Introduction

The transporter associated with antigen processing (TAP) is a key factor in the HLA class I antigen presentation pathway. TAP translocates peptides derived mainly from proteasomal degradation from the cytosol into the lumen of the endoplasmic reticulum (ER), where these peptides are loaded onto HLA class I molecules. Peptides–HLA class I complex is transported to the cell surface to present the antigen to the cytotoxic T lymphocytes (CTL). In case of malignant transformation or viral infection of the cells, an additional set of peptides is presented to CTL, leading to an efficient elimination of the infected or malignant cells. TAP is a heterodimeric transmembrane protein that comprises the homologous subunits TAP1 and TAP2 (Kelly et al., 1992). Both subunits are essential and sufficient for peptide transport (Powis et al., 1991; Spies and DeMars, 1991; Meyer et al., 1994).

In many cancers, including malignant melanoma (MM), immune escape has been associated with ineffective antigen processing and presentation of tumor-specific peptides because of low to nondetectable levels of TAP1 and/or TAP2 (Kageshita et al., 1999; Kamarashev et al., 2001; Cresswell et al., 2001). Accordingly, suppression or loss of TAP function in tumors generally results in failure of recognition and elimination by epitope-specific effector CTLs (Chen et al., 1996; Alimonti et al., 2000; Evans et al., 2001). Recently, it was observed that restoration of TAP1 expression by transfection resurrects the processing and presentation of viral antigens and the melanoma-associated antigen. Immunization with B16F10 (murine MM cell line)/rTAP1-transfected cells generates CTLs that are capable of killing B16F10/rTAP1-transfected targets and B16F10 targets deficient in TAP1 (Zhang et al., 2003). However, it is still unclear whether the reduction of TAP1 and/or TAP2 resulting in the deficiency of HLA class I expression exists in human MM cell lines and whether the restoration of TAP expression in human MM cell lines enhances MM immune response. Therefore, in our study, we investigate the expression of TAP1 and TAP2 in human MM cell lines, compare their expression difference in MM cell line, A375 cells transfected with TAP1, TAP2, and TAP1+TAP2 DNA, and examine the effect of the restoration of TAP1 and TAP2 expression on HLA class I antigen surface expression in A375 cells and function of tumor antigen-specific CTLs.

Results

Downregulation of TAP1 and TAP2 in human melanoma cell lines

To verify the hypothesis that TAP contributes to deficiency of HLA class I expression exhibited by human MM cell lines, mRNA and protein expression analyses were performed. As shown in Figure 1a, reverse transcription-PCR analysis revealed that the expression levels of both TAP1 and TAP2 were much more reduced in human MM cell lines when compared to the positive control, melanocytes. Only traces of TAP1 and TAP2 could be detected in the MM cell line A375. As shown in Figure 1b, the reduction of TAP1 and TAP2 protein was demonstrated in three MM cell lines examined by western blotting, and HLA class I expression was reduced along with the decrease of TAP1 and TAP2 in the MM cell lines, especially A375 cells, the least expressions of TAP and HLA class I among MM cell lines examined by FACS in Figure 1c.

Figure 1.

Deficiency of surface HLA class I expression in melanoma cell lines was due to the TAP1 and TAP2 downregulation. (a) Expressions of TAP1 and TAP2 mRNA in three melanoma cell lines, A375, A875, and KZ28, and control melanocytes, using RT-PCR. (b) Expressions of TAP1 and TAP2 protein in three melanoma cell lines, A375, A875, and KZ28, and control melanocytes, using western blot. (c) Expressions of HLA class I antigen in three melanoma cell lines, A375, A875, KZ28, and control melanocytes, using flow cytometry.

Full figure and legend (67K)

Confocal fluorescent microscopic examination reveals that the translated TAP protein is located in the endoplasmic reticulum

For the visualization of the TAP protein, the EGFP gene was introduced at the N terminus of TAP1 and TAP2 in a plasmid construct to be translated as a fusion protein, EGFP-TAP1 and EGFP-TAP2. Each plasmid together with an ER marker (pDsRed2-ER vector) was transiently transfected into A375 cells, and the localizations of the expressed proteins were analyzed by confocal microscopy. A375 cells transfected with pDsRed2-ER vector showed ER distribution for the red fluorescence (Figure 2b and e), and cells transfected with the EGFP-TAP showed the green fluorescence (Figure 2a and d). The localizations of EGFP-TAP1 and EGFP-TAP2 overlapped with that of the ER marker, indicating that TAP protein localized subcellularly into the ER (Figure 2c and f).

Figure 2.

Intracellular expression and localization of transfected TAP protein confocal fluorescent microscopy to demonstrate the expression and distribution of TAP proteins. A375 cells were transfected with pDsRed2-ER (b, e), pEGFP-TAP1 (a), pEGFP-TAP2 (d), pEGFP-TAP1+pDsRed2-ER (c), and pEGFP-TAP2+pDsRed2-ER (f). (b) For the detection of endogenous calreticulin protein, red fluorescence was observed. (a) For the detection of EGFP protein, green fluorescence was noted. (c) Colocalization of EGFP and calreticulin was demonstrated by the yellow color in the combined image.

Full figure and legend (100K)

A375 cells transfected with TAP upregulate TAP1, TAP2, and HLA class antigen I surface expression

TAP1 and TAP2 protein expressions were examined in A375 cells transfected with TAP1, TAP2, and TAP1+TAP2 by western blot. TAP1 and TAP2 protein levels in A375 transfected TAP1 and TAP1+TAP2 were increased compared to the negative control group. TAP1 protein expression in A375 transfected with TAP1 had no significant difference in A375 transfected with TAP1+TAP2 (Figure 3a). TAP2 protein levels in A375 transfected TAP1, TAP2, and TAP1+TAP2 were increased compared to the negative control group. However, TAP1 protein expression in A375 transfected with TAP2 was not increased, and TAP2 protein expression in A375 transfected with TAP2 was less in A375 transfected with TAP1+TAP2 (Figure 3a). Then the effect of TAP expression on cell surface HLA class I antigen expression in TAP-transfected A375 cells was investigated. HLA class I surface expression on A375 cells was compared to surface expression on A375/TAP1, A375/TAP2, and A375/TAP1+TAP2 cells, using FACS analysis. A375/TAP1, A375/TAP2, and A375/TAP1+TAP2 cells exhibited significant expression of HLA class antigen I on the cell surface (Figure 3c). This was in contrast to A375 cells, which had only a small amount of HLA class antigen I. TAP1+TAP2-transfected A375 cells express the highest level of HLA class antigen I in the experimental groups. TAP1-transfected A375 cells express it higher than do the TAP2-transfected A375 cells.

Figure 3.

The transfection of A375 cells with TAP induces HLA class I antigen surface expression. (a) TAP1 and TAP2 expression was detected by western blot: lane 1, A375 cells; lane 2, TAP1+TAP2-transfected A375 cells; lane 3, TAP1-transfected A375 cells; and lane 4, TAP2-transfected A375 cells. (b) Surface HLA class I antigen expression is detected by flow cytometry.

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TAP expression restores antigen presentation and immunogenicity in A375 cells

Peripheral blood mononuclear cells (PBMCs) from HLA-A^*0201 donors were stimulated three times with a melanoma cell line loaded with Melan-A peptide analog (ELAGIGILTV). We performed a CTL assay using A375 cells transfected with the various DNA constructs (TAP1, TAP2, and TAP1+TAP2) as target cells and Melan-A-specific-CTL as effector cells. As shown in Figure 4, A375 cells transfected with TAP1 and TAP1+TAP2 generated significantly higher percentages of specific lysis at the 9:1 (19.30.7%, 25.52.1% vs 9.730.8%, P<0.001) and 27:1 (48.34.7%, 57.16.5% vs 16.43.5%, P<0.001) effector/target (E/T) ratios compared with A375 cells transfected with TAP2. Although A375 cells transfected with TAP1+TAP2 generated higher percentages of specific lysis than A375 cells transfected with TAP1, there was no significant difference between them (P>0.05). Our results suggested that A375 cells transfected with TAP1 and TAP1+TAP2 were capable of directly presenting antigen through the HLA class I pathway to CTL cells more efficiently than A375 cells transfected with TAP2.

Figure 4.

CTL assays to demonstrate enhanced antigen presentation through the HLA class I pathway in A375 cells transfected with TAP. CTL assays with various E/T ratios (E/T=1:1, 3:1, 9:1, 27:1) were performed as described in Materials and Methods. A375 cells transfected with various DNA constructs (TAP1, TAP2, and TAP1+TAP2) were used as target cells, whereas Melan-A-specific CD8+ T cells were used as effector cells. ^*P<0.01, ^**P<0.01.

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TAP expression increases Melan-A-specific IFN- and IL-12-producing CTL

Melan-A-specific CD8+ T cell lines were highly lytic against A375 cell lines transfected with TAP1/TAP1+TAP2 and they efficiently produced IFN upon stimulation with these cells, as illustrated by the high fraction of IFN-/CD8 double-positive cells (Figure 5). A strong correlation was found between the percentage of IFN-/CD8 double-positive cells and the percentage of lysis (Figure 5a). There was a similar approach between the percentage of IL-12-positive cells and the percentage of lysis (Figure 5b). A positive correlation was also found between the percentage of IFN--positive cells and the percentage of IL-12-positive cells (Figure 5c). These results indicated that TAP expression in A375 cells induced a Th1-type tumor-specific immune response.

Figure 5.

Immunization with A375 cells transfected with TAP increased Melan-A specific IFN- and IL-12-secreting CTL. (a) Correlation between lysis and IFN- production by Melan-A-specific CTL in response to an HLA-A^*0201 Melan-A-positive melanoma cell line. (b) Correlation between lysis and IL-12 production by Melan-A-specific CTL in response to an HLA-A^*0201 Melan-A-positive melanoma cell line. For the lysis experiment, effector and target cells were incubated at a 27:1 ratio. For IFN- production, effector and target cells were incubated at a 1:2 ratio in the presence of Brefeldin A before being fixed, permeabilized with saponin, stained with anti-IFN- antibody, and analyzed on a FACScan. (c) Correlation between IFN- and IL-12 production by Melan-A-specific CTL in response to an HLA-A^*0201 Melan-A-positive melanoma cell line.

Full figure and legend (90K)

Discussion

Our results showed that the expressions of TAP1 and TAP2 in three human MM cell lines were downregulated compared to the positive control, melanocytes, and the expressions of TAP1 and TAP2 in the human MM cell line A375 cells were the lowest among them. The view that TAP has an important function in class I assembly derives from the observation that it not only delivers peptides to the ER, but also it is involved in the formation of a large loading complex critical for class I maturation (Powis et al., 1991; Spies and DeMars, 1991; Meyer et al., 1994). Therefore, we examined the expression of HLA class I and found that its expression was reduced compared to melanocytes as well as TAP in human MM cell lines, especially A375 cells. These indicated that the deficiencies of HLA class I caused by the reduction of TAP in the antigen presentation pathway existed in human MM cell lines, especially A375 cells. These results are in line with the murine MM cell line B16F10, which is deficienct in components of the MHC class I antigen-processing pathway, including TAP, MHC class I antigen surface expression (Zhang et al., 2007). However, TAP expression has been associated with tumor-infiltrating lymphocytes, a characteristic of good clinical outcome (Zhang et al., 2003, Dissemond et al., 2003) and spontaneous regression of MM (Panagopoulos and Murray, 1983). Therefore, we transfected TAP1, TAP2, and TAP1+TAP2 DNA into A375 cells, which had the lowest levels of TAP1 and TAP2, and evaluated their effect on tumor immune reaction.

To determine whether the EGFP-N1 vector affected the subcellular localization of TAP, we linked the EGFP gene to the TAP1 gene or the TAP2 gene. Transfection and subsequent examination with a confocal fluorescent microscope revealed that A375 cells transfected with TAP/EGFP and pDsRed2-ER vector (an ER marker) showed ER localization of TAP by yellow color in the combined image (Figure 2c). We further detected the protein expression of TAP1 and TAP2 in A375 cells stably transfected with TAP1, TAP2, and TAP1+TAP2 DNA. Our results showed that not only the expressions of TAP1 but also the expressions of TAP2 were increased in A375 cells transfected with TAP1 DNA. The TAP1 protein expression in A375 transfected with TAP1 had no significant difference in A375 transfected with TAP1+TAP2. However, only the expression of TAP2, not TAP1, was increased in A375 cells transfected with TAP2 DNA, and TAP2 protein expression in A375 transfected with TAP2 was less in A375 transfected with TAP1+TAP2. In A375 cells, TAP1 expression increased the expression of TAP2, but not vice versa. Our results are in line with the observation that the expression level of TAP1 determines the number of functional heterodimeric TAP molecules in the ER (Zhu et al., 1999; Herzer et al., 2003). The role of TAP1 as an assembly platform for newly synthesized unstable TAP2 would explain these puzzling and so far poorly understood observations (Keusekotten et al., 2006). We examined HLA class I surface expression on A375 cells transfected with TAP1, TAP2, and TAP1+TAP2 DNA and found that TAP1+TAP2-transfected A375 cells expressed the highest level of HLA class I antigen in the experimental groups and that TAP1-transfected A375 cells expressed it higher than TAP2-transfected A375 cells. This indicates that the re-expression of TAP1 may stabilize the expression of TAP2 and thus increase the amount of antigenic peptide available for assembly onto HLA class I molecules in the ER.

In the case of A375 cells, TAP1 and TAP1+TAP2 gene transfer were able to resurrect the presentation of Melan-A peptide analog on HLA-A^*0201 antigens to allow for Melan-A-specific CTL killing. A375 cells transfected with TAP1 and TAP1+TAP2 DNA generated significantly higher percentages of specific lysis compared to the negative control and A375 cells transfected with TAP2 DNA. Vaccination by irradiated A375 cells expressing TAP1 and TAP2 greatly enhances CTL activity toward A375/TAP1 and A375/TAP1+TAP2 target cells. We observed that the percentage of IFN-/IL-12-positive cells were increased in TAP1 and TAP1+TAP2 groups. IFN- (Fathallah-Shaykh et al., 2000) and IL-12 (Coughlin et al., 1998; Duda et al., 2000) are examples of various other compounds that have been shown to exert an antitumor effect. We also observed that there was a strong correlation between the percentage of IFN-/IL-12-positive cells and the percentage of lysis. This indicated that IFN- and IL-12 might take part in CTL's killing of MM.

In summary, our results indicated that the deficiencies of HLA class I caused by reduction of TAP in the antigen presentation pathway existed in human MM cell lines, especially A375 cells. The re-expression of TAP1 might stabilize TAP2 in A375 cells, and the restoration of TAP has the potential to enhance antigen presentation and tumor-specific immunity.

Materials And Methods

Cell lines

The human MM cell lines A375, A875, and KZ28 were cultured in plastic flasks in DMEM (Gibco, Grand Island, NY) containing 10% heat-inactivated fetal calf serum (Gibco), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (15 mM), L-glutamine (2 mM), penicillin (100 IU ml^-1), and streptomycin (100 g ml^-1). Normal human epidermal melanocytes were isolated from neonatal foreskins. Subcutaneous fat was removed with dissecting scissors and the remaining tissue was cut into small pieces and incubated with trypsin (Invitrogen, San Diego, CA) overnight at 4 °C. The epidermal cells were inoculated in 12-well plates in melanocyte growth medium and maintained at 37 °C with 5% CO₂. Cultures were provided with fresh medium three times weekly and melanocytes were used at passages 3–4 for RNA and protein extraction. The institutional approval is not necessary.

Reverse Transcription-PCR and gel electrophoresis

Total RNA from frozen sections was extracted using the TRIzol reagent (Invitrogen) and was reverse-transcribed and synthesized to complementary DNA using reverse transcriptase (Promega, Madison, WI). Primers were as follows:

TAP1: 5'-TCTCCTCTCTTGGGGAGATG-3' (sense primer)

5'-GAGACATGATGTTACCTGTCTG-3' (antisense primer)

TAP2: 5'-CTCCTCGTTGCCGCCTTCT-3' (sense primer)

5'-TCAGCTCCCCTGTCTTAGTC-3'(antisense primer)

-Actin: 5'-AAGAGAGGCATCCTCACCCT-3' (sense primer)

5'-TACATGGCTGGGGTGTTGAA-3' (antisense primer)

TAP1, TAP2, and -actin fragment sizes were 280, 300, and 218 bp, respectively. Band intensities were quantitated by image analysis system (LEICA Q550I W, Bensheim, Germany), integral absorbency (IA) of PCR products was calculated, and IA=average absorbency area.

Plasmids construction

Human TAP1 and TAP2 were amplified by PCR from a PBMC complementary DNA library and subcloned into EGFP-based mammalian expression vectors (Clontech, Palo Alto, CA). For deletion mutant constructs, DNA sequences corresponding to different regions of TAP were amplified by PCR from the above-mentioned constructs and subcloned into the expression vectors. All constructs generated from PCR products were sequenced.

Transfection and confocal microscopy

A total of 2.5 l0⁵ A375 cells were seeded in 12-well plates and transfected with 10 ng of pDsRed2-ER vector (Clontech), which is designed for fluorescent labeling of the endoplasmic reticulum in living cells, and 30 ng of the indicated TAP1 or TAP2 construct by Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were removed from the plates with phosphate-buffered saline, and live cells were sandwiched between coverslips and glass slides and visualized for fluorescence using the appropriate lasers and filters to visualize EGFP and DsRed. Confocal images were acquired with a Laser Scanning Microscope FV500 (Olympus) confocal system and then processed using Adobe Photoshop (Adobe Systems, San Jose, CA).

Transfection and isolation of TAP stable transfectant clones

A375 cells were transfected with pEGFP-TAP1/TAP2/TAP1+TAP2 or empty an vector by Lipofectamine 2000. After 48 hours, the cells were selected with 400 g ml^-1 G418 (Invitrogen) for 2 weeks as a selective marker and the surviving colonies were cloned. The transfectants were maintained in DMEM containing 10% heat-inactivated fetal bovine serum, and 150 g of G418/ml^-1 at 37 °C in a humidified atmosphere of 5% CO₂.

Western blot

Protein concentrations were determined by BCA Protein Assay Kit (Pierce, Rockford, IL) and 20 g equal amounts of protein was loaded into a 15% polyacrylamide gel. Proteins were resolved and transferred to a nitrocellulose membrane using semi-dry transfer (Bio-Rad, Hercules, CA). After incubating the membranes in a blocking buffer, the membranes were incubated with TAP1, TAP2, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) polyclonal antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were visualized by chemiluminescence using a horseradish peroxidase-conjugated IgG antibodies and ECL kit (Pierce) according to the instructions of the manufacturer. Quantitative analysis of the blots was performed with an imaging densitometer.

Generation of Melan-A-specific effector CTL clones from PBMC

To generate HLA-A^*0201 antigen-restricted Melan-A-specific CTLs, Melan-A analog peptide (ELAGIGILTV) was synthesized by Peptide Technologies (Invitrogen) to a purity more than 99% by HPLC and amino-acid analysis.

Loading with Melan-A analog peptide (ELAGIGILTV) was performed by incubating stimulator cells with the peptide at 37 °C in a serum-free medium for 2 hours. Stimulator cells were washed twice to eliminate unbound peptide. Total PBMCs from HLA-A^*0201 donors were isolated using the Ficoll–Hypaque density–gradient method and then stimulated in 96-well culture plates with 2 10⁴ irradiated stimulator cells (10,000 rads) in DMEM containing 8% human serum supplemented with 5 ng ml^-1 IL-6 (Peprotech, London, UK) during the first stimulation and 10 U ml^-1 IL-2 (Peprotech) during the second and third stimulations at 7-day intervals.

Cytotoxicity assay for Melan-A-specific effector CTLs using transfected TAP A375 cells as target cells

CTL assays were performed by quantitative measurements of lactate dehydrogenase using CytoTox96 nonradioactive cytotoxicity assay kits (CytoTox96; Promega). Target cells suspended in 50 l DMEM supplemented with 3% fetal calf serum were placed on a 96-well plate, and effector cells (stimulated PBMC) suspended in the same medium were also added to the wells at various E/T ratios in a final volume of 200 l. Untransfected A375 cells were used as a negative control. After incubation for 4 hours at 37°C with 5% CO₂, 50 l supernatant from each well was transferred to an enzymatic assay plate. Then 50 l substrate mixture was added to each well of the enzymatic assay plate, followed by 30 minutes of incubation at room temperature. Finally, 30 l stop solution was added and the absorbance was recorded at 490 nm. The percentage of specific lactate dehydrogenase release was calculated by the following equation: % cytotoxicity=100[(A-B)/(C-D)], where A is the reading of experimental effector signal value, B is the effector spontaneous background signal value, C is maximum signal value from target cells, and D is the target spontaneous background signal value.

FACS assays

Surface expression of the HLA class I antigen was detected by indirect immunofluorescence. PE-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark) was used as the secondary antibody. The mean logarithmic fluorescence intensity was measured by a FACScan analyzer (Becton Dickinson, Mountain View, CA).

The production of IFN- and IL-12 by Melan-A-specific CTL clones, in response to target cells, was assessed by intracellular cytokine labeling using a method described by Jung et al. (Herzer et al., 2003). PE-cy5-conjugated anti-CD8, APC-conjugated anti-IL-12, and APC-conjugated anti-IFN- purified antibodies were purchased from BD Bioscience Pharmingen, San Diego, CA. T-cell clones were stimulated by Ag-expressing melanoma cells at the 1:2 ratio in DMEM 10% fetal calf serum in the presence of Brefeldin A (10 g ml^-1; Sigma-Aldrich, St Louis, MO). After 6 hours, the cells were fixed for 10 minutes at room temperature in a solution of phosphate-buffered saline 4% paraformaldehyde. After staining with anti-cytokine mAbs, the cells were resuspended in phosphate-buffered saline and 5,000 events were analyzed on a FACScan.

Statistical analysis

Statistical comparisons of mean values were performed using one-way analysis of variance, and statistical comparisons of correlations were performed using Bivariate correlations analysis. All P-values were determined from two-sided tests. A significance criterion of P<0.05 was used. In the figures, P-values are indicated by asterisks (^*): P<0.05, P<0.01. The statistical analysis was performed using SPSS 10.0 software (SPSS, Chicago, IL).

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

This project was supported by the Young Scientists Fund Program of National Science Fund Program from National Natural Science Foundation of China (30500437).