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Biomedical subjects

N Koch

Publications and source records attributed to N Koch.

At least 37 records · Page 2Linked to original sources
PubMed2000

Invariant chains with the class II binding site replaced by a sequence from influenza virus matrix protein constrain low-affinity sequences to MHC II presentation.

Presentation of antigenic peptides by MHC II molecules is required to initiate CD4 T(h) cell responses. Some peptides, however, because of low affinity for MHC II, are not efficiently presented. A segment of the MHC II chaperon molecule, invariant chain (Ii), is known to bind early in biosynthesis with low affinity to the peptide binding groove. Here we have exploited the properties of Ii to manipulate the MHC II-loading pathway and to present low-affinity sequences. We used a deletion mutant of Ii where the promiscuous binding site to MHC II, which is adjacent to the groove binding segment, was deleted. A recombinant Ii (rIi) chimera, derived from this construct, was made in which the class II binding segment was exchanged for wild-type or single amino acid substitution variants of an HLA-DR1-restricted sequence from influenza matrix protein (MAT), which leads to MHC II allotype-specific binding. This rIi was expressed in antigen-presenting cells (APC) and introduced the MAT sequence into the MHC II-processing pathway. As expected, rIiMAT elicited antigen-specific, DR1-restricted T cell cytokine production and proliferation. Significantly, rIiMAT, that binds the HLA-DR4 allele with low affinity, elicited DR4-restricted IL-2 production but not proliferation. In contrast, exogenously provided MAT peptide failed to elicit any responses from DR4-restricted T cells. Compatible results were obtained with a single amino acid substitution variant (MAT(T)), which binds with high affinity to DR4 but low affinity to DR1. We conclude that loading of MHC II with antigenic peptides from endogenously synthesized rIi chimeras allows presentation of low-affinity sequences that cannot be presented if provided exogenously as peptides. Ii fusion proteins containing low-affinity antigenic sequences might be useful for vaccination with tumor antigens to overcome deficiencies in antigen presentation.

Adult↗
PubMed1999

Sodium dodecyl sulfate-resistant HLA-DR "superdimer" bands are in some cases class II heterodimers bound to antibody.

The detection of dimers of dimers in MHC class II crystals has excited speculation about their possible functions in T cell Ag recognition. Biochemical evidence for the existence of DR superdimers falls short of proof and is controversial. To monitor B lymphoma cells for high m.w. complexes of HLA-DR molecules, membrane preparations and cell lysates were screened by one- and two-dimensional Western blotting. Under these conditions, in which DRalpha beta heterodimers were readily detected, no DR complexes with an (alpha beta)2-chain composition could be identified. Two mAbs (L243 and D1-12) immunoprecipitated high m.w. DR complexes suspected to be superdimers. However, biochemical analysis revealed that, rather than superdimers, these were SDS-stable complexes of DR in combination with the Abs. Thus, previous observations of HLA-DR superdimer bands may also reflect complexes of DR molecules with bound Ab.

Antibodies, Monoclonal↗
PubMed1999

Genetic polymorphism near HIV-1 reverse transcriptase resistance-associated codons is a major obstacle for the line probe assay as an alternative method to sequence analysis.

The performance of the line probe assay (LIPA) for the detection of mutations conferring resistance to nucleoside inhibitors of HIV-1 reverse transcriptase was evaluated in comparison with sequence analysis. The tests were undertaken on plasma samples from 63 patients (61 receiving combination therapy and 2 without treatment at the time of inclusion). In 27 cases (43%) which included codons 41, 69, 70, 74, 184 and 215, the sequence of the RT gene was distinct from the hybridization probes used in LIPA. Correspondingly, LIPA gave uninterpretable results in 15, 30 and 41% of cases for codons 184, 215 and 41, respectively. Overall, the concordance between LIPA and sequence analysis varied from 52% (codons 41 and 215) to 85% (codon 70). These data show that the polymorphism of the nucleotide sequence near resistance-associated codons is a major shortcoming of LIPA.

Codon↗
PubMed1999

C-terminal extension of the MHC class II-associated invariant chain by an antigenic sequence triggers activation of naive T cells.

In vitro and in vivo activation of T cells was investigated with invariant chain-antigen fusion protein. The CD4 T cell epitope amino acid 52-61 of hen egg lysozyme (HEL) was attached to the C-terminal end of invariant chain (Ii). Expression of this recombinant Ii HEL directs the T cell epitope to the class II processing pathway. Class II molecules of transfected antigen presenting cells (APC) are charged with this HEL epitope. The endogenously provided epitope competes with processing and presentation of exogenously added antigen. APC expressing recombinant Ii HEL stimulate a maximal IL-2 response of HEL-specific T hybridoma cells. Nonprofessional APC expressing recombinant Ii HEL and H2-Ak are also able to activate naive T cells from 3A9 TCR transgenic mice, a result not achieved with peptide pulsed APC. To elicit an in vivo immune response dendritic cells (DC) were transfected with rIi HEL cDNA: following immunization of CBA mice with transfected DC, a primary T cell response against the HEL epitope was induced. Thus the procedure described here could be used to introduce antigens into the class II processing pathway and to elicit T cell activation both in vitro and in vivo.

Alcohol Oxidoreductases↗
PubMed1999

Comparison of human immunodeficiency virus type 1 (HIV-1) protease mutations in HIV-1 genomes detected in plasma and in peripheral blood mononuclear cells from patients receiving combination drug therapy.

Detections of mutations in the protease gene of human immunodeficiency virus type 1 in plasma and peripheral blood mononuclear cells (PBMC) were sought in two matched populations of 23 individuals receiving combination drug therapy with or without protease inhibitors. In the control group (23 patients not receiving protease inhibitors), no primary resistance mutations were found. In contrast, primary resistance mutations (especially at codons M46, V82, and L90) were found in 16 of 23 patients (70%) treated with protease inhibitors. In 30% of the cases, these mutations were detected in plasma but not in PBMC.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Stable rearrangements of the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase in plasma viruses from patients receiving combination therapy.

OBJECTIVES: To study the genetic rearrangements of HIV-1 reverse transcriptase (RT) in circulating viruses from patients under combination therapy, and to determine the impact of these changes on the virological response to treatment. METHODS: Blood samples were extracted from total RNA and amplified by RT-PCR. The HIV-1 RT and protease genes were sequenced by fluorescent dye terminator cycle sequencing. RESULTS: Specific rearrangements in the RT coding region (between amino acids 66 and 71) were documented in nine patients. This region, which corresponds to a loop between the beta3 and beta4 strands of the fingers subdomain of RT, is involved in the interaction between the enzyme and the template primer. In vitro data with recombinant enzymes have shown the importance of this domain in the processive polymerization of HIV-1 RT. The rearrangements (eight deletions/insertions and one deletion with conservation of the reading frame) did not affect the overall secondary structure of the fingers subdomain, as assessed by the Garnier Osguthorpe Robson prediction method. The changes were generally stable over a follow-up of 10-12 months. With the exception of two cases, most of the patients of this study did not respond efficiently to antiretroviral therapy as assessed by measurements of plasma viraemia. Correspondingly, the RT and protease genes sequenced from these patients displayed numerous resistance-associated mutations. CONCLUSION: Functional and stable rearrangements in the beta3-beta4 hairpin of HIV-1 RT can be found in circulating viruses from patients under combination therapy. These rearrangements may affect the virological response to antiretroviral therapy by increasing the processivity of RT, an enzymatic parameter that reflects the fidelity of the polymerization process.

Amino Acid Sequence↗
PubMed1998

Specific interaction of HIV-1 and HIV-2 surface envelope glycoproteins with monolayers of galactosylceramide and ganglioside GM3.

Cellular glycosphingolipids mediate the fusion between some viruses and the plasma membrane of target cells. In the present study, we have analyzed the interaction of human immunodeficiency virus (HIV)-1 and HIV-2 surface envelope glycoproteins from distinct viral isolates with monolayers of various glycosphingolipids at the air-water interface. The penetration of the viral glycoproteins into glycosphingolipid monolayers was detected as an increase in the surface pressure. We found that HIV-1 recombinant gp120 (IIIB isolate) could penetrate into a monomolecular film of alpha-hydroxylated galactosylceramide (GalCer-HFA), while ceramides, GluCer, and nonhydroxylated GalCer were totally inactive. The glycoproteins isolated from HIV-1 isolates LAI and NDK and from HIV-2(ROD) could also interact with a GalCer-HFA monolayer, whereas gp120 from HIV-1(SEN) and HIV-1(89.6) did not react. These data correlated with the ability of the corresponding viruses to gain entry into the CD4(-)/GalCer+ cell line HT-29, demonstrating the determinant role of GalCer-HFA in this CD4-independent pathway of HIV-1 and HIV-2 infection. In contrast, all HIV-1 and HIV-2 glycoproteins tested were found to interact with a monolayer of GM3, a ganglioside abundantly expressed in the plasma membrane of CD4(+) lymphocytes and macrophages. A V3 loop-derived synthetic peptide inhibitor of HIV-1 and HIV-2 infection in both CD4(-) and CD4(+) cells could penetrate into various glycosphingolipid monolayers, including GalCer-HFA and GM3. Taken together, these data suggest that the adsorption of human immunodeficiency viruses to the surface of target cells involves an interaction between the V3 domain of the surface envelope glycoprotein and specific glycosphingolipids, i.e. GalCer-HFA for CD4(-) cells and GM3 for CD4(+) cells.

Amino Acid Sequence↗
PubMed1998

Identification of a sequence that mediates promiscuous binding of invariant chain to MHC class II allotypes.

The invariant chain (Ii) shows promiscuous binding to a great variety of MHC class II allotypes. In contrast, the affinities of the Ii-derived fragments, class II-associated Ii peptides, show large differences in binding to class II allotypes. The promiscuous association of Ii to all class II polypeptides therefore requires an additional contact site to stabilize the interaction to the polymorphic class II cleft. We constructed recombinant molecules containing the class II binding site of Ii (CBS) and tested their association with HLA-DR dimers. The CBS fused to the transferrin receptor mediates binding of transferrin receptor-CBS to class II dimers. Within the CBS, deletion of a sequence N-terminal to the groove-binding motif abolished binding of Ii to DR. A promiscuous class II binding site was identified by reinsertion of the N-terminal residues, amino acids 81-87, of Ii into an Ii mutant that lacks the groove-binding segment. DR allotype-dependent association of Ii was achieved by insertion of antigenic sequences. The promiscuous association, in contrast to the class II allotype-dependent binding of Ii, is important to prevent interaction of class II dimers to nascent polypeptides in the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1997

Exon 6 is essential for invariant chain trimerization and induction of large endosomal structures.

Invariant chain (Ii) is a transmembrane type II protein that forms a complex with the major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum (ER). The membrane proximal luminal region of Ii is responsible for the non-covalent association with MHC class II molecules. Chemical cross-linking in COS cells was used to study the effect of luminal and cytoplasmic deletions on trimerization of Ii. We demonstrate that trimerization of Ii is independent of the cytosolic tail of Ii, whereas residues 162-191 (the sequence encoded by exon 6) in the luminal part of Ii are essential for trimer formation. Immunofluorescence studies of the transfected luminal deletion constructs show that the amino acids encoded by exon 6 of Ii are also essential for the induction of large endosomal vesicles. The data suggest that Ii must be in a trimeric form to modify the endosomal pathway.

Amino Acid Sequence↗
PubMed1997

Granulocyte-macrophage colony-stimulating factor elevates invariant chain expression in immature myelomonocytic cell lines.

Invariant chain (Ii) plays an important role in major histocompatibility complex (MHC) class II antigen processing and presentation and is constitutively synthesized in B lymphocytes, in macrophages, dendritic cells and in some epithelial cells. It has been shown that interferon-gamma, tumour necrosis factor-alpha and interleukin-4 co-regulate Ii and MHC class II expression in various cell types. We describe here a novel regulation of Ii expression in macrophages. Treatment of the premature monocytic cell lines WEHI 265.1, M1 and WEHI-3B with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) strongly enhances Ii expression while class II expression is not induced. In contrast, GM-CSF did not enhance Ii in mature macrophage cell lines. The increase of Ii expression in WEHI 265.1 cells takes several days. This long induction time, and a difference in activity between GM-CSF-conditioned medium and GM-CSF, together suggest that GM-CSF stimulates WEHI 265.1 cells to secrete a factor that modulates Ii expression. These results may imply a class II-independent function of Ii, which we discuss in this paper.

Animals↗
PubMed1997

Both invariant chain isoforms Ii31 and Ii41 promote class II antigen presentation.

The invariant chain (Ii) gene encodes two differentially spliced variants Ii31 and Ii41. The Ii31 isotype is the dominant form expressed in all antigen-presenting cells (APC). Ii41 is differentially expressed and can be found in large quantities in Langerhans and dendritic cells. While a functional role of Ii in class II antigen presentation is now well established, a distinct role of the Ii isotypes remains controversial. We tested Ii31 and Ii41 L cell transfectants for antigen presentation of hen egg lysozyme (HEL) to T cell hybridomas. The result indicates that both Ii chains promote antigen presentation equally well. To test other APC than transfected L cells, we introduced a recombinant Ii41 gene into anti-deficient mouse line. There the transgene induces about one-third of total li expression of wild-type mice. Surface expression of class II molecules and the CD4 compartment which are deficient in Ii knock-out mice are restored in Ii41 transgenic mice. B lymphocytes from Ii41 transgenic mice and Ii31-expressing B lymphocytes from wild-type mice were used as APC for presentation of keyhole limpet hemacyanin and ovalbumin to T cell hybridomas. The results show that both Ii chains facilitate antigen presentation equally well.

Animals↗
PubMed1997

Co-expression of CXCR4/fusin and galactosylceramide in the human intestinal epithelial cell line HT-29.

OBJECTIVE: To detect the expression CXCR4/fusin in human intestinal epithelial cells and to assess its potential role in the pathway of HIV-1 infection mediated by the alternative gp120 receptor galactosylceramide (GalCer). METHODS: GalCer+ (HT-29, HT-29/CD4+) and GalCer- (Caco-2/Cl2, Cl14 and Cl14/CD4+) human intestinal cell lines were analysed for CXCR4/fusin expression using the monoclonal antibody (MAb) 12G5. This MAb was then evaluated for its ability to inhibit HIV-1 infection in permissive cells. HIV-1 infection was measured by detection of p24 antigen, polymerase chain reaction amplification, and cocultivation with CD4+ cells. RESULTS: CXCR4/fusin was detected on the surface of HT-29 and HT-29/CD4+, but not on Caco-2/Cl2, Cl14 and Cl14/CD4+ cells. Ninety per cent of CXCR4/fusin+ HT-29 and HT-29/CD4+ cells co-expressed GalCer. Infection of HT-29 cells by laboratory isolates of HIV-1 was inhibited by both anti-GalCer and anti-CXCR4/fusin MAbs. Expression of CD4 rendered HT-29 cells sensitive to HIV-1(89.6), a macrophage-tropic isolate that does not recognize GalCer. The 12G5 MAb blocked HIV-1 infection of HT-29/CD4+ cells. In contrast, the expression of HIV-1 receptors, i.e., CD4 GalCer or both, into CXCR4/fusin-negative intestinal cells did not confer sensitivity to HIV-1 infection. The resulting receptor-positive cell lines could, however, bind HIV-1, whereas the original cell lines could not. CONCLUSION: HIV-1 entry into human intestinal cells involves both GalCer and CXCR4/fusin. HIV-1 isolates such as 89.6 that are able to use CXCR4/fusin as coreceptor, but do not bind to GalCer, do not infect these cells. These data raise the possibility that CXCR4/fusin may function as a coreceptor for HIV-1 entry into CD4-/GalCer+ intestinal epithelial cells.

Antibodies, Blocking↗
PubMed1996

The invariant chain derived fragment CLIP is an efficient in vitro inhibitor of peptide binding to MHC class II molecules.

The invariant chain derived peptide CLIP inhibits association of peptides to the class II peptide binding site. Two DR3 specific peptides, the microbacterial heat shock protein 65 derived peptide hsp3-13 and the naturally occurring invariant chain derived peptide Ii131-149 were employed to study binding inhibition by CLIP (Ii82-102) in a series of combinations. Incubation of detergent solubilized DR polypeptides from Ii-free cells with 500 microM of synthetic CLIP almost completely prevents binding of 50 microM subsequently added DR3-specific peptides. When CLIP and the peptides were added simultaneously to DR3 molecules, binding of hsp3-13 was abolished, whereas binding of Ii131-149 was only partially blocked. This indicates apparent affinity differences of the peptides. The addition of CLIP to preformed DR-peptide complexes substantially reduced binding of hsp3-13,while there was little effect on the DR associated Ii131-149. The profound inhibitory ability of CLIP, which in vivo would diminish binding of antigenic peptides, suggests an intracellular mechanism that abrogates the persistence of the CLIP-DR complex. The HLA-DM molecules have been suggested as candidates for this function. The strong in vitro binding of the naturally occurring peptide Ii131-149 to DR3 may suggest that only limited amounts of this peptide are available in vivo for competition of exogenous peptide binding to class II molecules.

Amino Acid Sequence↗
PubMed1996

Synthesis of the very acid-sensitive Fmoc-Cys(Mmt)-OH and its application in solid-phase peptide synthesis.

S-4-methoxytrityl cysteine was synthesized and converted into the corresponding Fmoc-Cys(Mmt)-OH by its reaction with Fmoc-OSu. As compared to the corresponding Fmoc-Cys(Trt)-OH, the S-Mmt-function was found to be considerably more acid labile. Quantitative S-Mmt-removal occurs selectively in the presence of groups of the tert butyl type and S-Trt by treatment with 0.5-1.0% TFA. The new derivative was successfully utilized in the SPPS of Tyr1-somatostatin on 2-chlorotrityl resin. In this synthesis groups of the Trt-type were exclusively used for amino acid side-chain protection. Quantitative cleavage from the resin and complete deprotection was performed by treatment with 3% TFA in DCM-TES (95:5) for 30 min at RT. We observed no reduction of tryptophan under these conditions.

Chromatography, High Pressure Liquid↗
PubMed1995

Deletion of a C-terminal sequence of the class II-associated invariant chain abrogates invariant chains oligomer formation and class II antigen presentation.

The MHC class II-associated invariant chain (Ii) is involved in Ag processing and presentation. Physical association of MHC class II molecules with Ii and an effect of Ii on peptide loading to class II have been demonstrated, but to date these functions have not been related to a particular region of Ii. We investigated luminal deletion mutants of Ii and their role in Ag processing and presentation. IAk-expressing L cells were transfected with deletion mutants of the Ii gene and assayed for their ability to present hen egg lysozyme to three different T cell hybridomas. It is shown that the sequence aa 131-191 of Ii is important for the presentation of native hen egg lysozyme. In addition, this C terminal region is shown to be responsible for Ii oligomer formation. It is therefore conceivable that oligomer formation of Ii is a prerequisite for class II-restricted Ag processing and presentation.

Animals↗
PubMed1995

Acquisition of peptides by MHC class II polypeptides in the absence of the invariant chain.

Association of invariant chain with class II molecules has been suggested to inhibit binding of peptides that are available while the class II complex is present in the endoplasmic reticulum (ER) and subsequently transported to endosomes. We tested HLA-DR-transfected rat2 fibroblast cells lacking expression of invariant chain for their ability to form SDS stable class II dimers indicative of peptide binding in the class II cavity. No SDS-resistant class II dimers originating from short pulse-labeled immunoprecipitates can be identified. Prolonged ER retention of DR polypeptides by Brefeldin A treatment does not induce any stable class II dimers. In pulse-chase experiments, heat labile class II dimers are readily detectable after a 60-min chase, increasing in amounts by 4 h of chase. In vitro incubation of rat2DR cell lysate with DR3-binding peptides converts pulse-labeled class II molecules to SDS-resistant dimers. This indicates the ability of ER-resident DR dimers to bind peptides. Inhibition studies were conducted to define the intracellular site where stable class II complexes are formed in rat2DR cells. The lysosomotropic reagent chloroquine abrogates the appearance of SDS-resistant DR complexes in invariant chain-free rat2DR cells, which is consistent with the impact of chloroquine on peptide loading in other APCs. Leupeptin treatment strongly reduces the amount of heat-labile class II molecules but does not impair peptide loading when DR3-specific peptides were added to viable cells or to cell lysates. This result suggests that leupeptin inhibits intracellular degradation of polypeptides and thereby depletes the endocytic pathway of peptides available for class II binding.

Amino Acid Sequence↗
PubMed1995

Surface expression of the invariant chain (CD74) is independent of concomitant expression of major histocompatibility complex class II antigens.

Whether or not intracellular transport and surface expression of the invariant chain (Ii; CD74) occurs independent of the presence of major histocompatibility complex (MHC) class II molecules was examined by comparing the class II-negative mutant lymphoblastoid cell line 174 x CEM.T2 (T2) and its class II-positive parental cell line 174 x CEM.T1 (T1). We found a similar proportion of Ii being transported to the Golgi complex in T1 and T2, as monitored by the degree of sialic acid addition to glycan side chains of Ii. In agreement with this result, T1 and T2 expressed comparable amounts of Ii at the cell surface, as measured by flow cytometry. This indicates that, although not associated with class II molecules, a proportion of Ii is transported to the plasma membrane. Both in T1 and T2, surface Ii (sIi) was rapidly internalized with a half-life of 3-4 min, suggesting that some Ii enters the endocytic route via the cell surface after being internalized. Our data demonstrate transport of Ii on a route alternative to the endocytic pathway. This alternative route could also account for delivery of newly synthesized class II-Ii complexes to processing compartments in antigen-presenting cells.

Antigens, Differentiation, B-Lymphocyte↗