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. Author manuscript; available in PMC: 2014 Jan 24.
Published in final edited form as: J Immunol. 2010 Dec 3;186(1):312–322. doi: 10.4049/jimmunol.1001989

FIGURE 4.

FIGURE 4

LAGE-1–specific CD4+ T cell clones are high-affinity T cells that recognize naturally processed and presented epitopes. A, Each LAGE-1– specific CD4+ T cell clone (1 × 103), 90/76, 116/12, and 89/130, was incubated in 48-h IFN-γ ELISPOT assays in the presence of either L.DR cells pulsed with 3 µM peptide LAGE-197–114 (left), peptide LAGE-1104–121 (middle), or peptide LAGE-1b118–135 (right), respectively, or with autologous immature DCs (iDCs) loaded with cell lysates of COS-7 cells transfected with a plasmid encoding the LAGE-1b protein (pcDNA3-LAGE-1b), or with iDCs loaded with the recombinant LAGE-1a protein. As controls, each clone was tested against NY-ESO-1–derived peptides overlapping the LAGE-1 peptide sequences (i.e., NY-ESO-197–114 [left], NY-ESO-1104–121 [middle], or NY-ESO-1118–135 [right]), or against autologous iDCs loaded with cell lysates from COS-7 cells transfected with a plasmid encoding the NY-ESO-1 protein (pcDNA3-NY-ESO-1). B, Each LAGE-1–specific CD4+ T cell clone was incubated with relevant peptide without APCs or autologous iDCs loaded either with recombinant protein LAGE-1a or with cell lysates from LAGE-1+ melanoma cell line UPCI-MEL 285.1. As controls, CD4+ T cell clones were also incubated with autologous iDCs loaded either with the irrelevant LAGE-1 ORF2 protein or cell lysates from LAGE-1− melanoma cell UPCI-MEL 136.1. For A and B, each bar represents the mean spot number of triplicates ± SD, with 1 × 103 CD4+ T cells initially seeded per well. *p < 0.05. One representative experiment of three performed is depicted. C, Bulk LAGE-1–specific CD4+ T cells (1 × 104) obtained from MP1 (left), MP5 (middle), and MP3 (right), and depicted in Fig. 3, were incubated in 48-h IFN-γ ELISPOT assays in the presence of L.DR cells pulsed with relevant LAGE-1 peptide or NY-ESO-1 peptide. Each bar represents the mean spot number of triplicates ± SD, with 1 × 104 CD4+ T cells initially seeded per well. *p < 0.05. D, L.DR cells were pulsed with titrated doses of peptide LAGE-197–114, LAGE-1104–121, or peptide LAGE-1b118–135 and incubated in the presence of CD4+ T cell clone 90/76 (left), clone 116/12 (middle), or clone 89/130 (right), respectively, in 48-h IFN-γ ELISPOT assays as described in Materials and Methods. Data represent the mean spot number of triplicates, with 1 × 103 CD4+ T cells initially seeded per well. One representative experiment of three performed is depicted.