Christopher C. Kloss

CD19 CAR-targeted T cells induce long-term remission and B Cell Aplasia in an immunocompetent mouse model of B cell acute lymphoblastic leukemia.

Although many adults with B cell acute lymphoblastic leukemia (B-ALL) are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs) and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.

Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition

UNLABELLED Several studies have demonstrated that hematopoietic cells originate from endotheliumin early development; however, the phenotypic progression of progenitor cells during human embryonic hemogenesis is not well described. Here, we define the developmental hierarchy among intermediate populations of hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells (hESCs). We genetically modified hESCs to specifically demarcate acquisition of vascular (VE-cadherin) and hematopoietic (CD41a) cell fate and used this dual-reporting transgenic hESC line to observe endothelial to hematopoietic transition by real-time confocal microscopy. Live imaging and clonal analyses revealed a temporal bias in commitment of HPCs that recapitulates discrete waves of lineage differentiation noted during mammalian hemogenesis. Specifically, HPCs isolated at later time points showed reduced capacity to form erythroid/ megakaryocytic cells and exhibited a tendency toward myeloid fate that was enabled by expression of the Notch ligand Dll4 on hESC-derived vascular feeder cells. These data provide a framework for defining HPC lineage potential, elucidate a molecular contribution from the vascular niche in promoting hematopoietic lineage progression, and distinguish unique subpopulations of hemogenic endothelium during hESC differentiation. KEY POINTS Live imaging of endothelial to hematopoietic conversion identifies distinct subpopulations of hESC-derived hemogenic endothelium. Expression of the Notch ligand DII4 on vascular ECs drives induction of myeloid fate from hESC-derived hematopoietic progenitors.

Novel approaches to enhance the specificity and safety of engineered T cells.

AbstractT-cell therapies using engineered T cells show great promise for cancer immunotherapy, as illustrated by the CD19 paradigm. Much of the excitement about this approach, and second-generation CARs in particular, is due to the dramatic clinical results recently reported by a few centers, especially in acute lymphoblastic leukemia, and the applicability of this approach, in principle, to a wide range of cancers. Extending the use of CAR therapies to cancers other than B-cell malignancies will require selective tumor targeting with minimal or acceptable “on-target, off-tumor” effects. The identification of new CAR target antigens is thus one of the next big challenges to address. Recognizing the paucity of currently available tumor-specific targets, we have developed broadly applicable approaches to enhance the tumor selectivity and safety of engineered T cells. Here, we review 2 promising concepts. One is to improve tumor targeting based on combinatorial antigen recognition. The other uses receptors that provide antigen-specific inhibition, which we named iCARs, to divert T cells from the normal tissues one wants to protect.

Activation of the vascular niche supports leukemic progression and resistance to chemotherapy.

Understanding the intricate cellular components of the bone marrow microenvironment can lead to the discovery of novel extrinsic factors that are responsible for the initiation and progression of leukemic disease. We have shown that endothelial cells (ECs) provide a fertile niche that allows for the propagation of primitive and aggressive leukemic clones. Activation of the ECs by vascular endothelial growth factor (VEGF)-A provides cues that enable leukemic cells to proliferate at higher rates and also increases the adhesion of leukemia to ECs. Vascular endothelial growth factor A-activated ECs decrease the efficacy of chemotherapeutic agents to target leukemic cells. Inhibiting VEGF-dependent activation of ECs by blocking their signaling through VEGF receptor 2 increases the susceptibility of leukemic cells to chemotherapy. Therefore, the development of drugs that target the activation state of the vascular niche could prove to be an effective adjuvant therapy in combination with chemotherapeutic agents.

Lentiviral Transduction and Clonal Selection of hESCs with Endothelial‐Specific Transgenic Reporters

Generation of vascular endothelial cells (EC) from human embryonic stem cells (hESC) is a vital component of cell-based strategies for treatment of cardiovascular disease. Before hESC-derived ECs can be administered in therapeutic modalities, however, chemically defined culture conditions must be developed that reproducibly and robustly induce vascular differentiation. One approach to screening for culture conditions that support differentiation of hESCs to any cell type is their genetic modification with exogenous DNA sequence comprising a tissue-specific gene promoter driving reporters such as fluorescent protein or antibiotic drug resistance. The protocols herein provide instructions for the generation of clonal hESC lines containing a reporter transgene that is specifically expressed in vascular endothelial derivatives. Additionally, they demonstrate the methodology employed to assess vascular differentiation from clonal lines. Together, these protocols provide a solid foundation for study of vascular differentiation, and may also be applied, in principle, to studies of other specialized cell types derived from hESCs.

Production of BMP4 by endothelial cells is crucial for endogenous thymic regeneration

BMP4 produced by endothelial cells promotes thymic regeneration after acute damage by activating FOXN1 and its downstream targets. Regeneration circuits in the thymus Chemotherapy and radiation treatments in cancer patients damage a number of tissues and organs, including the thymus. Prolonged thymic damage can lead to T cell deficiency and increase susceptibility to the development of opportunistic infections and malignancies. Here, Wertheimer et al. have examined thymic regeneration in mice after sublethal total body radiation and document a critical role for bone morphogenetic protein 4 (BMP4) signaling in thymic regeneration. They found endothelial cells to be a critical source of BMP4 and propose that BMP4 produced by endothelial cells induces the expression of the transcription factor FOXN1 in thymic epithelial cells to promote thymic regeneration. These studies should eventually facilitate the development of treatment regimens to promote immune competence in patients undergoing chemotherapy and radiation treatments. The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4). ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as Dll4, a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.