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Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK [alpha] and [zeta] synergistically promote T cell maturation in the thymus. Absence of both DGK[alpha] and [zeta] (DGK[[alpha].sup.-/-][[zeta].sup.-/-]) results in a severe decrease in the number of [CD4.sup.+][CD8.sup.-] and [CD4.sup.-][CD8.sup.+] single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DG[[alpha].sup.-/-][[zeta].sup.-/-] mice. The developmental blockage in DGK[[alpha].sup.-/-][[zeta].sup.-/-] mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection. phosphatidic acid | signaling | tumorigenesis

T cell receptor (TCR) [Alpha] and [Delta] gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3[prime] of TCR [Delta] gene segments and 5[prime] of TCR [Alpha] joining gene segments within this locus. BEAD-1 blocked the ability of the TCR [Delta] enhancer ([E.sub.[Delta]]) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR [Alpha]/[Delta] locus into distinct regulatory domains controlled by [E.sub.[Delta]] and the TCR [Alpha] enhancer, and that it prevents [E.sub.[Delta]] from opening the chromatin of the TCR [Alpha] joining gene segments for VDJ recombination at an early stage of T cell development.

Previous studies have identified nuclear matrix attachment regions (MARs) that are closely associated with transcriptional enhancers in the IgH, Ig[Kappa], and T cell receptor (TCR) [Beta] loci, but have yielded conflicting information regarding their functional significance. In this report, a combination of in vitro and in situ mapping approaches was used to localize three MARs associated with the human TCR [Delta] gene. Two of these are located within the J [Delta] 3-C [Delta] intron, flanking the core TCR [Delta] enhancer (E [Delta]) both 5' and 3' in a fashion reminiscent of the Ig heavy chain intronic enhancer-associated MARs. The third is located about 20 kb upstream, tightly linked to D [Delta] 1 and D [Delta] 2. We have previously used a transgenic minilocus V(D)J recombination reporter to establish that E [Delta] functions as a developmental regulator of V(D)J recombination, and that it does so by modulating substrate accessibility to the V(D)J recombinase. We show here that the E [Delta]-associated MARs function synergistically with the core E [Delta] to promote V(D)J recombination in this system, as they are required for enhancer-dependent transgene rearrangement in single-copy transgene integrants.

Investigates the function of the blocking element alpha/delta 1 (BEAD-1) within the human T cell receptor (TCR) alpha/sigma segments. Enhancer-activity and enhancer-blocking in soft-agar colony forming assay adapted to the human T cell leukemia; Identification of enhancer-blocking activity; Cotransfection and cloning.

The mechanisms that control invariant natural killer T (iNKT)-cell development and function are still poorly understood. The mechanistic or mammalian target of rapamycin (mTOR) integrates various environmental signals/cues to regulate cell growth, proliferation, metabolism, and survival. We report here that ablation of mTOR complex 1 (mTORC1) signaling by conditionally deleting Raptor causes severe defects in iNKT-cell development at early stages, leading to drastic reductions in iNKT-cell numbers in the thymus and periphery. In addition, loss of Raptor impairs iNKT-cell proliferation and production of cytokines upon α-galactosylceramide stimulation in vitro and in vivo, and inhibits liver inflammation in an iNKT cell-mediated hepatitis model. Furthermore, Raptor deficiency and rapamycin treatment lead to aberrant intracellular localization and functional impairment of promyelocytic leukemia zinc-finger, a transcription factor critical for iNKT-cell development and effector programs. Our findings define an essential role of mTORC1 to direct iNKT-cell lineage development and effector function.

T-cell anergy is a state of T cells that is hyporesponsive to stimulation via the T-cell receptor and costimulatory molecules and is thought to be important for self-tolerance. How T-cell anergy is regulated is still poorly understood. We report here that tuberous sclerosis (TSC)1 is critical for T-cell anergy. Deficiency of TSC1 resulted in enhanced T-cell proliferation and cytokine production in the absence of cluster of differentiation (CD)28-mediated costimulation, accompanied by enhanced T-cell metabolism. Resistance of TSC1-deficient T cells to anergy is correlated with increased signaling through the mammalian target of rapamycin complex (mTORC)1 and can be reverted by treatment of these cells with mTORC1 inhibitor rapamycin. Expression of the inducible costimulator (ICOS) is increased in TSC1-deficient T cells, which can be inhibited by rapamycin. Simultaneous blockade of both CD28 and ICOS costimulation partially restored sensitivity of TSC1-deficient T cells to anergy induction. Together, our data indicate that TSC1 is crucial for T-cell anergy by inhibiting mTORC1 signaling through both ICOS-dependent and -independent mechanisms.

Analysis of the molecular etiologies of SCID has led to important insights into the control of immune cell development. Most cases of SCID result from either X-linked or autosomal recessive inheritance of mutations in a known causative gene. However, in some cases, the molecular etiology remains unclear. To identify the cause of SCID in a patient known to lack the protein-tyrosine phosphatase CD45, we used SNP arrays and whole-exome sequencing. The patient's mother was heterozygous for an inactivating mutation in CD45 but the paternal alleles exhibited no detectable mutations. The patient exhibited a single CD45 mutation identical to the maternal allele. Patient SNP array analysis revealed no change in copy number but loss of heterozygosity for the entire length of chromosome 1 (Chr1), indicating that disease was caused by uniparental disomy (UPD) with isodisomy of the entire maternal Chr1 bearing the mutant CD45 allele. Nonlymphoid blood cells and other mesoderm- and ectoderm-derived tissues retained UPD of the entire maternal Chr1 in this patient, who had undergone successful bone marrow transplantation. Exome sequencing revealed mutations in seven additional genes bearing nonsynonymous SNPs predicted to have deleterious effects. These findings are unique in representing a reported case of SCID caused by UPD and suggest UPD should be considered in SCID and other recessive disorders, especially when the patient appears homozygous for an abnormal gene found in only one parent. Evaluation for alterations in other genes affected by UPD should also be considered in such cases.