A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.

Many 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.

"Endocytic pH regulates cell surface localization of glycolipid antigen loaded CD1d complexes".

Invariant natural killer T (iNKT) cells recognize glycolipid antigens presented by CD1d, an antigen presenting protein structurally similar to MHC class I. Stimulation of iNKT cells by glycolipid antigens can induce strong immune responses in vivo, with rapid production of a wide variety of cytokines including those classically associated with either T helper type 1 (Th1) or type 2 (Th2) responses. Alterations in the lipid tails or other portions of CD1d-presented glycolipid ligands can bias the iNKT response towards production of predominantly Th1 or Th2 associated cytokines. However, the mechanism accounting for this structure-activity relationship remains controversial. The Th1-biasing glycolipids have been found to consistently form complexes with CD1d that preferentially localize to plasma membrane cholesterol rich microdomains (lipid rafts), whereas CD1d complexes formed with Th2-biasing ligands are excluded from these microdomains. Here we show that neutralization of endosomal pH enhanced localization of CD1d complexes containing Th2-biasing glycolipids to plasma membrane lipid rafts of antigen presenting cells (APC). Transfer of APCs presenting these "stabilized" CD1d/αGC complexes into mice resulted in immune responses with a more prominent Th1-like bias, characterized by increased NK cell transactivation and interferon-γ production. These findings support a model in which low endosomal pH controls stability and lipid raft localization of CD1d-glycolipid complexes to regulate the outcome of iNKT cell mediated responses.

Endocytic pH regulates cell surface localization of glycolipid antigen loaded CD1d complexes.

Invariant natural killer T (iNKT) cells recognize glycolipid antigens presented by CD1d, an antigen presenting protein structurally similar to MHC class I. Stimulation of iNKT cells by glycolipid antigens can induce strong immune responses in vivo, with rapid production of a wide variety of cytokines including those classically associated with either T helper type 1 (Th1) or type 2 (Th2) responses. Alterations in the lipid tails or other portions of CD1d-presented glycolipid ligands can bias the iNKT response towards production of predominantly Th1 or Th2 associated cytokines. However, the mechanism accounting for this structure-activity relationship remains controversial. The Th1-biasing glycolipids have been found to consistently form complexes with CD1d that preferentially localize to plasma membrane cholesterol rich microdomains (lipid rafts), whereas CD1d complexes formed with Th2-biasing ligands are excluded from these microdomains. Here we show that neutralization of endosomal pH enhanced localization of CD1d complexes containing Th2-biasing glycolipids to plasma membrane lipid rafts of antigen presenting cells (APC). Transfer of APCs presenting these "stabilized" CD1d/αGC complexes into mice resulted in immune responses with a more prominent Th1-like bias, characterized by increased NK cell transactivation and interferon-γ production. These findings support a model in which low endosomal pH controls stability and lipid raft localization of CD1d-glycolipid complexes to regulate the outcome of iNKT cell mediated responses.

Improving Mycobacterium bovis bacillus Calmette-Guèrin as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells.

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.