HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Erfurt, C. Articles by Schultz, E. S. Search for Related Content PubMed PubMed Citation Articles by Erfurt, C. Articles by Schultz, E. S.

Tumor-Reactive CD4⁺ T Cell Responses to the Melanoma-Associated Chondroitin Sulphate Proteoglycan in Melanoma Patients and Healthy Individuals in the Absence of Autoimmunity¹

Cornelia Erfurt^*, Zhaojun Sun, Ina Haendle^*, Beatrice Schuler-Thurner^*, Carlo Heirman, Kris Thielemans, Pierre van der Bruggen, Gerold Schuler^* and Erwin S. Schultz^2,

^* Department of Dermatology, University Hospital of Erlangen, Hartmannstrae 14, Erlangen, Germany; Ludwig Institute for Cancer Research, Brussels, Belgium; Laboratory of Physiology, Medical School, Free University of Brussels, Brussels, Belgium; and Department of Dermatology and Allergology, University Hospital Giessen and Marburg, Deutschhausstrae 9, Marburg, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
To avoid immune escape by down-regulation or loss of Ag by the tumor cells, target Ags are needed, which are important for the malignant phenotype and survival of the tumor. We could identify a CD4⁺ T cell epitope derived from the human melanoma-associated chondroitin sulfate proteoglycan (MCSP) (also known as high m.w.-melanoma-associated Ag), which is strongly expressed on >90% of human melanoma lesions and is important for the motility and invasion of melanoma cells. However, MCSP is not strictly tumor specific, because it is also expressed in a variety of normal tissues. Therefore, self tolerance should prevent the induction of strong T cell responses against these Ags by vaccination strategies. In contrast, breaking self tolerance to this Ag by effectively manipulating the immune system might mediate antitumor responses, although it would bear the risk of autoimmunity. Surprisingly, we could readily isolate CD4⁺ Th cells from the blood of a healthy donor-recognizing peptide MCSP_693–709 on HLA-DR11-expressing melanoma cells. Broad T cell reactivity against this Ag could be detected in the peripheral blood of both healthy donors and melanoma patients, without any apparent signs of autoimmune disease. In some patients, a decline of T cell reactivity was observed upon tumor progression. Our data indicate that CD4⁺ T cells are capable of recognizing a membrane glycoprotein that is important in melanoma cell function, and it may be possible that the sizable reactivity to this Ag in most normal individuals contributes to immune surveillance against cancer.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
On our search for a suitable target Ag for active-specific immunotherapy of melanoma, we focused in the present study on the human melanoma-associated chondroitin sulfate proteoglycan (MCSP),³ which is uniformly expressed on >90% of human melanoma lesions and cultured cells, and likely important for the malignant phenotype (1). MCSP represents a unique glycoprotein-proteoglycan complex, with a 250 kDa core glycoprotein to which, via serine residues, the >450 kDa proteoglycan component is attached (2). MCSP has been implicated for some time in numerous aspects of melanoma cell biology, including adhesion, spreading, and migration. In fact, melanoma cell adhesion, chemotactic responses to fibronectin, and cytoplasmatic spreading on extracellular matrix proteins have been shown to be inhibited by Abs in vitro (3, 4, 5). In addition, NG2, the rat homologue of MCSP, has been found to act as a cell-surface receptor anchoring type VI collagen to the cell surface, and by this way promoting cell spreading and cell motility (6). Engagement of NG2 can trigger the cytoskeletal rearrangements necessary for changes in cell morphology and motility (7). Association of NG2 with CD44 and ₄₁ integrin, cell-surface components involved in melanoma progression, mediates an anti-adhesive effect on melanoma cells and, therefore, increases the metastatic potential of melanoma cells (8). The ability of MCSP to enhance the migratory potential of melanoma cells is further supported by the finding that expression of MCSP is restricted to cell surface microspike domains on migrating melanoma cells in vitro (9).

Furthermore, MCSP has been shown to form a complex with membrane-type 3 matrix metalloproteinase (MT3-MMP), which may be a crucial step in activating MT3-MMP to promote melanoma cell invasion (10). Recent studies have defined a model in which MCSP binds MT3-MMP on the cell surface, resulting in increased proteolysis of the extracellular matrix and tumor invasion (11).

Meanwhile, the signal transduction pathways by which MCSP mediates its influence on cell adhesion, migration, and invasion have been analyzed in more detail. It appears that MCSP indirectly enhances FAK activation by an integrin-dependent mechanism and, in addition, causes activation of ERK, which may increase the proliferation of melanoma cells (12). Finally, there is accumulating evidence from in vitro data that NG2/MCSP plays an important role in tumor angiogenesis. Thus, NG2-positive tumors have been found to have significantly increased neovascularization rates and vascular volumes, and NG2 has been shown to sequester angiostatin, which normally inhibits endothelial cell proliferation and angiogenesis (13, 14).

With respect to the functional role that MCSP seems to play in the oncogenic behavior of melanoma cells, it is not surprising that MCSP is broadly expressed on >90% of melanoma lesions (15). However, MCSP expression has also been described in a variety of normal tissues (e.g., endothelial cells, activated pericytes, chondrocytes, smooth muscle cells, certain basal keratinocytes within the epidermis, as well as cells within the hair follicles) (16).

MCSP-targeted T cell-based immunotherapy, therefore, faces two problems. First, the induction of T cell responses by vaccination should be hampered by pre-existing tolerance. Second, in the case of induced T cell responses, autoimmunity might be a potential and serious drawback.

However, we could readily isolate CD4⁺ Th type 1 cells from the blood of a healthy donor that recognizes a peptide located in the core protein of MCSP on melanoma cells. Unequivocal T cell reactivity against this peptide could be detected in the peripheral blood of both healthy donors and melanoma patients in the absence of the clinical signs of autoimmunity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Experiments were performed with cells from human healthy donors and melanoma patients after having obtained informed consent and approval by the ethics committee of the Medical Faculty of the University of Erlangen-Nuremberg.

Cell lines, reagents, and Abs

EBV-transformed B (EBV-B) cell lines and tumor cell lines were cultured in RPMI 1640 medium (Cambrex) supplemented with 10% FCS (PAA Laboratories), 20 µg/ml gentamicin (PAA Laboratories), 2 mM L-Glutamine (PAA Laboratories), 10 mM HEPES (PAA Laboratories), and 10 mM sodium pyruvate (PAA Laboratories). Dendritic cells (DC) and CD4⁺ T cells were cultured in the same medium supplemented with 1% autologous plasma or 10% human serum, hereafter referred to as DC or T cell medium, respectively. Human IL-2 was purchased from Roche Diagnostic Systems; IL-4, IL-1, IL-6, and GM-CSF from Strathmann; IL-7 from BIOMOL; TNF- from Bender MedSystems; and PGE₂ from Pfizer. The following mouse anti-human mAbs were used for blocking experiments: anti-HLA-DQ (SPVL3) from Immunotech and anti-HLA-DP (B7/21) and anti-HLA-DR (L234) from BD Biosciences. For flow cytometric analysis, the following conjugated Abs were used: anti-CD4 (RPA-T4), anti-IFN- (25723.11), and respective mouse and rat isotype controls (all from BD Biosciences).

Peptides

Peptide LAQGSAMPILPANLSVETNAVGQDVSVLFRVTGALQFGELQK = MCSP_673–714 was selected by using a database, allowing the prediction of T cell epitopes for a given HLA class I or II molecule ((17); SYFPEITHI-Database: www.uni-tuebingen.de/uni/kxi/). The chosen peptide of 42 amino acid length (aa 673–714) covers most of the predicted HLA-DR binding motifs within the sequence of the MCSP core protein. All peptides were synthesized by conventional solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (F-moc) for transient NH₂-terminal protection and were characterized using mass spectrometry.

The pMFG Retrovirus encoding human invariant chain (Ii)-MCSP

A cDNA encoding aa 659–735 of the MCSP core protein (provided by Dr. G. Pluschke, Swiss Tropical Institute, Basel, Switzerland). was ligated downstream of the first 80 amino acids of the Ii, followed by an internal ribosomal entry site sequence derived from the encephalomyocarditis virus and by the sequence encoding a truncated form of the human low-affinity nerve growth factor receptor. Cell lines were also transduced with Ii-MAGE-3 as a control, as described previously (18).

DCs and CD4⁺ responder cells

DCs were generated from leukapheresis products from donor 4800, as described previously (19). In brief, PBMC were obtained by Ficoll density gradient centrifugation, and monocytes were isolated by plastic adherence and cultured in the presence of 800 U/ml GM-CSF and 250 U/ml IL-4 for 6 days to generate immature, monocyte-derived DCs. Maturation was induced by adding a cytokine mixture consisting of 400 U/ml IL-1, 1000 U/ml IL-6, 10 ng/ml TNF-, and 1 µg/ml PGE₂ (20). Mature DCs were harvested on day 7. CD4⁺ T lymphocytes were isolated from PBMC by negative selection using anti-CD8⁺, anti-CD14⁺, and anti-CD19⁺ mAbs coupled to magnetic microbeads (Miltenyi Biotec).

Generation of peptide-specific CD4⁺ T cell clones

Autologous immature DCs were loaded overnight with 10 µg/ml MCSP_673–714 peptide and matured after 6 h by adding a cytokine mixture, as mentioned above. CD4⁺ T cells (1 x 10⁵) were cocultured with 1 x 10⁴ peptide-loaded DCs in 100 µl T cell medium/round-bottom microwell, supplemented with IL-6 (1000 U/ml), IL-12 (20 ng/ml), and TNF- (5 ng/ml). The CD4⁺ T lymphocytes were restimulated on days 7, 14, and 21 with autologous DCs freshly loaded with peptide MCSP_673–714 overnight (10 µg/ml) and matured by the cytokine mixture, adding IL-2 (10 U/ml) and IL-7 (5 ng/ml). Aliquots of each microculture (4,000 cells) were assessed on day 30 for their capacity to produce IFN- when stimulated with 15,000 autologous EBV-B cells that were loaded overnight with 10 µg/ml of peptide MCSP_673–714 or with a 40 amino acid-peptide derived from MAGE-12 protein as a negative control. After 20 h of coculture in round-bottom microwells in T cell medium supplemented with IL-2 (25 U/ml), IFN- released in the supernatant was measured by ELISA using reagents from MedGenix. Microcultures that were tested positive were then cloned by limiting dilution, using irradiated, autologous, peptide-loaded EBV-B cells (10⁴ cells/round-bottom microwell) as stimulator cells and irradiated allogeneic LG2-EBV-B cells (10⁴ cells/well) as feeder cells in the presence of IL-2 (50 U/ml), IL-4 (5 U/ml), IL-7 (5 ng/ml), and PHA (125 ng/ml; Sigma-Aldrich).

Recognition assays with peptides

CD4⁺ T cells (4 x 10³/microwell) were cocultured with 15 x 10³ peptide-loaded EBV-B cells from donors with different HLA class II typing. Supernatants were harvested after 20 h, and IFN- production was measured by ELISA. To screen a set of truncated peptides, autologous EBV-B cells were incubated for 1 h in the presence of different peptides in decreasing concentrations (3 µg/ml to 0.001 µg/ml). In addition, cytokine secretion was measured in the supernatant after stimulation of the clone with peptide-loaded EBV-B cells by using the Cytometric Bead Assay from BD Biosciences. To identify the HLA restriction of the T cell clones, blocking of the Ag-induced production of IFN- was investigated using mAbs against HLA-DR, HLA-DQ, and HLA-DP. All mAbs were used at a final concentration of 5 µg/ml.

Recognition assay with tumor cells

Several HLA-matched (ER-MEL 3 and ER-MEL 4) or mismatched (MEL 397 and LB 1622 MEL) MCSP-expressing melanoma cell lines were seeded at 20 x 10³/flat-bottom microwell plates and incubated for 48 h to allow the formation of a monolayer. CD4⁺ T cells were then added (4 x 10³/microwell plates) and, after 20 h, IFN- secreted in the supernatants was measured by ELISA.

MCSP expression profile analysis by quantitative real-time PCR (qPCR)

Most total RNA from normal tissues, melanoma lesions, and cell lines was prepared by GITC/CsCl procedure (21), and measured by an OD value at 260 nm. Total RNA from liver, heart, brain, testis, and thymus were from Ambion. Reverse transcription was performed using 1 µg mRNA with oligo(dT18) as primer. Mock cDNA was obtained by omitting reverse transcriptase. Part of the reverse transcription reactions with RNA from normal tissue (1/40) and from tumors (1/400) were used for qPCR (Eurogentec). The primers were as follows: forward, 5' CCT CCg AAA ACg CAA CAA gA (20nt, 6735-6754, NCBI: X96753); reverse, 5' TTg CgA AAg gTC TCg gTg TC (20nt, 6830-6811). Specific 5'-FAM/3'-TAMRA labeled probe’s sequence was ad follows: 5' ACg TCC Agg TCC TgA CTg CCA AgC (24nt, 6767-6780), synthesized by Eurogentec. Thermal cycling and fluorescence monitoring were performed in an Applied Biosystems Prism 7700 sequence detector (Applied Biosystems). PCR conditions were 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. The probe corresponds to the last exon of MCSP, near the 3' end of the reading frame. Mock cDNA was used to detect residual genomic DNA. Eight samples contained residual genomic DNA, and the MCSP data were corrected by subtracting the number of copies of MCSP estimated with the mock cDNA from the number of copies of MCSP estimated with cDNA. 18S rRNA copies were estimated with SYBR green dye to detect the 18S rDNA amplicon accumulation. cDNA reverse transcribed with oligo(dT18) was used as a template, because preliminary experiments indicated that equivalent results were achieved with oligo(dT18) and random primers. A total of 1/20,000 of the cDNA product was taken for each amplification reaction. The primers used were as follows: forward, 5' TCg AAC gTC TgC CCT ATC AA and reverse, 5' gCT ATT ggA gCA ggA ATT ACC. PCR conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 60°C, followed by a one-cycle reaction for melting curve: 95°C for 20 s; 55°C for 20 s; increase from 55°C to 95°C in 20 min; and kept at 95°C for 20 s. The mRNA expression level of MCSP was expressed as relative unit per 10⁵ 18S rRNA copies.

Measurement of MCSP T cell reactivity by ELISPOT analysis

PBMC from healthy blood donors and melanoma patients (after having obtained informed consent) were plated in triplicates at 3 x 10⁵/flat-bottom 96-well in medium containing 10% heat-inactivated human serum, and stimulated with 10 µg/ml of peptide MCSP_693–708. After 20 h, wells were washed and incubated with biotinylated mAb to IFN- (7-B6-1; Mabtech) for 2 h. Final staining and computer-assisted analysis was done as described before (22). Responses were considered significant if a minimum of 10 spot-forming cells per well were detected, and additionally, this number was at least twice that in negative control wells. In addition, PBMC (2.5 x 10⁶ per 24-well) were once stimulated with peptide (10 µg/ml) in the presence of IL-2 (5 U/ml) and IL-7 (10 ng/ml) and the ELISPOT was performed on day 7.

In some experiments, CD4⁺ T cells were isolated from PBMC by negative selection using magnetic microbeads (Miltenyi Biotec) and subsequently divided into CD45RA⁺ and CD45RA^– T cells by positive selection. Both populations were stimulated with peptide for 1 week and tested for IFN- production following antigenic stimulation by ELISPOT as described above.

Intracellular cytokine staining

PBMC from healthy donors were stimulated with 10 µg/ml of peptide MCSP_693–708. On day 7, cells were restimulated with peptide or not or PHA (10 µg/ml) as a positive control in the presence of brefeldin-A (10 µg/ml; Sigma-Aldrich). After overnight stimulation, cells were first stained for CD4 and subsequently fixed with formaldehyde and permeabilized with saponine (fix/perm solution; BD Biosciences) to allow for intracellular staining with an anti-IFN- Ab or isotype control (BD Biosciences). Analysis was done by flow cytometry (FACScan and CellQuest Software, BD Biosciences).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
To identify a CD4⁺ T cell epitope of MCSP, CD4⁺ T cells of a healthy donor were stimulated with autologous DCs loaded with a 42 amino acid-long MCSP fragment (aa 673–714). This approach should allow the identification of naturally processed peptides as the DCs take up the long peptide, process, and present the incorporated T cell epitopes in the groove of their HLA class II molecules.

Generation of CD4⁺ T cell clones

A total of 96 microcultures were set up, each containing CD4⁺ T cells and autologous-stimulator DCs loaded with peptide MCSP_673–714 as stimulator cells. Responder cells were restimulated three times with DCs loaded with peptide and tested 10 days after the last restimulation for IFN- production after contact with autologous EBV-B cells loaded either with peptide MCSP_673–714 or with a control peptide. Three positive microcultures were cloned by limiting dilution. Stably growing T cell clones were obtained from microculture C2. Additional experiments were performed with CD3⁺ CD4⁺ T cell clone C2.25, hereafter referred to as clone 25. Clone 25 specifically recognized autologous EBV-B cells loaded with the MCSP peptide (Fig. 1a).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. CD4+ T cell clone 25 recognizes a MCSP peptide. Autologous EBV-B cells from donor 4800 were loaded overnight with peptide MCSP673–714 or with a control peptide, washed, and used as stimulator cells (a). Alternatively, EBV-B cells were pulsed for 1 h with a panel of truncated peptides (b) or with different concentrations of peptide VGQDVSVLFRVTGALQ (c). IFN- production was measured after overnight coculture by ELISA. Values represent means of triplicate determinations; bars, SD. Finally, CD4+ T cells were stimulated with autologous EBV-B cells loaded with peptide, and after 20 h IFN-, TNF-, IL-4, and IL-10 secreted in the supernatant were measured using a flow cytometry-based multiplexed assay (d).

To determine the core epitope recognized by clone 25 within the 42-mer, a set of 16-mer peptides, overlapping each other by 12 amino acids, was tested for recognition. Clone 25 recognized two overlapping 16-mer peptides, ETNAVGQDVSVLFRVT and VGQDVSVLFRVTGALQ = MCSP_693–708 (data not shown). In a second step, a set of truncated peptides derived from the sequence of the 16-mer peptide recognized best by the clone was tested to define the fine specificity of clone 25. Truncation of either D at the N terminus or A at the C terminus resulted in loss of recognition by the T cell clone (Fig. 1b). Peptide titration experiments with the truncated peptides revealed that the 16-mer peptide VGQDVSVLFRVTGALQ was most efficiently recognized by clone 25. Significant T cell stimulation was only seen when using peptide concentrations >300 nM (Fig. 1c). Peptide recognition was abolished in the presence of an anti-HLA-DR Ab, and testing several EBV-B cell lines with known HLA class II typings revealed that the peptide was presented to clone 25 by HLA-DR11 (data not shown). To analyze the cytokine profile of clone 25, the concentration of different cytokines produced after stimulation with the 16-mer peptide was measured using a flow cytometry-based multiplexed assay. Clone 25 showed a TH1 cytokine profile producing IFN- and TNF-, but no IL-4 and only low level of IL-10 (Fig. 1d).

The antigenic peptide is naturally processed and presented

Importantly, clone 25 did not only recognize EBV-B cells loaded with peptide, but also HLA-DR11⁺ EBV-B cells transduced with a retroviral construct, retro-IiMCSP, which encodes a truncated Ii-fused with MCSP, demonstrating that recognition was really not directed against a contaminant in the batch of peptide (Fig. 2). EBV-B cells transduced with retro-IiMAGE-3, as a negative control, were not recognized.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. The MCSP peptide is naturally processed and presented. EBV-B cell lines (4800-EBV, autologous HLA-DR11+ EBV B cells; MVGS EBV, allogeneic HLA-DR11+ EBV B cells; and MMDH-EBV, allogeneic HLA-DR11-EBV-B cells) were transduced with a retrovirus coding for an Ii-MCSP fusion protein or an Ii-MAGE-3 protein as a control. Clone 25 was cocultured with stimulator cells for 20 h, and IFN- production by CD4+ T cells was measured after overnight coculture by ELISA. Values represent means of triplicate determinations; bars, SD.

View larger version (11K): [in this window] [in a new window]   FIGURE 3. Direct recognition of tumor cells. MCSP-positive melanoma cell lines expressing HLA-DR11 or not were used to stimulate clone 25. IFN- production by CD4+ T cells was measured after overnight coculture by ELISA. Values represent means of triplicate determinations; bars, SD.

As the expression profile of tumor Ags is of crucial interest with respect to their clinical use in immunotherapy qPCR for MCSP was established to systematically analyze the mRNA expression profile in a set of malignant and normal tissues (Fig. 4). As expected, we found a strong mRNA expression in melanoma cell lines and metastases from cutaneous and uveal melanoma, but there was also a significant, albeit lower, expression in normal tissues such as lung, heart, kidney, and thymus. It should be mentioned that these data reflect the expression of mRNA in the tested tissues, which does not necessarily correlate with the protein expression.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Expression profile of MCSP. Oligo(dT18) primer was used to reverse transcribe total RNA. Expression of MCSP was expressed as relative unit per 105 18S rRNA copies. MCSP expression of samples (*) containing residual genomic DNA was corrected as described in Materials and Methods.

Given the expression of MCSP in normal tissues, peripheral T cell tolerance against this Ag might impede the induction of Ag-specific T cell responses. However, we could readily generate T cells from the blood of a healthy donor who did not show any signs of autoimmune disease. Therefore, we set out to detect the T cell reactivity against the identified epitope in the blood of healthy individuals and melanoma patients by ELISPOT analysis. Although, we could not detect ex vivo responses, after one in vitro stimulation, 12 of 14 tested healthy donors showed significant IFN- secretion upon peptide stimulation exceeding >300 spots in six donors (Fig. 5). To confirm the T cell responses detected by ELISPOT, and to characterize them in more detail, we additionally performed intracellular cytokine staining. Fig. 6 shows the IFN- production by CD4⁺ T cells from two healthy individuals upon stimulation with the MCSP_693–708 peptide. To further define the origin of MCSP-reactive T cells, CD4⁺ T cells from healthy donors were isolated and subsequently separated in CD45RA⁺ and CD45RA^– cells by use of magnetic beads. We could detect MCSP reactivity by ELISPOT analysis only in the CD45RA^– population, suggesting that these T cells descend from the memory pool (Fig. 7). In a supplemental experiment, we tried to estimate the precursor frequency of MCSP-reactive CD4⁺ T cells in the blood of a healthy donor. We found 8 of 96 microwells (each set up with 1 x 10⁵ PBMC) showing T cell reactivity after 1 week of peptide stimulation, suggesting a precursor frequency of at least 8 x 10^–7 PBMC or 1.6–2.6 x 10^–6 CD4⁺ T cells (data not shown).

View larger version (39K): [in this window] [in a new window]   FIGURE 5. T cell reactivity in healthy donors. PBMC from healthy donors were stimulated with peptide MCSP693–708, and after 1 week, IFN- production upon peptide stimulation was measured by ELISPOT. Responses were considered significant if a minimum of 10 spot forming cells per well were detected, and additionally, this number was at least twice that in negative control wells. Values represent means of triplicate determinations.

View larger version (38K): [in this window] [in a new window]   FIGURE 6. Intracellular IFN- staining of PBMC from healthy donors stimulated with the MCSP.DR11 peptide. PBMC from two healthy donors (A and B) were stimulated with peptide MCSP693–708, and after 1 week, IFN- production by PBMC upon restimulation with peptide or PHA as a positive control was analyzed by intracellular cytokine staining and flow cytometric analysis.

View larger version (12K): [in this window] [in a new window]   FIGURE 7. MCSP reactive T cells descend from the CD45RA– memory pool. CD4+ T cells from healthy donors were divided into CD45RA+ and CD45RA– fractions by magnetic bead sorting and stimulated with peptide MCSP693–708. After 1 week, IFN- production upon stimulation with peptide or not was detected by ELISPOT.

View larger version (35K): [in this window] [in a new window]   FIGURE 8. T cell reactivity in melanoma patients. PBMC from melanoma patients were stimulated with peptide MCSP693–708, and after 1 week, IFN- production upon peptide stimulation was measured by ELISPOT. Responses were considered significant if a minimum of 10 spot forming cells per well were detected, and additionally, this number was at least twice that in negative control wells. Values represent means of triplicate determinations.

View larger version (18K): [in this window] [in a new window]   FIGURE 9. MCSP reactivity is declining upon tumor progression. The course of the T cell reactivity against the MCSP.DR11 peptide is demonstrated by serial ELISPOT analysis for patients 40L-58 (a) and 40L-54 (b). For comparison, T cell reactivity against other tumor Ags (MAGE-3.DR11/MAGE-3.DR13), which were used for vaccination with peptide-pulsed DC are shown to demonstrate that there was not a complete suppression of the immune system.

To rule out any potential effects of the DC vaccination on the observed MCSP T cell reactivity, we finally tested a comparable cohort of melanoma patients who had not been vaccinated. Two of 10 patients showed a significant T cell reactivity against peptide MCSP_693–708, which concurred with the 20% of respective patients observed in the vaccinated group, and thus was again in sharp contrast to the 85% reactivity occurring in healthy individuals (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
MCSP is broadly expressed in melanoma and appears to be functionally relevant for adhesion, migration, invasion, and proliferation of melanoma cells, making it an interesting target Ag for immunotherapy of melanoma (16). However, MCSP has been shown to be also expressed in a number of normal tissues, raising concerns of the induction of autoimmunity when immunizing with this Ag. Nevertheless, we could readily generate T cells from the blood of a healthy donor, who did not show any signs of autoimmunity. Moreover, there was a broad T cell reactivity against MCSP in the blood of 12 healthy donors of 14 tested, again without any signs of autoimmunity.

To have a better idea of the expression profile of MCSP, we screened a number of tissues by qPCR and found MCSP to be overexpressed in melanoma as compared with normal tissues, a situation which we know from several other tumor Ags, which are successfully targeted in Ab-based cancer therapy such as Her2, CD20, or VEGF (23, 24, 25). In this context, it should be mentioned that targeting MCSP in melanoma patients with unlabelled or radiolabelled anti-MCSP Ab 9.2.27 has not been associated with significant normal-organ-related accumulation or toxicity (26, 27, 28).

With qPCR, we could detect MCSP transcripts in a variety of normal tissues, including the thymus. Expression of MCSP has, in addition, been demonstrated on the protein level in some normal tissues (16). Despite this expression in normal tissues, CD4⁺ T cells seem to have escaped deletion in the thymus. These T cells should only show low to intermediate affinity to their Ag and, in fact, significant activation of the MCSP-specific T cells was only seen when using peptide concentrations >300 nM. However, the T cells could recognize MCSP- and HLA-DR11-expressing tumor cells without the need for exogenous peptide loading. These findings suggest that a significant number of MCSP-reactive, potentially tumor Ag-reactive Th cells with low to intermediate avidity circulate in the blood, which might be further activated and expanded by immunization strategies. These helper T cells should be capable to recognize tumor cells overexpressing MCSP, but do probably not recognize normal cells, which have a significant lower Ag expression. In addition, with respect to a vaccination approach with the CD4⁺ Th cell peptide described in this study, the risk of autoimmune pathology would be even more unlikely, because most somatic cells do not express HLA class II molecules under normal conditions.

Our findings are, to some extent, reminiscent of studies on T cell immunity to the ubiquitously expressed p53, where significant p53-specific Th responses have been detected in mice and patients with colorectal cancer (29, 30, 31). In addition, p53-specific CTL responses could be demonstrated in mice and humans, but at least in a mouse model, tolerance could be shown to be operative on the CTL level (32). We do not know whether this is also true for MCSP, but CTL responses in humans directed against wild-type MCSP have not been described so far. However, immunization of HLA-A2/Kb transgenic mice with MCSP cDNA-transfected DCs elicited a CD8⁺ CTL response specific for HLA-A2-binding MCSP peptides (33).

Another important aspect of our immunomonitoring results is the observation that the T cell responses in melanoma patients were infrequent and weaker as compared with normal healthy individuals, and that, at least in some patients, there was a clear correlation between disease progression and the loss or decline of MCSP T cell reactivity. This phenomenon raises the question of whether the precursor T cells are rendered anergic by a growing tumor load or trapped within the tumor and, thus, are no longer detectable in the peripheral blood. We cannot rule out that MCSP-reactive T cells may localize at the tumor site, but when rapid-growing melanoma metastases are excised, in most cases we do not see any lymphocytic infiltrate, indicating that the tumor often grows much faster than the immune system can counteract. Furthermore, our data indicate that the decline of MCSP-specific T cell reactivity is not due to a general immunosuppression caused by the tumor as we could still detect T cell reactivity to Ags other than MCSP in these patients. Be that as it may, MCSP-specific helper T cell reactivity seems to be associated with a favorable clinical course, suggesting a protective role against melanoma.

Taken together, we feel that MCSP represents a promising target Ag for T cell-based immunotherapy of melanoma.

	Acknowledgments

 
We thank S. Emmerling, E. Müller, and C. Klotz for their excellent technical assistance.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Two of the authors (G.S. and E.S.S.) have applied for a patent related to the work that is described in the present study.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from the Wilhelm-Sander-Stiftung, the Deutsche Forschungsgemeinschaft (Schu 1264 2-3 and SFB 643 Project C1), and the European Union (Cancerimmunotherapy, contract no. 518234).

² Address correspondence and reprint requests to Dr. Erwin S. Schultz, Department of Dermatology and Allergology University Hospital Giessen and Marburg, Deutschhausstrae 9, Marburg, Germany. E-mail address: eschultz{at}med.uni-marburg.de

³ Abbreviations used in this paper: MCSP, melanoma-associated chondroitin sulfate proteoglycan; MT3-MMP, membrane-type 3 matrix metalloproteinase; EBV-B, EBV-transformed B; DC, dendritic cells; Ii, invariant chain; qPCR, quantitative real-time PCR; LN, lymph nodes.

Received for publication June 16, 2006. Accepted for publication April 6, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Natali, P. G., K. Imai, B. S. Wilson, A. Bigotti, R. Cavaliere, M. A. Pellegrino, S. Ferrone. 1981. Structural properties and tissue distribution of the antigen recognized by the monoclonal antibody 653.40S to human melanoma cells. J. Natl. Cancer Inst. 67: 591-601. [Medline]
2. Ross, A. H., G. Cossu, M. Herlyn, J. R. Bell, Z. Steplewski, H. Koprowski. 1983. Isolation and chemical characterization of a melanoma-associated proteoglycan antigen. Arch. Biochem. Biophys. 225: 370-383. [Medline]
3. Iida, J., A. M. Meijne, R. C. Spiro, E. Roos, L. T. Furcht, J. B. McCarthy. 1995. Spreading and focal contact formation of human melanoma cells in response to the stimulation of both melanoma-associated proteoglycan (NG2) and 4 1 integrin. Cancer Res. 55: 2177-2185. [Abstract/Free Full Text]
4. de Vries, J. E., G. D. Keizer, A. A. te Velde, A. Voordouw, D. Ruiter, P. Rumke, H. Spits, C. G. Figdor. 1986. Characterization of melanoma-associated surface antigens involved in the adhesion and motility of human melanoma cells. Int. J. Cancer. 38: 465-473. [Medline]
5. Harper, J. R., R. A. Reisfeld. 1983. Inhibition of anchorage-independent growth of human melanoma cells by a monoclonal antibody to a chondroitin sulfate proteoglycan. J. Natl. Cancer Inst. 71: 259-263. [Medline]
6. Nishiyama, A., W. B. Stallcup. 1993. Expression of NG2 proteoglycan causes retention of type VI collagen on the cell surface. Mol. Biol. Cell 4: 1097-1108. [Abstract]
7. Fang, X., M. A. Burg, D. Barritt, K. Dahlin-Huppe, A. Nishiyama, W. B. Stallcup. 1999. Cytoskeletal reorganization induced by engagement of the NG2 proteoglycan leads to cell spreading and migration. Mol. Biol. Cell 10: 3373-3387. [Abstract/Free Full Text]
8. Burg, M. A., K. A. Grako, W. B. Stallcup. 1998. Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells. J. Cell. Physiol. 177: 299-312. [Medline]
9. Garrigues, H. J., M. W. Lark, S. Lara, I. Hellstrom, K. E. Hellstrom, T. N. Wight. 1986. The melanoma proteoglycan: restricted expression on microspikes, a specific microdomain of the cell surface. J. Cell Biol. 103: 1699-1710. [Abstract/Free Full Text]
10. Iida, J., D. Pei, T. Kang, M. A. Simpson, M. Herlyn, L. T. Furcht, J. B. McCarthy. 2001. Melanoma chondroitin sulfate proteoglycan regulates matrix metalloproteinase-dependent human melanoma invasion into type I collagen. J. Biol. Chem. 276: 18786-18794. [Abstract/Free Full Text]
11. Herlyn, M., M. Padarathsingh, L. Chin, M. Hendrix, D. Becker, M. Nelson, Y. DeClerck, J. McCarthy, S. Mohla. 2002. New approaches to the biology of melanoma: a workshop of the National Institutes of Health Pathology B Study Section. Am. J. Pathol. 161: 1949-1957. [Free Full Text]
12. Yang, J., M. A. Price, C. L. Neudauer, C. Wilson, S. Ferrone, H. Xia, J. Iida, M. A. Simpson, J. B. McCarthy. 2004. Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms. J. Cell Biol. 165: 881-891. [Abstract/Free Full Text]
13. Ozerdem, U., K. A. Grako, K. Dahlin-Huppe, E. Monosov, W. B. Stallcup. 2001. NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis. Dev. Dyn. 222: 218-227. [Medline]
14. Chekenya, M., M. Hjelstuen, P. O. Enger, F. Thorsen, A. L. Jacob, B. Probst, O. Haraldseth, G. Pilkington, A. Butt, J. M. Levine, R. Bjerkvig. 2002. NG2 proteoglycan promotes angiogenesis-dependent tumor growth in CNS by sequestering angiostatin. FASEB J. 16: 586-588. [Free Full Text]
15. Real, F. X., A. N. Houghton, A. P. Albino, C. Cordon-Cardo, M. R. Melamed, H. F. Oettgen, L. J. Old. 1985. Surface antigens of melanomas and melanocytes defined by mouse monoclonal antibodies: specificity analysis and comparison of antigen expression in cultured cells and tissues. Cancer Res. 45: 4401-4411. [Abstract/Free Full Text]
16. Campoli, M. R., C. C. Chang, T. Kageshita, X. Wang, J. B. McCarthy, S. Ferrone. 2004. Human high molecular weight-melanoma-associated antigen (HMW-MAA): a melanoma cell surface chondroitin sulfate proteoglycan (MSCP) with biological and clinical significance. Crit. Rev. Immunol. 24: 267-296. [Medline]
17. Rammensee, H., J. Bachmann, N. P. Emmerich, O. A. Bachor, S. Stevanovic. 1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213-219. [Medline]
18. Schultz, E. S., B. Lethe, C. L. Cambiaso, J. Van Snick, P. Chaux, J. Corthals, C. Heirman, K. Thielemans, T. Boon, P. van der Bruggen. 2000. A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4⁺ cytolytic T lymphocytes. Cancer Res. 60: 6272-6275. [Abstract/Free Full Text]
19. Thurner, B., C. Roder, D. Dieckmann, M. Heuer, M. Kruse, A. Glaser, P. Keikavoussi, E. Kampgen, A. Bender, G. Schuler. 1999. Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. J. Immunol. Methods 223: 1-15. [Medline]
20. Jonuleit, H., U. Kuhn, G. Muller, K. Steinbrink, L. Paragnik, E. Schmitt, J. Knop, A. H. Enk. 1997. Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions. Eur. J. Immunol. 27: 3135-3142. [Medline]
21. Davis, L. G., M. D. Dibner, J. F. Battey. 1986. Guanidine Isothiocyanate Preparation of Total RNA Elsevier, New York.
22. Thurner, B., I. Haendle, C. Roder, D. Dieckmann, P. Keikavoussi, H. Jonuleit, A. Bender, C. Maczek, D. Schreiner, P. von den Driesch, et al 1999. Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J. Exp. Med. 190: 1669-1678. [Abstract/Free Full Text]
23. Hurwitz, H., L. Fehrenbacher, W. Novotny, T. Cartwright, J. Hainsworth, W. Heim, J. Berlin, A. Baron, S. Griffing, E. Holmgren, et al 2004. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N. Engl. J. Med. 350: 2335-2342. [Abstract/Free Full Text]
24. Forstpointner, R., M. Dreyling, R. Repp, S. Hermann, A. Hanel, B. Metzner, C. Pott, F. Hartmann, F. Rothmann, R. Rohrberg, et al 2004. The addition of rituximab to a combination of fludarabine, cyclophosphamide, mitoxantrone (FCM) significantly increases the response rate and prolongs survival as compared with FCM alone in patients with relapsed and refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood 104: 3064-3071. [Abstract/Free Full Text]
25. Slamon, D. J., B. Leyland-Jones, S. Shak, H. Fuchs, V. Paton, A. Bajamonde, T. Fleming, W. Eiermann, J. Wolter, M. Pegram, et al 2001. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N. Engl. J. Med. 344: 783-792. [Abstract/Free Full Text]
26. Oldham, R. K., K. A. Foon, A. C. Morgan, C. S. Woodhouse, R. W. Schroff, P. G. Abrams, M. Fer, C. S. Schoenberger, M. Farrell, E. Kimball, et al 1984. Monoclonal antibody therapy of malignant melanoma: in vivo localization in cutaneous metastasis after intravenous administration. J. Clin. Oncol. 2: 1235-1244. [Abstract]
27. Buraggi, G. L., L. Callegaro, G. Mariani, A. Turrin, N. Cascinelli, A. Attili, E. Bombardieri, G. Terno, G. Plassio, M. Dovis, et al 1985. Imaging with 131I-labeled monoclonal antibodies to a high-molecular-weight melanoma-associated antigen in patients with melanoma: efficacy of whole immunoglobulin and its F(ab')₂. Cancer Res. 45: 3378-3387. [Abstract/Free Full Text]
28. Siccardi, A. G., G. L. Buraggi, L. Callegaro, G. Mariani, P. G. Natali, A. Abbati, M. Bestagno, V. Caputo, L. Mansi, R. Masi, et al 1986. Multicenter study of immunoscintigraphy with radiolabeled monoclonal antibodies in patients with melanoma. Cancer Res. 46: 4817-4822. [Abstract/Free Full Text]
29. van der Burg, S. H., K. de Cock, A. G. Menon, K. L. Franken, M. Palmen, A. Redeker, J. Drijfhout, P. J. Kuppen, C. van de Velde, L. Erdile, et al 2001. Long lasting p53-specific T cell memory responses in the absence of anti-p53 antibodies in patients with resected primary colorectal cancer. Eur. J. Immunol. 31: 146-155. [Medline]
30. van der Burg, S. H., A. G. Menon, A. Redeker, K. L. Franken, J. W. Drijfhout, R. A. Tollenaar, H. H. Hartgrink, C. J. van de Velde, P. J. Kuppen, C. J. Melief, R. Offringa. 2003. Magnitude and polarization of P53-specific T-helper immunity in connection to leukocyte infiltration of colorectal tumors. Int. J. Cancer. 107: 425-433. [Medline]
31. Zwaveling, S., M. P. Vierboom, S. C. Ferreira Mota, J. A. Hendriks, M. E. Ooms, R. P. Sutmuller, K. L. Franken, H. W. Nijman, F. Ossendorp, S. H. Van Der Burg, et al 2002. Antitumor efficacy of wild-type p53-specific CD4⁺ T-helper cells. Cancer Res. 62: 6187-6193. [Abstract/Free Full Text]
32. Theobald, M., J. Biggs, J. Hernandez, J. Lustgarten, C. Labadie, L. A. Sherman. 1997. Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes. J. Exp. Med. 185: 833-841. [Medline]
33. Peng, L., E. Ko, W. Luo, X. Wang, P. A. Shrikant, S. Ferrone. 2006. CD4-dependent potentiation of a high molecular weight-melanoma-associated antigen-specific CTL response elicited in HLA-A2/Kb transgenic mice. J. Immunol. 176: 2307-2315. [Abstract/Free Full Text]

This article has been cited by other articles:

				Y. Sun, M. Song, E. Jager, C. Schwer, S. Stevanovic, S. Flindt, J. Karbach, X. D. Nguyen, D. Schadendorf, and K. Cichutek Human CD4+ T Lymphocytes Recognize a Vascular Endothelial Growth Factor Receptor-2-Derived Epitope in Association with HLA-DR Clin. Cancer Res., July 1, 2008; 14(13): 4306 - 4315. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Erfurt, C. Articles by Schultz, E. S. Search for Related Content PubMed PubMed Citation Articles by Erfurt, C. Articles by Schultz, E. S.