Distinct sequence of negative or positive selection implied by thymocyte T-cell receptor densities

Recent evidence suggests that positive and negative selection of thymocytes bearing alpha beta T-cell receptors occurs during the predominant double-positive (CD4+CD8+) stage. But the sequence or stage at which positive or negative selection occurs during thymocyte maturation has not been well defined. Here we use transgenic mice to show that the CD4+CD8+ stage might be further subdivided into CD3lo (low) and CD3in (intermediate) stages. The CD3in stage could represent T cells that have been positively selected, as this stage is dependent on the presence of the appropriate major histocompatibility complex restriction element. In addition, we use two different tolerizing antigens to show that negative selection may occur either before or after this CD3in stage.     

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection

Thymocytes from mice lacking the CD3delta chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4- CD8- double-negative to a CD4+ CD8+ double-positive stage. However, CD3delta-/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3delta-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3delta maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3delta rescues both ERK activation and positive selection in CD3delta-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3zeta chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3delta-/- thymocytes may derive from defective tyrosine phosphorylation of CD3zeta in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.     

Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition.

In a longitudinal study of HIV seropositive patients, there were fluctuations in the specificity of cytotoxic T cells for the virus. This was matched by variability in proviral gag DNA epitope sequences in the lymphocytes of these patients. Some of these viral variants are not recognized by autologous T cells. Accumulation of such mutations in T-cell antigenic targets would provide a mechanism for immune escape.     

Positive selection of T-lymphocytes induced by intrathymic injection of a thymic epithelial cell line.

T lymphocytes recognize antigens as peptide fragments associated with molecules encoded by the major histocompatibility complex (MHC) and expressed on the surface of antigen-presenting cells. In the thymus, T cells bearing alpha beta receptors that react with the MHC molecules expressed by radioresistant stromal elements are positively selected for maturation. In (A x B-->A) bone marrow chimaeras, T cells restricted to the MHC-A haplotype are positively selected, whereas MHC-B-reactive thymocytes are not. We investigated whether the introduction of particular thymic stromal elements bearing MHC-B molecules could alter the fate of B-reactive T cells in these (A x B-->A) chimaeras. Thymic epithelial cell (TEC) lines expressing H-2b were introduced by intrathymic injection into (H-2b/s-->H2s) bone marrow chimaeras and we measured their ability to generate H-2b-restricted cytotoxic T-lymphocytes (CTLs). We report here that one TEC line, 427.1, was able positively to select CTLs specific for influenza and vesicular stomatitis virus antigens in association with class I H-2b molecules. In addition, line 427.1 can process cytoplasmic proteins for presentation to H-2Kb- and H-2Db-restricted CTLs. Thus, a TEC line capable of normal class I MHC antigen processing and presentation in vitro can induce positive selection after intrathymic injection.     

Sarcoma viruses carrying ras oncogenes induce differentiation-associated properties in a neuronal cell line

The growth-promoting and/or differentiation-blocking activities of Kirsten (Ki-MSV) or Harvey murine sarcoma virus (Ha-MSV) on various types of cells in vitro are well documented. Here we report an unexpected effect of these viruses on a rat phaeochromocytoma cell line, PC12. PC12 cells, which multiply indefinitely in growth medium, are known to respond to nerve growth factor (NGF) by cessation of cell division and expression of several properties resembling those of differentiated sympathetic neurones. We have found that Ki- and Ha-MSV mimic some, if not all, of the activities of NGF in PC12 cells, and there is evidence that the viral oncogenes, v-Ki-ras and v-Ha-ras, are responsible for this phenomenon. This system may be of value for studying the mechanism of action of the v-ras genes as well as the regulatory mechanism of growth and differentiation in neuronal cells.     

The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration.

Three dominant mutations of mec-4, a gene needed for mechanosensation, cause the touch-receptor neurons of Caenorhabditis elegans to degenerate. With deg-1, another C. elegans gene that can mutate to induce neuronal degeneration and that is similar in sequence, mec-4 defines a new gene family. Cross-hybridizing sequences are detectable in other species, raising the possibility that degenerative conditions in other organisms may be caused by mutations in similar genes. All three dominant mec-4 mutations affect the same amino acid. Effects of amino-acid substitutions at this position suggest that steric hindrance may induce the degenerative state.     

A mutant protein kinase C that can transform fibroblasts

Expression of normal protein kinase C (PKC) isoenzymes in fibroblasts has been shown to alter growth regulation but has failed to induce complete transformation of the recipient cells. Here we report on a murine ultraviolet-induced fibrosarcoma cell line which has an unusual PKC subcellular distribution with 87% of the PKC activity associated with the membrane. We have cloned and sequenced the alpha-PKC complementary DNA from ultraviolet-induced-fibrosarcoma cells and from mouse Balb/c brain and found four point mutations in the fibrosarcoma PKC, of which three are in the highly conserved regulatory domain and one is in the conserved region of the catalytic domain. Expression of this mutant alpha-PKC gene in normal Balb/c 3T3 fibroblasts results in a fibrosarcoma-like PKC membrane localization and in cell transformation, as judged by their formation of dense foci, anchorage-independent growth and ability to induce solid tumours when inoculated into nude mice. By contast, transfectants expressing the normal alpha-PKC cDNA do not display a morphology typical of malignant transformed cells and fail to induce tumours in vivo. These findings demonstrate that point mutations in the primary structure of PKC modulate enzyme function and are responsible for inducing oncogenicity.     

Inefficient positive selection of T cells directed by haematopoietic cells.

Intrathymic differentiation of alpha beta TCR+ T cells depends on positive selection of CD4+CD8+ thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows the maturation of only those T cells capable of restricted antigen recognition in the context of the hosts' MHC alleles. Studies of normal or T-cell receptor-transgenic mice engrafted with MHC-different bone marrow or thymuses support the conclusion that positive selection is directed by MHC molecules expressed on non-haematopoietic cells, presumably thymic epithelial cells. Here we, present contrary evidence that class I MHC molecules expressed by haematopoietic cell types direct positive selection of CD8+ T cells, though at a reduced rate compared with positive selection directed by thymic epithelial cells. The identity of cell types that direct positive selection bears directly on mechanistic models of the process, including the idea that thymic epithelial cell MHC molecules uniquely present specialized peptides that mediate positive selection, and the notion that thymic epithelial cells express unique differentiation-inducing cell surface molecules.     

Extrathymic positive selection of alpha beta T-cell precursors in nude mice.

T lymphocytes expressing alpha beta T-cell receptors with sufficient affinity to major histocompatibility complex (MHC) molecules expressed on thymus epithelial cells are positively selected and mature to functional T cells. But several studies have demonstrated that athymic nude mice grafted with MHC-incompatible thymuses developed T cells specific for nude host rather than thymic MHC. We examined this paradox by analysing the specificity of T lymphocytes derived from nude mice. We report here that nude T lymphocyte precursors transferred to allogeneic SCID (severe combined immunodeficiency) mice with a functioning thymus (but lacking T or B cells) generated host MHC-restricted effector T cells but also contained T cells restricted to donor MHC. If nude T cells were depleted from nude lymphohaemopoietic donor cells before or after transfer, only host MHC-specific T cells matured. The results may explain the unusual MHC specificities of nude T lymphocytes described in earlier studies and demonstrate two separate differentiation steps: in nude mice, T cells may be positively selected for self-MHC restriction specificity extrathymically; then a functional thymus is required for efficient T cell maturation.     

In vitro selection of RNA molecules that bind specific ligands.

Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.     

逼近纳什均衡的动态蜂窝选择方案

在异构蜂窝选择博弈中,纳什均衡对应终端的蜂窝选择结果可以让系统获得相对较优的性能,然而在动态的网络场景中,终端会不断地移动,导致此前选择的结果偏离原纳什均衡,采用传统搜索纳什均衡的方法无法非常快速地重新逼近新的纳什均衡。文章提出一种逼近纳什均衡的动态蜂窝选择方案,借助设计的经验公式,快速调整终端的蜂窝选择策略,使整个系统始终保持逼近纳什均衡。     

A suppressor T-lymphocyte cell line for autoimmune encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a model for the in vitro and in vivo study of T-cell activation. It is an autoimmune disease mediated by T lymphocytes of the helper T-cell (Th) subset. After sensitization to guinea-pig myelin basic protein in complete Freund's adjuvant, Lewis rats develop an autoimmune response to central nervous system (CNS) myelin basic protein, manifested clinically as paralysis and histologically by a perivascular mononuclear cell infiltrate of the CNS parenchyma. Suppressor cell regulation of EAE has long been suspected because Lewis rats, which spontaneously recover from active disease, are resistant to reinduction of active EAE, even though effector T-cell lines can be rescued from these recovered rats. Using cyclosporin A, an immunosuppressive agent believed to inhibit Th cell function, suppressor T-cell (Ts) lines have now been generated from recovered Lewis rats. These Ts cells, when admixed with guinea pig myelin basic protein-specific Th cells, will prevent the adoptive transfer of EAE. The Ts cells appear to be CD4+, which explains previous observations that CD8+ lymphocytes are not important in the recovery of EAE in the rat. This is the first direct demonstration of Ts-cell regulation of EAE.     

A mirror-symmetric cell division that orchestrates neuroepithelial morphogenesis

The development of cell polarity is an essential prerequisite for tissue morphogenesis during embryogenesis, particularly in the development of epithelia. In addition, oriented cell division can have a powerful influence on tissue morphogenesis. Here we identify a novel mode of polarized cell division that generates pairs of neural progenitors with mirror-symmetric polarity in the developing zebrafish neural tube and has dramatic consequences for the organization of embryonic tissue. We show that during neural rod formation the polarity protein Pard3 is localized to the cleavage furrow of dividing progenitors, and then mirror-symmetrically inherited by the two daughter cells. This allows the daughter cells to integrate into opposite sides of the developing neural tube. Furthermore, these mirror-symmetric divisions have powerful morphogenetic influence: when forced to occur in ectopic locations during neurulation, they orchestrate the development of mirror-image pattern formation and the consequent generation of ectopic neural tubes.     

A motif in the alphabeta T-cell receptor controls positive selection by modulating ERK activity

Positive selection allows thymocytes that recognize an individual's own major histocompatibility complex (self-MHC) molecules to survive and differentiate, whereas negative selection removes overtly self-reactive thymocytes. Although both forms of thymic selection are mediated by the alphabeta T-cell receptor (TCR) and require self-MHC recognition, an important question is whether they are controlled by distinct signalling cascades. We have shown that mutation of an essential motif within the TCR alpha-chain-connecting peptide domain (alpha-CPM) profoundly affects positive but not negative selection. Using transgenic mice expressing a mutant alpha-CPM TCR we examined the contribution of several mitogen-activated protein kinase (MAPK) cascades to thymic selection. Here we show that in thymocytes expressing a mutant alpha-CPM receptor, a positively selecting peptide failed to activate the extracellular signal-regulated kinase (ERK), although other MAPK cascades were induced normally. The defect in ERK activation was associated with impaired recruitment of the activated tyrosine kinases Lck and ZAP-70, phosphorylated forms of the TCR component CD3zeta and the adaptor protein LAT to detergent-insoluble glycolipid-enriched microdomains (DIGs). Therefore, an intact DIG-associated signalosome is essential for sustained ERK activation, which leads to positive selection.