Biomedical subjects

G Moldenhauer

Publications and source records attributed to G Moldenhauer.

At least 37 records · Page 2Linked to original sources

PubMed1998

A role for HLA-DO as a co-chaperone of HLA-DM in peptide loading of MHC class II molecules.

In B cells, the non-classical human leukocyte antigens HLA-DO (DO) and HLA-DM (DM) are residents of lysosome-like organelles where they form tight complexes. DM catalyzes the removal of invariant chain-derived CLIP peptides from classical major histocompatibility complex (MHC) class II molecules, chaperones them until peptides are available for loading, and functions as a peptide editor. Here we show that DO preferentially promotes loading of MHC class II molecules that are dependent on the chaperone activity of DM, and influences editing in a positive way for some peptides and negatively for others. In acidic compartments, DO is engaged in DR-DM-DO complexes whose physiological relevance is indicated by the observation that at lysosomal pH DM-DO stabilizes empty class II molecules more efficiently than DM alone. Moreover, expression of DO in a melanoma cell line favors loading of high-stability peptides. Thus, DO appears to act as a co-chaperone of DM, thereby controlling the quality of antigenic peptides to be presented on the cell surface.

Alleles↗

PubMed1998

Bispecific antibodies increase T-cell stimulatory capacity in vitro of human autologous virus-modified tumor vaccine.

The production and functional testing of two new bispecific (bs) hybrid antibodies [Abs; bs Ab hemagglutinin-neuraminidase (HN) x CD3 and bs Ab HN x CD28] designed for cancer vaccine modification are described. They allow distinct modifications of the human tumor cell vaccine ATV-NDV, an autologous tumor cell vaccine already modified by infection with Newcastle disease virus. The bs Abs use the viral HN molecule as a common foreign anchoring molecule for attachment to the tumor cells and allow the introduction of anti-CD3 or anti-CD28 T-cell-stimulatory molecules. The bs Abs attached to tumor target cells were able to cross-link CTL effector cells and up-regulate T-cell activation markers on autologous cancer patient-derived CD4 and CD8 T lymphocytes. This strategy of combining a cellular vaccine with a bs Ab is highly specific, quick, and economical and has broad-range applications. Five ng or less of target cell-bound bs Ab HN x CD28 were effective at augmenting T-cell-mediated antitumor cytotoxicity.

Antibodies, Bispecific↗

PubMed1998

Antibodies TC-12 ("unique") and TH-111 (CD96) characterize T-cell acute lymphoblastic leukemia and a subgroup of acute myeloid leukemia.

To extend the panel of monoclonal antibodies useful for immunophenotyping of acute leukemias, two new reagents, TC-12 and TH-111, were developed. TC-12 was found "unique," and TH-111 was assigned to the recently defined CD96 cluster. Both reagents show little reactivity with blood and bone marrow nucleated cells but define a major (TH-111: 78.3%) or an important (TC-12: 45.6%) subset of T-cell acute lymphoblastic leukemia (ALL). In addition, in acute myeloid leukemia (AML), the expression of TC-12 was found in 64 (20.2%) of 317 and TH-111 in 97 (29.1%) of 333 of these patients. TC-12 positivity in AML was virtually restricted to the Fab subtypes M0, M1, M2, and M6. In the group of immature AML characterized by the coexpression of CD7 as well as CD117 and CD34 positivity, leukemic blasts frequently disclosed the TC-12 and TH-111 antigen. Although the TC-12 antigen could not be determined, TH-111 immunoprecipitated the TACTILE (CD96) antigen and, when expressed, was found to be associated with the transferrin receptor. These reagents may help not only to define and dissect T-cell ALL, but also to characterize a subgroup of immature AML at the divergence of T-cell and myeloid lineage.

Animals↗

PubMed1997

HLA-DM stabilizes empty HLA-DR molecules in a chaperone-like fashion.

HLA-DM (DM) is a non-classical major histocompatibility complex (MHC) class II molecule that interacts with classical MHC II molecules in acidic compartments. During this association DM is supposed to catalyze the release of invariant chain (II)-derived CLIP peptides thereby rendering the peptide binding groove accessible for antigenic peptide loading. However, in situations of peptide scarcity the fate of these DM:DR complexes is not known. We could show that DR molecules incubated at lysosomal pH in the absence of peptide rapidly undergo functional inactivation and aggregation. In the presence of DM, however, empty DR molecules were shown to be stabilised and kept receptive for peptide loading, with the degree of the stabilising effect of DM varying for different DR alleles. In addition, in lysosomal compartments a considerable fraction of DM was found to be stably associated with empty DR alpha beta dimers thereby preserving their functionality. Upon encounter with antigenic peptide the DM-associated DR molecules could be rapidly loaded, whereupon they did no longer bind to DM. Thus, DM seems to act as a dedicated class II-specific chaperone that rescues uncharged alpha beta dimers. In view of the suggested shortage of self-peptides in the loading compartment, empty class II molecules that are kept receptive for loading by the chaperone function of DM may enable the antigen processing system to respond promptly to the challenge by newly entering antigens.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1997

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures.

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15-25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80-150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20 degrees C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.

Escherichia coli↗

PubMed1997

HLA-DM acts as a molecular chaperone and rescues empty HLA-DR molecules at lysosomal pH.

HLA-DM (DM) is a nonclassical MHC class II molecule that interacts with classical MHC II molecules in acidic compartments. During this association DM is supposed to catalyze the release of invariant chain (Ii)-derived CLIP peptides, as well as other peptides bound with low kinetic stability. Here we provide evidence that in lysosomal compartments of B cells a considerable fraction of DM is stably associated with empty DR alphabeta dimers, thereby preventing their functional inactivation and aggregation. Upon encounter with cognate peptide, the DM-associated DR molecules can be rapidly loaded and no longer bind to DM. Thus, DM seems to act as a dedicated class II-specific chaperone. In view of the suggested shortage of DM-resistant self-peptides in the loading compartment, empty class II molecules that are chaperoned by DM may enable the antigen-processing system to respond promptly to the challenge by newly entering antigens.

Alleles↗

PubMed1997

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity.

Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30-fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.

Amino Acid Sequence↗

PubMed1997

Reverse transcriptase-polymerase chain reaction (RT-PCR)-controlled immunomagnetic purging of breast cancer cells using the magnetic cell separation (MACS) system: a sensitive method for monitoring purging efficiency.

A modified reverse transcriptase-polymerase chain reaction (RT-PCR) technique was established with the aim of monitoring the tumor cell contamination in peripheral blood stem cells harvested from breast cancer patients. In an experimental approach, single cell suspensions of different breast cancer cell lines were mixed to normal peripheral blood mononuclear cells in order to 1) determine the sensitivity of tumor cell detection within PBMC and 2) compare polymerase chain reaction in its capacity of monitoring the efficiency of immunomagnetic purging using the magnetic cell separation (MACS) system to immunocytochemical staining. Several target sequences were assessed for their indicative potential and specificity allowing the detection of breast cancer cells by RT-PCR. Among the sequences evaluated, epithelial growth factor receptor (EGF-R) mRNA and Cytokeratin 19 mRNA were shown to be highly specific and sensitive markers for the detection of breast cancer cells within normal peripheral blood mononuclear cells and for the evaluation of the efficiency in immunomagnetic purging. In addition, we were able to show that the MACS is a potent and efficient tool for the selection of tumor cells from peripheral blood mononuclear cells, thus establishing its value for clinical scale immunomagnetic purging.

Breast Neoplasms↗

PubMed1996

Editing of the HLA-DR-peptide repertoire by HLA-DM.

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non-classical MHC class II molecule HLA-DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self-peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA-DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self-peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high-performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR-associated self-peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high-stability MHC class II-peptide complexes by kinetic proofreading before these complexes are presented to T cells.

Amino Acid Sequence↗

PubMed1996

Rapid detection of recombinant antibody fragments directed against cell-surface antigens by flow cytometry.

Cloning the correct genes coding for antibody variable domains (especially VL kappa) from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from the myeloma cell line. Indeed, four different VL genes were obtained after the amplification of immunoglobulin genes by PCR from the hybridoma HD37, which produces an antibody against the human CD19 B cell differentiation antigen. Most of the variants (eight out of 15) were derived from the kappa chain of the myeloma MOPC-21. For the rapid functional evaluation of recombinant antibody fragments against cell surface antigens, we established an efficient expression and detection system. First, deleted and mutated genes were eliminated by a colony screening procedure. Bacteria from picked colonies were then induced and grown in the presence of 0.4 M sucrose to increase the accumulation of soluble scFv in the periplasm (5-10 micrograms per ml of bacterial shake-tube culture). Finally, the cell-specific binding of scFv in crude periplasmic extracts was detected by flow cytometry. This procedure facilitated the efficient cloning of a functional anti-CD19 VH/VL combination from the hybridoma cDNA.

Amino Acid Sequence↗

PubMed1996

Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM.

The nonclassical major histocompatibility complex class II molecule HLA-DM (DM) has recently been shown to play a central role in the class II-associated antigen presentation pathway: DM releases invariant chain-derived CLIP peptides (class II-associated invariant chain protein peptide) from HLA-DR (DR) molecules and thereby facilitates loading with antigenic peptides. Some observations have led to the suggestion that DM acts in a catalytic manner, but so far direct proof is missing. Here, we investigated in vitro the kinetics of exchange of endogenously bound CLIP for various peptides on DR1 and DR2a molecules: we found that in the presence of DM the peptide loading process follows Michaelis-Menten kinetics with turnover numbers of 3-12 DR molecules per minute per DM molecule, and with KM values of 500-1000 nM. In addition, surface plasmon resonance measurements showed that DM interacts efficiently with DR-CLIP complexes but only weakly with DR-peptide complexes isolated from DM-positive cells. Taken together, our data provide evidence that DM functions as an enzyme-like catalyst of peptide exchange and favors the generation of long-lived DR-peptide complexes that are no longer substrates for DM.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1996

Ecto-sialyltransferase of human B lymphocytes reconstitutes differentiation markers in the presence of exogenous CMP-N-acetyl neuraminic acid.

The existence of an ecto-sialyltransferase (ecto-ST) on B lymphocytes with increasing activity at late maturation stages is shown using a novel flow cytometric enzyme assay. This ecto-ST is effective in reconstituting different surface glycoconjugates on desialylated B cells in the presence of exogenous CMP-NeuAc. We found that this ecto-ST is distinct in its activity from soluble ST released into the culture supernatant. Surface sialylation was independent of the amount of ST secreted into the culture supernatant and followed different kinetics than sialylation of exogenous substrate by soluble ST. Four human B-cell lines representing different maturation stages were analyzed for secreted and ecto-ST activity. The myeloma cell line U266 and the lymphoblastoid cell line JOK-1 showed higher activity of both ST forms than the acute lymphoblastic leukemia B-cell line Nalm-6. ST activity in culture supernatants of U266, JOK-1, and Nalm-6 cells consisted predominantly of the alpha 2,6 ST type with specificity for N-linked oligosaccharides. As an exception, the myeloma cell line IM-9, deficient of alpha 2,6 ST activity, secreted only small amounts of ST and showed low activity of ecto-ST. Sialylation of surface-expressed glycoconjugates by ecto-ST was measured by incubating B-cell lines in the presence of fluorescent CMP-sialic acid. Surface structures labeled with fluorescent sialic acid under this condition were visualized by confocal laser scanning microscopy and fluorescent label was quantitatively assessed by flow cytometric analysis on live cells. Incubation of cells in acidified culture medium, to release possibly receptor-bound ST, did not alter the intensity of cell surface sialylation. Inhibition of internalization and membrane traffic by various approaches (reduced incubation temperature and chloroquine or brefeldin A treatment) did not block surface sialylation. Together, these observations point to cell surface sialylation in B lymphocytes mediated by a cell surface-expressed ecto-ST distinct from the secreted ST form. On desialylated JOK-1 cells, ecto-ST in the presence of exogenous CMP-NeuAc was able to resialylate the B-cell surface sialoglycans CDw75 and HB-6 and major surface glycoproteins of B cells, such as HLA class I and II antigens, transferrin receptor, and surface IgM. In contrast, cell surface glycans of coincubated desialylated erythrocytes were not sialylated by the B-cell ecto-ST. Ecto-alpha 2,6 ST of B cells may be involved in the sialylation of distinct differentiation glycan antigens.

Antigens, CD↗

PubMed1996

Glycoprotein incorporation and HIV-1 infectivity despite exchange of the gp160 membrane-spanning domain.

We have examined the role of the membrane-anchoring domain of the HIV-1 glycoproteins in viral glycoprotein function, glycoprotein incorporation, and viral infectivity. For this purpose, we initially exchanged the entire membrane-spanning region with that from a cellular glycoprotein (CD22). Subsequently, the strictly conserved arginine in the central position of the transmembranal alpha-helix was replaced by a neutral residue (R696 --> I696). We have further examined the requirements within the cytoplasmic C-terminus for glycoprotein incorporation and replaced this region of gp160 with the long cytoplasmic C-terminus (118 amino acids) from CD22. Our results show that the specific amino acid sequence of the membrane-spanning region of gp160 is not necessary for viral infectivity, thus making it unlikely that this region is specifically involved in membrane fusion, in glycoprotein incorporation, or in infectivity of the cell lines tested. In contrast, recombinant gp160 with the CD22 C-terminal region, although present at the cell surface and membrane fusion-competent, was excluded from incorporation into particles. This could indicate that steric exclusion, and no pseudotyping, occurs when the heterologous, cytoplasmic C-terminal region is too long and not fitting.

Amino Acid Sequence↗

PubMed1996

Early detachment of colon carcinoma cells during CD95(APO-1/Fas)-mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44.

Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferon-stimulated HT-29 and COLO 205 colon carcinoma cells by cross-linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about 1 h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS-PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95-triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.

Apoptosis↗

PubMed1996

Activation of T cell cytotoxicity against autologous common acute lymphoblastic leukemia (cALL) blasts by CD3xCD19 bispecific antibody.

To develop an effective immunotherapy for B-lineage acute lymphoblastic leukemia (ALL), bispecific monoclonal antibodies (bsAb) were raised by cell fusion of two hybridoma cell lines secreting CD3 and CD19 antibodies. The resulting bispecific antibody contains two different specificities within a single antibody molecule. One binding site (CD3) activates the T cells via the T cell receptor complex, whereas the second binding site (CD19) targets the cytolytic T cells to malignant B cells. Leukemic blasts from children with B-lineage ALL showed stable and strong CD19 expression. CD3xCD19 bsAb were used to activate peripheral blood mononuclear cells (PBMC) from healthy donors or from patients with ALL during remission. Cytotoxic activity against autologous ALL cells by PBMC was induced upon addition of 100 ng/ml CD3xCD19 bsAb after 3 days of preincubation. Costimulation through CD28 increased T cell proliferation to some extent, but did not increase cytotoxic activity of PBMC against leukemic blasts. We present evidence for an effective and specific activation of resting human T lymphocytes by CD3xCD19 bsAb in vitro. Activation of cytotoxic effector T cells is feasible by preincubation with bsAb CD3xCD19 alone and does not rely on additional external costimulation. Thus, targeting of T cell cytotoxicity towards leukemic blasts via CD3xCD19 bsAb may represent a promising strategy for immunotherapy of B-lineage ALL.

Antibodies, Bispecific↗

PubMed1995

Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes.

Previously described primers for PCR amplification of variable immunoglobulin (Ig) genes were based on gene sequences. To include the large number of amino acid sequences of antibodies whose DNA has not been sequenced and to ensure a maximal fit to rearranged human Ig variable region genes, we have made a comprehensive comparison of both protein and nucleotide sequences. The resulting set of 15 primers was able to amplify a wide range of rearranged antibody variable region genes. Restriction sites included in the primers facilitate cloning of the PCR products into various expression vectors. Sequence analyses of PCR-amplified cDNA derived from a polyclonal B cell population showed that maximal enrichment is obtained for highly represented variable Ig gene subgroups. Rarely occurring V kappa 4 and V lambda 5 subgroups were not detected. Rearranged Ig variable region genes from each of 19 human B cell lines were also amplified. Comparisons to germline sequences allowed the allocation of rearranged genes to the original Ig genes. This primer set should be very useful for generating large repertoires of rearranged V genes and for amplifying genes of individual B cell clones.

Amino Acid Sequence↗

PubMed1995

Immediate effects of reversible HTLV-1 tax function: T-cell activation and apoptosis.

The tax protein of Human T-cell leukemia virus type 1 (HTLV-1) is important for the transforming properties of this virus in vitro and is considered to be responsible for the early stages of leukemogenesis in infected hosts. To address the early consequences of HTLV-1 tax function, we have constructed fusion proteins containing tax sequence either aminoterminal (taxER) or carboxy-terminal (ERtax) of the hormone binding domain of the human estrogen receptor (ER). Addition of estrogen or the antagonist hydroxytamoxifen to Jurkat T-cells expressing these constructs led to the trans-activation or responsive promoters and upregulation of cell surface markers CD28, CD69 and CD5 but not CD25 (IL2R-alpha subunit) or B7 (ligand for CD28). Additional stimulation of the T-cell receptor CD3 complex, led to the upregulation of CD25. B7 was upregulated by concomittent activation of ERtax and CD3 or CD28 pathways. These events were in part reversible upon withdrawal of hormone and inactivation of ERtax. Severe inhibition of proliferation, and apoptosis was observed with cells which had been subjected to short term (3 days) activation of the tax fusion proteins and the CD3 complex. Induction of ERtax activity for longer than 3 days promoted cell death independently of CD3 stimulation. Co-stimulation through the CD28 cell surface molecule did not suppress induction of apoptosis.

Apoptosis↗

PubMed1995

APO-1 (CD95)-dependent and -independent antigen receptor-induced apoptosis in human T and B cell lines.

Certain B and T cell lines respond to activation signals, e.g. through the antigen receptor, by undergoing apoptotic cell death. In T cells it has been recently shown that TCR-mediated apoptosis involves APO-1/Fas(CD95) receptor-ligand interaction. To investigate whether the TCR-CD3 complex can trigger alternative apoptosis pathways we generated subclones of the T cell line Jurkat which were completely resistant towards APO-1-mediated apoptosis. These JurkatR cells differed phenotypically from sensitive parental JurkatS cells only by the lack of APO-1 protein expression. Although JurkatR cells responded normally to anti-CD3 stimulation by expression of APO-1 ligand they failed to undergo anti-CD3-induced apoptosis. Thus, in Jurkat cells APO-1-mediated apoptosis was the main, and might be the only, mechanism for anti-CD3-induced cell death. However, BL-60 B cells, highly sensitive to anti-IgM-induced apoptosis, did not use the APO-1 receptor-ligand system because they failed to express APO-1 ligand mRNA. Taken together, our results suggest that malignant T and B cell lines may use APO-1 receptor-ligand-dependent and -independent antigen receptor-induced apoptosis pathways respectively. Similarly, differential pathways may be used by T and B cell subsets.

Apoptosis↗