Article Figures & Data
Figures
- Fig. 1.
Recognition of the NY-ESO-1 119–143 peptide and NY-ESO-1+ melanoma cell lines by CD4+ T cells of an HLA-DRB1*0701+/DRB4*0101+ normal donor. CD4+ T cells from an HLA-DRB1*0701+/DRB4*0101+ normal donor underwent three rounds of in vitro stimulation with autologous DCs pulsed with the NY-ESO-1 119–143 peptide as described in “Materials and Methods.” One thousand of the resulting responder CD4+ T cells were incubated in a 20-h IFN-γ ELISPOT assay in the presence of L.DR7 or L.DR53 cells pulsed with peptides NY-ESO-1 119–143, NY-ESO-1 121–138, NY-ESO-1 123–137, or NY-ESO-1 115–132 (10 μg/ml). The CD4+ T cells were also incubated in the presence of the HLA-DRB1*0701+/DRB4*0101+ melanoma cell line, MEL 591.8, and the HLA-DRB1*0701−/DRB4*0101+ melanoma cell line, MEL 527.1, +/− anti-HLA-DR (L243) antibodies or +/− anti-HLA-A, B, C (W6/32) antibodies. IFN-γ spots were developed and counted by computer-assisted video image analysis. Each column represents the mean spot number of triplicates ± SD (bars) with 103 CD4+ T cells initially seeded per well (P values of 0.05 were considered significant and are indicated by ∗). Data from one representative experiment of three performed are depicted.
- Fig. 2.
Recognition of the NY-ESO-1 119–143 peptide and NY-ESO-1+ melanoma cell lines by CD4+ T cells of an HLA-DRB1*1101+ normal donor. CD4+ T cells from an HLA-DRB1*1101+ normal donor underwent three rounds of in vitro stimulation with autologous DCs pulsed with the NY-ESO-1 119–143 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 DRB1*1101+ B-lymphoblastoid cells pulsed with peptides NY-ESO-1 119–143, NY-ESO-1 121–138, NY-ESO-1 123–137, or NY-ESO-1 115–132 peptide (10 μg/ml) and the NY-ESO-1+, DR11+ melanoma cell line, MEL 591.8, +/− anti-HLA-DR antibodies (L243) or +/− anti-HLA-A, B, C antibodies (w6/32). IFN-γ spots were developed and counted by computer-assisted video image analysis. Each column represents the mean spot number of triplicates ± SD (bars) with 104 CD4+ T cells initially seeded per well (P = 0.05 was considered significant and is indicated by ∗). Data from one representative experiment of two performed are depicted.
- Fig. 3.
Recognition of the NY-ESO-1 119–143 peptide and the autologous NY-ESO-1+ melanoma cell line by CD4+ T cells of an HLA-DRB1*0101+ melanoma patient. CD4+ T cells from a long-lived HLA-DRB1*0101 patient with melanoma, UPCI 285, underwent three rounds of in vitro stimulation with autologous DCs pulsed with the NY-ESO-1 119–143 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 L.DR1 cells pulsed with peptides NY-ESO-1 119–143, NY-ESO-1 121–138, NY-ESO-1 123–137, or Melan-A/MART-1 51–73 NY-ESO-1 123–137 or Melan-A/MART-1 51–73 (10 μg/ml) and the autologous melanoma cell line, MEL 285.1, +/− anti-HLA-DR antibodies (L243) or +/− anti-HLA-A, B, C antibodies (W6/32). IFN-γ spots were developed and counted by computer-assisted video image analysis. Each column represents the mean spot number of triplicates ± SD (bars) with 104 CD4+ T cells initially seeded per well (P = 0.05 was considered significant and is indicated by ∗). Data from one representative experiment of two performed are depicted.
- Fig. 4.
CD4+ T cells from an HLA-DRB1*0701+/DRB4*0101+ normal donor stimulated with autologous DCs loaded with the NY-ESO-1 protein recognize the NY-ESO-1 119–143 peptide and the DRB4*0101-matched melanoma cell line, MEL 285.1. Total peripheral blood mononuclear cells from an HLA-DRB1*0701+/DRB4*0101+ normal 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 L.DR7 pulsed with peptides (10 μg/ml) and the DR53+, NY-ESO-1+ UPCI-MEL 285.1 cells. IFN-γ spots were developed and counted by computer-assisted video image analysis. Each column represents the mean spot number of triplicates ± SD (bars) with 104 CD4+ T cells initially seeded per well (P = 0.05 was considered significant and is indicated by ∗).
- Fig. 5.
CD4+ T cells from an HLA-DRB1*0701+//DRB4*0101+ normal donor recognize autologous DCs loaded with the ESO-1 protein. CD4+ T cells were isolated from the peripheral blood of an HLA-DRB1*0701+/DRB4*0101+ normal donor and stimulated in vitro with autologous DCs pulsed with the NY-ESO-1 119–143 peptide. Five thousand CD4+ T cells 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) as well as unloaded DCs. IFN-γ spots were developed and counted by computer-assisted video image analysis. Each column represents the mean spot number of triplicates ± SD (bars). P = 0.05 was considered significant and is indicated by ∗.
- Fig. 6.
Peptide NY-ESO-1 119–143 stimulates Th2-type CD4+ T cells of an HLA-DRB1*0401/DRB4*0101+ melanoma patient. CD4+ T cells from an HLA-DRB1*0401+/DRB4*0101+ melanoma patient underwent at least three rounds of in vitro stimulation with autologous DCs pulsed with the NY-ESO-1 119–143 peptide. Ten thousand of the resulting responder CD4+ T cells were incubated in a 48-h IL-5 ELISPOT assay in the presence of T2.DR4 cells pulsed with peptides NY-ESO-1 119–143 or Melan-A/MART-1 51–73 (10 μg/ml) and the autologous melanoma cells, MEL 527.1; MEL 527.1 cells +/− anti-HLA-DR antibodies (L243), or UPCI-MEL 527.1 cells +/− anti-HLA-A, B, C antibodies (W6/32). IL-5 spots were developed and counted by computer-assisted video image analysis. Each column represents the mean spot number of triplicates ± SD (bars) with 104 CD4+ T cells initially seeded per well. Data from one representative experiment of three performed are depicted. P = 0.05 was considered significant and is indicated by ∗.
Tables
- Table 1
Binding capacities to immunopurified HLA-DR molecules NY-ESO-1 peptides
IC50s are expressed in nm and have been evaluated from at least three independent experiments. The biotinylated peptides used at indicated concentrations in these assays are the following: HA 306–318 (PKYVKQNTLKLAT) for DR1 (1 nm), DR4 (30 nm), DR11 (40 nm), and DRB5 (10 nm); MT 2–16 (AKTIAYDEEARRGLE) for DR3 (200 nm); YKL (AAYAAAKAAALAA) for DR7 (5 nm); B1 21–36 (TERVRLVTRHIYNREE) for DR13 (500 nm); A3 152–166 (EAEQLRRAYLDGTGVE) for DR15 (30 nm); Lo1 191–120 (ESWGAVWRIDTPDKLTGPFT) for DRB3 (10 nm); and E2/E168 (AGDLLAIETDKATI) for DRB4 (3 nm). IC50s for the active peptides are <1000 nm and are indicated in bold (19) .
Allele frequency Alleles DR1 DR3 DR4 DR7 DR11 DR13 DR15 DRB3 DRB5 DRB4 Peptides NY-ESO-1 119–143 20 >10,000 12 49 5 >10,000 40 800 43 125 NY-ESO-1 119–133 220 >10,000 13 63 1630 >10,000 400 1,830 8,330 1,830 NY-ESO-1 121–138 65 >10,000 20 13 38 >10,000 >10,000 3,000 600 >10,000 NY-ESO-123–137 18 >10,000 27 11 11 >10,000 >10,000 1,250 775 >10,000 NY-ESO-1 129–143 1,670 >10,000 1050 333 550 >10,000 8,000 >10,000 550 175
Volume 62, Issue 1
