Synthesis of a Versatile Building Block for the Preparation of 6-N-Derivatized α-Galactosyl Ceramides: Rapid Access to Biologically Active Glycolipids

A concise route to the 6-azido-6-deoxy-α-galactosyl-phytosphingosine derivative 9 is reported. Orthogonal protection of the two amino groups allows elaboration of 9 into a range of 6-N-derivatized α-galactosyl ceramides by late-stage introduction of the acyl chain of the ceramide and the 6-N-group in the sugar head-group. Biologically active glycolipids 6 and 8 have been synthesized to illustrate the applicability of the approach

Leishmania donovani-induced expression of signal regulatory protein α on Kupffer cells enhances hepatic invariant NKT-cell activation

Signal regulatory protein α (SIRPα) and its cognate ligand CD47 have been documented to have a broad range of cellular functions in development and immunity. Here, we investigated the role of SIRPα–CD47 signalling in invariant NKT (iNKT) cell responses. We found that CD47 was required for the optimal production of IFN-γ from splenic iNKT cells following exposure to the αGalCer analogue PBS-57 and in vivo infection of mice with Leishmania donovani. Surprisingly, although SIRPα was undetectable in the liver of uninfected mice, the hepatic iNKT-cell response to infection was also impaired in CD47−/− mice. However, we found that SIRPα was rapidly induced on Kupffer cells following L. donovani infection, via a mechanism involving G-protein-coupled receptors. Thus, we describe a novel amplification pathway affecting cytokine production by hepatic iNKT cells, which may facilitate the breakdown of hepatic tolerance after infection

Identification of an IL-17–producing NK1.1neg iNKT cell population involved in airway neutrophilia

Invariant natural killer T (iNKT) cells are an important source of both T helper type 1 (Th1) and Th2 cytokines, through which they can exert beneficial, as well as deleterious, effects in a variety of inflammatory diseases. This functional heterogeneity raises the question of how far phenotypically distinct subpopulations are responsible for such contrasting activities. In this study, we identify a particular set of iNKT cells that lack the NK1.1 marker (NK1.1neg) and secrete high amounts of interleukin (IL)-17 and low levels of interferon (IFN)-γ and IL-4. NK1.1neg iNKT cells produce IL-17 upon synthetic (α-galactosylceramide [α-GalCer] or PBS-57), as well as natural (lipopolysaccharides or glycolipids derived from Sphingomonas wittichii and Borrelia burgdorferi), ligand stimulation. NK1.1neg iNKT cells are more frequent in the lung, which is consistent with a role in the natural immunity to inhaled antigens. Indeed, airway neutrophilia induced by α-GalCer or lipopolysaccharide instillation was significantly reduced in iNKT-cell–deficient Jα18−/− mice, which produced significantly less IL-17 in their bronchoalveolar lavage fluid than wild-type controls. Furthermore, airway neutrophilia was abolished by a single treatment with neutralizing monoclonal antibody against IL-17 before α-GalCer administration. Collectively, our findings reveal that NK1.1neg iNKT lymphocytes represent a new population of IL-17–producing cells that can contribute to neutrophil recruitment through preferential IL-17 secretion

CD160 serves as a negative regulator of NKT cells in acute hepatic injury

[EN] CD160 and BTLA both bind to herpes virus entry mediator. Although a negative regulatory function of BTLA in natural killer T (NKT) cell activation has been reported, whether CD160 is also involved is unclear. By analyzing CD160−/− mice and mixed bone marrow chimeras, we show that CD160 is not essential for NKT cell development. However, CD160−/− mice exhibit severe liver injury after in vivo challenge with α-galactosylceramide (α-GalCer). Moreover, CD160−/− mice are more susceptible to Concanavalin A challenge, and display elevated serum AST and ALT levels, hyperactivation of NKT cells, and enhanced IFN-γ, TNF, and IL-4 production. Lastly, inhibition of BTLA by anti-BTLA mAb aggravates α-GalCer-induced hepatic injury in CD160−/− mice, suggesting that both CD160 and BTLA serve as non-overlapping negative regulators of NKT cells. Our data thus implicate CD160 as a co-inhibitory receptor that delivers antigen-dependent signals in NKT cells to dampen cytokine production during early innate immune activationSIWe thank the NIH Tetramer Core Facility for providing PBS 57 ligand loaded CD1d Tetramers. Further, we thank the staffs of Gyerim Experimental Animal Resource Center for animal care and technical assistance. K.-M. Lee was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future planning (NRF-2016M3A9B6948342, NRF- 2017R1A2B3004828, and NRF-2018M3A9D3079288). S.-J. Kim was supported by the Korea Health Industry Development Institute (KHIDI-HI14C2640) grant funded by Korea Government. S.-J. Ha was supported by a grant from the NRF (NRF- 2018R1A2A1A05076997). T.-J. Kim was additionally supported by a grant from the NRF (NRF-2016R1A6A3A04009698

Downregulation of BTLA on NKT Cells Promotes Tumor Immune Control in a Mouse Model of Mammary Carcinoma

[EN] Natural Killer T cells (NKT cells) are emerging as critical regulators of pro- and anti-tumor immunity, both at baseline and in therapeutic settings. While type I NKT cells can promote anti-tumor immunity, their activity in the tumor microenvironment may be limited by negative regulators such as inhibitory immune checkpoints.We observed dominant expression of B- and T-lymphocyte attenuator (BTLA) on type I NKT cells in polyoma middle T oncogene-driven (PyMT) murine autochthonous mammary tumors. Other immune checkpoint receptors, such as programmed cell death 1 (PD-1) were equally distributed among T cell populations. Interference with BTLA using neutralizing antibodies limited tumor growth and pulmonary metastasis in the PyMT model in a therapeutic setting, correlating with an increase in type I NKT cells and expression of cytotoxic marker genes. While therapeutic application of an anti-PD-1 antibody increased the number of CD8+ cytotoxic T cells and elevated IL-12 expression, tumor control was not established. Expression of ZBTB16, the lineage-determining transcription factor of type I NKT cells, was correlated with a favorable patient prognosis in the METABRIC dataset, and BTLA levels were instrumental to further distinguish prognosis in patents with high ZBTB16 expression. Taken together, these data support a role of BTLA on type I NKT cells in limiting anti-tumor immunitySIThe authors thank Praveen Mathoor and Margarete Mijatovic for excellent technical assistance. The authors are supported by Deutsche Forschungsgemeinschaft (SFB 1039 TP B04 and B06; FOR 2438), Deutsche Krebshilfe (70112451), and Else Kröner Fresenius-Foundation (Graduate school Translational Research Innovation—Pharma (TRIP); and Else Kröner Fresenius Graduate School

PLZF Controls the Expression of a Limited Number of Genes Essential for NKT Cell Function

Natural killer (NKT) T cells exhibit tissue distribution, surface phenotype, and functional responses that are strikingly different from those of conventional T cells. The transcription factor PLZF is responsible for most of these properties, as its ectopic expression in conventional T cells is sufficient to confer to them an NKT-like phenotype. The molecular program downstream of PLZF, however, is largely unexplored. Here we report that PLZF regulates the expression of a surprisingly small set of genes, many with known immune functions. This includes several established components of the NKT cell developmental program. Expression of the transcriptional regulators Id2, previously shown to be required for iNKT cell survival in the liver and c-Maf, which shapes the NKT cytokine profile, was compromised in PLZF-deficient cells. Ectopic expression of c-Maf complemented the cells’ defect in producing IL-4 and IL-10. PLZF also induced a program of cell surface receptors which shape the NKT cell’s response to external stimuli, including the costimulatory receptor ICOS and the cytokine receptors IL12rb1 and IL18r1. As an ensemble, the known functions of the molecules whose expression is affected by PLZF explain many defects observed in $PLZF^{−/−}$ NKT cells

Loss of Effector and Anti-Inflammatory Natural Killer T Lymphocyte Function in Pathogenic Simian Immunodeficiency Virus Infection

Chronic immune activation is a key determinant of AIDS progression in HIV-infected humans and simian immunodeficiency virus (SIV)-infected macaques but is singularly absent in SIV-infected natural hosts. To investigate whether natural killer T (NKT) lymphocytes contribute to the differential modulation of immune activation in AIDS-susceptible and AIDS-resistant hosts, we compared NKT function in macaques and sooty mangabeys in the absence and presence of SIV infection. Cynomolgus macaques had significantly higher frequencies of circulating invariant NKT lymphocytes compared to both rhesus macaques and AIDS-resistant sooty mangabeys. Despite this difference, mangabey NKT lymphocytes were functionally distinct from both macaque species in their ability to secrete significantly more IFN-γ, IL-13, and IL-17 in response to CD1d/α-galactosylceramide stimulation. While NKT number and function remained intact in SIV-infected mangabeys, there was a profound reduction in NKT activation-induced, but not mitogen-induced, secretion of IFN-γ, IL-2, IL-10, and TGF-β in SIV-infected macaques. SIV-infected macaques also showed a selective decline in CD4+ NKT lymphocytes which correlated significantly with an increase in circulating activated memory CD4+ T lymphocytes. Macaques with lower pre-infection NKT frequencies showed a significantly greater CD4+ T lymphocyte decline post SIV infection. The disparate effect of SIV infection on NKT function in mangabeys and macaques could be a manifestation of their differential susceptibility to AIDS. Alternately, these data also raise the possibility that loss of anti-inflammatory NKT function promotes chronic immune activation in pathogenic SIV infection, while intact NKT function helps to protect natural hosts from developing immunodeficiency and aberrant immune activation

Paucity of CD4+ Natural Killer T (NKT) Lymphocytes in Sooty Mangabeys Is Associated with Lack of NKT Cell Depletion after SIV Infection

Lack of chronic immune activation in the presence of persistent viremia is a key feature that distinguishes nonpathogenic simian immunodeficiency virus (SIV) infection in natural hosts from pathogenic SIV and HIV infection. To elucidate novel mechanisms downmodulating immune activation in natural hosts of SIV infection, we investigated natural killer T (NKT) lymphocytes in sooty mangabeys. NKT lymphocytes are a potent immunoregulatory arm of the innate immune system that recognize glycolipid antigens presented on the nonpolymorphic MHC-class I-like CD1d molecules. In a cross-sectional analysis of 50 SIV-negative and 50 naturally SIV-infected sooty mangabeys, ligand α-galactosylceramide loaded CD1d tetramers co-staining with Vα24-positive invariant NKT lymphocytes were detected at frequencies ≥0.002% of circulating T lymphocytes in approximately half of the animals. In contrast to published reports in Asian macaques, sooty mangabey NKT lymphocytes consisted of CD8+ and CD4/CD8 double-negative T lymphocytes that were CXCR3-positive and CCR5-negative suggesting that they trafficked to sites of inflammation without being susceptible to SIV infection. Consistent with these findings, there was no difference in the frequency or phenotype of NKT lymphocytes between SIV-negative and SIV-infected sooty mangabeys. On stimulation with α-galactosylceramide loaded on human CD1d molecules, sooty mangabey NKT lymphocytes underwent degranulation and secreted IFN-γ, TNF-α, IL-2, IL-13, and IL-10, indicating the presence of both effector and immunoregulatory functional capabilities. The unique absence of CD4+ NKT lymphocytes in sooty mangabeys, combined with their IL-10 cytokine-secreting ability and preservation following SIV infection, raises the possibility that NKT lymphocytes might play a role in downmodulating immune activation in SIV-infected sooty mangabeys

THE EXPRESSION AND FUNCTION OF GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEINS IN INVARIANT NATURAL KILLER T (/NKT) CELLS

Invariant natural killer T (/NKT) cells are a unique lymphocyte subpopulation with potent immunomodulatory properties. They can produce copious amounts of pro- and/or anti-inflammatory cytokines, yet the mechanisms governing the type of immune responses they elicit are not fully understood. Conventional T cell activation can be achieved or augmented by glycosylphosphatidylinositol (GPI)-anchored proteins. Whether this is also true for /NKT cells is essentially unexplored. I hypothesized that ligation of GPI-anchored proteins such as Thy-1 and CD55 will enhance / NKT cell activation leading to robust cytokine production. Using flow cytometry, mouse /NKT cells were found to constitutively express Thy-1. Thy-1 engagement combined with classical TCR stimulation led to /NKT cell activation and cytokine production as measured by ELISA and RT-qPCR. Similarly, human /NKT cells expressed CD55, the cross-linking of which enhanced TCR-mediated activation. Overall, I have demonstrated that GPI-anchored proteins play a significant role in the magnitude of /NKT cell responses

Homeostatic Regulation of Marginal Zone B Cells by Invariant Natural Killer T Cells

Marginal zone B cells (MZB) mount a rapid antibody response, potently activate naïve T cells, and are enriched in autoreactive B cells. MZBs express high levels of CD1d, the restriction element for invariant natural killer T cells (iNKT). Here, we examined the effect of iNKT cells on MZB cell activation and numbers in vitro and in vivo in normal and autoimmune mice. Results show that iNKT cells activate MZBs, but restrict their numbers in vitro and in vivo in normal BALB/c and C57/BL6 mice. iNKT cells do so by increasing the activation-induced cell death and curtailing proliferation of MZB cells, whereas they promote the proliferation of follicular B cells. Sorted iNKT cells can directly execute this function, without help from other immune cells. Such MZB regulation by iNKTs is mediated, at least in part, via CD1d on B cells in a contact-dependent manner, whereas iNKT-induced proliferation of follicular B cells occurs in a contact- and CD1d-independent manner. Finally, we show that iNKT cells reduce ‘autoreactive’ MZB cells in an anti-DNA transgenic model, and limit MZB cell numbers in autoimmune-prone (NZB×NZW)F1 and non-obese diabetic mice, suggesting a potentially new mechanism whereby iNKT cells might regulate pathologic autoimmunity. Differential regulation of follicular B cells versus potentially autoreactive MZBs by iNKT cells has important implications for autoimmune diseases as well as for conditions that require a rapid innate B cell response