A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells.

Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure-activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.

Optimizing NKT cell ligands as vaccine adjuvants.

NKT cells are a subpopulation of T lymphocytes with phenotypic properties of both T and NK cells and a wide range of immune effector properties. In particular, one subset of these cells, known as invariant NKT cells (iNKT cells), has attracted substantial attention because of their ability to be specifically activated by glycolipid antigens presented by a cell surface protein called CD1d. The development of synthetic α-galactosylceramides as a family of powerful glycolipid agonists for iNKT cells has led to approaches for augmenting a wide variety of immune responses, including those involved in vaccination against infections and cancers. Here, we review basic, preclinical and clinical observations supporting approaches to improving immune responses through the use of iNKT cell-activating glycolipids. Results from preclinical animal studies and preliminary clinical studies in humans identify many promising applications for this approach in the development of vaccines and novel immunotherapies.

Evaluation of importance of Toll-like receptor 4 in acute Streptococcus pneumoniae sinusitis in mice.

OBJECTIVES: To investigate the effect of RC-527, a synthetic toll-like receptor 4 (TLR4) agonist, on stimulating the immune response before acute Streptococcus pneumoniae sinusitis in a mouse model, and to determine the importance of TLR4 in modulating the response to S. pneumoniae. Toll-like receptor 4 agonists have been shown to induce protective innate immune responses when administered before some bacterial or viral challenges in mice. DESIGN: We intranasally inoculated BALB/c, TLR4 complex-deficient C3H/HeJ, and wild-type C3H/HeOuJ mice with S. pneumoniae 24 hours after treatment with 10 or 1 microg of RC-527 or vehicle. Bacterial counts from nasal lavage culture and the cell markers GR1, CD11b, CD3, CD4, and CD8 in sinus tissue were quantified at postinoculation days 2, 5, and 14. MAIN OUTCOME MEASURE: Immune response induced by RC-527. RESULTS: Treatment with RC-527 induced an immune response through TLR4, as demonstrated by recruitment of phagocytes in uninfected wild-type C3H/HeOuJ mice, but not in TLR4 complex-deficient C3H/HeJ mice. The immune response was also demonstrated by a significant increase of CD3+, CD4+, and CD8+ T cells in infected and uninfected wild-type C3H/HeOuJ mice, but not in TLR4 complex-deficient C3H/HeJ mice. However, the enhancement of the immune response induced by the TLR4 agonist showed a limited effect on bacterial clearance. CONCLUSIONS: Our studies in mice suggest that stimulation of TLR4 plays a minor role in the overall response to S. pneumoniae infection of the upper airway, and stimulating this receptor before infection does not significantly enhance the immune response of immunocompetent mice to clear S. pneumoniae infection.

Interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta inhibit cyclic AMP-induced Schwann cell differentiation.

Schwann cells differentiate in vivo in response to contact with axons, and cAMP simulates some of these aspects of differentiation in vitro, particularly morphologic changes and expression of certain phenotypic molecules. Unfractionated inflammatory cytokines inhibit cAMP-induced Schwann cell expression of galactolipids (Gal). We sought to identify which cytokines were responsible for this inhibition and to determine whether other phenotypic indicators of Schwann cell differentiation were also affected. Neonatal rat Schwann cells were incubated in vitro with 1 mM 8 Bromo cAMP (8 Br cAMP) with or without the addition of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or transforming growth factor-beta (TGF-beta). Cells were then examined for morphologic changes and for expression of surface Gal and low-affinity nerve growth factor receptor (NGFRp75), employing indirect immunofluorescence. 8 Br cAMP induced Schwann cell upregulation of Gal, downregulation of NGFRp75, and the cells became enlarged and somewhat amorphous and irregular in appearance. Cells treated with IFN-gamma or TNF-alpha alone were more bipolar and more evenly distributed on coverslips than were control cells, whereas TGF-beta alone induced elongated cells often in a swirling pattern. None of the cytokines alone induced upregulation of Gal or downregulation of NGFRp75. TNF-alpha, IFN-gamma, and TGF-beta inhibited the 8 Br cAMP-induced morphologic changes, as well as the upregulation of Gal and downregulation of NGFRp75. The other cytokines had no effects on Gal or NGFRp75 expression. Thus, these three cytokines, which are present in inflammatory lesions in the peripheral nervous system, are capable of inhibiting Schwann cell differentiation.