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Published May 2017 | Accepted Version + Supplemental Material
Journal Article Open

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

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^+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.

Additional Information

© 2017 Macmillan Publishers Limited. Received 02 December 2016. Accepted 03 March 2017. Published online 03 April 2017. We thank J. Scholes and F. Codrea at the UCLA Broad Stem Cell Research Center (BSCRC) Flow Cytometry Core for assistance with FACS sorting, R. Chan for assistance with specimen processing, C. Parekh (Children's Hospital Los Angeles) for generous assistance with thymus samples, M. Sehl (UCLA) for assistance with MPB collection, and A. Cooper (UCLA) for helpful advice and discussion. We thank I. Antoshechkin (Millard and Muriel Jacobs Genetics and Genomics Laboratory, Caltech), who developed the method for, and who assisted with, TCR sequencing analysis, A. Ribas (UCLA) for the NY-ESO-1 and MART-1 TCR constructs, J. Zuniger-Pflucker (University of Toronto) for OP9-DL1 cells, L. Coulombel for MS-5 cells and J. Chute (UCLA) for U266 cells. This work was supported by the NIH (grants R01 AG049753 (G.M.C.), 1R21AI119927 (G.M.C. and A.M.-H.), P01 HL073104 (G.M.C. and D.B.K.) and T32HL066992 (C.S.S.)), the Tower Cancer Research Foundation (C.S.S.), a UCLA BSCRC Innovation award (G.M.C. and D.B.K.) and a BSCRC Clinical Fellowship (C.S.S.). M.T.B. and D.B. are supported by Prostate Cancer Foundation Challenge Award 15CHAL02, and M.T.B. is the recipient of a Jane Coffin Childs Postdoctoral Fellowship. Core services were supported by the UCLA Jonsson Comprehensive Cancer Center shared facility (TPCL, grant 5P30CA016042), the UCLA Immunogenetics Center, the UCLA Center for AIDS Research Virology Core Lab and the UCLA AIDS Institute (grant 5P30 AI028697), and the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech. These authors contributed equally to this work. Gay M Crooks & Amélie Montel-Hagen. Author Contributions: C.S.S. and A.M.-H. designed and performed experiments, analyzed data, prepared figures and co-wrote the manuscript; C.H. performed histological experiments and, with B.C., assisted with in vivo experiments; S.L. assisted with ATO analysis and T cell functional assays; K.K. assisted with ATO cultures; Y.Z. performed human specimen processing and cultured the cell lines; E.H.G. and D.B.K. provided critical reagents and conceptual advice and edited the manuscript; M.T.B. and D.B. devised the approach for, and performed, TCR repertoire sequencing analysis and provided critical reagents; and G.M.C. and A.M.-H. co-directed the project and co-wrote the manuscript. Competing financial interests: Kite Pharma, Inc. is supporting the preclinical research of the ATO system at UCLA with G.M.C. as principal investigator. Kite Pharma, Inc. also holds an exclusive license to certain intellectual property that relates to the ATO system. Data availability. The data that support the findings of this study are available from the corresponding author upon request. Sequences for the TCR-encoding genes from this study are available in the NCBI Gene Expression Omnibus repository under accession number GSE94968.

Attached Files

Accepted Version - nihms857420.pdf

Supplemental Material - nmeth.4237-S1.pdf

Supplemental Material - nmeth.4237-S2.pdf

Supplemental Material - nmeth.4237-SF1.jpg

Supplemental Material - nmeth.4237-SF2.jpg

Supplemental Material - nmeth.4237-SF3.jpg

Supplemental Material - nmeth.4237-SF4.jpg

Supplemental Material - nmeth.4237-SF5.jpg

Supplemental Material - nmeth.4237-SF6.jpg

Supplemental Material - nmeth.4237-SF7.jpg

Supplemental Material - nmeth.4237-SF8.jpg

Supplemental Material - nmeth.4237-SF9.jpg

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