Project description:The appended raw files, csv files and other documents were deposited into the public domain in support for the publication "Development and characterization of a dendritic cell internalization assay contributing to the immunogenicity risk evaluation of biotherapeutics" by Michel Siegel, Aman Padamsey, Anna-Lena Bolender, Patrick Hargreaves, Axel Ducret, Johannes Fraidling, Katharina Hartmann, Cary M. Looney, Olivier Rohr, Tim Hickling, Thomas Kraft, Celine Marban-Doran. The abstract reads as follows: Assessing immunogenicity risks during the development of biotherapeutics is crucial. Given the complexity of immunogenicity - influenced by myriad biological, immunological, and patient-specific factors - Individual risk assessments are poorly predictive, necessitating a holistic approach to immunogenicity risk assessment. Anti-drug antibody production starts with the internalization of drugs by antigen presenting cells such as dendritic cells, which present drug-derived peptides to CD4+ T cells as peptide-MHC-II complexes. Assessing dendritic cell function is common in preclinical immunogenicity risk assessments, including presentation of potential T cell epitopes using the MAPPs assay. However, other aspects of dendritic cell biology are often overlooked. To better understand the dendritic cell contribution to immunogenicity, we developed two flow cytometry-based assays: the dendritic cell internalization assay and the dendritic cell activation assay. Our assay addresses two issues with existing methods for measuring internalization into antigen presenting cells; lack of specificity for the cellular compartment of internalization or the recycling of internalized antibodies, and the increased risk of aggregation, when using a large payload for the detection of antibodies, that would lead to activation of irrelevant scavenger receptors in the context of dendritic cell internalization. We also developed a DC activation assay, improving on various aspects of its relevance for immunogenicity risk assessment compared to previously published protocols. Additionally, we identified DC-SIGN (CD209) and CD80 as crucial for understanding dendritic cell activation mechanisms leading to immunogenicity. To evaluate the performance of these two assays, we used a set of marketed therapeutic antibodies. The dendritic cell internalization assay revealed differences in internalization rates for marketed therapeutic antibodies, even those targeting the same antigen (e.g. PCSK9-targeting antibodies: bococizumab, evolocumab and alirocumab or TNF-targeting antibodies: adalimumab and infliximab), potentially accounting for differential immunogenicity liabilities. The DC activation assay also showed different patterns of DC activation between bococizumab, which rapidly accumulates within the lysosomes, and the other PCSK9-targeting antibodies (evolocumab and alirocumab), giving new insights into specific immunogenicity profiles. Overall, this study provides valuable information for future risk assessments of therapeutic antibodies in development. doi: 10.3389/fimmu.2024.1406804 The data deposited here were used to generate the supplementary figure 3.

Project description:Genome wide association studies of schizophrenia encompassing the major histocompatibility locus (MHC) were highly significant following genome wide correction. This broad region implicates many genes including the MHC complex class II. Within this interval we examined the expression of two MHC II genes (HLA-DPA1 and HLA-DRB1) in brain from individual subjects with schizophrenia (SZ), bipolar disorder (BD), major depressive disorder (MDD), and controls by differential gene expression methods. A third MHC II mRNA, CD74, was studied outside of the MHC II locus, as it interacts within the same immune complex. HLA-DPA1 and CD74 were both reduced in hippocampus, amygdala, and dorsolateral prefrontal cortex regions in SZ and BD compared to controls by specific qPCR assay. We found several novel HLA-DPA1 mRNA variants spanning HLA-DPA1 exons 2-3-4 as suggested by an exon microarray study. The intronic rs9277341 SNP was a significant cis expression quantitative trait locus (eQTL) that was associated with the total expression of HLA-DPA1 in five brain regions. A biomarker study of MHC II mRNAs was conducted in SZ, BD, MDD, and control lymphoblastic cell lines (LCL) by qPCR assay of 87 subjects. There was significantly decreased expression of HLA-DPA1 and CD74 in BD, and trends for reductions in SZ in LCLs. The discovery of multiple splicing variants in brain for HLA-DPA1 is important as the HLA-DPA1 gene is highly conserved, there are no reported splicing variants, and the functions in brain are unknown. Future work on the function and localization of MHC Class II proteins in brain will help to understand the role of alterations in neuropsychiatric disorders. The HLA-DPA1 eQTL is located within a large linkage disequilibrium block that has an irrefutable association with schizophrenia. Future tests in a larger cohort are needed to determine the significance of this eQTL association with schizophrenia. Our findings support the long held hypothesis that alterations in immune function are associated with the pathophysiology of psychiatric disorders. There were 20 anterior cingulate postmortem brain samples that were extracted for total RNA, and analyzed using Affymetrix Exon Array (bipolar disorder subjects n = 9, controls n = 11).

Project description:Genome wide association studies of schizophrenia encompassing the major histocompatibility locus (MHC) were highly significant following genome wide correction. This broad region implicates many genes including the MHC complex class II. Within this interval we examined the expression of two MHC II genes (HLA-DPA1 and HLA-DRB1) in brain from individual subjects with schizophrenia (SZ), bipolar disorder (BD), major depressive disorder (MDD), and controls by differential gene expression methods. A third MHC II mRNA, CD74, was studied outside of the MHC II locus, as it interacts within the same immune complex. HLA-DPA1 and CD74 were both reduced in hippocampus, amygdala, and dorsolateral prefrontal cortex regions in SZ and BD compared to controls by specific qPCR assay. We found several novel HLA-DPA1 mRNA variants spanning HLA-DPA1 exons 2-3-4 as suggested by an exon microarray study. The intronic rs9277341 SNP was a significant cis expression quantitative trait locus (eQTL) that was associated with the total expression of HLA-DPA1 in five brain regions. A biomarker study of MHC II mRNAs was conducted in SZ, BD, MDD, and control lymphoblastic cell lines (LCL) by qPCR assay of 87 subjects. There was significantly decreased expression of HLA-DPA1 and CD74 in BD, and trends for reductions in SZ in LCLs. The discovery of multiple splicing variants in brain for HLA-DPA1 is important as the HLA-DPA1 gene is highly conserved, there are no reported splicing variants, and the functions in brain are unknown. Future work on the function and localization of MHC Class II proteins in brain will help to understand the role of alterations in neuropsychiatric disorders. The HLA-DPA1 eQTL is located within a large linkage disequilibrium block that has an irrefutable association with schizophrenia. Future tests in a larger cohort are needed to determine the significance of this eQTL association with schizophrenia. Our findings support the long held hypothesis that alterations in immune function are associated with the pathophysiology of psychiatric disorders.

Project description:Pluripotent stem cells, including human embryonic stem (hES) and induced pluripotent stem (hiPS) cells, have been regarded as useful sources for cell?based transplantation therapy. However immunogenicity of the cells remains the major determinant for successful clinical application. We report the examination of several hES cell lines (NTU1 and H9), hiPS cell lines, and their derivatives (including stem cell?derived hepatocytes) for the expression of major histocompatibility complex (MHC), natural killer (NK) cell receptor (NKp30, NKp44, NKp46) ligand, immune?related genes, human leukocyte antigen (HLA) haplotyping, and the effects in functional mixed lymphocyte reaction (MLR). Flow cytometry showed lower levels (percentages and fluorescence intensities) of MHC class I (MHC?I) molecules, β2?microglobulin and HLA?E in undifferentiated stem cells, but the levels were increased after co?treatment with interferon gamma and/or in vitro differentiation. Antigen presenting cell markers (CD11c, CD80 and CD86) and MHC?II (HLA?DP, DQ and DR) remained low throughout the treatments. Recognitions of stem cells/derivatives by NK lysis receptors were lower or absent. Activation of responder lymphocytes was significantly lower by undifferentiated stem cells than by allogeneic lymphocytes in MLR, but differentiated NTU1 hES cells induced a cell number?dependent lymphocyte proliferation comparable with that by allogeneic lymphocytes. Interestingly activation of lymphocytes by differentiated hiPS cells or H9 cells became blunted at higher cell numbers. Real?time RT?PCR showed significant differential expression of immune privilege genes (TGF?β2, Arginase 2, Indole 1, GATA3, POMC, VIP, CALCA, CALCB, IL?1RN, CD95L, CR1L, Serpine 1, HMOX1, IL6, LGALS3, HEBP1, THBS1, CD59 and LGALS1) in pluripotent stem cells/derivatives when compared to somatic cells. It is concluded that pluripotent stem cells/derivatives are predicted to be immunogenic, though evidences suggest some levels of potential immune privilege. In addition, differential immunogenicity may exist between different pluripotent stem cell lines and their derivatives

Dataset Information

Expanding the MAPPs assay to accommodate MHC-II pan re-ceptors for improved predictability of potential T cell epitopes

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets