Enhancement of HLA class II restricted antigen presentation by mannose-receptor-mediated uptake.
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Antigens↗
Biomedical subjects
F Koning
Publications and source records attributed to F Koning.
Celiac disease and malignancy: an immunological basis?
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Celiac Disease↗
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↗
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↗
The generation of SDS-stable HLA DR dimers is independent of efficient peptide binding.
MHC class II molecules can exist in two conformations which can be distinguished on the basis of their stability in SDS. The formation of SDS-stable dimers has been shown to correlate with persistent expression of antigen MHC class II-peptide complexes by murine antigen-presenting cells. HLA DR molecules contain either a Val or a Gly at position 86 of the beta chain, which is located in a conserved and prominent hydrophobic pocket in the peptide binding site. Here we show that Val86-containing DR molecules more frequently select peptides which induce the formation of SDS-stable dimers than Gly86 variants. Using analogues of the influence virus haemagglutinin epitope 307-319 we found that the replacement of the aromatic hydrophobic anchor residue (Tyr)at position 309 by amino acids with an aliphatic hydrophobic side chain resulted in the specific formation of high numbers of SDS-stable Val86-DR but not Gly86-DR dimers. These results indicate that the fit between the first anchor residue and the hydrophobic pocket around Dr beta 86 plays a critical role in the formation of SDS-stable DR dimers. Synthetic analogues of naturally processed DR-associated peptides displayed promiscuity in their capacity to bind to several DR specificities and in their ability to induce the SDS-stable conformation. However, no correlation was observed between binding capacity and the ability to induce the SDS-stable conformation. Since it has been shown that SDS stability can relate to the kinetics of peptide-MHC class II interactions, the definition of the requirements for the formation of SDS-stable HLA class II molecules may be important for the design of effective peptide-based immunomodulation protocols.
Amino Acid Sequence↗
Human gamma delta T-cells in the epithelium of the gut and in the inflamed synovial tissue preferentially express the V gamma 8 T-cell receptor chain.
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Arthritis, Rheumatoid↗
Identification of a graft versus host disease-associated human minor histocompatibility antigen.
Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1-positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation.
Amino Acid Sequence↗
Distribution of HLA class II molecules in epidermal Langerhans cells in situ.
We performed immunoelectron microscopic studies to investigate the expression of HLA class II molecules in Langerhans cells (LC). In the epidermis, LC expressed class II molecules on the plasma membrane of the dendrites, resulting in a class II positive reticulo-epithelial network, but not on the surface of the cell body. In contrast, isolated LC as well as activated LC in situ displayed an even and strong expression of class II molecules on their entire cell surface. Therefore, isolation and/or activation results in redistribution and up-regulation of class II molecules on the cell surface. In addition, double-labeling experiments were carried out with monoclonal antibodies to class II antigens and to LAMP-1, CD63 and alpha-glucosidase, specific markers for organelles of the endosomal/lysosomal system. The results show expression of class II molecules on intracellular, electron-dense vesicular structures, and co-localization of class II molecules and the markers for late endosomes and early lysosomes in human LC in situ. Expression of these markers was not found on Birbeck granules, an LC-specific organelle.
Animals↗
Identification of two distinct function gamma delta TCR complexes on the surface of a human T cell clone.
In this study we describe the expression of two T cell receptor (TCR) gamma chains on the surface of a human T cell clone isolated from the peripheral blood. Each gamma chain was part of an independent and functional TCR. The dual receptor T cell clone (and all subclones derived from this clone) had stable expression of this phenotype. Immunoprecipitation studies revealed the expression of non-disulfide linked TCRs by this V gamma 4+V gamma 9+V delta 1+ T cell clone, which was in agreement with the finding that both V gamma gene transcripts were rearranged to C gamma 2-associated joining elements. Both gamma chains were derived from productive rearrangements of different (allelic) genes coding for a V gamma 4+ and a V gamma 9+ gamma-chain, and both were coupled to a V delta 1+ delta chain. Incubation of this V gamma 4+V gamma 9+V delta 1+ T cell clone with TCR gamma-chain-specific MoAbs rapidly induced an increase in intracellular Ca++, indicating that both gamma-chains are functional. Furthermore, this clone responded to stimulation with S. aureus derived superantigens. We suggest therefore that exogenous (super)antigens can trigger dual receptor T cells resulting in activation of these T cells.
Amino Acid Sequence↗
DR4Dw4/DR53 molecules contain a peptide from the autoantigen calreticulin.
Rheumatoid arthritis (RA) occurs more frequently in HLA-DR4+ individuals than in those who do not express this MHC class II molecule. Although the role of this genetic factor in the immunopathology of this autoimmune disease is unclear, the association of RA with HLA-DR4 may indicate that DR4 molecules present autoantigen(s) to T cells. Here we report the analysis of naturally processed peptides, eluted from a mixture of HLA-DR4Dw4 (DRB1*0401) and DR53 (DRB4*0101) molecules isolated from an RA patient-derived EBV-transformed B cell line. Several (size variants of) self-peptides originating from the autologous molecules HLA-A2, HLA-Cw9, HLA-B62, HLA-DR4Dw4 and HLA-DR53, were identified. We also found a sequence that has no homology to any protein in the SwissProt protein sequence databank, and a peptide identical to an internal fragment of the autoantigen calreticulin. The association of the identified peptides with cells expressing HLA-DR4Dw4/DR53 was confirmed by peptide binding analysis. In agreement with previously described peptide binding motifs for DR4Dw4, most peptides contained an aromatic residue (Phe, Tyr, Trp) at relative position i and a small hydroxyl-containing residue (Ser, Thr) at i + 5. Our findings indicate that in RA patient-derived EBV-transformed B cells DR4Dw4/DR53 molecules present a peptide from the autoantigen calreticulin. Interestingly, autoantibodies against calreticulin have been found in various rheumatic diseases, including rheumatoid arthritis. Thus, the analysis of HLA class II-bound peptides can lead to the identification of putative T helper epitopes, which might be involved in the immunopathology of autoimmune diseases.
Amino Acid Sequence↗
T-cell receptor usage of interleukin-2-responsive peripheral gamma delta T cells.
The majority of human peripheral gamma delta T cells express the V gamma 9 gene in combination with the V delta 2 gene. The diversity of this subset of gamma delta T cells is limited by a preferential usage of the J gamma P gene segment and a highly distinctive junctional motif of the T-cell receptor (TCR) delta chain. We and others have observed that peripheral blood derived V gamma 9+V delta 2+ gamma delta T cells of healthy individuals are activated after stimulation with interleukin-2 (IL-2) in vitro, but only a small percentage of gamma delta T cells subsequently proliferates. To assess whether the proliferating, IL-2-responsive gamma delta T cells represent a selective group of T cells, we have analysed TCR junctional features of IL-2-responsive gamma delta T cells. Out of 30 individuals studied, nine were identified as IL-2-responders and three as IL-2-hyperresponders. The TCR V(D)J gene usage from IL-2 stimulated peripheral blood lymphocytes of these IL-2-(hyper)responsive individuals was analysed. The results showed that in most individuals gamma delta T cells polyclonally expanded after stimulation with IL-2. In two IL-2-hyperresponder individuals, however, a monoclonal expansion of a particular V gamma 9+V delta 2+ gamma delta T cell was found. In one of these individuals, this V gamma 9+V delta 2+ T-cell clone expressed a very rare gamma delta TCR type because of the presence of an Ala within the junctional region at a conserved position relative to V delta framework residues (delta 97), which is very infrequently used by peripheral blood V gamma 9+V delta 2+ cells. This particular clonotype could also be detected in unstimulated PBL samples taken from that individual, and made up for 30% of the total peripheral gamma delta T-cell pool. These data indicate that in general IL-2-responsive V gamma 9+V delta 2+ gamma delta T cells represent a polyclonal population, reflecting in vivo stimulation with multiple antigens or superantigens. In contrast, monoclonal expansions of gamma delta T cells after stimulation with IL-2 can also occur, which may be related to an in vivo stimulation by one particular antigen, rendering this gamma delta T-cell type dominant in the peripheral blood.
Base Sequence↗
High expression of V gamma 8 is a shared feature of human gamma delta T cells in the epithelium of the gut and in the inflamed synovial tissue.
We have analyzed the V-gene usage in gamma delta T cells of the human gut and joint by using a new mAb (B18) specific for V gamma 8 of human TCR-gamma delta+ T cells. The B18+ population constituted a minor subset of the gamma delta T cells in peripheral blood (PB) of healthy persons (6 +/- 5%) and only 1 of 35 gamma delta T cell clones analyzed was positive. In contrast, the B18+ subset was a dominant gamma delta T cell population among intraepithelial lymphocytes (IEL) derived from the human intestine (74 +/- 29, p < 0.002), and two of three IEL clones from patients with coeliac disease were B18+. Interestingly, a higher proportion of B18+ gamma delta T cells was found in the synovial fluid of patients with rheumatoid arthritis (RA) (21 +/- 18%, 0.02 < p < 0.05) compared with normal PB. Furthermore, the B18+ subset was more frequent among IL-2-expanded gamma delta T cells (42 +/- 20%) derived from synovial tissue than among IL-2-expanded cells derived from synovial fluid (p < 0.002) and PB from RA patients (p < 0.02) as well as normal PB (p < 0.002). The V-gene usage of 13 gamma delta T cell clones from the synovial fluid of arthritic patients was analyzed. All B18+ clones (n = 7) expressed mRNA for V gamma 8 together with mRNA for V delta 1 (n = 5) or mRNA for V delta 3 (n = 2). None of the B18- clones expressed V gamma 8 (n = 6). We conclude that the gamma delta T cell that expresses V gamma 8, together with mainly V delta 1, is a major gamma delta T cell subset among the IEL of the gut and a highly frequent subset in the synovial tissue of patients with RA. This subset may correspond to the mouse V gamma 7+ IEL, which has a high degree of amino acid sequence homology with the human V gamma 8 protein.
Adult↗
Identification of an HLA-DQ2 peptide binding motif and HLA-DPw3-bound self-peptide by pool sequencing.
Molecules of the major histocompatibility complex (MHC) present antigenic peptides to T cells. Sequencing peptide pools eluted from MHC class I molecules has established allele-specific peptide binding motifs. We applied pool sequencing to analyze human MHC class II-bound peptides and found that HLA-DQ2-eluted peptides predominantly contained lysine, isoleucine, and phenylalanine at relative position i, i + 3 and i + 8, respectively. These residues putatively represent anchor residues for MHC binding. Analysis of a heterogeneous HLA-DPw3/DPw4-eluted peptide pool yielded a sequence matching an epitope from the endogenous enzyme glyceraldehyde-3-phosphate dehydrogenase. This self-peptide and a partially identical, known allo-epitope bound specifically to DPw3 and DR13 molecules, suggesting the sharing of a binding motif. In particular, the presence of an arginine at relative position 4 appeared important for binding to these HLA class II specificities. Thus, pool sequencing is applicable for the analysis of MHC class II-eluted peptides.
Amino Acid Sequence↗
Functional and molecular characterization of B cell-responsive V delta 1+ gamma delta T cells.
Cells expressing the V delta 1+ gene segment are a minor gamma delta T cell population in human peripheral blood but predominate in epithelial and (inflamed) tissues. The characteristic dendritic-like morphology of these gamma delta T cells is consistent with their putative immune surveillance role in epithelia. Their function, however, remains unknown. We and others previously reported that a subset of V delta 1+ gamma delta T cells proliferates after stimulation with Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCL), but not with fresh peripheral blood-derived B cells. These responses were independent of the type of T cell receptor (TcR) gamma chain co-expressed with the V delta 1 chain. The in vivo relevance of this LCL-mediated activation as well as the nature of the stimulatory ligand on the LCL is not well established. In this study, we tested the proliferative response of V delta 1+ LCL-responsive T cells against non-EBV-transformed B cells, activated through CD40 by murine EL4 B5 cells, and to a panel of B cell lines differing in the expression of EBV nuclear antigen proteins and adhesion/co-stimulatory molecules. The role of the Epstein-Barr virus-derived antigen in the induction of this response could be excluded as the activated (non-EBV-transformed) peripheral blood B cells were also able to induce a proliferative response in the LCL-responsive V delta 1+ T cells. Therefore, the stimulatory ligand on B cells is of cellular rather than of viral origin, and its expression is up-regulated upon activation of B cells. The expression of B7 and CD39 molecules on the surface of activated B cells appeared to be crucial since antibodies to these structures could block the induction of proliferation of the V delta 1+ T cells. Finally, we investigated the diversity of the responding V delta 1+ gamma delta T cell clones by sequence analysis of the TcR delta junctional regions. No restricted V-D-J sequences were found among the LCL-responsive V delta 1+ T cell clones, arguing strongly against a mono- or oligoclonal V delta 1+ gamma delta T cell response to LCL. These findings may explain the presence of polyclonally activated V delta 1+ T cells in inflamed tissues where activated B cells are often present.
Adenosine Triphosphatases↗
Functional human epidermal Langerhans cells that lack Birbeck granules.
Birbeck granules (BG) are cytoplasmic organelles that are only found in Langerhans cells (LC). The function of BG is still unclear, although it has been claimed that they are actively involved in receptor-mediated endocytosis and participate in the antigen-processing/presenting function of LC. We have identified a healthy white 29-year-old man whose LC completely lack the presence of BG as determined by electronmicroscopic studies. This was observed repeatedly using skin biopsy specimens taken from several places on the body during a period of 2.5 years. The absence of BG in these LG was documented further by the lack of staining with a BG-specific monoclonal antibody. Despite the complete lack of BG, LC were present in normal numbers, had all the usual morphologic characteristics, and were CD1a and human leukocyte antigen (HLA) class II positive. Two observations indicate that these BG-negative LC display normal antigen-presenting capacity. First, the individual could be sensitized by the hapten diphenylcyclopropenone. This was accompanied by a strong increase in the cell surface expression of HLA class II antigens on his LC, suggesting LC activation. Second, his epidermal cells elicited a normal positive response in an allogeneic mixed epidermal cell lymphocyte reaction. Together these observations strongly suggest that BG are not a prerequisite for normal LC function in vivo and in vitro.
Adult↗
Immobilized staphylococcal enterotoxin A is sufficient to induce T cell proliferation.
We investigated the role of HLA-DR molecules in T cell stimulation by staphylococcal enterotoxin A (SEA). Previous results with immobilized purified HLA-DR preincubated with peptide showed that peptide-specific T cell clones were able to bind to and proliferate in response to purified HLA-DR/peptide complexes in the absence of antigen presenting cells. We report here that two human T cell clones (1 alpha beta and 1 gamma delta T cell clone) and a murine T cell hybridoma were each activated by immobilized purified HLA-DR4Dw4 preincubated with SEA. Furthermore, immobilized SEA in the absence of HLA-DR4Dw4 also stimulated the human T cell clones. The proliferative response of the human T cell clones was inhibited by CD3-reactive monoclonal antibodies, indicating that the T cell receptor (TCR)/CD3 reacts with SEA. These observations suggest that the HLA-DR in the complex functions only to immobilize SEA and that an interaction between the TCR and HLA-DR is not necessary for SEA-driven T cell stimulation. Finally, the assays described here could provide a method for defining and distinguishing the SEA binding sites for MHC class II and TCR.
Animals↗
Isolation of an HLA-A2.1 extracted human minor histocompatibility peptide.
Purified HLA-A2.1 molecules obtained by affinity chromatography of 6 x 10(10) Epstein Barr virus (EBV)-transformed B lymphocytes were used in an attempt to isolate the human HLA-A2.1-restricted minor histocompatibility (H) peptides H-Y and HA-2. Fraction 18 of the high-performance liquid chromatography (HPLC)-separated HLA-A2.1 peptide pool was found to contain the natural HA-2 peptide. An HA-2-specific, HLA-A2.1-restricted cytotoxic T lymphocyte clone lysed HLA-A2.1+ HA-2- EBV-transformed B lymphocyte cell lines reproducibly and in a concentration-dependent fashion in the presence of fraction 18, but not in the presence of other HPLC fractions. By contrast, H-Y sensitizing activity was not found in any fraction. Amino acid sequencing of peptide fraction 18 revealed a mixture of peptides with maximal length of nine amino acids, in which the presence of Leu at positions 2 and 9 was dominant. Surprisingly, the HA-2 peptide could not be mimicked by any of the peptide mixtures synthesized according to the amino acid sequences found in fraction 18. Our failure to obtain the actual amino acid sequence of the human minor H peptide HA-2 from a peptide pool with the established pattern for binding to HLA-A2.1 may indicate that this CTL defined minor H peptide does not represent an abundant HLA-A2.1 binding peptide.
Amino Acid Sequence↗
HLA-DR beta chain residue 86 controls DR alpha beta dimer stability.
Major histocompatibility complex class II molecules exist in two forms, which can be distinguished on the basis of their stability in sodium dodecyl sulfate (SDS) as SDS-stable and SDS-unstable alpha beta dimers. The ratio of stable vs. unstable alpha beta dimers varies between murine H-2 alleles and isotypes, but the molecular basis for this observation is unknown. Here we show that for the human HLA-DRB1 and HLA-DRB3 gene products this ratio is controlled by the valine/glycine dimorphism at position 86. Haplotypes coding for DR beta chains with a valine at position 86 express higher numbers of stable dimers compared to similar haplotypes expressing DR beta chains with a glycine at that position. Reverse-phase high-performance liquid chromatography analysis of iodinated peptides, which were eluted from DR dimers with either a DRB1*1101 or a DRB1*1104 beta chain which differ only at position 86, indicated that these DR dimers contain (partially) distinct sets of peptides. The valine/glycine dimorphism is highly conserved, present in most HLA-DR alleles and influences peptide-binding. Analysis of the occurrence of the Val86 and the Gly86 gene products revealed that these are not equally present in the population. Depending on the DR specificity either the Val86 of Gly86 allelic variant is favored. Thus, the natural, highly conserved dimorphism at HLA-DR beta chain position 86 influences peptide selection. The dimorphism is therefore likely to influence antigen presentation and forms the molecular basis for the observed differences in stability of Val86- and Gly86-containing DR dimers in the presence of SDS.
Alleles↗