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

Nat Commun. 2021 May 28;12(1):3236. doi: 10.1038/s41467-021-23331-5.


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 IFNγ 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / metabolism
  • Adenosine A2 Receptor Antagonists / pharmacology
  • Animals
  • CRISPR-Cas Systems / genetics
  • Cell Engineering / methods
  • Cell Line, Tumor / transplantation
  • Disease Models, Animal
  • Female
  • Gene Editing
  • Gene Expression Regulation, Neoplastic / immunology
  • Gene Knockdown Techniques
  • Gene Knockout Techniques
  • Humans
  • Immunotherapy, Adoptive / methods*
  • Lymphocytes, Tumor-Infiltrating / immunology
  • Mice
  • Mice, Transgenic
  • Neoplasms / genetics
  • Neoplasms / immunology
  • Neoplasms / therapy*
  • RNA, Small Interfering / metabolism
  • RNA-Seq
  • Receptor, Adenosine A2A / genetics*
  • Receptor, Adenosine A2A / metabolism
  • Receptor, ErbB-2 / genetics
  • Receptors, Chimeric Antigen / immunology
  • Receptors, Chimeric Antigen / metabolism
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Signal Transduction / immunology
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism
  • T-Lymphocytes / transplantation*
  • Tumor Escape / drug effects
  • Tumor Escape / genetics


  • ADORA2A protein, human
  • Adenosine A2 Receptor Antagonists
  • Adora2a protein, mouse
  • RNA, Small Interfering
  • Receptor, Adenosine A2A
  • Receptors, Chimeric Antigen
  • ERBB2 protein, human
  • Receptor, ErbB-2
  • Adenosine