Biomedical subjects

F Momburg

Publications and source records attributed to F Momburg.

At least 55 records · Page 3Linked to original sources

PubMed1994

Selectivity of MHC-encoded peptide transporters from human, mouse and rat.

Major histocompatibility complex (MHC) class I molecules present peptides from degraded intracellular antigens to CD8+ T cells. These peptides are translocated in an ATP-dependent fashion into the lumen of the endoplasmic reticulum (ER) for binding to class I molecules by means of the MHC-encoded transporters associated with antigen processing, TAP1 and TAP2. These are members of a family of proteins containing an ATP-binding cassette and form heterodimers in the ER membrane. Defects in the genes encoding TAP1 or TAP2 account for impaired class I assembly and antigen presentation in several human and rodent cell lines. Whereas MHC class I molecules select peptides according to binding motifs, it is not clear to what extent the TAP1-TAP2 transporters have peptide sequence and length specificity. Previous studies of the rat MHC class I molecule RT1Aa, suggested a specific conveyance of peptides by rat TAP1-TAP2. Here we substitute the amino- and carboxy-terminal and the penultimate amino-acid residues of model peptides to show that these residues influence the efficiency of transport. Human TAP and rat TAPa translocated peptides with hydrophobic and basic C termini, whereas mouse TAP and rat TAPu preferred peptides with hydrophobic C termini. This pattern correlates with the predominant peptide binding profiles of mouse and human class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Resistance to natural killer cell lysis conferred by TAP1/2 genes in human antigen-processing mutant cells.

The human Ag-processing-defective cell line 721.174, and its derivative T2, have a large homozygous deletion in the MHC class II region encompassing the TAP1 and TAP2 as well as the LMP2 and LMP7 genes. A similar, but smaller defect encompassing the TAP1 gene is observed in the 721.134 mutant cell line. With a few recent exceptions, these lines are unable to present endogenous Ag to class I-restricted CD8+ CTL. However, restoration of Ag processing and presentation to CD8+ cytotoxic T cells can be achieved by transfecting TAP1 and TAP2 genes into the T2 and .174 cells, or the TAP1 gene into .134 cells. In contrast to their resistance to MHC class I-restricted CTL-mediated lysis, T2 and .174 as well as .134 cells are highly sensitive to lysis by human NK cells. In the present study, we demonstrate the reversal of the NK-sensitive phenotype of these mutant cell lines upon transfection of TAP genes. The induced NK cell-resistant phenotype, studied in detail with TAP1/2-transfected T2 cells, appeared independent of the MHC class I haplotype of the NK cell donors and was also observed with xenogeneic NK cells. The resistance to NK cell lysis induced by re-expression of TAP1/2 seemed to be independent of the origin of the TAP genes; both human and rat transporters efficiently conferred NK cell resistance. Transfection of single TAP genes to T2 or .174 cells, whether TAP1 or TAP2, did not markedly affect NK cell susceptibility. This indicates that an intact TAP1/2 dimer, and thus efficient peptide translocation into the ER, is necessary for rendering T2 and .174, as well as .134 cells, resistant to NK cell-mediated lysis. Proper translocation of peptides across the ER membrane and loading to class I molecules may represent critical events in rendering an NK-resistant phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Presentation of viral antigens restricted by H-2Kb, Db or Kd in proteasome subunit LMP2- and LMP7-deficient cells.

In the class II region of the major histocompatibility complex (MHC(, four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP1 and TAP2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP2 and LMP7) code for subunits of the proteasome. While TAP1 and TAP2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP1/2 and LMP2/7 genes, it was recently shown that expression of TAP1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP2/7, we transfected T2 cells with TAP1, TAP2 and either of the H-2Kb, Db or Kd genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP1/2 gene products, presented all tested viral antigens restricted through either the H-2Kb, Db or Kd class I molecules. We conclude that the proteasome subunits LMP2/7 as well as other gene products in the MHC class II region, except from TAP1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.

Animals↗

PubMed1994

Peptide selection by MHC-encoded TAP transporters.

With the discovery of MHC-encoded peptide transporters (TAP) came the identification of a new class of molecules within the immune system. TAP belongs to a large family of ATP-binding, multimembrane-spanning transporters that are expressed in a diversity of cells, from prokaryotic to mammalian, and show specificity for a variety of different substrates. TAP represents the solution to a major topological problem in immunology, namely the translocation of peptides, generated by cytosolic degradation of antigens, into the lumen of the endoplasmic reticulum where they associate with newly synthesized MHC class I molecules. A novel assay allows us to determine the requirements for the TAP-mediated peptide transport. First results indicate that TAP preselects peptides according to sequence and length in a way that is compatible with the characteristics of peptides isolated from class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Peptide transporters and antigen processing.

Our knowledge about intracellular pathways involved in the presentation of antigens was considerably broadened with the recent discovery of peptide transporters encoded in the major histocompatibility complex. The transporter associated with antigen processing (TAP) belongs to an evolutionary conserved family of multimembrane-spanning translocators that bind ATP and show specificity for a variety of different substrates. TAP mediates the translocation of peptides, generated by cytosolic degradation of protein antigens, into the lumen of the endoplasmic reticulum where they bind to newly synthesized MHC class I molecules. A novel assay has been employed to elucidate the details of TAP-mediated peptide transport. The results indicate that TAP selects peptides of sequence and length according to the requirements of MHC class I molecules in different species.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Folding and assembly of major histocompatibility complex class I heterodimers in the endoplasmic reticulum of intact cells precedes the binding of peptide.

Major histocompatibility complex (MHC) class I molecules are heterotrimers consisting of a polymorphic H chain, beta 2-microglobulin (beta 2m) and peptide. Peptides are thought to associate early during biosynthesis but the order of assembly of class I molecules from their component subunits in intact cells is not settled. We have studied the assembly of MHC class I molecules in intact cells with or without peptide transporters. MHC class I H chain/beta 2m heterodimers can be efficiently recovered only 4 min after translation and are preceded by a folding intermediate. Approximately 2 min after their formation, the class I heterodimers are loaded with peptides resulting in stable class I heterotrimers. In these in vivo studies, no evidence was obtained that peptide binding to the H chain preceded the association with beta 2m. In contrast, nonassembled class I H chains could be recovered immediately after translation, but this pool did not participate in the formation of class I molecules.

Biological Transport↗

PubMed1993

Epitope-specific enhancement of antigen presentation by invariant chain.

The MHC class II-associated invariant chain (Ii) is involved in the intracellular sorting of class II molecules to the endocytic pathway where peptides from processed exogenous antigens are bound, and thereby Ii is thought to enhance antigen presentation. Here we demonstrate that presentation of only one out of five epitopes of a given antigen is augmented by Ii. We have compared the presentation of five different epitopes derived from hen egg white lysozyme (HEL) to Ak-restricted T hybridomas by rat-2 fibroblasts transfected with A alpha k and A beta k (RKK) and RKK cells supertransfected with the mouse invariant chain (RKKI). Only the presentation of the HEL epitope 46-61 was enhanced whereas the presentation of the HEL epitopes 25-43, 34-45, 112-124, and 116-129 was unchanged or even slightly diminished in RKKI cells. The presentation of the epitopes 25-43 and 34-45 was virtually insensitive to the lysosomotropic reagent chloroquine. Brefeldin A (BFA), which inhibits protein egress from the endoplasmic reticulum, blocked the presentation of all epitopes tested in RKKI cells. In contrast, in Ii-negative RKK cells only the presentation of the epitope HEL(46-61) was inhibited by BFA and the presentation of the epitopes 25-43 and 34-45 was only slightly impaired. These findings suggest that Ii may target class II molecules to selected endosomal subcompartments involved in the processing of different peptides derived from an endocytosed antigen. As a result, the enhancement of the class II-restricted presentation in Ii expressing cells appears to be epitope specific rather than antigen specific.

Animals↗

PubMed1993

Processing of endogenously synthesized hen egg-white lysozyme retained in the endoplasmic reticulum or in secretory form gives rise to a similar but not identical set of epitopes recognized by class II-restricted T cells.

To study the processing and presentation of endogenously synthesized Ag to class II MHC-restricted T cells, hen egg lysozyme (HEL), either tagged with a peptide that confers retention in the endoplasmic reticulum (HEL.KDEL), or in the secretory form (HELs), was stably expressed in LK-35.2 B hybridoma cells. Presentation of HEL peptides bound to class II molecules was assessed by activation of specific T cell hybridomas recognizing seven different epitopes derived from exogenous HEL. The presentation of endogenously synthesized HEL was not caused by reuptake of secreted of shed Ag. All the HEL epitopes examined were efficiently presented after processing of endogenous HEL by HELs-transfected LK-35.2 cells. Processing of HEL tagged with KDEL, however, gave rise to presentation of only six of the seven HEL epitopes. The epitope included in the HEL sequence 112-124 was not presented by HEL.KDEL-transfected B cells. In addition, two of the four T cell hybridomas recognizing HEL 116-129 together with I-Ak molecules were not activated by HEL.KDEL, and three other epitopes were presented with lower efficiency as compared with HELs. Thus, endogenously synthesized HEL in secretory form gives rise to a set of class II-binding epitopes indistinguishable from exogenous HEL, whereas endoplasmic reticulum-retained HEL generates a similar but not identical set of epitopes. The endosomal protease inhibitor leupeptin prevented presentation of the epitope 108-116, but not 46-61, both by HELs and HEL.KDEL transfected cells, indicating a requirement for endosomal processing in both cases. In addition, the presentation of peptides derived from endogenously synthesized, either secretory or endoplasmic reticulum-retained HEL, could be inhibited by lysosomotropic amines, further indicating that the intracellular route of class II molecules presenting peptides derived from endogenous Ag intersects the acidic endosomal compartment.

Amino Acid Sequence↗

PubMed1993

Selective and ATP-dependent translocation of peptides by the MHC-encoded transporter.

Major histocompatibility complex (MHC) class I molecules present peptides derived from nuclear and cytosolic proteins to CD8+ T cells. These peptides are translocated into the lumen of the endoplasmic reticulum (ER) to associate with class I molecules. Two MHC-encoded putative transporter proteins, TAP1 and TAP2, are required for efficient assembly of class I molecules and presentation of endogenous peptides. Expression of TAP1 and TAP2 in a mutant cell line resulted in the delivery of an 11-amino acid oligomer model peptide to the ER. Peptide translocation depended on the sequence of the peptide, was adenosine triphosphate (ATP)-dependent, required ATP hydrolysis, and was inhibited in a concentration-dependent manner.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

TAP2-defective RMA-S cells present Sendai virus antigen to cytotoxic T lymphocytes.

The murine antigen-processing-defective mutant cell line RMA-S is leaky in the presentation of certain endogenously synthesized minor histocompatibility and viral antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). The viral antigens include influenza virus nucleoprotein, vesicular stomatitis virus (VSV) nucleocapsid and Rauscher murine leukemia virus (MuLV) antigen. Here we demonstrate Sendai virus antigen presentation by the HAM2 (murine TAP2, transporter associated with antigen presentation type 2)-defective RMA-S cell line and compare antigen presentation after restoration of the defect by murine TAP1/2 gene transfection. Kinetic studies revealed that RMA-S cells required 2-3 h longer incubation and approximately 10 times higher doses of Sendai virus to reach the same level of killing as the RMA parental line. After transfection of RMA-S cells with the murine TAP1/2 gene, Sendai virus antigen presentation was restored to levels of the RMA wild-type line with regard to time of virus infection and dose of virus needed for sensitizing target cells. The presentation of Sendai virus antigen in RMA-S cells was sensitive to brefeldin A (BFA), suggesting that the presentation was mediated via the endogenous pathway. Our findings confirmed leakiness of antigen presentation in RMA-S cells and extended it to Sendai virus. The results underscored the role for intact expression of the TAP 1/2 molecules for efficient MHC class I-mediated antigen presentation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild-type p53.

The central role of the p53 tumor suppressor gene product in oncogenesis is gradually being clarified. Point mutations in the p53 tumor suppressor gene are common in most human cancers and are often associated with p53 protein overexpression. Overexpressed wild-type or mutant determinants of the p53 protein thus represent an attractive target for immunotherapy of cancer directed against a structure involved in malignant transformation. An important step towards this goal is identification of epitopes of p53 that can be recognized by human cytotoxic T lymphocytes. We identified peptides of (mutant) p53 capable of binding to HLA-A2.1 in an in vitro assay. These HLA-A2.1-binding peptides were utilized for in vitro induction of primary cytotoxic T lymphocyte responses using a human processing-defective cell line (174CEM.T2) as antigen-presenting cell. These cells display "empty" HLA class I surface molecules, that can efficiently be loaded with a single peptide. We obtained CD8+ cytotoxic T lymphocyte clones capable of specifically lysing target cells loaded with wild-type or tumor-specific mutant p53 peptides. This strategy allows the in vitro initiation of human cytotoxic T lymphocyte responses against target molecules of choice.

Amino Acid Sequence↗

PubMed1993

Differential effect of transporter Tap 2 gene introduction into RMA-S cells on viral antigen processing.

The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line RMA-S (H-2b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA-S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H-2d) or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Cell biology of antigen presentation.

MHC class I molecules present degradation products derived from intracellular proteins, whereas MHC class II molecules generally present peptides derived from extracellular or surface proteins. Recent insights into the cell biology of MHC class I and II molecules explain this difference.

Animals↗

PubMed1993

Preferential antibody targeting to small lymphoma metastases in the absence of the primary tumour.

Targeting of spontaneous liver metastases of the ESb.MP murine lymphoma was achieved with anti-CD2 monoclonal antibody (MAb) 12-15A, which does not react with normal liver tissue. Using quantitative autoradiography on whole body sections of animals that had received a standard dose of 1.1 MBq of 125I-labelled monoclonal antibody, metastases accumulated up to > 90% of the injected dose per gram (id/g). The average uptake of primary tumour lesions was at a low level of 24 Bq/mg (corresponding to 2.2% id/g) because of highly non-uniform accumulation, while metastatic lesions were all above 50 Bq/mg. Uptake was particularly pronounced in animals tested after resection of the primary tumour: 85% of metastases showed levels above 300 Bq/mg, which was the upper limit of uptake in metastases of non-resected animals. These findings demonstrate the potential of the antibody approach with regard to attacking residual metastatic lesions after debulking.

Animals↗

PubMed1993

Intracellular distribution of the MHC class II molecules and the associated invariant chain (Ii) in different cell lines.

Intracellular localization and routing of MHC class II molecules and the associated invariant chain (Ii) were studied using rat-2 cells and HeLa cells stably transfected with the murine class II genes A alpha kA beta k (Ak) alone or supertransfected with human Ii or a truncated delta 20Ii lacking the endosomal sorting signal within Ii. We show that Ii and delta 20Ii are able to bind the class II molecules and replace the highly homologous murine Ii in class II sorting. Expression of transfected Ii or delta 20Ii also result in more efficient transport of class II molecules from the ER. There are, however, cell type specific differences in the intracellular routing of the class II molecules. The endosomal sorting signal within Ii is required to target class II molecules to endosomes in HeLa cells, whereas class II molecules alone enter endosomes in rat-2 cells. Class II molecules cannot direct the associated delta 20Ii to endosomes, suggesting that they follow a direct route to the cell surface and the majority of class II may enter endosomes by internalization. This is also supported by the long lived presence of class II molecules in endosomes after arresting protein transport with cycloheximide or brefeldin A. Class II molecules expressed alone or together with Ii are located in the whole endosomal pathway in rat-2 cells. However, full length Ii is required to target the class II molecules to vesicles that are more chloroquine sensitive than vesicles containing class II molecules alone. Our results thus indicate that Ii is responsible for endosomal sorting of class II molecules in HeLa cells, whereas in rat-2 cells the presence of Ii leads to an altered endosomal distribution.

Animals↗

PubMed1992

Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation.

Major histocompatibility complex (MHC) class I molecules bind and deliver peptides derived from endogenously synthesized proteins to the cell surface for survey by cytotoxic T lymphocytes. It is believed that endogenous antigens are generally degraded in the cytosol, the resulting peptides being translocated into the endoplasmic reticulum where they bind to MHC class I molecules. Transporters containing an ATP-binding cassette encoded by the MHC class II region seem to be responsible for this transport. Genes coding for two subunits of the '20S' proteasome (a multicatalytic proteinase) have been found in the vicinity of the two transporter genes in the MHC class II region, indicating that the proteasome could be the unknown proteolytic entity in the cytosol involved in the generation of MHC class I-binding peptides. By introducing rat genes encoding the MHC-linked transporters into a human cell line lacking both transporter and proteasome subunit genes, we show here that the MHC-encoded proteasome subunit are not essential for stable MHC class I surface expression, or for processing and presentation of antigenic peptides from influenza virus and an intracellular protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Anergy induced by thymic medullary epithelium.

Thymocytes can be rendered tolerant by non-deletional mechanisms upon interaction with major histocompatibility complex (MHC) antigens on thymic epithelium. Whether the epithelial cells in the cortex or medulla could mediate this effect was not clear so far. To address this question, a transgenic mouse was generated in which the bovine keratin IV promoter was used to control expression of the alloantigen Kb. In the periphery the Kb transgene was expressed on a subset of keratinocytes. In the thymus expression was restricted to a subpopulation of medullary epithelial cells. No expression was found in the cortex. Such a tissue distribution has been reported for the keratin IV molecule demonstrating the faithfulness of the promoter used here. To follow the fate of the Kb-reactive thymocytes, this mouse was mated with another transgenic mouse expressing an anti-Kb T cell receptor (TcR). In the double-transgenic mice the CD8+CD4- thymocytes were not deleted but they were found to be anergic as assayed by their failure to be activated in vitro by either Kb-positive spleen cells or by cross-linked anti-TcR antibodies. These observations establish that expression of an MHC class I antigen in the thymic medullary epithelium is sufficient to induce anergy in the mature CD8+CD4- thymocyte population.

Animals↗

PubMed1992

Distinct mechanisms of extrathymic T cell tolerance due to differential expression of self antigen.

Self-reactive T lymphocytes escaping thymic tolerance induction can be rendered non-responsive by contact with antigens in the periphery. In order to determine the parameters controlling peripheral tolerance induction we followed the fate of one well-defined self-reactive T cell population in three different mice expressing the self antigen in various nonlymphoid tissues outside the thymus. This was achieved by crossing anti-Kb T cell receptor (TCR) transgenic mice with transgenic animals expressing the Kb antigen exclusively on hepatocytes or keratinocytes or neuroectodermal cells. Due to this differential expression clonotype+, anti-Kb reactive T cells were found to exist at three different levels of tolerance. These levels were distinct with regard to downregulation of TCR and CD8 molecules, and the requirements for reexpression of TCR in vitro. This differential induction of peripheral tolerance suggest that some tissues are more likely to be affected in autoimmune diseases than others.

Animals↗