Project description:The purpose of this study was to identify mRNA transcripts encoding tissue restricted cell surface proteins on human islet tissue that, in turn, might serve as markers for determination of healthy, transplanted and/or diseased islet cell mass. Using oligonucleotide microarrays and clinical grade human islets, we obtained the gene expression profiles of cultured normal islet tissue and compared them to profiles of islets treated with interferon alpha 2 beta, islet-depleted exocrine pancreas, pooled whole pancreas, pooled liver and pooled kidney tissue. Data set filtering and comparisons of gene expression patterns revealed a small set of genes corresponding to transmembrane or membrane associated proteins with limited tissue distributions (present in Islets of Langerhans and, often, central or peripheral nervous system tissues, but absent from exocrine pancreas, liver, kidney and other tissues) with possible utility as novel islet markers. Under the influence of IFN alpha, some of these transcripts show differential expression as confirmed by real time PCR. In addition, we found significant differential expression of two sets of transcripts expressed in islets but with broad tissue distributions, non classical MHC class I b (HLA-G and F) and MHC Class II locus (e.g. HLA-DR alpha and HLA-DQ alpha and beta).
Project description:Accurate prediction of antigen presentation by Human Leukocyte Antigen (HLA) class II molecules is crucial for rational development of immunotherapies and vaccines targeting CD4 T cell activation. So far, most prediction methods for HLA class II antigen presentation have focused on HLA-DR due to limited availability of immunopeptidomics data for HLA-DQ and HLA-DP, while not taking into account alternative peptide binding modes. Here, we present an update to the NetMHCIIpan prediction method which closes the performance gap between all three HLA class II loci. We accomplish this by first integrating large immunopeptidomics datasets describing the HLA class II specificity space across loci using a refined machine learning framework that accommodates inverted peptide binders. Next, we apply targeted immunopeptidomics assays to generate novel data that covers additional HLA-DP specificities. The final method, NetMHCIIpan-4.3, achieves high accuracy and molecular coverage across all HLA class II allotypes.
Project description:Alternative splicing (AS) within the β cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We employed a computational strategy to prioritize pathogenic splicing events in human islets treated with IL-1β + IFN-γ as an ex vivo model of T1D and coupled this analysis with a k-mer based approach to predict RNA binding proteins involved in AS events. In total, 969 AS events were identified in cytokine-treated islets, with the majority (44.8%) involving a skipped exon. AS events occurred with high frequency in MHC Class II-related mRNAs, and targeted qPCR validated reduced inclusion of Exon5 in the MHC Class II gene HLA-DMB, while RNA FISH confirmed HLA-DMB splicing in pancreatic sections from human T1D donors. Together, these data suggest that dynamic control of AS plays a role in the β cell response to inflammatory signals during T1D evolution.
Project description:Gene expression analysis of molecules with known function in HLA class II antigen processing and presentation. Various hematopoietic cell types and (cytokine pre-treated) non-hematopoietic cells that are targeted in Graft-versus-Leukemia reactivity and Graft-versus-Host Disease were collected. Expression was compared between the different hematopoietic and non-hematopoietic cell types for the Invariant chain, HLA-DMA, HLA-DMB, HLA-DOA and HLA-DOB genes. The data show that the Invariant chain, HLA-DMA, HLA-DMB and HLA-DOA genes are expressed in all or the majority of cell types with HLA class II surface expression, whereas expression of the HLA-DOB gene is restricted to professional antigen presenting B-cells and mature dendritic cells. Total RNA was isolated from various hematopoietic cell types isolated (and cultured) from (G-CSF mobilized) peripheral blood from five different individuals and from (IFN-g pre-treated) fibroblasts cultured from skin biopsies from four different patients transplanted with allogeneic hematopoietic stem cells.
Project description:Gene expression analysis of molecules with known function in HLA class II antigen processing and presentation. Various hematopoietic cell types and (cytokine pre-treated) non-hematopoietic cells that are targeted in Graft-versus-Leukemia reactivity and Graft-versus-Host Disease were collected. Expression was compared between the different hematopoietic and non-hematopoietic cell types for the Invariant chain, HLA-DMA, HLA-DMB, HLA-DOA and HLA-DOB genes. The data show that the Invariant chain, HLA-DMA, HLA-DMB and HLA-DOA genes are expressed in all or the majority of cell types with HLA class II surface expression, whereas expression of the HLA-DOB gene is restricted to professional antigen presenting B-cells and mature dendritic cells.
Project description:Classical antigen processing/presentation mechanisms lead to the presentation of antigenic peptides derived from endogenous and exogenous sources for MHC class I and class II molecules, respectively. We show here that, unlike other class II, prevalent HLA-DP molecules whose chains encode Gly84 (DP84Gly) constitutively present endogenous peptides.
Project description:The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) poses a major challenge in hemophilia A (HA) treatment. The formation of FVIII inhibitors is a CD4+ T-cell-dependent mechanism which includes anti- gen presenting cells (APC), B- and T-helper lymphocytes. APC present FVIII-derived peptides on major histocompatibility complex class II (MHC-II) to CD4+ T cells. We previously established a mass spectrometry-based approach to delineate the FVIII repertoire presented on HLA-DR and HLA-DQ. In this study, specific attention was directed towards the identification of FVIII peptides presented on HLA-DP. A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocyte-derived dendritic cells (moDC) from HLA-typed healthy donors. Using this method, we iden- tified 176 to 1,352 different HLA-DP presented peptides per donor, including 26 different FVIII-derived peptides. The most frequently presented peptides derived from the A3 and C2 domains of FVIII. Comparison of the FVIII repertoire presented on HLA-DP with that presented on HLA-DR revealed considerable overlap but also suggested preferential presentation of specific peptides on either HLA-DR or HLA-DP. Fourteen FVIII peptides presented on HLA-DP were synthesized and evalu- ated for their binding ability to the commonly expressed HLA-DP4 molecule which is highly prevalent in the Caucasian population. Peptide binding studies showed that 7 of 14 peptides competed with a reference peptide to HLA-DP4. Interest- ingly, an A3 domain-derived peptide bound with high affinity to HLA-DP4, positioning this peptide as a prime candidate for the development of novel peptide-based tolerogenic strategies for FVIII inhibitors.
Project description:Interferon (IFN)-α is the earliest cytokine signature observed in individuals at risk for type 1 diabetes (T1D), but its effect on the repertoire of HLA Class I (HLA-I)-bound peptides presented by pancreatic β-cells is unknown. Using immunopeptidomics, we characterized the peptide/HLA-I presentation in in-vitro resting and IFN-α-exposed β-cells. IFN-α increased HLA-I expression and peptide presentation, including neo-sequences derived from alternative mRNA splicing, post-translational modifications - notably glutathionylation - and protein cis-splicing. This antigenic landscape relied on processing by both the constitutive and immune proteasome. The resting β-cell immunopeptidome was dominated by HLA-A-restricted ligands. However, IFN-α only marginally upregulated HLA-A and largely favored HLA-B, translating into a major increase in HLA-B-restricted peptides and into an increased activation of HLA-B-restricted vs. HLA-A-restricted CD8+ T-cells. A preferential HLA-B hyper-expression was also observed in the islets of T1D vs. non-diabetic donors, and islet-infiltrating CD8+ T-cells from T1D donors were reactive to some HLA-B-restricted granule peptides. Thus, the inflammatory milieu of insulitis may skew the autoimmune response toward epitopes presented by HLA-B, hence recruiting a distinct T-cell repertoire that may be relevant to T1D pathogenesis.
Project description:In the context of HLA-DP-mismatched allogeneic stem cell transplantation, mismatched HLA-DP alleles can provoke profound allo-HLA-DP-specific immune responses from the donor T-cell repertoire leading to graft-versus-leukemia effect and/or graft-versus-host disease in the patient. The magnitude of allo-HLA-DP-specific immune responses has been shown to depend on the specific HLA-DP disparity between donor and patient and the immunogenicity of the mismatched HLA-DP allele(s). HLA-DP peptidome clustering (DPC) was developed to classify the HLA-DP molecules based on similarities and differences in their peptide-binding motifs. To investigate a possible categorization of HLA-DP molecules based on overlap of presented peptides, we identified and compared the peptidomes of the thirteen most frequently expressed HLA-DP molecules. Our categorization based on shared peptides was in line with the DPC classification. We found that the HLA-DP molecules within the previously defined groups DPC-1 or DPC-3 shared the largest numbers of presented peptides. However, the HLA-DP molecules in DPC-2 segregated into two subgroups based on the overlap in presented peptides. Besides overlap in presented peptides within the DPC groups, a substantial number of peptides was also found to be shared between HLA-DP molecules from different DPC groups, especially for groups DPC-1 and -2. The functional relevance of these findings was illustrated by demonstration of cross-reactivity of allo-HLA-DP-reactive T-cell clones not only against HLA-DP molecules within one DPC group, but also across different DPC groups. The promiscuity of peptides presented in various HLA-DP molecules and the cross-reactivity against different HLA-DP molecules demonstrate that these molecules cannot be strictly categorized in immunogenicity groups.