Mahmood Mohtashami

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.

Three-dimensional architecture of the thymus is required to maintain delta-like expression necessary for inducing T cell development.

The three-dimensional microarchitecture of the thymus plays a unique role in directing T cell lineage commitment and development. This is supported by the fact that, in contrast to fetal thymic organ cultures, thymic stromal cell monolayer cultures (TSMC) fail to support T lymphopoiesis. Nevertheless, OP9-DL1 cell monolayer cultures induce T lineage commitment and differentiation. Thus, the inability of TSMC to support T lymphopoiesis may be due to a loss of Notch ligand expression and/or function during culture. In this study, we report that, in contrast to fetal thymic organ cultures, TSMC fail to maintain expression of the Notch ligands, Delta-like (Dll) 1 and Dll4, and concomitantly lose the ability to support T lymphopoiesis. Importantly, ectopic re-expression of Dll1 or Dll4 is sufficient to restore the ability of TSMC to support T lymphopoiesis. These findings demonstrate that maintenance of endogenous Dll1 or Dll4 expression by thymic stromal cells is required for the commitment and differentiation of T cells in the absence of a three-dimensional microenvironment.

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.

Analysis of the mutant Drosophila N‐ethylmaleimide sensitive fusion‐1 protein in comatose reveals molecular correlates of the behavioural paralysis

NEM-sensitive fusion protein (NSF) is an ATPase required for many intracellular membrane trafficking steps. Recent studies have suggested that NSF alters the conformation of the SNAP receptors (SNAREs) to permit their interaction, or to uncouple them after they interact. Most organisms have a single NSF gene product but Drosophila express two highly related isoforms, dNSF-1 and dNSF-2. dNSF-1 is encoded by the gene comatose (comt), first identified as the locus of a temperature-sensitive paralytic mutation. Here we show that dNSF-1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation revealed that dNSF-1 was enriched in a vesicle fraction along with the synaptic vesicle protein synaptotagmin. comt flies maintained at the non-permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)-resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF-1 from cytosol and membrane fractions into a Triton X-100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlated with the irreversibility of this translocation. Interestingly, while dNSF-1 also translocates in comt(TP7) larvae, there is no associated neurophysiological phenotype at the neuromuscular junction (nmj) or accumulation of SDS-resistant complexes in the CNS. Together, these results suggest that dNSF-1 is required for adult neuronal function, but that in the larval nmj function may be maintained by other isoforms.

Analysis of the mutant Drosophila N-ethylmaleimide sensitive fusion-1 protein in comatose reveals molecular correlates of the behavioural paralysis: dNSF-1 functions in the adult nervous system

NEM‐sensitive fusion protein (NSF) is an ATPase required for many intracellular membrane trafficking steps. Recent studies have suggested that NSF alters the conformation of the SNAP receptors (SNAREs) to permit their interaction, or to uncouple them after they interact. Most organisms have a single NSF gene product but Drosophila express two highly related isoforms, dNSF‐1 and dNSF‐2. dNSF‐1 is encoded by the gene comatose (comt), first identified as the locus of a temperature‐sensitive paralytic mutation. Here we show that dNSF‐1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation revealed that dNSF‐1 was enriched in a vesicle fraction along with the synaptic vesicle protein synaptotagmin. comt flies maintained at the non‐permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)‐resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF‐1 from cytosol and membrane fractions into a Triton X‐100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlated with the irreversibility of this translocation. Interestingly, while dNSF‐1 also translocates in comtTP7 larvae, there is no associated neurophysiological phenotype at the neuromuscular junction (nmj) or accumulation of SDS‐resistant complexes in the CNS. Together, these results suggest that dNSF‐1 is required for adult neuronal function, but that in the larval nmj function may be maintained by other isoforms.

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 TCRαβ(+), 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.

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.

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.

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.

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

Significance Expression of a productively rearranged T-cell receptor (TCR)-β chain induces a program of αβ T-lineage differentiation, whereas thymocytes that productively rearranged TCR-γ and TCR-δ typically give rise to γδ-lineage T cells. However, given that all three TCR gene loci simultaneously undergo gene rearrangements, the possibility exists that a developing thymocyte may express a γδ-TCR together with a TCR-β or pre-TCR complex, and it is not clear to what this outcome would give rise in terms of T-lineage differentiation. Our findings point to a striking conclusion, in that rather than transmitting signals that exclusively promote αβ-lineage commitment/differentiation, the pre-TCR can function in concert with the γδ-TCR to promote γδ commitment/differentiation, a result that supports a signal strength model of αβ/γδ-lineage choice. Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, 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-β 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.