IL-1, IL-4, and IFN-gamma differentially regulate cytokine production and cell surface molecule expression in cultured human thymic epithelial cells.

We investigated the response of purified and cloned human thymic epithelial cells (TEC) to IL-1, IL-4, and IFN-gamma stimulation in vitro. IL-1 alpha strongly up-regulated the production of granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), IL-6, and IL-8, as measured by specific immunoenzymetric assays and by increased steady state mRNA levels. IL-4 or IFN-gamma did not induce these cytokines in TEC but in a sustained and dose-dependent manner down-regulated the IL-1-induced GM-CSF protein and mRNA levels. Only IFN-gamma, and not IL-4, suppressed the IL-1-induced G-CSF and IL-8 production, as shown at both the protein and mRNA levels. The inhibition was dose dependent, sustained for at least 96 h, and more pronounced for G-CSF than for IL-8. In contrast, both IL-4 and IFN-gamma enhanced the IL-1-induced IL-6 production. IL-4 and IFN-gamma had additive effects to increase IL-6 secretion and to more completely suppress the IL-1-induced GM-CSF. Analyses of cell surface molecules showed that intercellular adhesion molecule 1 (ICAM-1) expression on TEC was increased by IL-1 or IFN-gamma. IL-4 slightly down-regulated constitutive ICAM-1 levels but did not significantly modify the levels of expression induced by either IL-1 or IFN-gamma. MHC class II expression was induced by IFN-gamma but not by IL-1 or IL-4. The combination of IL-1 and IL-4 with IFN-gamma did not alter the levels of class II MHC Ag induced by IFN-gamma. In conclusion, TEC cytokine production and cell surface molecule expression are differentially regulated via a complex cytokine network. Our data suggest that developing T cells provide, in part, the signals controlling the function of their supporting stroma.     

Complex rearrangements within the human J delta-C delta/J alpha-C alpha locus and aberrant recombination between J alpha segments.

We have examined DNA rearrangements within a 120 kb cloned region of the human T cell receptor J delta-C delta/J alpha-C alpha locus. Three types of pattern emerge from an analysis of T cell lines and clones. Firstly, cells with two rearrangements within J delta-C delta; secondly, cells with one rearrangement within J delta-C delta and one or more J alpha rearrangements, and finally, cells with rearrangements within J alpha and consequential deletion of the delta locus. Further analysis by cloning of rearrangements within the J alpha locus show that, in addition to V alpha-J alpha joins, J alpha-J alpha aberrant recombinations occur and rearrangement data indicate that such events are frequent. A model is presented to account for such recombinations.     

Whole-genome multiple displacement amplification from single cells

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.     

Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes

It has been demonstrated previously that lymphocytes of donor CF (HLA-A29,w33; B7,14) are not recognized by the HLA-B7-specific CTL clone HG-31. This report presents a structural comparison of the HLA-B7 antigen of donor CF with a "normal" HLA-B7 antigen, derived from the cell line JY. Isoelectric focusing showed that CF HLA-B7 heavy chains were more acidic than JY HLA-B7 heavy chains by the equivalent of a single charge. High pressure liquid chromatography and ion exchange chromatography comparisons of double-labeled tryptic peptides revealed a single detectable difference, which corresponded to the tryptic peptide spanning residues 112 to 121 on the HLA-B7 heavy chain. Although the complete amino acid sequence of this peptide was not obtained, the partial sequence indicates a substitution of an unidentified amino acid for tyrosine at position 116 of the heavy chain. This residue is found to vary among HLA specificities and to be altered in many H-2Kb mutants.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.     

Polymorphisms within the HLA-DRw6 haplotype. I. Restriction fragment length variation and its correlation with serology

The Dw6/DRw6 complex, one of the MHC class II specificities that can be defined by cellular techniques and by serology, probably has one or more immunoregulatory functions. To obtain information on the molecular structure of the DRw6 region, we studied several DRw6 homozygous cell lines, of which three were of consanguineous origin. DNA-DNA hybridization comprised the use of seven restriction enzymes in combination with three DR beta cDNA probes. The obtained results were compared with similar analyses of an HLA homozygous cell panel, expressing DR1-w8 specificities. This comparison indicated that in DRw6 homozygous individuals the coding potential for DR beta chains resembles closely that of all other DR specificities, thus identifying DRw6 as a regular DR region. In addition, we found a restriction fragment length pattern unique for DRw6, indicating the possibility to type for DRw6 by DNA-DNA hybridization. Comparisons within the DRw6 cell panel revealed the occurrence of several HLA class II DNA subtypes. These subdivisions partly correlated with serologically obtained reaction patterns. No correlation, however, could be observed between the different DNA subtypes and cellular reaction patterns as obtained by MLC and T cell cytotoxicity.     

Differential recognition by human cytotoxic T cell clones of human M1 fibroblasts transfected with an HLA-B7 gene (JY150) suggests the existence of two different HLA-B7 alleles in the cell line JY (HLA-A2,2;B7,7;Cw-,-;DR4,w6)

We have used a panel of human HLA-B7-specific CTL clones to identify an HLA-B7 gene (JY150) transfected into human M1 fibroblasts (M1/B7). Only a subset of the CTL clones recognized the M1/B7 cells, whereas all CTL clones recognized the donor of the B7 gene, the cell line JY (HLA-A2,2;B7,7;Cw-,-;DR4,w6). Analysis of the fine specificity of these CTL clones was performed by testing the reactivity on M1 cells transfected with an HLA-B27K gene and on a panel of cell lines typed for HLA-B7 subtypes (variants). These results, combined with one-dimensional IEF analysis of the M1/B7 cells and the B7 subtypes, indicated that the differential recognition by the CTL clones of the transfected gene was not caused by aberrant expression of the gene itself or due to the absence of critical accessory molecules on the M1 fibroblast cells. Our data suggest that the widely used HLA-B7 reference cell line JY is not homozygous at the HLA-B locus, but contains two different B7 alleles encoding the B7.2 and B7.4 subtypes.