c-Jun overexpression in CAR T cells induces exhaustion resistance

Lynn RC, Weber EW, Sotillo E, Gennert D, Xu P, Good Z, Anbunathan H, Lattin J, Jones R, Tieu V, Nagaraja S, Granja J, de Bourcy CFA, Majzner R, Satpathy AT, Quake SR, Monje M, Chang HY, Mackall CL
Source: Nature
Publication Date: (2019)
Issue: 576(7786): 293-300
Research Area:
Immunotherapy / Hematology
Basic Research
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

CRISPR–Cas9 gene knockout was performed by transient Cas9/gRNA (RNP) complex electroporation using the P3 Primary Cell 4D-Nucleofector X Kit S (Lonza). On day 4 of culture, HA-28z CAR T cells were counted, pelleted and resuspended in P3 buffer at 1.5 × 10^6 –2 × 10^6 cells per 20 µl reaction. 3.3 ug Alt-R .Sp. Cas9 protein (IDT) and 40 pmol chemically modified synthetic sgRNA (Synthego) (2:1 molar ratio gRNA:Cas9) per reaction was pre-complexed for 10 min at room temperature to create ribonucleoprotein complexes (RNP). A 20-µl cell suspension was mixed with RNP and electroporated using the EO-115 protocol in 16-well cuvette strips. Cells were recovered at 37?°C for 30 min in 200 µl T cell medium.


Chimeric antigen receptor (CAR) T cells mediate anti-tumour efects in a small subset of patients with cancer , but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal diferentiation and improved anti-tumour potency in fve diferent mouse tumour models in vivo. We conclude that a functional defciency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.