Critical roles of the miR-17∼92 family in thymocyte development, leukemogenesis, and autoimmunity.

Thymocyte development requires precise control of PI3K-Akt signaling to promote proliferation and prevent leukemia and autoimmune disorders. Here, we show that ablating individual clusters of the miR-17∼92 family has a negligible effect on thymocyte development, while deleting the entire family severely impairs thymocyte proliferation and reduces thymic cellularity, phenocopying genetic deletion of Dicer. Mechanistically, miR-17∼92 expression is induced by Myc-mediated pre-T cell receptor (TCR) signaling, and miR-17∼92 promotes thymocyte proliferation by suppressing the translation of Pten. Retroviral expression of miR-17∼92 restores the proliferation and differentiation of Myc-deficient thymocytes. Conversely, partial deletion of the miR-17∼92 family significantly delays Myc-driven leukemogenesis. Intriguingly, thymocyte-specific transgenic miR-17∼92 expression does not cause leukemia or lymphoma but instead aggravates skin inflammation, while ablation of the miR-17∼92 family ameliorates skin inflammation. This study reveals intricate roles of the miR-17∼92 family in balancing thymocyte development, leukemogenesis, and autoimmunity and identifies those microRNAs (miRNAs) as potential therapeutic targets for leukemia and autoimmune diseases.

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.

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.

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.

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.

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.

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.

NOTCH1 signaling establishes the medullary thymic epithelial cell progenitor pool during mouse fetal development.

The cortical and medullary thymic epithelial cell (cTEC and mTEC) lineages are essential for inducing T cell lineage commitment, T cell positive selection and the establishment of self-tolerance, but the mechanisms controlling their fetal specification and differentiation are poorly understood. Here, we show that notch signaling is required to specify and expand the mTEC lineage. Notch1 is expressed by and active in TEC progenitors. Deletion of Notch1 in TECs resulted in depletion of mTEC progenitors and dramatic reductions in mTECs during fetal stages, consistent with defects in mTEC specification and progenitor expansion. Conversely, forced notch signaling in all TECs resulted in widespread expression of mTEC progenitor markers and profound defects in TEC differentiation. In addition, lineage-tracing analysis indicated that all mTECs have a history of receiving a notch signal, consistent with notch signaling occurring in mTEC progenitors. These data provide strong evidence for a requirement for notch signaling in specification of the mTEC lineage.

Chronic virus infection drives CD8 T cell-mediated thymic destruction and impaired negative selection.

Chronic infection provokes alterations in inflammatory and suppressive pathways that potentially affect the function and integrity of multiple tissues, impacting both ongoing immune control and restorative immune therapies. Here we demonstrate that chronic lymphocytic choriomeningitis virus infection rapidly triggers severe thymic depletion, mediated by CD8 T cell-intrinsic type I interferon (IFN) and signal transducer and activator of transcription 2 (Stat2) signaling. Occurring temporal to T cell exhaustion, thymic cellularity reconstituted despite ongoing viral replication, with a rapid secondary thymic depletion following immune restoration by anti-programmed death-ligand 1 (PDL1) blockade. Therapeutic hematopoietic stem cell transplant (HSCT) during chronic infection generated new antiviral CD8 T cells, despite sustained virus replication in the thymus, indicating an impairment in negative selection. Consequently, low amounts of high-affinity self-reactive T cells also escaped the thymus following HSCT during chronic infection. Thus, by altering the stringency and partially impairing negative selection, the host generates new virus-specific T cells to replenish the fight against the chronic infection, but also has the potentially dangerous effect of enabling the escape of self-reactive T cells.

RBPJ-dependent Notch signaling initiates the T cell program in a subset of thymus-seeding progenitors.

T cell specification and commitment require Notch signaling. Although the requirement for Notch signaling during intrathymic T cell development is known, it is still unclear whether the onset of T cell priming can occur in a prethymic niche and whether RBPJ-dependent Notch signaling has a role during this event. Here, we established an Rbpj-inducible system that allowed temporal and tissue-specific control of the responsiveness to Notch in all hematopoietic cells. Using this system, we found that Notch signaling was required before the early T cell progenitor stage in the thymus. Lymphoid-primed multipotent progenitors in the bone marrow underwent Notch signaling with Rbpj induction, which inhibited development towards the myeloid lineage in thymus-seeding progenitors. Thus, our results indicated that the onset of T cell differentiation occurred in a prethymic setting, and that Notch played an important role during this event.

Generation and function of progenitor T cells from StemRegenin-1-expanded CD34+ human hematopoietic progenitor cells.

Broader clinical application of umbilical cord blood (UCB), as a source of hematopoietic stem/progenitor cells (HSPCs), is limited by low CD34+ and T-cell numbers, contributing to slow lymphohematopoietic recovery, infection, and relapse. Studies have evaluated the safety, feasibility, and expedited neutrophil recovery associated with the transplantation of CD34+ HSPCs from ex vivo expansion cultures using the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1). In a phase 1/2 study of 17 patients who received combined SR1-expanded and unexpanded UCB units, a considerable advantage for enhancing T-cell chimerism was not observed. We previously showed that progenitor T (proT) cells generated in vitro from HSPCs accelerated T-cell reconstitution and restored immunity after hematopoietic stem cell transplantation (HSCT). To expedite immune recovery, we hypothesized that SR1-expanded HSPCs together with proT cells could overcome the known T-cell immune deficiency that occurs post-HSCT. Here, we show that SR1-expanded UCB can induce >250-fold expansion of CD34+ HSPCs, which can generate large numbers of proT cells upon in vitro differentiation. When compared with nonexpanded naive proT cells, SR1 proT cells also showed effective thymus-seeding and peripheral T-cell functional capabilities in vivo despite having an altered phenotype. In a competitive transfer approach, both naive and SR1 proT cells showed comparable thymus-engrafting capacities. Single-cell RNA sequencing of peripheral CD3+ T cells from mice injected with either naive or SR1 proT cells revealed functional subsets of T cells with polyclonal T-cell receptor repertoires. Our findings support the use of SR1-expanded UCB grafts combined with proT-cell generation for decreasing T-cell immunodeficiency post-HSCT.

Close Quarters Can Be a Good Fit for Stem Cells to Become T Cells.

Human pluripotent stem cells (PSCs) could potentially provide a potentially unlimited supply of T cells. In this issue of Cell Stem Cell, Montel-Hagen et al. (2019) take advantage of a three-dimensional mouse stromal cell-based system that combines mesoderm specification with organoid-induced hematopoietic differentiation to enable commitment, selection, and maturation of conventional human T cells.

In vitro-generated MART-1-specific CD8 T cells display a broader T-cell receptor repertoire than ex vivo naïve and tumor-infiltrating lymphocytes.

The differentiation of human hematopoietic stem cells into CD8 T cells can be achieved in vitro with the OP9-DL4 system. We considered that in the absence of medullary thymic epithelial cells, which serve to restrict the breath of the T-cell receptor (TCR) repertoire by expressing tissue-restricted antigens, a distinct repertoire would be generated in vitro. To test this notion, we compared the TCR-Vα/Vß (TRAV/TRBV) gene usage of major histocompatibility complex-restricted antigen (MART-1)-specific T cells generated in vitro to that from ex vivo naïve T cells and tumor-infiltrating lymphocytes (TILs) using high-throughput DNA sequencing. In contrast to naïve T cells and TILs, which showed the expected narrow TRAV repertoire, in vitro-generated MART-1-specific T cells used almost all TRAV gene families and displayed unique CDR3 lengths. Our work demonstrates that the OP9-DL4 system supports the creation of a broad antigen-specific TCR repertoire, suggesting that T cells generated in vitro may undergo a different set of selection events that otherwise constrains the TCR repertoire of thymus-derived T cells.

Generation and molecular recognition of melanoma-associated antigen-specific human γδ T cells.

Antigen recognition by T cells bearing αß T cell receptors (TCRs) is restricted by major histocompatibility complex (MHC). However, how antigens are recognized by T cells bearing γδ TCRs remains unclear. Although γδ T cells can recognize nonclassical MHC, it is generally thought that recognition of antigens is not MHC restricted. Here, we took advantage of an in vitro system to generate antigen-specific human T cells and show that melanoma-associated antigens, MART-1 and gp100, can be recognized by γδ T cells in an MHC-restricted fashion. Cloning and transferring of MART-1-specific γδ TCRs restored the specific recognition of the initial antigen MHC/peptide reactivity and conferred antigen-specific functional responses. A crystal structure of a MART-1-specific γδ TCR, together with MHC/peptide, revealed distinctive but similar docking properties to those previously reported for αß TCRs, recognizing MART-1 on HLA-A*0201. Our work shows that antigen-specific and MHC-restricted γδ T cells can be generated in vitro and that MART-1-specific γδ T cells can also be found and cloned from the naïve repertoire. These findings reveal that classical MHC-restricted human γδ TCRs exist in the periphery and have the potential to be used in developing of new TCR-based immunotherapeutic approaches.