CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy

Giuffrida L, Sek K, Henderson MA, Lai J, Chen AXY, Meyran D, Todd KL, Petley EV, Mardiana S, Mølck C, Stewart GD, Solomon BJ, Parish IA, Neeson PJ, Harrison SJ, Kats LM, House IG, Darcy PK, Beavis PA
Source: Nat Commun.
Publication Date: (2021)
Issue: 12(1): 3236
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
T cell, mouse, stim
Species: mouse
Tissue Origin: blood
Culture Media:
4D-Nucleofector® X-Unit

Briefly, per 1 × 10^6 activated human T cells or 20 × 10^6 murine splenocytes to be electroporated, 37 pmoles recombinant Cas9 (IDT) and 270 pmoles sgRNA (Synthego) were combined and incubated for 10 min at RT to generate Cas9/sgRNA RNP. Human T cells (1 × 10^6) or murine T cells (20 × 10^6) were resuspended in 20 µL P3 Buffer (Lonza), combined with RNP and electroporated using a 4D-Nucleofector (Lonza) using pulse code E0115 and DN100 for human and mouse T cells, respectively.


Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFN? and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.