Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2022)

T cells and Type 2 innate lymphoid cells (ILC2) are closely related lymphoid lineages which share certain developmental and transcriptional programs, including a requirement for Notch and IL-7 signaling during differentiation. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of human pluripotent stem cells (PSCs) to mature αβT cells. Unexpectedly, PSCs transduced with a CD19-targeted chimeric antigen receptor (CAR) resulted in ILC2-biased lymphopoiesis and a block in T cell differentiation in ATOs. PSC-derived CAR-ILC2s expressed classical ILC2 markers and gene expression, and surprisingly responded to both cytokine stimuli and antigen-induced CAR signaling. Mechanistically, single cell RNA-seq of early lymphoid precursors in ATOs revealed evidence of CAR activation in ILC2 precursors, and rational modulation of CAR signaling could restore generation of functional CAR-T cells from PSCs or, conversely, divert CAR-T cell differentiation to ILC2 and, to a lesser extent, NK/ILC1 lineages. Taken together, our findings shed light on human T and ILC2 lineage development and provide a framework for applying CAR technology to the generation of multiple lymphoid lineages from PSCs.

    Cover page: Strength of Chimeric Antigen Receptor Signaling Determines T versus Type 2 Innate Lymphoid Cell Lineage Differentiation from Pluripotent Stem Cells
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2018)
    Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFRβ), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies.
      Cover page: Transcriptionally and Functionally Distinct Mesenchymal Subpopulations Are Generated from Human Pluripotent Stem Cells
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2019)
      The ability to generate T cells from pluripotent stem cells (PSCs) has the potential to transform autologous T cell immunotherapy by facilitating universal, off-the-shelf cell products. However, differentiation of human PSCs into mature, conventional T cells has been challenging with existing methods. We report that a continuous 3D organoid system induced an orderly sequence of commitment and differentiation from PSC-derived embryonic mesoderm through hematopoietic specification and efficient terminal differentiation to naive CD3+CD8αβ+ and CD3+CD4+ conventional T cells with a diverse T cell receptor (TCR) repertoire. Introduction of an MHC class I-restricted TCR in PSCs produced naive, antigen-specific CD8αβ+ T cells that lacked endogenous TCR expression and showed anti-tumor efficacy in vitro and in vivo. Functional assays and RNA sequencing aligned PSC-derived T cells with primary naive CD8+ T cells. The PSC-artificial thymic organoid (ATO) system presented here is an efficient platform for generating functional, mature T cells from human PSCs.
        Cover page: Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2020)
        We report a serum-free, 3D murine artificial thymic organoid (M-ATO) system that mimics normal murine thymopoiesis with the production of all T cell stages, from early thymic progenitors to functional single-positive (CD8SP and CD4SP) TCRαβ and TCRγδ cells. RNA sequencing aligns M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATOs initiated with Rag1-/- marrow produce the same differentiation block as seen in the endogenous thymus, and Notch signaling patterns in M-ATOs mirror primary thymopoiesis. M-ATOs initiated with defined hematopoietic stem cells (HSCs) and lymphoid progenitors from marrow and thymus generate each of the downstream differentiation stages, allowing the kinetics of T cell differentiation to be tracked. Remarkably, single HSCs deposited into each M-ATO generate the complete trajectory of T cell differentiation, producing diverse TCR repertoires across clones that largely match endogenous thymus. M-ATOs represent a highly reproducible and efficient experimental platform for the interrogation of clonal thymopoiesis from HSCs.
          Cover page: In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2023)
          Generation of chimeric antigen receptor (CAR) T cells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. Understanding how CARs affect T cell differentiation from PSCs is important for this effort. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of PSCs to T cells. Unexpectedly, PSCs transduced with a CD19-targeted CAR resulted in diversion of T cell differentiation to the innate lymphoid cell 2 (ILC2) lineage in ATOs. T cells and ILC2s are closely related lymphoid lineages with shared developmental and transcriptional programs. Mechanistically, we show that antigen-independent CAR signaling during lymphoid development enriched for ILC2-primed precursors at the expense of T cell precursors. We applied this understanding to modulate CAR signaling strength through expression level, structure, and presentation of cognate antigen to demonstrate that the T cell-versus-ILC lineage decision can be rationally controlled in either direction, providing a framework for achieving CAR-T cell development from PSCs.
            Cover page: Strength of CAR signaling determines T cell versus ILC differentiation from pluripotent stem cells
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2017)
            Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCR-αβ+ single-positive CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.
              Cover page: Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids
              • Article
              • Peer Reviewed
              UCLA Previously Published Works (2022)
              Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.
                Cover page: Purine nucleoside phosphorylase enables dual metabolic checkpoints that prevent T cell immunodeficiency and TLR7-dependent autoimmunity
                Top