SL327, a small-molecule inhibitor of ERK 1/2 phosphorylation attenuates T-cell alloactivation in vitro, and cucurbitacin I and cucurbitacin E, small-molecule inhibitors of STAT-3 phosphorylation, can attenuate T-cell alloactivation in vitro and GVHD in vivo. (A) A total of 10⁵ RBC-lysed B6 splenic T cells were incubated for 24 hours with plate-bound anti-CD3 and anti-CD28 antibody (0.5 μg/mL concentration for both antibodies) in the presence of log-10 dilutions of SL327 (range: 100 ng to > 0.01 ng) and ³H thymidine incorporation was measured. One of 4 independent experiments. (B) A total of 10⁵ RBC-lysed B6 splenic T cells (responders) were incubated with 10⁵ irradiated and RBC-lysed B6 (syngeneic stimulators) or BALB/c splenic T cells (allogeneic stimulators) in a mixed leukocyte reaction. Log-10 dilutions of SL327 (range: 50 ng to > 0.05 ng) were added on day 0, and ³H thymidine was added on day 4 to measure proliferation. One of 4 independent experiments. (C) A total of 10⁵ RBC-lysed B6 splenic T cells (responders) were incubated with 10⁵ irradiated and RBC-lysed B6 (syngeneic stimulators) or BALB/c splenic T cells (allogeneic stimulators) in a mixed leukocyte reaction. Log-10 dilutions of cucurbitacin I (range: 50 nM to > 1 nM) were added on day 0, and ³H thymidine was added on day 4 to measure proliferation. One of 4 independent experiments. (D) Mixed leuckocyte reaction was performed as in panel A, with log-10 dilutions of cucurbitacin E (range: 5 nM to > 0.1 nM). Representative data from 1 of 4 independent experiments. (E) Whole B6 splenocytes (∼60-100 × 10⁶ cells) were incubated with 5 nM cucurbitacin E or control (DMSO) for 1 hour at 37°C before adoptive transfer into lethally irradiated (850 cGy) BALB/c mice. Recipient spleens were analyzed at 24 hours for donor T cells and expression of CD25. Representative data from 1 of 4 independent experiments. (F) Experiment was performed as in panel A and median fluorescence intensity of CD69 was determined. N = 8 per group, combined data from 4 independent experiments. (G) B6 splenocytes (6 × 10⁷ cells) were incubated with 5 nM cucurbitacin I or control (DMSO) for 1 hour at 37°C before adoptive transfer into lethally irradiated (850 cGy) BALB/c mice. Recipients also received 5 × 10⁶ T cell–depleted B6 marrow, and were monitored and analyzed for survival, weight loss, and clinical signs of GVHD. Combined data from 2 independent experiments, with N = 20 per group for groups receiving splenocytes. (H,I) Mice were transplanted as in panel G and evaluated for weight loss from baseline and semiquantitative clinical GVHD scores on day 6 after allo-BMT (N = 8 per group). (J) Mice were transplanted and total splenic cellularity was assessed by hemacytometer on day 6. Cells were analyzed by flow cytometry, and donor CD4 and CD8 T cells were defined as H-2K^b–positive (N = 8 per group). (K) Mice were transplanted and total splenic cellularity was assessed by hemacytometer on day 6. Percentages of donor CD25⁺FoxP3⁺ regulatory T cells as a percentage of donor CD4 T cells was determined by intracellular staining and flow cytometry (N = 8 per group). Error bars in panels indicate standard error of the difference (SED).