Tetramer analysis of IDO5-specific T cells.

<p><i>(</i><i>a</i><i>),</i> An example of IDO5-specific, CD8 T-cell enriched PBMC from a renal cell carcinoma patient visualised by flow cytometry staining using the tetramer complex HLA-A2/IDO5-PE, and CD8-allophycocyanin after one <i>in vitro</i> stimulation with IDO5 peptide. As a negative control, cells were stained with the tetramer complex HLA-A2/HIV-1 pol_476–484-PE, and CD8-allophycocyanin. <i>(</i><i>b</i><i>),</i> PBMC from healthy donors or from patients with breast cancer, melanoma cancer or renal cell carcinoma were stained with the tetramer complex HLA-A2/IDO5 or HLA-A2/HIV-1 pol_476–484 and analyzed by flow cytometry either <i>ex vivo</i> or after one <i>in vitro</i> peptide stimulation. The dotted lines illustrate that IDO5-tetramer positive cells are detectable both <i>ex vivo</i> and <i>in vitro</i> in the same patients. <i>(</i><i>c</i><i>),</i> An example of CD45RA and CD28 phenotype analysis of IDO5-tetramer/CD8 gated cells from CD8 T-cell enriched PBMC from a renal cell carcinoma patient visualised <i>ex vivo</i> by flow cytometry. For comparison, the cells were stained with isotype matched controls <i>(</i><i>d</i><i>),</i> An example of an IL-2 expanded TIL culture from a melanoma patient visualised by flow cytometry staining using the tetramer complex HLA-A2/IDO5-PE, and CD8-APC-Cy7. As a negative control, the TIL were stained with the tetramer complex HLA-A2/HIV-1 pol_476–484-PE, and CD8-APC-Cy7. <i>(</i><i>e</i><i>),</i> As a positive control of the IDO5 tetramer, an IDO5-specific T-cell clone (RBS35) was stained with the HLA-A2/HIV-1 pol_476–484-PE and HLA-A2/IDO5-PE tetramers.