Thymus aging and immune reconstitution, progresses and challenges.

Thymus is a primary lymphoid organ essential for the development of T lymphocytes. Age-related thymic involution is a prominent feature of immune senescence. The thymus undergoes rapid growth during fetal and neonatal development, peaks in size before puberty and then begins to undergo a decrease in cellularity with age. Dramatic changes occur with age-associated thymic involution. The most prominent features of thymic involution include: (i) epithelial structure disruption, (ii) adipogenesis, and (iii) thymocyte development arrest. There is a sex disparity in thymus aging. It is a multifactorial process controlled and regulated by a series of molecules, including the transcription factor FOXN1, fibroblast and keratinocyte growth factors (FGF and KGF, respectively), sex steroids, Notch signaling, WNT signaling, and microRNAs. Nevertheless, there is still no satisfactory evolutionary or physiological explanation for age-associated thymic involution, and understanding the precise mechanism(s) for thymus aging remains challenging. Sustained thymic regeneration has yet to be achieved by sex steroid ablation. Recent preclinical studies indicate that long-term thymic reconstitution can be achieved via adoptive transfer of in vitro-generated progenitor T (proT) cells, and improvements in the methods for the generation of human proT cells make this an attractive approach. Future clinical applications may rely on new applications integrating proT cells, cytokine support and sex-steroid inhibition treatments.

Recent Thymic Emigrants Require RBPJ-Dependent Notch Signaling to Transition into Functionally Mature Naive T Cells.

Recent thymic emigrant (RTE) cells are nascent T cells that continue their post-thymic maturation in the periphery and dominate T cell immune responses in early life and in adults having undergone lymphodepletion regimens. However, the events that govern their maturation and their functionality as they transition to mature naive T cells have not been clearly defined. Using RBPJind mice, we were able to identify different stages of RTE maturation and interrogate their immune function using a T cell transfer model of colitis. As CD45RBlo RTE cells mature, they transition through a CD45RBint immature naive T (INT) cell population that is more immunocompetent but shows a bias toward IL-17 production at the expense of IFN-γ. Additionally, the levels of IFN-γ and IL-17 produced in INT cells are highly dependent on whether Notch signals are received during INT cell maturation or during their effector function. IL-17 production by INT cells showed a total requirement for Notch signaling. Loss of Notch signaling at any stage of INT cells resulted in an impaired colitogenic effect of INT cells. RNA sequencing of INT cells that had matured in the absence of Notch signals showed a reduced inflammatory profile compared with Notch-responsive INT cells. Overall, we have elucidated a previously unknown INT cell stage, revealed its intrinsic bias toward IL-17 production, and demonstrated a role for Notch signaling in INT cell peripheral maturation and effector function in the context of a T cell transfer model of colitis.

Development and function of FOXP3+ regulators of immune responses.

The Forkhead Box P3 (FOXP3) protein is an essential transcription factor for the development and function of regulatory T cells (Tregs), involved in the maintenance of immunological tolerance. Although extensive research over the last decade has investigated the critical role of FOXP3+ cells in preserving immune homeostasis, our understanding of their specific functions remains limited. Therefore, unveiling the molecular mechanisms underpinning the up- and downstream transcriptional regulation of and by FOXP3 is crucial for developing Treg-targeted therapeutics. Dysfunctions in FOXP3+ Tregs have also been found to be inherent drivers of autoimmune disorders and have been shown to exhibit multifaceted functions in the context of cancer. Recent research suggests that these cells may also be involved in tissue-specific repair and regeneration. Herein, we summarize current understanding of the thymic-transcriptional regulatory landscape of FOXP3+ Tregs, their epigenetic modulators, and associated signaling pathways. Finally, we highlight the contributions of FOXP3 on the functional development of Tregs and reflect on the clinical implications in the context of pathological and physiological immune responses.

Induction of Human T Cell Development In Vitro with OP9-DL4-7FS Cells Expressing Human Cytokines.

For nearly a generation now, OP9-DL1 and OP9-DL4 cells have provided an efficient and reliable cell system to generate T cells from mouse and human hematopoietic stem cells (HSCs) and pluripotent stem cells. OP9-DL1 and OP9-DL4 were originally derived from the OP9 mouse bone marrow stromal cell line, which was transduced to ectopically express Delta-like 1 or 4 proteins, respectively. OP9-DL cells mimic the thymic microenvironment in that when cocultured with mouse or human (h) HSCs, they interact with and activate Notch receptors present on HSCs, required for T cell differentiation. The HSC/OP9-DL cocultures require additional cytokines that are necessary for survival and proliferation of hematopoietic cells. For hHSCs, these factors are interleukin-7 (IL-7), stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L) that are normally exogenously added to the cocultures. In this chapter, we describe methods for establishing a novel and improved version of OP9-DL4 cells, called OP9-DL4-7FS cells that circumvent the addition of these costly cytokines, by transducing OP9-DL4 cell line to express human IL-7, FLT3L, and SCF (7FS). Herein, we describe the protocol for the generation of OP9-DL4-7FS cells and the conditions for OP9-DL4-7FS/hHSC coculture to support T cell lineage initiation and expansion while comparing it to the now "classic" OP9-DL4 coculture. The use of OP9-DL4-7FS cell system will provide an improved and cost-effective method to the commonly used OP9-DL/HSC coculture for studying both mouse and human T cell development.

Regulation of the Signal-Dependent E Protein HEBAlt Through a YYY Motif Is Required for Progression Through T Cell Development.

The E protein transcription factors E2A and HEB are critical for many developmental processes, including T cell development. We have shown that the Tcf12 locus gives rise to two distinct HEB proteins, with alternative (HEBAlt) and canonical (HEBCan) N-terminal domains, which are co-expressed during early T cell development. While the functional domains of HEBCan have been well studied, the nature of the HEBAlt-specific (Alt) domain has been obscure. Here we provide compelling evidence that the Alt domain provides a site for the molecular integration of cytokine signaling and E protein activity. Our results indicate that phosphorylation of a unique YYY motif in the Alt domain increases HEBAlt activity by 10-fold, and that this increase is dependent on Janus kinase activity. To enable in vivo studies of HEBAlt in the T cell context, we generated ALT-Tg mice, which can be induced to express a HA-tagged HEBAlt coding cassette in the presence of Cre recombinases. Analysis of ALT-Tg mice on the Vav-iCre background revealed a minor change in the ratio of ISP cells to CD8+ SP cells, and a mild shift in the ratio of T cells to B cells in the spleen, but otherwise the thymus, spleen, and bone marrow lymphocyte subsets were comparable at steady state. However, kinetic analysis of T cell development in OP9-DL4 co-cultures revealed a delay in early T cell development and a partial block at the DN to DP transition when HEBAlt levels or activity were increased. We also observed that HEBCan and HEBAlt displayed significant differences in protein stability that were resolved in the thymocyte context. Finally, a proteomic screen identified STAT1 and Xpo1 as potential members of HEBAlt-containing complexes in thymocytes, consistent with JAK-induced activation of HEBAlt accompanied by translocation to the nucleus. Thus, our results show that the Alt domain confers access to multiple layers of post-translational control to HEBAlt that are not available to HEBCan, and thus may serve as a rheostat to tune E protein activity levels as cells move through different thymic signaling environments during T cell development.

Thymus Reconstitution in Young and Aged Mice Is Facilitated by In Vitro-Generated Progenitor T Cells.

The prolonged lag in T cell recovery seen in older patients undergoing hematopoietic stem cell transplant (HSCT), after chemo-/radiotherapy, can lead to immune dysfunction. As a result, recovering patients may experience a relapse in malignancies and opportunistic infections, leading to high mortality rates. The delay in T cell recovery is partly due to thymic involution, a natural collapse in the size and function of the thymus, as individuals age, and partly due to the damage sustained by the thymic stromal cells through exposure to chemo-/radiotherapy. There is a clear need for new strategies to accelerate intrathymic T cell reconstitution when treating aged patients to counter the effects of involution and cancer therapy regimens. Adoptive transfer of human progenitor T (proT) cells has been shown to accelerate T cell regeneration in radiation-treated young mice and to restore thymic architecture in immunodeficient mice. Here, we demonstrate that the adoptive transfer of in vitro-generated proT cells in aged mice (18-24 months) accelerated thymic reconstitution after treatment with chemotherapy and gamma irradiation compared to HSCT alone. We noted that aged mice appeared to have a more limited expansion of CD4-CD8- thymocytes and slower temporal kinetics in the development of donor proT cells into mature T cells, when compared to younger mice, despite following the same chemo/radiation regimen. This suggests a greater resilience of the young thymus compared to the aged thymus. Nevertheless, newly generated T cells from proT cell engrafted aged and young mice were readily present in the periphery accelerating the reappearance of new naïve T cells. Accelerated T cell recovery was also observed in both aged and young mice receiving both proT cells and HSCT. The strategy of transferring proT cells can potentially be used as an effective cellular therapy in aged patients to improve immune recovery and reduce the risk of opportunistic infections post-HSCT.

Realization of the T Lineage Program Involves GATA-3 Induction of Bcl11b and Repression of Cdkn2b Expression.

The zinc-finger transcription factor GATA-3 plays a crucial role during early T cell development and also dictates later T cell differentiation outcomes. However, its role and collaboration with the Notch signaling pathway in the induction of T lineage specification and commitment have not been fully elucidated. We show that GATA-3 deficiency in mouse hematopoietic progenitors results in an early block in T cell development despite the presence of Notch signals, with a failure to upregulate Bcl11b expression, leading to a diversion along a myeloid, but not a B cell, lineage fate. GATA-3 deficiency in the presence of Notch signaling results in the apoptosis of early T lineage cells, as seen with inhibition of CDK4/6 (cyclin-dependent kinases 4 and 6) function, and dysregulated cyclin-dependent kinase inhibitor 2b (Cdkn2b) expression. We also show that GATA-3 induces Bcl11b, and together with Bcl11b represses Cdkn2b expression; however, loss of Cdkn2b failed to rescue the developmental block of GATA-3-deficient T cell progenitor. Our findings provide a signaling and transcriptional network by which the T lineage program in response to Notch signals is realized.

Thymic Microenvironment: Interactions Between Innate Immune Cells and Developing Thymocytes.

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

Monoallelic Heb/Tcf12 Deletion Reduces the Requirement for NOTCH1 Hyperactivation in T-Cell Acute Lymphoblastic Leukemia.

Early T-cell development is precisely controlled by E proteins, that indistinguishably include HEB/TCF12 and E2A/TCF3 transcription factors, together with NOTCH1 and pre-T cell receptor (TCR) signalling. Importantly, perturbations of early T-cell regulatory networks are implicated in leukemogenesis. NOTCH1 gain of function mutations invariably lead to T-cell acute lymphoblastic leukemia (T-ALL), whereas inhibition of E proteins accelerates leukemogenesis. Thus, NOTCH1, pre-TCR, E2A and HEB functions are intertwined, but how these pathways contribute individually or synergistically to leukemogenesis remain to be documented. To directly address these questions, we leveraged Cd3e-deficient mice in which pre-TCR signaling and progression through ß-selection is abrogated to dissect and decouple the roles of pre-TCR, NOTCH1, E2A and HEB in SCL/TAL1-induced T-ALL, via the use of Notch1 gain of function transgenic (Notch1ICtg) and Tcf12+/- or Tcf3+/- heterozygote mice. As a result, we now provide evidence that both HEB and E2A restrain cell proliferation at the ß-selection checkpoint while the clonal expansion of SCL-LMO1-induced pre-leukemic stem cells in T-ALL is uniquely dependent on Tcf12 gene dosage. At the molecular level, HEB protein levels are decreased via proteasomal degradation at the leukemic stage, pointing to a reversible loss of function mechanism. Moreover, in SCL-LMO1-induced T-ALL, loss of one Tcf12 allele is sufficient to bypass pre-TCR signaling which is required for Notch1 gain of function mutations and for progression to T-ALL. In contrast, Tcf12 monoallelic deletion does not accelerate Notch1IC-induced T-ALL, indicating that Tcf12 and Notch1 operate in the same pathway. Finally, we identify a tumor suppressor gene set downstream of HEB, exhibiting significantly lower expression levels in pediatric T-ALL compared to B-ALL and brain cancer samples, the three most frequent pediatric cancers. In summary, our results indicate a tumor suppressor function of HEB/TCF12 in T-ALL to mitigate cell proliferation controlled by NOTCH1 in pre-leukemic stem cells and prevent NOTCH1-driven progression to T-ALL.

DL4-µbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system.

T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-µbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αß T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-µbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.

Ontogenic timing, T cell receptor signal strength, and Notch signaling direct γδ T cell functional differentiation in vivo.

γδ T cells form an integral arm of the immune system and are critical during protective and destructive immunity. However, how γδ T cells are functionally programmed in vivo remains unclear. Here, we employ RBPJ-inducible and KN6-transgenic mice to assess the roles of ontogenic timing, T cell receptor (TCR) signal strength, and Notch signaling. We find skewing of Vγ1+ cells toward the PLZF+Lin28b+ lineage at the fetal stage. Generation of interleukin-17 (IL-17)-producing γδ T cells is favored during, although not exclusive to, the fetal stage. Surprisingly, Notch signaling is dispensable for peripheral γδ T cell IL-17 production. Strong TCR signals, together with Notch, promote IL-4 differentiation. Conversely, less strong TCR signals promote Notch-independent IL-17 differentiation. Single-cell transcriptomic analysis reveals differential programming instilled by TCR signal strength and Notch for specific subsets. Thus, our results precisely define the roles of ontogenic timing, TCR signal strength, and Notch signaling in γδ T cell functional programming in vivo.

Cutting Edge: TCR-ß Selection Is Required at the CD4+CD8+ Stage of Human T Cell Development.

T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR ß-chain in mediating ß-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRß rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR ß-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRß- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR ß-chain during human T cell development.

High-Oxygen Submersion Fetal Thymus Organ Cultures Enable FOXN1-Dependent and -Independent Support of T Lymphopoiesis.

T cell development is effectively supported in fetal thymus organ cultures (FTOCs), which places thymus lobes atop an air-liquid interface (ALI) culture system. The direct exposure to air is critical for its success, as fetal thymus lobes placed in low oxygen submersion (LOS)-FTOCs fail to support thymocyte development. However, submersion cultures performed in the presence of high concentration of ambient oxygen (60~80%) allow for normal thymocyte development, but the underlying mechanism for this rescue has remained elusive. Here, we show that FOXN1 expression in thymic epithelial cells (TECs) from LOS-FTOCs was greatly reduced compared to conventional ALI-FTOCs. Consequently, the expression of important FOXN1 target genes, including Dll4 and Ccl25, in TECs was extinguished. The loss of DLL4 and CCL25 interrupted thymocyte differentiation and led to CD4+CD8+ cells exiting the lobes, respectively. High oxygen submersion (HOS)-FTOCs restored the expression of FOXN1 and its target genes, as well as maintained high levels of MHCII expression in TECs. In addition, HOS-FTOCs promoted the self-renewal of CD4-CD8-CD44-CD25+ cells, allowing for the continuous generation of later stage thymocytes. Forced FOXN1 expression in TECs rescued thymocyte developmental progression, but not cellularity, in LOS-FTOCs. Given that oxidative stress has been reported to accelerate the onset of age-associated thymic involution, we postulate that regulation of FOXN1 by oxygen and antioxidants may underpin this biological process.

The E protein-TCF1 axis controls γδ T cell development and effector fate.

TCF1 plays a critical role in T lineage commitment and the development of αß lineage T cells, but its role in γδ T cell development remains poorly understood. Here, we reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element in the Tcf7 locus, and this axis regulates both γδ T lineage commitment and effector fate. Indeed, the level of TCF1 expression plays an important role in setting the threshold for γδ T lineage commitment and modulates the ability of TCR signaling to influence effector fate adoption by γδ T lineage progenitors. This finding provides mechanistic insight into how TCR-mediated repression of E proteins promotes the development of γδ T cells and their adoption of the interleukin (IL)-17-producing effector fate. IL-17-producing γδ T cells have been implicated in cancer progression and in the pathogenesis of psoriasis and multiple sclerosis.

A 2020 View of Thymus Stromal Cells in T Cell Development.

The thymus is an intricate primary lymphoid organ, wherein bone marrow-derived lymphoid progenitor cells are induced to develop into functionally competent T cells that express a diverse TCR repertoire, which is selected to allow for the recognition of foreign Ags while avoiding self-reactivity or autoimmunity. Thymus stromal cells, which can include all non-T lineage cells, such as thymic epithelial cells, endothelial cells, mesenchymal/fibroblast cells, dendritic cells, and B cells, provide signals that are essential for thymocyte development as well as for the homeostasis of the thymic stroma itself. In this brief review, we focus on the key roles played by thymic stromal cells during early stages of T cell development, such as promoting the homing of thymic-seeding progenitors, inducing T lineage differentiation, and supporting thymocyte survival and proliferation. We also discuss recent advances on the transcriptional regulation that govern thymic epithelial cell function as well as the cellular and molecular changes that are associated with thymic involution and regeneration.

E2A regulates neural ectoderm fate specification in human embryonic stem cells.

E protein transcription factors are crucial for many cell fate decisions. However, the roles of E proteins in the germ-layer specification of human embryonic stem cells (hESCs) are poorly understood. We disrupted the TCF3 gene locus to delete the E protein E2A in hESCs. E2A knockout (KO) hESCs retained key features of pluripotency, but displayed decreased neural ectoderm coupled with enhanced mesoendoderm outcomes. Genome-wide analyses showed that E2A directly regulates neural ectoderm and Nodal pathway genes. Accordingly, inhibition of Nodal or E2A overexpression partially rescued the neural ectoderm defect in E2A KO hESCs. Loss of E2A had little impact on the epigenetic landscape of hESCs, whereas E2A KO neural precursors displayed increased accessibility of the gene locus encoding the Nodal agonist CRIPTO. Double-deletion of both E2A and HEB (TCF12) resulted in a more severe neural ectoderm defect. Therefore, this study reveals critical context-dependent functions for E2A in human neural ectoderm fate specification.

Thymic Engraftment by in vitro-Derived Progenitor T Cells in Young and Aged Mice.

T cells play a critical role in mediating antigen-specific and long-term immunity against viral and bacterial pathogens, and their development relies on the highly specialized thymic microenvironment. T cell immunodeficiency can be acquired in the form of inborn errors, or can result from perturbations to the thymus due to aging or irradiation/chemotherapy required for cancer treatment. Hematopoietic stem cell transplant (HSCT) from compatible donors is a cornerstone for the treatment of hematological malignancies and immunodeficiency. Although it can restore a functional immune system, profound impairments exist in recovery of the T cell compartment. T cells remain absent or low in number for many months after HSCT, depending on a variety of factors including the age of the recipient. While younger patients have a shorter refractory period, the prolonged T cell recovery observed in older patients can lead to a higher risk of opportunistic infections and increased predisposition to relapse. Thus, strategies for enhancing T cell recovery in aged individuals are needed to counter thymic damage induced by radiation and chemotherapy toxicities, in addition to naturally occurring age-related thymic involution. Preclinical results have shown that robust and rapid long-term thymic reconstitution can be achieved when progenitor T cells, generated in vitro from HSCs, are co-administered during HSCT. Progenitor T cells appear to rely on lymphostromal crosstalk via receptor activator of NF-κB (RANK) and RANK-ligand (RANKL) interactions, creating chemokine-rich niches within the cortex and medulla that likely favor the recruitment of bone marrow-derived thymus seeding progenitors. Here, we employed preclinical mouse models to demonstrate that in vitro-generated progenitor T cells can effectively engraft involuted aged thymuses, which could potentially improve T cell recovery. The utility of progenitor T cells for aged recipients positions them as a promising cellular therapy for immune recovery and intrathymic repair following irradiation and chemotherapy, even in a post-involution thymus.