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.
Highlights
In response to the constant antigenic stimulation caused by chronic viral infections, or cancer cell antigens, cytotoxic T lymphocytes (CTLs) often become “exhausted” with sustained expression of inhibitory receptors and a distinct transcriptional state
Engineering patient autologous T-cells to express chimeric antigen receptors (CARs) using replication-deficient viruses has led to long-term remission of B-cell neoplasms in some leukemia patients[7,8], but these too may be susceptible to checkpoint inhibition
Immune-related adverse events are frequently observed in patients who receive antibodies that act on immune checkpoints, occurring in up to 90% and 70% of patients that are treated with anti-CTLA44 or anti-PD-1/programmed death-ligand 1 (PD-L1) antibodies[12,13], respectively
Summary
In response to the constant antigenic stimulation caused by chronic viral infections, or cancer cell antigens, cytotoxic T lymphocytes (CTLs) often become “exhausted” with sustained expression of inhibitory receptors and a distinct transcriptional state. In this state, CTLs fail to perform their main function of killing their target cells[1]. Engineering patient autologous T-cells to express chimeric antigen receptors (CARs) using replication-deficient viruses has led to long-term remission of B-cell neoplasms in some leukemia patients[7,8], but these too may be susceptible to checkpoint inhibition In light of these developments, immunotherapy is playing an ever greater role in the cancer treatment, alongside the traditional treatments of surgery, radiotherapy and chemotherapy[9]. Our results underscore a potential application of isolating antigen-specific CTLs from patients, in vitro gene editing and expansion, and transfusion back into patients for treatments of cancers and/or chronic infections
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