Rapamycin decreases lymphoproliferation. (A-D) Serial ultrasounds were performed every 2 weeks to document lymph node volume in cubic millimeters and splenic area in square millimeters, comparing rapamycin-treated with untreated mice. Treated mice showed a statistically significant (P = .05) decrease in lymph node volume after 2 weeks of treatment when compared with control mice (E). Treated mice also showed a statistically significant (P = .03) decrease in splenic area by 4 weeks of treatment when compared with control mice (F). No statistical difference existed between groups at initiation of treatment. Bars represent mean lymph node volume or splenic areas from mice at each time point and error bars represent SEM. Average normal mouse spleen size in unaffected animals was 10 to 20 mm². (A) Representative example of a lymph node prior to treatment with rapamycin. (B) The same node from panel A after 4 weeks of treatment. Initial volume of the node was 114 mm³ and the volume after treatment was 19 mm³, representing an 80% reduction in size. (C) In contrast to panels A-B, a lymph node of a mouse prior to treatment with vehicle. (D) The same node from panel C after 4 weeks. Initial volume of this node was 230 mm³ and the volume after exposure to vehicle was 1242 mm³, representing a 600% increase in size.

Rapamycin decreases DNTs. CBA-lpr^cg mice were randomized to treatment with rapamycin 5 mg/kg/day versus vehicle control. Retro-orbital bleeds were performed every 2 weeks to assess absolute DNTs/mm³. Treated mice showed a statistically significant decrease in absolute DNTs compared with control by 4 weeks of treatment. No statistical difference existed between groups at initiation of treatment. Bars represent mean absolute DNT count from mice at each time point; error bars represent SEM. Normal mouse absolute DNT count range, 30 to 150/mm³.9,18

Rapamycin decreases autoantibody production. MRL-lpr mice were randomized to treatment with rapamycin, MMF, or control. Retro-orbital bleeds were obtained every 2 weeks to measure mouse anti-dsDNA IgG-specific antibodies in sera by quantitative ELISA. Mice treated with rapamycin had a statistically significant decrease in average titer levels compared with MMF (0.3 μg/mL vs 5 μg/mL; P < .001) and control (0.3 μg/mL vs 8 μg/mL; P = .001) after 4 weeks of treatment. Figure 6 depicts results of antibody titers for each mouse over time with 4 mice in each treatment group. Results are normalized to titer at initation of treatment for each mouse. Error bars represent SEM.

Rapamycin decreases lymphoproliferation. CBA-lpr^cg mice were randomized to treatment with rapamycin versus vehicle control. After 4 weeks of treatment a decrease in adenopathy is visually apparent.

Ultrasound accurately estimates organ volume. In order to ensure that ultrasound measurements reflected actual measurements, lymph node volumes from CBA-lpr^cg mice at death (calculated as 4/3π × radius height × radius width × radius length of lymph node using caliper) were compared with volume calculated on Vevo 660 ultrasound. Linear regression analysis demonstrated a statistically significant correlation. Dotted lines indicate 95% confidence intervals.

Rapamycin is superior to MMF. CBA-lpr^cg mice were randomizd to treatment with rapamycin, MMF, or control. Serial ultrasounds were performed every 2 weeks to document lymph node volume in cubic millimeters. Rapamycin-treated mice showed a statistically significant (P = .04) decrease in lymph node volume after 6 weeks of treatment when compared with MMF-treated mice. Bars represent mean lymph node volume from mice at each time point; error bars represent SEM. P values depict comparisons of rapamycin and MMF treatment by 2-tailed t test.

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.