Inhibition of FLT3 signaling targets DCs to ameliorate autoimmune disease

Abstract

Autoimmune diseases often result from inappropriate or unregulated activation of autoreactive T cells. Traditional approaches to treatment of autoimmune diseases through immunosuppression have focused on direct inhibition of T cells. In the present study, we examined the targeted inhibition of antigen-presenting cells as a means to downregulate immune responses and treat autoimmune disease. Dendritic cells (DCs) are the central antigen-presenting cells for the initiation of T cell responses, including autoreactive ones. A large portion of DCs are derived from hematopoietic progenitors that express FLT3 receptor (CD135), and stimulation of the receptor via FLT3 ligand either in vivo or in vitro is known to drive expansion and differentiation of these progenitors toward a DC phenotype. We hypothesized that inhibition of FLT3 signaling would thus produce an inhibition of DC-induced stimulation of T cells, thereby inhibiting autoimmune responses. To this end, we used small-molecule tyrosine kinase inhibitors targeted against FLT3 and examined the effects on DCs and their role in the promulgation of autoimmune disease. Results of our studies show that inhibition of FLT3 signaling induces apoptosis in both mouse and human DCs, and thus is a potential target for immune suppression. Furthermore, targeted inhibition of FLT3 significantly improved the course of established disease in a model for multiple sclerosis, experimental autoimmune encephalomyelitis, suggesting a potential avenue for treating autoimmune disease.

Fig. 1.

Mature DCs express FLT3, and CEP-701 inhibits its phosphorylation. Shown is a Western blot analysis of murine BM DCs demonstrating that mature DCs express high levels of FLT3 and that CEP-701 effectively inhibits its activation. Relative quantities of phospho-FLT3 are shown in the densitometry graph, normalized to total FLT3.

Fig. 2.

Treatment with FLT3 TKI induces apoptosis in mature mouse and human DCs. (A) DC survival was measured after exposure to CEP-701. Mature DCs were harvested and replated in the absence or presence of CEP-701 (0, 5, or 50 nM). CD11c⁺ cells were then assessed for MHC class II expression and Annexin V binding. CEP-701 induced apoptosis in DCs generated either in GM-CSF/IL-4 alone or in the combination of GM-CSF/IL-4 and FL. Shown are representative FACS plots (from four experiments). (B) Graph of the averages and SEM. (C) Treatment with FLT3 TKI induces apoptosis in mature human DCs. Shown are the effects of CEP-701 on human DCs. Either DMSO vehicle or CEP-701 was added to the cultures for 48 h, and then the cells were stained for CD11c and Annexin V binding. As was the case with the murine DCs, a significant increase in Annexin V⁺ cells was observed in the cultures exposed to CEP-701. Shown are representative plots from two separate studies. (D Left) FLT3 inhibition decreases proliferation of T cells through its effect on DCs. For these experiments, BM DCs from BALB/c mice were treated with CEP-701 or vehicle control, then plated with C57BL/6 splenocytes at the ratios of DC:spleen shown. After 3 days, proliferation of T cells was determined. As shown, CEP-701 significantly decreased proliferation. (D Right) Cytokine analysis for IFN-γ and TNF-α secretion was also conducted, and these results show a corresponding decrease in cytokine secretion for these inflammatory cytokines, shown as a percentage of control cytokine secretion in vehicle-treated cultures from the 1:10 DC:T cell ratio group. (E) FLT3 TKI does not directly inhibit T cells. Shown is the absence of a direct effect of FLT3 TKI on T cells. T cells were plated on anti-CD3-coated plates, followed by the addition of anti-CD28 antibody. As shown, the FLT3 TKI had no significant effect on direct stimulation of T cells. These studies were repeated twice.

Fig. 3.

Treatment of mice with FLT3 TKI decreases CD11c but not CD3 or B220 populations. (A) Spleens from BALB/c mice treated with CEP-701 or vehicle control, twice a day for 5 days, were harvested and analyzed by FACS for immune cell constitution. Shown is the FACS analysis for the presence of T cells (CD3⁺/DX5^-), NK cells (CD3^-, DX5⁺), NK T cells (CD3⁺, DX5⁺), B cells (B220⁺, CD11c^-), or DCs (myeloid, CD11c^hi/B220^-, or plasmacytoid CD11c^lo/B220⁺). As shown, whereas this treatment led to a decrease in NK and DC populations (FLT3 expressing populations), no significant change was observed in the B or T cell (not FLT3 expressing) populations. (B) In vivo treatment of murine DCs with CEP-701 leads to a loss of all major DC subsets in the lymph nodes and spleen. Shown are the relative percentages of some of the subsets, compared with controls (set at 100%). No significant change was observed in total cellularity or in CD11b⁺ CD11c^- macrophages.

Fig. 4.

Treatment of mice with FLT3 TKI decreases an in vivo autoreactive immune response. Transgenic HA-expressing mice (termed HA137) were injected with HA-specific CD4⁺ T cells (6.5), which express the congenic T cell marker thy1.1, to determine the effect of CEP-701 on expansion of autoreactive T cells. In untreated mice, the transferred transgenic T cells expand and induce an autoimmune process. For these experiments, HA137 mice were treated with CEP-701, then analyzed for expansion of the 6.5 T cells. As shown, the CEP-701 treatment led to a decrease in the autoreactive T cell expansion, as measured by thy1.1. Shown are the data from two separate experiments, with six mice per group.

Fig. 5.

Inhibition of FLT3 ameliorates established EAE. (A) EAE was induced in C57BL/6 mice, which were then injected twice daily with either vehicle control or CEP-701. Ten mice per group were treated in the study shown. Shown is the time course of one (of three similar) experiments. (B) Treatment with CEP-701 decreases demyelination. Shown is the histological staining on paraffin-embedded spinal cord sections of CEP-701-treated and vehicle control-treated mice after induction of EAE. As shown, significant demyelination (loss of blue stain) is found in MOG-vaccinated animals (Upper) but not in MOG-vaccinated and CEP-701-treated mice (Lower).

Fig. 6.

Treatment with CEP-701 does not inhibit the ability to respond to an infectious challenge. C57BL/6 mice were pretreated for 5 days with either vehicle control or CEP-701, then injected i.p. with an LD₂₀ of attenuated L. monocytogenes bacteria. Mice were followed for survival. This study was repeated with similar findings.