The molecular interactions provided by the thymic microenvironment that predicate T cell development remain obscure. Here, we show that a bone marrow stromal cell line ectopically expressing the Notch ligand Delta-like-1 loses its ability to support B cell lymphopoiesis, but acquires the capacity to induce the differentiation of hematopoietic progenitors into CD4 CD8 double- and single-positive T cells. Both gammadelta-TCR(+) and alphabeta-TCR(+) T cells are generated, and CD8(+) TCR(hi) cells produce gamma-interferon following CD3/TCR stimulation. These results establish that expression of Delta-like-1 on stromal cells provides key signals for the induction of T cell lineage commitment, stage-specific progenitor expansion, TCR gene rearrangement, and T cell differentiation in the absence of a thymus. Thus, it is likely that Delta-like-1/Notch interactions by the thymus underpin its unique ability to promote lineage commitment and differentiation of T cells.

The nature of early T lineage progenitors in the thymus or bone marrow remains controversial. Here we assess lineage capacity and proliferative potential among five distinct components of the earliest intrathymic stage (DN1, CD25(-)44(+)). All of these express one or more hemato-lymphoid lineage markers. All can produce T lineage cells, but only two of them display kinetics of differentiation, proliferative capacity, and other traits consistent with being canonical T progenitors. The latter also appeared limited to producing cells of the T or NK lineages, while B lineage potential derived mainly from the other, less typical T progenitors. In addition to precisely defining canonical early progenitors in the thymus, this work reconciles conflicting results from numerous groups by showing that multiple progenitors with a DN1 phenotype home to the thymus and make T cells, but possess different proliferative potentials and lineage capacities.

Embryonic stem cells (ESCs) have the potential to serve as a renewable source of transplantable tissue-specific stem cells. However, the molecular cues necessary to direct the differentiation of ESCs toward specific cell lineages remain obscure. Here we report the successful induction of ESC differentiation into mature functional T lymphocytes with a simple in vitro coculture system. The directed differentiation of ESCs into T cells required the engagement of Notch receptors by Delta-like 1 ligand (DL1) expressed on the OP9-DL1 stromal cell line. We found a normal program of T cell differentiation in ESC-OP9-DL1 cell cocultures. ESC-derived T cell progenitors effectively reconstituted the T cell compartment of immunodeficient mice, enabling an effective response to a viral infection. These findings provide a powerful tool for the molecular analysis of T cell development and open new avenues for the development of immunotherapeutic approaches using defined sources of stem cells.

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25− (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor–ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.

Relapse remains a leading cause of death after allogeneic hematopoietic cell transplantation (HCT) for patients with high-risk leukemias. The potentially beneficial donor T-cell-mediated graft-versus-leukemia (GVL) effect is often mitigated by concurrent graft versus host disease (GVHD). Providing T-cells that can selectively target Wilms’ Tumor Antigen 1 (WT1), a transcription factor over-expressed in leukemias that contributes to the malignant phenotype, represents a potential opportunity to promote anti-leukemic activity without inducing GVHD. HLA A*0201-restricted WT1-specific donor-derived CD8+ cytotoxic T-cell (CTL) clones were administered post-HCT to 11 relapsed or high-risk leukemia patients without any evidence of on-target toxicity. The last four treated patients received CTL clones generated with exposure to IL-21 as a means to prolong in vivo CTL survival, as IL-21 can limit terminal differentiation of antigen-specific T-cells generated in vitro. Transferred cells exhibited direct evidence of anti-leukemic activity in 2 patients: a transient response in one patient with advanced progressive disease and the induction of a prolonged remission in a patient with minimal residual disease (MRD). Additionally, three treated patients at high risk for relapse post-HCT survive without leukemia relapse, GVHD or additional anti-leukemic treatment. CTL generated in the presence of IL-21, which were transferred in these latter three patients and the patient with MRD, all remained detectable long-term and maintained/acquired in vivo phenotypic and functional characteristics associated with long-lived memory CD8+ T-cells. This study supports expanding efforts to immunologically target WT1, and provides insights into the requirements necessary to establish potent persistent T-cell responses in patients.

The first checkpoint during T cell development, known as β selection, requires the successful rearrangement of the TCR-β gene locus. Notch signaling has been implicated in various stages during T lymphopoiesis. However, it is unclear whether Notch receptor-ligand interactions are necessary during β selection. Here, we show that pre-TCR signaling concurrent with Notch receptor and Delta-like-1 ligand interactions are required for the survival, proliferation, and differentiation of mouse CD4−CD8− thymocytes to the CD4+CD8+ stage. Furthermore, we address the minimal signaling requirements underlying β selection and show a hierarchical positioning of key proximal signaling molecules. Collectively, our results demonstrate an essential role for Notch receptor-ligand interactions in enabling the autonomous signaling capacity of the pre-TCR complex.

Relapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of death in acute myeloid leukemia (AML) patients entering HCT with poor-risk features. 1-3 When HCT does produce prolonged relapse-free survival (RFS), it commonly reflects graft-versus-leukemia (GVL) effects mediated by donor T cells reactive with antigens on leukemic cells. 4 As graft T cells have not been selected for leukemia-specificity and frequently recognize proteins expressed by many normal host tissues, GVL is often accompanied by morbidity and mortality from graft-versus-host disease (GVHD). 5 Thus, AML relapse risk might be more effectively reduced with T cells expressing receptors (TCRs) that target selected AML antigens. 6 We therefore isolated a high

SUMMARY Pancreatic ductal adenocarcinomas (PDA) erect physical barriers to chemotherapy and induce multiple mechanisms of immune suppression, creating a sanctuary for unimpeded growth. We tested the ability of T cells engineered to express an affinity-enhanced T cell receptor (TCR) against a native antigen to overcome these barriers in a genetically engineered model of autochthonous PDA. Engineered T cells preferentially accumulate in PDA and induce tumor cell death and stromal remodeling. However, tumor-infiltrating T cells become progressively dysfunctional, a limitation successfully overcome by serial T cell infusions that resulted in a near-doubling of survival without overt toxicities. Similarly engineered human T cells lyse PDA cells in vitro, further supporting clinical advancement of this TCR-based strategy for the treatment of PDA.