Solubilization of α-galactosylceramide in aqueous medium: Impact on Natural Killer T cell activation and antitumor responses.

The potent antitumor effect of α-galactosylceramide (α-GalCer) is based on its recognition by invariant Natural Killer T cells (iNKT) after its capture and presentation by antigen presenting cells including dendritic cells (DCs). Synthetic α-GalCer has already been tested in advanced cancer patients but no or only moderate clinical responses were obtained. To optimize α-GalCer efficacy, we have postulated that alternative formulations impacting its molecular organization in aqueous medium could modify DC uptake and iNKT-based immune responses. To this end, we have developed two strategies: (1) the formulation of α-GalCer in non-cationic liposomes and (2) the synthesis of a water-soluble α-GalCer analogue by anchoring a polyethyleneglycol moiety on its sugar head. The biological activities of these new preparations were compared to that induced by the classically used Polysorbate 20 α-GalCer micelles. Both formulations retained their uptake by DCs and activated iNKT cells both in vitro and in vivo. Despite a lower cytokine production, the formulations induced a potent immune response able to control lung murine carcinoma. In conclusion, it is possible to increase α-GalCer solubility in aqueous solution without limiting its antitumor properties.

Activation of invariant Natural Killer T lymphocytes in response to the α-galactosylceramide analogue KRN7000 encapsulated in PLGA-based nanoparticles and microparticles.

Invariant Natural Killer T (iNKT) cells have potent immunostimulatory activities that could be exploited for human therapies. The high-affinity CD1d antigen α-galactosylceramide analogue KRN7000 (KRN) activates a cascade of anti-tumor effector cells and clinical studies have already had some initial success. To improve the efficacy of the treatment, strategies that aim to vectorize KRN would be valuable. In this study, we intended to characterize and compare the effect of KRN encapsulated in poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs, 90nm) and microparticles instead of macroparticles (MPs, 715nm) on the iNKT cell response. Our data show that whatever the size of the particles, vectorized KRN induced potent primary activation of iNKT cells in vitro and in vivo. We show that endocytosis of PLGA-based particles by dendritic cells is mediated by a clathrin-dependent manner and that this event is important to stimulate iNKT cells. Finally, we report that KRN vectorized in NPs and MPs exhibited different behaviours in vivo in terms of iNKT cell expansion and responsiveness to a recall stimulation. Collectively, our data validate the concept that KRN encapsulated in PLGA-based particles can be used as delivery systems to activate iNKT cells in vitro and in vivo.

Benzophenone-containing fatty acids and their related photosensitive fluorescent new probes: design, physico-chemical properties and preliminary functional investigations.

Hydrophobic photoaffinity labeling is a powerful strategy to identify hydrophobic segments within molecules, in particular membrane proteins. Here we report the design and synthesis of a novel family of fluorescent and photosensitive lipid tools, which have a common amino acid scaffold functionalized by three groups: (i) a first fatty acid chain grafted with a photoactivatable benzophenone moiety (Fatty Acid BenzoPhenone, FABP), (ii) a second fatty acid chain to ensure anchoring into a half-bilayer or hydrophobic environment, and (iii) a fluorescent carboxytetramethylrhodamine headgroup (CTMR) to detect the photolabeled compound. We present data of the synthesis and characterization of three lipid tools whose benzophenone ring is situated at various distances from the central scaffold. We could therefore establish structure/properties relationships dependent upon the depth of insertion of benzophenone into the membrane. Our lipid tools were extensively characterized both physico- and bio-chemically, and we assessed their functionality in vitro using bacterioRhodopsin (bR). We thus provide the scientific community with novel and reliable tools for the identification and study of hydrophobic regions in proteins.

Spleen-resident CD4+ and CD4- CD8α- dendritic cell subsets differ in their ability to prime invariant natural killer T lymphocytes.

One important function of conventional dendritic cells (cDC) is their high capacity to capture, process and present Ag to T lymphocytes. Mouse splenic cDC subtypes, including CD8α(+) and CD8α(-) cDC, are not identical in their Ag presenting and T cell priming functions. Surprisingly, few studies have reported functional differences between CD4(-) and CD4(+) CD8α(-) cDC subsets. We show that, when loaded in vitro with OVA peptide or whole protein, and in steady-state conditions, splenic CD4(-) and CD4(+) cDC are equivalent in their capacity to prime and direct CD4(+) and CD8(+) T cell differentiation. In contrast, in response to α-galactosylceramide (α-GalCer), CD4(-) and CD4(+) cDC differentially activate invariant Natural Killer T (iNKT) cells, a population of lipid-reactive non-conventional T lymphocytes. Both cDC subsets equally take up α-GalCer in vitro and in vivo to stimulate the iNKT hybridoma DN32.D3, the activation of which depends solely on TCR triggering. On the other hand, and relative to their CD4(+) counterparts, CD4(-) cDC more efficiently stimulate primary iNKT cells, a phenomenon likely due to differential production of co-factors (including IL-12) by cDC. Our data reveal a novel functional difference between splenic CD4(+) and CD4(-) cDC subsets that may be important in immune responses.