Positive selection determines T cell receptor V beta 14 gene usage by CD8+ T cells.

We report here a mAb, 14-2, reactive with TCRs that include V beta 14. The frequency of V beta 14+ T cells varies with CD4 and CD8 subset and is controlled by the H-2 genes. Thus CD8+ T cells from H-2b mice include approximately 2.3% V beta 14+ T cells while CD8+ T cells from mice expressing K kappa include greater than 8% V beta 14+ T cells. In all strains examined, 7-8% of CD4+ T cells express V beta 14. The frequent usage of V beta 14 in CD8+ T cells of K kappa-expressing mice is a result of preferential positive selection of V beta 14+ CD8+ T cells as demonstrated by analysis of radiation chimeras. These studies demonstrate that H-2-dependent positive selection occurs in unmanipulated mice. Furthermore, the results imply that positive selection, and possibly H-2 restriction, can be strongly influenced by a V beta domain, with some independence from the beta-junctional sequence and alpha chain

Positive selection of V beta 8+ CD4-8- thymocytes by class I molecules expressed by hematopoietic cells.

A small subset of T cells of mature phenotype express the alpha/beta T cell receptor, but not CD4 and CD8 coreceptors (alpha/beta double-negative [DN] cells). The repertoire of V beta usage of alpha/beta DN cells is strongly biased towards V beta 8 expression, suggesting that the formation of the population is subject to selection. We now report that deficiency of class I expression leads to a strongly depressed frequency of V beta 8+ DN cells, but has little effect on V beta 8- DN cells. Studies of hematopoietic chimeras between class I+ and class I- mice demonstrated that expression of class I molecules by hematopoietic cells is necessary and sufficient for selection of most V beta 8 DN cells. The lack of a role for class I expression by thymic epithelial cells suggests that the mechanism of selection of these cells by class I differs significantly from the mechanism of selection of conventional T cells. Models to explain the selection of these cells as well as their possible function in vivo are discussed

Predominant utilization of V beta 8+ T cell receptor genes in the H-2Ld- restricted cytotoxic T cell response against the immediate-early protein pp89 of the murine cytomegalovirus

Cytotoxic T cell responses to the murine Cytomegalovirus (MCMV) were elicited in BALB/c mice (H-2d) by infectious virus. Eight days after infection, MCMV-primed local lymph node T cells were either depleted for T cells expressing a V beta 8+ TCR or separated into V beta 8+ and V beta 8- subpopulations by a cell sorter using the mAb F23.1. T cells were then expanded in vitro under limiting dilution conditions in the presence of IL-2 and in the absence of viral Ag to avoid selection by Ag in vitro. Frequencies of CTL precursors specific for the Immediate- Early-Ag 1 of MCMV and restricted to H-2Ld were determined. L cells of the endogenous haplotype H-2k cotransfected with the genes for MCMV-IE 1 and H-2Ld were used as target cells. Detection of a CTL response required previous priming of the animals by infection in vivo (less than 1/10(6) for nonimmunized animals). In primed animals CTL precursors of this specificity and restriction were three to fivefold more frequent in the V beta 8+ population (1/9.900 to 1/22.300) than in the V beta 8- population (1/57.000 to 1/87.200). Control experiments showed that frequencies were not influenced by the treatment with the anti-V beta 8-antibody and the fluorescein-labeled anti-Ig itself. V beta 8+ and V beta 8- T cells did not reveal any frequency differences when several other responses were determined (TNP-specific self- restricted CTL precursor; Th cells specific for keyhole limpet hemocyanin or Listeria monocytogenes)

The polarized expression of Na+,K+-ATPase in epithelia depends on the association between beta-subunits located in neighboring cells

The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta(1)-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta(1)-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta(1)-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta(1)-subunit forces CHO-beta cells to coexpress endogenous a-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog P,-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells

Pertussis Toxin-sensitive Activation of Phospholipase C by the C5a and fMet-Leu-Phe Receptors

Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC beta 2 and PLC beta 3 through the Gbeta gamma subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC beta 2 and PLC beta 3 were shown to be activated by the beta gamma subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC beta 3 or recombinant PLC beta 3 in transfected COS-7 cells. However, both receptors can clearly activate PLC beta 2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC beta 2 probably through the Gbeta gamma subunits. These findings were further corroborated by the results that PLC beta 3 could only be slightly activated by Gbeta 1gamma 1 or Gbeta 1gamma 5 in the cotransfection assay, whereas the Gbeta gamma subunits strongly activated PLC beta 2 under the same conditions. PLC beta 3 can be activated by Galpha q, Galpha 11, and Galpha 16 in the cotransfection assay. In addition, the Ggamma 2 and Ggamma 3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gbeta gamma -mediated activation of PLC beta 2, were able to inhibit C5a or fMLP-mediated activation of PLC beta 2. These Ggamma mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gbeta gamma subunits are involved in PLC beta 2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC beta 3 by Gbeta gamma may not be the primary pathway for the receptors

Requirement of the NPXY motif in the integrin beta 3 subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo.

The NPXY sequence is highly conserved among integrin beta subunit cytoplasmic tails, suggesting that it plays a fundamental role in regulating integrin-mediated function. Evidence is provided that the NPXY structural motif within the beta 3 subunit, comprising residues 744-747, is essential for cell morphological and migratory responses mediated by integrin alpha v beta 3 in vitro and in vivo. Transfection of CS-1 melanoma cells with a cDNA encoding the wild-type integrin beta 3 subunit, results in de novo alpha v beta 3 expression and cell attachment, spreading, and migration on vitronectin. CS-1 cells expressing alpha v beta 3 with mutations that disrupt the NPXY sequence interact with soluble vitronectin or an RGD peptide, yet fail to attach, spread, or migrate on immobilized ligand. The biological consequences of these observations are underscored by the finding that CS-1 cells expressing wild-type alpha v beta 3 acquire the capacity to form spontaneous pulmonary metastases in the chick embryo when grown on the chorioallantoic membrane. However, migration-deficient CS-1 cells expressing alpha v beta 3 with mutations in the NPXY sequence lose this ability to metastasize. These findings demonstrate that the NPXY motif within the integrin beta 3 cytoplasmic tail is essential for alpha v beta 3-dependent post-ligand binding events involved in cell migration and the metastatic phenotype of melanoma cells

TGF beta 1 attenuates expression of prolactin and IGFBP-1 in decidualized endometrial stromal cells by both SMAD-dependent and SMAD-independent pathways

Background: Decidualization (differentiation) of the endometrial stromal cells during the secretory phase of the menstrual cycle is essential for successful implantation. Transforming Growth Factor beta 1 (TGF beta 1) canonically propagates its actions via SMAD signalling. A role for TGF beta 1 in decidualization remains to be established and published data concerning effects of TGF beta 1 on markers of endometrial decidualization are inconsistent. Methodology/Principal Findings: Non-pregnant endometrial stromal cells (ESC) and first trimester decidual stromal cells (DSC) were cultured in the presence or absence of a decidualizing stimulus. Incubation of ESCs with TGF beta 1 (10 ng/ml) down-regulated the expression of transcripts encoding the decidual marker proteins prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP-1) and tissue factor (TF). TGF beta 1 also inhibited secretion of PRL and IGFBP-1 proteins by ESCs and surprisingly this response preceded down-regulation of their mRNAs. In contrast, DSCs were more refractory to the actions of TGF beta 1, characterized by blunted and delayed down-regulation of PRL, IGFBP-1, and TF transcripts, which was not associated with a significant reduction in secretion of PRL or IGFBP-1 proteins. Addition of an antibody directed against TGF beta 1 increased expression of IGFBP-1 mRNA in decidualised cells. Knockdown of SMAD 4 using siRNAs abrogated the effect of TGF beta 1 on expression of PRL in ESCs but did not fully restore expression of IGFBP-1 mRNA and protein. Conclusions/Significance: TGF beta 1 inhibits the expression and secretion of decidual marker proteins. The impact of TGF beta 1 on PRL is SMAD-dependent but the impact on IGFBP1 is via an alternative mechanism. In early pregnancy, resistance of DSC to the impact of TGF beta 1 may be important to ensure tissue homeostasis