Programmed death-1 (PD-1):PD-ligand 1 interactions inhibit TCR-mediated positive selection of thymocytes

Positive selection during thymocyte development is driven by the affinity and avidity of the TCR for MHC-peptide complexes expressed in the thymus. In this study, we show that programmed death-1 (PD-1), a member of the B7/CD28 family of costimulatory receptors, inhibits TCR-mediated positive selection through PD-1 ligand 1 (PD-L1):PD-1 interactions. Transgenic mice that constitutively overexpress PD-1 on CD4+CD8+ thymocytes display defects in positive selection in vivo. Using an in vitro model system, we find that PD-1 is up-regulated following TCR engagement on CD4+CD8+ murine thymocytes. Coligation of TCR and PD-1 on CD4+CD8+ thymocytes with a novel PD-1 agonistic mAb inhibits the activation of ERK and up-regulation of bcl-2, both of which are downstream mediators essential for positive selection. Inhibitory signals through PD-1 can overcome the ability of positive costimulators, such as CD2 and CD28, to facilitate positive selection. Finally, defects in positive selection that result from PD-1 overexpression in thymocytes resolve upon elimination of PD-L1, but not PD-1 ligand 2, expression. PD-L1-deficient mice have increased numbers of CD4+CD8+ and CD4+ thymocytes, indicating that PD-L1 is involved in normal thymic selection. These data demonstrate that PD-1:PD-L1 interactions are critical to positive selection and play a role in shaping the T cell repertoire.     

NABP1, a novel RORgamma-regulated gene encoding a single-stranded nucleic-acid-binding protein

RORgamma2 (retinoid-related orphan receptor gamma2) plays a critical role in the regulation of thymopoiesis. Microarray analysis was performed in order to uncover differences in gene expression between thymocytes of wild-type and RORgamma-/- mice. This analysis identified a novel gene encoding a 22 kDa protein, referred to as NABP1 (nucleic-acid-binding protein 1). This subsequently led to the identification of an additional protein, closely related to NABP1, designated NABP2. Both proteins contain an OB (oligonucleotide/oligosaccharide binding) motif at their N-terminus. This motif is highly conserved between the two proteins. NABP1 is highly expressed in the thymus of wild-type mice and is greatly suppressed in RORgamma-/- mice. During thymopoiesis, NABP1 mRNA expression is restricted to CD4+CD8+ thymocytes, an expression pattern similar to that observed for RORgamma2. These observations appear to suggest that NABP1 expression is regulated either directly or indirectly by RORgamma2. Confocal microscopic analysis showed that the NABP1 protein localizes to the nucleus. Analysis of nuclear proteins by size-exclusion chromatography indicated that NABP1 is part of a high molecular-mass protein complex. Since the OB-fold is frequently involved in the recognition of nucleic acids, the interaction of NABP1 with various nucleic acids was examined. Our results demonstrate that NABP1 binds single-stranded nucleic acids, but not double-stranded DNA, suggesting that it functions as a single-stranded nucleic acid binding protein.     

RasGRP1 transmits prodifferentiation TCR signaling that is crucial for CD4 T cell development

TCR signaling plays a governing role in both the survival and differentiation of bipotent double-positive thymocytes into the CD4(+) and CD8(+) single-positive T cell lineages. A central mediator of this developmental program is the small GTPase Ras, emitting cytoplasmic signals through downstream MAPK pathways and eventually affecting gene expression. TCR signal transduction orchestrates the activation of Ras by integrating at least two Ras-guanyl nucleotide exchange factors, RasGRP1 and Sos. In this study, we have characterized the relationship between RasGRP1 function and its potential roles in promoting ERK activity, cell survival, maturation, and lineage commitment. Investigations on RasGRP1(-/-) mice expressing a transgenic (Tg) MHC class II-restricted TCR revealed that the development of CD4 T cells expressing this Tg TCR is completely dependent on RasGRP1. Unexpectedly, a small number of functional CD8 single-positive thymocytes expressing the Tg MHC class II-restricted TCR exists in mutant mice. In addition, RasGRP1(-/-) double-positive thymocytes exhibit marked deficits in TCR-stimulated up-regulation of the positive selection marker CD69 and the antiapoptotic protein Bcl-2, whereas CD5 induction is unaffected. To evaluate the role of RasGRP1 in providing cellular survival signaling, we enforced Bcl-2 expression in RasGRP1(-/-) thymocytes. These studies demonstrate that RasGRP1 function cannot be fully complemented by Tg Bcl-2 expression. Therefore, we propose that RasGRP1 transmits differentiation signaling critically required for CD4 T cell development.     

Elevated Mcl-1 inhibits thymocyte apoptosis and alters thymic selection

T cells developing in the thymus undergo rigorous positive and negative selection to ensure that those exported to peripheral lymphoid organs bear T-cell receptors (TCRs) capable of reacting with foreign antigens but tolerant of self. At each checkpoint, whether a thymocyte survives or dies is determined by antiapoptotic and proapoptotic Bcl-2 family members. We used Mcl-1 transgenic (tg) mice to investigate the impact of elevated expression of antiapoptotic Mcl-1 on thymocyte apoptosis and selection, making a side-by-side comparison with thymocytes from BCL-2tg mice. Mcl-1 was as effective as Bcl-2 at protecting thymocytes against spontaneous cell death, diverse cytotoxic insults and TCR–CD3 stimulation-driven apoptosis. In three different TCR tg models, Mcl-1 markedly enhanced positive selection of thymocytes, as did Bcl-2. In H-Y TCR tg mice, elevated Mcl-1 and Bcl-2 were equally effective at inhibiting deletion of autoreactive thymocytes. However, in the OT-1tg model where deletion is mediated by a peripheral antigen whose expression is regulated by Aire, Mcl-1 was less effective than Bcl-2. Thus, the capacity of Mcl-1 overexpression to inhibit apoptosis triggered by TCR stimulation apparently depends on the thymocyte subset subject to deletion, presumably due to differences in the profiles of proapoptotic Bcl-2 family members mediating the deletion.     

Tid1 is required for T cell transition from double-negative 3 to double-positive stages

Tid1, a DnaJ cochaperone protein, is the mammalian homologue of the Drosophila tumor suppressor Tid56 whose antitumor function is most likely mediated through its capacity to regulate cell differentiation in imaginal discs. We suspected that the mammalian counterpart, tid1, may also be involved in regulating cell differentiation. To investigate this, we exploited the system of T cell development to examine whether tid1 plays a role in this well-defined process. Mice with tid1 specifically deleted in T cells developed thymic atrophy, with dramatic reduction of double-positive and single-positive thymocytes in the tid1(-/-) thymus. Although the subpopulations of tid1(-/-) double-negative (DN) 1-3 thymocytes were normal, the subpopulation of DN4 thymocytes was measurably smaller because of reduced proliferation and significant cell death. Immature tid1(-/-) thymocytes show normal VDJ beta-chain rearrangement and pre-TCR and CD3 expression in both DN3 and DN4 thymocytes, but in DN4 thymocytes, there was significantly reduced expression of the antiapoptotic bcl-2 gene. Restoring the expression level of Bcl-2 protein in tid1(-/-) thymus by introduction of a transgenic human bcl-2 gene resulted in reversal of the developmental defects in tid1(-/-) thymus. Together, these results demonstrate that tid1 is critical in early thymocyte development, especially during transition from the DN3 to double-positive stages, possibly through its regulation of bcl-2 expression, which provides survival signals.     

IAN family critically regulates survival and development of T lymphocytes

The IAN (immune-associated nucleotide-binding protein) family is a family of functionally uncharacterized GTP-binding proteins expressed in vertebrate immune cells and in plant cells during antibacterial responses. Here we show that all eight IAN family genes encoded in a single cluster of mouse genome are predominantly expressed in lymphocytes, and that the expression of IAN1, IAN4, and IAN5 is significantly elevated upon thymic selection of T lymphocytes. Gain-of-function experiments show that the premature overexpression of IAN1 kills immature thymocytes, whereas short hairpin RNA-mediated loss-of-function studies show that IAN4 supports positive selection. The knockdown of IAN5 perturbs the optimal generation of CD4/CD8 double-positive thymocytes and reduces the survival of mature T lymphocytes. We also show evidence suggesting that IAN4 and IAN5 are associated with anti-apoptotic proteins Bcl-2 and Bcl-xL, whereas IAN1 is associated with pro-apoptotic Bax. Thus, the IAN family is a novel family of T cell–receptor-responsive proteins that critically regulate thymic development and survival of T lymphocytes and that potentially exert regulatory functions through the association with Bcl-2 family proteins.     

Over-expression of a putative poplar glycosyltransferase gene, PtGT1, in tobacco increases lignin content and causes early flowering

Family 1 glycosyltransferases catalyse the glycosylation of small molecules and play an important role in maintaining cell homeostasis and regulating plant growth and development. In this study, a putative glycosyltransferase gene of family 1, PtGT1, was cloned from poplar (Populus tomentosa Carr.). Sequence analysis showed that this gene encodes a protein of 481 amino acid residues with a conserved PSPG box at its C-terminal, suggesting that it is active in the glycosylation of plant secondary products. The PtGT1 gene was expressed in poplar stems and leaves, with a particularly high expression level in elongating stems. Transgenic tobacco plants ectopically over-expressing PtGT1 were obtained and phenotypes were analysed. Wiesner and M?ule staining showed that stem xylem of transgenic tobacco plants stained more strongly than controls. Measurement of the Klason lignins showed much higher lignin content in the transgenic lines than in control plants. Furthermore, the ectopic over-expression of PtGT1 in tobacco resulted in an early flowering phenotype. These findings offer a possible starting point towards better understanding of the function of poplar PtGT1, and provide a novel strategy for lignin engineering and flowering control in plants through the genetic manipulation of a poplar glycosyltransferase gene.     

定位于染色体9p21-22的一个新基因UBAP1的克隆测序及在鼻咽癌中的表达分析

 在染色体 9p2 1 2 2鼻咽癌杂合性丢失 (lossofheterozygosity,LOH)高频区 ,应用EST介导的定位 侯选克隆策略 ,用RT PCR及Northern杂交检测了 2 2个表达序列标记 (expressedsequencetag ,EST)在鼻咽癌细胞株HNE1和原代培养的正常鼻咽上皮细胞中的表达差异 ,并对其中一个在鼻咽癌细胞株HNE1中表达下调的EST检测了在鼻咽癌活检组织中的表达 .用生物信息学方法获得其全长cDNA序列 ,GenBank登录号AF2 2 2 0 4 3.该基因cDNA全长 2 70 1bp ,其开放阅读框 (openreadingframe ,ORF)编码一个含 50 2个氨基酸、分子量为 55kD的碱性蛋白质 ,在蛋白羧基端含有 2个连续的重要UBA功能域 (ubiquitinassociateddomain) ,属于遍在蛋白相关蛋白家族的一个新成员 ,经国际人类基因命名委员会同意 ,将其命名为UBAP1 (ubiquitinassociatedprotein 1 ) .Northern表达分析显示UBAP1在所检测的人组织中广泛表达 ,但在人的心脏、骨骼肌及肝脏中的表达较强 .UBAP1基因在63 2 % ( 1 2 1 9)的鼻咽癌活检组织中表达下调 .UBAP1基因作为一个遍在蛋白相关蛋白家族的新成员 ,结合其在 9p的重要定位信息 ,有必要进一步研究其表达下调参与鼻咽癌发生发展的可能机制 .     

A novel plant-specific family gene, ROOT PRIMORDIUM DEFECTIVE 1, is required for the maintenance of active cell proliferation

Hypocotyl segments of Arabidopsis (Arabidopsis thaliana) produce adventitious roots in response to exogenously supplied auxin. root primordium defective 1 (rpd1) is a temperature-sensitive mutant isolated on the basis of impairment in this phenomenon. This study describes further phenotypic analysis of the rpd1 mutant and isolation of the RPD1 gene. When adventitious root formation was induced from the rpd1 explants at the restrictive temperature, cell proliferation leading to root promordia formation was initiated at the same time as in wild-type explants. However, development of the root primordia was arrested thereafter in the mutant. Temperature-shift experiments indicated that RPD1 exerts its function before any visible sign of root primordium formation. The expression patterns of the auxin-responsive gene DR5:beta-glucuronidase and the cytodifferentiation marker gene SCARECROW suggest that the rpd1 mutation interferes with neither axis formation nor cellular patterning at the initial stage of root primordium development. Taken together with the effect of the rpd1 mutation on callus cell proliferation, these data imply a role for RPD1 in prearranging the maintenance of the active cell proliferation during root primordium development. Positional cloning of the RPD1 gene revealed that it encodes a member of a novel protein family specific to the plant kingdom. Disruption of the RPD1 gene by a T-DNA insertion caused embryogenesis arrest at the globular to transition stages. This phenotype is consistent with the hypothesized function of RPD1 in the maintenance of active cell proliferation.     

Expression of cDNA clones encoding the thymocyte antigens CD1a, b, c demonstrates a hierarchy of exclusion in fibroblasts

The CD1 gene family encodes at least three proteins: CD1a, CD1b, and CD1c, which are coexpressed on cortical thymocytes and a number of T cell leukemias. On thymocytes, CD1a forms noncovalent complexes with CD1b and CD1c, and a disulfide-linked heterodimer with CD8. This report describes the isolation and characterization of cDNA clones encoding the CD1a, CD1b, and CD1c Ag. Cotransfection of the cDNA was used to investigate the formation of intermolecular heterodimers by CD1a with other members of the CD1 gene family and with CD8. No intermolecular heterodimers were observed during transient expression in COS cells. However, an exclusion hierarchy was observed between members of the CD1 gene family when two or more members of the family were cotransfected into COS cells.