Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as “sentinels” of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: Sensitivity of human thymocytes

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.

Host microarray analysis reveals a role for the Salmonella response regulator phoP in human macrophage cell death

Bacterial pathogens manipulate host cells to promote pathogen survival and dissemination. We used a 22,571 human cDNA microarray to identify host pathways that are affected by the Salmonella enterica subspecies typhimurium phoP gene, a transcription factor required for virulence, by comparing the expression profiles of human monocytic tissue culture cells infected with either the wild-type bacteria or a phoP∷Tn10 mutant strain. Both wild-type and phoP∷Tn10 bacteria induced a common set of genes, many of which are proinflammatory. Differentially expressed genes included those that affect host cell death, suggesting that the phoP regulatory system controls bacterial genes that alter macrophage survival. Subsequent experiments showed that the phoP∷Tn10 mutant strain is defective for killing both cultured and primary human macrophages but is able to replicate intracellularly. These experiments indicate that phoP plays a role in Salmonella-induced human macrophage cell death.

A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans

The Deleted in AZoospermia (DAZ) genes encode potential RNA-binding proteins that are expressed exclusively in prenatal and postnatal germ cells and are strong candidates for human fertility factors. Here we report the identification of an additional member of the DAZ gene family, which we have called BOULE. With the identification of this gene, it is clear that the human DAZ gene family contains at least three members: DAZ, a Y-chromosome gene cluster that arose 30–40 million years ago and whose deletion is linked to infertility in men; DAZL, the “father” of DAZ, a gene that maps to human chromosome 3 and has homologs required for both female and male germ cell development in other organisms; and BOULE, a gene that we propose is the “grandfather” of DAZ and maps to human chromosome 2. Human and mouse BOULE resemble the invertebrate meiotic regulator Boule, the proposed ortholog of DAZ, in sequence and expression pattern and hence likely perform a similar meiotic function. In contrast, the previously identified human DAZ and DAZL are expressed much earlier than BOULE in prenatal germ stem cells and spermatogonia; DAZL also is expressed in female germ cells. These data suggest that homologs of the DAZ gene family can be grouped into two subfamilies (BOULE and DAZL) and that members of the DAZ family evolved from an ancestral meiotic regulator, Boule, to assume distinct, yet overlapping, functions in germ cell development.

Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells

The effects of Escherichia coli heat-stable enterotoxin (ST) and uroguanylin were examined on the proliferation of T84 and Caco2 human colon carcinoma cells that express guanylyl cyclase C (GC-C) and SW480 human colon carcinoma cells that do not express this receptor. ST or uroguanylin inhibited proliferation of T84 and Caco2 cells, but not SW480 cells, in a concentration-dependent fashion, assessed by quantifying cell number, cell protein, and [3H]thymidine incorporation into DNA. These agonists did not inhibit proliferation by induction of apoptosis, assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dNTP-biotin nick end labeling of DNA fragments) assay and DNA laddering, or necrosis, assessed by trypan blue exclusion and lactate dehydrogenase release. Rather, ST prolonged the cell cycle, assessed by flow cytometry and [3H]thymidine incorporation into DNA. The cytostatic effects of GC-C agonists were associated with accumulation of intracellular cGMP, mimicked by the cell-permeant analog 8-Br-cGMP, and reproduced and potentiated by the cGMP-specific phosphodiesterase inhibitor zaprinast but not the inactive ST analog TJU 1-103. Thus, GC-C agonists regulate the proliferation of intestinal cells through cGMP-dependent mechanisms by delaying progression of the cell cycle. These data suggest that endogenous agonists of GC-C, such as uroguanylin, may play a role in regulating the balance between epithelial proliferation and differentiation in normal intestinal physiology. Therefore, GC-C ligands may be novel therapeutic agents for the treatment of patients with colorectal cancer.

A low threshold level of expression of mutant-template telomerase RNA inhibits human tumor cell proliferation

The ribonucleoprotein telomerase synthesizes telomeric DNA by copying an intrinsic RNA template. In most cancer cells, telomerase is highly activated. Here we report a telomerase-based antitumor strategy: expression of mutant-template telomerase RNAs in human cancer cells. We expressed mutant-template human telomerase RNAs in prostate (LNCaP) and breast (MCF-7) cancer cell lines. Even a low threshold level of expression of telomerase RNA gene constructs containing various mutant templates, but not the control wild-type template, decreased cellular viability and increased apoptosis. This occurred despite the retention of normal levels of the endogenous wild-type telomerase RNA and endogenous wild-type telomerase activity and unaltered stable telomere lengths. In vivo tumor xenografts of a breast cancer cell line expressing a mutant-template telomerase RNA also had decreased growth rates. Therefore, mutant-template telomerase RNAs exert a strongly dominant-negative effect on cell proliferation and tumor growth. These results support the potential use of mutant-template telomerase RNA expression as an antineoplastic strategy.

Functional cell surface expression of the anion transport domain of human red cell band 3 (AE1) in the yeast Saccharomyces cerevisiae.

We expressed the 52-kDa integral membrane domain (B3mem) of the human erythrocyte anion transporter (band 3; AE1) in a protease-deficient strain of the yeast Saccharomyces cerevisiae under the control of the inducible GAL10-CYC1 promoter. Immunoblots of total protein from transformed yeast cells confirmed that the B3mem polypeptide was overexpressed shortly after induction with galactose. Cell surface expression of the functional anion transporter was detected by using a simple transport assay to measure stilbene disulfonate-inhibitable chloride influx into intact yeast cells. The B3mem polypeptide was recycled and degraded by the cells with a half-life of approximately 1-3 hr, which led to a steady-state level of expression in exponentially growing cultures. Our data suggest that 5-10% of total B3mem is functionally active at the cell surface at any one time and that overexpression of this anion transport protein does not interfere with cell growth or survival. This is one of only a few reports of the functional expression of a plasma membrane transport protein in the plasma membrane of yeast cells and to our knowledge is the first report of red cell band 3-mediated anion transport at the plasma membrane of cDNA-transformed cells. The cell surface expression system we describe will provide a simple means for future study of the functional properties of band 3 by using site-directed mutagenesis.

Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations.

A small percentage of human T lymphocytes, predominantly CD8+ T cells, express receptors for HLA class 1 molecules of natural killer type (NK-R) that are inhibitory for T-cell antigen receptor (TCR)-mediated functions. In the present study, it is demonstrated that the various NK-R molecules typically expressed by NK cells are also expressed on periheral blood T lymphocytes. These CD3+ NK-R+ cells have a cell surface phenotype typical of memory cells as indicated by the expression of CD45RO and CD29 and by the lack of CD28 and CD45RA. Furthermore, by the combined use of anti-TCR V beta-specific antibodies and a semiquantitative polymerase chain reaction assay, the TCR repertoire in this CD3+ NK-R+ cell subset was found to be skewed; in fact, one or two V beta families were largely represented, and most of the other V beta s were barely detected. In addition, analysis of recombinant clones of the largely represented V beta families demonstrated that these V beta s were oligoclonally or monoclonally expanded.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.

We have generated a human 293-derived retroviral packaging cell line (293GPG) capable of producing high titers of recombinant Moloney murine leukemia virus particles that have incorporated the vesicular stomatitis virus G (VSV-G) protein. To achieve expression of the retroviral gag-pol polyprotein, the precise coding sequences for gag-pol were introduced into a vector which utilizes totally nonretroviral signals for gene expression. Because constitutive expression of the VSV-G protein is toxic in 293 cells, we used the tetR/VP 16 transactivator and teto minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G. After stable transfection of the 293GPG packaging cell line with the MFG.SnlsLacZ retroviral vector construct, it was possible to readily isolate stable virus-producing cell lines with titers approaching 10(7) colony-forming units/ml. Transient transfection of 293GPG cells using a modified version of MFG.SnlsLacZ, in which the cytomegalovirus IE promoter was used to drive transcription of the proviral genome, led to titers of approximately 10(6) colony-forming units/ml. The retroviral/VSV-G pseudotypes generated using 293GPG cells were significantly more resistant to human complement than commonly used amphotropic vectors and could be highly concentrated (> 1000-fold). This new packaging cell line may prove to be particularly useful for assessing the potential use of retroviral vectors for direct in vivo gene transfer. The design of the cell line also provides at least theoretical advantages over existing cell lines with regard to the possible release of replication-competent virus.

p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex.

Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand. The gene is also necessary for repair of damaged DNA in yeast. We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer. The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1. Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed. This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1. Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo. Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.

Human CD100, a novel leukocyte semaphorin that promotes B-cell aggregation and differentiation.

Herein we describe the molecular characterization of the human leukocyte activation antigen CD100 and identify it as the first semaphorin, to our knowledge, in the immune system. Semaphorins have recently been described as neuronal chemorepellants that direct pioneering neurons during nervous system development. In this study we demonstrate that CD100 induces B cells to aggregate and improves their viability in vitro. We show that CD100 modifies CD40-CD40L B-cell signaling by augmenting B-cell aggregation and survival and down-regulating CD23 expression. Thus, these results suggest that semaphorins as exemplified by CD100 also play a functional role in the immune system.

Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.

Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.