Project description:To study immune responses in the context of human allogeneic graft rejection, we chose the Hu-PBL-NSG-MHCnull humanized mouse (Brehm et al., 2019). NOD-scid IL-2 receptor subunit γ (IL2rg)null (NSG) immunocompromised mice that lack murine MHC class I and II, were transplanted (under the kidney capsule, n=12) with 5M SC-islets (HLA-A2 positive), followed by human PBMC injection (termed ‘hPi-mice’; 50M/mouse, n=6) from healthy unmatched donors (HLA-A2 negative). The lack of murine MHC allowed us to monitor the graft function for prolonged durations without the risk of xenogeneic graft vs host disease (GVHD). Half of the SC-islet transplanted cohort (n=6 mice) was used as the control, without PBMC injection (Figure 1A). Since graft elimination by PBMCs is incomplete and residual endocrine cells remain in the hPi-mice grafts, we retrieved the SC-islet grafts for single cell RNA sequencing (scRNA-seq) analysis. These samples, along with pre-injected PBMCs as controls, were used for 10x Genomics mRNA expression library preparation and Illumina sequencing.
Project description:The goal of the experiment is to describe the overall transcriptomic alterations and signaling pathways activated by T cells upon transplantation in NSG or NSG-HLA-A2/HHD mice. PBMCs and CD3/CD28 activated T cells serve as negative and positive controls of these pathways activation, respectively. PBMCs from 4 different healthy volunteer donors were used. PBMCs from each donor were either used to sort T cells (by FACS) immediately after Ficoll isolation, or immediately stimulated in vitro or injected into mice.
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:Here we analysed the immunopeptidomes of 6 HLA-A2-positive triple negative breast cancer (TNBC) samples by nano-ultra performance liquid chromatography tandem mass spectrometry (nUPLC-MS2).
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:The study investigated presentation of HLA A2 restricted H3.3K27M neopeptide using immunopeptidomics followed by DDA and/or targeted multiple monitoring reaction (MRM).
Project description:The loading of high affinity peptides onto nascent class I MHC (MHC-I) molecules is facilitated by chaperones, including the class I-specific chaperone TAP-binding protein-related (TAPBPR). TAPBPR features a loop (amino acids 24-35) that projects towards the empty MHC-I peptide binding groove and rests above the F pocket. The 24-35 loop is much shorter in the closely related homologue tapasin, and therefore may be partly responsible for the unique antigen editing properties of TAPBPR. Previously we reported a deep mutational scan of human TAPBPR focused on the 24-35 loop, and determined the relative effects of single amino acid mutations on binding and peptide-mediated release of the murine H2-Dd MHC-I allomorph. Here, we extend our studies to determine the mutational landscape of the 24-35 loop when TAPBPR binds a human MHC-I allomorph, HLA-A*02:01. The data highlights how TAPBPR affinity can be increased or decreased for different MHC-I allomorphs by tuning the electrostatic complementarity of the 24-35 loop for surfaces on the rim of the peptide-binding groove. By changing the selection pressure from HLA-A2 binding to HLA-A2 loading and processing, we find that TAPBPR is reasonably tolerant of mutations in the 24-35 loop for efficient peptide-MHC-I processing and surface trafficking.
