CD28 COSTIMULATION OF PROLIFERATION IN MURINE SPLENIC T CELLS IS SENSITIVE TO INHIBITION BY SP100030, A NOVEL T CELL-SPECIFIC DUAL INHIBITOR OF NF-κB AND AP-1

101 Introduction: SP100030 (SP) is the only known T cell-specific dual inhibitor of both NF-κB and AP-1, and its inhibition of these transcription factors suppresses cytokine production in T cell lines. Our goal has been to investigate the in vivo efficacy of SP and to define its mechanisms of action on normal T cells. We previously showed that SP suppressed host vs graft murine popliteal lymph node hyperplasia, rejection of murine heart allografts, and inhibited murine splenocytes proliferationin vitro by suppressing IL-2 production. Now we show that SP inhibits not only CD3- or Ca2+/PMA-triggered T cell proliferation but also CD28-mediated costimulation of cell proliferation in vitro, and eventually blocks cell cycle progression of T cells at the G0-G1 stage. Methods: (1) Cell culture: C3H splenic T cells were isolated by positive selection using anti-thy 1.2 microbeads. Cells were stimulated with plateadherent anti-CD3 mAb ± soluble anti-CD28 mAb or different combinations of PMA, ionomycin (INM) and anti-CD28 mAb. The cells were incubated with or without drugs (SP or cyclosporine, CsA) at 37 °C in 5% humidified CO2. (2) Cell proliferation assay: After 30 hrs of culture, cells were pulsed for 12 hrs with 3H-TdR and incorporated3 H-TdR was measured at 42 hr. (3) PCNA and cell cycle analysis: Intracellular expression of PCNA and DNA content in the cells were simultaneously measured using flow cytometry at 42 hr. Results: Both SP and CsA suppressed CD3-triggered T cell proliferation by >90%. Addition of CD28 costimulation to CD3 triggering augmented T cell proliferation by 24 fold at the highest concentration of CsA, but only 6 fold at the highest concentration of SP. Although T cell proliferation induced with PMA+INM was inhibited by >90% by both SP and CsA, stimulation both by PMA+CD28 and by PMA+INM+CD28 was inhibited by >90% by SP, but not at all by CsA. Inhibition of 3H-TdR incorporation by SP or CsA correlated inversely with PCNA expression and the number of cells in S+G2/M phase; both drugs increased the number of apoptotic cells and cells in G0 phase. Conclusions: (1) CD28 costimulation for CD3-triggered T cell proliferation is sensitive to inhibition by SP, but resistant to inhibition by CsA. (2) CD28 costimulation is sensitive to inhibition by SP, but insensitive to inhibition by CsA when T cells were stimulated with PMA alone or PMA plus INM. (3) SP as well as CsA blocks T cell cycle progression at the G0-G1 stage. Therefore SP inhibits not only CD3- and Ca2+/PMA-triggered T cell proliferation but also CD28-mediated costimulation resistant to CsA, eventually blocking cell cycle progression of T cells at the G0-G1 stage.