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E Lacy

Publications and source records attributed to E Lacy.

At least 37 records · Page 2Linked to original sources
PubMed1994

The onset of Fas expression parallels the acquisition of CD8 and CD4 in fetal and adult alpha beta thymocytes.

Fas is an apoptosis-related cell surface molecule whose defective transcription results in the lpr defect and autoimmunity. Recent analysis of Fas mRNA and protein expression in normal mice showed high expression in the thymus, on activated T cells, and on 5-10% of peripheral T cells. To investigate the role of Fas in the thymus, we analyzed its expression in fetal and adult thymocyte subsets. Fas was not expressed on fetal nor adult CD8-CD4- (double-negative, DN) T cell precursors. The earliest precursors that expressed low levels of FAS were the immediate precursors of DP thymocytes that bear the CD44-CD25-CD8loCD4loTCRlo phenotype. Other DN cells that expressed Fas appeared to be either non-T cells or mature alpha beta + DN thymocytes. The onset of Fas expression followed the onset of expression of CD8 and CD4 and Fas expression reached its peak in CD8+CD4+ double-positive (DP) thymocytes. Both single-positive (SP) subsets were largely Fas+ (CD8 SP < CD4 SP) but expressed lower levels of Fas than DP cells. However, a majority (> 60%) of the most mature HSA(lo) SP cells (2-5% of all SP thymocytes) were Fas- and the remainder of the HSA(lo) SP cells was Fas(lo). We observed two main differences between Fas expression on fetal versus adult thymocytes. First, up to 90% of fetal gamma delta + DN cells expressed high levels of Fas, in contrast to the very low expression (< 7% Fas+ cells) among adult gamma delta + thymocytes. Second, whereas virtually all adult DP cells were Fas+, up to 75% of fetal day 16 DP cells were Fas-.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Transcription of the interleukin 4 gene is regulated by multiple promoter elements.

Activation of T helper cell 1 (Th1) and Th2 results in transcription of the interleukin 2 (IL-2) and IL-4 cytokine genes, respectively. Whereas many of the regulatory elements and factors responsible for IL-2 transcription in T cells are well defined, little is known about parallel mechanisms that drive transcription of the IL-4 gene. Here we have analyzed the murine IL-4 promoter, both in vivo and in a Th2 clone. 3 kb of IL-4 upstream sequence is shown to be sufficient to achieve tissue-specific and inducible expression of a thymidine kinase reporter gene in vivo in a manner that mirrors the expression of endogenous IL-4. Tissue-specific and inducible expression is also demonstrated in a Th2 clone, but not in a B cell line. Deletional and mutational analysis of the IL-4 promoter demonstrated that sequences from -100 to -28 were necessary for a transcriptional response to Concanavalin A or anti-CD3 monoclonal antibody. An overlapping, yet smaller region, spanning the sequences from -60 to -28 bp was shown to be required for the response to ionomycin. Mutation of an 8-bp region from -43 to -35 of the IL-4 promoter completely abrogated IL-4 gene transcription in response to all stimuli tested. In addition, our results show that the effects of the immunosuppressive agent Cyclosporin A map to the same DNA sequences as the positive control elements. These results identify DNA sequences that are functionally important for the control of IL-4 gene transcription both in vivo and in vitro. Although these sequences are highly conserved in the human and murine IL-4 genes, they are largely not present in the IL-2 enhancer complex. Thus, cytokine-specific cis-acting elements may be one mechanism by which these two cytokine genes are differentially regulated.

Animals↗
PubMed1993

Reconstitution of the subclass-specific expression of CD4 in thymocytes and peripheral T cells of transgenic mice: identification of a human CD4 enhancer.

During thymic maturation, CD4-CD8-TCR- immature thymocytes differentiate through a CD4+CD8+TCRlo intermediate into two functionally distinct mature T cell subsets: helper T cells expressing CD4 and a major histocompatibility complex (MHC) class II-restricted T cell receptor (TCR), and cytotoxic T cells expressing CD8 and and MHC class I-restricted TCR. The mutually exclusive expression of CD4 and CD8 is maintained in the periphery during expansion of these mature T cell subsets. To elucidate the mechanisms controlling CD4 and CD8 expression on differentiating thymocytes and mature peripheral T cells, we have examined the expression of human CD4 gene constructs in the lymphoid tissues of transgenic mice. Our analyses demonstrate that sequences contained within or closely linked to the human CD4 gene are sufficient to reconstitute the appropriate regulation of human CD4 expression on all thymocyte and mature peripheral T cell subsets. Specifically, appropriate developmental regulation was dependent on two sets of sequences, one contained within a 1.3-kb restriction fragment located 6.5 kb upstream of the human CD4 gene, and the other present within or immediately flanking the gene. Nucleotide sequence analysis identified the 1.3-kb restriction fragment as the likely human homologue of an enhancer found 13 kb upstream of the mouse CD4 transcription initiation site. The human CD4 transgenic mice provide a useful system for the identification and characterization of additional sequence elements that participate in human CD4 gene regulation and for the elucidation of regulatory mechanisms governing the developmental program mediating the maturation of the CD4+ and CD8+ peripheral T cell subsets.

Animals↗
PubMed1993

Transcriptional diversity at the duplicated human CD8 beta loci.

In order to understand the structural organization of the human CD8 beta locus, genomic clones containing CD8 beta sequences were isolated and analyzed. Physical linkage of these clones with the CD8 alpha locus using pulsed-field electrophoresis revealed a duplication of the CD8 beta locus. CD8B-1 lies 35 kb upstream from the CD8 alpha locus and contains eight exons, including four alternatively spliced cytoplasmic exons. The CD8B-2 gene contains six exons and is at present unlinked to CD8B-1. Analysis of sequences upstream to the leader exon of the CD8B-1 and CD8B-2 genes revealed a GC-rich promoter which lacks canonical "CCAAT" and "TATA" motifs, but which has sites for multiple transcriptional activators and three additional elements which are conserved in the murine CD8 beta promoter. Seven unique CD8 beta cDNA isoforms were isolated and characterized, which derive from alternative splicing of the transmembrane and/or cytoplasmic exons. Three cDNA are membrane spanning, while the remaining four isoforms lack a transmembrane region and are potentially secreted. These transcripts are differentially expressed in the thymus and in the periphery. Transfection experiments in murine fibroblasts confirmed that the membrane CD8 beta isoforms could be expressed as heterodimers with the CD8 alpha chain. The regulated expression of multiple CD8 beta cytoplasmic isoforms and their potential role in T lymphocyte signal transduction is discussed.

Alternative Splicing↗
PubMed1993

The lack of CD8 alpha cytoplasmic domain resulted in a dramatic decrease in efficiency in thymic maturation but only a moderate reduction in cytotoxic function of CD8+ T lymphocytes.

The glycoprotein CD8 is believed to play an important role in the maturation and function of MHC class I-restricted T lymphocytes. CD8 has been proposed to function as a co-receptor of the TcR to participate in signal transduction, possibly through its cytoplasmic domain that binds to protein tyrosine kinase p56lck. A T cell-specific transgene encoding CD8 alpha truncated at the cytoplasmic domain ("tailless CD8 alpha"), was introduced into CD8 alpha-deficient mice. This animal model was used to study the role of the CD8 cytoplasmic domain in T cell ontogeny and function. "Tailless CD8 alpha" was expressed on the cell surface of thymocytes and peripheral T cells. A small population of peripheral CD4- T cells (6% of T lymphocytes) was found to have cell surface expression of "tailless CD8 alpha" and endogenous CD8 beta, indicating that these cells may belong to the CD8+ T cell lineage. A consistent result was obtained from CD8 alpha-deficient mice bearing the "tailless CD8 alpha" and the MHC class I-restricted 2C TcR transgenes. A small population of CD4- T cells expressing CD8 beta, the "tailless CD8 alpha" and the 2C TcR transgenes was present in the periphery of these mice in a selecting background, but was absent in a deleting background. When "tailless CD8 alpha" mice were infected with lymphocytic choriomeningitis virus (LCMV), the peripheral CD8+ CD4- T cell subset expanded dramatically and a significant LCMV-specific cytolytic activity was detected. The results suggest that the cytoplasmic portion of CD8 alpha is not absolutely required but dramatically enhances the efficiency of thymic maturation of CD8+ T cells. The lack of CD8 alpha cytoplasmic domain in peripheral CD8+ T cells does not abolish the generation of cytotoxicity in response to an in vivo LCMV infection, although the cytolytic activity is slightly reduced compared to that in control mice.

Animals↗
PubMed1992

Species specificity and augmentation of responses to class II major histocompatibility complex molecules in human CD4 transgenic mice.

Murine T cell responses to human class II major histocompatibility complex (MHC) molecules were shown to be a minimum of 20-70-fold lower than responses to allogeneic molecules. Transgenic mice expressing slightly below normal (75-95%) or very high (250-380%) cell surface levels of human CD4 were utilized to determine whether this was due to a species-specific interaction between murine CD4 and class II molecules. Human CD4 was shown to function in signal transduction events in murine T cells based on the ability of anti-human CD4 antibody to synergize with suboptimal doses of anti-murine CD3 antibody in stimulating T cell proliferation. In mice expressing lower levels of human CD4, T cell responses to human class II molecules were enhanced up to threefold, whereas allogeneic responses were unaltered. In mice expressing high levels of human CD4, responses to human class II molecules were enhanced at least 10-fold, whereas allogeneic responses were between one and three times the level of normal responses. The relatively greater enhancement of the response to human class II molecules in both lines argues for a preferential interaction between human CD4 and human class II molecules. In mice expressing lower levels of human CD4, responses to human class II molecules were blocked by antibodies to CD4 of either species, indicating participation by both molecules. In mice expressing high levels of human CD4, responses to both human and murine class II molecules were almost completely blocked with anti-human CD4 antibody, whereas anti-murine CD4 antibody had no effect. However, anti-murine CD4 continued to synergize with anti-CD3 in stimulating T cell proliferation in these mice. Thus, overexpression of human CD4 selectively impaired the ability of murine CD4 to assist in the process of antigen recognition. The ability of human CD4 to support a strong allogeneic response under these conditions indicates that this molecule can interact with murine class II molecules to a significant extent. Despite the fact that human CD4 appeared to be the only functional coreceptor in these mice, responses to human class II molecules were still much lower than those to murine class II alloantigens. This indicates that species-specific interactions between class II molecules and CD4 expressed on peripheral T cells are not sufficient to account for the low xenogeneic response and that intrinsic differences in T cell receptor structures or the need for species specificity in the interaction between CD4 and class II molecules during positive selection are also important.

Animals↗
PubMed1992

CD8 surface levels alter the fate of alpha/beta T cell receptor-expressing thymocytes in transgenic mice.

The mature T cell receptor (TCR) repertoire is established on the basis of discriminative events involving binding of the TCR alpha and beta chains and CD4 or CD8 on immature thymocytes to major histocompatibility complex (MHC)/self-peptide complexes expressed in the thymus. To ask whether the strength of the interaction between a CD8/TCR complex and a MHC/self-peptide ligand plays a pivotal role in deciding the fate of a maturing thymocyte, we generated lines of transgenic mice that express distinct and elevated levels of CD8 alpha, approximately 2, 3, and 6-10 times. These lines were then crossed to a transgenic line expressing the class I-restricted TCR, 2C. We found that thymocytes expressing the 2C TCR in combination with the highest levels of CD8 were deleted on the H-2 Kb background that is normally positively selecting for the 2C TCR. In contrast, thymocytes coexpressing the 2C TCR and moderately elevated levels of CD8 were selected for maturation. These results demonstrate directly that CD8 levels can affect the developmental fate of a maturing thymocyte and argue in support of an affinity model for thymocyte selection.

Animals↗
PubMed1992

Genomic structure of the locus associated with an insertional mutation in line 4 transgenic mice.

In line 4 transgenic mice, six to eight copies of a 50-kb lambda recombinant clone are arranged in a head-to-tail tandem array on chromosome 3. Embryos homozygous for the transgene become arrested in their development on Day 5 of gestation shortly after implantation. The insertion site was cloned using a small segment of the transgene as a probe. Comparison of the insertion site with the wildtype locus indicates that a 22-kb deletion of host DNA has occurred in line 4 mice. Restriction enzyme analyses showed that neither the tandem array nor the flanking chromosomal DNA had any detectable rearrangements. Sequencing of the junctions between host and foreign DNA, however, revealed insertions of small fragments of DNA of unknown origin as well as an insertion of a DNA segment derived from another region of the transgene. Therefore, disruption of an essential gene in the line 4 transgenic mouse may have been caused by the insertion of 300-400 kb of foreign DNA or a deletion of sequences in the host genome.

Animals↗
PubMed1991

Influenza A-specific, HLA-A2.1-restricted cytotoxic T lymphocytes from HLA-A2.1 transgenic mice recognize fragments of the M1 protein.

Previous studies have indicated that in transgenic mice expressing human class I MHC molecules, it is difficult to demonstrate a significant CTL response to a viral Ag in the context of the transgenic molecule. In this paper, a procedure is reported for the isolation of influenza-specific murine CTL restricted by the human class I molecule HLA-A2.1. The principal specificity of such CTL is for a fragment of the influenza M1 protein that has been previously shown to be immunodominant for human HLA-A2.1-restricted CTL. CTL of this specificity were also established through the use of peptide-pulsed rather than virus-infected stimulators. The dependence of murine CTL recognition upon peptide length and HLA-A2 structure was established to be similar to that previously reported for human CTL. However, the fine specificity of CTL maintained on virus-infected stimulators was somewhat different from that of CTL maintained with M1 peptide. This suggests that differences in surface density or peptide structure between peptide-pulsed and virus-infected stimulators may result in the outgrowth of T cells with different receptor structures. The immunodominance of the M1 peptide determinant in both mice and humans suggests that species-specific differences in TCR structure, Ag-processing systems, and self-tolerance are of less importance than limitations on the ability of antigenic peptides to bind to appropriate class I molecules. These results thus establish the utility of the transgenic system for the identification of human class I MHC-restricted T cell epitopes.

Amino Acids↗
PubMed1991

Ectopic expression of a c-kitW42 minigene in transgenic mice: recapitulation of W phenotypes and evidence for c-kit function in melanoblast progenitors.

The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor that is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and adult life, and they result from the partial or complete loss of c-kit function. The W42 allele is a W mutation with severe effects in both the homozygous and the heterozygous states. Previous analysis of the W42 allele identified a missense mutation in an essential amino acid of the c-kitW42 kinase domain that abolishes the in vitro kinase activity of the c-kitW42 protein but does not affect its normal expression. These results suggested that the c-kitW42 allele was a dominant negative mutation within the context of c-kit-mediated signal transduction. To further explore the dominant negative characteristics of the W42 mutation, we have generated transgenic mice in which ectopic expression is driven by the human beta-actin promoter (hAP). Two mouse lines carrying the hAP-c-kitW42 transgene show an effect on pigmentation and the number of tissue mast cells. The patchy coat color pattern of the line 695 mice may reflect variable expression of the transgene in melanoblast progenitors and their descendants and, consequently, is indicative of a function for c-kit in early melanoblasts. Germ cell development and erythropoiesis, however, do not appear to be affected by the transgene. Mice expressing the c-kitW42 transgene therefore recapitulate some of the phenotypes of mice with W mutations. These results are therefore in agreement with the molecular basis of the W42 mutation and the dominant-negative characteristics of the c-kitW42 protein product.

Alleles↗
PubMed1990

Specific neuronal expression of human NGF receptors in the basal forebrain and cerebellum of transgenic mice.

Transgenic mice carrying multiple copies of the human NGF receptor gene have been generated. Using a monoclonal antibody specific for the human receptor, we have detected specific expression in cholinergic neurons in the basal forebrain and Purkinje cells in the cerebellum during the postnatal period. Expression in the PNS was exemplified by immunostaining of sympathetic and sensory neurons during an early embryonic age. Transection of the sciatic nerve in transgenic animals resulted in induction of human NGF receptors, indicating that the inserted gene can be appropriately regulated. These transgenic mice will provide an opportunity to study the elements regulating the NGF receptor. Furthermore, the ability to obtain specific expression in transgenic mice will permit directed expression of heterologous genes in discrete cells important in the cholinergic septal-hippocampal pathway and the PNS.

Animals↗
PubMed1990

Bovine alpha S1-casein gene sequences direct high level expression of active human urokinase in mouse milk.

We have produced a line of transgenic mice carrying a hybrid bovine alpha S1 casein/human urokinase gene. Bovine alpha S1-casein gene regulatory sequences specifically direct expression of the human urokinase gene in lactating mammary tissue from these mice. Urokinase is a 54 kD protein with 9 disulfide bonds that is normally synthesized in the kidney; however, the casein/urokinase transgenic mice secrete active human urokinase into their milk at concentrations of 1-2 mg/ml. The mice show no other abnormalities. They give birth to, and nurse, normal sized healthy litters. Thus it is possible to produce high concentrations of a large, cysteine rich, non-milk protein in the milk of transgenic animals. This line of transgenic mice provides a model for the eventual production of transgenic farm animals producing high levels of recombinant proteins in their milk.

Animals↗
PubMed1990

Structure of the T-cell antigen receptor: evidence for two CD3 epsilon subunits in the T-cell receptor-CD3 complex.

The T-cell antigen receptor (TCR) consists of heterodimeric glycoproteins (TCR alpha beta or gamma delta) that demonstrate homology with immunoglobulins. Noncovalently associated with the alpha beta (or gamma delta) heterodimer are at least five nonvariant proteins (CD3-gamma, -delta, -epsilon, -zeta, and -eta), which together comprise the TCR-CD3 complex. The stoichiometry of the antigen receptor has been assumed to be either alpha beta gamma delta epsilon zeta zeta or alpha beta gamma delta epsilon zeta eta. In this paper we provide several lines of evidence that support the notion that the mature TCR-CD3 complex on the cell surface contains two CD3-epsilon polypeptide chains. Transfection of two murine T cell-T cell hybridomas with the human DNA encoding CD3-epsilon protein demonstrated that both murine and human CD3-epsilon chains were present within the same TCR-CD3 complex. Analysis of thymocytes isolated from transgenic mice that expressed high copy numbers of the human CD3-epsilon gene showed that the heterologous human CD3-epsilon subunits were coexpressed with murine CD3-epsilon in the same TCR-CD3 complex. Since CD3-epsilon was shown to form disulfide-linked homodimers both in human and murine T cells, the two CD3-epsilon subunits present in the TCR-CD3 complex were in direct contact with one another. The presence of two CD3-epsilon polypeptide chains in close proximity to one another in the TCR-CD3 complex may have important implications for its assembly and its signal transduction mechanisms.

Animals↗
PubMed1989

Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation.

One action of cyclosporin A thought to be central to many of its immunosuppressive effects is its ability to inhibit the early events of T lymphocyte activation such as lymphokine gene transcription in response to signals initiated at the antigen receptor. Cyclosporin A was found to specifically inhibit the appearance of DNA binding activity of NF-AT, AP-3, and to a lesser extent NF-kappa B, nuclear proteins that appear to be important in the transcriptional activation of the genes for interleukin-2 and its receptor, as well as several other lymphokines. In addition, cyclosporin A abolished the ability of the NF-AT binding site to activate a linked promoter in transfected mitogen-stimulated T lymphocytes and in lymphocytes from transgenic mice. These results indicate that cyclosporin A either directly inhibits the function of nuclear proteins critical to T lymphocyte activation or inhibits the action of a more proximal member of the signal transmission cascade leading from the antigen receptor to the nucleus.

Base Sequence↗
PubMed1989

Alternatively spliced mRNA encodes a secreted form of human CD8 alpha. Characterization of the human CD8 alpha gene.

We have determined the organization and nucleotide sequence of the gene encoding the human T cell surface glycoprotein CD8 alpha. This gene spans approximately 8 kb and is organized into six exons which encode separate functional domains of the protein. Exon 1 encodes the 5' untranslated region and leader peptide, exon II the Ig V-like region, exon III the hinge-like region, exon IV the transmembrane domain, and exons V and VI the cytoplasmic tail. Alternative splicing that excludes nucleotide sequences from exon IV results in a transcript which encodes a secreted form of the protein. This transcript accounts for approximately 15% of the total CD8 alpha mRNA in human T cell leukemia lines and in normal human tissues. Secreted CD8 alpha protein can be detected in culture supernatants of T cell leukemia lines and PHA-stimulated PBMC by immunoprecipitation with the anti-CD8 alpha mAb OKT8 or with a polyclonal rabbit antiserum specific for the 28 amino acid cytoplasmic domain of CD8 alpha. The secreted CD8 alpha protein forms homodimers; when analyzed by SDS-PAGE, the protein migrates with an apparent molecular mass of 27 or 54 kDa under reducing or non-reducing conditions, respectively. Human secreted CD8 alpha may serve an immunoregulatory role for the interactions of T cells with their targets in vivo.

Amino Acid Sequence↗
PubMed1989

Cytotoxic T cell responses in HLA-A2.1 transgenic mice. Recognition of HLA alloantigens and utilization of HLA-A2.1 as a restriction element.

Previous studies have indicated that the frequency of murine CTL precursors (CTLp) for human class I molecules is one to two orders of magnitude lower than that for murine class I alloantigens, and that this is due to species-specific structural differences between these molecules. Transgenic mice expressing the human class I MHC Ag HLA-A2.1 were used to examine changes in the frequency of class I HLA-specific precursors after T cell differentiation in an HLA-A2.1 positive environment. The HLA-A2.1 gene product was expressed at levels comparable to those of the endogenous H-2Db molecule in thymus, bone marrow, and spleen. By limiting dilution analysis, it was observed that the frequencies of CTLp in transgenic mice responding to the human alloantigens HLA-B7 or HLA-A2.2 were comparable to or lower than those in normal C57BL/6 mice, regardless of whether the Ag was presented on human or murine cells. Thus, expression of a human class I molecule in these animals did not result in an expansion of the number of CTLp specific for other human class I Ag. In addition, the frequency of HLA-A2.1-restricted, influenza specific CTLp was substantially lower than the frequency of H-2b restricted CTLp, indicating a poor utilization of HLA-A2.1 as a restricting element. Finally, the frequencies of CTLp for HLA-A2.1 expressed on syngeneic murine tumor cells were decreased significantly. Thus, expression of HLA-A2.1 in these animals appeared to induced tolerance to this Ag. Interestingly, however, these mice were not tolerant to the HLA-A2.1 molecule expressed on human cells. This indicates that the HLA-A2.1 associated epitopes expressed on murine and human cells differ and suggests that, under these circumstances, HLA-A2.1 acts as a restricting element for human nominal Ag. These results are discussed in the context of current models of T cell repertoire development.

Animals↗