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The Journal of Immunology, 2004, 172: 7206-7211.
Copyright © 2004 by The American Association of Immunologists

Identification of an SSX-2 Epitope Presented by Dendritic Cells to Circulating Autologous CD4+ T Cells1

Maha Ayyoub*, Charles S. Hesdorffer{dagger}, Genevieve Metthez*, Stefan Stevanovic{ddagger}, Gerd Ritter§, Yao-Tseng Chen, Lloyd J. Old§, Daniel Speiser||, Jean-Charles Cerottini# and Danila Valmori2,*

* Ludwig Institute Clinical Trial Center and {dagger} Division of Medical Oncology, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032; {ddagger} Institute for Cell Biology, Department of Immunology, University of Tubingen, Tubingen, Germany; § Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY 10021; Department of Pathology, Weill Medical College of Cornell University, New York, NY 10021; || Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital, Lausanne, Switzerland; and # Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Accumulating evidence supports the requirement for both tumor-specific CD8+ and CD4+ T cell responses for efficient tumor rejection to occur. Because of its expression in different tumor types, the cancer/testis Ag encoded by the synovial sarcoma X breakpoint 2 (SSX-2) gene is among the most relevant candidates for the development of generic cancer vaccines. The immunogenicity of SSX-2 has been previously corroborated by detection of specific humoral and CD8+ T cell responses in cancer patients. In this study we report identification of the first CD4+ T cell epitope encoded by SSX-2. The identified epitope mapped to the 19–34 region of the protein and was recognized by CD4+ T cells from an Ag-expressing melanoma patient in association with HLA-DPB1*0101. The absence of detectable response in healthy donors and other patients suggests that SSX-2-specific CD4+ T cells in the responder patient had been previously expanded in vivo in response to the autologous tumor. The epitope did not appear to be presented on the surface of tumor cells at levels sufficient to allow direct recognition. In contrast, it was efficiently presented by autologous dendritic cells, supporting the concept that processing by professional APC is the main pathway through which the CD4+ T cell immunoresponse to tumor Ags occurs in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability of the T cell arm of the antitumor immune response to distinguish tumor cells from normal tissues with exquisite specificity provides the basis for the development of T cell-based cancer immunotherapy. This specific recognition is the result of the preferential or exclusive expression of some Ags in tumors compared with normal tissues. Several categories of Ags with more or less tumor-restricted expression have been identified during the last decade. Most of them correspond to nonmutated self-Ags with tissue-restricted expression, although tumor-specific, mutated Ags have also been identified (1). Tissue-specific differentiation Ags, such as Melan-A or gp100 (2, 3), expressed by both normal cells of the melanocytic lineage and malignant melanoma cells and often spontaneously immunogenic in melanoma patients have been extensively studied. The group of tumor Ags most relevant for the development of generic cancer vaccines, however, is that of the so-called cancer/testis Ags (CTA)3 (4), whose gene expression is developmentally regulated, being mostly restricted to gametogenic cells, but silent in adult normal cells. Possibly as the result of activation of a common gametogenic protein expression program in cancer cells (5), CTA are expressed in variable proportions of tumors of different histological types.

Numerous MHC class I-restricted epitopes recognized by tumor-reactive CD8+ T cells and specific for Ags in each of the groups listed above have been identified. Interestingly, spontaneous CD8+ T cell responses directed against several of these epitopes have been detected in cancer patients (6, 7). In contrast, the identification of MHC class II-restricted epitopes recognized by tumor Ag-specific CD4+ T cells has proven more difficult, possibly because of the relatively low frequency of the latter and/or the lack of effective identification methods (8). Lately, however, most likely because of some important technical advances, the identification of CD4+ T cell epitopes derived from tumor Ags, including CTA, has been reported with increasing frequency (9, 10).

Because most nonhemopoietic tumors express MHC class I, but not class II, molecules, it has been assumed that the predominant antitumor, T cell-mediated effector mechanism in vivo is direct recognition of tumor cells by tumor Ag-specific CD8+ T lymphocytes (CTL). CTL can indeed directly and efficiently lyse tumor cells, sometimes resulting in in vivo regression of large tumor masses. It is, however, becoming increasingly clear that both tumor Ag-specific CD8+ and CD4+ T cell responses are essential for efficient immune response to tumors to occur in vivo (11). The multiple roles that tumor Ag-specific CD4+ T cells can potentially play in mediating antitumor functions are being progressively unveiled. These involve different mechanisms going from providing help for both priming and maintenance of tumor Ag-specific CD8+ T cells to activation of B cells for production of tumor Ag-specific Abs, to more direct effects in the effector phase of tumor rejection. The identification of CD4+ T cell epitopes toward which spontaneous responses arise in cancer patients is of particular interest because it provides the opportunity to analyze such responses and the underlying molecular mechanisms in vivo.

Synovial sarcoma X breakpoint 2 (SSX-2) is a classical CTA belonging to a multigene family mapping to chromosome X. Some family members, including SSX-2, are expressed in a wide variety of tumors (12, 13, 14). The SSX-2-encoding gene was initially described as one of two partner genes found in a recurrent chromosomal translocation in synovial sarcoma (15, 16) and then identified as a tumor Ag by SEREX analysis of serum from a melanoma patient. The potential spontaneous immunogenicity of the SSX-2 Ag was initially suggested by detection of specific Abs in 10% of melanoma patients (13). By analyzing CD8+ T lymphocytes from an SSX-2-expressing melanoma patient, we identified an epitope mapping to the 41–49 region of the SSX-2 protein and recognized by tumor-reactive CD8+ T lymphocytes in association with the MHC class I allele HLA-A2 (17, 18). Importantly, in a recent survey of the CD8+ T cell response to SSX-241–49 in HLA-A2+ melanoma patients, we detected specific responses more frequently in Ag-expressing patients compared with patients with nonexpressing tumors and healthy donor controls (19). Importantly, whereas a large functional avidity of Ag recognition and tumor reactivity was found among isolated SSX241–49-specific CD8+ T cells, those isolated from both tumor-infiltrating and circulating lymphocytes of patients bearing SSX-2-expressing tumor lesions uniformly exhibited high functional avidity of Ag recognition and tumor reactivity. These findings indicate that spontaneous T cell responses to SSX-2 frequently occur in Ag-expressing melanoma patients, encouraging the search for additional MHC class I- and class II-restricted epitopes in this patient population.

In this study, by analyzing the CD4+ T cell response to peptides spanning the SSX-2 protein sequence in circulating lymphocytes from an SSX-2-expressing melanoma patient, we identified the first CD4+ T cell epitope encoded by SSX-2. SSX-2-specific CD4+ T cells failed to directly recognize Ag-expressing tumor cells even in the presence of the appropriate MHC class II restriction element, but were stimulated by Ag-loaded autologous dendritic cells (DC), indicating that the CD4+ T cell response to SSX-2 had occurred as a consequence of processing and presentation of exogenously captured Ag by autologous professional APCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and tissue culture

Peripheral blood was obtained from healthy donors (Swiss Blood Bank) and melanoma patients after obtaining informed consent (Lausanne University Hospital, Lausanne, Switzerland). Melanoma cell lines and anti-HLA-DR (D1.12), -DP (B7.21.3), and -DQ (BT3/4) Abs were provided by Dr. D. Rimoldi (Ludwig Institute for Cancer Research, Lausanne, Switzerland). Cell lines were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated FCS. The culture medium for lymphocytes was IMDM (Life Technologies, Gaithersburg, MD) supplemented with 8% heat-inactivated pooled human serum (CTL medium), recombinant human (rh) IL-2 (Glaxo, Geneva, Switzerland), and rhIL-7 (BioSource International, Camarillo, CA).

Generation of SSX-2-specific CD4+ T cells

In vitro stimulation of CD4+ T cells was conducted as described previously for CD8+ T cells. Briefly, 1–2 x 106 CD4+ T cells highly enriched (>90%) from PBMC by MACS using a miniMACS device (Miltenyi Biotec, Auburn, CA) were stimulated with a mixture containing peptides spanning the entire SSX-2 protein sequence (18) (2 µM each) in the presence of irradiated autologous cells from the CD4– fraction in CTL medium containing rhIL-2 (10 U/ml) and rhIL-7 (10 ng/ml) and were cultured for 2–3 wk before being tested. CD4+ T cells secreting cytokines in response to peptide stimulation were isolated by cytokine-guided flow cytometry cell sorting using the cytokine secretion detection kit (Miltenyi Biotec) and were cloned by limiting dilution culture in the presence of PHA (Sigma-Aldrich, Steinheim, Germany), allogeneic irradiated PBMC, and rhIL-2 as previously described (6). Clones were subsequently expanded by periodic (every 3–4 wk) stimulation under the same conditions.

Molecular HLA-DPB1 typing, HLA-DP and SSX-2 plasmids, and transient transfection

Molecular HLA-DPB1 typing was performed using the high resolution SSP UniTray kit (Pel-Freez Biologicals, Rogers, AR) according to the manufacturer’s instructions. The SSX-2-encoding cDNA was cloned into the pcDNA3.1 vector. Tumor cells were transiently transfected with plasmids using FuGENE according to the manufacturer’s instructions (Roche, Rotkreuz, Switzerland).

Generation of DC, recombinant proteins, tumor lysates, and Ag loading

Monocyte-derived DC were generated by isolating CD14+ monocytes from PBMC by MACS using a miniMACS device (Miltenyi Biotec). The population obtained (containing >95% CD14+ cells) was cultured in CTL medium containing 1000 U/ml rhGM-CSF (BD PharMingen, San Diego, CA) and 1000 U/ml rhIL-4 (BD PharMingen) for 6 days. At the end of the culture period, the DC preparation contained >90% HLA-DR+ CD83+ cells. SSX-2 and NY-ESO-1 proteins were expressed in Escherichia coli as full-length proteins with a six-histidine tag at the N terminus (20). The proteins were purified from washed and solubilized inclusion bodies by nickel chelate affinity chromatography (Chelating Sepharose FF; Amersham Pharmacia Biotech, Piscataway, NJ) using a pH gradient. Proteins were eluted in 8 M urea, 100 mM phosphate, and 10 mM Tris at pH 4.5. The purified proteins were reactive with anti-NY-ESO-1 and anti-SSX-2 mAbs by Western blot analysis; purity was >80% by SDS-PAGE. Tumor cells (2 x 105) were lysed in 200 µl of RPMI 1640 by 10 cycles of rapid freezing-thawing. Where indicated, DC were incubated with proteins (5 µg/ml) or lysates (at the equivalent of three tumor cells per DC) for 12 h and washed before use in the stimulation or Ag recognition assay.

Ag recognition assays

For intracellular cytokine secretion detection, T cells were incubated with APC at a 1:1 T cell:APC ratio for 4–6 h in the absence or the presence of peptides at the indicated dose. One hour after the beginning of the incubation, brefeldin A (20 µg/ml; Sigma-Aldrich) was added to inhibit cytokine secretion. At the end of the incubation period, cells were stained with anti-CD4 mAb for 20 min at 4°C and fixed. Cells were then permeabilized using saponin (Sigma-Aldrich; 0.1% in PBS/5% FCS), stained by incubation with mAb against IFN-{gamma} or IL-2 (BD PharMingen), and analyzed by flow cytometry. Data analysis was performed using CellQuest software. For detection of cytokine secretion in the culture supernatant, T cells (10,000) were incubated with stimulating cells (15,000/well) in 96-well, round-bottom plates in 200 µl/well CTL medium containing 20 U/ml rhIL2. After 24-h incubation at 37°C, culture supernatants were collected, and the content of IFN-{gamma} was determined by ELISA (BioSource Europe, Fleurus, Belgium). IFN-{gamma} ELISPOT assay was performed as described previously (18), using nitrocellulose-lined, 96-well microplates (MAHA S45; Millipore, Bedford, MA) and an IFN-{gamma} ELISPOT kit (DIACLONE, Besancon, France). Stimulator cells (5 x 104/well) were added together with T cells (4 x 104/well) and peptide (2 µM) where indicated. Spots were counted using a stereomicroscope with a magnification of x10.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Assessment of SSX-2-specific CD4+ T cell responses in circulating lymphocytes of Ag-expressing melanoma patients

Enriched CD4+ T cells from PBMC samples from five melanoma patients with detectable SSX-2 expression in their tumor lesions were stimulated in vitro with a peptide mix containing 15 20- to 22-aa-long peptides spanning the SSX-2 protein sequence and overlapping by 10 aa (18). Two to 3 wk after a single in vitro stimulation, culture aliquots were stimulated with submixtures, each composed of three peptides (P1–3, P4–6, etc.). The presence of specific CD4+ T cells was monitored by intracellular staining with cytokine-specific Abs (IFN-{gamma} and IL-2; Fig. 1). For one patient (LAU 672), one peptide submixture (P1–3, containing peptides SSX-2 1–22, 13–34, and 25–46) stimulated a significant proportion of IFN-{gamma}- and IL-2-secreting CD4+ T cells compared with controls containing either no peptide or other peptide mixtures (Fig. 1). Assessment of the reactivity of the culture from patient LAU 672 to single peptides in the submixture P1–3 revealed that SSX-213–34 was the active peptide, whereas no significant activity was detected in response to peptides SSX-21–22 and SSX-225–46 (not shown). After a second cycle of in vitro stimulation, the proportion of SSX-213–34-specific CD4+ T cells in the culture from this patient significantly increased (Table I). No specific responses were detected in the other four melanoma patients analyzed (Table I and data not shown) after both one and two cycles of in vitro stimulation. Peptide SSX-213–34-specific CD4+ T cells were isolated from the culture by cytokine secretion-guided flow cytometry cell sorting and were cloned under limiting dilution conditions. The obtained clonal populations were used for additional experiments.



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  		FIGURE 1. Detection of SSX-2-specific CD4+ T cells in peptide-stimulated cultures. A, The presence of specific CD4+ T cells in the culture from patient LAU 672 was assessed by intracellular staining with anti-IFN-{gamma} (left panel) or anti-IL-2 Abs (right panel) after stimulation with autologous PBMC alone (upper panels) or with the peptide submixture P1–3 (containing SSX-2 peptides 1–22, 13–34, and 25–46). Numbers in upper right quadrants are the percentage of cytokine-producing cells among CD4+ T cells. Cells in the lower left quadrants correspond to APCs. B, Data were obtained for all peptide submixtures.

 

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  		Table I. Assessment of SSX-213–34-specific CD4+ T cell responses in circulating lymphocytes of Ag-expressing melanoma patients and healthy donors

 
Mapping of the minimal peptide optimally recognized by SSX-2-specific CD4+ T cells

To more precisely define the SSX-2-derived peptide optimally recognized by specific CD4+ T cells from patient LAU 672, we analyzed the relative capacities of peptide SSX-213–34 extended or truncated variants to stimulate IFN-{gamma} secretion by specific clonal T cells (clone 3C8). As illustrated in Fig. 2, both extension and truncation of peptide SSX-213–34 C terminus resulted in decreased peptide recognition. Truncation of the first six amino acids at the N terminus did not significantly affect recognition. In contrast, truncation of two additional N-terminal amino acids resulted in a 10-fold reduction of peptide activity. Thus, among analyzed peptides, SSX-219–34 was the minimal peptide optimally recognized by SSX-2-specific CD4+ T cells. Similar results were obtained using another CD4+ T cell clone (not shown).



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  		FIGURE 2. Determination of the minimal sequence optimally recognized by SSX-2-specific CD4+ T cells. Synthetic peptides extended or truncated at the SSX-213–34 N or C terminus were used to determine the minimal length of the epitope recognized by SSX-2-specific CD4+ T cells (clone 3C8). Serial dilutions of each peptide were incubated with EBV LAU 149 and SSX-2-specific CD4+ T cells. IFN-{gamma} secretion was determined by ELISA in the culture supernatant after 24 h of culture.

 
SSX-213–34 is recognized by specific CD4+ T cells in the context of different HLA-DP alleles

To identify the restriction element used by SSX-2-specific CD4+ T cells, recognition of peptide SSX-213–34 was conducted in the presence of Abs that specifically block the recognition of Ags restricted by different MHC class II elements (HLA-DR, -DP, or -DQ). As illustrated in Fig. 3A, anti-HLA-DP Abs abolished the ability of SSX-2-specific CD4+ T cells to recognize peptide 13–34. In contrast, no significant inhibition was observed using anti-HLA-DR or anti-HLA-DQ Abs. To attempt to establish the HLA-DP-presenting allele(s), we first analyzed the frequency at which healthy donors were able to present the SSX-2 epitope to CD4+ T cells. We obtained presentation by three of 14 PBMC analyzed, suggesting a frequency of the presenting allele(s) in the test population (Caucasian) of ~20% (not shown). Presentation was further assessed using HLA-DP-typed APC (Fig. 3B). Peptide presentation was obtained using either DPB1*0101/0401 or DPB1*0301/0401, but not DPB1*0401/0402 expressing APC, indicating that the peptide can be recognized by T cells in the context of the DPB1*0101 and DPB1*0301 alleles. In addition, one of the three presenting healthy donors expressed DPB1*1301/1401, suggesting that one of these alleles could also be able to present peptide SSX-213–34 to specific CD4+ T cells. As illustrated in Fig. 3C, DPB1*0101/0401, DPB*0301/0401, and DPB1*1301/1401 APC were able to present peptide SSX-213–34 to specific CD4+ T cells. In all cases Ag recognition was specifically inhibited by anti-HLA-DP-specific, but not by anti-HLA-DR-specific, Abs. No recognition was detected with DPB1*0401/0402 APCs, which were used as an internal control.



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  		FIGURE 3. Recognition of SSX-213–34 by specific CD4+ T cells in the context of HLA-DP. A, Intracellular IFN-{gamma} secretion by SSX-2-specific CD4+ T cells (clone 3C8) upon stimulation with peptide SSX-213–34 (2 µM) was assessed in both the absence and the presence of anti-HLA-DR, -DP, or -DQ Abs. Cells in the lower left quadrants correspond to APCs (EBV). B and C, The ability of APC bearing different HLA-DP alleles to present peptide SSX-213–34 to specific CD4+ T cells (clone 3C8) was assessed by intracellular IFN-{gamma} secretion.

 
The T cell epitope recognized by SSX-2-specific CD4+ T cells from LAU 672 is not presented by tumor cells, but is efficiently processed and presented by professional APCs

To assess whether the T cell epitope recognized by SSX-2-specific CD4+ T cells from LAU 672 is naturally presented on the surface of tumor cells, we selected two lines: Me 260 (HLA-DPB*0301) expressed SSX-2 (18), but was not recognized by CD4+ T cells in the absence of exogenously added peptide even after treatment with IFN-{gamma} for 48 h, which resulted in increased expression of HLA-DP (Fig. 4A and not shown), and T465A (HLA-DPB*0101) was SSX-2 negative, but expressed HLA-A2 (19). Transfection of T465A cells with SSX-2 did not result in recognition by specific CD4+ T cells, whereas CD8+ T cells specific for peptide SSX-241–49 were able to recognize SSX-2-transfected T465A cells (Fig. 4B). We then assessed the ability of professional APC to process the SSX-2 Ag and present the 13–34 epitope to specific CD4+ T cells. As illustrated in Fig. 4C, EBV cells from patient LAU 149 were able to efficiently process the SSX-2 protein and present the relevant epitope to SSX-213–34-specific CD4+ T cells. The clone was not significantly stimulated by paraformaldehyde-fixed LAU 149 EBV pulsed with the SSX-2 protein, by SSX-2-pulsed EBV cells unable to present the peptide (not shown), or by EBV LAU 149 pulsed with NY-ESO-1 protein. Specific Ag presentation was also obtained using LAU 672 autologous DC (Fig. 4, D and E) loaded with either the SSX-2 protein or a lysate of SSX-2-expressing tumor cells (SK-MEL-37). Processing of exogenous SSX-2 protein by DC did not result in recognition of the CD8+ T cell epitope 41–49 by specific clonal CD8+ T cells (clone B3.4; Fig. 4D).



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  		FIGURE 4. The T cell epitope recognized by SSX-2-specific CD4+ T cells from LAU 672 is not presented by tumor cells, but is efficiently processed and presented by professional APCs. A, Recognition of Me 260 cells by CD4+ T cells (4 x 104/well) was tested by ELISPOT in the absence or the presence of exogenously added peptide SSX-213–34. Where indicated, cells were treated with IFN-{gamma} for 48 h. B, Recognition of T465A cells was assessed as described in A, as well as upon transfection with an SSX-2-encoding plasmid (*). The CD8+ T cell clone B3.4 specific for peptide SSX-241–49 was used as an internal control. C, The ability of EBV LAU 149 to process the SSX-2 protein and present the relevant epitope to specific CD4+ T cells was assessed by ELISPOT after 12-h incubation with soluble recombinant SSX-2 protein at the indicated dose, followed by washing. {blacksquare}, Results obtained with paraformaldehyde-fixed EBV. NY-ESO-1 protein was used as an internal control. D and E, Processing and presentation of SSX-2 Ag by DC after incubation with soluble recombinant SSX-2 protein (or NY-ESO-1 protein as a negative control) or with lysates from tumor cell lines SK-MEL-37 (SSX-2 expressing) and NA8 (SSX-2 negative) was assessed by ELISPOT (D) as well as by ELISA measurement of IFN-{gamma} secretion in the culture supernatant (E).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Clinical trials of vaccination of cancer patients with MHC class I-restricted peptides derived from tumor Ags are presently ongoing worldwide. Although the results of some of these trials have shown evidence for the immunogenicity of the selected peptides and formulations together with episodic clinical responses, their overall outcomes have failed to meet investigators’ expectations. Among others, one of the evocated explanations for the relatively poor outcome of tumor vaccines composed of MHC class I-restricted peptides alone is the lack of simultaneous stimulation of CD4+ T cells that may be required for generating and sustaining vigorous antitumor immunity (11, 21, 22). In addition, the role of tumor Ag-specific CD4+ T cells in promoting cross-presentation of MHC class I-restricted epitopes by DC has recently been emphasized by numerous studies. CD4+ T cell-mediated mechanisms of the observed inhibition of tumor growth in the absence of CD8+ T cells have also begun to be unveiled (23, 24, 25, 26, 27). Thus, the interest of tumor immunologists toward tumor Ag-specific CD4+ T cells has been recently renewed because of the need for incorporating MHC class II-restricted tumor Ag-derived sequences into tumor vaccines.

The identification of CD4+ T cell epitopes from tumor Ags, however, has proven considerably more challenging than that of their CD8+ T cell counterparts. A major difficulty in the isolation of Ag-specific CD4+ T cells, including those specific for tumor Ags, has been the absence of methods that allow their direct detection and isolation from mixed lymphocyte populations. Whereas analysis and isolation of CD8+ T cells have been tremendously boosted by the development of fluorochrome-labeled soluble multimeric peptide/MHC class I complexes, those of the corresponding peptide/MHC class II complexes have met with technical difficulties in refolding and staining efficiency combined with low in vivo frequencies of CD4+ T cells (8, 28).

Considerable efforts have been made in the development of alternative approaches, including, among others, elution of MHC class II-bound peptides from tumor cells (29), peptide purification from tumor cell lysates (30), or more sophisticated molecular approaches based on targeting tumor Ags to the endogenous Ag presentation pathway (31). As a consequence of these efforts, the rate of success has lately considerably improved, and several tumor Ag-derived CD4+ T cell epitopes, including some derived from CTA, have been identified (11). Most of the procedures used in these studies, however, are technically cumbersome.

In this study we have used a mixture composed of 20- to 22-aa-long peptides spanning the SSX-2 protein sequence and overlapping by 10 aa to stimulate enriched CD4+ T lymphocytes from an SSX-2-expressing melanoma patient with autologous irradiated CD4– cells. Peptide submixtures and then single peptides were used for screening the cultures for the presence of specific T cells together with autologous PBMC as a source of APC. A reactive peptide in the active mixture was identified, and specific CD4+ T cells were isolated using a cytokine secretion-based, cell-sorting procedure and were cloned by mitogen stimulation under limiting dilution conditions.

The fact that no response to the identified epitope was detected using the same methodology in normal donors expressing the appropriate MHC class II restriction element suggests that the specific response detected in patient LAU 672 was most likely the result of a spontaneous response to his autologous SSX-2-expressing tumor. It is noteworthy that we have previously found a spontaneous response to the CD8+ T cell epitope SSX-241–49 in both circulating and tumor infiltrating lymphocytes of patient LAU 672. No specific responses to the SSX-2-spanning peptide mixture were detected using the same method in the case of four additional melanoma patients bearing SSX-2-expressing tumors, including two (LAU 149 and LAU 343) whose APC were able to present the SSX-213–34 epitope to specific CD4+ T cells. The clinical relevance of the identified epitope should be addressed in the future in studies performed in a large patient population.

The recognition of the identified SSX-2 epitope by specific CD4+ T cells was HLA-DP restricted. HLA-DP-restricted recognition has, until recently, been poorly characterized. Importantly, however, a single DP allele mismatch has been shown to be sufficient for triggering acute graft-vs-host disease after bone marrow transplantation (32). In support of the importance of DP-restricted immune responses, including those against tumors, two tumor Ag-derived epitopes (from MAGE-A3 and NY-ESO-1) recognized by CD4+ T cells in association with the HLA-DP4 molecule have been recently identified (10, 33). HLA-DP4 alleles (DPB1*0401 and DPB1*0402) are the most frequently expressed in the worldwide population. The two alleles share a very similar binding motif and, together with DPB1*0201, have recently been proposed to define a supertype of peptide binding specificity (34) characterized by the common presence of a G at position {beta}86 that is located in a pocket of the molecule critical for peptide binding. Recognition of SSX-213–34 by CD4+ T cells from patient LAU 672 was restricted by at least two other HLA-DP molecules. These molecules, HLA-DP1 and DP3, are, after DP4, the most prevalent DP alleles. Interestingly, they are characterized together with DP9 by the presence of D at position {beta}86 and may possibly define a separate supertype of peptide binding specificity.

SSX-213–34-specific CD4+ T cells from patient LAU 672 failed to recognize SSX-2+ tumor cells expressing the presenting restriction allele, indicating that the identified epitope was not expressed at levels sufficient to allow direct recognition of tumor cells even in the case of good MHC class II expression. However, professional APC were able to process the native Ag and present the relevant epitope to specific CD4+ T cells. Thus, it is likely that processing and presentation of tumor-derived SSX-2 Ag by autologous professional APC through the exogenous pathway were the mechanisms through which this spontaneous CD4+ T cell response to the autologous tumor occurred in vivo.

In conclusion, in this study we have reported identification of the first described CD4+ T cell epitope from the CTA SSX-2. These findings confirm the previously observed immunogenicity of SSX-2 in Ag-expressing cancer patients and encourage the onset of clinical trials of vaccination with SSX-2 immunogenic molecules. The method used for identification of the epitope, based on the use of synthetic peptides spanning the protein sequence, is simple and can be used for the simultaneous identification of CD4+ and CD8+ T cell epitopes. The same approach will be instrumental for monitoring upcoming vaccination trials.


   Acknowledgments
 
We thank Dr. Donata Rimoldi for kindly providing the tumor cell lines. We are deeply grieved by the recent loss of our dear colleague, Dr. P. Batard, who assisted us with the flow cytometry immunofluorescence cell-sorting experiments.


   Footnotes
 
1 This work was supported by the Ludwig Institute for Cancer Research (to D.V. and M.A.), the Cancer Research Institute (to D.V. and M.A.), and a grant from the Breast Cancer Alliance (to C.H.). Back

2 Address correspondence and reprint requests to Dr. Danila Valmori, Ludwig Institute Clinical Trial Center, Division of Medical Oncology, Department of Medicine, Columbia University College of Physicians and Surgeons, 650 West 168th Street, Black Building Room 20-22, New York, NY 10032. E-mail address: valmori{at}cancercenter.columbia.edu Back

3 Abbreviations used in this paper: CTA, cancer/testis Ag; DC, dendritic cell; rh, recombinant human; SSX-2, synovial sarcoma X breakpoint 2. Back

Received for publication November 4, 2003. Accepted for publication March 31, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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