Perifosine Affects Phenotype and Function of Human Myeloid Dendritic Cells

Abstract 3911

Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agent which targets cell membranes and inhibits Akt activation. Perifosine inhibits multiple myeloma (MM) cell growth in vitro and in vivo. Currently perifosine is under phase III clinical evaluation in MM. Although perifosine has shown significant direct antitumor effects, its effect on immune system has not yet been clarified. Dendritic cells (DCs) play a crucial role in immune system via mediating antigen-specific immune responses as well as regulating the innate and adaptive immunity through secreted cytokines. In this study, effect of perifosine on phenotype, antigen uptake, processing and presentation, and cytokine production on human monocyte-derived dendritic cells (DCs) were evaluated at clinically relevant concentrations (2.5μM to 10μM). The effect of perifosine on survival of DCs was tested by annexin V and PI staining. We observed that up to 48 hours of perifosine treatment had no effect on viability of both immature DCs and DCs during maturation by LPS (100ng/ml) or poly(IC) (25μg/ml) (>90%). Alteration of DC phenotype by perifosine was further examined by flow cytometry. Our results demonstrated that perifosine treatment led to a dose-dependent downregulation of surface antigen expression, associated with costimulation (CD40), antigen presentation (HLA-ABC) and maturation (CD83) on both immature and mature DCs at 24 and 48 hours. We next evaluated whether perifosine affected the antigen uptake capacity by immature DCs, using various sizes and forms of antigens. Compared to control, 24 hours-perifosine (10μM) treatment significantly reduced uptake capacity of both protein antigens (Alexa Fluor 488-conjugated 45-KDa protein A and 20-KDa protein G) and polysaccharide antigen (Alexa Fluor 488-conjugated 40-KD dextran) by immature DCs. Importantly, we systematically investigated the impact of perifosine on DCs involved in both classic (MHC-mediated) and non-classic (CD1d-mediated) antigen specific immune responses and report a significant impairment upon perifosine treatment. Perifosine inhibited DC-mediated T cell activation. Following 24 hours treatment, control or perifosine (10μM)-treated immature DCs were pulsed with tetanus toxoid (0.5μg/ml) overnight in the presence of LPS, and then used to stimulate autologous T cells. T cell response was inhibited as evidenced by significantly reduced IFN-gamma production detected by ELISA (control vs treatment = 15106 pg/ml vs 8332 pg/ml). We also confirmed that perifosine pretreatment of freshly isolated blood myeloid DCs led to significant inhibition of allogenic T cell immune responses (control vs treatment = 982 pg/ml vs 605 pg/ml). In addtion to presenting peptide antigens, DCs also present glycolipids to activate invariant NKT (iNKT) cells. Our data demonstrated that perifosine impairs DCs-mediated iNKT cell activation. The production of both Th1-type cytokines and Th2-type cytokines by iNKT cells were significantly repressed upon perifosine (10μM)-treated alpha-GalCer-pulsing DCs, compared to control (IFN-gamma production (control vs treatment= 11631 pg/ml vs 6768 pg/ml) and IL-4 production (control vs treatment= 1285 pg/ml vs 783 pg/ml). Since DCs play a crucial regulatory role via cytokine production, we next determined IL-12p70 and IL-10 secretion by LPS-induced DCs with and without perifosine treatment. Compared to controls, perifosine treatment at 24 hours significantly inhibited LPS-induced-IL-12p70 production by DCs (control vs treatment = 192 pg/ml vs 166 pg/ml (2.5μM), 111 pg/ml (5μM) and 45 pg/ml (10μM)), as well as inhibited IL-10 production (control vs treatment = 472 pg/ml vs 371 pg/ml (2.5μM), 306 pg/ml (5μM) and 179 pg/ml (10μM)). Mechanism study revealed that RelB, a component of NF-kappaB signaling pathway, was downregulated in immature DCs by 18 hours of perifosine treatment (10μM). We have also evaluated the effect of perifosine on the expression of IDO, a repressor of DCs activation. However, we did not observe the significant alteration of perifosine treatment compared to the control. In summary, our preclinical data suggest that perifosine is able to affect both immature and mature DCs and could contribute to inhibition of DC-mediated immune responses, indicating a need to monitor immune functions in patients under the Akt inhibitor treatment.