Evidence that T cells can down-regulate the immune response by producing or consuming certain cytokines or by lysing APCs or Th cells has been provided in various systems. However, the generation and characterization of suppressor T cell lines have met with limited success. Here we show that xenospecific suppressor T cells can be generated by in vitro stimulation of human T cells with pig APCs. Similar to allospecific suppressors, these xenospecific suppressor T cells carry the CD8+ CD28− phenotype and react to MHC class I Ags expressed by the APCs used for priming. TCR spectratyping of T suppressor cells showed oligoclonal usage of TCR-Vβ families, indicating that xenostimulation of CD8+ CD28− T cells results in Ag-driven selection of a limited Vβ repertoire. Xenospecific T suppressor cells prevent the up-regulation of CD154 molecules on the membrane of Th cells, inhibiting their ability to react against the immunizing MHC class II xenoantigens. The mechanism of this suppression, therefore, appears to be blockade of CD154/CD40 interaction required for efficient costimulation of activated T cells.