GILT Expression in Human Melanoma Cells Enhances Generation of Antigenic Peptides for HLA Class II-Mediated Immune Recognition

Abstract

Melanoma is an aggressive skin cancer that has become increasingly prevalent in western populations. Current treatments such as surgery, chemotherapy, and high-dose radiation have had limited success, often failing to treat late stage, metastatic melanoma. Alternative strategies such as immunotherapies have been successful in treating a small percentage of patients with metastatic disease, although these treatments to date have not been proven to enhance overall survival. Several melanoma antigens (Ags) proposed as targets for immunotherapeutics include tyrosinase, NY-ESO-1, gp-100, and Mart-1, all of which contain both human leukocyte antigen (HLA) class I and class II-restricted epitopes necessary for immune recognition. We have previously shown that an enzyme, gamma-IFN-inducible lysosomal thiol-reductase (GILT), is abundantly expressed in professional Ag presenting cells (APCs), but absent or expressed at greatly reduced levels in many human melanomas. In the current study, we report that increased GILT expression generates a greater pool of antigenic peptides in melanoma cells for enhanced CD4+ T cell recognition. Our results suggest that the induction of GILT in human melanoma cells could aid in the development of a novel whole-cell vaccine for the enhancement of immune recognition of metastatic melanoma.

Keywords: Ag presenting cells (APCs); CD4+ T cells; cathepsins; gamma-IFN-inducible lysosomal thiol-reductase (GILT); human leukocyte antigen (HLA) class II; melanoma.

Figure 1

GILT insertion influences the expression of the components of the class II pathway and CD80/CD86 molecules in human melanoma cells. (A) Western blot analysis of whole cell lysates from J3.DR4.vec, J3.DR4.GILT, DM-331.vec and DM-331.GILT cells showing protein expression of GILT, HLA-DR, Ii, and HLA-DM. β-actin was utilized as a loading control. (B) Densitometric analysis was performed using β-actin as a reference protein band to quantitate relative protein expression. Data are representative of at least three separate experiments. Significant differences were calculated by student’s t test; * p < 0.05, ns = not significant. (C) J3.DR4 cells transfected with empty vector or GILT were simultaneously stained with primary antibodies against CD80 and CD86 proteins, followed by Alexa488-labeled secondary antibody, as described in the methods. Slides were analyzed by Leica TCS SP5 confocal laser scanning microscope using Las-AF software. Representative confocal microscopy images of J3.DR4.vec and J3.DR4.GILT cells indicate increased levels of CD80 and CD86 expression (green) with nuclear staining DAPI (blue), overlapped (arrows) in the right hand corners. Bar = 29.9 μm.

Figure 2

GILT colocalizes with cysteinyl and aspartyl cathepsins and enhances their activities in human melanoma cells. (A) J3.DR4.GILT cells were stained with either rabbit anti-CatB or CatD, and co-stained with goat anti-GILT, followed by rhodamine-conjugated anti-rabbit IgG and FITC-conjugated anti-goat IgG antibodies. Live images were acquired using a Leica TCS SP2 AOBS confocal system and processed with Leica software. The colocalization of GILT (green) with CatB (upper panel) and CatD (lower panel) is indicated by the yellow staining and arrows. (B–D) Cell lysates obtained from J3.DR4.vec, J3.DR4.GILT, DM-331.vec, and DM-331.GILT cells wBar ere analyzed for cathepsins B, S and D activities as described in the methods. Enhanced activities of cysteinyl/asparrtyl cathepsins were detected in melanoma cells expressing GILT, as compared to cells lacking GILT. The experiments were repeated at least three times and results are expressed as mean fluorescence unit ± SEM of triplicate wells.

Figure 3

Mass spectroscopy and fragmentation of cysteinylated versus reduced Igκ_188–203 peptides. (A) Cysteinylation of the κ_188–203 peptide was determined by the detection of ionized species at 954 m/z. (B) GILT catalyzed the reduction of cysteinylated-κ_188–203 as ionized species at 894 detected. (C) Extracted ion chromatograph of peptides generated by J3.DR4 and J3.DR4.GILT cells. LC-MS mass spectrometry was performed on supernatant obtained from J3.DR4 and J3.DR4.GILT cells fed with IgG. The data suggest that J3.DR4.GILT cells generated a greater pool of reduced peptides.

Figure 4

Percent occupation of cysteine oxidation in supernatants obtained from cells with or without GILT. (A) J3.DR4 and J3.DR4.GILT cells were fed with IgG in serum-free HBSS for 4 h. Supernatants were collected by centrifugation and analyzed by mass spectroscopy as described in the methods. Extracted ion chromatograph (XIC) of the peptide ALVLIAFAQYLQQCPFEDHVK identified in supernatants obtained from cells±GILT; 831.66 is the mass of the +3 charge state for the peptide ALVLIAFAQYLQQCPFEDHVK with the cysteine chemically modified by iodoacetamide (+57); 828.25 is the +3 charge state of ALVLIAFAQYLQQCPFEDHVK, which contains a tri-oxidized cysteine; 1205.4 is +3 charge state of the peptide ALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK, which contains a tri-oxidized cysteine and 1 trypsin missed cleavage. The percent occupation of oxidation was calculated using the area under the curve from the XIC (the total area of the oxidized peptides divided by the total area of the peptide ×100). Cells expressing GILT had a 40.7% tri-oxidized cysteine, while cells lacking GILT had a significantly higher level of oxidation with 77.9% of the peptide being tri-oxidized. (B) Tandem MS spectra (MS/MS) of the reduced and oxidized forms of the peptides ALVLIAFAQYLQQCPFEDHVK and ALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK, with the cysteine detected as either carbamidomethylated or tri-oxidized. The top spectra identified ALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK with the cysteine tri-oxidized with an Xcorr value of 5.39. The site of oxidation was confirmed by the presence of the b14 ions 1610.8 (+1) and 805.9 (+2) and the y18 ion 805.9 (+2). The middle tandem MS spectrum was identified as ALVLIAFAQYLQQCPFEDHVK with a trioxidized cysteine and an Xcorr value of 3.36. The site of oxidation was confirmed by the presence of the b14 ions 1610.8 (+1) and 805.9 (+2) and the y8 ions 1022 (+1) and 511.7 (+2). The bottom MS/MS spectra was identified with an Xcorr of 4.78 as the peptide ALVLIAFAQYLQQCPFEDHVK, with the cysteine carbamidomethylated. The modification detected by mass spectral analysis was localized by the presence of b14 ions 1619 (+1) and 810 (+2), and the y8 ion 516(+2).

Figure 4

Percent occupation of cysteine oxidation in supernatants obtained from cells with or without GILT. (A) J3.DR4 and J3.DR4.GILT cells were fed with IgG in serum-free HBSS for 4 h. Supernatants were collected by centrifugation and analyzed by mass spectroscopy as described in the methods. Extracted ion chromatograph (XIC) of the peptide ALVLIAFAQYLQQCPFEDHVK identified in supernatants obtained from cells±GILT; 831.66 is the mass of the +3 charge state for the peptide ALVLIAFAQYLQQCPFEDHVK with the cysteine chemically modified by iodoacetamide (+57); 828.25 is the +3 charge state of ALVLIAFAQYLQQCPFEDHVK, which contains a tri-oxidized cysteine; 1205.4 is +3 charge state of the peptide ALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK, which contains a tri-oxidized cysteine and 1 trypsin missed cleavage. The percent occupation of oxidation was calculated using the area under the curve from the XIC (the total area of the oxidized peptides divided by the total area of the peptide ×100). Cells expressing GILT had a 40.7% tri-oxidized cysteine, while cells lacking GILT had a significantly higher level of oxidation with 77.9% of the peptide being tri-oxidized. (B) Tandem MS spectra (MS/MS) of the reduced and oxidized forms of the peptides ALVLIAFAQYLQQCPFEDHVK and ALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK, with the cysteine detected as either carbamidomethylated or tri-oxidized. The top spectra identified ALVLIAFAQYLQQCPFEDHVKLVNEVTEFAK with the cysteine tri-oxidized with an Xcorr value of 5.39. The site of oxidation was confirmed by the presence of the b14 ions 1610.8 (+1) and 805.9 (+2) and the y18 ion 805.9 (+2). The middle tandem MS spectrum was identified as ALVLIAFAQYLQQCPFEDHVK with a trioxidized cysteine and an Xcorr value of 3.36. The site of oxidation was confirmed by the presence of the b14 ions 1610.8 (+1) and 805.9 (+2) and the y8 ions 1022 (+1) and 511.7 (+2). The bottom MS/MS spectra was identified with an Xcorr of 4.78 as the peptide ALVLIAFAQYLQQCPFEDHVK, with the cysteine carbamidomethylated. The modification detected by mass spectral analysis was localized by the presence of b14 ions 1619 (+1) and 810 (+2), and the y8 ion 516(+2).

Figure 5

Percent occupation of methionine oxidation in supernatants obtained from cells with or without GILT. Cells (J3.DR4 and J3.DR4.GILT) were fed with IgG in serum-free HBSS for 4 h. Supernatants obtained were analyzed by mass spectroscopy. (A) Extracted ion chromatograph (XIC) of the peptide AVMDDFAAFVEK identified in cell supernatants obtained from J3.DR4 and J3.DR4.GILT cell; 672.5 was identified as the reduced form of peptide AVMDDFAAFVEK, and determined to be the +2 charge state. The oxidized form of this peptide, 680.5 (+2 charge state), was below the quantitation limit as a peak was unable to be extracted to calculate the area. Based on the areas of the unoxidized form of this peptide, there is no significant change in oxidation of methionines in J3.DR4.GILT supernatant. (B) Tandem MS spectra (MS/MS) of the reduced (672.1) and oxidized (680.1) forms of the peptide AVMDDFAAFVEK. The top spectra represent the oxidized form of the peptide AVMDDFAAFVEK. This peptide was identified with an Xcorc value of 3.15. Localization of the oxidized amino acid was determined by the presence of the b3 ion at 318.3 and the y10 ions at 1188.5 (+1) and 594.7 (+2). The bottom spectrum was identified as the reduced peptide AVMDDFAAFVEK with an Xcorr of 3.53.

Figure 5

Percent occupation of methionine oxidation in supernatants obtained from cells with or without GILT. Cells (J3.DR4 and J3.DR4.GILT) were fed with IgG in serum-free HBSS for 4 h. Supernatants obtained were analyzed by mass spectroscopy. (A) Extracted ion chromatograph (XIC) of the peptide AVMDDFAAFVEK identified in cell supernatants obtained from J3.DR4 and J3.DR4.GILT cell; 672.5 was identified as the reduced form of peptide AVMDDFAAFVEK, and determined to be the +2 charge state. The oxidized form of this peptide, 680.5 (+2 charge state), was below the quantitation limit as a peak was unable to be extracted to calculate the area. Based on the areas of the unoxidized form of this peptide, there is no significant change in oxidation of methionines in J3.DR4.GILT supernatant. (B) Tandem MS spectra (MS/MS) of the reduced (672.1) and oxidized (680.1) forms of the peptide AVMDDFAAFVEK. The top spectra represent the oxidized form of the peptide AVMDDFAAFVEK. This peptide was identified with an Xcorc value of 3.15. Localization of the oxidized amino acid was determined by the presence of the b3 ion at 318.3 and the y10 ions at 1188.5 (+1) and 594.7 (+2). The bottom spectrum was identified as the reduced peptide AVMDDFAAFVEK with an Xcorr of 3.53.

Figure 6

GILT expression enhances HLA class II-mediated antigen presentation and CD4+ T cell recognition of tumors by producing a greater pool of functional epitopes. (A,B) Human melanoma cell lines J3.DR4.vec, J3.DR4.GILT, DM-311.vec, and DM-311.GILT were incubated with whole Igκ Ag overnight at 37 °C. Cells were then washed, and co-cultured with the κ_188–203 peptide-specific CD4+ T cell hybridoma line (2.18a) for 24 h. The T cell production of IL-2 in the culture supernatant was measured by ELISA. The production of IL-2 is used as an indication of Ag presentation and T cell recognition. Data are expressed as the mean ± SD, shown in pg/mL of triplicate wells of at least three independent experiments. * p < 0.01. (C,D) Melanoma cell lines J3.DR4.vec, J3.DR4.GILT, DM-331.vec, and DM-331.GILT were incubated overnight with whole Igκ Ag in HBSS at 37 °C. Cell supernatants were transferred to paraformaldehyde fixed J3.DR4.vec (C) and DM-331.vec (D) cells overnight at 37 °C. Supernatants from GILT-expressing cells were also incubated with L-cystine (290 μM) as described. Cells were then washed and co-cultured with the κ_188–203 peptide-specific CD4+ T cell hybridoma 2.18a for 24 h. The T cell production of IL-2 was quantitated by ELISA. Data are representative of at least three separate experiments. * p < 0.001.

Figure 7

GILT expression is detected in tumor-infiltrating cells and is lost in human metastatic melanoma. Human melanoma patient samples TB#8933, TB#9111, and TB#7812 were minced into cubes (approx. 1 mm) and placed into T75 flasks with complete DMEM containing 10% FBS and 1 mg/mL collagenase. These tumor tissues were incubated at 37 °C for 24 h, washed, suspended in complete DMEM with no collagenase in the medium, and cultured for at least 14 days as described in the methods. (A) Samples from day 0, day 7 and day 14 were subjected to western blot analysis. Western blot analysis showing differential GILT expression at day 0 (high) and day 7 (low), and the expression was lost at day 14 in metastatic tumors. J3 cells transfected with GILT (J3.GILT) were used as a positive control. (B) Densitometric analyses were performed using β-actin as a reference protein band to quantitate relative GILT protein expression in human metastatic melanoma at different time points. Data are representative of at least three separate experiments. * p < 0.001. (C) Immunohistochemical analyses of TB#7812 cultures for tyrosinase (melanoma), vimentin (fibroblast) and CD11c (dendritic cells) markers at day 0 and day 14. Bar = 100 μM.