NKT cell subsets can be divided based on CD4 and NK1.1 expression and tissue of origin, but the developmental and functional relationships between the different subsets still are poorly understood. A comprehensive study of 19 cytokines across different NKT cell subsets revealed that no two NKT subpopulations exhibited the same cytokine profile, and, remarkably, the amounts of each cytokine produced varied by up to 100-fold or more among subsets. This study also revealed the existence of a population of CD4 ؊ NK1.1 ؊ NKT cells that produce high levels of the proinflammatory cytokine IL-17 within 2-3 h of activation. On intrathymic transfer these cells develop into mature CD4 ؊ NK1.1 ؉ but not into CD4 ؉ NK1.1 ؉ NKT cells, indicating that CD4 ؊ NK1.1 ؊ NKT cells include an IL-17-producing subpopulation, and also mark the elusive branch point for CD4 ؉ and CD4 ؊ NKT cell sublineages.cytokines ͉ CD1d ͉ thymus ͉ T cell

SUMMARY The role of the microenvironment in T cell acute lymphoblastic leukemia (T-ALL), or any acute leukemia, is poorly understood. Here we demonstrate that T-ALL cells are in direct, stable contact with CXCL12-producing bone marrow stroma. Cxcl12 deletion from vascular endothelial, but not perivascular, cells impeded tumor growth, suggesting a vascular niche for T-ALL. Moreover, genetic targeting of CXCR4 in murine T-ALL after disease onset led to rapid, sustained disease remission, and CXCR4 antagonism suppressed human T-ALL in primary xenografts. Loss of CXCR4 targeted key T-ALL regulators, including the MYC pathway, and decreased leukemia initiating cell activity in vivo. Our data identify a T-ALL niche, and suggest targeting CXCL12/CXCR4 signaling as a powerful therapeutic approach for T-ALL.

Summary Plasma sphingosine-1-phosphate (S1P) regulates vascular permeability, and plasma and lymph S1P guide lymphocyte egress from lymphoid organs. S1P is made intracellularly, and little is known about how S1P is delivered into circulatory fluids. Here we find that mice without the major facilitator superfamily transporter Spns2 have a profound reduction in lymph S1P, but only a minor decrease in plasma S1P. Spns2-deficient mice have a redistribution of lymphocytes from the spleen to lymph nodes and a loss of circulating lymphocytes, consistent with normal egress from the spleen directed by plasma S1P and blocked egress from lymph nodes directed by lymph S1P. Spns2 is needed in endothelial cells to supply lymph S1P and support lymphocyte circulation. As the first differential requirement for lymph and blood S1P to our knowledge, Spns2 may be an attractive target for immune suppressive drugs.

The maize transposable element Activator (Ac) has been exploited as an insertional mutagen to disrupt, clone, and characterize genes in a number of plant species. To develop an Ac-based mutagenesis platform for maize, a large-scale mutagenesis was conducted targeting the pink scutellum1 locus. We selected 1092 Ac transposition events from a closely linked donor Ac, resulting in the recovery of 17 novel ps1 alleles. Multiple phenotypic classes were identified corresponding to Ac insertions in the 59-UTR and coding region of the predicted Ps1 gene. To generate a stable allelic series, we employed genetic screens and identified 83 germinally heritable ps1 excision alleles. Molecular characterization of these excision alleles revealed a position-dependent bias in excision allele frequencies and the predominance of 7-and 8-bp footprint products. In total, 19 unique ps1 excision alleles were generated in this study, including several that resulted in weak mutant phenotypes. The analysis of footprint alleles suggests a model of Ac excision in maize that is consistent with recent in vitro studies of hAT element excision. Importantly, the genetic and molecular methods developed in this study can be extended to generate novel allelic variation at any Ac-tagged gene in the genome.

Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator β-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. β-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and β-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of β-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of β-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/β-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of β-catenin activity. Our analyses in ICG001-treated mice confirmed a role for β-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and β-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of β-catenin to NKT cell functions.