TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4-8+ T cells.

The transporter associated with the antigen processing 1 (TAP1) gene encodes a subunit for a transporter, presumed to be involved in the delivery of peptides across the endoplasmic reticulum membrane to class I molecules. We have generated mice with a disrupted TAP1 gene using embryonic stem cell technology. TAP1-deficient mice are defective in the stable assembly and intracellular transport of class I molecules and consequently show severely reduced levels of surface class I molecules. These properties are strikingly similar to those described for the TAP2 mutant cell line RMA-S. Cells from the TAP1-deficient mice are unable to present cytosolic antigens to class I-restricted cytotoxic T cells. As predicted from the near absence of class I surface expression, TAP1-deficient mice lack CD4-8+ T cells.     

T cell-activating protein on murine lymphocytes

A functional T cell surface molecule, T cell-activating protein (TAP), has been identified on murine lymphocytes. TAP is a protein with an apparent molecular mass of 10-12 kilodaltons (kDa) nonreduced, 16-17 kDa reduced. Cross-linking of TAP can result in profound activation of T lymphocytes to produce lymphokines and to enter the cell cycle. Furthermore, antibody binding to TAP can modulate antigen-driven T cell stimulation. Current data suggest that TAP is physically distinct from the T cell receptor complex. On unstimulated lymphocytes, TAP is expressed on T cells and defines heterogeneity within the major T cell subsets. Its profile of expression is rapidly altered on cell activation. Ontologically, it is first detected in the thymus, where it is found on both the most immature and the most mature cell subsets, and it is functional on both. Together, these TAP+ cells constitute a small fraction of thymocytes. TAP expression, however, defines the immunocompetent compartment of the thymus, and thus could be involved in functional maturation. Finally, the gene controlling TAP expression has been mapped, and is tightly linked to a locus of hematopoietic antigens (Ly-6). TAP is molecularly distinct from these antigens. Furthermore, all of these proteins show a markedly distinct developmental regulation in their cell surface expression.     

Internalization of phosphatidylinositol-anchored lymphocyte proteins. I. Documentation and potential significance for T cell stimulation

Several proteins that are anchored to the surface of T lymphocytes via a phosphatidylinositol (PI) moiety can initiate cell stimulation upon cross-linking. Inasmuch as these proteins do not traverse the plasma membrane, it is not clear how they are capable of signaling across the membrane. Herein we report two distinct sets of experiments that examine the consequence of cross-linking PI-anchored molecules on murine T cells. We first analyzed the fate of antibody cross-linked TAP (Ly-6A.2) and Thy-1 molecules on T-T hybrids. Using an assay to measure receptor-mediated endocytosis, an intracellular accumulation of 125I labeled anti-TAP and anti-Thy-1 mAb was documented that was specific and Ag dependent. The internalization of these molecules was confirmed by cytotoxicity assays using antibody-toxin conjugates, and electron microscopic studies. Although the PI-anchored proteins lack a cytoplasmic domain that is necessary for the internalization of many receptors, they nevertheless can be induced to enter the cell upon cross-linking. The rate of entry of cross-linked TAP and Thy-1 into cells was shown to be 10 and 2% per hour, respectively, which is considerably less than that observed for the transferrin receptor or TCR/CD3 complex. To assess whether the internalization of TAP and Thy-1 might be of importance in their ability to stimulate T cells, we attempted to cross-link these molecules under conditions where the mAb or its cross-linked complex can not enter the cell. We observed that anti-TAP and anti-Thy-1 mAb conjugated to a cell impermeant matrix fail to stimulate T cells. This loss of stimulatory activity was observed with multiple T-T hybridomas and mAb over a wide titration of antibody concentration and was independent of the mAb isotype. Results from experiments with anti-Ig cross-linking of the mAb-PI anchored protein complex suggested that the loss of T cell stimulation upon mAb immobilization is not simply due to an alteration in the degree of antibody cross-linking. These findings were generalized to three distinct PI-anchored proteins: TAP, Thy-1, and Ly6C on normal T cells. When the same cells were stimulated through the TCR/CD3 complex, only immobilized mAb are stimulatory. These results demonstrate a marked difference in the cross-linking requirements for stimulating T cells through PI-anchored molecules in contrast to the transmembrane TCR complex. Furthermore, these findings raise the possibility that molecular internalization of Ab-PI-anchored complexes may be necessary in signaling through these molecules.     

Reversion of the transformed phenotype of B16 mouse melanoma: involvement of an 83 kd cell surface glycoprotein in specific growth inhibition

Treating B16 mouse melanoma cells with monoclonal antibody NORM-2 reduces cell growth in tissue culture, agar, and syngeneic mice. We show that the NORM-2 antibody recognizes an integral 83 kd glycoprotein that is mobile in the plane of the plasma membrane of B16 melanoma cells. Expression of the glycoprotein is reduced under conditions that inhibit B16 growth, such as low serum, high cell density, and addition of transforming growth factor-beta. The glycoprotein reappears during S phase, when growth-arrested cells are restimulated. The NORM-2 antigen appears to be involved in growth regulation of B16 melanoma cells both in vitro and in vivo.     

Identification of the components of the murine T cell antigen receptor complex

In addition to the alpha and beta chains of the MHC class II restricted antigen receptor, monoclonal anti-receptor antibodies coprecipitate four polypeptides that appear to be noncovalently associated with the alpha-beta dimer of murine T cells. Included in the murine T cell antigen receptor complex are two glycoproteins of 25 kd (gamma) and 21 kd (delta) and two nonglycosylated polypeptides of 26 kd (epsilon) and 16 kd (zeta). The epsilon chain appears to possess an intrachain disulfide bond and zeta exists in the complex as a disulfide-linked homodimer. The delta chain is phosphorylated on a serine residue in response to T cell activation with antigen. In contrast, both delta and epsilon are phosphorylated in response to treatment of the T cells with phorbol 12-myristate 13-acetate. These polypeptides may play a role in the transduction of the signal(s) in T cell activation.     

Stimulation of T cells via the TAP molecule, a member in a family of activating proteins encoded in the Ly-6 locus

T-cell activating protein, TAP, is a Ly-6 encoded 12,000 dalton glycoprotein involved in the activation of murine T cells. TAP is distinct from other known surface activating structures, such as the T cell receptor/T3 complex and Thy-1. This study investigates the mechanism of activation via the TAP molecule. Soluble alpha TAP monoclonal antibody (MAb) is sufficient for T cell activation. This, however, requires cross-linking because F(ab) monovalent antibody is not stimulatory unless cross-linked by a second antibody. Immediately after cross-linking, there is a rapid influx of calcium, which is comparable with concanavalin A or T cell receptor triggered responses. Subsequently, interleukin 2 (IL 2) is produced, and IL 2 receptors (IL 2R) are expressed. TAP-stimulated T cell proliferation is driven by this autocrine pathway because it is inhibited by alpha IL 2R MAb. Thus TAP-mediated activation appears to share a common final pathway with other mitogenic stimuli. A nonactivating alpha TAP MAb was observed to stimulate T cells upon additional cross-linking. Given this observation, we asked whether other Ly-6 linked proteins might share similar activating potential. We show that at least three distinct Ly-6 linked molecules are capable of stimulating T hybrid clones and/or heterogeneous T lymphocytes. Thus it appears that the Ly-6 locus encodes a family of activating cell surface molecules.     

The expression, function, and ontogeny of a novel T cell-activating protein, TAP, in the thymus

The TAP molecule is an allelic 12,000 m.w. membrane protein that participates in T cell activation. This report analyzes the expression, function, and ontogeny of this molecule in the thymus. TAP is expressed on a small subset (10 to 20%) of thymocytes which is distinct from its expression on a majority (70%) of peripheral T lymphocytes. In the adult thymus, the majority of the TAP-bearing thymocytes are cortisone-resistant, Thy-1+, TL-, J11D-, and PNA-, which localizes TAP expression to medullary thymocytes. Cortical thymocytes do not bear this determinant. Parallel functional studies demonstrated that TAP+ thymocytes are required for Con A and MLR responsiveness. Anti-TAP MAb plus PMA specifically induces proliferation of mature thymocytes comparable in magnitude to the Con A response. These results demonstrate that TAP expression defines the immunocompetent thymocyte compartment and, further, that this molecule is functional on these cells. The ontogeny of TAP expression was also analyzed. TAP is expressed early in fetal thymic development at a time when most T cell markers (except Thy-1 and the iL2-R) are absent. The small sub-population of adult L3T4- and Lyt-2- thymocytes, which resemble early fetal thymocytes, also express TAP. These early thymocytes are capable of being activated through the TAP molecule. The implications of these findings for T cell development and, in particular, the relationship of TAP to T cell receptor expression and acquisition of immunocompetence are discussed.     

T cell receptor/CD3 negative variants are unresponsive to stimulation through the Ly-6 encoded molecule, TAP

TAP is a phosphatidylinositol-anchored Ly-6-encoded, glycoprotein that is involved in the activation of murine T-inducer cells. Anti-TAP antibodies can stimulate T cells directly and also synergize with Ag stimulation. To determine the relationship between TAP and TCR-mediated activation, we have derived TCR/CD3 loss variants of functional T hybrid clones. T-T hybrid variants that have lost TCR expression are not responsive to anti-TAP stimulation. Isolation and analysis of revertant clones, obtained from one of the TCR/CD3-negative mutant cell lines, demonstrate a concordant re-expression of the TCR/CD3 complex and responsiveness to anti-TAP stimulation. A similar loss of responsiveness to TAP stimulation is observed after antibody-induced modulation of the TCR/CD3 complex from the surface of T cell hybrids. In contrast, both the TCR/CD3 loss variants and TCR/CD3-modulated T-T hybrids remain fully responsive to stimulation with a calcium ionophore and phorbol esters. This structure-function correlation has been observed repeatedly in independent isolations of variants, and with TCR/CD3 modulated cells, from two different T-T hybridomas. Given the apparent functional interrelationship between TAP and the TCR/CD3 complex, we have also analyzed if these molecules were significantly associated on the T cell surface. Antibody-induced modulation of the TCR/CD3 complex does not affect the cell surface expression of TAP molecules. Moreover, the expression of these two sets of proteins is also independent as evidenced by the selective loss of either set of these proteins in the TCR/CD3 expression mutants.     

Biosynthesis and membrane topography of the neural cell adhesion molecule L1.

The biosynthesis and membrane topography of the neural cell adhesion molecule L1 have been studied in cerebellar cell cultures by metabolic labeling and immunoprecipitation. Pulse and pulse-chase experiments with [35S]methionine show that L1 is synthesized in its high mol. wt. form, the 200 kd component. The lower mol. wt. components with 40, 80 and 140 K apparent mol. wts. can be generated by proteolysis in intact cellular membranes. Peptide maps generated by protease treatment of L1 isolated from adult mouse brain show that the 80 and 140 kd components are related to the 200 kd component, but not to each other. The 200, 80 and 40 kd components can be biosynthetically phosphorylated. The 140 kd component is not phosphorylated and not released from the surface membrane during tryspinization. The phosphorylated amino acid is serine. In the presence of tunicamycin the 200 kd component is synthesized as a 150 kd protein. Pulse-chase experiments in the presence of tunicamycin indicate that the carbohydrate moieties are predominantly N-glycosidically linked and that the contribution of O-glycosylation is minimal. The carbohydrate moieties are of the complex type as shown by treatment with endoglycosidase H. Since monensin inhibits processing of the carbohydrate moieties, the 200 kd component appears to be transported to the surface membrane via the Golgi apparatus.     

Tapasin interacts with the membrane-spanning domains of both TAP subunits and enhances the structural stability of TAP1 x TAP2 Complexes

The transporter associated with antigen processing (TAP) proteins are involved in transport of peptides from the cytosol into the endoplasmic reticulum. Two subunits, TAP1 and TAP2, are necessary and sufficient for peptide binding and peptide translocation across the endoplasmic reticulum membrane. TAP1 and TAP2 contain an N-terminal hydrophobic membrane-spanning region and a C-terminal nucleotide binding domain. Tapasin is an endoplasmic reticulum resident protein that has been found associated with the TAP subunits and shown to increase expression levels of TAP. Here we investigated TAP-tapasin interactions and their effects on TAP function in insect cells. We show tapasin binding to both TAP1 and TAP2 and to the corresponding nucleotide binding domain-exchanged chimeras as well as to a truncated TAP1.TAP2 complex containing just the membrane-spanning regions of TAP1 and TAP2. However, tapasin interactions with either the truncated TAP construct containing just the nucleotide binding domain are not observed. Tapasin is not required for high affinity peptide binding to TAP1.TAP2 complexes, and in fact, the presence of tapasin slightly reduces the affinity of TAP complexes for peptides. However, at near physiological temperatures, both tapasin and nucleotides stabilize the peptide binding site of TAP1.TAP2 complexes against inactivation, and enhanced thermostability of both TAP subunits is observed in the presence of tapasin. The enhanced structural stability of TAP1.TAP2 complexes in the presence of tapasin might explain the observations that tapasin increases TAP protein expression levels in mammalian cells.     

Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment

The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.     

Tobacco reduces membrane HLA class I that is restored by transfection with transporter associated with antigen processing 1 cDNA

HLA class I molecules are recognized by CTL that eliminate virally infected and malignantly transformed cells presenting foreign peptide-a process termed immunosurveillance. Many tumors have reduced levels of membrane HLA class I. Tumor cells with mutations that reduce HLA class I avoid immunosurveillance and continue to proliferate. As tobacco use can induce tumors, we examined the effect of tobacco extracts on membrane HLA class I. These studies show that culture of cells in media containing tobacco extracts reduces membrane HLA class I, but not other proteins, on primary keratinocytes and other cell types. Culture in tobacco extracts, but not extracts of other substances, reduces TAP1 protein, but does not reduce expression of HLA class I H chain, L chain, or the housekeeping protein beta-actin. The reduction of TAP1 protein occurs within 4 h and is dose-dependent. Culture in tobacco extracts reduces TAP1 protein abundance, but not steady-state mRNA abundance. Tobacco-treated cells show defects in HLA class I biosynthesis similar to those found in TAP1-deficient cell lines. Transfection with TAP1 cDNA restores TAP1 protein abundance, HLA class I biosynthesis, and cell surface expression. Combined, these data show that culture in tobacco extracts reduces TAP1 protein abundance and membrane HLA class I levels. Reduction in membrane HLA class I could permit subsequent malignant transformation of cells to be undetected by the immune system.     

Evidence for an interaction between the cell surface and intermediate filaments in cultured fibroblasts

Intermediate filaments (IF) were found in close proximity to the plasma membrane in substrate attached baby hamster kidney cells (BHK-21) and chick embryo fibroblasts (CEF) as well as cells removed from their substrate in the absence of trypsin. However, in cells removed with trypsin, it appeared that IF had retracted away from the membrane. In cells with abundant extracellular matrix (ECM), colchicine induced massive cables of IF, which appeared to interact with specialized areas of the inner plasma membrane. In cells lysed to extract most microfilaments and cytoplasmic constituents, the intact IF network which remained was closely associated with the ECM. From these ultrastructural observations it was concluded that IF interact in some way with a "cell membrane complex" defined as comprising the plasma membrane and molecules attached to its inner and outer surfaces. In order to investigate the possibility that components of the membrane complex may co-isolate with IF, native intermediate filaments (NIF) were prepared. In addition to the structural subunits and other associated polypeptides, a approximately 220 kd species which reacted specifically with antibodies directed against the ECM protein fibronectin (FN) was observed; 220 kd was still present after NIF were isolated under pH conditions where FN is more soluble, suggesting that its presence was not simply due to the coprecipitation of two insoluble proteins. Immunofluorescence and immunogold localization confirmed that FN is a component of the cell membrane complex with which IF appeared to interact.     

Loss of fibronectin among the selective surface protein changes associated with p-formaldehyde promoted membrane vesiculation

p-formaldehyde-promoted membrane vesiculation of preiodinated cultures lead to the release of surface proteins of about 68 kd in normal and transformed rat liver epithelial cells and rat fibroblasts, concurrent with a marked decrease in surface-associated fibronectin. Membrane vesiculation in the presence of soybean trypsin inhibitor permitted the detection of an 18 kd surface protein in membrane vesicles and the increased expression of a 70 kd external component in normal but not in transformed epithelial cells.Our results show that the membrane vesiculation process is associated both with the release and selective degradation of specific cell surface proteins in a process which may involve surface protease activation. Our data also suggest the potential of the chemical vesiculation process as a probe to monitor differences in surface topography between different cell types.     

Suppression of BCG cell wall-induced delayed-type hypersensitivity by pretreatment with killed BCG: induction of nonspecific suppressor T cells by the adjuvant portion (MDP) and of specific suppressor T cells by the antigen portion (TAP)

We showed previously that antigen-nonspecific suppressor T cells induced by i.v. injection of heat-killed bacillus Calmette-Guérin (BCG) were involved in suppression of delayed-type hypersensitivity (DTH). We suggested that the adjuvant portion of BCG might be involved in the induction of these cells. In this report, we show that BCG cell wall-induced DTH responses in mice pretreated with muramyl dipeptide (MDP), a minimum adjuvant constituent of BCG, were suppressed nonspecifically. In addition, we show that pretreatment with tuberculin active peptide (TAP), the antigenic peptide from Mycobacterium tuberculosis, induces antigen-specific suppression of DTH responses. In both instances, suppression was shown to be due to non-adherent cells that act to inhibit elicitation of DTH. Furthermore, using the macrophage migration inhibition assay, an in vitro correlate of DTH, we found that antigen-nonspecific and antigen-specific suppressor T cells were induced by the injection of MDP and TAP, respectively. Thus, suppressor T cells induced by the adjuvant and antigen portions of BCG may act by interfering with the lymphokine-dependent mechanisms by which DTH effector T cells elicit DTH.     

Exploring the minimal functional unit of the transporter associated with antigen processing

TAP, an ABC transporter in the ER membrane, provides antigenic peptides derived from proteasomal degradation to MHC class I molecules for inspection by cytotoxic T lymphocytes at the cell surface so as to trace malignant or infected cells. To investigate the minimal number of transmembrane segments (TMs) required for assembly of the TAP complex based on hydrophobicity algorithms and alignments with other ABC transporters we generated N-terminal truncation variants of human TAP1 and TAP2. As a result, a 6+6 TM core-TAP complex represents the minimal functional unit of the transporter, which is essential and sufficient for heterodimer assembly, peptide binding, and peptide translocation into the ER. The TM1 of both, core-TAP1 and core-TAP2 are critical for heterodimerization of the complex.     

A novel nucleoskeletal-like protein located at the nuclear periphery is required for the life cycle of Saccharomyces cerevisiae.

In order to study the role of nucleoskeletal components for nuclear and cell division in the budding yeast Saccharomyces cerevisiae, we have employed a combined biochemical/genetic approach. We have identified a peripheral nuclear protein which appears to be located both at the nuclear membrane and the spindle pole body. The gene has been cloned and subsequently shown to be essential for cell growth. The DNA sequence of the gene has been determined. As deduced from the nucleotide sequence, the gene potentially codes for a novel 86 kd protein with a highly repetitive and conserved nine amino acid sequence motive in the middle part of the protein. The flanking amino- and carboxy-terminal regions have similarities to intermediate filaments and calcium binding proteins, respectively. It appears that the 86 kd protein is a regulated nucleoskeletal-like protein (NSP1) involved in the process of nuclear and/or cell division. The affinity-purified antibody against the yeast NSP1 protein stained the nucleus and centrosomes of mammalian MDCK (Madin Darby canine kidney) cells in indirect immunofluorescence.     

Leishmania antigens are presented to CD8+ T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo

CD8+ T cells are generated in response to Leishmania major (Lm) or Toxoplasma gondii parasitic infections, indicating that exogenously delivered Ag can be processed for presentation by MHC class I molecules. We show that presentation of Lm nucleotidase (NT)-OVA is TAP independent in vivo and in vitro, and is inhibited by chloroquine, but not by proteasome inhibitors. In contrast, the presentation of T. gondii P30-OVA relies on the TAP/proteasome pathway. Presentation of OVA- or rNT-OVA-coated beads also bypassed TAP requirement above a certain Ag threshold. TAP was also dispensable for the presentation of wild-type Lm Ags to primed CD8+ T cells in vitro. Finally, in vivo priming of CD8+ T cells involved in acquired resistance to Lm was not compromised in TAP-deficient mice. Thus, Leishmania Ags appear to be confined to an intraphagosomal processing pathway that requires higher concentrations of Ags, suggesting that these parasites may have evolved strategies to impair the efficient endoplasmic reticulum-based, TAP-dependent cross-presentation pathway to avoid or delay CD8+ T cell priming.