Limiting dilution analysis (LDA) determines the frequency of survivin reactive CD4+CD25- T cells. Serially diluted, purified CD4+CD25- T cells were stimulated with autologous DC:survivin in the presence of exogenous IL-2. Each LDA was performed with a minimum of 10 replicates per cell concentration. (A) Log-fraction plot of the LDA of survivin specific CD4+CD25- T cells both before and after expansion with DC:survivin. The slope represents log-active cell fraction, bold lines represent frequency estimates, and non-bold lines show 95% CIs based on the likelihood ratio test of single-hit model. One out of 44,907 cells were estimated to respond to the survivin peptide pool before expansion (Before). Separately, CD4+CD25- T cells from the same donor were expanded using DC:survivin and exogenous IL-2 for 12 days. Cells were collected, enumerated, and rested without cytokines for 2 days. Repeat LDA after expansion demonstrated enrichment for survivin specific T cells to 1 out of 383 (After), p < 0.0001. Representative of 2 independent experiments from separate healthy donors. (B) LDA was validated by labeling un-stimulated CD4+CD25- T cells with CTV (cell trace violet) prior to expansion with DC:survivin and exogenous IL-2. After 12 days, T cells were flow sorted into CD4+CTV- (replicated) and CD4+CTV+ (non-replicated), then rested for 2 days without cytokines. 2.5×103 CD4+ T cells were stimulated with DC:survivin or DC:HIV (irrelevant) peptide pool for 24 hours. * = p < 0.05 by t test, error bars indicate the standard deviation. Representative of two independent experiments from separate healthy donors.