Sandrine Aspeslagh

Pharmacological sensitivity of ATP release triggered by photoliberation of inositol-1,4,5-trisphosphate and zero extracellular calcium in brain endothelial cells

Recently, ATP has gained much interest as an extracellular messenger involved in the communication of calcium signals between cells. The mechanism of ATP release is, however, still a matter of debate. In the present study we investigated the possible contribution of connexin hemichannels or ion channels in the release of ATP in GP8, a rat brain endothelial cell line. Release of ATP was triggered by photoactivation of InsP3 or by reducing the extracellular calcium concentration. Both trigger protocols induced ATP release significantly above baseline. InsP3‐triggered ATP release was completely blocked by α‐glycyrrhetinic acid (α‐GA), the connexin mimetic peptides gap 26 and 27, and the trivalent ions gadolinium and lanthanum. ATP release triggered by zero calcium was, in addition to these substances, also blocked by flufenamic acid (FFA), niflumic acid, and NPPB. Gap 27 selectively blocked zero calcium‐triggered ATP release in connexin‐43 transfected HeLa cells, while having no effect in wild‐type and connexin‐32 transfected cells. Of all the agents used, only α‐GA, FFA and NPPB significantly reduced gap junctional coupling. In conclusion, InsP3 and zero calcium‐triggered ATP release show major similarities but also some differences in their sensitivity to the agents applied. It is suggested that both stimuli trigger ATP release through the same mechanism, which is connexin‐dependent, permeable in both directions, potently blocked by connexin mimetic peptides, and consistent with the opening of connexin hemichannels. J. Cell. Physiol. 197: 205–213, 2003© 2003 Wiley‐Liss, Inc.

Galactose-modified iNKT cell agonists stabilized by an induced fit of CD1d prevent tumour metastasis

Invariant natural killer T (iNKT) cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. Here, we report the structure and function of a novel class of aromatic α‐galactosylceramide structurally related glycolipids with marked Th1 bias in both mice and men, leading to superior tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1‐biasing glycolipid, α‐C‐GalCer, whose CD1d binding follows a conventional key‐lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose‐modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.

Rationale for anti-OX40 cancer immunotherapy

Immune checkpoint blockade with antagonistic monoclonal antibodies (mAbs) targeting B7 immunoglobulin superfamily molecules (CTLA-4, PD-1, and PD-L1) generate long lasting anti-tumour immune responses translating into clinical benefit across many cancer types. However, many patients are primarily resistant to immune checkpoint blockade -based monotherapy and many others will eventually relapse. Therefore, new immunostimulatory targets are needed to overcome primary and secondary resistance to immunotherapy. Besides the B7 co-inhibitory receptors, the tumour necrosis factor receptor superfamily contains many other immune checkpoints, which could become the next generation immunomodulators. Among them stands OX40 (CD134), a co-stimulatory molecule that can be expressed by activated immune cells. Several anti-OX40 agonistic monoclonal antibodies are currently tested in early phase cancer clinical trials. Accumulating preclinical evidence supports their clinical development. However, conflicting results and controversies between in vitro and in vivo data point to the need for comprehensive ancillary studies to be performed in upcoming clinical trials to better understand the mechanism of action of anti-OX40 mAbs-based therapy.

Activated iNKT Cells Promote Memory CD8+ T Cell Differentiation during Viral Infection

α-galactosylceramide (α-GalCer) is the prototypical lipid ligand for invariant NKT cells. Recent studies have proposed that α-GalCer is an effective adjuvant in vaccination against a range of immune challenges, however its mechanism of action has not been completely elucidated. A variety of delivery methods have been examined including pulsing dendritic cells with α-GalCer to optimize the potential of α-GalCer. These methods are currently being used in a variety of clinical trials in patients with advanced cancer but cannot be used in the context of vaccine development against pathogens due to their complexity. Using a simple delivery method, we evaluated α-GalCer adjuvant properties, using the mouse model for cytomegalovirus (MCMV). We measured several key parameters of the immune response to MCMV, including inflammation, effector, and central memory CD8+ T cell responses. We found that α-GalCer injection at the time of the infection decreases viral titers, alters the kinetics of the inflammatory response, and promotes both increased frequencies and numbers of virus-specific memory CD8+ T cells. Overall, our data suggest that iNKT cell activation by α-GalCer promotes the development of long-term protective immunity through increased fitness of central memory CD8+ T cells, as a consequence of reduced inflammation.

Enhanced TCR Footprint by a Novel Glycolipid Increases NKT-Dependent Tumor Protection

NKT cells, a unique type of regulatory T cells, respond to structurally diverse glycolipids presented by CD1d. Although it was previously thought that recognition of glycolipids such as α-galactosylceramide (α-GalCer) by the NKT cell TCR (NKTCR) obeys a key–lock principle, it is now clear this interaction is much more flexible. In this article, we report the structure–function analysis of a series of novel 6′′-OH analogs of α-GalCer with more potent antitumor characteristics. Surprisingly, one of the novel carbamate analogs, α-GalCer-6′′-(pyridin-4-yl)carbamate, formed novel interactions with the NKTCR. This interaction was associated with an extremely high level of Th1 polarization and superior antitumor responses. These data highlight the in vivo relevance of adding aromatic moieties to the 6′′-OH position of the sugar and additionally show that judiciously chosen linkers are a promising strategy to generate strong Th1-polarizing glycolipids through increased binding either to CD1d or to NKTCR.

Preclinical evaluation of invariant natural killer T cells in the 5T33 multiple myeloma model.

Immunomodulators have been used in recent years to reactivate host anti-tumor immunity in several hematological malignancies. This report describes the effect of activating natural killer T (NKT) cells by α-Galactosylceramide (α-GalCer) in the 5T33MM model of multiple myeloma (MM). NKT cells are T lymphocytes, co-expressing T and NK receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain. We followed iNKT numbers during the development of the disease in both 5T33MM mice and MM patients and found that their numbers dropped dramatically at the end stage of the disease, leading to a loss of total IFN-γ secretion. We furthermore observed that α-GalCer treatment significantly increased the survival of 5T33MM diseased mice. Taken together, our data demonstrate for the first time the possibility of using a preclinical murine MM model to study the effects of α-GalCer and show promising results of α-GalCer treatment in a low tumor burden setting.

Bacterial CD1d–Restricted Glycolipids Induce IL-10 Production by Human Regulatory T Cells upon Cross-Talk with Invariant NKT Cells

Invariant NKT (iNKT) cells and CD4+CD25+FOXP3+ regulatory T cells (Tregs) are important immune regulatory T cells with Ag reactivity to glycolipids and peptides, respectively. However, the functional interplay between these cells in humans is poorly understood. We show that Tregs suppress iNKT cell proliferation induced by CD1d-restricted glycolipids, including bacterial-derived diacylglycerols, as well as by innate-like activation. Inhibition was related to the potency of iNKT agonists, making diacylglycerol iNKT responses very prone to suppression. Cytokine production by iNKT cells was differentially modulated by Tregs because IL-4 production was reduced more profoundly compared with IFN-γ. A compelling observation was the significant production of IL-10 by Tregs after cell contact with iNKT cells, in particular in the presence of bacterial diacylglycerols. These iNKT-primed Tregs showed increased FOXP3 expression and superior suppressive function. Suppression of iNKT cell responses, but not conventional T cell responses, was IL-10 dependent, suggesting that there is a clear difference in mechanism between the Treg-mediated inhibition of these cell types. Our data highlight a physiologically relevant interaction between human iNKT and Tregs upon pathogen-derived glycolipid recognition that has a significant impact on the design of iNKT cell–based therapeutics.

Synthesis and evaluation of amino-modified α-GalCer analogues

Alpha-GalCer analogues featuring a phytoceramide 3- and 4-amino group have been synthesized. A Mitsunobu reaction involving phthalimide was employed for the introduction of the amino groups at the 3- and 4-positions of suitable phytosphingosine-derived precursors. The influence of these modifications on the interaction with the T-cell receptor of NKT cells was investigated, as well as the capacity of the amino-modified analogues to induce a cytokine response after in vivo administration.

TILs in Head and Neck Cancer: Ready for Clinical Implementation and Why (Not)?

The assessment of tumor infiltrating lymphocytes (TILs) has recently emerged as a prognostic biomarker in several solid tumors. Quantification and subtyping of TILs reflects the immune response in the tumor microenvironment, contributing to either tumoral immune attack or escape and thereby affecting outcome. Despite the growing evidence of its value as prognosticator, TILs analysis has not yet found its way to daily clinical practice. The aim of this review is to evaluate whether the current knowledge on TILs in head and neck cancer justifies its clinical implementation. Therefore, we summarize the data on TILs in squamous cell cancer of the head and neck with focus on the most important subsets (T lymphocytes and more specifically CD8+ cytotoxic T cells and FoxP3+ regulatory T cells) and site-specific characteristics such as Human Papilloma Virus infection. In addition, we discuss methodological problems and pitfalls that can account for discordant findings and that may hamper inclusion of TILs assessment in routine practice of pathologists and oncologists.

Invariant natural killer T cells in rheumatic disease: a joint dilemma

Invariant natural killer T (iNKT) cells are an innate T-cell lineage known to recognize a range of endogenously derived and exogenously derived glycolipid antigens. Advances in our understanding of this T-cell subset have enabled researchers to investigate the immunomodulatory activity of iNKT cell ligands in experimental models of diseases such as cancer, allergy and chronic inflammatory joint disease. To a large extent, the ability of iNKT cells to regulate such disease models has been ascribed to their capacity to promote a polarized cytokine environment, which is understood to skew adaptive immune responses. In this Review, we discuss the current understanding of how iNKT-cell polarization is regulated and relate this basic theory to the proposed role for iNKT cells in models of rheumatologic disease.