Distinct phenotype and function of anergic CD8+ T cells produced by Treg-cell suppression.

Four conditions of cultured CD8+ T cells were analyzed with Affymetrix HG-U133-Plus-2.0 microarrays. Peripheral blood samples were obtained from healthy individuals (all of them had CMV-IgG Ab). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient with Ficoll-Paque (GE Healthcare). All donors provided written informed consent before sampling according to the Declaration of Helsinki. The present study was approved by the institutional ethics committee of Osaka University. CD8+ T cells were purified from PBMCs using CD8 Microbeads (Miltenyi Biotec). Treg cells were sorted as CD25highCD4+ T cells (highest 3% of CD25+CD4+ T cells) with FACS Aria II and Regulatory T cell Isolation kit (Miltenyi Biotec) from PBMCs. Antigen-specific CD8+ T cells were induced as described previously with some modifications. Melan-A-specific CD8+ T cells were detectable by MHC/peptide tetramers (Tet+CD8+ T cells). Tet+CD8+ T cells induced at CD8+ T cells: Treg cells ratio, 1:0.5 and 1:0 were sorted by FACSAria II (BD Bioscience), and subjected to Gene Chip Human Genome U133 Plus 2.0 Array. The data were preprocessed with RMA (Robust Multichip Average) normalization. Genes reportedly associated with CD8+ T-cell dysfunction such as anergy and exhaustion were selected. These gene expressions were compared among 4 groups, Tet(+) with Treg, Tet(+) w/o Treg, Tet(-) with Treg and Tet(-) w/o Treg.