SummaryMany hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α+ DEC-205+ dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α+ dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

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.

Background:Immune suppression in the tumour microenvironment remains a major limitation to successful immunotherapy of cancer. In the current study, we analysed whether the natural killer T cell-activating glycolipid α-galactosylceramide could overcome immune suppression and improve vaccination against metastatic breast cancer.Methods:Mice with metastatic breast cancer (4T1 model) were therapeutically treated with a Listeria monocytogenes-based vaccine expressing tumour-associated antigen Mage-b followed by α-galactosylceramide as separate agents, or as a complex of α-galactosylceramide stably incorporated into Listeria-Mage-b. Effects on metastases, tumour weight, toxicity and immune responses were determined.Results:Sequential treatments of mice with established 4T1 breast carcinomas using Listeria-Mage-b followed by α-galactosylceramide as a separate agent was highly effective at reducing metastases, but was accompanied by severe liver toxicity. In contrast, combined therapy using Listeria-Mage-b modified by incorporation of α-galactosylceramide resulted in nearly complete elimination of metastases without toxicity. This was associated with a significant increase in the percentage of natural killer T cells in the spleen, and an increase in natural killer cell activity and in T cell responses to Mage-b.Conclusions:Our results suggest that direct incorporation of α-galactosylceramide into a live bacterial vaccine vector is a promising non-toxic new approach for the treatment of metastatic breast cancer.

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

Natural Killer T cells (NKT cells) are a subpopulation of T lymphocytes with unique phenotypic properties and a remarkably broad range of immune effector and regulatory functions. One subset of these cells, known as invariant NKT cells (iNKT cells), has become a significant focus in the search for new and better ways to enhance immunotherapies and vaccination. These unconventional T cells are characterized by their ability to be specifically activated by a range of foreign and self-derived glycolipid antigens presented by CD1d, an MHC class I-related antigen presenting molecule that has evolved to bind and present lipid antigens. 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 the basic background biology of iNKT cells that is relevant to their potential for improving immune responses, and summarize recent work supporting the further development of glycolipid activators of iNKT cells as a new class of vaccine adjuvants.