Targeting Notch signaling in autoimmune and lymphoproliferative disease

Abstract

Patients with autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosis (SLE) have T-cell dysregulation and produce abnormal, activated T lymphocytes and an atypical peripheral T-cell population, termed double negative T cells (DNTs). T-cell functions, including DNT transition in T-cell development and T-cell activation, are critically dependent on Notch signaling. We hypothesized that inhibiting Notch signaling would be effective in ALPS and SLE by reducing the production of abnormal DNTs and by blocking aberrant T-cell activation. We tested this hypothesis using murine models of ALPS and SLE. Mice were randomized to treatment with the notch pathway inhibitor (gamma-secretase inhibitor), N-S-phenyl-glycine-t-butyl ester (DAPT), or vehicle control. Response to treatment was assessed by measurement of DNTs in blood and lymphoid tissue, by monitoring lymph node and spleen size with ultrasound, by quantifying cytokines by bead-array, by ELISA for total IgG and anti-double-stranded DNA (dsDNA) specific antibodies, and by histopathologic assessment for nephritis. We found a profound and statistically significant decrease in all disease parameters, comparing DAPT-treated mice to controls. Using a novel dosing schema, we avoided the reported toxicities of gamma-secretase inhibitors. Inhibiting the Notch signaling pathway may thus present an effective, novel, and well-tolerated treatment for autoimmune and lymphoproliferative diseases.

Figure 1

Notch inhibition decreases lymphoproliferation. CBA-lpr^cg mice were randomized to treatment with DAPT 5mg/kg per day versus vehicle control (6 treated, 9 control). After 6 weeks of treatment, a decrease in disease burden is visually apparent. Shown here are a control animal at (A) initiation and (B) after 42 days of vehicle, and a treated mouse at (C) initiation of DAPT treatment and (D) after 42 days of treatment. Serial ultrasounds were performed every 2 weeks to document lymph-node volume (mm³) and splenic area in (mm²). Treated mice showed a statistically significant (P = .009) decrease in lymph-node volume after 2 weeks of treatment compared with control mice (E). Treated mice also showed a statistically significant (P = .001) decrease in splenic area by 2 weeks of treatment compared with control mice (F). No statistical difference existed between groups at initiation of treatment. Bars represent mean lymph node volume (E) or splenic area (F) from mice at each time point, and error bars represent SEM.

Figure 2

Notch inhibition decreases DNTs. (A) Response of DNTs in peripheral blood to GSI in 6 treated and 13 contorl CBA-lpr^cg mice. Retro-orbital bleeds were performed every 2 weeks to assess absolute DNTs/mm³, comparing mice treated with DAPT and vehicle control (absolute DNTs: WBC in mm³ × % CD3⁺/TCRα/β⁺/CD4⁻/CD8⁻ cells) Serial absolute DNT measurements were normalized to the untreated baseline for each mouse. Treated mice showed a statistically significant decrease in absolute DNTs compared with controls by 6 weeks of treatment. Bars represent normalized mean absolute double negative T-cell counts from mice at each time point, and error bars represent SEM. (B) Average number of DNTs in lymph nodes and spleens (defined as total cell count × % CD3⁺/TCRα/β⁺/CD4⁻/CD8⁻ cells), comparing GSI-treated and control animals, showing a statistically significant decrease in both. Error bars represent SEM.

Figure 3

DNTs are more sensitive to Notch inhibition than other cell populations. The effect of DAPT was assessed on different cell populations in mouse spleens at sacrifice. (A) Absolute number of cells in different lymphocyte populations (absolute cell count in spleen × % of lymphocyte subset by flow cytometry), comparing 5 treated to 7 control animals. Percentages listed on X-axis represent mean percentage reduction of cells in treated animals compared with control. Error bars represent SEM. There was a 68% decrease in the average number of total cells comparing treated to control animals. A profound decrease (81%, P = .007) was found in the DNT cell compartment. A less significant decrease was found in CD4⁺ (45%), CD8⁺ (20%), and non–T-cell (CD3⁻) (59%) lymphocytes. (B) Relative percentage of each lymphocyte population in mouse spleens comparing treated to control animals.

Figure 4

Notch inhibition decreases autoantibody production. MRL-lpr mice were randomized to treatment with DAPT or vehicle (4 treated, 4 control). Retro-orbital bleeds were obtained every 2 weeks to measure mouse total IgG (anti-IgG2a) and mouse anti-dsDNA IgG specific antibodies in sera by quantitative ELISA. Mice treated with DAPT had a statistically significant decrease in average titer levels compared with control for both antibodies (P < .001) as determined by ANOVA. Error bars represent SD.

Figure 5

Gamma-secretase inhibitors improve nephropathy. MRL-lpr mice were randomized to treatment with DAPT or vehicle. Eight weeks after starting therapy, mice were killed and kidneys were assessed for disease. Kidney disease burden was scored 0 to 5 on 4 measures: (1) nephropathy; (2) glomerulonephritis; (3) cortical lymphoid proliferation (LP); and (4) medullary lymphoid proliferation (LP). Higher scores represent more severe disease. (A) Average total cumulative score for 4 control and 6 treated animals. Colored portions of the bars depict the individual disease measures. Treated mice demonstrated a reduction in all 4 disease parameters compared with control and a statistically significant reduction in total kidney disease (P = .007). Error bars depict SD of total kidney disease score. (B-E) Kidney pathology by H&E stain. Treated animals have healthy kidneys with limited disease (B,C). In contrast, control animals have pronounced lymphocytic infiltration in cortex (D) and medulla (E). In addition, control animals have damaged glomeruli and proteinaceous material in renal tubules. Images were captured using a Zeiss Axiovert 40C light microscope (Carl Zeiss, Thornwood, NJ) equipped with an apochromatic 10×/0.25 NA objective lens and a Nikon 995 camera (Nikon, Melville, NY).

Figure 6

DAPT decreases activated Notch protein, transcription of downstream effectors, T cell proliferation, and cytokines. (A,B) Lymph node cells were harvested from mice after 4 days of treatment with DAPT or control. Panel A depicts immunoblot of activated Notch protein (Notch 1C) showing significantly decreased activated protein in 2 representative treated animals compared with 2 controls. In addition, RNA was isolated to assess down-stream targets of Notch1C activated transcription, including Hes1 and Deltex1. (B-tubulin was used as internal control.) Panel B depicts RT-PCR of Hes1 and Deltex1, comparing a representative control and treated animal. Error bars represent SD. (C,D) CBA-lpr^cg mice were treated with DAPT or control for 3 months (5 treated; 3 control). At killing, lymph node cells, splenocytes, and thymocytes were harvested, supported in vitro, and stimulated with mitogen. After 24 hours, supernatants were collected and cytokines assessed by cytometric bead array. After 48 hours, proliferation was measured by MTT. Panel C depicts proliferation, comparing treated to control. Bars represent mean of the treated/control (untreated) cells (relative absorbance of triplicate cultures, as described in “T-cell functional assays”); error bars represent SD. Panel D depicts results of cytokine bead array, demonstrating elevated levels of IL-10 and MCP-1 and a marked and statistically significant decrease in treated animal. Error bars depict SEM. Similar results were found with MRL mice.