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Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility.Cell Stem Cell Apr 2019Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M...
Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Here, we show two genome-editing strategies for making immunocompatible donor iPSCs. First, we generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, we generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade T cells and NK cells in vitro and in vivo. We estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world's population, greatly facilitating iPSC-based regenerative medicine applications.
Topics: Animals; CRISPR-Cas Systems; Cell Line; Female; Gene Editing; HLA Antigens; Histocompatibility; Humans; Induced Pluripotent Stem Cells; Male; Mice; Mice, Inbred NOD
PubMed: 30853558
DOI: 10.1016/j.stem.2019.02.005 -
Nature Nov 2021The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by...
The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.
Topics: Animals; Antigens, Neoplasm; Cell Line; Cell Line, Tumor; Cross Reactions; Cross-Priming; Female; HLA Antigens; Homeodomain Proteins; Humans; Immunotherapy; Interferon-gamma; Mice; Neoplasms; Oncogene Proteins; Receptors, Chimeric Antigen; T-Lymphocytes; Transcription Factors
PubMed: 34732890
DOI: 10.1038/s41586-021-04061-6 -
Immunity Oct 2019Increasing evidence indicates CD4 T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that...
Increasing evidence indicates CD4 T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that will be presented by human leukocyte antigen class II molecules (HLA-II), hindering efforts to optimally target them therapeutically. Obstacles include inaccurate peptide-binding prediction and unsolved complexities of the HLA-II pathway. To address these challenges, we developed an improved technology for discovering HLA-II binding motifs and conducted a comprehensive analysis of tumor ligandomes to learn processing rules relevant in the tumor microenvironment. We profiled >40 HLA-II alleles and showed that binding motifs were highly sensitive to HLA-DM, a peptide-loading chaperone. We also revealed that intratumoral HLA-II presentation was dominated by professional antigen-presenting cells (APCs) rather than cancer cells. Integrating these observations, we developed algorithms that accurately predicted APC ligandomes, including peptides from phagocytosed cancer cells. These tools and biological insights will enable improved HLA-II-directed cancer therapies.
Topics: Algorithms; Alleles; Antigen Presentation; Antigen-Presenting Cells; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; Cancer Vaccines; Datasets as Topic; Epitope Mapping; HLA Antigens; HLA-D Antigens; Histocompatibility Antigens Class II; Humans; Immunotherapy; Mass Spectrometry; Neoplasms; Protein Binding; Protein Interaction Domains and Motifs; Software
PubMed: 31495665
DOI: 10.1016/j.immuni.2019.08.012 -
Frontiers in Immunology 2021This review provides insight into the role of engineered T-cell receptors (TCRs) in immunotherapy. Novel approaches have been developed to boost anticancer immune... (Review)
Review
This review provides insight into the role of engineered T-cell receptors (TCRs) in immunotherapy. Novel approaches have been developed to boost anticancer immune system, including targeting new antigens, manufacturing new engineered or modified TCRs, and creating a safety switch for endo-suicide genes. In order to re-activate T cells against tumors, immune-mobilizing monoclonal TCRs against cancer (ImmTAC) have been developed as a novel class of manufactured molecules which are bispecific and recognize both cancer and T cells. The TCRs target special antigens such as NY-ESO-1, AHNAK or ERBB2 to boost the efficacy of anticancer immunotherapy. The safety of genetically modified T cells is very important. Therefore, this review discusses pros and cons of different approaches, such as ImmTAC, Herpes simplex virus thymidine kinase (HSV-TK), and inducible caspase-9 in cancer immunotherapy. Clinical trials related to TCR-T cell therapy and monoclonal antibodies designed for overcoming immunosuppression, and recent advances made in understanding how TCRs are additionally examined. New approaches that can better detect antigens and drive an effective T cell response are discussed as well.
Topics: Animals; Antigen Presentation; Antigens, Neoplasm; Genetic Engineering; HLA Antigens; Humans; Immunotherapy, Adoptive; Neoplasms; Precision Medicine; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; T-Lymphocytes
PubMed: 33859650
DOI: 10.3389/fimmu.2021.658753 -
Nature Biotechnology Aug 2017Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients....
Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8 T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.
Topics: Animals; Female; Graft Rejection; HLA Antigens; Humans; Killer Cells, Natural; Mice; Pluripotent Stem Cells; Transplants
PubMed: 28504668
DOI: 10.1038/nbt.3860 -
Nature Jul 2017T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual. Here we define the minimal...
T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.
Topics: Adolescent; Algorithms; Amino Acid Sequence; Crystallography, X-Ray; Epitopes, T-Lymphocyte; HLA Antigens; Humans; Ligands; Models, Molecular; Mycobacterium tuberculosis; Receptors, Antigen, T-Cell; Substrate Specificity
PubMed: 28636589
DOI: 10.1038/nature22976 -
Cancer Immunology Research Aug 2019Human tumor xenograft models do not replicate the human immune system and tumor microenvironment. We developed an improved humanized mouse model, derived from fresh cord...
Human tumor xenograft models do not replicate the human immune system and tumor microenvironment. We developed an improved humanized mouse model, derived from fresh cord blood CD34 stem cells (CD34 HSC), and combined it with lung cancer cell line-derived human xenografts or patient-derived xenografts (Hu-PDX). Fresh CD34 HSCs could reconstitute detectable mature human leukocytes (hCD45) in mice at four weeks without the onset of graft-versus-host disease (GVHD). Repopulated human T cells, B cells, natural killer (NK) cells, dendritic cells (DC), and myeloid-derived suppressor cells (MDSC) increased in peripheral blood, spleen, and bone marrow over time. Although cultured CD34 HSCs labeled with luciferase could be detected in mice, the cultured HSCs did not develop into mature human immune cells by four weeks, unlike fresh CD34 HSCs. , reconstituted T cells, obtained from the tumor-bearing humanized mice, secreted IFNγ upon treatment with phorbol myristate acetate (PMA) or exposure to human A549 lung tumor cells and mediated antigen-specific CTL responses, indicating functional activity. Growth of engrafted PDXs and tumor xenografts was not dependent on the human leukocyte antigen status of the donor. Treatment with the anti-PD-1 checkpoint inhibitors pembrolizumab or nivolumab inhibited tumor growth in humanized mice significantly, and correlated with an increased number of CTLs and decreased MDSCs, regardless of the donor HLA type. In conclusion, fresh CD34HSCs are more effective than their expanded counterparts in humanizing mice, and do so in a shorter time. The Hu-PDX model provides an improved platform for evaluation of immunotherapy.
Topics: Animals; Antigens, Neoplasm; Biomarkers, Tumor; Disease Models, Animal; Female; Flow Cytometry; HLA Antigens; Hematopoietic Stem Cells; Humans; Immunity; Lung Neoplasms; Male; Mice; T-Lymphocytes; Tumor Microenvironment; Xenograft Model Antitumor Assays
PubMed: 31186248
DOI: 10.1158/2326-6066.CIR-18-0874 -
Current Opinion in Organ Transplantation Aug 2020Transplant pathology contributes substantially to personalized treatment of organ allograft recipients. Rapidly advancing next-generation human leukocyte antigen (HLA)... (Review)
Review
PURPOSE OF REVIEW
Transplant pathology contributes substantially to personalized treatment of organ allograft recipients. Rapidly advancing next-generation human leukocyte antigen (HLA) sequencing and pathology are enhancing the abilities to improve donor/recipient matching and allograft monitoring.
RECENT FINDINGS
The present review summarizes the workflow of a prototypical patient through a pathology practice, highlighting histocompatibility assessment and pathologic review of tissues as areas that are evolving to incorporate next-generation technologies while emphasizing critical needs of the field.
SUMMARY
Successful organ transplantation starts with the most precise pratical donor-recipient histocompatibility matching. Next-generation sequencing provides the highest resolution donor-recipient matching and enables eplet mismatch scores and more precise monitoring of donor-specific antibodies (DSAs) that may arise after transplant. Multiplex labeling combined with hand-crafted machine learning is transforming traditional histopathology. The combination of traditional blood/body fluid laboratory tests, eplet and DSA analysis, traditional and next-generation histopathology, and -omics-based platforms enables risk stratification and identification of early subclinical molecular-based changes that precede a decline in allograft function. Needs include software integration of data derived from diverse platforms that can render the most accurate assessment of allograft health and needs for immunosuppression adjustments.
Topics: HLA Antigens; Histocompatibility Testing; Humans; Organ Transplantation; Precision Medicine; Tissue Donors; Transplantation Immunology; Transplantation, Homologous; Transplants
PubMed: 32520786
DOI: 10.1097/MOT.0000000000000772 -
Genes and Immunity Apr 2022Uveitis is the most common form of intraocular inflammatory disease and is a significant cause of visual impairment worldwide. Aetiologically, uveitis can also be... (Review)
Review
Uveitis is the most common form of intraocular inflammatory disease and is a significant cause of visual impairment worldwide. Aetiologically, uveitis can also be classified into infectious uveitis and non-infectious uveitis. The common non-infectious forms of uveitis include acute anterior uveitis (AAU), Behçet's disease (BD), Vogt-Koyanagi-Harada (VKH) disease, birdshot chorioretinopathy (BSCR), sarcoid uveitis. In addition, a few monogenic autoinflammatory disorders can also cause uveitis, such as Blau Syndrome and haploinsufficiency of A20 (HA20). Although the exact pathogenesis of non-infectious uveitis is still unclear, it is well-recognised that it involves both genetic and environmental risk factors. A hallmark of uveitis is its strong associations with human leucocyte antigens (HLA). For examples, AAU, BD and BSCR are strongly associated with HLA-B27, HLA-B51, and HLA-A29, respectively. In uveitis studies, multiple GWAS have successfully been conducted and led to identification of novel susceptibility loci, for example, IL23R has been identified in BD, VKH and AAU. In this review, we summarize the latest progress on the genetic associations of both HLA and non-HLA genes with major forms of uveitis, including AAU, BD, VKH, BSCR, sarcoid uveitis, Blau Syndrome and HA20, and potential future research directions.
Topics: Arthritis; Behcet Syndrome; HLA Antigens; HLA-B27 Antigen; Humans; Sarcoidosis; Synovitis; Uveitis; Uveitis, Anterior
PubMed: 35379982
DOI: 10.1038/s41435-022-00168-6 -
Nature Biotechnology Oct 2020CD4 T cells are critical to fighting pathogens, but a comprehensive analysis of human T-cell specificities is hindered by the diversity of HLA alleles (>20,000) and the...
CD4 T cells are critical to fighting pathogens, but a comprehensive analysis of human T-cell specificities is hindered by the diversity of HLA alleles (>20,000) and the complexity of many pathogen genomes. We previously described GLIPH, an algorithm to cluster T-cell receptors (TCRs) that recognize the same epitope and to predict their HLA restriction, but this method loses efficiency and accuracy when >10,000 TCRs are analyzed. Here we describe an improved algorithm, GLIPH2, that can process millions of TCR sequences. We used GLIPH2 to analyze 19,044 unique TCRβ sequences from 58 individuals latently infected with Mycobacterium tuberculosis (Mtb) and to group them according to their specificity. To identify the epitopes targeted by clusters of Mtb-specific T cells, we carried out a screen of 3,724 distinct proteins covering 95% of Mtb protein-coding genes using artificial antigen-presenting cells (aAPCs) and reporter T cells. We found that at least five PPE (Pro-Pro-Glu) proteins are targets for T-cell recognition in Mtb.
Topics: Alleles; Amino Acid Sequence; CD4-Positive T-Lymphocytes; Cluster Analysis; Epitopes, T-Lymphocyte; HLA Antigens; Humans; Mycobacterium tuberculosis; Receptors, Antigen, T-Cell; Software
PubMed: 32341563
DOI: 10.1038/s41587-020-0505-4