Vorinostat unmasks MAEL to enhance DC vaccine-induced CTL killing in hepatocellular carcinoma, potentiated by TIGIT checkpoint inhibition.

The immunobiology of hepatocellular carcinoma in humans and mice: Basic concepts and therapeutic implications

BackgroundHuman hepatocellular carcinoma (HCC) cells express WT1 and/or carcinoembryonic antigen (CEA) as potential targets for the induction of antitumor immunity. In this study, generation of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) by fusions of dendritic cells (DCs) and HCC cells was examined.MethodsHCC cells were fused to DCs either from healthy donors or the HCC patient and investigated whether supernatants derived from the HCC cell culture (HCCsp) influenced on the function of DCs/HCC fusion cells (FCs) and generation of CTL and Treg.ResultsFCs coexpressed the HCC cells-derived WT1 and CEA antigens and DCs-derived MHC class II and costimulatory molecules. In addition, FCs were effective in activating CD4+ and CD8+ T cells able to produce IFN-γ and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism. However, HCCsp induced functional impairment of DCs as demonstrated by the down-regulation of MHC class I and II, CD80, CD86, and CD83 molecules. Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated Streptococcus pyogenes. Interestingly, fusions of immature DCs generated in the presence of HCCsp and allogeneic HCC cells promoted the generation of CD4+ CD25high Foxp3+ Treg and inhibited CTL induction in the presence of HCCsp. Importantly, up-regulation of MHC class II, CD80, and CD83 on DCs was observed in the patient with advanced HCC after vaccination with autologous FCs. In addition, the FCs induced WT1- and CEA-specific CTL that were able to produce high levels of IFN-γ.ConclusionThe current study is one of the first demonstrating the induction of antigen-specific CTL and the generation of Treg by fusions of DCs and HCC cells. The local tumor-related factors may favor the generation of Treg through the inhibition of DCs maturation; however, fusion cell vaccination results in recovery of the DCs function and induction of antigen-specific CTL responses in vitro. The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer.

Severely impaired CTL killing is a feature of the neurological disorder Niemann-Pick disease type C1

Objective To investigate the improvement of cytotoxic T lymphocyte (CTL) anti-tumor immune response by recombinant adeno-associated virus (rAAV) mediated HLA-A2 gene transduction of hepatocellular carcinoma cells. Methods The AFP specific CTL anti-hepatocarcinoma response was induced by rAAV carrying human alpha fetoprotein (AFP) gene (rAAV-AFP) transduction of human dendritic cells (DC) in vitro. The rAAV carrying human HLA-A2 gene (rAAV-HLA-A2) was used to transduce hepatocellular carcinoma cells to improve the expression level of HLA-A2 molecules in vitro and enhance the sensitivity of hepatoma cells to rAAV-AFP transduced DC vaccine-induced AFP-specific CTL immunoreaction. Results rAAV-AFP transduced DC vaccine could effectively induce AFP-specific anti-hepatocarcinoma CTL immune response. rAAV-HLA-A2 can increase the expression of HLA-A2 molecules in hepatocellular carcinoma cells and enhance the sensitivity of hepatocellular carcinoma cells to rAAV-AFP transduced DC vaccine-induced CTL immunoreaction. Conclusion Increasing the expression of HLA-A2 molecules in hepatocellular carcinoma cells by rAAV-HLA-A2 is an effective way to enhance the immune response of CTL against hepatocellular carcinoma, which provides a new solution for active immunotherapy for hepatocellular carcinoma patients with positive AFP expression but with low HLA-class I molecular expression. Key words: Adenoviridae; alpha-Fetoproteins; Dendritic cells; Liver neoplasms; human leukocyte antigen-A2

Objective To explore the cytotoxicity of the cytotoxic T lymphocyte (CTL) induced by SW480 sonicate sensitized dendritic cells (DC) on the colon cancer cell line SW480. Methods PBMC were separated from the HLA-A*0201 donor and DC were cultured with rhGM-CSF, rhIL-4 and rhTNF-α. The same donor's primary CTL were stimulated by DC loaded with SW480 sonicate. The cytotoxicity of CTL on SW480 (HLA-A*0201 positive) and K562 (HLA-A*0201 negative) was determined by the MTT method. Results The cytotoxicity of the CTL on SW480 was stronger than that on K562 (P ＜0.05). Conclusion The DC vaccine can stimulate specific CTL which can trigger cytotoxic activity on the target cells and this cytotoxicity is related to MHC restriction. Key words: Neoplasms; Cancer vaccines; Dendritic cell; T-lymphocyte, cytotoxic; Immunologic; SW480

To fuse human hepatocellular carcinoma (HCC) cells with mature monocyte-derived dendritic cells (FastDC) and to observe in vitro the function of the fused cells in stimulating autologous T cells proliferation and inducing HCC-specific cytotoxic T lymphocyte (CTL) response. CD14(+) cells were isolated and purified from the peripheral blood of a healthy HLA-A2 blood donor and cultured in fresh dendritic cell complete medium for 24 h, then proinflammatory mediators were supplemented for another 24 h, thus generating mature dendritic cell (FastDCs). The FastDCs were fused with human HCC cells of the line HCCLM3 to generate novel dendritoma. T cells were isolated from selected CD14(-) cells and then divided into 4 groups to be stimulated with dendritoma cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells respectively for 96 hours. 18 hours before the end of cultivation (3)H-TdR was added into the culture fluid. Scintillation counter was used to measure the cpm values. CD8(+)T cells were isolated from CD14(-) cells, and added with different stimulating cells radiated by (60)Co and IL-2, IL-6, and IL-7. The values of IFN-gamma in the supernatants of the culture fluid of CD8(+)T cells with dendritoma cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells was measured. HCCLM3, K562, HLE, self monocytes labeled with Na(2)(51)CrO(4) were added with effector cells, gamma-scintillation counter was used to measure the cpm value so as to calculate the killing ability of CTL. The CTLs activated by dendritoma cells specifically killed the HCCLM3 cells in the context of MHC class I and acted less vigorously against the control target cells. The CTLs activated by dendritoma cells were stronger in killing HCCLM3 cells than DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells (all P < 0.05). The killing activity was decreased on the HCCLM3 cells incubated with anti-HLA-ABC antibody. Three, five, and seven days after co-cultivation the value of IFN-gamma in the supernatants of the culture fluid of CD8(+)T cells with fused cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells increased gradually, especially in the supernatants of the culture fluid of CD8(+)T cells with dendritoma cells (400 pg/ml +/- 60 pg/ml 3 days after, 1030 pg/ml +/- 160 pg/ml 5 days after, and 1260 pg/L +/- 180 pg/L 7 days after). The novel dendritomas formed with HCCLM3 cells and mature FastDCs from healthy human peripheral blood CD14(+) monocytes are potent stimulators for CD8(+)T cells in inducing HCCLM3 cell-specific lysis. With shorter time required for in vitro DC development, the rapid method of generation of dendritoma is more economic and may represent a new strategy for immunotherapy of hepatocellular carcinoma.

Although quantitative insights into the killing behaviour of Cytotoxic T Lymphocytes (CTLs) are necessary for the rational design of immune-based therapies, CTL killing function remains insufficiently characterised. One established model of CTL killing treats CTL cytotoxicity as a Poisson process, based on the assumption that CTLs serially kill antigen-presenting target cells via delivery of lethal hits, each lethal hit corresponding to a single injection of cytotoxic proteins into the target cell cytoplasm. Contradicting this model, a recent in vitro study of individual CTLs killing targets over a 12-hour period found significantly greater heterogeneity in CTL killing performance than predicted by Poisson-based killing. The observed killing process was dynamic and varied between CTLs, with the best performing CTLs exhibiting a marked increase in killing during the final hours of the experiments, along with a “burst killing” kinetic. Despite a search for potential differences between CTLs, no mechanistic explanation for the heterogeneous killing kinetics was found. Here we have used stochastic simulations to assess whether target cells might require multiple hits from CTLs before undergoing apoptosis, in order to verify whether multiple-hitting could explain the late onset, burst killing dynamics observed in vitro. We found that multiple-hitting from CTLs was entirely consistent with the observed killing kinetics. Moreover, the number of available targets and the spatiotemporal kinetics of CTL:target interactions influenced the realised CTL killing rate. We subsequently used realistic, spatial simulations to assess methods for estimating the hitting rate and the number of hits required for target death, to be applied to microscopy data of individual CTLs killing targets. We found that measuring the cumulative duration of individual contacts that targets have with CTLs would substantially improve accuracy when estimating the killing kinetics of CTLs.

Although quantitative insights into the killing behaviour of Cytotoxic T Lymphocytes (CTLs) are necessary for the rational design of immune-based therapies, CTL killing function remains insufficiently characterised. One established model of CTL killing treats CTL cytotoxicity as a Poisson process, based on the assumption that CTLs serially kill antigen-presenting target cells via delivery of lethal hits, each lethal hit corresponding to a single injection of cytotoxic proteins into the target cell cytoplasm. Contradicting this model, a recent in vitro study of individual CTLs killing targets over a 12-hour period found significantly greater heterogeneity in CTL killing performance than predicted by Poisson-based killing. The observed killing process was dynamic and varied between CTLs, with the best performing CTLs exhibiting a marked increase in killing during the final hours of the experiments, along with a "burst killing" kinetic. Despite a search for potential differences between CTLs, no mechanistic explanation for the heterogeneous killing kinetics was found. Here we have used stochastic simulations to assess whether target cells might require multiple hits from CTLs before undergoing apoptosis, in order to verify whether multiple-hitting could explain the late onset, burst killing dynamics observed in vitro. We found that multiple-hitting from CTLs was entirely consistent with the observed killing kinetics. Moreover, the number of available targets and the spatiotemporal kinetics of CTL:target interactions influenced the realised CTL killing rate. We subsequently used realistic, spatial simulations to assess methods for estimating the hitting rate and the number of hits required for target death, to be applied to microscopy data of individual CTLs killing targets. We found that measuring the cumulative duration of individual contacts that targets have with CTLs would substantially improve accuracy when estimating the killing kinetics of CTLs.

Dendritic cells fused with allogeneic hepatocellular carcinoma cell line compared with fused autologous tumor cells as hepatocellular carcinoma vaccines

MAGE antigens: therapeutic targets in hepatocellular carcinoma?

Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: Perspectives and challenges

Enhancement of dendritic cell-based vaccine potency by anti-apoptotic siRNAs targeting key pro-apoptotic proteins in cytotoxic CD8 + T cell-mediated cell death

Objective To observe the effect of immunotherapy on hepatocellular carcinoma (HCC) by melanoma-associated antigen-A3 (MAGE-A3) activated dendritic cells (DC)-induced antigen-specific CD8+ cytotoxic T lymphocytes (CTL) and offer help to HCC treatment. Methods After MAGE-A3 activated DC biomarkers, interleukin (IL)-12, and IL-10 in DC were tested. MAGE-A3-DC was used to induce antigen-specific CD8+ CTL. The apoptosis of L02 cells and HepG2 cells was detected after treatment with CD8+ CTL. Interferon (IFN)-γ of CD8+ CTL was detected by enzyme-linked immunospot assay (ELISPOT). Subcutaneous tumor model of mouse HCC was established. The tumor volume of different groups was measured. Tumor tissue was further observed by pathological sections. Results MAGE-A3 protein stimulation significantly increased the levels of Human leukocyte antigen-DR (HLA-DR), DC83, DC86 and DC80 markers on the surface of DC (97.45±2.58 vs. 48.33±1.68, 92.81±2.36 vs. 52.92±1.90, 94.00±3.01 vs. 53.97±2.05, 92.16±1.87 vs. 34.13±1.32; t=35.680, P=0.013; t=29.440, P=0.021; t=24.580, P=0.012; t=56.690, P=0.009). The IL-12 level of MAGE-A3-DC was significantly higher than that of DC [(338.44±18.15) pg/ml vs. (243.23±16.56) pg/ml; t=8.670, P=0.005], but IL-10 level of MAGE-A3-DC was significantly lower than that of DC [(207.21±10.89) pg/ml vs. (327.58±14.36) pg/ml; t=14.930, P=0.009]. The apoptosis of L02 cells treated with MAGE-A3-DC-CD8+ CTL was similar to that after treatment with DC-CD8+ CTL [(9.10±1.40)% vs. (9.71±1.58)%; t=0.650, P=0.120]. The apoptostic rate of HepG2 cells treated with MAGE-A3-DC-CD8+ CTL was significantly higher than that after treatment with DC-CD8+ CTL [(58.84±5.27)% vs. (9.63±1.61)%; t=19.970, P=0.008]. After therapy, tumor sizes V/V0 in MAGE-A3-DC-CD8+ CTL group decreased significantly as compared with those of phosphate buffer (PBS) group and DC-CD8+ CTL group (5.43±1.22 vs. 21.81±2.01 vs. 22.85±2.40; t=22.030, P=0.010; t=20.460, P=0.012). Hematoxylin and eosin (HE) staining showed that nuclear condensation, increased cell gap, vacuoles, and edema were observed in tumor tissues after treatment with MAGE-A3-DC-CD8+ CTL, but pathological changes of tumor tissues were not observed in PBS group and DC-CD8+ CTL group. Conclusion MAGE-A3 protein can stimulate the maturation of DC that induce the production of MAGE-A3 specific CD8+ CTL. MAGE-A3-DC-CD8+ CTL have specific tumor killing ability. Key words: Carcinoma, hepatocellular; Immunotherapy; Melanoma-associated antigen-A3; Dendritic cells; Cytotoxic T lymphocytes

Objective To investigate the safety and effectiveness of tumor antigen-specific cytotoxic T lymphocytes (CTL) combined with dendritic cells (DC) for primary hepatocellular carcinoma (HCC).Methods The tumor antigen-specific dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs) were induced from peripheral blood mononuelear cells of patients with HCC in vitro using cytokines such as recombinant human granulocyte-macrophage colony stimulating factor(rhGM-CSF) and so on and then were pulsed with tumor antigen.The tumor antigen specific DC-CTL cells thus obtained were infused back to individual patients.Altogether 30 patients undertook such treatment.The changes of signs and symptomsliver function,AFP,the subsets of lymphocytes in peripheral blood and life quality were assessed after treatment.Results The patients' liver function improved greatly (P ＜ 0.05),the level of AFP decreased significantly(985.2 ng/ml vs 630.7 ng/ml),there were significant differences (P ＜ 0.05).The percentages of CD3 +,CD3 + CD8 +,and CD3 + CD4 + increased remarkably (P ＜ 0.05).The symptoms,signs and quality of life of most patients significantly improved.Conclusions Infusion of tumor antigen-specific DC-CTL cells for patients with advanced primary hepatocellular carcinoma is safe and effective. Key words: Cytotoxic T lymphocyte (CTL) ; Dendritic cells ; Primary hepatocellular carcinoma; Safety ; Effectiveness