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*
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, University Hospital, Lausanne, Switzerland;
Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen, Germany;
Medizinische Klinik I, Universität des Saarlandes, Homburg, Germany; and
Ludwig Institute for Cancer Research, Lausanne Branch, and
¶ Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serological identification of Ags by recombinant expression cloning (SEREX), a method based on the cloning of genes encoding proteins recognized by high titer IgG Abs from cancer patients’ sera, has led to the identification of a series of Ags including the SSX, NY-ESO, and SCP CT Ag families (3, 4, 5, 6, 7, 8). Spontaneous humoral immune responses in cancer patients indicate that SEREX-defined proteins are immunogenic and may thus represent interesting targets for immunotherapy of cancer. Because CTLs represent a major arm of the immune response against cancer, the elicitation of a specific CTL response against tumor Ags is one of the main aims of current immunotherapy trials. Therefore, it is crucial to define CD8+ T cell epitopes in SEREX-defined Ags. However, despite the large number of SEREX-defined Ags, no CD8+ T cell epitopes have been characterized thus far in SEREX proteins other than NY-ESO-1 (9, 10).
HOM-MEL-40 was cloned in the initial SEREX study using serum from a metastatic melanoma patient (6). The sequence of the HOM-MEL-40 gene was identical with that of SSX-2, identified as one of the two genes involved in the translocation t(X;18)(p11.2;q11.2) found in 70% of synovial sarcomas (7, 11). Expression analysis performed by Northern blot and RT-PCR demonstrated the presence of SSX-2 transcripts in a significant proportion of human cancers, as for instance in melanomas (35%), head and neck cancers (35%), lymphomas (36%), and colon carcinomas (12%), but not in normal tissues with the exception of testis (7, 12). These findings allowed the classification of SSX-2 as a CT Ag. In addition, Abs against SSX-2 were found in 10% of patients with melanoma (7). Altogether these data highlighted SSX-2 as a potentially interesting target for immunotherapy of a large number of malignancies. However, the cellular immune response against SSX-2 in cancer patients has not been documented and no SSX-2-derived T cell epitopes have been identified.
A simple approach used to identify CD8+ T cell epitopes in a given protein is based on the prediction of peptides displaying the appropriate MHC class I binding motif and on the subsequent in vitro generation of CTLs against the predicted peptides (13). This approach, named reverse immunology, has been applied to a number of tumor-associated proteins but has led, in several instances, to the generation of peptide-specific CTLs that are unable to lyse Ag-presenting tumor cells (14, 15). This was shown to be mainly due to the fact that these peptides are normally not efficiently processed by the cellular machinery (16, 17). Recently, an improved reverse immunology strategy has been described (18). In that study, in vitro digestions of synthetic oligopeptides with purified proteasome led to the identification of cleavage products the C termini of which were compatible with the peptide-binding motifs for particular HLA class I molecules. The likelihood that the candidate HLA class I ligands were indeed processed in vivo was high, because it is now accepted that proteasomes generally generate the exact C termini of MHC class I-restricted antigenic peptides (19). Synthetic nonapeptides displaying the identified C termini were then used to stimulate PBMC and were shown to elicit a CTL response (18). Validation of this approach was provided by the demonstration that the derived CTL were able to lyse target cells expressing both the studied protein and the appropriate MHC restriction element. Although this strategy was a clear improvement over the original motif-based peptide prediction, the authors did not determine whether a natural immune T cell response against any of the identified peptide tumor Ags took place in vivo.
Here we associated the in vitro proteasomal degradation assay of overlapping SSX-2 peptides with the prediction of HLA-A2-binding peptides based on anchor residues to identify a natural T cell response in a tumor-infiltrated lymph node (TILN) of a melanoma patient seropositive for SSX-2 and whose tumor expressed SSX-2. We report the identification of an HLA-A2-restricted peptide derived from SSX-2 against which a natural CD8+ T cell response was detectable.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epitope prediction was performed as described (20). Briefly, potential HLA-A*0201 ligands from the sequence of SSX-2 were selected using a matrix pattern suitable for the calculation of nonamer or decamer peptides fitting to the HLA-A*0201 motif. Such motif predictions are available on our web page at http://www.syfpeithi.de.
Peptides were synthesized in an automated peptide synthesizer 432A (Applied Biosystems, Weiterstadt, Germany) following the N-(9-fluorenyl)methoxycarbonyl-/tert-butanol strategy. After removal from the resin by treatment with trifluoroacetic acid (TFA)-phenol-ethanedithiol-thioanisole-water (90:3.75:1.25:2.5:2.5 by volume) for 1 or 3 h (arginine-containing peptides), peptides were precipitated from methyl-tert-butyl ether, washed once with methyl-tert-butyl ether and twice with diethyl ether, and resuspended in water before lyophilization. Synthesis products were analyzed by HPLC (Varian star; Zinsser, Munich, Germany) and MALDI-TOF mass spectrometry (G2025A; Hewlett-Packard, Waldbronn, Germany). Peptides of <80% purity were purified by preparative HPLC.
Cell lines and clones
Surgically resected metastatic LN from patient LAU 50 was finely minced with needles in sterile RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS. Cell suspensions were placed in 24-well tissue culture plates (Costar, Cambridge, MA) in 2 of IMDM (Life Technologies, Basel, Switzerland) supplemented with 0.24 mM aspartic acid, 0.55 mM arginine, 1.5 mM glutamine, 8% pooled human serum (CTL medium), 100 U/ml human recombinant (hr) IL-2 (Glaxo, Geneva, Switzerland; kindly provided by Dr. M. Nabholz, Institut Suisse de Recherches Experimentales sur le Cancer, Epalinges, Switzerland), and 10 ng/ml hrIL-7 (Biosource International, Camarillo, CA). Cells were cultured 2–3 wk before FACS or ELISPOT. SSX-2-specific CTL clones were derived from TILN by limiting dilution cultures in the presence of irradiated allogeneic PBMC, PHA, and hrIL-2 as described elsewhere after isolation of specific cells by multimer guided cell sorting. Clones were derived from wells with a probability of clonality higher than 90% according to single-hit Poisson distribution. Melanoma cell lines Me 275 and Me 260 were established at the Ludwig Institute for Cancer Research, Lausanne Branch, from surgically excised melanoma lesions from patients LAU 50 and LAU 149, respectively. SK-MEL-37 and SK-MEL-23 cells were kindly provided by Dr. Y. T. Chen (Ludwig Institute for Cancer Research, New York Branch, New York, NY). Tumor cell lines (Me 290, Me 260, SK-MEL-23, and SK-MEL-37) and COS-7 cells were maintained in DMEM and the HLA-A2+ human mutant cell line CEMx721.T2 (T2 thereafter) in RPMI 1640 supplemented with 10% FCS.
Proteasome purification and peptide digestion
Proteasomes were purified from outdated human blood as previously described (17). Synthetic peptide (2 nmol) was digested with 16 µg purified proteasome for 10, 20, 40, and 80 min at 37°C. The reactions were stopped by adding 2% TFA. After lyophilization, the samples were analyzed by MALDI-TOF as described (17). The identification of proteolytic fragments was based on digestion times that were variable for each peptide. However, the time point was selected so that the mixture still contained relative excess of precursor peptides, thereby avoiding overdigestion of the precursors. Each peptide precursor was digested at least twice, using two different batches of purified proteasome, and yielded similar proteolytic products, confirming that the results obtained by this assay are reproducible.
PCR analysis
RNA extraction from cell lines was performed with Trizol reagent (Life Technologies, Basel, Switzerland). cDNA synthesis was performed as previously described (21), and aliquots (equivalent to 100 ng of RNA) were used for different PCR using a Qiagen HotStar Taq polymerase Master Kit (Basel, Switzerland). SSX-2 was amplified as described (22). Actin was amplified on each cDNA sample to assess the quality and quantity of input RNA.
IFN-
ELISPOT assay
IFN- ELISPOT assay was performed in nitrocellulose-lined
96-wells microplates (MAHA S45; Millipore, Bedford, MA) using a IFN-
ELISPOT kit (DIACLONE, Besançon, France) according to the
manufacturer’s instructions with minor modifications. Plates were
coated overnight with human IFN--specific Ab and washed six times.
T2 cells (5 x 104/well) were then added
together with the indicated number of responder T cells and peptide (1
µM where indicated). After incubation for 20 h at 37°C, cells
were removed and plates were developed with a second (biotinylated)
human IFN--specific Ab and streptavidin-alkaline phosphatase. Spots
were counted using BIOREADER 2000 (BIO SYS, Frankfurt, Germany).
Tetramer staining and flow cytometry immunofluorescence analysis
A2/SSX-241–49 multimeric complexes were synthesized as described (23). Cells were stained with PE-labeled multimers (0.25 µg/105 cells) in 20 µl PBS, 5% FCS during 20 min at 25°C, then 20 µl of a 1/25 dilution of anti-CD8FITC mAb (BD Biosciences, San Jose, CA) were added and incubated for an additional 30 min at 4°C. Cells were washed once in the same buffer, analyzed, and/or sorted by flow cytometry (FACScan or FACSVantage SE; BD Biosciences). Data analysis was performed using Cell Quest software.
Chromium release assay
Ag recognition was assessed using target cells labeled with 51Cr for 1 h at 37°C and washed three times. Labeled target cells (1000 cells in 50 µl) were then added to effector cells (100 µl) at the indicated E:T ratios in V-bottom microwells in the presence or absence of antigenic peptide (50 µl) at the indicated concentrations. Chromium release was measured in supernatant (100 µl) harvested after 4 h incubation at 37°C. The percent specific lysis was calculated as: 100 x [(experimental - spontaneous release)/(total - spontaneous release)].
Transient transfection and IFN- release assay
SSX-2 and SSX-4 encoding cDNA was cloned in pcDNA3.1 plasmid
(Invitrogen; Life Technologies, Basel, Switzerland). COS-7 cells were
transiently transfected with SSX-2- or SSX-4-encoding plasmids and with
a plasmid encoding HLA-A2 using FuGENE reagent according to the
manufacturer’s instructions (Roche Diagnostics, Rotkreuz,
Switzerland). Transfected cells were then tested for their
ability to stimulate the release of IFN- by CTL clones. In brief,
CTL (25,000/well) were added to transfected cells (10,000/well) in
96-well flat-bottom plates in 200 µl/well IMDM supplemented with 10%
human serum and 20 U/ml hrIL2. After a 24-h incubation at 37°C,
supernatants were collected, and the IFN- content was determined by
ELISA (BioSource Europe, Fleurus, Belgium).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We tested whether the in vitro proteasomal digestion of the 188-aa
tumor Ag SSX-2 could be used to restrict the number of candidate
epitopes among peptides predicted to bind to HLA-A2 molecules. To this
end, a set of 14 peptides 22 aa long and 1 with 20 aa, which covered
the complete SSX-2 protein sequence (Fig. 1
), was synthesized so that each peptide
overlapped with the preceding one by 10 aa. These peptides were
incubated with standard proteasome purified from human erythrocytes,
and the digested products were subsequently analyzed by mass
spectrometry. A summary of the identified cleavage sites is shown in
Fig. 1. The number of SSX-2 nonamer and decamer peptides predicted to
bind to HLA-A2 molecules (i.e., with a score of >15) was 29 (data not
shown). This binding prediction is mainly based on the identity of the
anchor residues and has been described elsewhere
(http://www.syfpeithi.de; Ref. 20). Of 17
HLA-A2-restricted high or intermediate binding affinity nonamers and 12
decamers predicted in the SSX-2 protein, the C terminus of only 8
nonamers and 4 decamers was generated by purified proteasome (Table I). Taken together, these results
indicate that <50% of the predicted high or intermediate binding
affinity HLA-A2-restricted nonamers and <35% of the predicted
decamers are likely to be generated intracellularly by proteasomal
degradation.
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The accumulation of T lymphocytes specific for a given tumor Ag in
melanoma lesions is one of the main features of a natural immune
response against this Ag (24).
HLA-A*0201+ patient LAU 50 was operated on for
a primary malignant melanoma in 1992, s.c. metastases in 1993, and
lymph node metastases in 1995 and has remained tumor free since then.
While screening a series of patients’ sera for the presence of
anti-SSX-2 Abs, a sample obtained in 1999 from LAU 50 was found
positive (data not shown). RT-PCR of RNA from LAU 50 metastatic lesion
excised in 1993 showed that the tumor expressed SSX-2 (data not shown).
SSX-2 was also expressed in the tumor cell line Me 275 derived from the
TILN excised in 1995 (Fig. 4A). As previously reported
(10, 25, 26), CTL clones specific for several tumor Ags
(NY-ESO-1157–165,
Camel1–11, and
MAGE-A10254–262) have been derived from this
TILN. On the basis of these data, we performed an ELISPOT to assess the
recognition of the above predicted SSX-2-derived peptides (Table I
) by
the short term-cultured TILN population obtained from LAU 50 in 1995.
As shown in Table I, peptides SSX-241–49
(KASEKIFYV) and SSX-240–49 (MKASEKIFYV) induced
the production of IFN- by cells from the TILN (69 and 39 spots,
respectively). The other 11 predicted peptides gave only 3 to 12 spots
per well, comparable with the background given by the control
HIV-derived peptide. Because the nonamer peptide
SSX-241–49 has a higher HLA-A2-binding score
than the decamer peptide SSX-240–49 (Table I),
we synthesized PE-labeled HLA-A2 multimers containing peptide
SSX-241–49 and used them to stain the TILN
population. As shown in Fig. 2A, 0.64% of
CD8+ cells in the TILN population stained clearly
positive. Altogether these data demonstrate the presence of
SSX-241–49-specific CD8+ T
cells within the TILN obtained from LAU 50.
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SSX-241–49 specific clones were obtained by
limiting dilution cloning of
multimer+CD8+ T cells
isolated by multimer guided flow cytometry cell sorting from the TILN
of patient LAU 50. Clone LAU 50 E2.4 was specifically stained by
A2/SSX-241–49 multimers but not by irrelevant
multimers (Fig. 2B). The specificity of this clone was
further confirmed in a 51Cr release assay. It
lysed T2 cells incubated with peptide SSX-241–49
(with an apparent avidity of 
10 pM) but not with an irrelevant
peptide (Fig. 3A, left).
Decapeptide SSX-240–49 was also recognized with
an apparent avidity of 100 pM. Whether peptide
SSX-240–49 binds to HLA-A2 and is recognized by
CTLs as such or is first converted to SSX-241–49
by degradation by serum proteases was not determined.
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A). The undigested
peptide SSX-237–58 was recognized by clone LAU
50 E2.4 only at a very high concentration. In contrast, the same
peptide preparation was recognized 100 times more efficiently after a
30-min incubation with proteasome, showing that proteasome contributed
to the formation of the CTL clone-defined epitope.
Although these data showed that CTL could recognize a product generated
by the proteasome in vitro, it was crucial to assess whether
SSX-241–49-specific T cells were also able to
recognize endogenously produced A2/SSX-241–49
complexes. To this end, COS-7 cells were transiently transfected with
plasmids encoding HLA-A2 and either SSX-2 or SSX-4 and tested for their
capacity to induce IFN- production by
SSX-241–49-specific clone LAU 50 E2.4. As shown
in Fig. 3B, the CTL clone produced high levels of IFN- in
the presence of cells cotransfected with plasmids encoding HLA-A2 and
SSX-2 but not HLA-A2 and SSX-4, a gene homologue to SSX-2 but which
differs by 2 aa within the antigenic peptide region. Untransfected
cells or cells expressing either HLA-A2 or SSX-2 alone were not
recognized.
To further verify the relation between the expression of SSX-2 and
HLA-A2 and the recognition by SSX-241–49
specific T cells, we used a panel of melanoma cell lines that were
characterized for SSX-2 expression by RT-PCR (Fig. 4A) and HLA-A2 expression by
Ab staining and flow cytometry analysis (data not shown). The cell
lines were tested for recognition by
anti-SSX-241–49 clone LAU 50 E2.4 (Fig. 4B). SSX-2+ and
A2+ autologous (Me 275) and allogeneic
(SK-MEL-37) tumor cell lines were efficiently lysed by the CTL clone,
in both the absence and presence of exogenously added peptide.
SSX-2-A2+ SK-MEL-23 cells
were not lysed unless peptide SSX-241–49 was
added exogenously. Finally, SSX-2+
A2- Me 260 cells were not lysed by the CTL
clone, neither in the presence nor in the absence of synthetic
peptide.
Similar results, in terms of peptide recognition as well as recognition
of transfected cells and tumor cell lines, were obtained with two other
independent CTL clones (having different TCR 
-chain variable domains
as assessed by mAb staining and flow cytometry analysis) derived from
the same TILN (data not shown). These results clearly demonstrate that
a spontaneous T cell response against SSX-2 can take place in cancer
patients and identify SSX-241–49 as an
HLA-A2-restricted CD8+ T cell epitope.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SSX-2 gene was cloned as encoding a target of IgG Abs from a melanoma patient in the first SEREX study in 1995. It was also identified as one of the two genes involved in the translocation t(X;18)(p11.2;q11.2) observed in synovial sarcomas in 1994 (6, 7, 11). Expression analysis performed by Northern blot and RT-PCR demonstrated the presence of SSX-2 transcripts in a significant proportion of human cancers, including melanomas (35%), head and neck cancers (35%), lymphomas (36%), and colon carcinomas (12%), but not in normal tissues with the exception of testis (12, 22). In contrast to synovial sarcoma, no translocation implicating SSX-2 was observed in those malignancies. Anti-SSX-2 Abs were found in 10 of 89 patients with melanoma but not in 41 apparently healthy controls (7). Here we demonstrate for the first time that SSX-2 can elicit a spontaneous CD8+ T cell response in addition to Abs. This response was detected in short term-cultured TILN of a patient with malignant melanoma who developed an anti-SSX-2 humoral response and whose tumor expressed SSX-2 mRNA. However, the correlation among seropositivity, SSX-2 expression in tumors, and T cell responses remains to be investigated. Indeed, in the case of NY-ESO-1, the only other SEREX-defined Ag for which a cellular immune response has been reported, CD8+ T cell responses were found in virtually all seropositive patients tested but also in a smaller proportion of seronegative patients (10). SSX-2 is a member of the SSX multigene family composed of 5 genes the products of which share 77–91% sequence homology (12). All SSX genes are expressed in testis but not in other normal tissues, and all, with the exception of SSX-3, were found to be expressed in tumors (12, 22). Based on their high degree of homology, it would be of interest to study the immunogenicity of the other members of the SSX family that are expressed in tumors. SSX-1 and SSX-2 genes are both implicated in the translocation t(X;18)(p11.2;q11.2) observed in synovial sarcomas. This translocation event results in the expression of a fusion protein containing the 78 C-terminal amino acids of the SSX polypeptide (27), thus excluding the CTL epitope identified in our study. Whether this fusion protein is immunogenic and can therefore represent a potential target for immunotherapy of synovial sarcomas is unknown.
The original method used to identify peptide tumor Ags relied on the isolation of autologous tumor-specific CTL from cancer patients. These CTL were then used to screen Ag-negative target cells either transfected with cDNA libraries derived from tumor cells or pulsed with peptides eluted from MHC class I molecules purified from tumor cells (1, 2, 28). Because CTL used to identify these epitopes are tumor reactive, the antigenicity and proper processing of such epitopes are not questionable. More recently, a new strategy has been tested. This strategy, named reverse immunology, allows prediction, in silico, of the potential HLA class I ligands from any protein sequence based on the identification of appropriately positioned anchor residues specific for a given HLA class I allele (13, 20). The peptides that were predicted to bind to a given HLA class I molecule with the highest affinity were selected, and their immunogenicity was tested. Although this method allowed the identification of a number of promising peptides, several of the latter were not produced by tumor cells as demonstrated by the poor recognition of cells expressing the corresponding protein of interest endogenously (15, 16, 17). Hence, a crucial step for a correct application of this latter method is the demonstration that the generated CTL can recognize target cells expressing the antigenic protein. Regarding peptide SSX-241–49, the accumulation of specific T cells within melanoma lesions strongly supported the antigenicity and immunogenicity of the peptide. Moreover, the fact that specific CTL clones derived from TILN, which recognized the peptide with high apparent avidity, were able to recognize the endogenously produced epitope in tumor cell lines and in transfected COS-7 cells further confirmed the adequate production of the epitope by the cellular processing machinery. In contrast to the approach recently applied by Kessler et al. (18), in which they narrowed the number of high affinity HLA-A2 ligands derived from the tumor Ag PRAME by performing in vitro proteasomal digestions of peptides encompassing candidate peptides, we took the inverse approach, namely to identify among the peptides with C termini that were generated by proteasome those that were predicted to bind HLA-A2 with high or intermediate affinity. Because the C termini of >50% of the nonamers and 65% of decamers predicted to bind HLA-A2 with high or intermediate affinity were not generated by the proteasome in vitro, we were able to restrict the synthesis of peptides to be tested. In addition, our approach allows the simultaneous identification of potential ligands for various HLA class I molecules.
The majority of mammalian cells contain the standard proteasome.
However, lymphoid cells (including myeloid-derived dendritic cells)
or cells exposed to IFN- express the immunoproteasome, which differs
from the standard proteasome by the presence of different catalytic
subunits. Evidence indicates that such exchange results in a reduced
production of several peptide tumor Ags (29, 30). Whereas
digestion of PRAME peptides was performed using immunoproteasome
(18), we used standard proteasome to digest the SSX-2
precursor peptides. Because the tumor cell environment may differ in
terms of IFN- concentrations, it is possible that the proteasome
composition and, consequently, the type of peptide tumor Ags produced
may vary. However, preliminary experiments show that tumor cells
cultured in presence of IFN- are recognized by
SSX-241–49 specific CTL clones, suggesting that
this peptide can be generated by both standard and
immunoproteasomes.
Peptide SSX-241–49 is a new candidate peptide for immunotherapy of a wide range of human malignancies. However, the immunogenicity of this peptide in cancer patients (with malignant melanoma and other malignancies) and the efficiency of the peptide-induced CTL to recognize tumor cells should be determined before introducing peptide KASEKIFYV into clinical trials. Peptides homologous to SSX-241–49 in the other SSX proteins differ by only 1 or 2 aa from peptide KASEKIFYV. We are currently investigating whether peptides derived from the same region of other SSX family members could also constitute T cell epitopes. In the case of MAGE-A1 and MAGE-A3 tumor Ags, two homologous but not identical peptides derived from the same region (MAGE-A1160–168 and MAGE-A3168–176) were shown to constitute two independent HLA-A1-restricted epitopes that stimulate distinct CTL repertoires (31). The other potential SSX-2-derived epitopes predicted in this study were not recognized by TILN from patient LAU 50. We are currently investigating the recognition of these peptides by TIL(N)s from other cancer patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Maha Ayyoub, Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Hôpital Orthopédique, Niveau 5, Avenue Pierre-Decker 4, 1005 Lausanne, Switzerland. E-mail address: Maha.Ayyoub{at}chuv.hospvd.ch
3 Current address: Johannes Gutenberg Universitaet Mainz, Med Klinik III, Mainz, Germany.
4 D.S. and F.L. equally contributed to this work.
5 Abbreviations used in this paper: CT, cancer-testis; SEREX, serological identification of Ags by recombinant expression cloning; TILN, tumor-infiltrated lymph node; TFA, trifluoroacetic acid; hr, human recombinant.
Received for publication October 26, 2001. Accepted for publication December 12, 2001.
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) of SSX-241–49 synthetic
peptide (0.1 µM). HLA-A2 expression was assessed by mAb staining and
flow cytometry analysis (data not shown).
), SSX-240–49 (
) or
MelanA26–35 A27L (
) or 30 (
) min at 37°C; the reaction was stopped by addition
of 2% TFA; and samples were lyophilized, resuspended in medium to a
concentration of 1 µM of the precursor peptide at time 0, and added
in 3-fold dilution steps to T2 cells. B, Recognition of
transfected COS-7 cells. Cells transfected with plasmids encoding
HLA-A2, SSX-2, or SSX-4 (as indicated) were cocultured with effector
cells at an E:T ratio of 2.5:1. The production of IFN-