Enhancement of specific T-lymphocyte responses by monocyte-derived dendritic cells pulsed with E2 protein of human papillomavirus 16 and human p16INK4A

Preparation and purification of HPV16 E2 and p16INK4a proteins using Escherichia coli BL21 system

The vectors pTrcHisA-HPV16 E2 and pTrcHisA-p16INK4a were transformed using heat shock into competent Escherichia coli BL21 cells and the resulting cells were used for protein expression (Servinsky et al., 2016; Inoue, Nojima & Okayama, 1990). The bacteria were cultivated for 24 h in Luria-Bertani (LB) broth containing 100 μg/ul of ampicillin at 37 °C. Expression of p16INK4a and HPV16 E2 proteins was induced by adding isopropyl thiogalactoside (Thermo Scientific, Waltham, MA, US). Proteins were extracted using B-PERR Bacteria Protein Extraction Reagent (Thermo Scientific, Waltham, MA, US). The desired proteins were purified by using HisPur Cobalt Purification Kit (Thermo Scientific, Waltham, MA, US) and removed the endotoxin by using Pierce High Capacity Endotoxin Removal Resin (Thermo Scientific, Waltham, MA, US). Protein concentration was measured using Bradford’s method (Kruger, 1994). The identities of the purified proteins were confirmed by SDS-PAGE and western blot analysis using anti-histidine antibody, anti-HPV16 E2 antibody, and anti-p16INK4A antibody (Li et al., 2017).

Immunogenic epitope prediction of HPV16 E2 and p16INK4a proteins for HLA class I and II binding

The common HLA class I and II molecules in the Thai population were identified at http://www.allelefrequencies.net/. Possible binding epitopes of HLA class I and II for HPV16 E2 and p16INK4a proteins were predicted using two databases, SYFPEITHI and NetMHCpan version 4.0 (Nielsen et al., 2010; Nielsen & Andreatta, 2016). The prediction was performed using 9-meric amino-acid residues for HLA class I and 15-meric amino-acid residues for HLA class II epitopes, respectively.

Isolation of CD14⁺ peripheral blood mononuclear cell and cultivation of MDCs

Peripheral blood was collected from five healthy volunteers who had given written consent. The study was approved by the Human Ethics Research Committee of Khon Kaen University (No. HE611307). The peripheral blood mononuclear cell (PBMC) were separated by centrifugation with Lymphoprep (Stemcell technologies, Oslo, Norway, Europe) (Müller et al., 1993). CD14⁺ monocytes were isolated from PBMC with CD14 magnetic particles to which an anti-human CD14 monoclonal antibody was conjugated and using the IMag separator (BD Biosciences, San Jose, CA, USA). The 95% purity of CD14⁺ monocytes was confirmed using flow cytometry. The CD14⁺ monocytes of each volunteer were separately cultured in RPMI-1640 medium (Gibco, Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher, Waltham, MA, USA), 100 U/ml recombinant human GM-CSF (Peprotech, Rocky Hill, NJ, USA), 50 U/ml recombinant human IL-4 (Peprotech, Rocky Hill, NJ, USA), 100 U/mL penicillin and 100 μg/mL streptomycin for 5–6 days (Murray et al., 2015; Steevels & Meyaard, 2011; Xiao et al., 2018). This cultivation method enhanced the differentiation of monocytes into immature MDCs. The CD14⁻ cells remaining after CD14⁺ isolation were cultured in RPMI-1640 supplemented with 5% FBS, 5% autologous serum, recombinant human IL-2 (20 IU/mL) (Peprotech, Rocky Hill, NJ, USA), 100 U/mL penicillin and 100 μg/mL streptomycin for T-lymphocyte proliferation (Xiao et al., 2018).

HPV16 E2 and p16INK4a proteins stimulate immature MDCs

The immature MDCs were seeded into 96-well plates and stimulated to become mature MDCs by using HPV16 E2 or p16INK4a at various concentrations; 0, 50, 100, 150, 250 and 500 ng/mL (added on day 5 and incubated for 48 h). The stimulated MDCs were harvested, washed and stained with FITC-HLA-DR, PE-CD80, APC-CD86 and PE-Cy7-CD83-tagged monoclonal antibodies (BD Pharmingen, San Jose, CA, USA) and analyzed using flow cytometry (FACScanto II, BD Biosciences, San Jose, CA, USA) (Xiao et al., 2018).

Evaluation the role of MAPK pathways on maturation and activation of MDCs

The role of MAPK pathways in the maturation of MDCs was determined by adding three specific kinase inhibitors into immature MDCs at day 5. The specific kinase inhibitors were p38 MAPK inhibitor (SB203580, 50 μM), ERK inhibitor (PD98059, 50 μM), and JNK inhibitor (SP600125, 20 μM). A total of 2 h after addition of these inhibitors, HPV16 E2 and/or p16INK4a were added and culture was continued for a further 48 h. The cells were collected and examined for CD83 expression by flow cytometry. The secretion of IL-12 and IL-1β was determined by ELISA (Peprotech, Rocky Hill, NJ, USA) (Xiao et al., 2018).

Specific protein-pulsed MDCs stimulate autologous T-lymphocyte proliferation and IFN-γ secretion

Immature MDCs were seeded into 96-well U-bottom plates and cultured in RPMI-1640 containing 10% FBS, 100 U/ml rhGM-CSF, 50 U/ml rhIL-4, 100 U/mL penicillin, and 100 μg/mL streptomycin. The immature MDCs were further stimulated with HPV16 E2, p16INK4a, or HPV16 E2/p16INK4a at the determined optimal concentration for 24 h. The protein-pulsed MDCs were washed and co-cultured with autologous T lymphocytes in the ratio of protein-pulsed MDCs:T cells at 1:1, 1:5 and 1:10. The supernatants were harvested after 24 h of co-culture for determination of IFN-γ secretion by ELISA. The cells were harvested for staining with FITC-CD3, APC-CD4, and PerCP-Cy5.5-CD8-tagged monoclonal antibodies (BD Pharmingen, San Jose, CA, USA) and intracellular IFN-γ monoclonal antibodies (BD Pharmingen, San Jose, CA, USA) and detected using flow cytometry (Xiao et al., 2018).

Evaluation of HLA blockade on stimulation of T lymphocytes by protein-pulsed MDCs

Immature MDCs were seeded into 96-well U-bottom plates and cultured in RPMI-1640 containing 10% FBS, 100 U/ml rhGM-CSF, 50 U/ml rhIL-4, 100 U/mL penicillin and 100 μg/mL streptomycin. The immature MDCs were further stimulated with HPV16 E2, p16INK4a, or HPV16 E2/p16INK4a at the determined optimal concentration for 24 h. Then, protein-pulsed MDCs were washed and blockade antibodies added. These were mouse anti-human HLA-ABC monoclonal antibody (eBioscience, San Diego, CA, USA) and mouse anti-human HLA-DR, DP, DQ monoclonal antibodies (BD Pharmingen, San Jose, CA, USA) for 2 h (Rahman et al., 2017). The protein-pulsed MDCs were washed and co-cultured with autologous T lymphocytes in the ratio of protein-pulsed MDCs:T cells at 1:1, 1:5 and 1:10. The supernatants were harvested after 24 h of co-culture for determination of IFN-γ secretion by ELISA. The cells were harvested for staining with FITC-CD3, APC-CD4 and PerCP-Cy5.5-CD8-tagged monoclonal antibodies (BD Pharmingen, San Jose, CA, USA) and intracellular IFN-γ monoclonal antibodies (BD Pharmingen, San Jose, CA, USA) and detected using flow cytometry (Xiao et al., 2018; Rahman et al., 2017).

Statistical analysis

Experiments were performed independently three times. The data were analyzed using analysis of variance and differences among the experimental groups tested using Tukey’s multiple-comparison test implemented in Prism version 5. The results are shown as the mean and standard deviation (SD). The symbols * and # indicate statistical significance, at p < 0.05, in comparisons with controls or between groups, respectively.

Frequency of common HLA class I and II alleles in the Thai population and prediction of the immunogenic epitopes of HPV16 E2 and p16INK4a proteins

Online database information about common HLA class I and class II alleles in Thailand was based on data from 16,807 people. The three most common HLA class I alleles were HLA-A*02, HLA-A*11 and HLA-A*24 with frequencies of 29.2, 27.7 and 17.3%, respectively. The three most common HLA class II alleles were HLA-DRB1*15, HLA-DRB1*12 and HLA-DRB1*04 with frequencies of 17.5, 16.9 and 14.4%, respectively (Table 1).

The prediction of HLA class I and class II epitopes for HPV16 E2 and p16INK4a proteins was performed using two databases, the 9-meric amino-acid residues and 15-meric amino-acid residues were predicted for HLA class I and class II alleles, respectively. The HPV16 E2 protein (365 amino-acid residues) has immunogenic epitopes for both types of HLA. Fig. 1A shows common immunogenic epitopes of HPV16 E2 protein for HLA class I alleles including HLA-A*02:01, HLA-A*11:01, HLA-A*24:02, HLA-B*15:01 and HLA-B*40:01, and for HLA class II alleles including

HLA-DRBI*04:01 and HLA-DRBI*15:01. Similarly, the p16INK4a protein (156 amino-acid residues) also presents immunogenic epitopes for both types of HLA including HLA-A*02:01, HLA-A*11:01, HLA-A*24:02, HLA-B*15:01, HLA-B*40:01, HLA-DRBI*04:01 and HLA-DRBI*15:01 (Fig. 1B). This suggests that these two proteins can be presented by antigen-presenting cells via HLA class I and class II molecules.

Production of HPV16 E2 and p16INK4a protein in Escherichia coli BL21

HPV16 E2 and p16INK4a proteins expressed in E. coli BL21 were purified and eluted twice. Identities of the proteins were confirmed by western blot using specific antibodies for polyhistidine and HPV16 E2, respectively. We found the purified p16INK4a protein with a polyhistidine tag was of the expected size, 18 kDa (Fig. 1C). The purified HPV16 E2-polyhistidine tagged protein was 43 kDa (Fig. 1D). Therefore, these proteins were further used for stimulating of MDCs and T lymphocytes.

HPV16 E2 and p16INK4a proteins induced phenotypic maturation of MDCs

We determined the optimum concentrations of HPV16 E2 and p16INK4a proteins for stimulating the maturation of MDCs. Evaluation of the expression level of CD83, which is a marker of mature DCs, was done after stimulating CD14⁺ monocytes with HPV16 E2 and/or p16INK4a at various concentrations; 0, 50 100, 150, 250 and 500 ng/mL (treated on day 5 and cultured for 48 h). We observed that CD14⁺ monocytes cultured with rhGM-CSF and rhIL-4 can be induced to develop to immature monocyte-derived DC (Fig. 2B) when compared to negative control (CD14⁺ monocytes without rhGM-CSF and rhIL-4) (Fig. 2A). The immature DCs were stimulated with HPV16 E2 and p16INK4a proteins resulting in their differentiation into mature DCs with more cytoplasmic protrusions (Figs. 2C and 2D). However, their phenotypic were not significantly observed.

Both the percentage of positive cells and mean fluorescence intensity (MFI) of CD83⁺ MDCs increased in a dose-dependent manner after stimulation with HPV16 E2 protein to peak at a concentration of 250 ng/mL (Figs. 2C and 2D). For p16INK4a, percentage of positive cells and MFI of CD83⁺ MDCs peaked at 150 ng/mL (Figs. 2E and 2F). Therefore, the optimum concentration of HPV16 E2 and p16INK4a proteins for further experiments were 250 ng/mL and 150 ng/mL, respectively (Figs. 2I and 2J).

HPV16 E2 and p16INK4a proteins stimulate the maturation and activation of MDCs via MAPK pathway

To confirm whether HPV16 E2 and p16INK4a proteins stimulated immature MDCs via the MAPK pathway, immature MDCs were treated with p38 MAPK, ERK and JNK inhibitors in separate experiments prior to stimulation with the determined optimum concentrations of HPV16 E2 and/or p16INK4a. We found that the inhibitors significantly decreased the percentage of CD83⁺ MDCs in cultures stimulated by HPV16 E2 or p16INK4a proteins (Figs. 3A and 3B). Secretion levels of IL-1β did not significantly differ between all treatment groups (Fig. 3C). However, secretion of IL-12 was significantly lower in experiments where MAPK inhibitors were administered in conjunction with p16INK4a relative to p16INK4a alone (Fig. 3D). In contrast, IL-12 levels were not suppressed when MAPK inhibitors were administered in conjunction with HPV16E2 or HPV16 E2 together with p16INK4a (Fig. 3D). These findings suggest that the activation of MDCs by HPV16 E2 and/or p16INK4a proteins is via the MAPK pathway.

MDCs pulsed with HPV16 E2 and p16INK4a stimulate CD4⁺ and CD8⁺ T lymphocytes to produce IFN-γ

To determine the capability of MDCs to process immunogenic epitopes, the populations of CD4⁺ and CD8⁺ T lymphocytes were examined. MDCs pulsed with HPV16 E2 and/or p16INK4a were co-cultured with IL-2-cultured T lymphocytes in ratios of 1:1, 1:5 and 1:10. Percentages of CD4⁺ and CD8⁺ T lymphocytes were not significantly different in all ratios (Figs. 4A and 4B). Interestingly, the percentage of CD4⁺ IFN-γ⁺ T lymphocytes dramatically increased ratio-wise after co-culture with MDCs pulsed with HPV16 E2 and/or p16INK4a (Fig. 4C). The percentage of CD8⁺ IFN-γ⁺ T lymphocytes did not significantly differ between treatments at ratios of 1:5 and 1:10. However, at the ratio of 1:1, the percentage of these lymphocytes was significantly greater when MDCs were pulsed with HPV16 E2 or p16INK4a relative to the combination of HPV16 E2 and p16INK4a (Fig. 4D). This indicates that HPV16 E2 might exhibit higher immunogenicity than the combination of HPV16 E2 and p16INK4a. The activation of these proteins can be triggered via CD4⁺ or CD8⁺ lymphocytes. We further confirmed the secretion of IFN-γ using ELISA. Production of IFN-γ was higher in co-cultures where MDCs had been pulsed with HPV16 E2 rather than p16INK4a, and varied according to the ratio. The combination of HPV16 E2 and p16INK4a significantly increased the production of IFN-γ at ratios of 1:5 and 1:10, while HPV16 E2 alone strongly induces the production of IFN-γ in a ratio-dependent manner (Fig. 4E). This suggests that the activation of T lymphocytes by HPV16 E2 and p16INK4a for IFN-γ production might be processed by CD4⁺ and CD8⁺ T lymphocytes.

CD4⁺ and CD8⁺ T lymphocytes co-response to combination of HPV16 E2 and p16INK4a pulsed MDCs

To determine the effectiveness of HPV16 E2 and/or p16INK4a in stimulation of CD4⁺ or CD8⁺ T lymphocytes, HLA blockade antibodies were specifically applied in co-culture conditions at a ratio of 1:5. We found inhibition of IFN-γ production in both HLA class I and II blockade in co-culture with MDCs pulsed with a combination of HPV16 E2 and p16INK4a (Fig. 4F). This effect was not apparent when the MDCs were pulsed with either HPV16 E2 alone or p16INK4a alone (Fig. 4F). Suggesting that the stimulation with combination of HPV16 E2 and p16INK4a clearly enhances response of both CD4⁺ and CD8⁺ T lymphocytes.