Article Figures & Data
Figures
- Fig. 1.
Recognition of the NY-ESO-1119–143 peptide and UPCI-MEL 527.1 cell line by CD4+ T cells of an HLA-DRB1*0401 normal donor. CD4+ T cells from an HLA-DRB1*0401 donor underwent three rounds of in vitro stimulation with autologous DCs pulsed with the NY-ESO-1119–143 peptide, as described in “Materials and Methods.” Ten thousand of the resulting responder CD4+ T cells were incubated in a 20-h IFN-γ ELISPOT assay in the presence of T2. DR4 cells were pulsed with the NY-ESO-1119–143 peptide or Melan-A/MART-1 51–73 peptide (10 μg/ml), UPCI-MEL 527.1 cells ± anti-HLA-DR antibodies (L243), or UPCI-MEL 527.1 cells ± anti-HLA-A,B,C antibodies (W6/32). IFN-γ spots were developed and counted by computer-assisted video image analysis. Columns, mean spot numbers of triplicates with 104 CD4+ T cells initially seeded/well; bars, SD. Data from one representative experiment of three performed are depicted.
- Fig. 2.
Recognition of the NY-ESO-1119–143 and NY-ESO-1119–133 peptides and the NY-ESO-1+, DR4+ cell line, UPCI-MEL 527.1 cell line by the CD4+ T-cell clone 10/4. CD4+ T cells were isolated from the peripheral blood of patient UPCI-MEL 527 and stimulated in vitro with autologous DCs pulsed with the NY-ESO-1119–143 peptide. The CD4+ T cells that recognize not only APCs pulsed with the NY-ESO-1119–143 peptide but also the DR4+, NY-ESO-1+, melanoma cell line UPCI-MEL 527 were cloned by limiting dilution as described in “Materials and Methods.” One thousand CD4+ T cells from clone 10/4 were incubated in a 20-h IFN-γ ELISPOT assay (A) or in a 20-h IFN-γ ELISA (B) in the presence of T2. DR4 cells pulsed with peptides (10 μg/ml), UPCI-MEL 527.1 cells ± anti-HLA-DR antibodies (L243), UPCI-MEL 527.1 cells ± anti-HLA-A,B,C antibodies (W6/32) are shown. The DR4+, NY-ESO-1− melanoma cell line UPCI-MEL 136.1 unpulsed or pulsed with the NY-ESO-1119–143 peptide were added as targets in the assays. Data from one representative experiment of three performed are depicted. Bars, SD.
- Fig. 3.
Lysis of the NY-ESO-1+, DR4+ cell line UPCI-MEL 527.1 by clone 10/4. The melanoma cells were preincubated for 48 h with IFN-γ prior to the lysis assay to up-regulate HLA-DR4 expression. Unlabeled T2.DR4 cells or NY-ESO-1119–143-pulsed T2.DR4 (50,000/well) were added as cold-target inhibitors to suppress lysis. Chromium release was measured after 4 h. We show the data from one representative experiment of two performed; bars, SD.
- Fig. 4.
Reactivity of CD4+ T-cell clone 10/4 to titered doses of peptide NY-ESO-1119–143. T2.DR4 cells were pulsed with different concentrations of the NY-ESO-1119–143 peptide and used as target cells in a 20-h IFN-γ ELISPOT assay (A). Alternatively, T2.DR4 cells were similarly pulsed with NY-ESO-1119–143 peptide and incubated in the presence of the CD4+ T cell clone 10/20 in a 20-h IFN-γ ELISA assay (B). The data represent the average of triplicate cultures; bars, SD. The data were obtained from one representative experiment of three performed, with comparable results observed in each assay.
- Fig. 5.
Recognition of the NY-ESO-1119–143 and NY-ESO-1123–137 peptides and the autologous melanoma cell line, UPCI-MEL 527.1, by the CD4+ T-cell clone 11/4. CD4+ T cells were isolated from the peripheral blood of patient UPCI-MEL 527 and stimulated in vitro with autologous DCs pulsed with the NY-ESO-1119–143 peptide. The CD4+ T cells that recognize not only APCs pulsed with the NY-ESO-1119–143 peptide but also the DR4+, NY-ESO-1+ autologous melanoma cell line UPCI-MEL 527 were cloned by limiting dilution as described in“ Materials and Methods.” One thousand CD4+ T cells from clone 11/4 were incubated in a 20-h IFN-γ ELISPOT assay (A) or in a 20-h IFN-γ ELISA (B) in the presence of T2.DR4 cells pulsed with peptides (10 μg/ml), UPCI-MEL 527.1 cells ± anti-HLA-DR antibodies (L243), UPCI-MEL 527.1 cells ± anti-HLA-A,B,C antibodies (W6/32). The DR4+, NY-ESO-1− melanoma cell line UPCI-MEL 136.1 unpulsed or pulsed with the NY-ESO-1119–143 peptide were added as targets in the assays. Data from one representative experiment of three performed are depicted. Bars, SD.
- Fig. 6.
Reactivity of CD4+ T-cell clone 11/4 to titered doses of the NY-ESO-1119-143 and NY-ESO-1123–137 peptides. T2.DR4 cells were pulsed with different concentrations of either the NY-ESO-1119–143 peptide (•) or the NY-ESO-1123–137 peptide (○) and used as target cells in a 20-h IFN-γ ELISPOT assay (A). Alternatively, T2.DR4 cells were similarly pulsed with each peptide and incubated in the presence of the CD4+ T-cell clone 10/20 in a 20-h IFN-γ ELISA assay (B). The data represent the average of triplicate cultures; bars, SD. The data were obtained from one representative experiment of three performed, with comparable results observed in each assay.
- Fig. 7.
Lysis of the autologous melanoma cell line UPCI-MEL 527.1 by clone 11/4. CD4+ T cells from clone 11/4 were incubated in the presence of autologous melanoma cells, UPCI-MEL 527, ± anti-HLA-DR antibodies (L243), ± anti-HLA-A,B,C antibodies (W6/32). The melanoma cells were preincubated for 48 h with IFN-γ prior to the lysis assay to up-regulate HLA-DR4 expression. Unlabeled T2.DR4 cells or NY-ESO-1119–143-pulsed T2.DR4 (50,000/well) were added as cold-target inhibitors to suppress antigen-specific lysis. Chromium release was measured after 4 h. We show the data from one representative experiment of two performed. Bars, SD.
- Fig. 8.
Clone 11/4 recognizes autologous DCs loaded with the ESO-1 protein. Five thousand cells of T-cell clone 11/4 were incubated in a 20-h IFN-γ ELISPOT assay in the presence of DCs loaded either with the ESO-1 protein or the SSX protein (30 μg/ml) and unloaded DCs. IFN-γ spots were developed and counted by computer-assisted video image analysis. Columns, mean spot numbers of triplicates; bars, SD.
- Fig. 9.
CD4+ T cells stimulated with autologous DCs loaded with the NY-ESO-1 protein recognize the NY-ESO-1119–143 peptide and the autologous melanoma cell line UPCI-MEL527.1. Total PBMCs from an HLA-DRB1*0401 donor underwent three rounds of in vitro stimulation with autologous DCs loaded with the NY-ESO-1 protein as described in “Materials and Methods.” Ten thousand of the resulting responder T cells were incubated in a 20-h IFN-γ ELISPOT assay in the presence of T2.DR4 cells pulsed with the potential NY-ESO-1 DR4-binding peptides including NY-ESO-1119–143 peptide or Melan-A/MART-151–73 peptide (10 μg/ml). The DR4+, NY-ESO-1+ UPCI-MEL 527.1 cells and the DR4+, NY-ESO-1− UPCI-MEL 136.1 cells were included as targets in the assays. IFN-γ spots were developed and counted by computer-assisted video image analysis. Columns, mean spot numbers of triplicates with 104 CD4+ T cells initially seeded per well; bars, SD.
Tables
- Table 1
HLA-DRB1*0401-binding NY-ESO-1 peptides predicted by the peptide binding algorithm
Peptide binding sequences are provided using single-letter amino acid designation. Peptide binding algorithm (11 , 12) scores for the highest-scoring embedded 9-mer sequences (underlined) are indicated for each peptide evaluated.
NY-ESO-1 peptides: amino acid residues Sequences Algorithm score, 0–10 Peptides tested in the study 119–143 PGVLLKEFTVSGNILTIRLTAADHR + 119–133 PGVLLKEFTVSGNIL + 123–137 LKEFTVSGNILTIRL + 129–143 SGNILTIRLTAADHR + 122–130 LLKEFTVSG 9 126–134 FTVSGNILT 4 132–140 ILTIRLTAA 4 144–169 QLQLSISSCLQQLSLLMWITQCFLPV + 147–155 LSISSCLQQ 3 153–161 LQQLSLLMW 3 158–166 LLMWITQCF 6 161–170 WITQCFLPV 3 158–180 LLMWITQCFLPVFLAQPPSGQRR + 161–170 WITQCFLPV 3 162–170 FLPVFLAQP 5 166–174 VFLAQPPSG 6 169–177 FLAQPPSGQ 4
Volume 60, Issue 17
