Cholangiocarcinoma is a malignancy of bile duct epithelia with an increasing in incidence rate worldwide. Surgery is the only curative treatment, while adjuvant chemotherapy and radiotherapy render poor responses. Cell-based immunotherapy is a potential strategy for cholangiocarcinoma treatment. However, variation of tumor antigens in cholangiocarcinoma leads to the ineffectiveness of cell-based immunotherapy. In this study, we examined the activation of effector T-cells by dendritic cells pulsed with protein lysate or total RNA from cholangiocarcinoma cell lines for their cytolytic activity against cholangiocarcinoma. Broad-spectrum antigen types with respect to RNA antigen sources were obtained from combination of three cholangiocarcinoma cell lines (KKU-213, KKU-100, and KKU-055). Compared with protein lysate-pulsed dendritic cells, total RNA-pulsed dendritic cells induced anti-tumor effector T-cell response with higher killing ability to KKU-100 and KKU-213 cells compared with protein lysate-pulsed dendritic cells. Moreover, pooled messenger RNA from three cholangiocarcinoma cell lines significantly increased the specific killing capacity of activated lymphocytes against KKU-213 cells. These results suggest that activation of anti-tumor effector T-cells against cholangiocarcinoma by RNA-pulsed dendritic cells is more effective than that by protein lysate-pulsed dendritic cells. In addition, pulsing dendritic cells with pooled messenger RNA from multiple cell lines enhanced the efficacy of a cellular immune response against cholangiocarcinoma.

Cellular immunotherapy is a promising new therapeutic approach for hepatocellular carcinoma (HCC), which has a high recurrence rate, irrespective of the treatment administered. In this study, we attempted to improve the cytolytic activity of effector T-lymphocytes against HCC. T-lymphocytes were activated by monocyte-derived dendritic cells (DCs) pulsed with cell lysate or RNA prepared from HCC cell lines. Monocytes were activated for differentiation into DCs by treatment with the IL4 and GM-CSF. DCs were pulsed with cell lysate or RNA prepared from a single cell line or combinations of two or three HCC cell lines, and then co-cultured with autologous T-lymphocytes with the intent of creating specific cytotoxicity. We discovered that DCs pulsed with total RNA effectuated greater T-lymphocyte function than DCs pulsed with total cell lysate, as evidenced by greater cytolytic activities against HCC target cells. The percentage of Huh7, HepG2, and SNU449 cell apoptosis at effector:target ratio of 10:1 was 42.6 ± 4.5% (p = 0.01), 33.6 ± 3.1% (p = 0.007), and 21.4 ± 1.4% (p < 0.001), respectively. DCs pulsed with pools of antigens prepared from three cell lines improved the cytolytic function of effector T-lymphocytes by approximately two-fold (p < 0.001), which suggests that this approach be further developed and applied for adoptive transfer treatment of HCC.

Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms.