High Throughput T Epitope Mapping and Vaccine Development
Figure 3
Frequency of specific T cells and culture size. The frequency of antigen specific T cells must be kept in mind when scaling down culture conditions. Reasonable frequencies for specific T cells approximately range from 1% to 0.1% for memory responses to recall protein antigens. Responses to individual epitopes may be at lower frequencies. Frequencies of specific T cells in the naïve repertoire may be 0.01%. Panels represent 1000 lymphocytes containing 1 specific cell (0.1%, left panel) or 10 specific cells (1%, right panel). In case 1000 cells containing 1 specific cell are seeded in a microwell, the specific T-cell may or may not be dispensed according to Poissonian distribution and the signal provided by one single cell may not be detected. In case 1000 cells containing 1% specific cells are seeded, 10 specific cells can provide a detectable positive signal. Therefore, it can be assumed that the smallest number of cells per well should be 103 cells. This reasoning applies to any T-cell assay adapted to 384 or 1536 well plates. Obviously, for very low frequency responses, more cells need to be seeded, but in this case the intrinsic sensitivity of the assay should be checked to establish a frequency threshold. Unfortunately, reliable cellular standards are not available, unless established antigen specific T-cell lines are used, as discussed in [108].
