Generation of antigen-specific mature T cells from RAG1-/-RAG2-/-B2M-/- stem cells by engineering their microenvironment

Patrick C Chang; Xuegang Yuan; Alexandre Zampieri; Chloe Towns; Sang Pil Yoo; Claire Engstrom; Steven Tsai; Christopher R Robles; Yuhua Zhu; Shawn Lopez; Amelie Montel-Hagen; Christopher S Seet; Gay M Crooks

doi:10.1038/s41551-023-01146-7

Generation of antigen-specific mature T cells from RAG1^-/-RAG2^-/-B2M^-/- stem cells by engineering their microenvironment

Nat Biomed Eng. 2023 Dec 7. doi: 10.1038/s41551-023-01146-7. Online ahead of print.

Authors

Patrick C Chang^{1

2}, Xuegang Yuan^#², Alexandre Zampieri^#², Chloe Towns², Sang Pil Yoo^{1

2}, Claire Engstrom², Steven Tsai³, Christopher R Robles⁴, Yuhua Zhu², Shawn Lopez², Amelie Montel-Hagen², Christopher S Seet^{3

5

6

7}, Gay M Crooks^{8

9

10

11

12}

Affiliations

¹ Molecular Biology Interdepartmental Program, University of California, Los Angeles (UCLA), Los Angeles, CA, USA.
² Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
³ Division of Hematology-Oncology, Department of Medicine, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
⁴ Department of Computational Medicine, UCLA, Los Angeles, CA, USA.
⁵ Molecular Biology Institute, UCLA, Los Angeles, CA, USA.
⁶ Broad Stem Cell Research Center, UCLA, Los Angeles, CA, USA.
⁷ Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA.
⁸ Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA. GCrooks@mednet.ucla.edu.
⁹ Molecular Biology Institute, UCLA, Los Angeles, CA, USA. GCrooks@mednet.ucla.edu.
¹⁰ Broad Stem Cell Research Center, UCLA, Los Angeles, CA, USA. GCrooks@mednet.ucla.edu.
¹¹ Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA. GCrooks@mednet.ucla.edu.
¹² Department of Pediatrics, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA. GCrooks@mednet.ucla.edu.

^# Contributed equally.

PMID: 38062131
DOI: 10.1038/s41551-023-01146-7

Abstract

Pluripotent stem cells (PSCs) are a promising source of allogeneic T cells for off-the-shelf immunotherapies. However, the process of differentiating genetically engineered PSCs to generate mature T cells requires that the same molecular elements that are crucial for the selection of these cells be removed to prevent alloreactivity. Here we show that antigen-restricted mature T cells can be generated in vitro from PSCs edited via CRISPR to lack endogenous T cell receptors (TCRs) and class I major histocompatibility complexes. Specifically, we used T cell precursors from RAG1^-/-RAG2^-/-B2M^-/- human PSCs expressing a single TCR, and a murine stromal cell line providing the cognate human major histocompatibility complex molecule and other critical signals for T cell maturation. Possibly owing to the absence of TCR mispairing, the generated T cells showed substantially better tumour control in mice than T cells with an intact endogenous TCR. Introducing the T cell selection components into the stromal microenvironment of the PSCs overcomes inherent biological challenges associated with the development of T cell immunotherapies from allogeneic PSCs.

Abstract

Grants and funding