Invariant Natural Killer T cell (iNK Tcell) have an innate immunity-like rapidity of response and the capacity to modulate effector functions of other cells. We show that the BTB-ZF transcriptional regulator, PLZF, is specifically expressed in iNKT cells. iNKT cells develop in the absence of PLZF, but lack many innate T cell features. PLZF deficient iNKT cells accumulate in the lymph nodes rather than in the liver and do not have an activated phenotype or express NK markers. PLZF deficient iNKT cells do not secrete high levels of IL-4 and IFNγ upon activation, however some cells produce either IL-4 or IFNγ, but not both. PLZF, therefore, is an iNKT cell specific transcription factor that is necessary for full functionality.Nearly all hematopoietic cells mature in the bone marrow. In contrast, multipotent progenitor T cells leave the bone marrow and home to the thymus, where signals from stromal cells are required for commitment to the T lineage 1 . Once directed into the T lineage, the cells undergo a rigorous selection process that eliminates more than 95% of the candidate T cells. Full maturation requires the expression of a T cell receptor (TCR) that binds self-peptide:self-MHC complexes with sufficient avidity. At some point during development, T cells are directed into one of several distinct T cell lineages such as CD4 single positive "helper" cells, CD8 single positive "killer" cells or CD4 + CD25 + regulatory cells. Commitment to these various lineages defines the specialized functions of the cell, which is critical since each cell type plays an essential and distinct role for host defense. The genes responsible for directing multipotent T cell progenitors into the various lineages are largely unknown 2 .Correspondence and requests for materials should be addressed to D.B.S (Email: santangd@mskcc.org). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAmong the various lineages of T cells, invariant Natural Killer T cells (iNKT cells) have several unique phenotypic traits such as the expression of receptors typically associated with Natural Killer cells (NK cells), the constitutive expression of activation markers and extremely restricted TCR diversity 3 . iNKT cells express an identical TCRα chain and most use a TCRβ chain that utilizes the Vβ8.2 gene segment. This TCR confers specificity to the non-MHC encoded self-molecule, CD1d, which binds and presents glycolipids rather than the typical peptide cargo presented by conventional MHC molecules.iNKT cells are also functionally distinct. Of particular interest is their ability to secrete large quantities of a variety of cytokines only minutes after activation via the TCR 3 . The rapid response of these cells, the conserved nature of the TCR and their indirect ability to modulate the function of many different cell types of the immune system has led to the appreciation that iNKT cells lay at a functional cusp between the innate and adaptive immune systems 4 . The broad range of cytokines released by iNKT cells results in th...

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Activation of ␤-catenin has been causatively linked to the etiology of colon cancer. Conditional stabilization of this molecule in pro-T cells promotes thymocyte development without the requirement for pre-TCR signaling. We show here that activated ␤-catenin stalls the developmental transition from the double-positive (DP) to the single-positive (SP) thymocyte stage and predisposes DP thymocytes to transformation. ␤-Catenin-induced thymic lymphomas have a leukemic arrest at the early DP stage. Lymphomagenesis requires Rag activity, which peaks at this developmental stage, as well as additional secondary genetic events. A consistent secondary event is the transcriptional up-regulation of c-Myc, whose activity is required for transformation because its conditional ablation abrogates lymphomagenesis. In contrast, the expression of Notch receptors as well as targets is reduced in DP thymocytes with stabilized ␤-catenin and remains low in the lymphomas, indicating that Notch activation is not required or selected for in ␤-catenininduced lymphomas. Thus, ␤-catenin activation may provide a mechanism for the induction of T-cell-acute lymphoblastic leukemia ( IntroductionThe canonical ␤-catenin/TCF-LEF signaling is stimulated by wnts, a family of secreted cysteine-rich glycoproteins that bind to cell surface Frizzled receptors. In unstimulated cells, newly synthesized ␤-catenin is captured by a large cytoplasmic complex consisting of the tumor suppressor adenomatous polyposis coli (APC), the constitutively active kinase glycogen synthase kinase 3␤ (GSK-3␤), and Axin. In this complex, ␤-catenin is phosphorylated by GSK-3␤ at 4 N-terminal serine and threonine residues and targeted for degradation. [1][2][3][4] Activation of the Wnt/␤-catenin cascade results in inhibition of the constitutive activity of GSK-3␤ 5 by the cytoplasmic protein Dishevelled (Dvl). [6][7][8][9] Consequently, ␤-catenin is no longer phosphorylated and can accumulate in the cytoplasm and nucleus. Once in the nucleus, ␤-catenin binds to members of the TCF/LEF family of transcription factors, the most downstream components of the Wnt-signaling pathway. For reviews, see van de Wetering 10 and Staal and Clevers. 11 The Wnt/␤-catenin signaling cascade has been implicated in multiple stages of hematopoietic development. It was proposed that Wnt signaling controls the self-renewal of hematopoietic stem cells (HSCs). 12 More recently it was shown that deregulated activation of this pathway enforced cell cycle entry in HSCs leading to the exhaustion of the long-term stem cell pool and a multilineage developmental block. 13,14 In the thymus, loss-and gain-of-function studies have indicated that at least 2 stages of thymopoiesis require Wnt/␤-catenin signaling. Thymocytes express the TCF-1 and LEF-1 effectors of the canonical Wnt/␤-catenin signaling pathway. Ablation of TCF-1 activity affects all proliferating stages of thymocyte development including the CD44 ϩ CD25 ϩ DN2 and the pre-TCR-dependent CD44 Ϫ CD25 Ϫ DN4 and CD8 ϩ TCR␤ Ϫ immature single-positive (IS...

Constitutive and cell-autonomous signals emanating from the pre-T-cell receptor (pre-TCR) promote proliferation, survival and differentiation of immature thymocytes. We show here that induction of pre-TCR signaling resulted in rapid elevation of c-Myc protein levels. Cremediated thymocyte-specific ablation of c-Myc in CD25 ؉ CD44 ؊ thymocytes reduced proliferation and cell growth at the pre-TCR checkpoint, resulting in thymic hypocellularity and a severe reduction in CD4 ؉ CD8 ؉ thymocytes. In contrast, c-Myc deficiency did not inhibit pre-TCRmediated differentiation or survival. IntroductionImmature T cells progress through a number of well-defined developmental stages in the thymus. Commitment of bone marrowderived T-cell progenitors [1][2][3][4][5][6] to the T-cell lineage requires stimulation of Notch signaling, 2,3,7,8 and results in the up-regulation of CD25 (double-negative [DN] 2 subset). Survival and proliferation of thymocytes at this stage is supported by cytokines such as interleukin-7 (IL-7) and stem cell factor (SCF). The subsequent CD44 Ϫ CD25 ϩ (DN3) stage is marked by the rearrangement of the T-cell receptor (TCR) ␤, ␥, and ␦ loci. Productive TCR␤ gene rearrangements and synthesis of TCR␤ chains lead to the surface assembly of a functional pre-TCR, comprising the TCR␤ as well as the invariant pT␣ chain and CD3 subunits. Signals emanating from the pre-TCR promote survival and proliferation of immature thymocytes as well as their differentiation to the CD4 ϩ CD8 ϩ double-positive (DP) stage, effectively instructing immature thymocytes to the ␣␤ T-cell lineage. 9 Despite recent progress in understanding pre-TCR signaling many questions remain unanswered. It is currently known that the pre-TCR is constitutively localized in plasma membrane glycoprotein-enriched microdomains (GEMs) from where it signals in a cell autonomous manner. 10,11 Proximal events of pre-TCR signaling include the phosphorylation of Lck and Zap70. Assembly of the pre-TCR and activation of this pathway is accompanied by a biphasic calcium mobilization, which appears to be regulated by cytoplasmic IP 3 and plasma membrane store-operated calcium channels (SOCs), resulting in nuclear factor (NF) of activated T cells (NFAT) and NFB activation. 12 These findings, however, are not sufficient to explain the multiplicity of events following the onset of pre-TCR signaling. Recent evidence both from loss-and gain-of-function approaches indicates that several other genes and signaling pathways are involved in the pre-TCR checkpoint. These include kinases such c-Fyn, 13 Csk, 14 and Pim1, 15 and adaptor proteins such as LAT and SLP-76. 16 Several transcription factors were also shown to be essential at this developmental stage, such as Ikaros, 17 E2A, 18 Runx2,21 Multiple findings indicate the involvement of a number of signaling cascades, including Notch, 22,23 Wnt, 24 and Hedgehog. 25 The interactions between these pathways, however, the signals that mediate their activation and their orchestration with respect to pre-TCR signaling are cur...