Differential sensitivity of T cell activation vs deletion

It has been previously shown from studies using super-antigen or TCR transgenic mice that deletion of double positive (DP) thymocytes is more sensitive than activation of mature T cells. In this study we quantitate the magnitude of this difference in sensitivity in identical culture conditions. We used the 3A9 transgenic mouse which expresses a transgenic T cell receptor (TCR) specific for hen egg lysozyme (HEL) peptide 48-61 on I-A^k. The thymus of this mouse contains roughly 35% mature CD4 single positive(SP) and 45% immature DP. 1x10⁶ thymocytes were placed into a well, with 1x10⁵ B-lymphomas transfected with I-A^k which have been pulsed with HEL 48-61 overnight and washed. After 20 hours in culture the viable cells are counted, and then stained for CD4, CD8, and CD69. Deletion is then measured as % of control DP cells, and activation is measured as % of maximum mean fluorescence of CD69 on CD4 SP cells. A monoclonal antibody specific for I-A^k-48-61 peptide-MHC complex was used to estimate the number of complexes per antigen presenting cell. Our results indicate that negative selection of DP thymocytes is a highly sensitive process that requires 10-15 fold less antigen than activation of mature CD4 T cells.