Mechanism of action of rapamycin in ALPS. (A) Rapamycin down-regulates phospho-S6. Lymph node cells were harvested from a mouse (no. 1) both before (before tx) and after (after tx) treatment with rapamycin for 3 days. Lymph node cells were also harvested from a mouse treated with rapamycin for 4 weeks (no. 3, long tx) compared with a mouse treated with vehicle for 4 weeks (no. 2, control). Immunoblot of phospho-S6 (Ser 235/236) (top bands), total S6 (middle bands), and β-tubulin (bottom bands) from the mice demonstrates a correlation between biochemical and clinical response to rapamycin in the mice. Lymph cells from treated mice had down-regulation of phospho-S6 compared with lymph cells from untreated mice. (B) Rapamycin down-regulates phospho-Bad. Lymph node cells were harvested from a mouse treated with rapamycin for 4 weeks and a mouse treated with vehicle for 4 weeks. Immunoblot of phospho-Bad (Ser 112; top bands), total Bad (middle bands), and β-tubulin (bottom bands) from the mice demonstrates down regulated phospho-Bad in the rapamycin-treated cells. Results for phospho-Bad (Ser 136) were similar and are not shown. (C) Rapamycin induces caspase-dependent apoptosis in cultured murine ALPS lymphocytes. T cells from lymph node biopsy of CBA-lpr^cg mice were maintained in culture. Aliquots of 10⁵ lymphocytes were exposed to 100 ng/mL rapamycin for 48 hours and were assessed for apoptosis by flow cytometric staining for annexin V. In addition, prior to treating with rapamycin, aliquots of cells were pretreated for 2 hours with a pancaspase inhibitor, a caspase-9 inhibitor, or a caspase-8 inhibitor. Data represent percentage of apoptotic (annexin V–positive/7-AAD–negative) cells determined by flow cytometric analysis.