Increased antitumor efficacy of PD-1-deficient melanoma-specific human lymphocytes

J Immunother Cancer. 2020 Jan;8(1):e000311. doi: 10.1136/jitc-2019-000311.


Background: Genome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far, PDCD1 editing has been successfully tested mainly in chimeric antigen receptor T (CAR-T) cells and human primary T cells. Nonetheless, for patients with solid tumors, the adoptive transfer of effector memory T cells specific for tumor antigens remains a relevant option, and the use of high avidity T cells deficient for programmed cell death-1 (PD-1) expression is susceptible to improve the therapeutic benefit of these treatments.

Methods: Here we used the transfection of CAS9/sgRNA ribonucleoproteic complexes to edit PDCD1 gene in human effector memory CD8+ T cells specific for the melanoma antigen Melan-A. We cloned edited T cell populations and validated PDCD1 editing through sequencing and cytometry in each T cell clone, together with T-cell receptor (TCR) chain's sequencing. We also performed whole transcriptomic analyses on wild-type (WT) and edited T cell clones. Finally, we documented in vitro and in vivo through adoptive transfer in NOD scid gamma (NSG) mice, the antitumor properties of WT and PD-1KO T cell clones, expressing the same TCR.

Results: Here we demonstrated the feasibility to edit PDCD1 gene in human effector memory melanoma-specific T lymphocytes. We showed that PD-1 expression was dramatically reduced or totally absent on PDCD1-edited T cell clones. Extensive characterization of a panel of T cell clones expressing the same TCR and exhibiting similar functional avidity demonstrated superior antitumor reactivity against a PD-L1 expressing melanoma cell line. Transcriptomic analysis revealed a downregulation of genes involved in proliferation and DNA replication in PD-1-deficient T cell clones, whereas genes involved in metabolism and cell signaling were upregulated. Finally, we documented the superior ability of PD-1-deficient T cells to significantly delay the growth of a PD-L1 expressing human melanoma tumor in an NSG mouse model.

Conclusion: The use of such lymphocytes for adoptive cell transfer purposes, associated with other approaches modulating the tumor microenvironment, would be a promising alternative to improve immunotherapy efficacy in solid tumors.

Keywords: CD8-Positive T-Lymphocytes; cell engineering; costimulatory and inhibitory T-cell receptors; immunotherapy, adoptive; melanoma.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Female
  • Gene Editing
  • Humans
  • Immunotherapy, Adoptive / methods*
  • Melanoma / immunology*
  • Melanoma / therapy*
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • Programmed Cell Death 1 Receptor / deficiency*
  • Programmed Cell Death 1 Receptor / genetics
  • Programmed Cell Death 1 Receptor / immunology
  • Random Allocation
  • T-Lymphocytes, Cytotoxic / immunology*
  • Transfection
  • Xenograft Model Antitumor Assays


  • PDCD1 protein, human
  • Programmed Cell Death 1 Receptor