Biology subjects

Nakauchi, Y.

Publications and source records attributed to Nakauchi, Y..

7 recordsLinked to original sources

bioRxiv2026

Single cell multi-omics enables high-resolution identification and functionalpurification of human acute myeloid leukemia stem cells

In human acute myeloid leukemia (AML), a sub-population of leukemia stem cells (LSCs) drive disease initiation, therapeutic resistance, and relapse. However, the lack of reliable markers to distinguish LSCs from bulk leukemia cells has impeded progress in studying LSC pathogenesis and developing meaningful LSC-specific diagnostics and therapeutics. Existing LSC gene signatures, derived from bulk populations, cannot definitively identify LSCs at single-cell resolution. To address this, we analyzed large patient cohorts with bulk gene expression data and single-cell multi-omic assays to identify a prognostic gene signature that is specifically enriched in a clinically adverse AML sub-population. Using this signature, we defined and prospectively isolated CD34+CD90-CLL1-CD69+CD53- immunophenotypic LSCs that are significantly enriched for LSC content based on limiting dilution xenotransplantation assays. Our findings demonstrate the power of single-cell multi-omics to precisely identify a clinically relevant LSC population and establish a clear framework for future translational research in AML. Key PointsO_LISingle cell multi-omics identifies human AML LSCs at high resolution. C_LIO_LIHOPX and SOCS2 co-expression (hrLSC2) defines a prognostic gene signature in de novo acute myeloid leukemia. C_LIO_LIhrLSC2 marks an AML subpopulation (iLSCs) with a distinct immunophenotype. C_LIO_LIiLSCs can be purified using flow cytometry and are significantly enriched for LSCs. C_LI

cancer biology↗

bioRxiv2025

Donor-Targeted Anti-HLA-A2 Antibody Shows Nanogram-Level Efficacy in a Novel GVHD Mouse Model

Acute graft-versus-host disease (GVHD) remains a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), with limited treatment options for steroid-refractory cases. Current therapies broadly suppress immune responses, increasing infection risk and impairing graft function. We hypothesized that selective depletion of donor immune cells through HLA allele-specific targeting could offer a safer and more precise alternative. To test this, we developed a monoclonal antibody (AN7) against HLA-A2 by immunizing HLA-A24 transgenic mice with recombinant HLA-A2 tetramers, followed by hybridoma screening. AN7 specifically recognized HLA-A2 and HLA-A68, and was reformatted into murine IgG2a and human chimeric IgG1 recombinant antibodies. These retained binding specificity and mediated robust complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) against HLA-A2+ target cells. To evaluate therapeutic potential, we established a fully MHC-mismatched allo-HSCT model using HLA-A2 transgenic donor bone marrow in lethally irradiated BALB/c recipients. A single ultra-low dose of AN7 (1-100 ng) administered post-transplant significantly improved survival and reduced GVHD while preserving donor hematopoiesis. These findings highlight the potential of allele-specific antibody-mediated clearance as a mechanistically distinct approach to donor immune modulation in GVHD.

immunology↗

bioRxiv2025

Development of iPSC-Derived T Cells Targeting EGFR Neoantigens in Non-Small Cell Lung Cancer

A long-sought goal of cancer immunotherapy is to mass-produce T cells that specifically target tumor neoantigens. One decisive challenge is the identification of neoantigens derived from cancer driver genes. Here, we identify T cells that recognize the NSCLC-associated EGFR C797S mutation, which confers resistance to current inhibitors and is linked to poor prognosis. To overcome limitations in T cell availability, we reprogrammed EGFR C797S-specific T cells into induced pluripotent stem cells (iPSCs) and re-differentiated them into CD8 T cells. These iPSC-derived T cells specifically recognized the EGFR C797S mutation and effectively killed cancer cells expressing this mutation. Our findings underscore the potential of targeting driver mutation-derived neoantigens for immunotherapy and demonstrate that iPSC-derived T cells can mediate antitumor effects. Collectively, this approach combining neoantigen identification with T cell reprogramming may offer a promising strategy for targeting drug-resistant tumors.

immunology↗

bioRxiv2024

DNMT3AR882H Is Not Required for Disease Maintenance in Primary Human AML, but Is Associated With Increased Leukemia Stem Cell Frequency

Genetic mutations are being thoroughly mapped in human cancers, yet a fundamental question in cancer biology is whether such mutations are functionally required for cancer initiation, maintenance of established cancer, or both. Here, we study this question in the context of human acute myeloid leukemia (AML), where DNMT3AR882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the DNA methylation landscape to initiate leukemia. We developed CRISPR-based methods to directly correct DNMT3AR882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3AR882 mutations were largely dispensable for disease maintenance. Replacing DNMT3AR882 mutants with wild-type DNMT3A did not impair the ability of AML cells to engraft in vivo, and minimally altered DNA methylation. Taken together, DNMT3AR882 mutations are initially necessary for AML initiation, but are largely dispensable for disease maintenance. The notion that initiating oncogenes differ from those that maintain cancer has important implications for cancer evolution and therapy. STATEMENT OF SIGNIFICANCEUnderstanding which driver mutations are required for cancer initiation, maintenance, or both phases remains poorly understood. Here, we uncover that highly prevalent pre-leukemic DNMT3A mutations are only required during disease initiation, but become dispensable after leukemic transformation, uncovering the context-specific role of this driver mutation with important therapeutic implications.

cancer biology↗

bioRxiv2024

A single cell framework identifies functionally and molecularly distinct multipotent progenitors in adult human hematopoiesis

Hematopoietic multipotent progenitors (MPPs) regulate blood cell production to appropriately meet the biological demands of the human body. Human MPPs remain ill-defined whereas mouse MPPs have been well characterized with distinct immunophenotypes and lineage potencies. Using multiomic single cell analyses and complementary functional assays, we identified new human MPPs and oligopotent progenitor populations within Lin-CD34+CD38dim/lo adult bone marrow with distinct biomolecular and functional properties. These populations were prospectively isolated based on expression of CD69, CLL1, and CD2 in addition to classical markers like CD90 and CD45RA. We show that within the canonical Lin-CD34+CD38dim/loCD90CD45RA-MPP population, there is a CD69+ MPP with long-term engraftment and multilineage differentiation potential, a CLL1+ myeloid-biased MPP, and a CLL1-CD69-erythroid-biased MPP. We also show that the canonical Lin-CD34+CD38dim/loCD90-CD45RA+ LMPP population can be separated into a CD2+ LMPP with lymphoid and myeloid potential, a CD2-LMPP with high lymphoid potential, and a CLL1+ GMP with minimal lymphoid potential. We used these new HSPC profiles to study human and mouse bone marrow cells and observe limited cell type specific homology between humans and mice and cell type specific changes associated with aging. By identifying and functionally characterizing new adult MPP sub-populations, we provide an updated reference and framework for future studies in human hematopoiesis.

cancer biology↗

bioRxiv2023

AML/T cell interactomics uncover correlates of patient outcomes and the key role of ICAM1 in T cell killing of AML

T cells are important for the control of acute myeloid leukemia (AML), a common and often deadly malignancy. We observed that some AML patient samples are resistant to killing by human engineered cytotoxic CD4+ T cells. Single-cell RNA-seq of primary AML samples and CD4+ T cells before and after their interaction uncovered transcriptional programs that correlate with AML sensitivity or resistance to CD4+ T cell killing. Resistance-associated AML programs were enriched in AML patients with poor survival, and killing-resistant AML cells did not engage T cells in vitro. Killing-sensitive AML potently activated T cells before being killed, and upregulated ICAM1, a key component of the immune synapse with T cells. Without ICAM1, killing-sensitive AML became resistant to killing to primary ex vivo-isolated CD8+ T cells in vitro, and engineered CD4+ T cells in vitro and in vivo. Thus, ICAM1 on AML acts as an immune trigger, allowing T cell killing, and could affect AML patient survival in vivo. SIGNIFICANCEAML is a common leukemia with sub-optimal outcomes. We show that AML transcriptional programs correlate with susceptibility to T cell killing. Killing resistance-associated AML programs are enriched in patients with poor survival. Killing-sensitive, but not resistant AML activate T cells and upregulate ICAM1 that binds to LFA-1 on T cells, allowing immune synapse formation which is critical for AML elimination. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/558911v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@84e2e1org.highwire.dtl.DTLVardef@4b2a40org.highwire.dtl.DTLVardef@c2d1f3org.highwire.dtl.DTLVardef@65db2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Genetically Corrected RAG2-SCID Human Hematopoietic Stem CellsRestore V(D)J-Recombinase and Rescue Lymphoid Deficiency

Recombination-activating genes (RAG1 and RAG2) are critical in lymphoid cell development and function for initiating the V(D)J-recombination process to generate polyclonal lymphocytes with broad antigen-specificity. Clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCID), causing life-threatening infections and death early in life in the absence of hematopoietic cell transplantation (HCT). Haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phases of the cell cycle at the early stages of T and B cell development, underscoring that a direct gene correction would capture the precise temporal expression of the endogenous gene, is a promising therapeutic approach for RAG1/2-deficiencies. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) in healthy donors and one RAG2-SCID patient. V(D)J recombinase activity was restored following gene correction of RAG2-SCID-derived HSPCs, resulting in the development of TCR {beta} and {gamma}{delta} CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of the CDR3 length and preserved usage of distal TRAV genes. We confirmed in vivo rescue of B-cell development, with normal IgM surface expression and a significant decrease in CD56bright NK cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit all RAG2-deficient patients. KEY POINTSO_LIHuman hematopoietic stem cells can be corrected to restore endogenous RAG2 gene expression while preserving durable engraftment potential. C_LIO_LIGene-corrected RAG2 locus restores V(D)J recombination in RAG2-SCID patient stem cells, promoting T and B-cells receptor formation. C_LI

immunology↗