Orthotopic replacement of T-cell receptor a- and ß-chains with preservation of near-physiological T-cell function.

Authors:
Schober K, Müller TR, Gökmen F, Grassmann S, Effenberger M, Poltorak M, Stemberger C, Schumann K, Roth TL, Marson A, Busch DH.
In:
Source: Nat Biomed Eng
Publication Date: (2019)
Issue: 10: 01
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Regenerative medicine
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
CD4+, human
Species: human
Tissue Origin: blood
CD8+, human
Species: human
Tissue Origin: blood
Platform:
4D-Nucleofector® X-Unit
Experiment

Frozen enriched CD4+ and CD8+ T cells were thawed and rested in RPMI + 50 IU ml-1 IL-2 overnight. CD4+ and CD8+T cells mixed in a 1:1 ratio were then activated for two days with CD3/CD28 Expamer (Juno Therapeutics), 300 IU ml-1 IL-2, 5 ng ml-1 IL-7 and 5 ng ml-1 IL-15 per ml RPMI for 1 × 106 T cells. Expamer stimulus was removed by incubation with 1 mM d-biotin. Cells were electroporated (pulse code EH115) with Cas9ribonucleoprotein and DNA templates in Nucleofector Solution (20 µl per million
T cells; Lonza) with a 4D Nucleofector X unit (Lonza). After electroporation, cellswere cultured in RPMI with 180 IU ml-1 IL-2 before analysis.

Abstract

Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of a- and ß-chains combined with orthotopic TCR placement leads to accurate aß-pairing and results in TCR regulation similar to that of physiological T cells.