Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that functions to attenuate T cell activation. In this study, we knocked out (KO) PD-1 in cytotoxic T lymphocytes (CTLs) using CRISPR-Cas9 system to evaluate its effect on the anti-tumor activity of the CTLs against multiple myeloma (MM). Results show that PD-1 KO CTLs facilitate apoptosis and caspase activation of the co-cultured MM cells and enhanced MM cell death by 36% compared with the control. PD-1 KO also increased TNF-α and IFN-γ secretion of the CTLs by 2.4 and 1.9-fold respectively. The effectiveness of PD-1 KO in enhancing anti-tumor activity of the CTLs was verified in vivo using mouse xenograft model. The xenografted mice treated with PD-1 KO CTLs demonstrated repressed MM tumor growth and prolonged survival compared with the control group. We conclude that CRISPR-Cas9 is an efficient system to knock out PD-1 from CTLs and PD-1 KO could significantly enhance the anti-tumor activity of CTLs.
Abstract. The aim of the present study was to explore the clinicopathological and prognostic significance of long non-coding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) expression in human colorectal cancer (CRC). Expression levels of lncRNA CCAT2 in CRC, adjacent non-tumor and healthy colon mucosa tissues were detected by quantitative polymerase chain reaction. The disease-free survival and overall survival rates were evaluated using the Kaplan-Meier method, and multivariate analysis was performed using Cox proportional hazard analysis. The expression level of lncRNA CCAT2 in CRC tissues was increased significantly compared with adjacent normal tissues or non-cancerous tissues. CCAT2 expression was observed to be progressively increased between tumor-node-metastasis (TNM) stages I and IV. A high level of CCAT2 expression was revealed to be associated with poor cell differentiation, deeper tumor infiltration, lymph node metastasis, distance metastasis, vascular invasion and advanced TNM stage. Compared with patients with low levels of CCAT2 expression, patients with high levels of CCAT2 expression had shorter disease-free survival and overall survival times. Multivariate analyses indicated that high CCAT2 expression was an independent poor prognostic factor. Therefore, increased lncRNA CCAT2 expression maybe a potential diagnostic biomarker for CRC, and an independent predictor of prognosis in patients with CRC.
◥Viral-based chimeric antigen receptor-engineered T (CAR T)cell manufacturing has potential safety risks and relatively high costs. The nonviral minicircle DNA (mcDNA) is safer for patients, cheaper to produce, and may be a more suitable technique to generate CAR T cells. In this study, we produced mcDNA-based CAR T cells specifically targeting prostate stem cell antigen (PSCA; mcDNA-PSCA-CAR T cells). Our results showed that mcDNA-PSCA-CAR T cells persisted in mouse peripheral blood as long as 28 days and demonstrated more CAR T-cell infiltration, higher cytokine secretion levels, and better antitumor effects. Together, our results suggest that mcDNA-CAR can be a safe and cost-effective platform to produce CAR T cells.
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