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

Enhancement of Adjuvant Functions of Natural Killer T Cells Using Nanovector Delivery Systems: Application in Anticancer Immune Therapy

Type I natural killer T (NKT) cells have gained considerable interest in anticancer immune therapy over the last decade. This “innate-like” T lymphocyte subset has the unique ability to recognize foreign and self-derived glycolipid antigens in association with the CD1d molecule expressed by antigen-presenting cells. An important property of these cells is to bridge innate and acquired immune responses. The adjuvant function of NKT cells might be exploited in the clinics. In this review, we discuss the approaches currently being used to target NKT cells for cancer therapy. In particular, we highlight ongoing strategies utilizing NKT cell-based nanovaccines to optimize immune therapy

Targeted Delivery of α-Galactosylceramide to CD8α + Dendritic Cells Optimizes Type I NKT Cell–Based Antitumor Responses

International audienceImmunotherapy aiming at enhancing innate and acquired host immunity is a promising approach for cancer treatment. The invariant NKT (iNKT) cell ligand α-galactosylceramide (α-GalCer) holds great promise in cancer therapy, although several concerns limit its use in clinics, including the uncontrolled response it promotes when delivered in a nonvectorized form. Therefore, development of delivery systems to in vivo target immune cells might be a valuable option to optimize iNKT cell-based antitumor responses. Using dendritic cell (DC)-depleted mice, DC transfer experiments, and in vivo active cell targeting, we show that presentation of α-GalCer by DCs not only triggers optimal primary iNKT cell stimulation, but also maintains secondary iNKT cell activation after challenge. Furthermore, targeted delivery of α-GalCer to CD8α(+) DCs, by means of anti-DEC205 decorated nanoparticles, enhances iNKT cell-based transactivation of NK cells, DCs, and γδ T cells. We report that codelivery of α-GalCer and protein Ag to CD8α(+) DCs triggers optimal Ag-specific Ab and cytotoxic CD8(+) T cell responses. Finally, we show that targeting nanoparticles containing α-GalCer and Ag to CD8α(+) DCs promotes potent antitumor responses, both in prophylactic and in therapeutic settings. Our data may have important implications in tumor immunotherapy and vaccine development

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

International audienceInvariant 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

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

International audienceThe 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

CD4⁺ and CD4⁻ cDC are equivalent in their ability to activate CD4⁺ T lymphocytes.

<p>(A) Splenic CD4⁺ and CD4⁻ cDC were sorted on the basis of CD11c, CD11b, CD8 and CD4 expression. The presence of contaminating plasmacytoid DC and CD8α⁺ cDC precursors in the CD4⁻ cDC fraction was evaluated by using anti-Siglec-H and Sirp-α mAbs, respectively. (B, C) Both cDC subsets were sensitized with graded doses of OVA peptide (B) or whole OVA (C) and co-cultured for 3 and 5 days with naive CD4⁺ T cells purified from OT-II mice. The production of IFN-γ and IL-13 was quantified by ELISA. Results represent the mean ± SD of a representative experiment out of two.</p

CD4⁺ and CD4⁻ cDC are equivalent in their ability to activate CD8⁺ T lymphocytes.

<p>(A, B) CD4⁺ and CD4⁻ cDC subsets were sensitized with graded doses of OVA peptide (A) or whole OVA (B) and co-cultured for 3 and 5 days with naive CD8⁺ T cells purified from OT-I mice. The production of IFN-γ was quantified by ELISA. Results represent the mean ± SD of a representative experiment out of two.</p

CD4⁺ and CD4⁻ cDC differ <i>in vitro</i> in their capacity to activate iNKT cells.

<p>(A, C) Sorted CD4⁺ and CD4⁻ cDC were exposed to graded doses of α-GalCer and then co-cultured for 48 h with sorted iNKT cells (A) or with the iNKT cell hybridoma DN32.D3 (C). Cytokine production was quantified by ELISA. Results represent the mean ± SD of 3 (A) or 2 (C) independent experiments. (B) CD1d expression on CD4⁺ and CD4⁻ cDC was assessed by flow cytometry. Of note, the staining with the isotype control was identical on both cDC subsets. For clarity, the isotype control on the CD4⁺, but not CD4⁻, cDC subset is shown. Shown is a representative experiment out of three. (D) Sorted CD4⁺ and CD4⁻ cDC were exposed, or not (medium), to α-GalCer (100 ng/ml) and then co-cultured for 6 h with sorted iNKT cells. RNAs were prepared and IL-12p35 (<i>Il12p35</i>) mRNA copy numbers were measured by quantitative RT-PCR. Data are normalized to expression of <i>Gapdh</i> and are expressed as fold increase over average gene expression in vehicle-treated cDC. Of note, the basal level of IL-12p40 transcript in <i>ex vivo</i> sorted cDC is relatively elevated (Ct: 25–26) (Ct of <i>gapdh</i>: 20, Ct of <i>il12p35</i>: 31–32). Data represent the mean ± SD (triplicates) of an experiment out of two performed. (E) α-GalCer-loaded cDC subsets were co-cultured for 48 h with sorted iNKT cells in the presence of a neutralizing IL-12 Ab or an isotype control Ab. Shown is a representative experiment (mean ± SD) out of three performed. * p<0.05; ** p<0.01; *** p<0.001.</p

Key role for respiratory CD103(+) dendritic cells, IFN-γ, and IL-17 in protection against Streptococcus pneumoniae infection in response to α-galactosylceramide.

International audienceBACKGROUND: Exogenous activation of pulmonary invariant natural killer T (iNKT) cells, a population of lipid-reactive αβ T lymphocytes, with use of mucosal α-galactosylceramide (α-GalCer) administration, is a promising approach to control respiratory bacterial infections. We undertook the present study to characterize mechanisms leading to α-GalCer-mediated protection against lethal infection with Streptococcus pneumoniae serotype 1, a major respiratory pathogen in humans. METHODS AND RESULTS: α-GalCer was administered by the intranasal route before infection with S. pneumoniae. We showed that respiratory dendritic cells (DCs), most likely the CD103(+) subset, play a major role in the activation (IFN-γ and IL-17 release) of pulmonary iNKT cells, whereas alveolar and interstitial macrophages are minor players. After challenge, S. pneumoniae was rapidly (4 hours) eliminated in the alveolar spaces, a phenomenon that depended on respiratory DCs and neutrophils, but not macrophages, and on the early production of both IFN-γ and IL-17. Protection was also associated with the synthesis of various interferon-dependent and IL-17-associated genes as revealed by transcriptomic analysis. CONCLUSIONS: These data imply a new function for pulmonary CD103(+) DCs in mucosal activation of iNKT cells and establish a critical role for both IFN-γ and IL-17 signalling pathways in mediating the innate immune response to S. pneumoniae