In vitro statin treatment reduces T-cell expansion in response to alloantigen in vitro and in vivo. (A) In vitro expansion of CFSE- (A) or luciferase-labeled (B) CD4⁺/CD8⁺ T cells (4:1, FVB/N, 2 × 10⁵ T cells/ flat-bottomed, 96-well plate, 200 μL per well) in the presence of CD3/CD28 stimulation (2 μg/mL or 5 μg/mL each) for 48 hours in complete media (cRPMI) and the indicated statins (10 μM). Afterward, cells were washed and analyzed by FACS for CFSE dilution. cRPMI indicates no statin; AT, atorvastatin; SI, simvastatin; FLU, fluvastatin; LO, lovastatin; Unstim, no CD3/CD28 mAb stimulation. T cells expand upon in vitro stimulation (upper panel). AT, SI, FLU, and LO reduce T-cell expansion significantly as measured by levels of CFSE dilution. (B) The same experimental in vitro conditions as described in panel A were employed. Expansion of luciferase transgenic T cells was quantified in photons/second/cm². All statins reduce in vitro T-cell expansion significantly as compared with cRPMI (P < .01). (C) Survival of mice receiving TCD-BM (●, n = 10) with T cells exposed to CD3/CD28 stimulation as described in panel A in cRPMI alone (▵, n = 10) or with FLU (○, n = 10) or AT (■, n = 10). Survival of Balb/c recipients was significantly higher when T cells were exposed to AT or FLU as compared with cRPMI (■ vs ▵, P = .003; and ○ vs ▵, P = .004). All Balb/c recipients (H-2^d) underwent transplantation as described in “aGVHD model.” Data from 2 independent experiments are combined. (D) Expansion of luciferase-labeled (luc⁺) T cells was quantified in emitted photons over total body area at serial time points after BMT in mice receiving TCD-BM (●, n = 10) with T cells exposed to cRPMI (▵, n = 10), fluvastatin (FLU, ○, n = 10), or atorvastatin (AT, ■, n = 10). Signal intensity is significantly higher in animals receiving T cells exposed to cRPMI only as compared with FLU- and AT-treated animals (▵ vs ○, P < .001; and ▵ vs ■, P < .001). Data from 2 independent experiments are combined.