Thymocyte Subsets Research Articles

T-ALL Oncogenes Function in the Context of Pre- and Post T-Cell Commitment Programs

Human T-cell development is less well understood than its murine counterpart. We previously described the transcriptional landscape of 11 immature human T-cell developmental stages developed from hematopoietic stem cells on the OP9-DL1 system that match those of ex vivo flow-sorted murine and human thymocyte subsets. We defined a gene signature comprising of 547 genes that distinguishes pre- from post- αβ T-cell commitment stages. Commitment is marked by loss of dim CD44 expression of early CD7+CD5+CD45dim cells, before acquiring CD1a surface expression. Unlike uncommitted CD44dimCD1a- thymocytes,CD44-CD1a- post-committed thymocytes undergo first TCR rearrangements while having lost myeloid-, B- or NK-lineage potential.1We used this gene signature to study whether genetic or transcription-based subtypes of human T-cell acute lymphoblastic leukemia are connected to pre- or post-commitment T-cell development.Clustering of human T-ALL using our 547 pre- and post-commitment gene signature distinguished four T-ALL clusters that were highly similar to the four subgroups that we identified before using an unsupervised cluster analysis. This previous analysis distinguished immature/ETP ALL, TLX, Proliferative and TALLMO subtypes, strongly correlated with MEF2C or MEF2C -activating abnormalities, HOXA -activating or TLX3 rearrangements, NKX2-1 or TLX1 rearrangements, and aberrations affecting members of the TAL1 and/or LMO2 gene families, respectively.2 Further inspection of the original unsupervised gene expression signature revealed that nearly half of all genes overlapped with genes that were expressed at pre- or post-commitment. This implies strong dependency or collaboration of specific oncogene-driven pathogenic programs and pre- or post-commitment programs. In fact, Immature/ETP-ALL and TLX subtypes are each strongly characterized by expressing a pre-commitment program, whereas the Proliferative and TALLMO subtypes are associated with the post-commitment program. Interestingly, the TLX subgroup that has preferentially been associated with γδ-lineage arrest implies that normal γδ T-cell development does not transit through the αβ T-cell commitment point before branching off to the γδ-lineage. Consistent with this, rearrangements of the BCL11B locus that impact on critical levels of the haploinsufficient BCL11B transcription factor for T-cell commitment function are nearly exclusively associated with these ‘pre-commitment’ ETP-ALL and TLX subgroups. In contrast, TCR-driven chromosomal aberrations were almost exclusively found in the post-commitment Proliferative and TALLMO subtypes.We then further explored whether particular networks or signaling routes as captured by specific Hallmark gene sets are activated in pre- or post commitment programs and associated with particular T-ALL subtypes. We identified hallmark gene sets connected to interferon gamma and inflammatory responses that are activated in ETP-ALL, as well as signaling of the KRAS, the IL2-STAT5 and the IL6-JAK-STAT3 pathways including CD44 . For the TLX subgroup, hallmark gene sets included NOTCH signaling, coagulation, angiogenesis and myogenesis. These data are in agreement with activating mutations in IL7R-JAK-STAT5B and RAS pathways that are most prevalent in ETP ALL and TLX subtypes. The TLX subtype is particularly associated with strongly-activating NOTCH mutations in line with the requirement of strong NOTCH signaling for γδ T-cell development. Hallmark gene sets for the Proliferative group include E2F and MYC targets, G2M checkpoint and glycolysis. Surprisingly, we did not identify specific Hallmark gene sets for the TALLMO subtype, implying that it may include patients that have arrested at several distinct stages of αβ development.In conclusion, our data demonstrate that T-ALL oncogenes can exert their pathogenic effects only in the context of particular T-cell developmental programs.1Canté-Barrett, K. et al. Front Immunol 2017; 8:32. doi: 10.3389/fimmu.2017.000322Homminga, I. et al. Cancer Cell 2011; 19(4). doi: 10.1016/j.ccr.2011.02.008 DisclosuresNo relevant conflicts of interest to declare.

Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

CCR4 and CCR7 differentially regulate thymocyte subset localization with distinct outcomes for central tolerance

Abstract Central tolerance is established as T cells mature in the thymus by the combined effects of clonally deleting autoreactive thymocytes or inducing their diversion to the Treg lineage. To ensure T cell tolerance to diverse self-antigens, thymocytes must efficiently enter the medulla and scan heterogeneous antigen presenting cells, which display the majority of the peptidome in a sparse mosaic. While it is widely accepted that CCR7 contributes to central tolerance by promoting thymocyte medullary localization and rapid migration therein, the contribution of CCR4 to these processes remains unclear. For example, it is not known whether CCR4 is simply redundant to CCR7 in promoting medullary entry or if it plays a distinct role in central tolerance. Here, we report that early post-positive selection thymocytes undergo chemotaxis towards CCR4 ligands, while progressively mature cells migrate towards CCR7 ligands. Synchronized positive selection assays reveal that CCR4 is upregulated within hours of positive selection signaling, while CCR7 upregulation is delayed. 2-photon microscopy shows that CCR4 facilitates entry of early post-positive selection cells into the thymic medulla, while CCR7 promotes medullary accumulation of mature subsets. These findings suggest that CCR4 and CCR7 have non-redundant roles in negative selection. Consistent with this possibility, flow cytometric analysis reveals that CCR4 deficiency results in a selective decline in early-stages of negative selection, while CCR7 deficiency impairs only late-stage negative selection. Our data support a new model in which CCR4 and CCR7 mediate medullary entry of different thymocyte subsets and play distinct roles in enforcing central tolerance.

Immune Reconstitution and Thymic Involution in the Acute and Delayed Hematopoietic Radiation Syndromes.

Lymphoid lineage recovery and involution after exposure to potentially lethal doses of ionizing radiation have not been well defined, especially the long-term effects in aged survivors and with regard to male/female differences. To examine these questions, male and female C57BL/6 mice were exposed to lethal radiation at 12 wk of age in a model of the Hematopoietic-Acute Radiation Syndrome, and bone marrow, thymus, spleen, and peripheral blood examined up to 24 mo of age for the lymphopoietic delayed effects of acute radiation exposure. Aged mice showed myeloid skewing and incomplete lymphocyte recovery in all lymphoid tissues. Spleen and peripheral blood both exhibited a monophasic recovery pattern, while thymus demonstrated a biphasic pattern. Naïve T cells in blood and spleen and all subsets of thymocytes were decreased in aged irradiated mice compared to age-matched non-irradiated controls. Of interest, irradiated males experienced significantly improved reconstitution of thymocyte subsets and peripheral blood elements compared to females. Bone marrow from aged irradiated survivors was significantly deficient in the primitive lymphoid-primed multipotent progenitors and common lymphoid progenitors, which were only 8-10% of levels in aged-matched non-irradiated controls. Taken together, these analyses define significant age- and sex-related deficiencies at all levels of lymphopoiesis throughout the lifespan of survivors of the Hematopoietic-Acute Radiation Syndrome and may provide a murine model suitable for assessing the efficacy of potential medical countermeasures and therapeutic strategies to alleviate the severe immune suppression that occurs after radiation exposure.

Abstract 5618: Evaluating anti-IL-2Ra in vivo efficacy on humanized knock-in mice

Abstract Interleukin-2 receptor alpha chain (also called as CD25) is a 55 kD glycoprotein, also known as IL-2Ra, Ly-43, p55, or Tac. It is expressed on activated T and B cells, thymocyte subset, pre-B cells, and T regulatory cells. IL-2Ra and IL-2Rβ (CD122), together with the common gamma chain (IL2Rg)(CD132), form the high affinity signaling IL-2 receptor. Its affinity for IL2 and cellular function are tightly regulated and vary in different cell types. The high frequency of IL-2Ra on the surface of many different haematological tumor cells is now well established and, apart from its prognostic significance, IL-2Ra may be present on leukemic stem cells and enable oncogenic signaling pathways in leukemic cells. Additionally, high IL-2Ra expression in activated circulating immune cells and Tregs is a factor that has already been exploited by IL2 immunotherapies for treatment of tumors and autoimmune disease. The relative clinical safety and efficacy of administering anti- IL-2Ra radioimmunoconjugates and immunotoxins in various haematological tumor indications has been established and clinical trials of a novel IL-2Ra-directed antibody drug conjugate are underway. Biocytogen has generated IL-2Ra humanized mouse for both in vitro functional validation of signaling pathway and in vivo efficacy evaluation of IL-2Ra antibodies. The targeting strategy is that the genomic DNA covering exons 2~6 of mouse IL-2Ra gene which encode the extracellular domain are replaced by the counterpart of human IL-2Ra genomic DNA covering exons 2~6. Human IL-2Ra is mainly detectable on the T cells in spleen of the IL-2Ra humanized mice. The humanized mice are highly similar to the wild-type mice in basal leukocyte subpopulations, including T/B cells, NK cells, DC, granulocytes and monocytes/macrophages. STAT-5 phosphorylation induced by mouse IL2 protein is blocked by anti-human IL-2Ra antibodies, suggesting that the functional pathways in IL-2Ra humanized mice work well. The colon cancer model established in IL-2Ra humanized mice is undergoing, anti-human IL-2Ra antibodies showed good efficacy in inhibiting tumor growth. Taken together, IL-2Ra humanized mice are a useful tool for in vivo efficacy evaluation of human IL-2Ra antibodies that can be advanced to human clinical trials. Citation Format: Yanan Guo, Xiaofei Zhou, Youhong Su, Gary Ng, Chaoshe Guo. Evaluating anti-IL-2Ra in vivo efficacy on humanized knock-in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5618.

Conventional and Computational Flow Cytometry Analyses Reveal Sustained Human Intrathymic T Cell Development From Birth Until Puberty.

The thymus is the organ where subsets of mature T cells are generated which subsequently egress to function as central mediators in the immune system. While continuously generating T cells even into adulthood, the thymus does undergo involution during life. This is characterized by an initial rapid decrease in thymic cellularity during early life and by a second age-dependent decline in adulthood. The thymic cellularity of neonates remains low during the first month after birth and the tissue reaches a maximum in cellularity at 6 months of age. In order to study the effect that this first phase of thymic involution has on thymic immune subset frequencies, we performed multi-color flow cytometry on thymic samples collected from birth to 14 years of age. In consideration of the inherent limitations posed by conventional flow cytometry analysis, we established a novel computational analysis pipeline that is adapted from single-cell transcriptome sequencing data analysis. This allowed us to overcome technical effects by batch correction, analyze multiple samples simultaneously, limit computational cost by subsampling, and to rely on KNN-graphs for graph-based clustering. As a result, we successfully identified rare, distinct and gradually developing immune subsets within the human thymus tissues. Although the thymus undergoes early involution from infanthood onwards, our data suggests that this does not affect human T-cell development as we did not observe significant alterations in the proportions of T-lineage developmental intermediates from birth to puberty. Thus, in addition to providing an interesting novel strategy to analyze conventional flow cytometry data for the thymus, our work shows that the early phase of human thymic involution mainly limits the overall T cell output since no obvious changes in thymocyte subsets could be observed.

CCR4 and CCR7 make distinct contributions to the spatial distribution and negative selection of different thymocyte subsets

Abstract Central tolerance mechanisms, including clonal deletion and Treg selection, are required to remove autoreactive T cells generated during thymocyte differentiation. To ensure T cell tolerance to diverse self-antigens, the majority of the peptidome is displayed in a sparse mosaic by medullary thymic epithelial cells and dendritic cells in the medulla. Thus, thymocytes must efficiently enter the medulla and scan multiple antigen presenting cells to ensure complete tolerance. We and others have previously reported that the chemokine receptors CCR4 and CCR7 are important regulators of medullary enrichment, rapid motility, and negative selection of post-positive selection thymocytes; however, it is not known whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, we report that early post-positive selection thymocytes undergo chemotaxis towards CCR4 ligands, while progressively mature cells migrate towards CCR7 ligands. Synchronized positive selection experiments reveal that CCR4 is upregulated within hours of receiving a positive selection signal, while CCR7 upregulation is delayed for about 24h. 2-photon microscopy reveals that CCR4 facilitates entry of early post-positive selection cells into the thymic medulla. These findings suggest that CCR4 and CCR7 promote distinct stages of negative selection. Consistent with this possibility, flow cytometric analysis reveals that CCR4 deficiency results in a selective decline in early-stage negative selection, while CCR7 deficiency impairs only late-stage negative selection. Altogether, our data suggest a new model in which CCR4 and CCR7 mediate medullary entry of distinct thymocyte subsets and contribute differentially to central tolerance.

Semaphorin-3A-Related Reduction of Thymocyte Migration in Chemically Induced Diabetic Mice

Background: Previous work revealed the existence of a severe thymic atrophy with massive loss of immature CD4⁺CD8⁺ thymocytes in animals developing insulin-dependent diabetes, chemically induced by alloxan. Furthermore, the intrathymic expression of chemokines, such as CXCL12, is changed in these animals, suggesting that cell migration-related patterns may be altered. One molecular interaction involved in normal thymocyte migration is that mediated by soluble semaphorin-3A and its cognate receptor neuropilin-1. Objectives: We investigated herein the expression and role of semaphorin-3A in the migratory responses of thymocytes from alloxan-induced diabetic mice. We characterized semaphorin-3A and its receptor, neuropilin-1, in thymuses from control and diabetic mice as well as semaphorin-3A-dependent migration of developing thymocytes in both control and diabetic animals. Methods: Diabetes was chemically induced after a single injection of alloxan in young adult BALB/c mice. Thymocytes were excised from control and diabetic individuals and subjected to cytofluorometry for simultaneous detection of semaphorin-3A or neuropilin-1 in CD4/CD8-defined subsets. Cell migration in response to semaphorin-3A was performed using cell migration transwell chambers. Results: Confirming previous data, we observed a severe decrease in the total numbers of thymocytes in diabetic mice, which comprised alterations in both immature (double-negative subpopulations) and mature CD4/CD8-defined thymocyte subsets. These were accompanied by a decrease in the absolute numbers of semaphorin-3A-bearing thymocytes, comprising CD4^–CD8^–, CD4⁺CD8⁺, and CD4^–CD8⁺ cells. Additionally, immature CD4^–CD8^– and CD4⁺CD8⁺ developing T cells exhibited a decrease in the membrane density of semaphorin-3A. The relative and absolute numbers of neuropilin-1-positive thymocytes were also decreased in diabetic mouse thymocytes compared to controls, as seen in CD4^–CD8^–, CD4⁺CD8⁺, and CD4^–CD8⁺ cell subpopulations. Functionally, we observed a decrease in the chemorepulsive role of semaphorin-3A, as revealed by transwell migration chambers. Such an effect was seen in all immature and mature thymocyte subsets. Conclusions: Taken together, our data clearly unravel a disruption in the normal cell migration pattern of developing thymocytes following chemically induced insulin-dependent diabetes, as ascertained by the altered migratory response to sempahorin-3A. In conceptual terms, it is plausible to think that such disturbances in the migration pattern of thymocytes from these diabetic animals may exert an impact in the cell-mediated immune response of these mice.

Qualitative Changes in Cortical Thymic Epithelial Cells Drive Postpartum Thymic Regeneration.

During gestation, sex hormones cause a significant thymic involution which enhances fertility. This thymic involution is rapidly corrected following parturition. As thymic epithelial cells (TECs) are responsible for the regulation of thymopoiesis, we analyzed the sequential phenotypic and transcriptomic changes in TECs during the postpartum period in order to identify mechanisms triggering postpartum thymic regeneration. In particular, we performed flow cytometry analyses and deep RNA-sequencing on purified TEC subsets at several time points before and after parturition. We report that pregnancy-induced involution is not caused by loss of TECs since their number does not change during or after pregnancy. However, during pregnancy, we observed a significant depletion of all thymocyte subsets downstream of the double-negative 1 (DN1) differentiation stage. Variations in thymocyte numbers correlated with conspicuous changes in the transcriptome of cortical TECs (cTECs). The transcriptomic changes affected predominantly cTEC expression of Foxn1, its targets and several genes that are essential for thymopoiesis. By contrast, medullary TECs (mTECs) showed very little transcriptomic changes in the early postpartum regenerative phase, but seemed to respond to the expansion of single-positive (SP) thymocytes in the late phase of regeneration. Together, these results show that postpartum thymic regeneration is orchestrated by variations in expression of a well-defined subset of cTEC genes, that occur very early after parturition.

A New Mechanism for T-ALL Leukemogenesis: Early Expression of Alpha-Beta TCR Occurs in a Natural Subset of CD4-CD8- Double Negative (DN) Thymocytes Where MHC Engagement May Drive NOTCH Mutation and Tumor Initiation

T cell lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from transformed thymocytes. Most human T-ALL involves hyperactive NOTCH signaling that is often caused by activating NOTCH mutations. However, the identification of specific molecular signals that might induce or select for mutation and transformation are incompletely understood. We report that an understudied low-frequency, natural thymocyte subset expresses αβ T cell antigen receptor (TCR) earlier than most cells in mice and humans; engagement of the early αβTCR by major histocompatibility complexes (MHC) can cause outgrowth of NOTCH1 mutant clones and T-ALL leukemogenesis in a mouse model of T-ALL. Assessment of 5 recent human T-ALL cases found one to present this unique CD4-CD8- double- negative (DN) stage as the earliest identifiable developmental stage. These studies present a model of T-ALL leukemogenesis that identifies (i) a natural cell stage of origin susceptible to transformation, (ii) a matching mouse model showing that a signaling receptor (αβTCR) and its ligand (MHC) drive leukemogeneis and outgrowth of tumors bearing activating NOTCH1 mutations, and (iii) a human case that presents with a tumor consistent with this model and mechanism. In past work, the pre-TCR has been shown to impact T-ALL development in mice (Campese et al, Blood 2006), but an oncogenic role for the mature αβTCR is less well characterized and somewhat surprising. This is because, although T-ALL tumor cells may express variable levels of surface αβTCR/CD3, the earliest cell stages that are thought to transform are also thought to precede stages with αβTCR expression. Most conventional αβ thymocytes rearrange TCRβ and TCRα loci in separate, ordered developmental stages. However, some thymocytes in the conventional pathway rearrange both at DN stage thus exhibiting 'precocious' αβTCR (PAT) expression. Importantly, these PAT cells are indeed part of the conventional αβ lineage, being a 'subset' only due to early αβTCR expression but without known distinction in ultimate immune function (Aifantis et al, JEM 2006). We found that ~0.01% of mouse and human thymocytes are such PAT cells at steady state. To interrogate the PAT thymocyte surface phenotype, we performed multi-parametric flow cytometry with Spanning-tree Progression Analysis of Density-normalized Events (SPADE). This revealed that PAT thymocytes constitute a DN subset that is not associated with other well-described unconventional DN thymocytes known to express αβTCR, consistent with as the expectation that PATs are part of the conventional developmental pathway. We observed that the OT1 TCR transgene is expressed in mice with parallel timing and level to the natural PAT subset, allowing use of this model to study antigen-dependent signaling and oncogenesis. In a cohort study, no T-ALL was observed in wild-type C57BL/6 or OT-1.β2M-/- mice (deficient in endogenous antigen presentation), but MHC-sufficient OT-1 mice developed PAT-stage-specific T-ALL with activating NOTCH1 mutations. Transplant experiments corroborated a requirement for antigen presentation and TCR signaling for tumor maintenance as transplanted tumors grew in MHC+ but not MHC-deficient mice. This predicted that PAT thymocytes might have an unusual ability to signal through αβTCR even without coreceptor expression. When cultured in the presence of either exogenously added β2M or antigen presenting cells, both untransformed and neoplastic PAT cells upregulated CD69 in response to the OT-1 antigenic peptide, OVA. Furthermore, ex vivo analysis of PAT cells from polyclonal C57BL/6 versus MHC-deficient mice showed intrinsic upregulation of TCR-signaling-dependent Nur77 in an MHC-dependent manner. These data revealed a unique ability of PAT cells to engage in co-receptor independent but antigen-dependent signaling. Microarray analysis showed that the gene expression profile of neoplastic PAT cells from OT-1 T-ALL most closely resembled that of conventional post β-selection DN thymocytes, in agreement with the natural PAT stage during normal T cell development. These data support a model in which transformation occurred in the naturally occurring αβ PAT thymocyte subset as cell-of-origin. Collectively, our data suggest that precocious αβTCR expression and coreceptor-independent antigen engagement can cause activating NOTCH mutation and T-ALL development. Disclosures No relevant conflicts of interest to declare.

Cre-loxP Reporter Mouse Reveals Stochastic Activity of the Foxp3 Promoter.

Mouse models that combine specific loxP-flanked gene sequences with Cre recombinase expressed from cell-regulated promoters have become important tools to investigate gene function. Critically however, expression of Cre recombinase may not always be restricted to the target cell or tissue of interest due to promiscuous activity of the driving promoter. Expression of Cre recombinase and, by extension, excision of the loxP-flanked gene may occur in non-target cells and may not be readily apparent. Here we report on the fidelity of Cre recombinase expressed from the il17a or Foxp3 promoters by combining them with a constitutively expressed floxed-stopped tdTomato reporter gene. Foxp3-driven Cre recombinase in F1 mice induced tdTomato red fluorescent protein in Treg cells but also in a range of other immune cells. Frequency of tdTomato expression was variable but positively correlated (p < 0.0001) amongst lymphoid (B cells and CD8 T cells) and blood-resident myeloid cells (dendritic cells, monocytes, neutrophils) suggesting stochastic activity of the Foxp3 promoter rather than developmental regulation in common ancestral progenitors. Interestingly, frequency of tdTomato+ dendritic cells, monocytes and neutrophils did not correlate with the tdTomato+ fraction in eosinophils, indicating that activity of the Foxp3 promoter in eosinophils occurred after the split from a common multipotent progenitor. When these F1 mice were crossed to achieve homozygosity of the promoter and reporter gene, a novel visually red phenotype was observed segregating amongst littermates. The red coloration was widespread and prevalent in non-immune tissues. Thymocytes examined from these red mice showed that all four subsets of immature thymocytes (CD4− CD8−) based on differential expression of CD25 and CD44 were expressing tdTomato. Finally, we show evidence of Foxp3 Cre recombinase independent tdTomato expression, suggesting germ line transmission of an activated tdTomato reporter gene. Our data highlights potential issues with conclusions drawn from using specifically the B6.129(Cg)-Foxp3tm4(YFP/Cre)Ayr/J mice.

Intrathymic adeno-associated virus gene transfer rapidly restores thymic function and long-term persistence of gene-corrected T cells

Patients with T-cell immunodeficiencies are generally treated with allogeneic hematopoietic stem cell transplantation, but alternatives are needed for patients without matched donors. An innovative intrathymic gene therapy approach that directly targets the thymus might improve outcomes. We sought to determine the efficacy of intrathymic adeno-associated virus (AAV) serotypes to transduce thymocyte subsets and correct the T-cell immunodeficiency in a zeta-associated protein of 70kDa (ZAP-70)-deficient murine model. AAV serotypes were injected intrathymically into wild-type mice, and gene transfer efficiency was monitored. ZAP-70-/- mice were intrathymically injected with an AAV8 vector harboring the ZAP70 gene. Thymus structure, immunophenotyping, T-cell receptor clonotypes, T-cell function, immune responses to transgenes and autoantibodies, vector copy number, and integration were evaluated. AAV8, AAV9, and AAV10 serotypes all transduced thymocyte subsets after in situ gene transfer, with transduction of up to 5% of cells. Intrathymic injection of an AAV8-ZAP-70 vector into ZAP-70-/- mice resulted in a rapid thymocyte differentiation associated with the development of a thymic medulla. Strikingly, medullary thymic epithelial cells expressing the autoimmune regulator were detected within 10days of gene transfer, correlating with the presence of functional effector and regulatory T-cell subsets with diverse T-cell receptor clonotypes in the periphery. Although thymocyte reconstitution was transient, gene-corrected peripheral T cells harboring approximately 1 AAV genome per cell persisted for more than 40weeks, and AAV vector integration was detected. Intrathymic AAV-transduced progenitors promote a rapid restoration of the thymic architecture, with a single wave of thymopoiesis generating long-term peripheral T-cell function.

IL-2 production by self-reactive CD4 thymocytes scales regulatory T cell generation in the thymus.

Regulatory T (T reg) cells, a specialized subset of CD4+ T cells, are essential to prevent fatal autoimmunity. Expression of the T reg lineage-defining transcription factor Foxp3, and therefore their differentiation in the thymus, is dependent upon T cell receptor (TCR) and interleukin-2 (IL-2) signaling. Here, we report that the majority of IL-2-producing cells in the thymus are mature CD4 single-positive (CD4SP) thymocytes and that continuous IL-2 production sustained thymic T reg cell generation and control of systemic immune activation. Furthermore, single-cell RNA sequencing analysis of CD4 thymocyte subsets revealed that IL-2 was expressed in self-reactive CD4SP thymocytes, which also contain T reg precursor cells. Thus, our results suggest that the thymic T reg cell pool size is scaled by a key niche factor, IL-2, produced by self-reactive CD4SP thymocytes. This IL-2-dependent scaling of thymic T reg cell generation by overall self-reactivity of a mature post-selection thymic precursor pool may likely ensure adequate control of autoimmunity.

Immunofluorescent characterization of innervation and nerve-immune cell neighborhood in mouse thymus.

The central nervous system impacts the immune system mainly by regulating the systemic concentration of humoral substances, whereas the peripheral nervous system (PNS) communicates with the immune system specifically according to local "hardwiring" of sympathetic/parasympathetic (efferent) and sensory (afferent) nerves to the primary and secondary lymphoid tissue/organs (e.g., thymus spleen and lymph nodes). In the present study, we use immunofluorescent staining of neurofilament-heavy to reveal the distribution of nerve fibers and the nerve-immune cell neighborhood inside the mouse thymus. Our results demonstrate (a) the presence of an extensive meshwork of nerve fibers in all thymic compartments, including the capsule, subcapsular region, cortex, cortico-medullary junction and medulla; (b) close associations of nerve fibers with blood vessels (including the postcapillary venules), indicating the neural control of blood circulation and immune cell dynamics inside the thymus; (c) the close proximity of nerve fibers to various subsets of thymocytes (e.g., CD4+, CD8+ and CD4+CD8+), dendritic cells (e.g., B220+, CD4+, CD8+ and F4/80+), macrophages (Mac1+ and F4/80+) and B cells. Our novel findings concerning thymic innervation and the nerve-immune cell neighborhood in situ should facilitate the understanding of bi-directional communications between the PNS and primary lymphoid organs. Since the innervation of lymphoid organs, including the thymus, may play essential roles in the pathogenesis and progression of some neuroimmune, infectious and autoimmune diseases, better knowledge of PNS-immune system crosstalk should benefit the development of potential therapies for these diseases.

PF460 SINGLE CELL RNASEQ REVEALS THE IN VIVO DIFFERENTIATION POTENTIAL AND TRANSCRIPTIONAL DYNAMICS OF IMMATURE HUMAN THYMOCYTES

Background:T cell development depends on the colonization of the thymus by multipotent progenitor cells from the bone marrow. There, these precursor cells are gradually reprogrammed into committed T cell progenitors that have lost the developmental potential towards other hematopoietic lineages. While lineage tracing experiments in mice have provided evidence to which non T‐cell lineages these early T cell precursors differentiate in vivo, it remains unclear in human which blood cell lineages can originate from early T cell precursors. In vitro differentiation experiments have revealed erythroid, myeloid (DC, monocyte, granulocyte) and lymphoid (B, T, NK) differentiation potential, but whether this occurs in vivo is not clear.Aims:While lineage tracing experiments in mice have provided evidence to which non T‐cell lineages these early T cell precursors differentiate in vivo, it remains unclear in human which blood cell lineages can originate from early T cell precursors. In vitro differentiation experiments have revealed erythroid, myeloid (DC, monocyte, granulocyte) and lymphoid (B, T, NK) differentiation potential, but whether this occurs in vivo is not clear.Methods:Therefore, we have performed single cell RNA sequencing on more than 70.000 human CD34+lin‐ thymocytes that comprise the most immature thymocyte fraction of the human pediatric thymus.Results:Analysis of this dataset showed that cell cycle constitutes the majority of variation in the dataset, indicating less heterogeneity than anticipated. Indeed, using a strategy in which both clustering and trajectory inference were used, only limited branching was observed towards other, non T‐cell hematopoietic lineages, indicating a discrepancy between reported developmental potential and in vivo cell fate. Using these branching events, commitment was traced to coincide with loss of CD44 expression, confirming recent findings. In addition, we used CITEseq to correlate previously defined immature thymocyte subsets to these newly defined developmental cell states.Summary/Conclusion:In conclusion, our work suggests that the in vivo differentiation of immature human thymocytes is much more restricted compared to what in vitro differentiation experiments have suggested. In addition, we have delineated the transcriptional events that define the transition from multipotency toward T‐cell lineage commitment at the single cell level and redefine immature thymocyte subsets in human.

TCR signal strength shapes functional imprinting during CD4 T cell development

Abstract Naïve CD4 and CD8ab T cells are selected in the thymus in response to low-avidity TCR interactions. Such weak TCR signals install a quiescent program that guides naïve T cells to the lymph nodes where they differentiate upon encounter of cognate antigen. Thymocytes expressing self-specific TCRs can be agonist-selected in response to a strong TCR signal and differentiate in the thymus to functionally committed double negative T cells, NKT cells or regulatory T cells. Although the role for strength of TCR activation is clear in instructing these different outcomes, the impact of self-recognition in the thymus on “naïve” T cells is unclear. Previous work demonstrated that negative selection of self-specific T cells is not all encompassing and that affinity for self could even enhance immune protection against pathogens. We have used Stim1fl/fl Stim2fl/fl conditional deletion mice that fail to sustain calcium entry into the cytosol upon TCR activation to assess the role of TCR strength during selection. We confirmed that agonist selection is drastically affected in these mice whereas numbers of conventional T cells are not. Interestingly, viSNE representation of multi-parameter flow cytometry revealed single cell heterogeneity among the CD4SP thymocyte subset that is controlled by Stim1/Stim2. This indicates that differential TCR activation levels give rise to phenotypically different CD4 T cells. Purification of a strongly signaled (Stim1/2-dependent) subset of CD4 thymocytes showed functional differences in terms of their ability to proliferate in response to TCR activation. Our work points to a role for self-recognition during T cell selection that shapes the response of individual CD4 T cells to TCR activation in the periphery.

22q11.2 Deletion Syndrome (DiGeorge) and Mutations in Forkhead Box N1 (FOXN1) cause a Thymic Hypoplasia through distinct Developmental Processes

Abstract Patients with 22q11.2 deletion syndrome and those with mutations in the Forkhead Box N1(FOXN1) transcription factor (Nude/SCID) can both present with a thymic hypoplasia that results in a severe T cell lymphopenia. In both clinical conditions, the thymic anlage fails to develop properly within the 3rdpharyngeal pouch during embryogenesis. We characterized the development of the thymus in mouse models of 22q11.2 deletion syndrome (22q11.2del) and a new set of mice with mutations in Foxn1that genocopied a SCID patient with novel compound heterozygous mutations in FOXN1. Both sets of mice develop hypoplastic thymic lobes. An analysis of thymopoiesis in embryos revealed distinct development problems. The hypoplastic thymii from the 22q11.2del mice were primarily sized restricted, with normal percentages of all thymocyte subsets apparent. This contrasted a severe deficiency of thymocytes due to the Foxn1mutations, which primarily affected the development and expansion of thymic epithelial cells. Comparative gene expression analyses of e13.5 fetal thymii revealed differentially regulated transcripts that define the basis of the hypoplasia. A dysregulated mesenchymal cell signature was apparent in the 22q11.2del model, which contrasted the epithelial transcript disruption due to the Foxn1mutations. These results suggest different strategies are necessary to correct the thymic tissue abnormalities in patients who present with thymic hypoplasias due to their congenital disorders.

Live-Cell FRET Imaging Reveals a Role of ERK Activity Dynamics in Thymocyte Motility

Thymocyte motility is key to orchestrating migration between different thymic microenvironments at the appropriate developmental stage. Several lines of evidence suggest that extracellular signal-regulated kinase (ERK) signaling pathways plays critical roles in T cell development. Nevertheless, the dynamics of ERK activity and its role in regulating cell motility remain largely unknown due to technical difficulties. Therefore, there is an increasing demand for genetic reporter systems that provide the information of thymocyte motility and ERK signaling under physiological condition in real time.To meet the demand, we developed transgenic mice expressing fluorescence resonance energy transfer (FRET) based biosensor for ERK incorporated into the ROSA26 locus conditionally. EKAREV is a genetically encoded intramolecular FRET biosensor for monitoring ERK activity in living cells. We introduced cDNAs of EKAREV into the ROSA26 locus to generate mouse lines named R26-EKAREV-NLSfl/+. In these mouse lines, EKAREV will be expressed in the nucleus after Cre-mediated excision of the loxP-flanked tdKeima-coding sequence. To study ERK activity dynamics in T cells, R26-EKAREV-NLSfl/+ were crossed with Lck-Cre mice to generate R26-EKAREV-NLSlck/+. R26-EKAREV-NLSlck/+ showed uniform and high-level expression of EKAREV in lymphocytes and allowed us to examine the ERK activity of T cells in vivo.After initial characterization of R26-EKAREV-NLSlck/+, we attempted to unravel the potential crosstalk between ERK activity dynamics and cell motility within the thymic microenvironment. Long-term in vivo imaging of thymus was difficult due to the anatomical location abutting the heart. To circumvent this problem, thymocytes obtained from R26-EKAREV-NLSlck/+ were overlaid on C57BL/6 (WT) thymic slices and observed under two-photon excitation microscopy. During the course of experiments, we noticed a negative correlation between ERK activity and migration speed of thymocytes. To confirm this observation, thymocytes were cultured in the presence or absence of an mitogen-activated protein kinase/ERK kinase (MEK) inhibitor prior to transfer onto thymic slices. Treatment of thymocytes with MEK inhibitor increased averaged migration speed in both double-positive (DP) and single-positive (SP) subsets. Moreover, time-lapse imaging of each subsets of thymocytes revealed that the deviation of ERK activity from the average of individual cells regulated cell motility in CD4-SP, while the absolute value of ERK activity regulated cell motility in DP and CD8-SP. Collectively, these results suggest that CD4-SP is unique in that the ERK activity dynamics negatively regulate cell motility.Which signal regulates ERK activity and cell motility of CD4-SP in the medulla? Interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) is known to regulate ERK activity and cell motility of T cells, but their direct relationship remains unknown in tissues. To examine this possibility, CD4-SP thymocytes were overlaid onto WT or MHC class II knockout (KO) thymic slices. When overlaid onto KO thymic slices, the average migration speed was significantly increased, indicating that CD4-SP cells arrest upon TCR-MHC II interaction. Moreover, the variance of ERK activity, but not the average of it, in CD4-SP cells on KO thymic slices was decreased, suggesting that TCR-MHC II interaction contribute the variance of ERK activity in CD4-SP.Our findings unravel that the deviation of ERK activity induced by TCR-MHC interactions negatively regulate cell motility of CD4-SP in the medulla. The live-cell FRET imaging of ERK activity will open a new window to understand the dynamic nature and the diverse functions of ERK signaling in T cell biology. DisclosuresTakaori-Kondo:Janssen Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding.

The role of IL-22 in T cell reconstitution after thymus damage induced by ionizing radiation

目的探索γ射线照射诱导胸腺损伤后胸腺内IL-22表达水平的变化趋势，并研究IL-22在胸腺损伤后T细胞免疫重建中的作用。方法建立非致死剂量γ射线照射诱导的小鼠胸腺损伤模型，分别设置正常对照组和全身照射（TBI）组，ELISA法检测小鼠胸腺及血浆中IL-22的含量；分别给予TBI组小鼠PBS或重组小鼠IL-22腹腔注射处理，设为TBI+PBS组和TBI+IL-22组，计数胸腺内总细胞和外周血白细胞含量，同时使用流式细胞术分析胸腺上皮细胞（thymic epithelial cells，TEC）、各阶段胸腺细胞以及外周血T细胞的含量，实时定量PCR分析胸腺中与TEC功能相关的基因Foxn1、Ccl25、Aire和Dll4的mRNA表达水平。结果①照射后小鼠胸腺内IL-22表达水平高于未经照射处理的正常对照小鼠（P值均<0.05）；②给予TBI组小鼠IL-22腹腔注射后，TBI+IL-22组小鼠胸腺内IL-22含量高于TBI+PBS组（P值均<0.05）；③照射后第14天，TBI+IL-22组胸腺内Foxn1、Ccl25、Aire和Dll4 mRNA表达水平均高于TBI+PBS组（P值均<0.05），同时，TBI+IL-22组胸腺总细胞计数[（5.93±3.19）×106/ml对（1.42±0.46）×106/ml，t=3.128，P=0.033]和外周白细胞计数[（3.08±0.94）×106/ml对（1.43±0.30）×106/ml，t=3.730，P=0.015]均高于TBI+PBS组。流式细胞术分析示，照射后第14天TBI+IL-22组小鼠胸腺内TEC和各胸腺细胞亚群含量均高TBI+PBS组（P值均<0.05），照射后第7天和14天TBI+IL-22组外周血T细胞含量均高于TBI+PBS组（P值均<0.05）。结论γ射线照射处理可导致小鼠胸腺内IL-22的含量升高，注射外源性IL-22可增加胸腺内IL-22含量。输注外源性IL-22可促进受损胸腺内TEC功能的修复，并增加各胸腺细胞亚群以及外周血中T细胞的数量。

Lack of Galectin-3 Disrupts Thymus Homeostasis in Association to Increase of Local and Systemic Glucocorticoid Levels and Steroidogenic Machinery.

Maintenance of thymus homeostasis is a delicate interplay involving hormones, neurotransmitters and local microenvironmental proteins, as well as saccharides, acting on both thymocytes and stromal cells. Disturbances in these interactions may lead to alterations on thymocyte development. We previously showed that galectin-3, a β-galactoside-binding protein, is constitutively expressed in the thymus, interacting with extracellular matrix glycoproteins and acting as a de-adhesion molecule, thus modulating thymocyte-stromal cell interactions. In this work, we aimed to investigate the participation of galectin-3 in the maintenance of thymus homeostasis, including hormonal-mediated circuits. For that, we used genetically engineered galectin-3-deficient mice. We observed that the thymus of galectin-3-deficient mice was reduced in mass and cellularity compared to wild-type controls; however, the proportions of different thymocyte subpopulations defined by CD4/CD8 expression were not changed. Considering the CD4−CD8− double-negative (DN) subpopulation, an accumulation of the most immature (DN1) stage was observed. Additionally, the proliferative capacity of thymocytes was decreased in all thymocyte subsets, whereas the percentage of apoptosis was increased, especially in the CD4+CD8+ double-positive thymocytes. As glucocorticoid hormones are known to be involved in thymus homeostasis, we evaluated serum and intrathymic corticosterone levels by radioimmunoassay, and the expression of steroidogenic machinery using real-time PCR. We detected a significant increase in corticosterone levels in both serum and thymus samples of galectin-3-deficient mice, as compared to age-matched controls. This was paralleled by an increase of gene transcription of the steroidogenic enzymes, steroidogenic acute regulatory protein (Star) and Cyp11b1 in thymus, 11β-Hydroxysteroid Dehydrogenase (Hsd11b1) in the adrenal, and Cyp11a1 in both glands. In conclusion, our findings show that the absence of galectin-3 subverts mouse thymus homeostasis by a mechanism likely associated to intrathymic and systemic stress-related endocrine circuitries, affecting thymocyte number, proliferation and apoptosis.