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.

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.

Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1.

The molecular and cellular signals that guide T-cell development from hematopoietic stem and progenitor cells (HSPCs) remain poorly understood. The thymic microenvironment integrates multiple niche molecules to potentiate T-cell development in vivo. Recapitulating these signals in vitro in a stromal cell-free system has been challenging and limits T-cell generation technologies. Here, we describe a fully defined engineered in vitro niche capable of guiding T-lineage development from HSPCs. Synergistic interactions between Notch ligand Delta-like 4 and vascular cell adhesion molecule 1 (VCAM-1) were leveraged to enhance Notch signaling and progenitor T-cell differentiation rates. The engineered thymus-like niche enables in vitro production of mouse Sca-1+cKit+ and human CD34+ HSPC-derived CD7+ progenitor T-cells capable of in vivo thymus colonization and maturation into cytokine-producing CD3+ T-cells. This engineered thymic-like niche provides a platform for in vitro analysis of human T-cell development as well as clinical-scale cell production for future development of immunotherapeutic applications.

Integration of T-cell receptor, Notch and cytokine signals programs mouse γδ T-cell effector differentiation.

γδ T-cells perform a wide range of tissue- and disease-specific functions that are dependent on the effector cytokines produced by these cells. However, the aggregate signals required for the development of interferon-γ (IFNγ) and interleukin-17 (IL-17) producing γδ T-cells remain unknown. Here, we define the cues involved in the functional programming of γδ T-cells, by examining the roles of T-cell receptor (TCR), Notch, and cytokine-receptor signaling. KN6 γδTCR-transduced Rag2-/- T-cell progenitors were cultured on stromal cells variably expressing TCR and Notch ligands, supplemented with different cytokines. We found that distinct combinations of these signals are required to program IFNγ versus IL-17 producing γδ T-cell subsets, with Notch and weak TCR ligands optimally enabling development of γδ17 cells in the presence of IL-1ß, IL-21 and IL-23. Notably, these cytokines were also shown to be required for the intrathymic development of γδ17 cells. Together, this work provides a framework of how signals downstream of TCR, Notch and cytokine receptors integrate to program the effector function of IFNγ and IL-17 producing γδ T-cell subsets.

Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals.

Developing thymocytes bifurcate from a bipotent precursor into αß- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) ß-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-ß and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.

Notch signals are required for in vitro but not in vivo maintenance of human hematopoietic stem cells and delay the appearance of multipotent progenitors.

All blood cell lineages start from hematopoietic stem cells (HSCs), which were recently shown to represent a heterogeneous group of cells. In mice, Notch signaling promotes the maintenance of "stemness" as well as the expansion of self-renewing HSCs in vitro. Additionally, human CD34(+) cells were shown to expand in vitro in response to Notch signals. However, it is unclear whether Notch directly affects all HSCs, and whether this role is relevant in vivo. Here, we developed culture conditions that support the maintenance of CD34(+)CD133(+)CD90(low)CD38(-)CD7(-)CD10(-)CD45RA(-) (CD90(low)) cells, phenotypically defined HSCs, as well as 2 early progenitor cells (CD34(+)CD38(-)CD7(-)CD10(-)CD45RA(int) [RA(int)] and CD34(+)CD38(-)CD7(-)CD10(-)CD45RA(hi) [RA(hi)]) that were functionally equivalent to multipotent progenitor-2 and lymphoid-primed multipotent progenitor, respectively, found in cord blood. Using a genetic approach, we show that Notch signals were required for HSC preservation, with cultured HSCs being equal to ex vivo HSC cells in their ability to reconstitute immunodeficient mice; however, dnMaml-transduced HSCs were not maintained in vitro. Interestingly, Notch signaling did not appear to be required for the self-renewal of human HSCs in vivo. Our findings support the notion that Notch signals maintain human HSCs in vitro that have hematopoietic-reconstituting ability in vivo and delay the appearance of 2 newly described early progenitor cells.

In Vitro T-Cell Generation From Adult, Embryonic, and Induced Pluripotent Stem Cells: Many Roads to One Destination.

T lymphocytes are critical mediators of the adaptive immune system and have the capacity to serve as therapeutic agents in the areas of transplant and cancer immunotherapy. While T cells can be isolated and expanded from patients, T cells derived in vitro from both hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (hPSCs) offer great potential advantages in generating a self-renewing source of T cells that can be readily genetically modified. T-cell differentiation in vivo is a complex process requiring tightly regulated signals; providing the correct signals in vitro to induce T-cell lineage commitment followed by their development into mature, functional, single positive T cells, is similarly complex. In this review, we discuss current methods for the in vitro derivation of T cells from murine and human HSPCs and hPSCs that use feeder-cell and feeder-cell-free systems. Furthermore, we explore their potential for adoption for use in T-cell-based therapies.

Transcriptional priming of intrathymic precursors for dendritic cell development.

Specialized dendritic cells (DCs) within the thymus are crucial for the deletion of autoreactive T cells. The question of whether these cells arise from intrathymic precursors with T-cell potential has been hotly debated, and the regulatory pathways and signals that direct their development remain unclear. Here, we compared the gene expression profiles of thymic DC subsets with those of four early thymic precursor subsets: early T-cell precursors (ETPs), double-negative 1c (DN1c), double-negative 1d (DN1d) and double-negative 1e (DN1e) subsets. We found that the DN1d subset expressed Spi-B, HEBCan, Ccr7 and Ccr4, similar to thymic plasmacytoid DCs, whereas the DN1e subset expressed Id2, Ccr7 and Ccr4, similar to thymic conventional DCs. The expression of Ccr7 and Ccr4 in DN1d and DN1e cells suggested that they might be able to migrate towards the medulla (low in Dll proteins) and away from the cortex (high in Dll proteins) where early T-cell development occurs. We therefore assessed the sensitivity of developing DC precursors to Dll-Notch signaling, and found that high levels of Dll1 or Dll4 were inhibitory to DC development, whereas medium levels of Dll4 allowed DC development but not myeloid development. To evaluate directly the lineage potential of the ETP, DN1d and DN1e subsets, we injected them into nonirradiated congenic hosts intrathymically or intravenously, and found that they were all able to form medullary DCs in vivo. Therefore, DN1d and DN1e cells are transcriptionally primed to home to the thymus, migrate into DC-permissive microenvironments and develop into medullary DCs.

Human proT-cells generated in vitro facilitate hematopoietic stem cell-derived T-lymphopoiesis in vivo and restore thymic architecture.

Hematopoietic stem cell transplantation (HSCT) is followed by a period of immune deficiency due to a paucity in T-cell reconstitution. Underlying causes are a severely dysfunctional thymus and an impaired production of thymus-seeding progenitors in the host. Here, we addressed whether in vitro-derived human progenitor T (proT)-cells could not only represent a source of thymus-seeding progenitors, but also able to influence the recovery of the thymic microenvironment. We examined whether co-transplantation of in vitro-derived human proT-cells with hematopoietic stem cells (HSCs) was able to facilitate HSC-derived T-lymphopoiesis posttransplant. A competitive transfer approach was used to define the optimal proT subset capable of reconstituting immunodeficient mice. Although the 2 subsets tested (proT1, CD34(+)CD7(+)CD5(-); proT2, CD34(+)CD7(+)CD5(+)) showed thymus engrafting function, proT2-cells exhibited superior engrafting capacity. Based on this, when proT2-cells were coinjected with HSCs, a significantly improved and accelerated HSC-derived T-lymphopoiesis was observed. Furthermore, we uncovered a potential mechanism by which receptor activator of nuclear factor κb (RANK) ligand-expressing proT2-cells induce changes in both the function and architecture of the thymus microenvironment, which favors the recruitment of bone marrow-derived lymphoid progenitors. Our findings provide further support for the use of Notch-expanded progenitors in cell-based therapies to aid in the recovery of T-cells in patients undergoing HSCT.

Cellular and molecular requirements for the selection of in vitro-generated CD8 T cells reveal a role for Notch.

Differentiation of CD8 single-positive (SP) T cells is predicated by the ability of lymphocyte progenitors to integrate multiple signaling cues provided by the thymic microenvironment. In the thymus and the OP9-DL1 system for T cell development, Notch signals are required for progenitors to commit to the T cell lineage and necessary for their progression to the CD4(+)CD8(+) double-positive (DP) stage of T cell development. However, it remains unclear whether Notch is a prerequisite for the differentiation of DP cells to the CD8 SP stage of development. In this study, we demonstrate that Notch receptor-ligand interactions allow for efficient differentiation and selection of conventional CD8 T cells from bone marrow-derived hematopoietic stem cells. However, bone marrow-derived hematopoietic stem cells isolated from Itk(-/-)Rlk(-/-) mice gave rise to T cells with decreased IFN-γ production, but gained the ability to produce IL-17. We further reveal that positive and negative selection in vitro are constrained by peptide-MHC class I expressed on OP9 cells. Finally, using an MHC class I-restricted TCR-transgenic model, we show that the commitment of DP precursors to the CD8 T cell lineage is dependent on Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout T cell differentiation, including the final step of CD8 SP selection.

Induction of T-cell development by Delta-like 4-expressing fibroblasts.

The thymus provides a unique environment for the induction of T-cell lineage commitment and differentiation, which is predicted by specific Notch ligand-receptor interactions on epithelial cells and lymphoid progenitors, respectively. Accordingly, a bone marrow-derived stromal cell line (OP9) ectopically expressing the Notch ligand Delta-like 1 (Dll1) or Dll4 (OP9-DL1 and OP9-DL4, respectively) gains the ability to recapitulate thymus-like function, supporting T-cell differentiation of both mouse and human progenitors. In this study, we extend these findings by demonstrating that, unlike the NIH3T3 cell line, mouse primary fibroblasts made to express Dll4 (mFibro-DL4) acquire the capacity to promote and support T-cell development from hematopoietic stem cells (HSCs) into TCRαß(+), CD4(+) and CD8(+) T-lineage cells. However, mFibro-DL4 cells showed a lower efficiency of T-cell generation than OP9-DL4 cells did. Nevertheless, progenitor T-cells (CD117(+) CD44(+) CD25(+)) generated in HSC/mFibro-DL4 co-cultures, when intravenously transferred into immunodeficient (Rag2(-/-) γc(-/-)) mice, home to the thymus, undergo differentiation, and give rise to mature T-cells that go on to populate the periphery. Surprisingly, primary human fibroblast cells expressing Dll4 showed very low efficiency in supporting human T-lineage differentiation, which could not be improved by blocking myelopoiesis. Nevertheless, mFibro-DL4 cells could support human T-cell lineage differentiation. Our results provide a functional framework for the induction of T-cell development using easily accessible fibroblasts made to express Dll4. These cells, which are amenable for in vitro applications, can be further utilized in the design of individualized systems for T-cell production, with implications for the treatment of immunodeficiencies.

Removal of myeloid cytokines from the cellular environment enhances T-cell development in vitro.

The majority of T-cell development occurs in the thymus. Thymic epithelial cells are specialized cells that express NOTCH ligands and secrete specific cytokines required for normal T-cell lymphopoiesis. It has been demonstrated that OP9 cells derived from macrophage colony-stimulating factor (M-CSF)-deficient mice can support T-cell development when transduced with a NOTCH ligand, Delta-like 1 (Dll1). In this report, we have tested CSF-deficient mouse fibroblasts transduced with Dll1 for their ability to support T-cell differentiation. The data provided here demonstrate that CSF-deficient fibroblasts expressing DLL1 can support T-cell development. Indeed, co-cultures with these fibroblasts produced more T-cell progenitors compared with OP9-DL1 cultures. Addition of myeloid cytokines to OP9-DL1 co-cultures significantly inhibited T-cell development while CSF-deficient DLL1(+) fibroblasts retained partial T-cell differentiation. Taken together, these data imply that their lack of myeloid cytokines allows DLL1(+) fibroblasts to more efficiently generate T-cells. Development of this fibroblast system suggests that there is potential for generating human T-cell precursors via co-culture with human fibroblasts expressing DLL1 or DLL4. These T-cell precursors could be used for treating immunodeficient patients.

Role of recycling, Mindbomb1 association, and exclusion from lipid rafts of δ-like 4 for effective Notch signaling to drive T cell development.

Intrathymic T cell development is predicated on the Notch1 ligand Delta-like (Dll) 4. However, both Dll4 and Dll1 can support T cell development in vitro. Endocytosis of Dll1 is important for Notch activation, whereas currently there is no evidence for the role of Dll4 endocytosis in T cell development. To elucidate this, we generated Dll4 constructs that modify or inhibit endocytosis. Our results show that targeting the intracellular domain affects Dll4's ability to induce Notch target gene expression, support efficient T cell development, and inhibit B cell development. Dll4 function relies on a combination of factors, which include strong Mindbomb1 (Mib1) association, ubiquitination, and internalization and recycling back to the cell surface, to engage Notch1 effectively. Distinct membrane localization and the Delta/Serrate/Lag2 (DSL) domain were important for Dll4 function. These features are consistent with a "recycling" model, but not in opposition to a "mechano-transduction" model, whereby Dll4 is able to engage Notch and create a pulling force required to activate signaling, leading to the induction of T-lineage development. Taken together, in contrast to Dll1, Dll4 does not localize to lipid rafts and shows stronger association with Mib1 and increased Notch1 uptake, which likely account for its superior ability to induce T cell development.

Dynamics of human prothymocytes and xenogeneic thymopoiesis in hematopoietic stem cell-engrafted nonobese diabetic-SCID/IL-2rγnull mice.

To model the developmental pattern of human prothymocytes and thymopoiesis, we used NOD-scid/γc(-/-) mice grafted with human umbilical cord blood CD34(+) hematopoietic progenitor cells (HPCs). Human prothymocytes developed in the murine bone marrow (BM) from multipotent CD34(++)CD38(lo)lineage(-) HPCs to CD34(++)CD7(+)CD2(-) pro-T1 cells that progressed in a Notch-dependent manner to CD34(+)CD7(++)CD2(+) pro-T2 cells, which migrated to the thymus. BM prothymocyte numbers peaked 1 mo after graft, dropped at mo 2, and persisted at low levels thereafter, with only a few CD34(+)CD7(lo) prothymocytes with limited T potential being detected by mo 5. As a consequence, thymopoiesis in this xenogeneic setting began by weeks 4-6, peaked at mo 3, and decreased thenceforth. Analyzing mice grafted at 2, 4 or 8, mo of age showed that in an "older" BM, prothymocyte differentiation was perturbed and resulted in CD34(+)CD7(lo) prothymocytes with limited T potential. Whereas the early drop in BM thymopoietic activity was related to a Notch-independent loss of T potential by CD34(++)CD38(lo)lineage(-) HPCs, the later age-dependent production decline of prothymocytes was linked to a more complex mix of cell-intrinsic and microenvironmental defects. Accordingly, and contrasting with what was observed with umbilical cord blood HPCs, CD34(+) HPCs from human adult BM displayed only marginal thymopoietic activity when grafted into young 2-mo-old NOD-scid/γc(-/-) mice. These data demonstrate that the developmental pattern of BM prothymocytes during human late fetal and early postnatal life can be reproduced in humanized mice, and they suggest that onset of human thymus involution relates to decreased colonization by prothymocytes.

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.

Direct comparison of Dll1- and Dll4-mediated Notch activation levels shows differential lymphomyeloid lineage commitment outcomes.

In the thymus, Notch signaling is essential for T lymphopoiesis, with Delta-like (Dll)4 uniquely involved in this process. However, using cocultures, either Dll4 or Dll1 were shown to support T lymphopoiesis. To address which Dll is more effective at inducing hematopoietic progenitor cells to give rise to T lineage cells in vitro, we generated OP9 cells expressing a series of incrementally discrete and equivalent levels of Dll1 or Dll4. In keeping with previous findings, OP9 cells expressing high levels of either Dll1 or Dll4 gave rise to T lineage cells with similar efficacy, and prevented the differentiation of B and myeloid-lineage cells. However, at limiting levels, Dll4 maintained its ability to inhibit B lineage choice and induce T lineage commitment and differentiation at lower levels than Dll1. This manifest property of Dll4 is evident despite lower levels of steady-state surface expression than Dll1 on OP9 cells. The heightened effectiveness of Dll4 over Dll1 also corresponded to the induction of Notch target genes, and inhibition of B and myeloid-specific transcription factors. Furthermore, we show that OP9 cells expressing levels of Dll4 equivalent to those present in thymic epithelial cells, as expected, gave rise to T lineage cells, but were also permissive for the differentiation of myeloid cells; whereas, still inhibiting B lymphopoiesis. Our findings show that Dll4 expressed at physiological levels on OP9 cells is functionally distinct from similarly expressed levels of Dll1, illustrating the unique properties of Dll4 in supporting the combined T lineage and specific myeloid-lineage outcomes that underpin its function within the thymus.

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.

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.

Inhibitor-2 induced M-phase arrest in Xenopus cycling egg extracts is dependent on MAPK activation.

The evolutionarily-conserved protein phosphatase 1 (PP1) plays a central role in dephosphorylation of phosphoproteins during the M phase of the cell cycle. We demonstrate here that the PP1 inhibitor inhibitor-2 protein (Inh-2) induces an M-phase arrest in Xenopus cycling egg extracts. Interestingly, the characteristics of this M-phase arrest are similar to those of mitogen-activated protein kinase (p42MAPK)-induced M-phase arrest. This prompted us to investigate whether Inh-2-induced M-phase arrest was dependent on activation of the p42MAPK pathway. We demonstrate here that MAPK activity is required for Inh-2-induced M-phase arrest, as inhibition of MAPK by PD98059 allowed cycling extracts to exit M phase, despite the presence of Inh-2. We next investigated whether Inh-2 phosphorylation by the MAPK pathway was required to induce an M-phase arrest. We discovered that while p90Rsk (a MAPK protein required for M-phase arrest) is able to phosphorylate Inh-2, this phosphorylation is not required for Inh-2 function. Overall, our results suggest a novel mechanism linking p42MAPK and PP1 pathways during M phase of the cell cycle.

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.