therefore less toxic alternative could be selected using convention techniques cd4 cd25 t cells were purified from normal donor pbmc using standard treg kits miltenyi with automacs the purified cells were stained for foxp3 and the percentage of foxp3 cells was determined using facs approximately 50 of the purified cd4 cd25 cells were also foxp3 prior to expansion purified cd4 cd25 cells were stimulated with anti cd3 cd28 beads in the presence of il 2 with various p70 s6 kinase inhibitors for two weeks as their population was allowed to undergo expansion the expansion was allowed to continue for a sufficient period of time to permit a sizeable portion of cells to be obtained but not for so long that unacceptable drift in the composition of the sample was realized the expression of foxp3 was determined using facs as shown in table 1 when no additive is used the composition of the culture drifts to lower percentages of foxp3 cells the most likely explanation of this observation is that the expansion of the foxp3 u2212 cells begins to out pace the expansion of the desired foxp3 cells in the example given after two weeks the composition of foxp3 cells had fallen to only 21 the addition of 100 nm rapamycin caused the cellular composition to be increased in the percentage of foxp3 expressing cells relative to its absence during the expansion process presumably due to inhibition of mtor applicants have discovered that p70 s6 kinase inhibits provide a benefit that is comparable with rapamycin inclusion of drb in the culture medium consistently increased in the percentage of foxp3 expressing cells relative to its absence other compounds with described p70 s6 inhibitory action were also tested to verify.