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J J Neefjes

Publications and source records attributed to J J Neefjes.

At least 55 records · Page 3Linked to original sources
PubMed1991

Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments.

Traffic of MHC molecules dictates the source of peptides that are presented to T cells. The intracellular distribution of MHC class I and class II molecules reflects the dichotomy in presentation of antigen from endogenous and exogenous origin, respectively. In human B lymphoblastoid cells, class I molecules are present in compartments constituting the biosynthetic pathway, whereas class II molecules enter structures related to lysosomes during their biosynthesis.

Antigens, CD↗
PubMed1991

Absence of preferential homologous H/L chain association in hybrid hybridomas.

Bispecific mAb contain two Ag-combining sites each composed of a different combination of H and L chains. The resulting ability to react with and cross-link two different Ag makes these molecules a novel tool for application in biology and medicine. Intact bispecific mAb can be made only by biologic means, e.g., by fusion of two established hybridomas. Appropriate assembly of bispecific mAb by these hybrid cells depends on H = L chain behavior: strong preferential homologous H-L pairing would benefit the yield of bispecific antibodies. We have analyzed the Ig species produced by eight hybrid hybridomas (quadromas). Quadroma-produced IgG was fractionated and characterized for H and L chain content. The Ag reactivities were verified by using ELISA and immunofluorescence. Preferential homologous pairing was seen only with a minority of H-L chain pairs; L chains associated on average in a random fashion with H chains. This indicates that in the B cells from which the parental hybridomas were obtained, no strong selection had occurred on H-L recombination. Our results extend recent biochemical competitive H-L reassociation experiments, where on average an at random association of L chains with H chains was found; evidently this random association occurs in our biologic system as well. For the biologic production of bispecific antibodies this means that only in a small number of cases the "ideal" producer will be met. From the viewpoint of generation of antibody diversity, our results favor a large freedom for combinatorial binding of H and L chains during B cell ontogeny.

Antibodies, Monoclonal↗
PubMed1991

Assembly and intracellular transport of major histocompatibility complex molecules.

Recent advances in our understanding of biosynthesis and assembly of MHC subunits are discussed. Intracellular traffic of MHC proteins is reviewed in the context of antigen presentation. While the overall picture of antigen presentation is now clear, much of the detail of the early stages of assembly of MHC products remains to be established. The degradative pathways that result in the peptides presented by MHC molecules (in particular those serving Class I molecules) have not yet been identified with certainty.

Animals↗
PubMed1990

Direct binding of peptide to empty MHC class I molecules on intact cells and in vitro.

MHC class I molecules devoid of peptide are expressed on the cell surface of the mouse mutant lymphoma cell line RMA-S upon culture at reduced temperature. Empty class I molecules are thermolabile at the cell surface and in detergent lysates, but can be stabilized by the addition of presentable peptide; peptide binding appears to be a rapid process. Furthermore, class I molecules on the surface of RMA-S (H-2b haplotype) cells cultured at 26 degrees C can efficiently and specifically bind iodinated peptide presented by H-2Kb. Binding of iodinated peptide is also observed at a lower level for nonmutant cells (RMA) cultured at 26 degrees C. These experiments underscore the role for peptide in maintenance of the structure of class I molecules and, more importantly, provide two assay systems to study the interactions of peptides with MHC class I molecules independent of the availability of T cells that recognize a particular peptide-MHC class I complex.

Animals↗
PubMed1990

Empty MHC class I molecules come out in the cold.

Major histocompatibility complex (MHC) class I molecules present antigen by transporting peptides from intracellularly degraded proteins to the cell surface for scrutiny by cytotoxic T cells. Recent work suggests that peptide binding may be required for efficient assembly and intracellular transport of MHC class I molecules, but it is not clear whether class I molecules can ever assemble in the absence of peptide. We report here that culture of the murine lymphoma mutant cell line RMA-S at reduced temperature (19-33 degrees C) promotes assembly, and results in a high level of cell surface expression of H-2/beta 2-microglobulin complexes that do not present endogenous antigens, and are labile at 37 degrees C. They can be stabilized at 37 degrees C by exposure to specific peptides known to interact with H-2Kb or Db. Our findings suggest that, in the absence of peptides, class I molecules can assemble but are unstable at body temperature. The induction of such molecules at reduced temperature opens new ways to analyse the nature of MHC class I peptide interactions at the cell surface.

Animals↗
PubMed1990

The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route.

We studied the intracellular traffic and subcellular distribution of MHC class I and class II antigens in comparison with a recycling surface glycoprotein, the transferrin receptor (Tfr), in the human lymphoblastoid cell line JY. No internalization was detectable for class I molecules. Class II molecules were internalized but did not recycle. In contrast, Tfr was found to internalize and recycle. The biosynthetic pathway of class II molecules differ from that of class I molecules in that it shows a delay (1-3 hr) in transport from trans-Golgi to cell surface: here it intersects the endocytic route. Immunoelectron microscopy using anti-MHC antibodies revealed the existence of vesicular structures that were intensely labeled for class II molecules. It is proposed that at this site combination of class II molecules with processed antigen could occur.

B-Lymphocytes↗
PubMed1990

N-linked glycan modification on antigen-presenting cells restores an allospecific cytotoxic T cell response.

The B6 anti-bm6 allospecific CTL response is strictly dependent on CD4+ cells when using LPS blasts as stimulator cells. Altering the N-linked carbohydrates on stimulator cells by use of the N-linked trimming glycosidase inhibitors 1-deoxymannojirimycin and swainsonine, or by treatment with bacterial neuraminidase, results in a restoration of the B6 anti-bm6 response in the absence of CD4+ cells. The extent of restoration is inversely correlated with the number of sialic acids present on N-linked glycans of stimulator cells.

Animals↗
PubMed1990

Intracellular interactions of transferrin and its receptor during biosynthesis.

The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during biosynthesis were studied in the human hepatoma cell line HepG2, which synthesizes both. Early during biosynthesis the Tfr monomer is converted to a disulfide-linked Tfr dimer. The Tfr monomer is not able to bind Tf, but Tf binding is observed as soon as the covalent Tfr dimer is formed and can take place in the ER. The Tf-Tfr complex is transported through the Golgi reticulum and trans-Golgi reticulum (TGR) and is ultimately delivered to an acidic compartment, where Tf releases its Fe3+. We did not observe conversion of Tf to apoTf in the TGR, showing that the part of the TGR passed by secreted Tf has a pH higher than 5.5. We conclude that when a ligand-receptor combination is synthesized by one and the same cell, ligand and receptor can interact during biosynthesis and be transported to the cell surface.

Cell Compartmentation↗
PubMed1989

Non-carrier-mediated uptake of the mannosidase I inhibitor 1-deoxymannojirimycin by K562 erythroleukemic cells.

A 3H label was introduced at the C-1 position of the mannosidase I inhibitor 1-deoxymannojirimycin (dMM) by catalytic hydrogenolysis of benzyl-2,3-O-isopropylidene-5-N-benzyl-6-O-benzyl-alpha-D-mannofurano side with 3H2. 1-[3H]dMM as well as its precursor 1-[3H]2,3-O-isopropylidene-dMM had identical Rf as the nonradioactive compounds on TLC. Furthermore, alpha 1-antitrypsin secreted by HepG2 cells was modified indistinguishably by treatment of the cells with dMM and 1-[3H]dMM. Thus, 1-[3H]dMM had chemical and biological properties identical with authentic dMM. Uptake of [14C]mannose by K562 cells could be inhibited by glucose but not by the mannose analogue dMM. Thus, dMM does not enter the cell through hexose transporter(s). Uptake of 1-[3H]dMM by K562 cells could not be inhibited by increasing concentrations of nonradioactive dMM (from 1-32,000 microM), showing transport of dMM into cells through nonfacilitated diffusion. Furthermore, uptake of 1-[3H]dMM by K562 cells was observed at 0 degrees C.

1-Deoxynojirimycin↗
PubMed1989

Specific immune responses restored by alteration in carbohydrate chains of surface molecules on antigen-presenting cells.

Two class I major histocompatibility (MHC) mutant mouse strains, H-2bm14 and H-2bm6, differ from the strain of origin C57BL/6 (B6, H-2b) in one and two amino acids of the H-2Db and H-2Kb molecule, respectively. The bm14 Db mutation results in specific failure of female bm14 mice to generate a cytotoxic T lymphocyte (Tc) response to the male-specific antigen H-Y. The allospecific Tc response of CD8+ B6T cells against bm6 Kb mutant spleen cells, in contrast to that against other Kb mutants, is absolutely CD4+ T helper cell dependent. Purified CD8+ T cells completely fail to respond. We now report that the inability to mount these specific immune responses is restored by the use of dendritic cells (DC) as antigen-presenting cells (APC). Comparison of MHC expression on various types of APC by cytofluorimetry and quantitative immunoprecipitation showed very high expression of class I and class II MHC molecules on DC. Strikingly, examination of class I and class II molecules by isoelectric focusing revealed qualitative differences as well. We show that the surface MHC class I molecules of DC are present in greater quantity and carry on average fewer sialic acids than the same molecules isolated from other APC types such as spleen cells, lipopolysaccharide blasts or concanavalin A blasts. That sialic acids on cell surface molecules, including MHC, may play a role in antigen presentation is suggested by our finding that removal of sialic acids, by neuraminidase, can restore specific responses to nonresponder APC as well.

Animals↗
PubMed1989

Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new anti-rat macrophage antibodies against a heterodimer of 160 and 95 kd (CD11/CD18).

A set of three monoclonal antibodies (MoAbs), ED1, ED2, and ED3, has been shown to recognize in situ different subsets of macrophages in the rat. This macrophage diversity can be correlated with differences in stage of differentiation of cells belonging to one lineage. The present study quantifies this antigen distribution in the macrophage fractions of several lymphoid organs provided by Percoll centrifugation. Four new MoAbs (ED4, ED7, ED8, and ED9) raised against macrophages are included in this study. The tissue distribution of each of the four new MoAbs is determined by immuno- and enzyme-histochemistry on cryostat sections. The MoAbs recognize distinct subpopulations of macrophages. The new MoAbs ED4, ED7, ED8, and ED9 recognize granulocytes and other unrelated cell types, as well as cells of the mononuclear phagocyte system. ED7 and ED8 recognize a surface heterodimer of Mr 160,000 and 95,000.

Animals↗
PubMed1989

Allele-specific down-regulation of MHC class I antigens in Burkitt lymphoma lines.

We have reported that Burkitt lymphomas (BL) that arise in HLA-A11 positive individuals are resistant to lysis by HLA-A11-specific and HLA-A11-restricted CTLs(10,11). Here we show that this phenomenon can be explained by a selective loss of the HLA-A11 polypeptide. The HLA-A11 negative phenotype is due to a regulatory phenomenon, rather than a structural defect, as proven by the ability to rescue expression of HLA-A11 in in vitro Epstein-Barr virus (EBV)-converted sublines of EBV negative BLs.

Alleles↗
PubMed1989

Influenza virus changes cell-surface glycoproteins including major histocompatibility complex determinants on lymphocytes.

The effect of influenza virus infection on the expression of major histocompatibility complex (MHC) antigens was investigated. Infection with influenza virus resulted in an increase of the binding of anti-MHC class I and class II antibodies to resting T cells. The binding of anti-MHC class II antibodies to activated T cells was increased approximately threefold. The binding of anti-MHC class I and class II antibodies to Epstein-Barr virus-transformed B cells appeared unaffected after influenza virus infection. Recombinant human interferon-alpha and/or -gamma added to T cells did not enhance the binding of anti-MHC antibodies. Biochemical analysis revealed no increase in the amount of class I and class II antigens as a consequence of viral infection, but a marked decrease in sialic acid content was found, most probably caused by the viral neuraminidase. Pulse-chase experiments suggest that the viral neuraminidase can catalyze the removal of sialic acids both en route to and at the cell surface. The absence of sialic acid residues can explain the increased binding of anti-MHC antibodies, because neuraminidase (clostridium perfringens) treatment of T and Epstein-Barr virus-transformed B cells resulted in a shift in both isoelectric point and antibody binding similar to that observed after influenza virus infection.

Antibodies, Monoclonal↗
PubMed1989

Biochemical evidence that the DLA-B locus codes for a class II determinant expressed on all canine peripheral blood lymphocytes.

We have analyzed the products of the canine DLA-B gene locus by biochemical and serological methods. Due to its ubiquitous expression on lymphocytes this antigen series was thought to be of class I nature. However, by two-dimensional gel electrophoresis and lysostrip experiments we could unequivocally identify the DLA-B antigens as typical class II gene products. The serological polymorphism is caused by the beta-chain, whereas the alpha-chain appears invariant. The presence of these class II gene products on almost all lymphocytes in the dog is in sharp contrast to the numerous reports on their restricted expression in other mammalia such as man and mouse.

Animals↗
PubMed1988

Allele and locus-specific differences in cell surface expression and the association of HLA class I heavy chain with beta 2-microglobulin: differential effects of inhibition of glycosylation on class I subunit association.

The assembly of HLA class I antigens, and the contribution of the single N-linked glycan to this process were examined. We observed a requirement for N-linked glycosylation in the proper assembly and surface expression of HLA-B locus products in particular, although considerable variation was seen within the allelic series of the HLA-A and B loci. We conclude that the single N-linked glycan can contribute in a major way to that conformation of the heavy (H) chain which is competent to associate with the light chain beta 2-microglobulin, and that the presence, rather than the type, of carbohydrate chain is important in this respect. The association of human class I H chains with beta 2-microglobulin shows biphasic kinetics, where an initially rapid phase is followed by a prolonged period during which no further association can be measured. It appears that HLA-C H chains are initially synthesized in amounts similar to HLA-A and B H chains, but associate inefficiently with beta 2-microglobulin, resulting in low expression of HLA-C at the cell surface. The individual stages of assembly and maturation of class I antigens including the transfer from Golgi to cell surface were found to display characteristic allelic variation.

1-Deoxynojirimycin↗
PubMed1988

Ras (proto)oncogene induces N-linked carbohydrate modification: temporal relationship with induction of invasive potential.

The effect of expression of the ras oncogene on protein glycosylation was studied. VSV G-protein and class I histocompatibility antigens were analysed to monitor ras-mediated changes in glycosylation. Transient expression of the c-Ha-ras oncogene, introduced into NIH 3T3 cells by the DEAE-dextran method, altered protein glycosylation within 25 h of transfection. The same result was obtained after dexamethasone-induced expression of p21-ras in stable NIH 3T3 transfectants containing either an activated Ha-ras oncogene or a normal N-ras proto-oncogene under control of the glucocorticoid-inducible MMTV promoter. The alteration of cell surface carbohydrates, induced by the ras (proto)oncogene and the subsequent acquisition of invasive potential, occurred prior to morphological transformation.

Animals↗
PubMed1988

Biochemical complexity of serum HLA class I molecules.

Human serum was found to contain a variety of class I-like molecules by Western blotting with anti-class I heavy chain reagents: major bands usually are observed around Mr 44,000, 40,000, and 35,000-37,000. HLA-A24-positive individuals are distinguished by higher serum levels of Mr 44,000 and 40,000 class I-like molecules than those found in HLA-A24-negative individuals. The Mr 44,000 serum molecules are probably intact class I molecules that have been shed from the cell membrane, because they contain both a transmembrane segment (TM), as deduced from detergent-binding experiments, and a cytoplasmic tail (CT), as inferred from reactivity with an antipeptide serum specific for the cytoplasmic domain of class I antigens (RaCT). The Mr 35,000 and 37,000 molecules contain neither a TM nor a CT region and therefore are probably proteolytic breakdown products of cellular and/or serum Mr 44,000 molecules, although the existence of Q10-like molecules in man cannot be ruled out. The Mr 40,000 molecules do not contain a TM region. Mr 40,000 molecules reactive with the RaCT serum were found in the minority (2/13) of sera tested. We conclude that alternative splicing resulting in a precise excision of the TM exon plays a minor role in the generation of serum HLA class I antigens.

Animals↗