Human CD4 restores normal T cell development and function in mice deficient in murine CD4.

The ability of a human coreceptor to function in mice was investigated by generating human CD4 (hCD4)-expressing transgenic mice on a mouse CD4-deficient (mCD4-/-) background. From developing thymocyte to matured T lymphocyte functions, hCD4 was shown to be physiologically active. By examining the expansion and deletion of specific V beta T cell families in mutated mice with and without hCD4, it was found that hCD4 can participate in positive and negative selection. Mature hCD4 single positive cells also were found in the periphery and they were shown to restore MHC class II-restricted alloreactive and antigen-specific T cell responses that were deficient in the mCD4 (-/-) mice. In addition, these hCD4 reconstituted mice can generate a secondary immunoglobulin G humoral response matching that of mCD4 wild-type mice. The fact that human CD4 is functional in mice and can be studied in the absence of murine CD4 should facilitate studies of human CD4 activity in general and human immunodeficiency virus 1 gp120-mediated pathogenesis in acquired immune deficiency syndrome specifically.

Locus control region function and heterochromatin-induced position effect variegation.

Human CD2 locus control region (LCR) sequences are shown here to be essential for establishing an open chromatin configuration. Transgenic mice carrying an hCD2 mini-gene attached only to the 3' CD2 transcriptional enhancer exhibited variegated expression when the transgene integrated in the centromere. In contrast, mice carrying a transgene with additional 3' sequences showed no variegation even when the latter integrated in centromeric positions. This result suggests that LCRs operate by ensuring an open chromatin configuration and that a short region, with no enhancer activity, functions in the establishment, maintenance, or both of an open chromatin domain.

Induction of insulitis by glutamic acid decarboxylase peptide-specific and HLA-DQ8-restricted CD4(+) T cells from human DQ transgenic mice.

Insulin-dependent diabetes mellitus in humans is linked with specific HLA class II genes, e.g., HLA-DQA1*0301/ DQB1*0302 (DQ8). To investigate the roles of HLA-DQ8 molecules and glutamic acid decarboxylase (GAD) in disease development, we generated DQ8(+)/I-Abo transgenic mice expressing functional HLA-DQ8 molecules and devoid of endogenous mouse class II. DQ8(+)/I-Abo mice produced antigen-specific antibodies and formed germinal centers after immunization with GAD65 peptides. Two GAD peptide-specific (247-266 and 509-528), DQ8 restricted Th1 CD4(+) T cell lines, were generated from immunized DQ8(+)/I-Abo mice. They induced severe insulitis after adoptive transfer into transgene positive (but not negative) mice who were treated with a very low dose of streptozotocin that alone caused no apparent islet pathology. In addition to CD4, islet mRNA from these mice also showed expression of CD8, IFNgamma, TNFalpha, Fas, and Fas ligand. Our data suggest that a mild islet insult in the presence of HLA-DQ8 bearing antigen-presenting cells promotes infiltration of GAD peptide reactive T cells into the islet.

Retroviral-mediated gene transfer. Applications in neurobiology.

There are now many examples of the successful expression of genes transduced by retroviruses in studies from outside the field of neuroscience. Retroviruses will undoubtedly also prove to be effective tools for neuro-scientists interested in expressing cloned neurotransmitter and receptor genes. There are also other less obvious applications of retroviruses, such as their insertional mutagenic effects, which may be useful in studies of the genetic factors and biochemical mechanisms involved in, for example, neurotoxicity. Strong cellular promoters have been identified by retroviral infection and subsequent rescue of the flanking genomic DNA. Retroviruses can be employed again to reintroduce these regulatory sequences back into cells. In this way the complexities of gene expression in the many subpopulations of neurons may be unraveled. Retroviruses can also serve as very useful genetic markers in studies of development and lineage relationships. Retroviruses may be used to efficiently transfer oncogenes into neuronal cells to create new cell lines. This application exploits one of the natural traits of retroviruses--oncogenesis--which led to their original discovery. Finally, there are neurotropic retroviruses that could serve as important vectors for delivering genes into neurons. Studying these retroviruses may lead to an understanding of how they cause neuropathologic changes in the CNS.

The GABAA receptor and its antibodies.

The GABAA/benzodiazepine receptor has been purified to homogeneity from bovine and rat cerebral cortex. Under optimum conditions, the purified receptor has been shown to possess four distinct drug-binding sites, for GABA, benzodiazepine, barbiturate and Cl- channel gating classes of ligands. The receptor is a multi-subunit membrane glycoprotein with an oligomeric size of 230,000 Da. It contains at least two subunits, alpha and beta, the first of which can be photoaffinity-labelled with the benzodiazepine, flunitrazepam. Polyclonal and monoclonal antibodies have been raised to the native receptor and both have been used in an immunological characterization of the receptor protein in detergent extracts and likewise in purified preparations from bovine cerebral cortex.

Reconstitution of the purified gamma-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex into phospholipid vesicles.

The purified gamma-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex has been reconstituted into phospholipid vesicles by a cholate dialysis procedure. The reconstituted receptor bound [3H]flunitrazepam at a single class of sites with dissociation constant Kd = 2.9 +/- 0.3 nM, an increase in affinity to the membrane level from the 4-fold weakening found in detergent solution. It also bound [3H]muscimol with a Kd for the high-affinity sites of approximately 50 nM. [35S]tert.-Butyl-bicyclophosphorothionate, for which there is evidence in membranes for binding to a channel gating site on this receptor, showed similar binding to the reconstituted receptor.

Identification of thymus specific and developmentally regulated genes by an improved version of the mRNA differential display technique.

During embryogenesis in mouse, the thymus is seeded by waves of hematopoietic stem cells that provide the first peripheral T lymphocytes after birth. It is known that embryo thymocytes and adult thymocytes have different phenotypic and functional features. The identification of genes expressed in the thymus only during embryogenesis would help to understand the molecular basis underlying these characteristics. We used the mRNA differential display technique to compare gene expression between thymus and kidney from embryo (171/2 days) and adult mice. This technique is the method of choice for comparing gene expression because it is able to display rapidly and simultaneously the mRNA complement from several different types of cells. The major drawback of the method is that it leads to the cloning of many false positives and therefore needs a high throughput method to screen for the truly differentially expressed cDNAs. We combined advantages from previously described methods in order to develop a new version of the mRNA differential display technique that is fast, cheap, and reliable. Instead of oligo dT priming, we used random hexameres for the reverse transcription of total RNA and 10-mer primers for the amplification of internal parts of the cDNAs. We obtained reproducible and clean patterns of discrete bands. We were able to easily identify DNAs differentially amplified between embryo and adult tissues (embryo specific; E 58.73), between thymus and kidney (thymus specific; Thy 52.54), or between embryo and adult thymus (embryo thymus specific; E Thy 58.73) cDNA fragments. After reamplification, cloning, and sequencing of these DNA fragments, it appeared that in most cases, one band corresponded to a single DNA sequence. On a northern blot, each of these candidate genes recognized a transcript that is differentially expressed as expected. Thus, we report an optimized, reproducible, and fast mRNA differential display method that overcomes the usual problems met with the originally described technique or its reported modifications.

In vitro negative selection of alpha beta T cell receptor transgenic thymocytes by conditionally immortalized thymic cortical epithelial cell lines and dendritic cells.

We have established conditionally immortalized thymic cortical epithelial cell lines from transgenic mice carrying a temperature-sensitive SV40 large T antigen. One of these cell lines expresses cortical markers and produces IL-1 alpha, IL-6, IL-7, and TGF-beta 1. These cells express class I major histocompatibility complex (MHC) constitutively and class II MHC upon induction with IFN-gamma. The cells appear to have a normal class I antigen presenting pathway since messages for both peptide transporter genes (TAP1, TAP2) were detected. The ability of these cortical epithelial cells to present peptide antigen was compared to that of thymic dendritic cells. In suspension culture with alpha beta T cell receptor (TcR) transgenic thymocytes, these epithelial cells and dendritic cells (pre-pulsed with peptide cognate for the transgenic TcR) caused down-regulation of CD4, CD8, and TcR in an antigen dose-dependent and MHC-restricted manner. CD4dullCD8dull cells were taken as evidence for negative selection because these cells contained apoptotic DNA. Concentration of peptide required for negative selection of thymocytes was similar between dendritic cells and cortical epithelial cells. In contrast, alpha beta TcR transgenic spleen cells were activated only by dendritic cells but not by cortical epithelial cells.

T cell activation results in physical modification of the mouse CD8 beta chain.

The T lymphocyte glycoprotein, CD8, is an essential component of the response of class I MHC-restricted T cells to Ag. CD8 is expressed on the surface of class I-restricted T cells as disulfide-bonded heterodimers and higher multimers of two distantly related polypeptides, alpha and beta. The CD8 alpha polypeptide, expressed in transfection studies as homodimers, is able to reproduce both the adhesive and stimulatory properties of CD8, leaving the function of the CD8 beta polypeptide unresolved. Herein we demonstrate that the CD8 beta polypeptide changes physically during T cell maturation and activation by reversibly altering its sialic acid content. These changes occur specifically on CD8 beta not -alpha, indicating that the primary role of the CD8 beta chain may be regulatory, influencing the physical structure of the CD8 complex, and suggesting a novel mechanism of controlling receptor/ligand interactions.

The GABAA/benzodiazepine receptor is a heterotetramer of homologous alpha and beta subunits.

The GABAA receptor has been purified to homogeneity from bovine cerebral cortex. Under stringent conditions of isolation, the GABAA receptor was shown to consist only of alpha (Mr 53 000) and beta (Mr 57 000) subunits. A densitometric scan of SDS-PAGE gels under reducing conditions showed that these subunits were present in a 1:1 ratio. A model of the receptor as a heterologous tetramer alpha 2 beta 2 is proposed. Monoclonal antibodies have been raised to the purified bovine GABAA receptor. One of these antibodies, 1A6, was shown to react with both the alpha and beta subunits of the purified receptor. The subunits were still positive in immunoblots following the removal of the carbohydrate moieties of the respective polypeptides by endoglycosidase F treatment. This antibody has been employed to demonstrate antigenic cross-reactivity between the GABAA receptors of three vertebrate species. It is further proposed that there is partial amino acid sequence homology between the alpha and beta polypeptides and hence that they are derived from a single ancestral gene.

Molecular size of the gamma-aminobutyric acidA receptor purified from mammalian cerebral cortex.

The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method.

Thymic selection and peptide-induced activation of T cell receptor-transgenic CD8 T cells in interleukin-2-deficient mice.

The requirement for interleukin-2 (IL-2) in repertoire selection and peripheral activation of CD8 T cells was tested in mice rendered IL-2 deficient by gene targeting and expressing a transgenic T cell receptor (TcR) (F5) specific for influenza nucleoprotein (NP) 366-374 + H-2Db. Positive selection of the transgenic F5 TcR into the CD8 compartment proceeded normally. Both in vivo and in vitro, the antigenic peptide induced depletion of immature thymocytes and proliferation of mature CD8 T cells regardless of the presence of an intact IL-2 gene. In contrast, cytotoxic T lymphocyte (CTL) activity was only generated by T cells from IL-2+ F5 transgenic mice. Exogenous IL-2 was able to fully restore the CTL response of IL-2-/- responder cells in vitro. Thus, both in vivo and in vitro, clonal expansion of CD8 T cells can proceed in the absence of IL-2, whereas in peptide-immunized F5 transgenic mice, induction of cytotoxic effector function is IL-2 dependent.

Antibodies recognising the GABAA/benzodiazepine receptor including its regulatory sites.

Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.

Expression in vivo of CD45RA, CD45RB and CD44 on T cell receptor-transgenic CD8+ T cells following immunization.

We used mice transgenic for a major histocompatibility complex class I-restricted T cell receptor to study the changes of phenotype in vivo which follow priming by antigen of CD8 T cells. We show that following priming with peptide, CD44 on CD8 T cells is up-regulated. The change of phenotype was relatively stable, as primed CD8 cells isolated from thymectomized mice 6 weeks after priming still expressed increased levels of CD44. CD8 T cells in these mice are still responsive to peptide and could represent long-lived primed cells. No down-regulation in vivo of the CD45RA or CD45RB isoforms was found, indicating that there is a differential regulation of the expression of CD44 and CD45RB by activated CD8 transgenic T cells. These results contradict earlier studies in vitro which showed that CD8 T cells which have been primed earlier belong to the CD45RA- or CD45RB- subset.

A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex.

The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.

Deletion analysis of the human CD2 gene locus control region in transgenic mice.

Genomic sequences located at the 3' flanking region of the human CD2 gene confer high level tissue-specific, position-independent expression of the gene when introduced in the germ line of mice. In order to further characterize these sequences a range of deletions, from the 3' end were produced and transgenic mice were generated with the human CD2 (hCD2) gene linked to these deleted fragments. This allowed us to establish the minimum sequences necessary for the copy-dependent transgene expression. 2.1 kb or 1.5 kb of 3' flanking sequences linked to a hCD2 mini-gene is sufficient to allow T-cell specific, copy-dependent, integration-independent expression in transgenic mice. 1.1 kb of 3' sequences results in the gene being expressed in a T-cell specific manner, but copy-dependent, integration-independent expression was not observed in a small number of transgenic animals. 0.2 or 0.5 kb of 3' flanking sequences were insufficient to allow expression above the level previously found with a human CD2 gene which lacked 3' flanking sequences. We conclude that the Locus Control Region (LCR) effect is caused by 1.5 kb of flanking sequences immediately 3' to the polyadenylation signal of the gene.

T cell unresponsiveness correlates with quantitative TCR levels in a transgenic model.

Two lines of transgenic mice were developed which differ in their level of expression of V beta 11. To determine the role of TCR density in tolerance induction, these mice were bred with I-E expressing mice and were investigated for tolerance induction. T cells expressing V beta 11 at high density are deleted by negative selection. T cells expressing < 10% of the normal TCR receptor density are not subject to negative selection and were not activated in vitro. There is a correlation in receptor density in both strains of mice with in vitro activation. These findings support the notion that there is a definable quantitative signal threshold which is critical for tolerance induction.