Restoration by IL-15 of MHC Class I Antigen-Processing Machinery in Human Dendritic Cells Inhibited by Tumor-Derived Gangliosides

Cells

Primary oral SCC cell lines PCI-4B and PCI-38 established at the University of Pittsburgh Cancer Institute and described previously (28) were cultured in RPMI 1640 supplemented with 2 mM l-glutamine, 50 μg/ml gentamicin sulfate, 10 mM HEPES, 5% FBS, 10 mM nonessential amino acids, and 1 mM sodium pyruvate, (Invitrogen Life Technologies). This medium is referred to as complete medium (CM).

DC were generated from PBMC of healthy donors. After gradient separation on Histopaque-1077 (Sigma-Aldrich) and lysis of RBC, PBMC were resuspended in CM (5 × 10⁶cells/ml) and incubated for 1 h at 37°C. Nonadherent cells were removed, and adherent monocytes were cultured in CM with 1000 U/ml recombinant human (rh)GM-CSF and 1000 U/ml rhIL-4 (PeproTech). On day 7, nonadherent cells were collected and analyzed. Their morphology and phenotype corresponded to immature DC characterized previously (18, 29). Percentage ranges for various surface markers in DC obtained from 10 donors were 72–99% for HLA-DR, 60–87% for CD86, 16–25% for CD80, 12–30% for CD83, 54–88% for CD40, and 2–20% for CD14.

DC (0.7–1.5 × 10⁶cells/well) were coincubated with 40 μg/ml disialoganglioside GD₃ (tested at a range of concentrations from 20 to 80 μg/ml) (Matreya) reconstituted in DMSO (Sigma-Aldrich) and added once a day for the first 3 days in culture. DC were also incubated with SCC cell lines PCI-4B or PCI-38 (25 × 10⁴cells/well) in CM separated by membrane inserts with 0.4-μm pore size (Transwell system) for the first 3 days in culture. As a control, DC were treated with medium alone, medium with DMSO, or coincubated with immortalized human keratinocytes HET-1A (CRL-2692; American Type Culture Collection) in the same way. To test the ability of IL-15 to restore the integrity of APM in DC in the tumor microenvironment, we added rhIL-15 (50 ng/ml) to DC generated in the presence of SCC cell lines and to control DC once per day for the last three consecutive days in culture.

To block ganglioside synthesis, tumor cells were cultured in the presence of inhibitor of glucosyl transferase d-threo-PPMP (1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol · HCl) (5 μM) (Matreya) once a day for 3 days.

Antibodies

mAbs to individual APM components were generated and characterized according to the methodology we described previously (18). The anti-calnexin mAb TO-5, anti-calreticulin mAb TO-11, anti-ERp57 mAb TO-2, and anti-tapasin mAb TO-3 were developed and characterized as described previously (30). The anti-MB1 mAb SJJ-3, anti-low m.w. protein (LMP2) mAb SY-1, anti-LMP7 mAb SY-3, and anti-LMP10 mAb TO-7 were developed and characterized using the strategy described elsewhere (30, 31). Briefly, the mAb-secreting hybridomas were derived from BALB/c mice immunized with synthetic peptides derived from the amino acid sequence of the native protein and with recombinant proteins. Abs of the desired specificity were identified by their specific binding to the immunizing peptides in ELISA. The specificity of the selected mAb was proven by their reactivity with the corresponding Ags when tested with lymphoid cell lysates with the appropriate phenotype in Western blotting. The specificity of anti-LMP2 mAb SY-1 was corroborated further by their lack of reactivity with a lysate of the T2 cell line, which does not express these molecules. For additional controls, rabbit anti-LMP2 and anti-LMP7 polyclonal Ab were purchased from Affinity Research Products. mAb W6/32 was produced by Dr. A. DeLeo (University of Pittsburgh, Pittsburgh, PA) using a hybridoma obtained from American Type Culture Collection. Labeled mAbs for staining of surface Ags on DC (anti-HLA-DR, -HLA-ABC, -CD80, -CD83, -CD86, -CD40, and -CD14) by flow cytometry were purchased from BD Biosciences Pharmingen. FITC-conjugated goat anti-mouse and goat anti-rabbit IgG Ab were purchased from The Jackson Laboratory. Anti-human disialoganglioside GD3 Ab were purchased from BD Biosciences Pharmingen.

Small interfering RNAs (siRNA) and nucleofection

siRNA for LMP10 and MB1 were purchased from Dharmacon. Nucleofection efficiencies were determined with 5 μg of plasmid DNA encoding the GFP using Human Dendritic Cell Nucleofector Kit (Amaxa) and varied between 40 and 60%. Nucleofection was conducted according to the manufacturer-optimized protocol for human DC (Amaxa). Briefly, 6-day-old DC were centrifuged, and 2 × 10⁶ cells were resuspended in Dendritic Cell Nucleofection Solution (Amaxa). Next, the cells were mixed with 5 μg of LMP10 or MB1 siRNA, transferred to the special cuvette, and inserted into the Nucleofector (program U-02). All of the samples were removed from the cuvette immediately after the program had been finished, transferred into the 6-well plates, and cultured in CM (0.25 × 10⁶ cells/ml) with 1000 U/ml rhGM-CSF and 1000 U/ml rhIL-4 for 12–18 h at 37°C.

Western blot

The protein expression of APM components in nucleofected and control DC was assessed using a Western blot technique. Cells were collected, washed in HBSS, and lysed in 100 μl of extraction buffer (1% Triton X-100, 0.1% SDS, 50 mM Tris, 150 mM NaCl, 0.6 mM PMSF, and 5 μg/ml leupeptin) for 30 min on ice. After centrifugation (9000 × g, 15 min, at 4°C), the protein concentration was determined in the supernatants by the Bradford method using the BioRad protein kit (Bio-Rad). Fifty micrograms of total proteins were dissolved in electrophoresis sample buffer, separated by 4–12% PAGE (NuPAGE), and transferred to a nitrocellulose membrane (NOVEX). The membrane was blocked with 0.5% nonfat milk, 0.1% Tween 20 (Fisher Scientific) in 20 mM Tris-HCl buffer (pH 7.2). LMP10 and MB1 were detected using the anti-LMP10 mAb TO-7 and anti-MB1 mAb SJJ-3 in a final concentration of 100 μg/ml and goat anti-mouse secondary mAb (IgG (H+L), 1/150,000 dilution; Pierce). Expression of β-actin was evaluated as a housekeeper control protein using mouse anti-human mAb (IgG1, 1/100,000 dilution; Sigma-Aldrich). Secondary Abs were the same as for APM components detection. The membrane was processed and treated with chemiluminescent reagents (Pierce), and the bands were visualized on a Kodak film (Eastman Kodak).

OVA-specific presentation by DC

To evaluate the capacity of DC to present Ags, 6-day-old DC, which had been incubated for the first 3 days with tumor cells, and control DC incubated with immortalized keratinocytes were pulsed overnight with OVA (Sigma-Aldrich) (500 μg/ml). Next, DC were washed twice and cultured for 96 h at 37°C, 5% CO₂ in 96-well plates in CM with autologous OVA-specific CD8⁺ T cells (1 × 10⁴ cells/well) at different DC:T ratios (1:100 to 1:1). At the end of incubation, T cell proliferation was measured by uptake of [³H]thymidine (1 μCi/well, 5 Ci/mmol; PerkinElmer Life Science) added for the last 16–18 h. Cultures were harvested on GF/C glass fiber filter paper (Whatman) using a MACH III microwell harvester (Tomtec). [³H]Thymidine incorporation was determined in a MicroBeta TriLux liquid scintillation counter (Wallac). Counts are expressed as cpm and presented as stimulation index (SI). SI was calculated as stimulated cpm divided by spontaneous cpm.

To generate autologous OVA-specific T cells, CD8⁺ T cells were isolated from PBMC using MACS paramagnetic purification system (Miltenyi Biotec) and cultured with weekly addition of irradiated autologous PBMC (1:1), OVA (Sigma-Aldrich; 500 μg/ml), and IL-2 (10 IU/ml) for 4 wk.

Flow cytometry analysis of DC and SCC cells

Intracellular staining for detection of APM protein expression in DC was performed with modifications, as described earlier (30). Briefly, DC were washed three times in PBS/1% BSA, fixed with 2% paraformaldehyde (PFA) at room temperature (RT) for 20 min, washed three times in PBS/1% BSA, resuspended in 10 ml of PBS containing 0.5% BSA, transferred to glass flasks, and treated in a microwave. Cells were then immediately chilled on ice for 10 min, washed three times in PBS-1% BSA, permeabilized at RT for 30 min with 100 μl/sample of PBS containing 1% BSA and 0.1% saponin (Sigma-Aldrich), and stained with an optimal amount of primary mAb at RT for the next 30 min. Murine IgG were used as a control. Cells were then washed three times with PBS containing 1% BSA and 0.1% saponin, and incubated with FITC-conjugated goat anti-mouse IgG Ab (1:100) (Jackson ImmunoResearch Laboratories) at RT for 30 min. Cells were washed three times again and fixed with 0.5% PFA.

For evaluation of ganglioside expression, tumor cells (PCI-4B and PCI-38) were trypsinized, washed in FACS medium (PBS containing 0.1% BSA and 0.1% NaN₃), fixed in 1% PFA (15 min on ice), followed by washing with FACS medium containing 0.1% saponin and staining with murine anti-human disialoganglioside GD3 Ab (BD Biosciences Pharmingen) for 30 min at 4°C. Murine IgG was used as a control. Next, tumor cells were washed with FACS medium containing 0.1% saponin and incubated with FITC-conjugated goat anti-mouse IgG Ab (1:100) (Jackson ImmunoResearch Laboratories) for 30 min at 4°C. Finally, cells were washed in FACS medium and fixed with 0.5% PFA.

Fluorescence was measured using a FACScan flow cytometer (BD Biosciences), and the data analysis was performed using the CellQuest Software (BD Biosciences). Results are expressed as percentage of positive cells and as mean fluorescence intensity (MFI).

HPLC for gangliosides in PCI-4B cells

To confirm ganglioside expression in SCC cells, total lipid extraction of tumor cells (10⁶ cells) was performed using chloroform:methanol, and gangliosides were isolated from the lipids by a diisopropyl ether/1-butanol partition and subsequent Sephadex G-25 gel filtration step (32). The water-soluble gangliosides were further fractionated by HPLC using a LiChrosorb-NH2 column (Merck). Gangliosides were eluted using a linear flow rate of 0.5 ml/min throughout, where solvent A was Acetonitrile-5 mM phosphate buffer, pH5.6 (83:17) and solvent B was Acetonitrile-20 mM phosphate buffer, pH5.6 (50:50). Individual bovine brain-derived gangliosides were also subjected to HPLC analysis to establish the retention times of known gangliosides (GM2, GM1, GD3, GD1a, GD1b, and GT1b).

Statistical analysis

The differences between groups were determined using the Student’s t test or the nonparametric Mann-Whitney U test after evaluation for normality. For multiple group comparison, ANOVA was used. For all statistical analyses, a p value of 0.05 was considered significant. Data are presented as mean ± SEM. All of the experiments were repeated at least three times.

Down-regulation by oral SCC cells and restoration by IL-15 of APM component expression in DC

Previously, we observed that the expression of some APM components was down-regulated in DC incubated with SCC cells. To confirm these findings, we evaluated the expression of MB1 (β5), LMP2, LMP7, LMP10, calnexin, calreticulin, ERp57, and tapasin in DC, which had been coincubated with SCC cells in a Transwell system, intracytoplasmatically stained with APM component-specific mAb, and analyzed by flow cytometry. As a control, DC were treated with medium alone or coincubated with immortalized keratinocytes. In 10 independent experiments with DC from 10 donors, expression of MB1, LMP2, LMP7, LMP10, and ERp57 was significantly and reproducibly decreased in DC generated in the presence of the PCI-4B or PCI-38 cell lines (Fig. 1⇓). For instance, the tumor-induced inhibition of APM components expression in DC was 44.8 ± 9.6% for MB1, 39.4 ± 8.7% for LMP2, 43.3 ± 6.2% for LMP7, 36.6 ± 6.7% for LMP10, and 39.8 ± 9.8% for ERp57. Similar results were obtained by analyzing the corresponding MFI values (Table I⇓). In contrast, expression of the calnexin, calreticulin, and tapasin was not significantly altered in the tumor cell-treated human DC.

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FIGURE 1.

HLA class I APM component down-regulation in human DC coincubated with oral SCC cell lines in vitro. DC precursors were cocultured with SCC cell lines PCI-4B or PCI-38 in a Transwell system for 72 h. Tumor-treated and control (keratinocyte-treated) DC were intracytoplasmatically stained with APM component-specific mAb to MB1, LMP2, LMP7, LMP10, ERp57, tapasin, calnexin, and calreticulin, and analyzed by flow cytometry as described in Materials and Methods. Expression of MB1, LMP2, LMP7, LMP10, and ERp57 was significantly down-regulated in SCC-treated DC (p < 0.05). Data represent the percentage of positive cells from 10 independent donors.

Because IL-15 has been shown to promote DC activation (25) and increased DC function and survival in the tumor microenvironment (33), we hypothesized that IL-15 might restore the integrity of APM in DC generated in the presence of tumor cells. To test this possibility, we added rhIL-15 (50 ng/ml) to DC generated in the presence of SCC cell lines and to control DC once per day for the last 3 consecutive days in cultures and measured the level of APM component expression in tumor cell-treated DC. Treatment with IL-15 restored expression of MB1, LMP2, LMP7, LMP10, and ERp57 in tumor cell-treated DC in the range from 53 to 98%, depending on the donor (Table I⇑). These results demonstrate that not all APM components but only specific components of the constitutive proteasome and immunoproteasome are selectively decreased in expression in DC coincubated with tumor cells. Restoration by IL-15 of APM component expression suggests the ability of this cytokine to regulate APM in DC.

Depletion of APM component mRNA in DC correlates with significant reduction of APM protein expression in DC and with reduced expression of surface MHC class I molecules

To prove that anti-APM component Abs specifically detect up- or down-regulation of Ag-processing pathway components in DC, using commercially available siRNA for LMP10 and MB1, we assessed expression of LMP10 and MB1 in DC nucleofected with specific APM component siRNA, control RNA encoding GFP and empty vector. Our results revealed that the level of DC transfection using DC protocol developed by the manufacturer (Amaxa) was between 40 and 60% based on GFP expression assessed by flow cytometry. Next, we have demonstrated that depletion of LMP10 mRNA in DC correlates with significant reduction of LMP10 protein expression in the same cells, assessed by Western blot with specific anti-APM Abs (Fig. 2⇓A). Importantly, down-regulation of LMP10 protein in DC induced by siRNA was associated with reduced expression of surface MHC class I molecules (Fig. 2⇓B). Similar results were obtained for DC nucleofected with MB1 siRNA.

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FIGURE 2.

Down-regulation of expression of APM components in DC nucleofected with siRNA correlates with reduction of the corresponding protein expression in DC and reduced expression of surface MHC class I molecules. The transfection efficiencies of DC nucleofection ranged between 40 and 60% based on GFP expression. A, The protein expression of APM components in DC nucleofected with LMP10 siRNA, control RNA encoding GFP, and empty vector was assessed using Western blot technique as described in Materials and Methods. B, LMP10 protein expression and expression of surface MHC class I molecules in nontreated DC, control transfected DC, and DC nucleofected with LMP10 siRNA was determined by flow cytometry as described in Fig. 1 legend. Results are expressed as MFI. The results of one representative of three independent experiments are shown.

Restoration by IL-15 of suppressed OVA-specific Ag presentation by DC generated in the presence of SCC cells

To test whether APM component down-regulation in DC generated in the presence of tumor cells has functional consequences of poor Ag presentation, we evaluated the ability of DC to present OVA to OVA-specific autologous CD8⁺ T cells. Six-day-old tumor cell-treated and control DC were pulsed with OVA (500 μg/ml) overnight, washed, and coincubated with OVA-specific autologous CD8⁺ T cells, and T cell proliferation was measured by uptake of [³H]thymidine. The ability of DC to induce proliferation of CD8⁺ T cells was significantly decreased when DC had been coincubated with PCI-4B cells, in comparison to DC coincubated with keratinocytes or in medium alone (Fig. 3⇓). For instance, at the E:T ratio 1:10, [³H]thymidine uptake was reduced by 76 ± 12% (p < 0.05) and at the ratio 1:1 × 60 ± 8% (p < 0.05). The ability of DC to induce proliferation of autologous CD8⁺ T cells was HLA class I restricted, because it was inhibited by the addition of anti-HLA class I mAb W6/32. Thus, coincubation of DC with SCC cells impairs DC ability to present OVA-derived peptides to autologous OVA-specific CD8⁺ T cells. Because it has been shown that DC generated in the presence of GM-CSF plus IL-15 prime potent CD8⁺ Tc1 responses in vivo (27), and because of the ability of IL-15 to reverse tumor cell-induced down-regulation of APM component expression in DC, we hypothesized that IL-15 might also restore DC Ag-presenting function. To test this possibility, IL-15 (50 ng/ml) was added to DC generated in the presence of SCC cell lines and to control DC once per day for the last 3 consecutive days in culture, and the ability of DC to present OVA Ag to OVA-specific autologous CD8⁺ T cells was measured. The addition of IL-15 caused a complete recovery of suppressed DC Ag-presenting activity (Fig. 3⇓). These results suggest that IL-15 is able to restore the SCC cell-induced inhibition of APM component expression and Ag-presenting function of DC.

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FIGURE 3.

IL-15 restores DC Ag-presenting capacity suppressed by oral SCC cells. DC were cocultured with SCC cell line PCI-4B (▾) or keratinocytes (▪) in Transwell system for 72 h. Then rhIL-15 (50 ng/ml) was added to DC generated in the presence of SCC cell lines () and control DC (▦) once per day for the last 3 consecutive days in culture. Tumor-treated and keratinocyte-treated 6-day-old DC were loaded with OVA overnight, then washed and cultured in 96-well plates in CM with autologous OVA-specific CD8⁺ T cells (1 × 10⁴ cells/well) for 96 h. Proliferation of T cells was measured by uptake of [³H]thymidine added for the last 16–18 h. Inhibition of the proliferation with anti-HLA class I W6/32 mAb indicates that DC-induced activation of CD8⁺ T cells was HLA class I restricted. The counts are presented as SI. Similar results were obtained with PCI-38 oral SCC cell line. Data represent the mean ± SEM of triplicate measurements from three independent experiments. ∗, p < 0.05.

Down-regulation by gangliosides and restoration by IL-15 of APM component expression in DC

It has been previously shown that gangliosides down-regulate maturation and function of APC (13, 14, 15, 16, 17). To test whether gangliosides are responsible for modifying APM component expression in DC, disialoganglioside GD₃ (40 μg/ml) was added once daily for the first 3 days in culture. As a control, DC were treated with DMSO, used as a solvent for gangliosides. Synthetic gangliosides significantly (p < 0.05) suppressed MB1, LMP2, LMP7, LMP10, and ERp57 expression in DC (Table II⇓). For instance, MB1 was down-regulated by 37 ± 7%, LMP2 × 53 ± 10%, LMP7 × 60 ± 14%, LMP10 × 41 ± 8%, and ERp57 × 21 ± 6% in DC treated with gangliosides vs control DC. Other tested APM components were not significantly inhibited. Furthermore, the treatment of DC with IL-15 after coincubation with gangliosides reversed the inhibitory effect of gangliosides on MB1, LMP2, LMP7, LMP10, and ERp57 expression in the range from 40 to 100%, depending on the donor and specific APM components (Table II⇓). Because synthetic gangliosides down-regulated similar APM components in DC as the SCC cell lines, and because IL-15 can restore these components expression in DC coincubated both with tumor cells or gangliosides, we hypothesized that tumor cell-induced APM component down-regulation in DC could be due to tumor cell-derived gangliosides.

GD₃ ganglioside expression by SCC cell lines PCI-4B and PCI-38

To test the hypothesis that tumor cell-induced APM component down-regulation in DC may be mediated by tumor cell-derived gangliosides, we first measured GD₃ ganglioside expression in the SCC cell lines PCI-4B and PCI-38. Neuroblastoma cell lines were used as a positive control (14). Flow cytometry and HPLC analysis showed that both cell lines express GD₃ ganglioside (Figs. 4⇓ and 5⇓).

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FIGURE 4.

GD₃ ganglioside expression on oral SCC cell lines. FACScan analysis was performed on PCI-38 and PCI-4B cells, using staining with primary purified mouse anti-human disialoganglioside GD₃ Ab and secondary (FITC)-conjugated goat anti-mouse IgG Ab. No differences between both cell lines were detected. The results of one representative of three independent experiments are shown.

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FIGURE 5.

HPLC detection of ganglioside expression in PCI-4B cells. A total lipid extraction of tumor cells was performed using chloroform:methanol, and gangliosides were isolated from the lipids by a diisopropyl ether/1-butanol partition and subsequent Sephadex G-25 gel filtration step. The water-soluble gangliosides were further fractionated by HPLC using a LiChrosorb-NH2 column. Gangliosides were eluted using a linear flow rate of 0.5 ml/min throughout, where solvent A was Acetonitrile-5 mM phosphate buffer, pH 5.6 (83:17) and solvent B was Acetonitrile-20 mM phosphate buffer, pH5.6 (50:50). Individual bovine brain-derived gangliosides were also subjected to HPLC analysis to establish the retention times of known gangliosides (GM2, GM1, GD3, GD1a, GD1b, and GT1b).

Role of tumor cell-derived gangliosides in APM component down-regulation in DC

To prove that tumor cell-derived gangliosides are indeed responsible for APM component down-regulation in DC coincubated with SCC cells, we blocked synthesis of gangliosides in tumor cells using enzymatic inhibition. Tumor cells were pretreated with an inhibitor of glucosyl transferase d-threo-PPMP for 3 days to block glycosphingolipid synthesis, including ganglioside synthesis (14). APM component down-regulation in DC coincubated with SCC cells pretreated with PPMP was significantly (p < 0.05) less than down-regulation in DC coincubated with nontreated SCC cells (Fig. 6⇓). Similar data were obtained for DC coincubated with both SCC cell lines PCI-4B and PCI-38. Thus, inhibition of ganglioside synthesis in tumor cells significantly decreased their inhibitory effect on APM component expression in DC, suggesting that the observed down-regulation of APM components is, at least in part, mediated by tumor cell-derived gangliosides.

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FIGURE 6.

Suppression of ganglioside synthesis in oral SCC cells significantly abolished tumor-induced inhibition of APM component expression in DC. SCC cell line PCI-4B was treated with PPMP for 3 days to block ganglioside synthesis. DC were coincubated with PPMP-treated (▦) and nontreated (▪) SCC cells in a Transwell system for 72 h. APM component expression in DC coincubated with treated and nontreated SCC cells was determined by flow cytometry as described in Fig. 1 legend. The results are presented as the percentage of inhibition of APM component expression in DC by PCI-4B cells. Similar results were obtained with PCI-38 cell line. Data represent the mean ± SEM from three independent experiments. ∗, p < 0.01.