The Role of RLIP76 in Dendritic Cell-Based Immunotherapies.

Antigen presentation is, perhaps, the most important function of the immune system for both initiating protective immune response against foreign and/or dangerously altered self structures and maintaining self-tolerance. Dendritic cells (DCs) together with monocytes and macrophages constitute the heterogeneous population of mononuclear phagocytic system and act as non-specific effector cells against microbes and tumors. The unique capacity of DCs to efficiently stimulate naïve T cells, B cells, NK and NKT cells, initiate primary immune response, and orchestrate the intimate interplay between the innate and adaptive arm of the immune system (Banchereau et al, 2000; Whiteside and Odoux, 2004) renders them the most potent professional antigen presenting cells (APCs) (Van Voorhis et al, 1983; Guermonprez et al, 2002). Ral-binding protein 1 (RALBP1), or RLIP76 (Cantor et al, 1995; Jullien-Flores et al, 1995; Park et al, 1995), is a member of the non-ATP-binding cassette (non-ABC) stress-responsive transporters which – in an ATP-dependent fashion – mediates signaling and transport of a wide array of amphiphilic organic substrates such as glutathione-electrophile thioethers (GS-E), genotoxic end-products of lipid peroxidation, e.g., 4-hydroxynonenal (4-HNE), metabolites of arachidonic acid (eicosanoids), natural antineoplastic agents and various xenobiotics. Although mainly involved in mediating efflux of agents normally toxic for the cell, RLIP76 has been identified as primary determinant of the rate of clathrin-coated pit-mediated endocytosis, one of the major ways of antigen uptake and processing by APCs. We, therefore, wanted to investigate the details of this connection, particularly, the significance of changes in cell surface expression of RLIP76 upon DC maturation for the efficiency of antigen uptake and presentation.

Using flow cytometry, we found varying cell surface expression of RLIP76 on non-activated (immature) monocyte-derived DCs of healthy individuals, while anti-RLIP76 antibody-treated DCs, even after subsequent delivery of the usual maturation signal, the bacterial lipopolysaccharide (LPS), clearly showed ‘maturation arrest’ as evidenced by low levels of typical activation markers and co-stimulatory molecules, such as CD83, HLA-DR, HLA-ABC, CD80, and CD38. In a functional assay, the antibody-treated DC also failed to fully stimulate allogeneic mixed lymphocyte reaction (allo-MLR) as compared to their untreated counterparts.

These exciting novel findings predict that expression of RLIP76 on DCs plays an important role in their maturation and that anti-RLIP76 antibodies inhibit this process. Earlier we have also demonstrated that intra-peritoneal administration of RLIP76-loaded proteoliposomes to mice produced increased tissue levels of RLIP76, and, given up to 3 days after irradiation, improved the survival of animals. The above findings led us to hypothesize that augmented presence of RLIP76 in DCs will contribute to improved humoral and cell-mediated immune response through enhanced immune potentiating capacity of DCs. To confirm the implication of these findings in DC-based immunotherapies of human malignancies, more extensive in vitro as well as in vivo experiments, using gene silencing and anti-sense DNA for manipulating expression levels of RLIP76 on DCs, are underway in our laboratory.

No relevant conflicts of interest to declare.