Objective: Cancer immunotherapy is to activate and develop cancer-specific effector cells, which will eradicate cancer cells by recognizing tumor associated antigens expressed on them. Development of cancer-specific effector cells necessitates three sequential signaling events, antigen-presenting cells (APCs) in which dendritic cells (DCs) capture and process cancer antigens from tumor cells, and then present antigenic peptides through major histocompatibility complex (MHC) molecules for recognition by TCR of a T cell. Our pilot study designed to activate dendritic cells (DCs) obtained from hepatocellular carcinoma patients to sensitize autologous T & B lymphocytes against HepG2 tumor antigens ex-vivo and the outcome of ribonuclease infusion to attenuate HepG2 cells to boost antigen presentation. Patients & Methods: Monocytes derived dendritic cell and lymphocytes obtained from patients finally diagnosed as HCC. Activated immune cells were pulsed by intact HepG2 cells or ribonuclease intracellularly infused attenuated HepG2 cells. Immunophenotyping analysis after exposure to tumor cells, T-cells, NK-cells, B-cells & dendritic cells, supernatant media levels of IL-12 & LDH by ELISA and CD44, TAP-2 & LMP-2 gene expressions by Real-Time PCR were analyzed. Results: Ribonuclease-attenuated HepG2 cells showed significant increase in terms of activated DCs (CD83+), activated mature DCs (CD83+CD86+), upregulation of CD44 gene expression and elevated LDH & IL-12. On the other hand, significant decreases in the markers of T-helper cells (CD3+CD4+), cytotoxic T-cells (CD3+CD8+), double positive T-cells (CD4+CD8+), activated T-cells (CD19-CD38+), natural killer cells (CD56+), mature DCs (CD86+), B-cells (CD19+), activated B-cells (CD19+CD38+) and downregulation of LMP2 gene expression. Conclusion: Using ribonuclease enzyme for attenuating HepG2 cells against activated immune cells showed declined allogeneic antigen presentation compared to unattenuated tumor cells and shift towards tumor associated antigens presentation (TAAs). Further studies are required to study TAAs presentation mechanism.
Keywords: Immunotherapy, antigen presentation, attenuated tumor cell, HepG2, Ribonuclease.
1. Introduction
The immune evasion in hepatocellular carcinoma (HCC) represents a main barrier to the delivery of effective immunotherapy (1). Therefore, overcoming the immune suppressive effect plays a pivotal role in generating an effect in cancer immunotherapy. While protocols vary, a common scheme in dendritic cell-based immunotherapy involves activation of dendritic cells (DCs) in order to increase antigen presentation and cellular immunity (2). Therefore, proper use of immune adjuvant is a central subject of the studies and the need for more effective therapeutic approaches with the lowest side effects remains imperative.
Dendritic cells (DCs) are the principal stimulators of primary immune reaction as they process internalized antigen, load peptide fragments onto Major histocompatibility (MHC) proteins and priming na”ve T cell to that antigen (3). Upon inflammation, monocytes differentiate into monocyte derived cells (MCs) which display many different functions from presenting the antigen to eliminating the pathogen (4). The function of DCs relies on maturation where inflammatory mediators enhancing differentiation of immature DC into matured immunogenic DCs. This process is associated with changes in morphology, reduced uptake of antigens and impaired antigen processing activity. Furthermore, mature DCs exhibit a strong co-stimulatory and T-lymphocytes activating capacity (5).
Antigen presentation process is a multi-step process with several hallmarks at each phase. Studying surface and molecular markers can aid in evaluating efficient immunotherapeutic tools. Gene expressions of transporter associated with antigen processing-2 (TAP-2) and large multifunctional proteases-2 (LMP-2) are located in antigen presenting cells, in which they play a crucial role in antigen presentation (6).
Ribonuclease enzyme is widely used in diagnosis as a method for screening intracellular proteins. Ribonuclease was introduced intracellularly in the way that the cells were fixated and permeabilized by a method does not alter the cell surface antigens expression (7). Our pilot study designed to activate DCs ex-vivo (obtained from HCC patients) by exposure to either attenuated HepG2 tumor cells (by infusing ribonuclease intracellularly) or unattenuated HepG2 tumor cells (control cells) and sensitize autologous T & B lymphocytes by activated DCs and studying the outcome of ribonuclease infusion on antigen presentation of HepG2 cells antigenicity. ‘
2. Methods
2.1. Isolation and preparation of DCs and lymphocytes
Peripheral blood (PB) samples were obtained from fifteen newly diagnosed Hepatocellular Carcinoma (HCC) patients selected from the outpatient clinic at the Egyptian National Cancer Institute (2015 ‘ 2016). A sampling of the PB was approved by the Institutional Board of Research of the Egyptian National Cancer Institute (NCI) and informed consent was obtained from all subjects (IRB No.: IRB00004025 and Approval No.: 201516031.3).
All procedures performed in our study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Mononuclear cells (MCs) were isolated from PB of patients by the Ficoll’ Hypaque density gradient centrifugation (Seromed, Biochrom KG, Berlin, Germany) and then cultured in RPMI 1640 medium (Sigma Aldrich, St Louis, MO, USA) containing 10% fetal calf serum (FCS) (Biochrome AG, Berlin, Germany), 1% 100 U/ml penicillin and 100 ”g/ml streptomycin (usual antibiotic for cell culture) (Biochrome AG, Berlin, Germany) for 4 h at 37oC in 5% CO2 atmosphere.
Non-adherent cells (lymphocytes) were transferred to be cultured in RPMI-1640 medium containing two cytokines: recombinant human Interleukin-2 (IL-2; 20 IU/ml) and recombinant human Interleukin-6 (IL-6; 10 IU/ml). These concentrations were the optimized values according to Ding et al. (2007) (8). Incubation was carried out at 37”C with 5% CO2 for 6 days. On day 3, culture medium including cytokines were semi-refreshed.
Adherent cells (monocytes) were cultured for 6 days in RPMI 1640 medium containing 10% FCS, recombinant human granulocyte monocyte – colony stimulating factor (hGM-CSF; 800 IU/ml) and recombinant human Interleukin-4 (rIL-4; 500 IU/ml) (purchased from Bio Basic Inc., Ontario, Canada). On day 3, culture medium including cytokines were semi-refreshed. On day 6, non-adherent and loosely adherent cells (non-viable dendritic cells) were discarded, viable immature dendritic cells (iDCs: well-attached cells) were harvested by using one ml of trypsin enzyme (purchased from Biowest Inc., Nuaill” ‘ France) to liberate the cells followed by neutralizing trypsin enzyme using complete RPMI medium. Washing of cells was performed later by using phosphate buffer saline (PBS) to discard the media and trypsin enzyme.
2.2. Culturing of HepG2 cells
Human hepatoma cell (HepG2; ATCC” HB-8065.1′) were cultured in RPMI 1640 medium supplemented with 10% FCS, antibiotic in atmosphere of 5% CO2 at 37oC.
2.3. Experimental design
Experimental design contains two groups, Group I which represents the control, in which iDCs and lymphocytes were exposed to unattenuated HepG2 cells. Group II represents the ribonuclease-attenuated HepG2 cells which would be further exposed to iDCs and subsequently to lymphocytes.
Attenuation of HepG2 cells was performed for the purpose of overcoming the immune suppressive role of HepG2 cells. Attenuation of HepG2 was achieved in two steps: first step (fixation step) was done by incubation HepG2 cells with paraformaldehyde (2%) for 10 minutes at room temperature, second step was permeabilizing its plasma membrane by tween-20 & phosphate buffer saline (PBS) and 5 ul of (10 mg/ml) ribonuclease A enzyme (QIAGEN, Hilden, Germany) was added immediately and incubation for 10 minutes at room temperature. Cells were washed well with PBS. Afterward, cells were ready to be used as attenuated HepG2 cells.
2.4. Activation and maturation of DCs
Obtained iDCs were activated by culturing in RPMI 1640 medium containing 10% FCS, antibiotic, tumor necrosis factor-alpha (TNF-alpha; 10 mg/ml) (purchased from Bio Basic Inc., Ontario, Canada), 400 IU/ml hGM-CSF and 250 IU/ml hIL-4 for 24 hours in the presence of each group of HepG2 cells separately. Afterward, propagated lymphocytes were added to the previous cocktail of cells in addition to 50 IU/ml hIL-2 and 50 IU/ml hIL-6 for 3 days. Cells were harvested by collecting all non-attached cells (which are DCs, lymphocytes and few HepG2 cells), those cells were subdivided into two main divisions; the first aliquot was instantaneously separated to measure the immune cells immunophenotyping and the second aliquot was instantly used to extract total RNA from cells for the objective of gene expressions analysis. The liquid culture media of the cells were used to quantity IL-12 & LDH concentrations.
2.5. Immunophenotypic analysis of immune cells
T-lymphocytes and Natural killer cells (NK-cells) were determined to elucidate effect of ribonuclease in attenuating tumor cells by measuring anti-CD3, anti-CD4, anti-CD8 and anti-CD56 (all purchased from DakoCytomation, M”nster, Germany). B-lymphocytes activity was determined by measuring anti-CD19 and activated B & T-lymphocytes were determined by anti-CD38 (both purchased from ebioscience, CA, USA). Anti-CD81, anti-CD83, anti-CD86 and anti-CD209 (DC-SIGN) were used to measure dendritic cells activity (all purchased from ebioscience, CA, USA). The procedures were followed regarding the manufacturer protocol of each monoclonal antibody. The analysis was performed by using EPICS flow cytometer (Backman coulter, CA, USA).
2.6. LDH release from necrotized HepG2 cells
Necrosis of HepG2 cells was determined by LDH ELISA kit (Cloud-Clone crop, REF: SEB864Hu, Houston, TC, USA). The procedures were followed regarding the manufacturer protocol.
2.7. Interleukin-12 secretion by activated DCs
IL-12 level in cultured media supernatant was measured by an ELISA kit (Human IL-12 P70, Quantikine ELISA, R&D systems, Minneapolis, USA). The procedures were followed regarding the manufacturer protocol.
2.8. Molecular analysis of gene expressions of CD44, TAP-2 & LMP-2
Total cellular RNA was extracted from all non-attached collected cells (which are DCs, lymphocytes and few HepG2 cells) by using (High Pure RNA Isolation Kit, Roche Diagnostics, Mannheim, Germany). In this step, the procedures were followed regarding the manufacturer protocol. Extracted total RNA was quantified by Nanodrop one (Thermofisher, WI, USA). Total RNA was reversely transcribed by High Capacity cDNA Reverse Transcription Kit (Applied bioscience, CA, USA), reverse transcription procedures were followed regarding the manufacturer protocol. Gene expression of CD44, TAP-2 and LMP-2 was estimated by RT2 SYBR Green ROX’ qPCR Mastermix (QIAGEN, Hilden, Germany). The machine used in Amplification: mini PikoReal (Ver.2.2) (Thermo scientific, Finland). Primers used in amplification process ((house-keeping gene GADPH), CD44, TAP-2 & LMP-2 (purchased from AlphaDNA, Montreal, Canada), (table 2). the procedures were followed regarding the manufacturer protocol of QIAGEN kit. ”Ct of each gene of the control group was initially calculated. The relative expression of gene transcripts was calculated by ”Cq. Finally, the ratios of target to reference gene were determined with the Pfaffl method (9).
Statistical Analysis
Data was firstly examined for their normality using Shapiro-Wilk test (10). Student’s t test was used to determine whether the group means were statistically significantly different (n=30). The correlations between different markers were calculated by Pearson’s correlation. All statistical tests were two-sided; a P value of less than 0.05 was considered statistically significant. All statistical analyses were done using SPSS software (version 24; Chicago, IL).
3. Results
Cocultured immune cells sensitized by HepG2 cells, were subjected to multilevel analysis. We examined the effect of ribonuclease on enhancing the antigenicity or decline the suppressive effect of tumor cells by infusing ribonuclease intracellularly into HepG2 cells.
Effect of ribonuclease – attenuated HepG2 cells on gene expression.
Attenuation of HepG2 cells by ribonuclease enzyme and subsequent pulsation to dendritic cells showed an upregulation of CD44 gene expressions (P<0.001) (% change 128.71%) and a downregulation of LMP-2 gene expression (P<0.001) (% change -20.79 %) compared to unattenuated pulsed HepG2 cells (fig.1).
Levels of dendritic cells markers due to ribonuclease treatment.
Dendritic cells pulsed by ribonuclease-attenuated HepG2 showed elevated level of CD83+ (P<0.001) (% change 35.69 %), elevated level of CD83+ CD86+ (P<0.01) (% change 43.17 %) and reduced level of CD86+ (P<0.01) (% change -30.77 %) compared to unattenuated pulsed HepG2 cells (fig.2).
Comparison of stimulated levels of T-cells & NK-cells between groups.
Significant reductions in all subsets of T-cells were observed due to ribonuclease effect on pulsed HepG2, where T-helper lymphocytes level (CD3+CD4+) was decreased (P<0.001) (% change -39.47 %), cytotoxic T-lymphocytes level was declined (CD3+CD8+) (P<0.05) (% change -34.99 %), double positive T-cells level was reduced (CD4+CD8+) (P<0.01) (% change -49.74 %) and NK-cells level was diminished (CD56+) (P<0.001) (% change -52.50 %) (fig.3).
Comparison of stimulated levels of B-cells and activated T-cell between groups.
Pulsing immune cells with ribonuclease-attenuated HepG2 lead to significant suppression in activated B-lymphocytes (CD19+CD38+) (P<0.001) (% change -68.91 %) and significant suppression in total B-lymphocytes population (CD19+) (P<0.001) (% change -44.07 %), meanwhile a significant elevation in activated T-cell marker (CD19-CD38+) (P<0.01) (% change 52.26 %) were observed compared to unattenuated control group (fig.4).
Comparison of secreted IL-12 levels and LDH in cultured media, between groups.
Significant elevation of LDH (P<0.001) (% change 95.97 %) and IL-12 (P<0.01) (% change 23.73 %) due to the effect ribonuclease effect on HepG2 cells (fig.5).
4. Discussion
Cancer immunotherapy is intended to boost immune cells, to fight against cancer cells whereas leaving normal cells untouched. Our study focused on using a novel method to attenuate tumor cells. We measured the expression of three pivotal genes for the objective of screening antigen presentation of tumor associated antigens (TAAs). Gene expression of CD44 in T-lymphocytes is a later stage for T-lymphocytes priming by antigen presenting cells (APC) (11). Meanwhile, gene expressions of (TAP-2) and (LMP-2) are lonely expressed in APC only (6). Allorecognition of antigens is well known to be mediated by CD4+ and CD8+ T cells recognizing either intact allo-MHC molecules or allopeptides bound to self-MHC molecules on recipient APCs (12), therefore the elevation of CD44 mRNA may refer to the phase proceeding CD4+ & CD8+ cells activation. Our results showed upregulation of CD44 along with the downregulation of LMP-2 gene expressions after attenuating tumor cells with ribonuclease, and this indicates that attenuation of HepG2 overcomes the suppressive action of tumor cells which in turn accelerate the antigen presentation of allogenic antigens and starting the presentation of low antigenic TAAs.
The levels of lymphocytes and mature DCs markers was in harmony with gene expressions pattern in unattenuated HepG2, in which the presence of immune activation was directed towards allogenic antigens. Conversely, attenuated HepG2 cells showed decreased immune cells counts accompanied with elevated activation markers (DCs and T-lymphocytes markers) were observed. The decrease in the immune cells count is a sign of fading of allogenic antigen presentation and initiating TAAs presentation (fig.2-4).
Various methods used to attenuate tumor cells, as our trial using ribonuclease enzyme, were used to rise the immune sensitization towards tumor cells, which include functionalized poly-lactic-co-glycolic acid nanoparticles (13), knockdown genes (14) and sensitization using polythylene glycol (15) resulted in enhanced activation of mature DCs (CD80+ & CD86+), activated proliferation of lymphocytes and activated cytotoxic T-lymphocytes (CTLs).
Attenuated pulsed HepG2 cells enhanced T-lymphocytes, natural killer cells (NK) and DCs stimulation (16-18). Likewise, Heat shocked HepG2 cell lysate was used to pulse PBMC derived DCs maturated with TNF-alpha, it was found that DCs mediated cytolysis of HepG2 cells when co-cultured with autologous CD4+ and/or CD8+ cells compared to pulsing DCs with lysate of non-heat shocked tumor cells. Then hyperthermia may play a role in improved cellular immune response (19). A trial used transfect DCs with mRNA of Heat Shock protein-70 and these DCs were injected in HCV-related HCC patients, showed a complete response in most of the patients approved by tumor killing activity of the cytotoxic T-lymphocytes in HCC patients (20).
When mature DCs and activated T-cells has pulsed by allogenic pool of TAAs from different HCC patients, DCs, cytotoxic T-cells and cytotoxicity were activated against HepG2 and Huh7 cell lines (21). However, the nature of these effective antigens was not specified.
Gene transduction in DCs showed better antigen presentation and cytotoxic effect against tumor cells. Zhou et al. (2016) transduced DCs with interferon-” & alpha-feto protein genes (22), Pardee et al. (2015) transduced DCs from HCC patients with genes of alpha-feto protein and IL-18 (25) and others used glypican gene in DCs transduction (23, 24). Wang et al. (2015) have transduced DCs with Glypican-3 gene (25) and finally Ren et al., (2008) have transduced DCs derived from CD34+ cell-enriched peripheral blood mononuclear cells with liposome coated AFP and mutant P53 (mtP53) fused gene (26). Significances of all these studies showed that there were elevations in T-cell markers (CD3+CD4+, CD3+CD8+), elevation in DCs markers (CD80+, CD86+), increase in LDH & IL-12 expressions.
With different stimulation, we obtained similar results in term of LDH and IL-12 levels and adverse results in the immunophenotypic analysis of immune cells by attenuating HepG2 tumor cells with ribonuclease. Ribonuclease attenuation to HepG2 cells may aid in blocking antigen presentation of allogenic antigens.
Xing et al. (2009) used antigen presentation by pulsating DCs & lymphocytes with dead HepG2 cells resulted in increased levels of DCs and CTLs markers (27). Similar results were obtained when used transfected immature DCs (IDCs) with total RNA of HepG2 tumor cells in vitro resulted in differentiation and maturation of DCS, and elicitation of specific T-cell responses. RNA-transfected DCs successfully generated specific T-cells that specifically recognize HCC cells (28, 29). These results were similar to ours in the concept of antigen presentation. However, DCs & lymphocytes response in our study depended on antigen presentation of unattenuated HepG2 cells in term of allogenic antigens and TAAs.
Aref et al. (2014) used four identified TAAs to be pulsed to DCs, stimulated CTLs (CD3+CD8+) and (CD3+CD4+). They found that DCs can recognize more than 100 epitopes in these cancer cells (30).
The only trial that emphasized the role of suppressor mediators in delaying or obstacle the antigen presentation was done by Pardee at al. (2014), where secreted mediator like AFP by tumor cells play a pivotal role in immune-suppressive action (31)Correspondingly to our study, attenuating HepG2 cells by ribonuclease may play a synergistic effect with the suppressive mediators of HepG2 tumor cell line against the allogenic activated immune cells.
Our results revealed significant direct correlations between (TAP-2 vs. CD19+; p<0.05), (TAP-2 vs. CD86+; p<0.001), (CD209+ vs. CD86+; P<0.001), (CD209+ vs. CD19+; P<0.001) and inverse correlation between (CD209+ vs. CD81+; P<0.05) which indicate the process of antigen presentation is realized by only mature DCs and B-lymphocytes (32, 33).
Using ribonuclease enzyme as a tool for attenuating tumor cells showed an effective method in antigen presentation. Although several clinical trials showed significant results in antigen presentation of TAAs, no one has determined the antigenic determinant responsible for tumor cell lysis. We suppose an answer to the previously asked question that, activated immune cells of HCC patients, pulsed by allogenous tumor attenuated cells by ribonuclease, is a safe method and showed a directed antigen presentation against TAAs.