Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

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Abstract

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+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly 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 naïve phenotypes, a diverse 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 loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies.

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Most cited references 42

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Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.

G J Brewer, J R Torricelli, E K Evege … (1993)

We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman, Brain Res 494: 65-74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement, B27, produced neuron survival above 60%, independent of plating density above 160 plated cells/mm2. For isolated cells (< 100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones.

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Exploiting the curative potential of adoptive T-cell therapy for cancer.

Christian Hinrichs, Steven Rosenberg (2014)

Adoptive T-cell therapy (ACT) is a potent and flexible cancer treatment modality that can induce complete, durable regression of certain human malignancies. Long-term follow-up of patients receiving tumor-infiltrating lymphocytes (TILs) for metastatic melanoma reveals a substantial subset that experienced complete, lasting tumor regression - and may be cured. Increasing evidence points to mutated gene products as the primary immunological targets of TILs from melanomas. Recent technological advances permit rapid identification of the neoepitopes resulting from these somatic gene mutations and of T cells with reactivity against these targets. Isolation and adoptive transfer of these T cells may improve TIL therapy for melanoma and permit its broader application to non-melanoma tumors. Extension of ACT to other malignancies may also be possible through antigen receptor gene engineering. Tumor regression has been observed following transfer of T cells engineered to express chimeric antigen receptors against CD19 in B-cell malignancies or a T-cell receptor against NY-ESO-1 in synovial cell sarcoma and melanoma. Herein, we review recent clinical trials of TILs and antigen receptor gene therapy for advanced cancers. We discuss lessons from this experience and consider how they might be applied to realize the full curative potential of ACT.

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Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions.

Zhili Zheng, M Mahon, Candace Johnson … (2008)

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3alpha and CDR2beta amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1(+)/HLA-A*02(+) tumor cell lines by TCR gene-modified CD4(+) T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3alpha chain was identified that enhanced Ag-specific reactivity in gene-modified CD4(+) and CD8(+) T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27-35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4(+) T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.

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Author and article information

Journal

Journal ID (nlm-journal-id): 101215604

Journal ID (pubmed-jr-id): 32338

Journal ID (nlm-ta): Nat Methods

Journal ID (iso-abbrev): Nat. Methods

Title: Nature methods

ISSN (Print): 1548-7091

ISSN (Electronic): 1548-7105

Publication date Nihms-submitted: 20 March 2017

Publication date (Electronic): 03 April 2017

Publication date (Print): May 2017

Publication date PMC-release: 03 October 2017

Volume: 14

Issue: 5

Pages: 521-530

Affiliations

[1 ]Division of Hematology-Oncology, Department of Medicine, David Geffen School of Medicine (DGSOM), University of California Los Angeles (UCLA)

[2 ]Department of Pathology & Laboratory Medicine, DGSOM, UCLA

[3 ]Division of Biology and Biological Engineering, California Institute of Technology, Pasadena

[4 ]Department of Microbiology, Immunology and Molecular Genetics, DGSOM, UCLA

[5 ]Division of Pediatric Hematology-Oncology, Department of Pediatrics, DGSOM, UCLA

[6 ]Broad Stem Cell Research Center, UCLA

[7 ]Jonsson Comprehensive Cancer Center, UCLA

Author notes

Corresponding Author Information: Gay M. Crooks, M.B., B.S., 610 Charles E. Young Drive, East, 3014 TLSB, Los Angeles, CA 90095, gcrooks@ 123456mednet.ucla.edu , Phone: (310) 206 0205, Fax: (310) 206 0356

[*]

Gay M. Crooks and Amélie Montel-Hagen are co-senior authors

Article

Manuscript ID: NIHMS857420

DOI: 10.1038/nmeth.4237

PMC ID: 5426913

PubMed ID: 28369043

SO-VID: 495daccc-42d0-41c7-92f2-e0db73ae47cc

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Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

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Abstract

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Most cited references 42

Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.

Exploiting the curative potential of adoptive T-cell therapy for cancer.

Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions.

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