells, a putative CD8mathrm{alpha alpha } T cell precursor population, exhibited several TCR features previously associated with thymic and peripheral ZNF683+ CD8mathrm{alpha alpha } T cells, including enrichment of large and positively charged complementarity-determining region 3 (CDR3) amino acids. Thus, the TCR repertoire may partially explain the decision between conventional vs. agonist selected thymocyte differentiation, an aspect of importance for the development of therapies for patients with immune-mediated diseases.

Divergent molecular events underlying initial T-cell commitment in human prenatal and postnatal thymus.

Intrathymic T-cell development is a coordinated process accompanied by dynamic changes in gene expression. Although the transcriptome characteristics of developing T cells in both human fetal and postnatal thymus at single-cell resolution have been revealed recently, the differences between human prenatal and postnatal thymocytes regarding the ontogeny and early events of T-cell development still remain obscure. Moreover, the transcriptional heterogeneity and posttranscriptional gene expression regulation such as alternative polyadenylation at different stages are also unknown. In this study, we performed integrative single-cell analyses of thymocytes at distinct developmental stages. The subsets of prenatal CD4-CD8- double-negative (DN) cells, the most immature thymocytes responsible for T-cell lineage commitment, were characterized. By comprehensively comparing prenatal and postnatal DN cells, we revealed significant differences in some key gene expressions. Specifically, prenatal DN subpopulations exhibited distinct biological processes and markedly activated several metabolic programs that may be coordinated to meet the required bioenergetic demands. Although showing similar gene expression patterns along the developmental path, prenatal and postnatal thymocytes were remarkably varied regarding the expression dynamics of some pivotal genes for cell cycle, metabolism, signaling pathway, thymus homing, and T-cell commitment. Finally, we quantified the transcriptome-wide changes in alternative polyadenylation across T-cell development and found diverse preferences of polyadenylation site usage in divergent populations along the T-cell commitment trajectory. In summary, our results revealed transcriptional heterogeneity and a dynamic landscape of alternative polyadenylation during T-cell development in both human prenatal and postnatal thymus, providing a comprehensive resource for understanding T lymphopoiesis in human thymus.