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F Koning

Publications and source records attributed to F Koning.

At least 37 records · Page 2Linked to original sources
PubMed1999

Human dendritic cells shed a functional, soluble form of the mannose receptor.

Human monocyte-derived dendritic cells (DC) use mannose receptor (MR)-mediated endocytosis for efficient antigen capture and targeting to the endosomal/lysosomal compartment. Active biosynthesis of the MR takes place in such cells. We now report that a substantial percentage (up to 20%) of these newly synthesized MR are secreted into the culture medium. The secretion of the soluble MR (sMR) was found to be proportional to the rate of synthesis. The addition of the inflammatory mediator lipopolysaccharide (LPS) to DC, known to induce maturation, strongly reduced MR synthesis, expression and shedding of the MR. The sMR is approximately 10 kDa smaller than the membrane-bound form, but contains an intact N-terminus, indicating the lack of the cytoplasmic and transmembrane region. The sMR appeared to be directly generated from the cell-bound form, indicative of proteolytic cleavage. Importantly, the sMR has maintained its mannose-binding properties since it was capable of binding a mannosylated ligand. The high amount of sMR released by DC and its ability to bind mannosylated ligand might indicate that this molecule plays a role in the transport of mannosylated proteins from the site of inflammation to other parts of the body. Whether that contributes to the generation of immune responses remains to be determined.

Cells, Cultured↗
PubMed1999

Definition of agonists and design of antagonists for alloreactive T cell clones using synthetic peptide libraries.

Alloreactive T cells form an important barrier for organ transplantation. To reduce the risk of rejection patients are given immunosuppressive drugs, which increase the chance of infection and the incidence of malignancies. It has been shown that a large proportion of alloreactive T cells specifically recognize peptides present in the groove of the allogeneic MHC molecule. This implies that it might be possible to modulate the alloresponse by peptides with antagonistic properties, thus preventing rejection without the side effects of general immunosuppression. Peptide antagonists can be designed on the basis of the original agonist, yet for alloreactive T cells these agonists are usually unknown. In this study we have used a dedicated synthetic peptide library to identify agonists for HLA-DR3-specific alloreactive T cell clones. Based on these agonists, altered peptide ligands (APL) were designed. Three APL could antagonize an alloreactive T cell clone in its response against the library-derived agonist as well as in its response against the original allodeterminant, HLA-DR3. This demonstrates that peptide libraries can be used to design antagonists for alloreactive T cells without knowledge about the nature of the actual allostimulatory peptide. Since the most potent agonists are selected, this strategy permits detection of potent antagonists. The results, however, also suggest that the degree of peptide dependency of alloreactive T cell clones may dictate whether a peptide antagonist can be found for such clones. Whether peptide antagonists will be valuable in the development of donor-patient-specific immunosuppression may therefore depend on the specificity of the in vivo-generated alloreactive T cells.

Amino Acid Sequence↗
PubMed1999

Get into the groove! Targeting antigens to MHC class II.

The activation of MHC class II-restricted helper T cells is paramount to adaptive immune responses. Vaccine development could therefore benefit from improved ways of targeting antigens into MHC class II molecules. In recent years, the natural pathways of MHC class II antigen presentation have been exploited to achieve this goal. First, antigenic proteins and peptides have been modified to facilitate receptor-mediated uptake by professional antigen-presenting cells. Second, DNA constructs containing specific targeting sequences have been used to direct endogenously synthesized antigens to the MHC class II compartments. Both strategies proved to be highly effective. We review these data and describe how this knowledge is currently applied to the design of vaccines that activate helper T cells in vivo.

Amino Acid Sequence↗
PubMed1998

Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin.

Celiac disease is a common severe intestinal disease resulting from intolerance to dietary wheat gluten and related proteins. The large majority of patients expresses the HLA-DQ2 and/or DQ8 molecules, and gluten-specific HLA-DQ-restricted T cells have been found at the site of the lesion in the gut. The nature of peptides that are recognized by such T cells, however, has been unclear so far. We now report the identification of a gliadin-derived epitope that dominantly is recognized by intestinal gluten-specific HLA-DQ8-restricted T cells. The characterization of such epitopes is a key step toward the development of strategies to interfere in mechanisms involved in the pathogenesis of celiac disease.

Amino Acid Sequence↗
PubMed1998

Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity.

Celiac disease (CD) is caused by gluten ingestion in susceptible individuals. Tissue transglutaminase (tTG)-specific Abs are characteristic of CD, and increased tTG activity has been observed in the jejunal biopsies of patients. Here we demonstrate that tTG selectively deamidates gluten peptides, which results in strongly enhanced T cell-stimulatory activity. To our knowledge, this is the first example of an enzymatic modification of a food protein that affects T cell recognition. Moreover, these modifications may lead to the amplification of gluten-specific T cell responses in the gut and consequently may be important for the development of CD.

Amides↗
PubMed1998

Increased HLA-DQ2-affinity of a synthetic gliadin peptide by acid-induced deamidation of glutamine residues.

Presentation of antigenic gliadin peptides by the HLA-DQ2 molecule is considered as a key event in celiac disease pathogenesis. Chemical deamidation of the side chains of glutamine residues might have a strong influence on gliadin peptide binding to the DQ2 molecule. Glutamine deamidation of A-gliadin peptide (45-56) under acidic conditions corresponding to the gastric environment was studied using RP-HPLC, Edman degradation, capillary electrophoresis and electrospray mass spectrometry. Deamidation resulted in peptides with increased DQ2-affinities as assessed in a cell-free binding assay.

Amides↗
PubMed1998

Altered peptide ligands and wild-type peptide induce indistinguishable responses of a human Th0 clone.

The response of the human influenza hemagglutinin (HA)-specific T helper clone HA1.7 to its wild-type (wt) HA307-319 peptide ligand and related altered peptide ligands (APL) was examined over a wide range of antigen concentrations. The time course of cytokine production and surface expression of CD40 ligand and CD3 was followed at the single-cell level by flow cytometry and compared with the induction of proliferation. We observed that the APL induced responses that were indistinguishable from those induced by the wt HA ligand, albeit at higher antigen densities. Moreover, the activation parameters were induced with identical kinetics. Blocking of CD4 co-ligation inhibited the recognition of the weakly stimulatory APL, but not of the wt HA307-319 peptide. Finally, in all cases the response of the T cell clone correlated with down-regulation of surface TCR/CD3 complexes. Together, these observations support a quantitative model of T cell activation.

Amino Acid Sequence↗
PubMed1998

Anti-alpha-gliadin antibodies (AGA) in the serum of coeliac children and controls recognize an identical collection of linear epitopes of alpha-gliadin.

Anti-gliadin antibodies can be found in the serum of patients with overt and subclinical coeliac disease, but also in that of some controls. The aim of the present study was to identify the linear epitopes of the alpha-gliadin molecule to which the humoral response is directed. Therefore, the IgG and IgA antibody reactivity against an overlapping set of synthetic peptides covering the entire sequence of alpha-gliadin was measured in the sera from patients with coeliac disease, from controls with elevated titres of anti-gliadin antibodies and from healthy children using an ELISA technique. The antibodies mainly recognize peptides derived from the N-terminal region of alpha-gliadin, containing the motif QPFXXQXPY. Reactivity was also detected against two other synthetic peptides, which do not contain this motif and represent a sequence encoded further to the C-terminal region of alpha-gliadin. Anti-gliadin antibodies in sera from patients with coeliac disease and from controls recognize the same linear epitopes. Thus, serological investigation of the specificity of these antibodies using a peptide ELISA does not allow discrimination between patients and controls.

Adolescent↗
PubMed1998

Strongly increased efficiency of altered peptide ligands by mannosylation.

Altered peptide ligands (APL) have the potential to modulate pathogenic T cell reactivity. High concentrations of APL, however, are required to achieve efficient blocking of the T cell response. We have therefore investigated whether improved delivery of APL to professional antigen-presenting cells (APC) can lead to more efficient application of such peptides. For this purpose APL were bis-mannosylated in order to facilitate their uptake by mannose receptor-positive dendritic cells (DC) in vitro. We present evidence that a 100- to 1000-fold lower concentration of bis-mannosylated APL was sufficient for complete blocking of the proliferative T cell response against the agonist peptide compared to the non-mannosylated APL. Moreover, bis-mannosylated APL were similarly effective in the inhibition of the T cell response against whole protein antigens. In contrast, unrelated, bis-mannosylated class II binding peptides were ineffective, indicating that the increased efficiency of the mannosylated APL was not due to competition for binding to class II molecules. Furthermore, a strong increase in the efficiency of presentation of APL was also observed when macrophages and peripheral blood mononuclear cells were used as APC. Thus, bis-mannosylation of APL greatly increases their potency to inhibit proliferative T cell responses. Moreover, it is likely that the use of bis-mannosylated APL will result in preferential presentation by mannose receptor-positive, professional APC. These results may be of relevance for more effective use of APL for immunoregulation in vivo.

Adjuvants, Immunologic↗
PubMed1998

The majority of HLA-DR3 alloreactive T cells is peptide specific, but does not recognize known DR3-bound sequences.

Rejection of transplants is frequently caused by activation of alloreactive T cells that recognize HLA/peptide differences between patient and graft. This T-cell response can be directed towards the HLA molecule, the HLA-bound peptide or towards a combination. More insight in the involvement of peptides in this process may help to find ways to avoid rejection using for example antagonist peptides. In recent years many naturally processed HLA-bound peptides have been identified. This raises the question of whether these, presumably abundant, peptides are involved in class II-specific allorecognition. To investigate this, we first determined the proportion of peptide-specific alloreactive T cells in the alloresponse against HLA-DR3. For this purpose we have tested a panel of DR3-specific alloreactive T-cell clones against a DM-mutant (i.e. peptide loading deficient) cell line. We found that 59 out of 64 alloreactive T-cell clones were dependent upon the presence of DM for an optimal response. However, only 2 DM-dependent T-cell clones recognize known peptide sequences. Thus we conclude that most DR3-specific alloreactive T-cell clones are peptide specific and that the currently known DR3-bound peptides are not the main target for allorecognition. Finally, we identified 4 T-cell clones that recognized the DM-mutant better than the wild-type cell line. The response against the wild-type cell line could not be restored with invariant chain derived peptides (CLIP). This provides additional evidence that DM can negatively select self-peptides other than CLIP, which can result in selection against peptides involved in allorecognition.

Amino Acid Sequence↗
PubMed1997

Efficient loading of HLA-DR with a T helper epitope by genetic exchange of CLIP.

The HLA class II-associated invariant chain (Ii)-derived peptide (CLIP) occupies the peptide binding groove during assembly in the endoplasmic reticulum, travels with HLA class II to endosomal compartments, and is subsequently released to allow binding of antigenic peptides. We investigated whether the exchange of CLIP with a known T helper epitope at the DNA level would lead to efficient loading of this helper epitope onto HLA class II. For this purpose, a versatile Ii-encoding expression vector was created in which CLIP can be replaced with a helper epitope of choice. Upon supertransfection of HLA-DR1-transfected 293 cells with an Ii vector encoding a known T helper epitope (HA307-319), predominantly length variants of this epitope were detected in association with the HLA-DR1 molecules of these cells. Moreover, this transfectant was efficiently recognized by a peptide-specific T helper clone (HA1.7). The results suggest that this type of Ii vector can be used to create potent class II+ cellular vaccines in which defined T cell epitopes are continuously synthesized.

Amino Acid Sequence↗
PubMed1997

Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DC) efficiently take up antigens by macropinocytosis and mannose receptor-mediated endocytosis. Here we show that endocytosis of mannose receptor-antigen complexes takes place via small coated vesicles, while non-mannosylated antigens were mainly present in larger vesicles. Shortly after internalization the mannose receptor and its ligand appeared in the larger vesicles. Within 10 min, the mannosylated and non-mannosylated antigens co-localized with typical markers for major histocompatibility complex class II-enriched compartments and lysosomes. In contrast, the mannose receptor appeared not to reach these compartments, suggesting that it releases its ligand in an earlier endosomal structure. Moreover, we demonstrate that mannosylation of protein antigen and peptides resulted in a 200-10,000-fold enhanced potency to stimulate HLA class II-restricted peptide-specific T cell clones compared to non-mannosylated peptides. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DC which may be applicable in vaccine design.

Amino Acid Sequence↗
PubMed1997

Mannose receptor mediated uptake of antigens strongly enhances HLA-class II restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DCs) use macropinocytosis and mannose receptor mediated endocytosis for the uptake of exogenous antigens. Here we show that the endocytosis of the mannose receptor and mannosylated antigen is distinct from that of a non-mannosylated antigen. Shortly after internalization, however, both mannosylated and non-mannosylated antigen are found in an MIIC like compartment. The mannose receptor itself does not reach this compartment, and probably releases its ligand in an earlier endosomal structure. Finally, we found that mannosylation of peptides strongly enhanced their potency to stimulate HLA class II-restricted peptide-specific T cell clones. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DCs which may be useful for vaccine design.

Antigen Presentation↗
PubMed1997

Unique peptide binding characteristics of the disease-associated DQ(alpha 1*0501, beta 1*0201) vs the non-disease-associated DQ(alpha 1*0201, beta 1*0202) molecule.

To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(alpha 1*0501, beta 1*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(alpha 1*0201, beta 1*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I alpha chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. AT p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(alpha 1*0201, beta 1*0202) molecule. This most striking difference between the two DQ molecules is the presence of and additional anchor residue at p3 for the DQ(alpha 1*0201, beta 1*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(alpha 1*0501, beta 1*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(alpha 1*0501, beta 1*0201) and DQ(alpha 1*0201, beta 1*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(alpha 1*0501, beta 1*0201) molecule should be helpful in the identification of CD-inducing epitopes.

Amino Acid Sequence↗
PubMed1996

Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96/GRP94.

Heat shock protein gp96 primes class I restricted cytotoxic T cells against antigens present in the cells from which it was isolated. Moreover, gp96 derived from certain tumors functions as an effective vaccine, causing complete tumor regressions in in vivo tumor challenge protocols. Because tumor-derived gp96 did not differ from gp96 isolated from normal tissues, a role for gp96 as a peptide carrier has been proposed. To test this hypothesis, we analyzed whether such an association of antigenic peptides with gp96 occurs in a well-defined viral model system. Here we present the full characterization of an antigenic peptide that endogenously associates with the stress protein gp96 in cells infected with vesicular stomatitis virus (VSV). This peptide is identical to the immunodominant peptide of VSV, which is also naturally presented by H-2Kb major histocompatibility complex class I molecules. This peptide associates with gp96 in VSV-infected cells regardless of the major histocompatibility com- plex haplotype of the cell. Our observations provide a biochemical basis for the vaccine function of gp96.

Animals↗
PubMed1996

Peptide binding characteristics of the coeliac disease-associated DQ(alpha1*0501, beta1*0201) molecule.

Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of the HLA-DQ2 (alpha1(*)0501, beta1(*)0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found to be derived from two self-proteins: in addition to several size-variants of the invariant chain (li)-derived CLIP peptide, a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this naturally processed epitope (MHClalpha46 - 63) were tested in a cell-free peptide binding competition assay to investigate the requirements for binding to DQ2. First, a core sequence of 10 amino acids within the MHClalpha46 - 63 peptide was identified. By subsequent single amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity of the peptides.

Amino Acid Sequence↗