Amplified T cell libraries as a new tool to measure frequencies of antigen-specific naive T cells in human adult blood and cord blood. (A) Schematic representation of the method. Library generation: naive CD4⁺ T cells are seeded at 2,000 cells/well in multiple wells containing irradiated allogeneic PBMC, PHA, and IL-2. The individual cultures are expanded into larger wells that make up the library of amplified T cell blasts. Screening of the library: T cell from individual cultures are collected, washed, and tested for their capacity to proliferate in response to various antigens (Ag) in the presence of autologous irradiated monocytes. (B) Libraries consisting of 192 polyclonal cell cultures, each derived from 2 × 10³ naive T cells, were prepared from 7 adult donors and screened for their capacity to proliferate in response to KLH or PA (both at 5 µg/ml). After 3 d, proliferation was measured after 16-h pulse with [³H]thymidine. Shown are delta cpm values. Each symbol illustrates one culture out of the 192 screened. In all graphs, background values <100 cpm were set to 100 cpm to represent all data points analyzed in the y-log scale. Dotted lines represent the cut-off value. The specificity of positive cultures was confirmed in a second independent experiment. None of the cultures recognized both antigens (not depicted). (C) Frequencies of naive CD4⁺ T cells specific for KLH and PA in the seven adult donors were calculated from the data shown in B. Bars represent 95% confidence intervals. (D) Frequencies with 95% confidence intervals of naive CD4⁺ T cells specific for KLH in 5 cord blood samples. (E) Libraries of naive CD4⁺ T cells were produced with different input cell numbers (1,000, 2,000, and 4,000 per well) and screened with TT. Shown are delta cpm values (left) and precursor frequencies with 95% confidence intervals (right). Dotted line represents the cut-off value. One representative experiment of two performed.