SSX-2–specific CD4⁺ T cells efficiently recognize the SSX-2 native antigen upon processing and presentation by professional APCs. Processing and presentation of SSX-2 antigen by DCs after incubation with recombinant SSX-2 protein (or NY-ESO-1 protein as negative control), or with lysates from tumor cell lines SK-MEL-37 (SSX-2–expressing) or NA8-MEL (SSX-2–negative), was assessed by intracellular staining with anti–IFN-γ antibodies (A) or by ELISA measurement of IFN-γ secretion in the culture supernatant (B).

Assessment of SSX-2_37–58–specific CD4⁺ T cells among circulating lymphocytes and tumor-infiltrated lymph nodes (TILN). (A) The presence of specific CD4⁺ T cells among peptide-stimulated PBMCs was assessed by intracellular staining with anti–IFN-γ antibodies after incubation in the absence or the presence of peptide SSX-2_37–58. (B) SSX-2_37–58–specific CD4⁺ T cells were similarly assessed in TILN, enriched by cytokine secretion–guided cell sorting, and expanded in vitro as polyclonal or monoclonal populations. Numbers in the upper right quadrants are the percentage of cytokine-producing cells among CD4⁺ T cells.

Detection of SSX-2–specific CD4⁺ T cells among circulating T lymphocytes from patient LAU 672 after stimulation with SSX-2–loaded autologous DCs. The presence of specific CD4⁺ T cells in the culture from patient LAU 672 was assessed by staining with phycoerythrin-labeled anti-CD4 mAB and intracellular staining with anti–IFN-γ mAb after incubation in the absence of added peptide or after stimulation with a pool containing all overlapping peptides spanning the SSX-2 protein, or subpools containing three peptides each, as indicated. Numbers in the upper right quadrants are the percentage of cytokine-producing cells among CD4⁺ T cells. Results for all subpools are summarized in Table 1. PE, phycoerythrin.

Determination of the minimal sequence optimally recognized by SSX-2–specific CD4⁺ T cells and assessment of cross-recognition of homologous peptides from other SSX antigens. (A and B) Synthetic peptides truncated at the N- or C- terminus of the SSX-2_37–58 sequence were used to determine the optimal length of the epitope recognized by SSX-2–specific CD4⁺ T cells. Peptide activity was calculated relative to that of SSX-2_37–58 in peptide-titration experiments. (C) Binding score and ranking of SSX-2_45–59–homologous peptides from other SSX antigens were calculated using the SYFPEITHI binding prediction program (http://www.syfpeithi.de). (D) Cross-recognition of 45–59 homologous peptides from SSX-1 and -5 by SSX-2–specific CD4⁺ T cells was assessed in peptide-titration experiments by ELISA measurement of IFN-γ secretion in the culture supernatant.

Identification of the active peptide and recognition in the context of HLA-DR. (A) Intracellular IFN-γ secretion by SSX-2–specific CD4⁺ T cells (clone 1B2) was assessed upon stimulation with a pool containing all overlapping peptides spanning the SSX-2 protein or single peptides in the active P4–6 subpool. (B) Peptide recognition was assessed either in the absence or in the presence of anti–HLA-DR, -DP, or -DQ antibodies. (C) The ability of homozygous EBV-transformed cell lines pulsed with serial dilutions of peptide SSX-2_37–58 to stimulate specific CD4⁺ T cells was assessed by ELISA measurement of IFN-γ secretion in the culture supernatant.

SSX-2–specific CD4⁺ T cells do not directly recognize SSX-2–expressing tumor cells. (A) Surface expression of HLA-DR on the melanoma cell line T567A was assessed using HLA-DR–specific mAb. (B) Recognition of T567A by SSX-2–specific CD4⁺ T cells was assessed by intracellular staining with anti–IFN-γ antibodies, in the absence or the presence of exogenously added peptide. Where indicated, cells were treated with IFN-γ (200 IU/ml) for 48 hours. Recognition was similarly assessed using a CD8⁺ T cell clone specific for the previously described HLA-A2–restricted CD8⁺ T cell epitope SSX-2_41–49. PerCP, peridinin chlorophyll protein. (C) Recognition of melanoma cell lines by SSX-2–specific CD4⁺ or CD8⁺ T cells, in the absence or the presence of exogenously added peptide, as indicated, was assessed by ELISA measurement of IFN-γ secretion in the culture supernatant. Prior to the test, tumor cells were treated with IFN-γ for 48 hours. Where indicated, tumor cells were transfected with a plasmid encoding full-length SSX-2. *The Me 279.1 tumor cell line had no surface expression of MHC class I molecules as assessed by staining with anti–HLA-ABC mAb.