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:Despite the increasing use of humanized mouse models to study new approaches of graft-versus-host disease (GVHD) prevention, the pathogenesis of xenogeneic GVHD (xGVHD) in these models remains misunderstood. The aim of this study is to describe this pathogenesis in NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice infused with human PBMCs and to assess the impact of the expression of HLA-A0201 by NSG mice cells (NSG-HLA-A2/HHD mice) on xGVHD and graft-versus-leukemia (GvL) effects, by taking advantage of next-generation technologies. We found that T cells recovered from NSG mice after transplantation had upregulated expression of genes involved in cell proliferation, as well as in TCR, co-stimulatory, IL-2/STAT5, mTOR and Aurora kinase A pathways. T cells had mainly an effector memory or an effector phenotype and exhibited a Th1/Tc1-skewed differentiation. TCRβ repertoire diversity was markedly lower both in the spleen and lungs (a xGVHD target organ) than at infusion. There was no correlation between the frequencies of specific clonotypes at baseline and in transplanted mice. Finally, expression of HLA-A0201 by NSG mice led to more severe xGVHD and enhanced GvL effects toward HLA-A2+ leukemic cells. Altogether our data demonstrate that the pathogenesis of xGVHD shares important features with human GVHD and that NSG-HLA-A2/HHD mice could serve as better model to study GVHD and GvL effects.
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.
