Secretion and differential localization of the proteolytic cleavage products Abeta40 and Abeta42 of the Alzheimer amyloid precursor protein in human fetal myogenic cells.

Abeta peptides are major components of the amyloid plaques that characterize Alzheimer's disease. These peptides are proteolytic cleavage products of the amyloid precursor protein (APP) and are generated by beta- and gamma-secretases. Here we show by multiparameter immunofluorescence imaging in muscle cells that localization of the Abeta40 and Abeta42 cleavage products reveals different myocyte types in a three-dimensional culture system. These myocyte types are heterogeneous by selective intracellular concentration of either Abeta40 or Abeta42 in vesicular structures, whilst only the Abeta40 peptide is secreted as indicated by Western blot analysis. This cellular pattern of APP proteolysis and Abeta peptide secretion correlates with lack of L-APP mRNA splice isoforms. Differential secretion and intracellular accumulation of Abeta peptides is characteristic for the early myocyte development and might be related to cell fusion.

A novel form of CD4 (L3T4) mRNA in the murine fetal liver results in cell-surface expression of the L3T4 antigen.

The expression of the L3T4 antigen during ontogeny in the murine fetal liver has been investigated in parallel by northern blot analysis and cytofluorometry. The L3T4 gene is transcribed in the murine fetal liver in two polyadenylated mRNA species with the size of 3.5 kb and 3.7 kb. Whereas the 3.5-kb mRNA is expressed from days 13 to 18 of gestation, expression of the 3.7-kb mRNA is found only from days 16 to 18 of gestation and thus appears to be developmentally regulated. Immunofluorescent staining of fractionated fetal liver cells from days 12 to 18 of gestation with the anti-L3T4 antibody (GK1.5) provides evidence that cell-surface expression of the L3T4 antigen on a subset of lympho-haematopoietic cells in the murine fetal liver is the product of a novel form of L3T4 mRNA with the size of 3.5 kb.

A novel subset of CD2-, CD3/T cell receptor alpha/beta+ human peripheral blood T cells. Phenotypic and functional characterization of interleukin 2-dependent CD2-CD3+ T cell clones.

It is generally believed that CD2 (T11, sheep erythrocyte receptor) is expressed on all human T cells. In the present study we have identified and characterized a minor subset of CD2- CD3/TCR alpha/beta+ T cells in the peripheral blood of healthy individuals. CD2-CD3+ T cells were enriched in PBMC depleted of plastic-adherent macrophages, E-rosetting (i.e., CD2+) T cells and surface Ig+ B cells. CD2-CD3+ T cells accounted for 0.1-0.8% of PBMC in six individuals. IL-2-dependent long-term clones of CD2-CD3+ T cells neither reacted with a panel of anti-CD2 mAbs nor expressed detectable levels of CD2 mRNA by Northern blot analysis. These clones, however, expressed a full-length TCR C beta mRNA and reacted with mAbs against TCR-alpha/beta, CD3, and CD4, and thus were mature T cells. CD2-CD3/TCR+ T cell clones could be triggered into proliferation, IL-2 production, and cytotoxic effector activity by anti-CD3 and anti-TCR mAbs. We conclude that (a) a minor subset of CD2-, CD3/TCR-alpha/beta+ T cells is present in normal peripheral blood; and (b) expression of CD2 at the level of protein and/or mRNA is not required for T cell signaling via the CD3/TCR molecular complex.

Rearrangement and expression of T cell antigen receptor and gamma genes during thymic development.

Rearrangement and expression of the T cell antigen receptor and the gamma genes during T cell ontogeny is a regulated process; the gamma genes are rearranged and expressed first, followed by the beta and then the alpha genes. Expression of both functional alpha and beta gene RNA first occurs at day 17 of gestation, along with the expression of T3 delta chain RNA. T cell antigen receptor gene rearrangements occur primarily or exclusively in the thymus, although some gamma gene rearrangements occur outside the thymus in fetal liver cells that may be committed T cell progenitors. There is no gross difference in the extent of beta and gamma gene rearrangements in the adult thymocyte subpopulations that were analyzed, despite the fact that some of these populations cannot respond to antigen and never emigrate from the thymus. Quantitative analysis of rearrangements in total adult thymocyte DNA shows that beta gene rearrangements generally occur on both chromosomal homologs, and that rearrangements occur preferentially to the J beta 2 gene segment cluster.

Idiotypic network regulations of immune responses to HLA.

The development of antiidiotypic autoimmunity with respect to HLA alloantigens provides an attractive explanation for the phenomenon of maternal tolerance to the fetus and for the tandem selection of two antagonistic traits, major histocompatibility complex polymorphism and alloreactivity. We have demonstrated that both T and B lymphocyte responses to allogeneic HLA antigens are subjected to feedback regulation by autologous antiidiotypic immunity. Idiotypic receptors for the alloantigen expressed by T lymphocytes induce antiidiotypic antibodies that are readily detectable in serum during pregnancy, and antiidiotypic T cells that can be revealed in the autologous mixed lymphocyte culture system. Such antiidiotypic T cells and antibodies inhibit specifically the alloimmune function of autologous T cell lines. Similarly, antiidiotypic antibodies (Ab2) to HLA antibody molecules (Ab1) block the binding of the latter to the immunizing HLA antigen. The prevalence of Ab2 over Ab1 during pregnancy may explain the maternal tolerance to the fetus.

Rearrangement and transcription of the beta-chain genes of the T-cell antigen receptor in different types of murine lymphocytes.

Rearrangements of T-cell receptor beta-chain genes are usually found on both chromosomal homologues, occurring by both deletional and non-deletional mechanisms. Two constant-region (C beta) genes have been identified previously and at least one is transcribed in every helper or cytotoxic T cell tested, but the choice of C beta gene expression is not correlated with the specialized functions of these T lymphocytes. By contrast, four of five suppressor T-cell hybridomas examined have deleted all known joining (J beta) gene segments and C beta genes and therefore may have antigen receptors encoded by different T-cell receptor gene families.

The structure, rearrangement and expression of D beta gene segments of the murine T-cell antigen receptor.

It has been postulated that the variable region of the beta-polypeptide of the murine T-cell antigen receptor is encoded by three distinct germ-line gene segments--variable (V beta), diversity (D beta) and joining (J beta)--that are rearranged to generate a V beta gene. Germ-line V beta and J beta gene segments have been isolated previously. Here we report the isolation and characterization of two germ-line D beta gene segments that have recognition signals for DNA rearrangement strikingly similar to those found in the three immunoglobulin gene families and in V beta and J beta gene segments. The D beta and J beta segments can join in the absence of V beta gene segment rearrangement and these rearranged sequences are transcribed in some T cells.

The T cell receptor beta chain genes are located on chromosome 6 in mice and chromosome 7 in humans.

Homologous clones that encode the beta chain of the T cell antigen receptor have been isolated recently from both murine and human cDNA libraries. These cDNA clones have been used in connection with interspecies hybrid cell lines to determine that the murine T cell receptor gene is located on chromosome 6 and the human gene on chromosome 7. In situ hybridization confirms these data and further localizes these genes to band B of chromosome 6 in the mouse and bands 7p13-21 in the human genome. The organization of the T cell antigen receptor J beta gene segments and C beta genes appears to be conserved, since very few intraspecies polymorphisms of restriction fragment length have been detected in either mouse or human DNA.

Modulation of T-cell antigen receptor on lymphocyte membrane.

A mouse monoclonal antibody, Mo. Ab. 108.45, detecting cell-surface determinants associated with the T-cell receptor for alloantigen was produced by immunizing mice with an alloreactive human T-cell clone and fusing the splenocytes with the NS1 plasmocytoma. This Mo. Ab. reacts with the immunizing T-cell clone but not with autologous or allogeneic lymphocytes, lymphoblasts or monocytes; stimulates the proliferation of the immunizing T cells in the absence of the alloantigen; inhibits the response to the specific stimulator; and precipitates a disulfied linked heterodimer composed of two distinct glycoproteins of molecular weights 40000 and 46000. The receptor molecule detected by Mo. Ab. 108.45 modulates on the surface of the cells, reaching the highest levels 5 days following exposure to the specific stimulator. The receptor-associated molecule detected by Mo. Ab. 108.45 was expressed by T-cell clones obtained independently in two different mixed lymphocyte cultures between the same responder and stimulator.

Monoclonal antibody directed against RNA polymerase II of Drosophila melanogaster.

Monoclonal antibodies were raised against purified RNA polymerase II ( or B) from Drosophila melanogaster. The antibody produced by one hybridoma cell clone was found to be directed against the two large subunits of the enzyme. The absence of antibodies directed against proteins possibly contaminating the antigens used for immunization allowed us to identify RNA polymerase unequivocally in interbands and puffs of polytene chromosomes. Within a single heat shock puff (87C1) RNA polymerase was found to be clustered in two separate areas suggesting two distinct regions of RNA polymerase activity in this puff.