Synthesis of C-5" and C-6"-modified α-GalCer analogues as iNKT-cell agonists

Alpha-Galactosyl Ceramide (α-GalCer) is a prototypical synthetic ligand of invariant natural killer T (iNKT) cells. Upon presentation by the MHC class I-like molecule CD1d, this glycolipid stimulates iNKT cells to secrete a vast amount of both pro-inflammatory Th1 and anti-inflammatory Th2 cytokines. Recently, we discovered that selected 6″-modified α-GalCer analogues may produce markedly Th1-biased responses due to the formation of either an additional anchor with CD1d or by establishing extra interactions with the T-cell receptor of iNKT cells. Here, we report a practical synthesis towards 6″-O-carbamate and galacturonamide analogues of α-GalCer and their evaluation as iNKT cell agonists in mice

A computational high-throughput screening approach of iNKT-agonists: a novel tool to find optimized iNKT cell ligands

Depending on the environment and the activating glycolipids, iNKT cells are known to induce T-helper 1 and/or T-helper 2 cytokines. This highly versatile nature makes these innate-like cells very interesting targets for immunomodulation. As many pathologies as well as physiological ageing are associated with altered immune responses, iNKT cells could play a role in new therapies. Many analogs of the glycolipid alpha-galactosylceramide (a-GalCer) are known to activate iNKT-cells through their interaction with CD1d-expressing antigen-presenting cells, inducing the release of Th1 and/or Th2 cytokines. The design of iNKT cell ligands with selective Th1 and Th2 properties requires refined structural insights. Therefore, the chemical space of 333 currently known iNKT activators, including several newly tested analogs, was visualized by more than 3000 chemical descriptors which were calculated for each individual analog. The immunological responses consisted of four different cytokines in five different test-systems. With these two information-sets, structure-activity models were developed using a system biology computational approach. We present highly sensitive and specific predictive models that can be further exploited as high-throughput instruments to in-silico screen potential glycolipids, thereby reducing the attrition rate

An in silico approach for modelling T-helper polarizing iNKT cell agonists

Many analogues of the glycolipid alpha-galactosylceramide (α-GalCer) are known to activate iNKT cells through their interaction with CD1d-expressing antigen-presenting cells, inducing the release of Th1 and Th2 cytokines. Because of iNKT cell involvement and associated Th1/Th2 cytokine changes in a broad spectrum of human diseases, the design of iNKT cell ligands with selective Th1 and Th2 properties has been the subject of extensive research. This search for novel iNKT cell ligands requires refined structural insights. Here we will visualize the chemical space of 333 currently known iNKT cell activators, including several newly tested analogues, by more than 3000 chemical descriptors which were calculated for each individual analogue. To evaluate the immunological responses we analyzed five different cytokines in five different test-systems. We linked the chemical space to the immunological space using a system biology computational approach resulting in highly sensitive and specific predictive models. Moreover, these models correspond with the current insights of iNKT cell activation by α-GalCer analogues, explaining the Th1 and Th2 biased responses, downstream of iNKT cell activation. We anticipate that such models will be of great value for the future design of iNKT cell agonists

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 a-galactosylceramide (alpha-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 alpha-GalCer with more potent antitumor characteristics. Surprisingly, one of the novel carbamate analogs, alpha-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

Tumor PD-L1 status and CD8+ tumor-infiltrating T cells : markers of improved prognosis in oropharyngeal cancer

Introduction: The aim of this study was to evaluate the expression of PD-L1 in oropharyngeal squamous cell carcinoma. Its relation with clinicopathological variables, tumor infiltrating lymphocytes and survival was also determined. Results: Positive PD-L1 status for the SP142 clone related with improved overall survival in oropharyngeal squamous cell carcinoma. Tumors heavily infiltrated by tumor infiltrating lymphocytes were also linked with better outcome, and this as well for the total number of tumor infiltrating lymphocytes as for the CD3(+) and CD8(+) T cell count. A Cox proportional hazard model proved that solely infiltrating CD8+ T cells exhibit a positive effect on overall survival (hazard ratio = 0.31 [0.14-0.70]; P = 0.0050) Materials and Methods: Formalin-fixed, paraffin-embedded tissue from oropharyngeal tumors of 99 patients was immunohistochemically stained for PD-L1 (SP142 and 22C3 clones), CD3, CD8 and FoxP3. Expression of PD-L1, CD3, CD8, FoxP3 and HPV status were correlated with clinicopathological variables. Overall survival was determined by a log-rank (Mantel-Cox) test whereas the Cox proportional hazard model was used for multivariate analysis. Conclusions: Our results demonstrate that CD8(+) T lymphocytes constitute an independent prognostic marker in patients diagnosed with oropharyngeal squamous cell carcinoma. PD-L1 positivity for SP142, but not for 22C3, also tends to have a positive effect on survival in oropharyngeal squamous cell carcinoma