Anneliese O. Speak

Invariant NKT cells reduce the immunosuppressive activity of influenza A virus–induced myeloid-derived suppressor cells in mice and humans

Infection with influenza A virus (IAV) presents a substantial threat to public health worldwide, with young, elderly, and immunodeficient individuals being particularly susceptible. Inflammatory responses play an important role in the fatal outcome of IAV infection, but the mechanism remains unclear. We demonstrate here that the absence of invariant NKT (iNKT) cells in mice during IAV infection resulted in the expansion of myeloid-derived suppressor cells (MDSCs), which suppressed IAV-specific immune responses through the expression of both arginase and NOS, resulting in high IAV titer and increased mortality. Adoptive transfer of iNKT cells abolished the suppressive activity of MDSCs, restored IAV-specific immune responses, reduced IAV titer, and increased survival rate. The crosstalk between iNKT and MDSCs was CD1d- and CD40-dependent. Furthermore, IAV infection and exposure to TLR agonists relieved the suppressive activity of MDSCs. Finally, we extended these results to humans by demonstrating the presence of myeloid cells with suppressive activity in the PBLs of individuals infected with IAV and showed that their suppressive activity is substantially reduced by iNKT cell activation. These findings identify what we believe to be a novel immunomodulatory role of iNKT cells, which we suggest could be harnessed to abolish the immunosuppressive activity of MDSCs during IAV infection.

Normal development and function of invariant natural killer T cells in mice with isoglobotrihexosylceramide (iGb3) deficiency

CD1d-restricted natural killer T (NKT) cells, expressing the invariant T cell antigen receptor (TCR) chain encoded by Vα14-Jα18 gene segments in mice and Vα24-Jα18 in humans [invariant NKT (iNKT) cells], contribute to immunoregulatory processes, such as tolerance, host defense, and tumor surveillance. iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4+/CD8+ cortical thymocytes. However, the identity of the endogenous lipid(s) responsible for positive selection of iNKT cells remains unclear. One candidate lipid proposed to play a role in positive selection is isoglobotrihexosylceramide (iGb3). However, no direct evidence for its physiological role has been provided. Therefore, to directly investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in iGb3 synthase [iGb3S, also known as α1–3galactosyltransferase 2 (A3galt2)]. These mice developed, grew, and reproduced normally and exhibited no overt behavioral abnormalities. Consistent with the notion that iGb3 is synthesized only by iGb3S, lack of iGb3 in the dorsal root ganglia of iGb3S-deficient mice (iGb3S−/−), as compared with iGb3S+/− mice, was confirmed. iGb3S−/− mice showed normal numbers of iNKT cells in the thymus, spleen, and liver with selected TCR Vβ chains identical to controls. Upon administration of α-galactosylceramide, activation of iNKT and dendritic cells was similar in iGb3S−/− and iGb3S+/− mice, as measured by up-regulation of CD69 as well as intracellular IL-4 and IFN-γ in iNKT cells, up-regulation of CD86 on dendritic cells, and rise in serum concentrations of IL-4, IL-6, IL-10, IL-12p70, IFN-γ, TNF-α, and Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to be an endogenous CD1d lipid ligand determining thymic iNKT selection.

Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation.

Invariant natural killer T (iNKT) cells are a subset of nonconventional T cells recognizing endogenous and/or exogenous glycolipid antigens in the context of CD1d molecules. It remains unclear whether innate stimuli can modify the profile of endogenous lipids recognized by iNKT cells on the surface of antigen-presenting cells (APCs). We report that activation of human APCs by Toll-like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells, as defined by IFN-γ secretion. APC-derived soluble factors further increase CD1d-restricted iNKT cell activation. Finally, using soluble tetrameric iNKT T cell receptors (TCR) as a staining reagent, we demonstrate specific up-regulation of CD1d-bound ligand(s) on TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of APCs resulting in iNKT cell activation and APC maturation underscores the role of iNKT cells in assisting priming of antigen-specific immune responses.

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of NPC1 disease.

Implications for invariant natural killer T cell ligands due to the restricted presence of isoglobotrihexosylceramide in mammals

Development of invariant natural killer T (iNKT) cells requires the presentation of lipid ligand(s) by CD1d molecules in the thymus. The glycosphingolipid (GSL) isoglobotrihexosylceramide (iGb3) has been proposed as the natural iNKT cell-selecting ligand in the thymus and to be involved in peripheral activation of iNKT cells by dendritic cells (DCs). However, there is no direct biochemical evidence for the presence of iGb3 in mouse or human thymus or DCs. Using a highly sensitive HPLC assay, the only tissue where iGb3 could be detected in mouse was the dorsal root ganglion (DRG). iGb3 was not detected in other mouse or any human tissues analyzed, including thymus and DCs. Even in mutant mice that store isoglobo-series GSLs in the DRG, we were still unable to detect these GSLs in the thymus. iGb3 is therefore unlikely to be a physiologically relevant iNKT cell-selecting ligand in mouse and humans. A detailed study is now warranted to better understand the nature of iNKT cell-selecting ligand(s) in vivo.

T cell fate and clonality inference from single-cell transcriptomes

We developed TraCeR, a computational method to reconstruct full-length, paired T cell receptor (TCR) sequences from T lymphocyte single-cell RNA sequence data. TraCeR links T cell specificity with functional response by revealing clonal relationships between cells alongside their transcriptional profiles. We found that T cell clonotypes in a mouse Salmonella infection model span early activated CD4+ T cells as well as mature effector and memory cells.

Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.

Beneficial effects of substrate reduction therapy in a mouse model of GM1 gangliosidosis.

GM1 gangliosidosis is an inherited neurodegenerative disorder caused by lysosomal beta-galactosidase deficiency, resulting in the storage of GM1 and GA1, primarily in the central nervous system. This disease typically afflicts infants and young children and there is currently no effective therapy. Substrate reduction therapy (SRT) could be of potential benefit. The imino sugars N-butyldeoxynojirimycin (NB-DNJ, miglustat, Zavesca) and N-butyldeoxygalactonojirimycin (NB-DGJ) used for SRT inhibit glucosylceramide synthase (GlcCerS) that catalyses the first committed step in glycosphingolipid biosynthesis. We have compared the efficacy and tolerability of NB-DNJ and NB-DGJ in the beta-galactosidase knockout mouse. NB-DGJ was better tolerated than NB-DNJ, due to intrinsic gastrointestinal tract dysfunction that was exacerbated by NB-DNJ. However, functional improvement was greatest with NB-DNJ treatment which may potentially be caused by novel anti-inflammatory properties of NB-DNJ.

Diverse Endogenous Antigens for Mouse NKT Cells: Self-Antigens That Are Not Glycosphingolipids

NKT cells with an invariant Ag receptor (iNKT cells) represent a highly conserved and unique subset of T lymphocytes having properties of innate and adaptive immune cells. They have been reported to regulate a variety of immune responses, including the response to cancers and the development of autoimmunity. The development and activation of iNKT cells is dependent on self-Ags presented by the CD1d Ag-presenting molecule. It is widely believed that these self-Ags are glycosphingolipids (GSLs), molecules that contain ceramide as the lipid backbone. In this study, we used a variety of methods to show that mammalian Ags for mouse iNKT cells need not be GSLs, including the use of cell lines deficient in GSL biosynthesis and an inhibitor of GSL biosynthesis. Presentation of these Ags required the expression of CD1d molecules that could traffic to late endosomes, the site where self-Ag is acquired. Extracts of APCs contain a self-Ag that could stimulate iNKT cells when added to plates coated with soluble, rCD1d molecules. The Ag(s) in these extracts are resistant to sphingolipid-specific hydrolase digestion, consistent with the results using live APCs. Lyosphosphatidylcholine, a potential self-Ag that activated human iNKT cell lines, did not activate mouse iNKT cell hybridomas. Our data indicate that there may be more than one type of self-Ag for iNKT cells, that the self-Ags comparing mouse and human may not be conserved, and that the search to identify these molecules should not be confined to GSLs.

CD8α⁺β⁻ and CD8α⁺β⁺ plasmacytoid dendritic cells induce Foxp3⁺ regulatory T cells and prevent the induction of airway hyper-reactivity.

Dendritic cells (DCs) control the balance between protection against pathogens and tolerance to innocuous or self-antigens. Here, we demonstrate for the first time that mouse plasmacytoid DCs (pDCs) can be segregated into three distinct populations, exhibiting phenotypic and functional differences, according to their surface expression of CD8α or CD8β as CD8α−β−, CD8α+β−, or CD8α+β+. In a mouse model of lung inflammation, adoptive transfer of CD8α+β− or CD8α+β+ pDCs prevents the development of airway hyper-reactivity. The tolerogenic features of these subsets are associated with increased production of retinoic acid, which leads to the enhanced induction of Foxp3+ regulatory T cells compared with CD8α−β− pDCs. Our data thus identify subsets of pDCs with potent tolerogenic functions that may contribute to the maintenance of tolerance in mucosal sites such as the lungs.