Figure 3. Inhibition of bright STAT5 phosphorylation among CD4⁺ alloresponders.

(A) Dose response of CAS 285986-31-4 (STAT5 inhibitor, 0–100 μM) in human allo-MLRs (DC:T cell ratio 1:30) treated once on day 0 with drug or DMSO diluent control. T-cell proliferation was determined by a colorimetric assay on day 4. The stimulation index was calculated by dividing the OD of the allo-MLR by the OD of T cells alone. (B, C) Representative histograms (using an identical gating strategy as in Fig. 2A) demonstrate the on-target selectivity of CAS 285986-31-4, impairing bright STAT5 activity, yet preserving STAT3 phosphorylation in 5 day allostimulated T cells. (B, C) Gates in the histograms define the (B) CD4⁺, pSTAT3⁺ and (C) CD4⁺, pSTAT5^Bright T cells. Graph data are the mean ± sd of results in 3 independent experiments. (D) STAT3 and (E) -5 phosphorylation by geometric MFI ratio, (F) the frequency of CD4⁺, pSTAT5^Bright T cells, and (G) the CD4⁺, pSTAT5^Bright:pSTAT3⁺ ratio by percentage of phosphorylation in CD4⁺ T cells harvested from CAS 285986-31-4- or DMSO-treated 5 day allogeneic cocultures. *P < 0.05, paired t test.