ABSTRACT
Immune checkpoint (IC) blockade and adoptive transfer of tumor-specific T-cells (ACT) are two major strategies to treat metastatic melanoma. Their combination can potentiate T-cell activation in the suppressive tumor microenvironment, but the autoimmune adverse effects associated with systemic injection of IC blockers persist with this strategy. ACT of tumor-reactive T-cells defective for IC expression would overcome this issue. For this purpose, PD-1 and TIGIT appear to be relevant candidates, because their co-expression on highly tumor-reactive lymphocytes limits their therapeutic efficacy within the tumor microenvironme,nt. Our study compares the consequences of PDCD1 or TIGIT genetic deletion on anti-tumor properties and T-cell fitness of melanoma-specific T lymphocytes. Transcriptomic analyses revealed down-regulation of cell cycle-related genes in PD-1KO T-cells, consistent with biological observations, whereas proliferative pathways were preserved in TIGITKO T-cells. Functional analyses showed that PD-1KO and TIGITKO T-cells displayed superior antitumor reactivity than their wild-type counterpart in vitro and in a preclinical melanoma model using immunodeficient mice. Interestingly, it appears that TIGITKO T-cells were more effective at inhibiting tumor cell proliferation in vivo, and persist longer within tumors than PD-1KO T-cells, consistent with the absence of impact of TIGIT deletion on T-cell fitness. Taken together, these results suggest that TIGIT deletion, over PD-1 deletion, in melanoma-specific T-cells is a compelling option for future immunotherapeutic strategies.
Subject(s)
Melanoma , Programmed Cell Death 1 Receptor , Receptors, Immunologic , Animals , Mice , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Melanoma/immunology , Melanoma/genetics , Melanoma/pathology , Melanoma/therapy , Gene Deletion , Tumor Microenvironment/immunology , Mice, Knockout , Mice, Inbred C57BL , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Cell Line, Tumor , Humans , Lymphocyte Activation/immunologyABSTRACT
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.