Genetic abrogation of immune checkpoints in antigen-specific cytotoxic T-lymphocyte as a potential alternative to blockade immunotherapy

Abstract

T cell function can be compromised during chronic infections or through continuous exposure to tumor antigens by the action of immune checkpoint receptors, such as programmed cell death protein 1 (PD-1). Systemic administration of blocking antibodies against the PD-1 pathway can restore T cell function, and has been approved for the treatment of several malignancies, although there is a risk of adverse immune-related side-effects. We have developed a method for generating gene knockouts in human antigen (Ag)-specific cytotoxic T-Lymphocyte (CTLs) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. Using this method, we generated several transduced CD4+ or CD8+ antigen-specific polyclonal CTL lines and clones, and validated gene modifications of the PD-1 gene. We compared these T-cell lines and clones with control groups in the presence of programmed death-ligand 1 (PD-L1) and observed improved effector functions in the PD1-disrupted cell group. Overall, we have developed a versatile tool for functional genomics in human antigen-specific CTL studies. Furthermore, we provide an alternative strategy for current cell-based immunotherapy that will minimize the side effects caused by antibody blockade therapy.

Self-Maintaining CD103+ Cancer-Specific T Cells Are Highly Energetic with Rapid Cytotoxic and Effector Responses

“…All in vitro functional experiments were conducted using clonally expanded cancer-specific CTLs that were generated from cancer patients, using a well-established human antigen-specific T-cell generation technique as previously described (20)(21)(22). A detailed flow chart of the generation of human cancer-specific CTLs is shown in Supplementary Fig.…”

Enriched HLA-E and CD94/NKG2A Interaction Limits Antitumor CD8+ Tumor-Infiltrating T Lymphocyte Responses

“…Antigen-specific T-cell lines were generated as described previously (29). Briefly, isolated lymphocytes from blood and tissues were stimulated with either cancer SSX2 41-49 -specific KV9 peptide (KASEKIFYV) or CMV pp65 495-503 -specific NV9 peptide (NLVPMVATV) and cultured in RPMI1640 supplemented with 10% v/v heat-inactivated human AB serum (National Blood Service), 2 mmol/L L-Glutamine and 1% v/v (500 U/mL) penicillin-streptomycin (Sigma-Aldrich), and recombinant human IL2 (PeproTech) at 37 C. After 14 days, antigenspecific T cells were purified with peptide-MHC Class I tetramer (HLA-A2 KV9 or HLA-A2 NV9) using BD Aria II (BD Biosciences) and later expanded in vitro.…”

“…An HLA-A2 þ Epstein-Barr virus-transformed B-cell line (BCL) was generated in the laboratory in 2005 and maintained in RPMI1640 supplemented with 10% v/v FCS (Sigma Aldrich), 2 mmol/L L-glutamine, and 1% v/v (500 U/mL) penicillinstreptomycin (Sigma-Aldrich) at 37 C. Surface expression of CD19 and HLA-A2 on this BCL was assessed regularly by flow cytometry staining. Lentiviruses expressing either HLA-E0101 or HLA-E0103 allele were generated using the three plasmids system by cotransfection of 293T cell line cultured in DMEM supplemented with 10% v/v FCS (Sigma-Aldrich), 2 mmol/L L-Glutamine, and 1% v/v (500 U/mL) penicillin-streptomycin (Sigma-Aldrich) at 37 C, as described previously (29). After 48 hours, supernatant containing lentivirus was collected, filtered, and later concentrated using Lenti-X Concentrator (Clontech) according to the manufacturer's instruction.…”

“…Cells isolated from tissues and PBMC were first stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific), then stained with conjugated antibodies, with incubation at each step for 20 minutes at 4 C. Tetramer staining was performed as described previously (29). Briefly, cells were stained with either HLA-A2 KV9 or HLA-A2 NV9 tetramer for 20 minutes at 37 C before continuing with above mentioned staining process.…”

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

“…22 In all cases, engineered CAR T cells exhibited enhanced tumor control in mouse models. Improved effector functions have also been reported following PDCD1 gene editing in virus-specific cytotoxic T lymphocytes (CTL) 23 24 and in myeloma-specific CTL. 25 In melanoma, the superior antitumor efficacy of PDCD1-edited human-specific CTL has not been formally documented so far.…”