Abstract
NY-ESO-1–specific CD4+ T cells are of interest for immune therapy against tumors, because it has been shown that their transfer into a patient with melanoma resulted in tumor regression. Therefore, we investigated how NY-ESO-1 is processed onto MHC class II molecules for direct CD4+ T cell recognition of melanoma cells. We could rule out proteasome and autophagy-dependent endogenous Ag processing for MHC class II presentation. In contrast, intercellular Ag transfer, followed by classical MHC class II Ag processing via endocytosis, sensitized neighboring melanoma cells for CD4+ T cell recognition. However, macroautophagy targeting of NY-ESO-1 enhanced MHC class II presentation. Therefore, both elevated NY-ESO-1 release and macroautophagy targeting could improve melanoma cell recognition by CD4+ T cells and should be explored during immunotherapy of melanoma.
Introduction
Cancer testis Ags are a unique class of tumor-associated Ags, because they are normally expressed in the adult male germ line, but not in other normal tissues, and are overexpressed in various malignancies. This selective expression makes them ideal candidates for immunotherapy (1). NY-ESO-1 is such a cancer testis Ag, which is overexpressed in at least 40% of melanomas and in many different other types of tumors. Furthermore, NY-ESO-1 spontaneously elicits humoral and cellular responses in many patients with cancer (2, 3). Therefore, among tumor-associated Ags, it is one of the most promising Ags for immunotherapy (4), and different vaccines using NY-ESO-1 peptides, full-length NY-ESO-1 protein, or NY-ESO-1 DNA are being evaluated in phase 2 clinical trials. Thus, Ag processing of NY-ESO-1 for T cell recognition should be explored in more detail to characterize how this Ag sensitizes tumor cells for targeting by the adaptive immune system.
Although the goal of many of these immunotherapeutic trials has been to mount a specific antitumoral CD8+ T cell response, many of these responses have been transient, and a long-lasting clinical benefit was only achieved in a minority of cases. There is now increasing evidence for an additional role of CD4+ T cell response in antitumoral immunity. The antitumor effect of CD4+ T cells can be direct by different cytotoxic mechanism of this leukocyte subset (5, 6) or indirect by enhancing both NK and CD8+ T cell responses, providing the so-called T cell help during priming and maintenance of long lasting memory CD8+ T cell response (7–9) and sustained NK cell reactivity (10). This CD4+ T cell help is in part mediated by IL-2 and IL-21 (11), which are critical cytokines for CD8+ T cell survival and NK cell activation. In addition, CD4+ T cells can also efficiently mature dendritic cells via CD40L-mediated engagement of CD40 on dendritic cells, and these potent APCs can then in turn stimulate CD8+ T cells and NK cells. For both direct and indirect antitumoral functions of CD4+ T cells, understanding how tumor cells can process tumor Ags that are recognized by CD4+ T cells is essential to enhance T cell responses during immunotherapeutic treatments.
In a recent proof-of-concept study, adoptive transfer of CD4+ T cells specific to the 157–170 epitope of NY-ESO-1 Ag markedly improved the clinical outcome of a patient with refractory metastatic melanoma (12). Indeed, the patient was in clinical durable remission for up to 2 years after the T cell transfer. Due to its promising features as a tumor Ag, we were particularly interested in the pathway by which the endogenously expressed NY-ESO-1157–170 epitope can gain access to MHC class II compartments for presentation to CD4+ T cells in melanoma cell lines. In addition, this particular epitope is of significant interest because it overlaps with an immunodominant CD8+ T cell epitope restricted by HLA-A2 (NY-ESO-1157–165) and is often used in immunotherapeutic trials.
We could show that melanoma cells that endogenously express NY-ESO-1, efficiently present the HLA-DP4–restricted NY-ESO-1157–170 epitope to clonal CD4+ T cells. Surprisingly, the pathway for the processing of this epitope results from intercellular transfer of the Ag between melanoma cells and is processed for MHC class II presentation after endocytosis. Indeed, we could show that NY-ESO-1–negative melanoma cell lines that have a moderate phagocytic activity can acquire and process NY-ESO-1 Ag either from neighboring cells, from exogenous necrotic material, or from cellular supernatant of NY-ESO-1–expressing tumor cells. Finally, to enhance NY-ESO-1 processing for MHC class II presentation, we constructed a fusion protein by coupling NY-ESO-1 with Atg8/LC3, an essential autophagy protein, to target NY-ESO-1 to autophagosomes. The fusion protein NYESO-LC3 could be delivered with very high efficiency to the MHC class II loading compartment, suggesting that macroautophagic delivery of this tumor Ag could serve as a new strategy to enhance antitumoral CD4+ T cell responses.
Materials and Methods
Target cell lines
Melanoma cell lines were generated from tumor biopsies of patients as previously described (13, 14). The human lung epithelium cell line A549 is a gift from Dr. Thomas Moran (Mount Sinai Hospital, New York, NY), and the A549 cell line was cultured in DMEM with 10% FCS (Sigma-Aldrich), 2 mmol glutamine, 110 μg/ml sodium pyruvate, and 2 μg/ml gentamicin. Melanoma cell lines were cultured in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FCS, 2 mmol glutamine, and 2 μg/ml gentamicin. Necrotic A549 cells were prepared by three cycles of freeze/thawing.
Generation of expression plasmids
KFERQ-GFP plasmid was constructed by inserting the PCR product of KFERQ into the pEGFP-C2 plasmid (Invitrogen). NY-ESO-1 Myc-His Tag plasmid was constructed by inserting the cDNA of human NY-ESO-1 sequence NM_0011327.1 (purchased from Origene) into the pEM.CMV-Myc-His vector (a gift from Dr. Cheolho Cheong, University of Montreal, Montreal, QC, Canada). The human lysosome-associated membrane protein 2 isoform A (Lamp2a) NM_002294.1 and Lamp2b NM_013995.1 plasmids were purchased from Origene (Rockville, MD). Rab7 wild-type and Rab7 DN T22N were a gift from Dr. Tamotsu Yoshimori (University of Osaka, Osaka, Japan). The NYESO-LC3 construct was generated by inserting the human NY-ESO-1 sequence NM_0011327.1 (purchased from Origene) into the pEGFP-C1 plasmid (Invitrogen). The cDNA of human MAP1LC3B sequence (NM_022818) was cloned from a human B-lymphoblastoid cell lines by RT-PCR with gene-specific primers.
Small interfering RNA–mediated gene silencing
For delivery of small interfering RNAs (siRNAs) into epithelial cell lines, siRNAs were transfected with Lipofectamine 2000 (Invitrogen) using 200 pmol siRNA plus 7 μl Lipofectamine/well in a six-well format. At 48 h after the transfection, cells were analyzed by Western blot for the expression of the cognate protein. The Atg12-specific siRNA was purchased from Dharmacon composed of Atg12 sense 5′-GUGGGCAGUAGAGCGAACAUdT-3′ and Atg12 antisense 5′-UCAUGUAGUAGCAAGUUGAUdT-3′. The Lamp2a N1 and N2 siRNA were purchased from Dharmacon composed of Lamp2a-N1 sense 5′-UGUAUAAGGACUAUAGUGAUU-3′ and antisense 3′−UUACAUAUUCCUGAUAUCACU-5′ and Lamp2a-N2 sense 5′-UGUAUAAGGACUAUAGUGAUU-3′ and antisense 3′-UUA CAUAUUCCUGAUAUCACU 5′.
Inhibitors
Melanoma cells were treated with different inhibitors. All inhibitors were purchased from Sigma-Aldrich or Calbiochem and used at the following concentration: chloroquine (CQ) at 50 μmol, lactacystin at 5, 10, and 20 μmol, E64 at 25 and 50 μmol, epoxomycin at 5 and 25 nmol, MG132 at 2.5, 5, and 10 μm, leupeptin at 25 μg/ml, and brefeldin A at 5 μg/ml.
T cell assays
The NY-ESO-1–specific CD4+ T cell clone was generated as previously described (14, 15). When necessary target cells were treated with 200 U/ml IFN-γ 48 h before the coculture to upregulate MHC class II expression, cells were then washed three times in RPMI 1640 before coculture with T cells to remove IFN-γ. Target cells were cocultured overnight with NY-ESO-1–specific T cells in 5% PHS medium (RPMI 1640 with 5% PHS plus glutamine plus gentamicin) in 96-well round-bottom plates (105 T cells/well plus indicated target cell numbers). After coculture, IFN-γ in culture supernatants was measured using the human IFN-γ ELISA kit from Mabtech according to the manufacturer’s instructions. Recombinant human IFN-γ (Mabtech) at concentrations of 30–2000 pg/ml was used as a standard. If IFN-γ levels in supernatants exceeded 2000 pg/ml, supernatants were diluted in 5% PHS medium, and IFN-γ was remeasured by ELISA. In each experiment, peptide-pulsed CD4+ T cells were used as a positive control (105 of CD4+ T cells pulsed with 10 μg/ml cognate peptide). Data are presented as concentration of IFN-γ (in pg/ml) or as percentage of IFN-γ secretion relative to the positive control (peptide-stimulated T cells). This normalization was performed if multiple T cell assays were pooled.
Lysate preparation, SDS-PAGE, and immunoblotting
Cells were lysed in ice-cold lysis buffer (50 mmol Tris-HCl [pH 8], 150 mmol NaCl, and 1% Nonidet P-40 with the complete protease inhibitor mixture from Roche for 10 min on ice [∼106 cells/200 μl]). Protein concentration was determined by BCA protein assay (Pierce). Protein samples were denatured at 95°C for 5 min in the presence of 4× SDS-PAGE-loading buffer (250 mmol Tris-HCl [pH 6.8], 40% glycerol, 8% SDS, 0.57 mol 2-ME, and 0.12% bromophenol blue). Equal amounts of protein were run on 12 or 15% SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane (Hybond-P; Amersham Biosciences). Primary Abs were visualized with HRP-conjugated goat anti-rabbit (The Jackson Laboratory) or anti-mouse IgG (Bio-Rad) and the ECL plus detection system (Amersham Biosciences).
Abs
The following primary Abs were used: NY-ESO-1 rabbit Ab (Spring Bioscience), Atg8/LC3 mouse mAb (clone 5F10; Nanotools), LAMP2 (clone H4B4) mouse mAb (eBioscience), and Histidine 6× Tag mouse mAb (clone 3D5) (Invitrogen). The following secondary Abs were used: HRP-conjugated goat anti-rabbit (The Jackson Laboratory) HRP-conjugated goat anti-mouse (Bio-Rad), and Alexa 555–conjugated goat anti-mouse Ab (Invitrogen).
Immunofluorescence microscopy
Cells were grown on microscopy cover glasses in 24-well plates. Cells were fixed in 3% paraformaldehyde in PBS for 15 min and permeabilized in 0.01% Triton X-100 in PBS for 3 min. Primary and secondary Abs were applied in PBS 0.1% saponin plus 10% of serum from the species of the secondary Ab for 45–60 min, followed by three 5-min washes in PBS. Finally, cells were stained with DAPI nucleic acid stain (Invitrogen-Molecular Probes) for 1 min, and cover glasses were mounted onto microscope slides using Aqua Polymount (Polysciences) or Prolong Gold antifade reagent (Invitrogen-Molecular Probes). All steps were carried out at room temperature. Cells were analyzed on an Olympus wide-field microscope with a 60× or 100×/1.4 numerical aperture oil immersion lens (Olympus), and pictures were processed with the ImageJ (National Institutes of Health) or the Metamorph Software (Universal Imaging Corporation) or on an inverted LSM 510 laser scanning confocal microscope (Zeiss Axiovert 200; Zeiss) with a 63 or 100×/1.4 numerical aperture oil immersion lens using a pinhole diameter of 1 Airy unit. Pictures were taken with the LSM 510 confocal software (Zeiss).
Phagocytosis assay
Cells were incubated at 37°C for 2 h with 0.8-μm FITC-labeled latex beads (Sigma-Aldrich): a ratio of 50 and 100 beads/cell was used. After incubation, cells were extensively washed, and the amount of beads per cell was evaluated by flow cytometry analysis.
Statistical evaluation
Statistical analyses were performed with the unpaired two-tailed Student t test. The p value of significant differences is reported. Plotted data represent mean plus SEM, unless otherwise stated.
Results
NY-ESO-1 is processed for MHC class II presentation via lysosomal degradation
In order to investigate Ag processing for MHC class II presentation, we first evaluated the localization of NY-ESO-1 in melanoma cell lines and their recognition by NY-ESO-1–specific T cell clones. Due to the paucity of NY-ESO-1–specific Abs for immune fluorescence microscopy, we used an MYC-HIS–tagged NY-ESO-1 construct, which we transfected into human melanoma cell lines. We found that transfected NY-ESO-1 is diffusely distributed in the cytosol and to a lesser extent in the nucleus (Fig. 1A). Because the expression of NY-ESO-1 was mainly cytosolic, we hypothesized that NY-ESO-1 could access MHC class II loading compartments by an endogenous processing pathway. To address this question, we used three different HLA-DP4–positive melanoma cell lines and tested their recognition by a NY-ESO-1–specific CD4+ T cell clone, reactive toward the 157–170 epitope restricted by HLA-DP4. Using IFN-γ secretion into the coculture supernatant as a readout, measured by ELISA assay, we could show that the NY-ESO-1–positive melanoma cells lines M6 and M29 efficiently present the NY-ESO-1157–170 epitope (Fig. 1B). In contrast, the NY-ESO-1–negative HLA-DP4–positive melanoma cell line M199 was not recognized. In order to address whether NY-ESO-1 would be processed for MHC class II presentation via lysosomal degradation, we incubated the M6 melanoma cell line with inhibitors of lysosomal degradation. Using CQ, a lysomotropic agent that neutralizes lysosomal acidification, we found that overnight treatment of melanoma target cells completely abrogated their recognition by the NY-ESO-1–specific CD4+ T cell clone (Fig. 1C). Having established that NY-ESO-1 protein is degraded in the lysosomal compartment for presentation on MHC class II molecules, we used more specific inhibitors for classes of lysosomal proteases to demonstrate the requirement of lysosomal NY-ESO-1157–170 processing. Leupeptin, which inactivates cysteine and serine proteases, including cathepsins S, L, and B, completely abrogated T cell recognition of the three melanoma cell lines (Fig. 1C). Moreover, brefeldin A, which blocks the transport of newly synthesized MHC class II molecules from the endoplasmic reticulum to the Golgi, also blocked T cell recognition (Fig. 1C) without affecting MHC class II surface expression (data not shown). These data suggest that newly synthesized and not recycling MHC class II molecules present NY-ESO-1 on melanoma cells.
FIGURE 1.
Cytosolic NY-ESO-1 is processed for MHC class II presentation by lysosomal degradation. (A) The human melanoma M6 cell line was transfected with MYC-HIS–tagged NY-ESO-1 or with a control plasmid. Localization of transfected NY-ESO-1 protein was investigated with an anti-HIS Ab in immune fluorescence microscopy. DAPI was used to stain nuclear DNA. One representative out of three experiments is shown. Scale bars, 40 μm. (B) IFN-γ secretion by the NY-ESO-1157–170–specific HLA-DP4–restricted CD4+ T cell clone in response to the NY-ESO-1–positive HLA-DP4–positive melanoma cell lines M6 and M29 and as a negative control to the NY-ESO-1–negative HLA-DP4–positive melanoma cell line M199. Secreted IFN-γ was assessed by ELISA assays from the coculture supernatant. Melanoma–T cell cocultures were performed at the indicated E:T cell ratios. The data represent the mean of four independent experiments. The y-axis represents the relative percentage of IFN-γ secretion reported to the maximal secretion of IFN-γ of the CD4+ T cell clone, pulsed with the cognate peptide only (10 μg/ml) and set to 100%. (C) As in (B), but targets were M6 melanoma cells treated overnight with leupeptin (LEU), brefeldin A (BFA), CQ, and lactacystin (LACT). The data represent the mean of three independent experiments. Statistical analysis was performed using an unpaired nonparametric t test (*p < 0.05, **p < 0.01). (D and E) Inhibition of lysosomal proteases leads to the accumulation of NY-ESO protein: (D) Western blot analysis of NY-ESO-1 levels in untreated (Control) M6 melanoma cells or after 24 h of treatment with different inhibitors: 50 μmol CQ, E64 at 50 and 25 μmol, and lactacystin (LACT) at 5 μmol. The data represent one of two experiments. The high molecular mass bands marked with an asterisk (*) are proteins that cross-react with the NY-ESO-1 antiserum. (E) Immunofluorescence analysis of M6 melanoma cell lines transfected with the MYC-HIS–tagged NY-ESO-1 plasmid and treated for 24 h with E64 and lactacystin. DAPI was used to stain nuclear DNA. Scale bars, 40 μm.
In parallel, the use of two different proteasome inhibitors, the lactacystin at 5, 10, and 20 μmol (Fig. 1C, Supplemental Fig. 1A, 1B) or the epoxomycin at 5 nmol (Supplemental Fig. 1C), did not significantly change the Ag-presentation assay. The use of these two inhibitors at a higher concentration was not possible due to their toxicity. In addition, we tested a third proteasome inhibitor, MG132, which was toxic to our cells, even at low concentrations (Supplemental Fig. 1D), and therefore could not be used in our Ag-presentation assay.
In confirmation with these Ag-presentation data, we observed by Western blot and immune fluorescence analysis the accumulation of NY-ESO-1 protein in M6 melanoma cells upon treatment with inhibitors of lysosomal degradation such as CQ and more strongly with E64 treatment, a specific inhibitor of cysteine proteases. In parallel, treatment of melanoma cells with lactacystin, an inhibitor of the proteasome, did not accumulate NY-ESO-1 protein in these cells (Fig. 1D, 1E, Supplemental Fig. 1A). Therefore, cathepsins of the cysteine protease family are involved in processing NY-ESO-1 Ag for MHC class II presentation.
In conclusion, from this first set of experiments, we could demonstrate that the NY-ESO-1 protein is a cytosolic Ag that is degraded by cysteine proteases in lysosomes. We also demonstrated that MHC class II presentation of the NY-ESO-1157–170 epitope requires newly synthesized MHC class II molecules and lysosomal processing.
Macroautophagy is not involved in NY-ESO-1157–170 processing
In a second set of experiments, we were interested in the endogenous pathway by which a cytosolic tumor Ag, namely NY-ESO-1, gains access to the lysosomal degradation pathway for MHC class II presentation. Two main autophagic pathways, which deliver cytoplasmic constituents for lysosomal degradation, have been described for intracellular Ag processing toward MHC class II presentation. These are macroautophagy and chaperone-mediated autophagy (16).
To test whether the NY-ESO-1 epitope was delivered to the lysosomal compartment by macroautophagy, we first investigated if melanoma cell lines exhibit constitutive formation of autophagosomes, the cell organelles used by this pathway to deliver cargo to lysosomes. To quantify macroautophagy, we made use of the specific autophagosome marker Atg8, of which LC3 is one of the mammalian protein isoforms. LC3 is an ubiquitin-like protein that is covalently coupled via its C terminus to a phospholipid in the newly forming inner and outer autophagosomal membranes and thus is specifically incorporated into autophagosomes (17). After fusion of autophagosomes with endosomes or lysosomes, intraluminal LC3 is rapidly degraded by lysosomal proteases. The more autophagosomes that are formed, the more LC3 is degraded in autolysosomes, and therefore, lysosomal turnover of LC3 is a valid measure for macroautophagic activity. To visualize lysosomal turnover of LC3 in melanoma cells by fluorescence microscopy, we transfected four melanoma cell lines (M29, M6, M199, and M45) with a GFP-LC3 fusion construct. GFP-LC3–transfected melanoma cell lines were treated with the lysosomal acidification inhibitor CQ to block lysosomal proteolysis and visualize the accumulation of GFP-LC3 in autolysosomes. As shown in Fig. 2A (top panel), GFP-LC3 strongly accumulated in cytosolic vesicles after 10 h of CQ treatment, suggesting that macroautophagy is constitutively active in melanoma cell lines. We extended this result to endogenous LC3, because we could demonstrate by immunoblotting that lipidated LC3 (LC3-II) accumulates upon CQ treatment of melanoma cell lines (data not shown).
FIGURE 2.
Constitutive macroautophagy of melanoma cells does not process NY-ESO-1 for MHC class II presentation. (A) Top panel, M6 melanoma stably transfected with the macroautophagy reporter construct GFP-LC3 were treated with CQ for 10 h. Bottom panel, M6-GFP-LC3 cells were transiently transfected with a siRNA control or an siRNA-targeting Atg12 (ATG12 SiRNA). Forty-eight hours later, autophagosomes accumulation was analyzed by fluorescence microscopy, after overnight treatment with CQ. DAPI was used to stain nuclear DNA. The data represent one of four independent experiments. Scale bars, 40 μm. (B) IFN-γ secretion by the NY-ESO-1157–170–specific HLA-DP4–restricted CD4+ T cell clone after coculture with M6 melanoma cells transfected with an siRNA control or a specific Atg12 siRNA (ATG12 SiRNA). Cocultures were performed 48 h after siRNA transfection at the indicated E:T ratios. The data represent the mean of four independent experiments. The y-axis represents the relative percentage of IFN-γ secretion reported to the maximal secretion of IFN-γ of the CD4+ T cell clone, pulsed with the cognate peptide only (10 μg/ml) and set to 100%. (C) Western blot analysis of Atg12 level in M6 melanoma cells 48 h posttransfection either with an siRNA control (si control) or a specific Atg12 siRNA (si ATG12).
Having established that macroautophagy is an active process in melanoma cell lines, we hypothesized that NY-ESO-1 Ag could access MHC class II compartment by delivery via autophagosomes. To test this hypothesis, we transfected the target melanoma cells with an siRNA specific for Atg12, another essential gene for autophagosome formation. As shown in Fig. 2A (bottom panel), this treatment reduced autophagosome accumulation in M6 melanoma cell lines treated with CQ. We also verified the downregulation of Atg12 expression by Western blot analysis (Fig. 2C). However, NY-ESO-1–specific CD4+ T cell recognition was not affected by blocking autophagosome formation in M6 melanoma cells (Fig. 2B). These findings suggest that melanoma cells constitutively perform macroautophagy, but that this pathway does not contribute to NY-ESO-1 processing for MHC class II presentation.
Chaperone-mediated autophagy does not process NY-ESO-1 for MHC class II presentation
Chaperone-mediated autophagy has been found to deliver endogenous autoantigens for MHC class II Ag processing (18). In this pathway, proteins that carry a KFERQ sequence motif bind to cytosolic chaperones including HSC70 and HSC90 members. The complex then docks to LAMP2a, which assists in the translocation of the substrate into the lysosomal lumen with the assistance of a lysosomal HSC70 member (17). By analyzing the NY-ESO-1 amino acid sequence, we could not detect a classical KFERQ like motif. Nevertheless, we investigated if this pathway could be involved in NY-ESO-1 processing for MHC class II presentation by using three different strategies. First, to achieve a loss-of-function experiment, we aimed to inhibit the expression of LAMP2a. Downregulation of LAMP2a expression by RNA interference (Fig. 3A) in M6 melanoma cells did not affect their recognition by the NY-ESO-1–specific CD4+ T cell clone (Fig. 3B). In parallel, to achieve a gain-of-function experiment, we overexpressed LAMP2a. However, this treatment did not improve NY-ESO-1157–170 processing for MHC class II presentation (Fig. 3C). Finally, we overexpressed a KFERQ-GFP construct to inhibit chaperone-mediated autophagy by substrate competition and therefore cause diminished translocation of other substrates of this pathway. Indeed, it had been shown that substrate proteins for chaperone-mediated autophagy show saturable binding to LAMP2a (19). However, overexpression of KFERQ-GFP did not affect the MHC class II presentation of the NY-ESO-1157–170 epitope (Fig. 3D). Taken together, these results suggest that NY-ESO-1 is not processed for MHC class II presentation by chaperone-mediated autophagy.
FIGURE 3.
NY-ESO-1 is not processed by chaperone-mediated autophagy for MHC class II presentation. (A) Western blot analysis of RNA silencing of Lamp2A in M6 melanoma cells at 48 h posttransfection. Different siRNA against Lamp2a were tested: Oligo N1, Oligo N2, Oligo N3, or a mix of N1 + N2 + N3. A scramble siRNA was used as a negative control. One representative out of three experiments is shown. (B) IFN-γ secretion by the NY-ESO-1157–170–specific HLA-DP4–restricted CD4+ T cell clone after coculture with M6 melanoma cells transfected with an siRNA specific for Lamp2A (Oligo N1) or with a control siRNA (Ctl SiRNA). Cocultures were performed 48 h after siRNA transfection at the indicated E:T ratios. The data represent one of four independent experiments. (C) IFN-γ secretion by the NY-ESO-1157–170–specific HLA-DP4–restricted CD4+ T cell clone after coculture with M6 melanoma cells transfected with Lamp2A, Lamp2B, or a control plasmid at the indicated E:T ratios. The data represent the mean of three independent experiments. Left panel, y-axis represents INF-γ secretion in picograms per milliliter. Right panel, y-axis represents the relative percentage of IFN-γ secretion reported to the maximal secretion of IFN-γ of the CD4+ T cell clone, pulsed with the cognate peptide only (10 μg/ml) and set to 100%. (D) IFN-γ secretion by the NY-ESO-1157–170–specific HLA-DP4–restricted CD4+ T cell clone after coculture with M6 melanoma cells transfected with a plasmid encoding for the chaperone-mediated autophagy substrate KFERQ-GFP or for the GFP only. Cocultures were performed at the indicated E:T ratios. One representative of two experiments is shown. Left panel, y-axis represents INF-γ secretion in picograms per milliliter. Right panel, y-axis represents the relative percentage of IFN-γ secretion reported to the maximal secretion of IFN-γ of the CD4+ T cell clone, pulsed with the cognate peptide only (10 μg/ml) and set to 100%.
Melanoma cells can present NY-ESO-1157–170 after intercellular Ag transfer
Because we ruled out macroautophagy and chaperone-mediated autophagy, the two most commonly discussed pathways in Ag processing for MHC class II presentation of endogenous cytosolic Ags, we next investigated if melanoma cells can take up Ag from neighboring cells for presentation on MHC class II molecules. To test this hypothesis, we first investigated if melanoma cells can have enough phagocytic activity for this pathway to be functional. The melanoma cell lines M6 and M199 were tested for their capacity to engulf fluorescent latex beads. Because beads contain a fixed number of fluorescent equivalents on their surface, the number of ingested beads per cell can be readily determined by flow cytometry. As shown in Fig. 4A, melanoma cells displayed a moderate phagocytic activity, with most cells taking up 1 to 2 beads (first peak 12% of the cells for a ratio of 100 beads/cell and 18% of the cells for an initial ratio of 500 beads/cell).
FIGURE 4.
NY-ESO-1 Ag is presented on MHC class II molecules of melanoma cells after intercellular Ag transfer. (A) The melanoma cell line M6 was cocultured for 2 h with the indicated stoichiometric ratios of FITC-coupled beads. Bead uptake was evaluated by flow cytometry. One representative of two experiments is shown. (B) IFN-γ secretion by the NY-ESO-1157–170–specific HLA-DP4–restricted CD4+ T cell clone after coculture with M199 cells pulsed for 24 h: with MHC class II–negative A549 necrotic cells transfected with NY-ESO-1 (A549-NY cells), with 5× concentrated supernatant from the corresponding A549 cells transfected with NY-ESO-1 (A549-NY SN), or with 5× concentrated supernatant from M6 melanoma cells endogenously expressing NY-ESO-1 (M6 SN). Data represent the mean of two independent experiments. (C) IFN-γ secretion by the NY-ESO-1157–170–specific HLA-DP4–restricted CD4+ T cell clone after coculture with M29 melanoma cell line pulsed with A549 necrotic cells transfected with a control plasmid or NY-ESO-1 plasmid. One representative of two experiments is shown. (D) IFN-γ secretion by the NY-ESO-1157–170–specific HLA-DP4–restricted CD4+ T cell clone, after coculture with M6 melanoma cell line transfected with the dominant-negative (DN) Rab7 T22N or with the wild-type (WT) Rab7, at the indicated E:T cell ratios. One representative of two experiments is shown. *p < 0.05.
We therefore decided to investigate if melanoma cells can engulf necrotic NY-ESO-1–expressing cells and process the NY-ESO-1157–170 epitope from endocytosed material for MHC class II presentation. We used necrotic human lung epithelial A549 cells, transfected with NY-ESO-1. M199 melanoma cells (DP4-positive NY-ESO negative) were then pulsed with freeze-thawed A549 NY-ESO-1–transfected cells at a 1:1 ratio. The A549 cells used in this assay as a source of necrotic material do not express MHC class II molecules even after IFN-γ stimulation, preventing them from directly presenting Ags to NY-ESO-1–specific CD4+ T cells. In parallel, we cocultured M199 cells with 5-fold concentrated supernatants of A549 NY-ESO-1–transfected cells or with 5-fold concentrated supernatants of M6 cells (expressing the NY-ESO-1 at an endogenous level). We found that NY-ESO-1–negative HLA-DP4–positive M199 melanoma cells present the NY-ESO-1157–170 epitope to CD4+ T cells after pulsing either with A549 NY-ESO-1 necrotic cells or with their concentrated supernatant (Fig. 4B). In contrast, the culture of M199 melanoma cells for 24 h with 5-fold concentrated supernatant of M6 cells did not significantly improve the recognition of M199 cells by the NY-ESO-1 157–170–specific clone (Fig. 4B).
In parallel, M29 and M6 melanoma cells, endogenously expressing NY-ESO-1, presented the NY-ESO-1157–170 epitope more efficiently upon overnight pulsing with necrotic NY-ESO-1–positive A549 cells (Fig. 4C). In parallel, we could replicate these results by coculturing overnight live A549-transfected cells with the M6 and M29 cell lines (data not shown), indicating that Ag transfer does not require active necrosis or apoptosis induction in the donor cells.
Finally, to test if we could block NY-ESO-1 presentation after intercellular Ag transfer in melanoma cells, we used a dominant-negative Rab7 mutant (Rab7 T22N). Rab7 is a small GTPase essential for fusion of late endosomes with lysosomes. Therefore, Rab7 is crucial for the delivery of phagocytosed Ags to lysosomal compartments, inducing the MHC class II loading compartment (20). We found that transfection of melanoma cells with a dominant-negative form of Rab7 (T22N) significantly reduced MHC class II presentation of NY-ESO-1157–170 to CD4+ T cells compared with targets transfected with the wild-type form of Rab7 (Fig. 4D). Therefore, melanoma cells can take up extracellular Ags via endocytosis and process them in their endosomal compartment for MHC class II presentation through a Rab7-dependent pathway.
Targeting NY-ESO-1 to autophagosomes significantly improves CD4+ T cell recognition of the NY-ESO-1157–170 epitope
Because endogenous NY-ESO-1 does not get processed via macroautophagy for MHC class II presentation, we investigated if its targeting to autophagosomes could enhance MHC class II presentation of NY-ESO-1. For this purpose, we engineered a fusion protein by coupling Atg8/LC3 to the C terminus of NY-ESO-1 (NYESO-LC3). Because Atg8/LC3 is degraded with the inner autophagosomal membrane in lysosomes, this should deliver NY-ESO-1 for lysosomal degradation and potentially enhance its MHC class II presentation. Indeed, NYESO-LC3, transfected into M199 cells (Fig. 5A), was 5–10-fold more efficiently presented to NY-ESO-1–specific CD4+ T cells compared with wild-type NY-ESO-1 (Fig. 5B). Thus, presentation of NY-ESO-1 on MHC class II molecules to CD4+ T cells can be significantly enhanced by targeting this Ag to autophagosomes.
FIGURE 5.
MHC class II presentation of NY-ESO-1 can be enhanced by targeting to autophagosomes. (A) Western blot analysis of NY-ESO-1 expression in M199 cells, 24 h after transfection with the wild-type NY-ESO-1 or with the autophagosome-targeted NY-ESO-1–LC3 fusion protein. (B) NY-ESO-1157–170–specific recognition of M199 melanoma cells transfected with autophagosome-targeted (NY-ESO-1-LC3) or wild-type (NY-ESO-1) Ag at the indicated E:T cell ratios. One representative experiment out of three is shown. Statistical analysis was performed using an unpaired nonparametric t test.
Discussion
The antitumoral CD4+ T cells response to melanoma Ags is attracting more and more interest, because of the need to improve immunotherapeutic protocols that are exclusively based on antitumoral CD8+ T cell responses. Indeed, in different experimental setups, it was shown that an exclusive tumor Ag-specific CD8+ T cell response in the absence of an antitumoral CD4+ T cell response fails to establish long-lasting T cell memory (21, 22). In addition, the secondary loss of antitumoral CD4+ T cells compromises secondary antitumor responses after a successful primary immunotherapy (23), reinforcing the idea of a necessary CD4+ T cell help.
In this context, understanding how CD4+ T cell epitopes are processed in tumor cells is of high interest to improve Ag presentation of MHC class II–restricted tumor-specific T cell epitopes for direct CD4+ T cell recognition in the tumor microenvironment.
Melanoma cells have been shown to present tumor Ags on MHC class II molecules (24–26), but the processing pathways of the respective ligands are often not fully understood and have not extensively been explored. In this study, we have identified a new pathway of Ag presentation of NY-ESO-1–derived MHC class II epitopes in melanoma cells. We have shown that melanoma cells can acquire this Ag by intercellular transfer and process it for MHC class II presentation. So far, different endogenous pathways for Ag processing of MHC class II epitopes have been described in nonprofessional APCs (19, 27–29).
We have ruled out two different autophagic pathways for NY-ESO-1 Ag presentation onto MHC class II molecules. In parallel, we did not find any evidence in our setup for the use of the MHC class I Ag-processing machinery (proteasome and TAP transporter) in NY-ESO-1 presentation on MHC class II molecules.
Unexpectedly, our findings identified the classical MHC class II Ag processing pathway to be involved in MHC class II presentation of NY-ESO-1 after its intercellular transfer. Indeed, we could show that this processing is dependent on newly synthesized MHC class II molecules because it is sensitive to brefeldin A. In parallel, the use of lysosomal inhibitors and serine protease inhibitors completely abrogated the Ag presentation of this epitope. Finally, the requirement for Rab7 revealed that MHC class II presentation of NY-ESO-1 requires endosomal maturation.
How do melanoma cells acquire Ags from neighboring cells or from necrotic material for the classical MHC class II Ag-processing pathway? The first possibility is the transfer of Ag via phagocytosis. The progression of melanoma cells toward the metastatic phenotype has been found to be associated with the acquisition of a phagocytic phenotype (30). Indeed, ezrin, a cytoskeleton-associated protein involved in “metastasis-related functions” (migration, capacity, phagocytic activity, and vesicular sorting), has been shown to enhance the phagocytic capacity of metastatic melanoma cells (31). This could render these melanoma cells more susceptible to CD4+ T cell recognition. A second possibility that could explain the transfer of NY-ESO-1 between melanoma cells is the melanosome transfer pathway, releasing these organelles for uptake by neighboring cells (32). Although NY-ESO-1 does not have a classical melanosome targeting sequence, we cannot completely rule out the presence of NY-ESO-1 in melanosomes. A third possibility could be the existence of gap junctions between melanocytes, gap junctions having already been implicated in intercellular Ag transfer (33). However, connexin 43 expression, the protein involved in gap junctions, is normally lost during melanoma progression. Finally, one last possibility could be that NY-ESO-1 protein is secreted in the extracellular milieu and endocytosed through a plasma membrane receptor. Indeed melanoma cells could efficiently present the Ag after coculture with cell-free concentrated supernatant. This last pathway was previously described for EBNA proteins in EBV-transformed lymphoblastoid cell lines (29).
Overall, we have shown that melanoma cells have a moderate phagocytic capacity and can acquire NY-ESO-1 Ag by intercellular transfer, but the precise mechanism underlying this transfer still needs to be characterized. In addition, the precise nature of the transferred Ag could be multiple because its source could be necrotic cells, live cells, or soluble cell-free supernatant.
Other studies have focused on the pathway of naturally processed MHC class II epitopes from NY-ESO-1 in tumor cells. Indeed, one group has identified the NY-ESO-195–106 DR01-restricted epitope as being processed by a proteasome and HSP90-dependent pathway (34). In the same line, the same group has identified the minimal NY-ESO-1159–170 epitope to be processed by a proteasomal/TAP-dependent and HSP90-independent pathway, in the context of ovarian cancer (35). In our setup, Ag presentation of the NY-ESO-1157–170 DP4-restricted epitope by the M6 or M199 melanoma cell lines was independent of the proteasome because the use of two different proteasome inhibitors (lactacystin and epoxomycin) did not impact its processing.
Therefore, different NY-ESO-1–derived CD4+ T cell epitopes might be processed by different pathways onto MHC class II molecules, and these pathways may vary depending on the tumor type.
The consequence of these unconventional Ag-processing pathways could promote different immune responses. One deleterious consequence is the possibility to generate Ag-specific CD4+ T cells that are anergic or of a regulatory phenotype. Because melanoma cells are not professional APCs, they lack some costimulatory molecules at their surface or preferentially express a subset of them involved in the induction of a regulatory T cell (Treg) phenotype. Indeed, a subset of melanoma cells, malignant melanoma initiating cells, has been shown to preferentially express B7.2 and programmed cell death-1, as well as MHC class II molecules and to be associated with the induction of tumor Ag–specific Tregs (36). Thus, tumor cells could, for example, convert CD4+CD25− T cells into Foxp3+CD4+CD25high cells (Tregs) by presenting MHC class II epitopes at their cell surface, leading to tumor evasion.
In contrast, one possible positive consequence of unconventional Ag presentation pathways is effective tumor immune surveillance. Indeed, if effector cytotoxic CD4+ T cells can directly recognize melanoma cells, they can actively participate in tumor eradication. This was particularly demonstrated for the NY-ESO-1157–170 epitope of interest in one case report for a patient having a metastatic melanoma for whom the exclusive infusion of NY-ESO-1157–170–specific CD4+ T cells resulted in tumor regression (12, 37). In this context, understanding in more detail how this epitope is presented at the cell surface of tumor cells could lead to the developing of new strategies to enhance tumor recognition and immune surveillance.
Supplementary Material
Acknowledgments
We thank Dr. Chae Gyu Park and Dr. Cheolho Cheong for great help in designing the cloning strategy for the following construct: the NY-ESO-1 Myc-His Tag plasmid. We also thank Dr. Monica Lee for help in designing the NY-ESO-1/LC3 construct.
This work was supported in part by the National Cancer Institute (R01CA108609) and the Swiss National Science Foundation (310030_126995). M.G. is supported by the Institute of Arthritis Research and the Carlos and Elsie de Reuter Foundation.
The online version of this article contains supplemental material.
- CQ
- chloroquine
- LAMP2A
- lysosome-associated membrane protein 2 isoform A
- LC3
- L-chain 3B
- siRNA
- small interfering RNA
- Treg
- regulatory T cell.
Disclosures
The authors have no financial conflicts of interest.
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