No abstract

The vertebrate thymus provides an inductive environment for T-cell development. Within the thymus, Notch signals are indispensable for imposing the T-cell fate on multipotential hematopoietic progenitors, but the downstream effectors that impart T-lineage specification and commitment are not well understood. Here we show that transcription factor, T-cell factor 1 (TCF-1), is a critical regulator in T-cell specification. TCF-1 is highly expressed in the earliest thymic progenitors, and its expression is upregulated by Notch signals. Most importantly, when TCF-1 is forcibly expressed in BM progenitors, it drives the development of T-lineage cells in the absence of T-inductive Notch1 signals. Further characterization of these TCF-1-induced cells revealed expression of many T-lineage genes, including T-cell specific transcription factors Gata3, Bcl11b, and components of the T-cell receptor. Our data suggest a model where Notch signals induce TCF-1, and TCF-1 in turn imprints the T-cell fate by upregulating expression of T-cell essential genes.

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies.

Key Points• Targeting of CD123 via CAR-engineered T cells results in rejection of human AML and myeloablation in mouse models.Many patients with acute myeloid leukemia (AML) are incurable with chemotherapy and may benefit from novel approaches. One such approach involves the transfer of T cells engineered to express chimeric antigen receptors (CARs) for a specific cell-surface antigen. This strategy depends upon preferential expression of the target on tumor cells. To date, the lack of AML-specific surface markers has impeded development of such CARbased approaches. CD123, the transmembrane a chain of the interleukin-3 receptor, is expressed in the majority of AML cells but is also expressed in many normal hematopoietic cells. Here, we show that CD123 is a good target for AML-directed CAR therapy, because its expression increases over time in vivo even in initially CD123 dim populations, and that human CD123-redirected T cells (CART123) eradicate primary AML in immunodeficient mice. CART123 also eradicated normal human myelopoiesis, a surprising finding because anti-CD123 antibody-based strategies have been reportedly well tolerated. Because AML is likely preceded by clonal evolution in "preleukemic" hematopoietic stem cells, our observations support CART123 as a viable AML therapy, suggest that CART123-based myeloablation may be used as a novel conditioning regimen for hematopoietic cell transplantation, and raise concerns for the use of CART123 without such a rescue strategy. (Blood. 2014;123(15):2343-2354

During wound healing in adult mouse skin, hair follicles and then adipocytes regenerate. Adipocytes regenerate from myofibroblasts, a specialized contractile wound fibroblast. Here we study wound fibroblast diversity using single-cell RNA-sequencing. On analysis, wound fibroblasts group into twelve clusters. Pseudotime and RNA velocity analyses reveal that some clusters likely represent consecutive differentiation states toward a contractile phenotype, while others appear to represent distinct fibroblast lineages. One subset of fibroblasts expresses hematopoietic markers, suggesting their myeloid origin. We validate this finding using single-cell western blot and single-cell RNA-sequencing on genetically labeled myofibroblasts. Using bone marrow transplantation and Cre recombinase-based lineage tracing experiments, we rule out cell fusion events and confirm that hematopoietic lineage cells give rise to a subset of myofibroblasts and rare regenerated adipocytes. In conclusion, our study reveals that wounding induces a high degree of heterogeneity among fibroblasts and recruits highly plastic myeloid cells that contribute to adipocyte regeneration.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19- leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.

Notch ligands and receptors have been implicated in helper T cell (Th cell) differentiation. Whether Notch signals are involved in differentiation of T helper type 1 (Th1) cells, Th2 cells, or both, however, remains unresolved. To clarify the role of Notch in Th cell differentiation, we generated mice that conditionally inactivate Notch signaling in mature T cells. Mice that lack Notch signaling in CD4+ T cells fail to develop a protective Th2 cell response against the gastrointestinal helminth Trichuris muris. In contrast, they exhibit effective Th1 cell responses and are able to control Leishmania major infection. These data demonstrate that Notch signaling is a regulator of type 2 immunity.

Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.