Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells

Invariant natural killer T cells (iNKT cells) are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled gene expression in iNKT cells during ontogeny and in peripheral subsets as part of the Immunological Genome Project. High-resolution comparative transcriptional analyses defined developmental and subset-specific programs of gene expression by iNKT cells. In addition, we found that iNKT cells shared an extensive transcriptional program with NK cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Notably, the program shared by NK cells and iNKT cells also operated constitutively in γδ T cells and in adaptive T cells after activation. Together our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes

Polyclonal mucosa-associated invariant T cells have unique innate functions in bacterial infection

Mucosa-associated invariant T (MAIT) cells are a unique population of αβ T cells in mammals that reside preferentially in mucosal tissues and express an invariant Vα paired with limited Vβ T-cell receptor (TCR) chains. Furthermore, MAIT cell development is dependent upon the expression of the evolutionarily conserved major histocompatibility complex (MHC) class Ib molecule MR1. Using in vitro assays, recent studies have shown that mouse and human MAIT cells are activated by antigen-presenting cells (APCs) infected with diverse microbes, including numerous bacterial strains and yeasts, but not viral pathogens. However, whether MAIT cells play an important, and perhaps unique, role in controlling microbial infection has remained unclear. To probe MAIT cell function, we show here that purified polyclonal MAIT cells potently inhibit intracellular bacterial growth of Mycobacterium bovis BCG in macrophages (MΦ) in coculture assays, and this inhibitory activity was dependent upon MAIT cell selection by MR1, secretion of gamma interferon (IFN-γ), and an innate interleukin 12 (IL-12) signal from infected MΦ. Surprisingly, however, the cognate recognition of MR1 by MAIT cells on the infected MΦ was found to play only a minor role in MAIT cell effector function. We also report that MAIT cell-deficient mice had higher bacterial loads at early times after infection compared to wild-type (WT) mice, demonstrating that MAIT cells play a unique role among innate lymphocytes in protective immunity against bacterial infection

NKT Cell Stimulation with α-Galactosylceramide Results in a Block of Th17 Differentiation after Intranasal Immunization in Mice

In a previous study we demonstrated that intranasal (i.n.) vaccination promotes a Th17 biased immune response. Here, we show that co-administration of a pegylated derivative of α-galactosylceramide (αGCPEG) with an antigen, even in the presence of Th17-polarizing compounds, results in a strong blocking of Th17 differentiation. Additional studies demonstrated that this phenomenon is specifically dependent on soluble factors, like IL-4 and IFNγ, which are produced by NKT cells. Even NK1.1 negative NKT cells, which by themselves produce IL-17A, are able to block Th17 differentiation. It follows that the use of αGCPEG as adjuvant would enable to tailor Th17 responses, according to the specific clinical needs. This knowledge expands our understanding of the role played by NKT cells in overall control of the cytokine microenvironment, as well as in the overall shaping of adaptive immune responses

Capturing the systemic immune signature of a norovirus infection: an n-of-1 case study within a clinical trial.

BACKGROUND: The infection of a participant with norovirus during the adaptive study of interleukin-2 dose on regulatory T cells in type 1 diabetes (DILT1D) allowed a detailed insight into the cellular and cytokine immune responses to this prevalent gastrointestinal pathogen. METHODS: Serial blood, serum and peripheral blood mononuclear cell (PBMC) samples were collected pre-, and post-development of the infection. To differentiate between the immune response to norovirus and to control for the administration of a single dose of aldesleukin (recombinant interleukin-2, rIL-2) alone, samples from five non-infected participants administered similar doses were analysed in parallel. RESULTS: Norovirus infection was self-limited and resolved within 24 hours, with the subsequent development of anti-norovirus antibodies. Serum pro- and anti-inflammatory cytokine levels, including IL-10, peaked during the symptomatic period of infection, coincident with increased frequencies of monocytes and neutrophils. At the same time, the frequency of regulatory CD4 + T cell (Treg), effector T cell (Teff) CD4 + and CD8 + subsets were dynamically reduced, rebounding to baseline levels or above at the next sampling point 24 hours later.  NK cells and NKT cells transiently increased CD69 expression and classical monocytes expressed increased levels of CD40, HLA-DR and SIGLEC-1, biomarkers of an interferon response. We also observed activation and mobilisation of Teffs, where increased frequencies of CD69 + and Ki-67 + effector memory Teffs were followed by the emergence of memory CD8 + Teff expressing the mucosal tissue homing markers CD103 and β7 integrin. Treg responses were coincident with the innate cell, Teff and cytokine response. Key Treg molecules FOXP3, CTLA-4, and CD25 were upregulated following infection, alongside an increase in frequency of Tregs with the capacity to home to tissues. CONCLUSIONS: The results illustrate the innate, adaptive and counter-regulatory immune responses to norovirus infection. Low-dose IL-2 administration induces many of the Treg responses observed during infection

Analysis of the CD1 Antigen Presenting System in Humanized SCID Mice

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34+ hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a+ cells with a dendritic morphology were present in the thymic medulla. CD1+ cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens

Identification of CD4−CD8− Double-Negative Natural Killer T Cell Precursors in the Thymus

BACKGROUND: It is well known that CD1d-restricted Valpha14 invariant natural killer T (NKT) cells are derived from cells in the CD4(+)CD8(+) double-positive (DP) population in the thymus. However, the developmental progression of NKT cells in the earlier stages remains unclear, and the possible existence of NKT cell presursors in the earlier stages than DP stage remains to be tested. PRINCIPAL FINDINGS: Here, we demonstrate that NKT cell precursors that express invariant Valpha14-Jalpha18 transcripts but devoid of surface expression of the invariant Valpha14 receptor are present in the late CD4(-)CD8(-) double-negative (DN)4 stage and have the potential to generate mature NKT cells in both in vivo and in vitro experimental conditions. Moreover, the DN4 population in CD1d knock-out (CD1dKO) mice was similar to those with an NKT cell potential in wild-type (WT) C57BL/6 (B6) mice, but failed to develop into NKT cells in vitro. However, these precursors could develop into NKT cells when co-cultured with normal thymocytes or in an in vivo experimental setting, indicating that functional NKT cell precursors are present in CD1dKO mice. CONCLUSIONS: Together, these results demonstrate that thymic DN4 fraction contains NKT cell precursors. Our findings provide new insights into the early development of NKT cells prior to surface expression of the invariant Valpha14 antigen receptor and suggest the possible alternative developmental pathway of NKT cells

Unique Interplay between Sugar and Lipid in Determining the Antigenic Potency of Bacterial Antigens for NKT Cells

Structural and biophysical studies reveal the induced-fit mechanism underlying the stringent specificity of invariant natural killer T cells for unique glycolipid antigens from the pathogen Streptococcus pneumoniae

Determination of Cellular Lipids Bound to Human CD1d Molecules

CD1 molecules are glycoproteins that present lipid antigens at the cell surface for immunological recognition by specialized populations of T lymphocytes. Prior experimental data suggest a wide variety of lipid species can bind to CD1 molecules, but little is known about the characteristics of cellular ligands that are selected for presentation. Here we have molecularly characterized lipids bound to the human CD1d isoform. Ligands were eluted from secreted CD1d molecules and separated by normal phase HPLC, then characterized by mass spectroscopy. A total of 177 lipid species were molecularly identified, comprising glycerophospholipids and sphingolipids. The glycerophospholipids included common diacylglycerol species, reduced forms known as plasmalogens, lyso-phospholipids (monoacyl species), and cardiolipins (tetraacyl species). The sphingolipids included sphingomyelins and glycosylated forms, such as the ganglioside GM3. These results demonstrate that human CD1d molecules bind a surprising diversity of lipid structures within the secretory pathway, including compounds that have been reported to play roles in cancer, autoimmune diseases, lipid signaling, and cell death

Linking Inflammation to Natural Killer T Cell Activation

Immune activation is often associated with inflammation, but inflammation's role in the expansion of antigen-specific immune responses remains unclear. This primer focuses on recent findings that show how specific natural killer T cells are activated by inflammatory messengers, thus illuminating the cellular and molecular links between immunity and inflammation

Spontaneous focal activation of invariant natural killer T (iNKT) cells in mouse liver and kidney

<p>Abstract</p> <p>Background</p> <p>Invariant natural killer T (iNKT) cells differ from other T cells by their hyperactive effector T-cell status, in addition to the expression of NK lineage receptors and semi-invariant T-cell receptors. It is generally agreed that the immune phenotype of iNKT cells is maintained by repeated activation in peripheral tissues although no explicit evidence for such iNKT cell activity <it>in vivo </it>has so far been reported.</p> <p>Results</p> <p>We used an interferon (IFN)-γ-inducible cytoplasmic protein, Irga6, as a histological marker for local IFN-γ production. Irga6 was intensely expressed in small foci of liver parenchymal cells and kidney tubular epithelium. Focal Irga6 expression was unaffected by germ-free status or loss of TLR signalling and was totally dependent on IFN-γ secreted by T cells in the centres of expression foci. These were shown to be iNKT cells by diagnostic T cell receptor usage and their activity was lost in both CD1 d and Jα-deficient mice.</p> <p>Conclusions</p> <p>This is the first report that supplies direct evidence for explicit activation events of NKT cells <it>in vivo </it>and raises issues about the triggering mechanism and consequences for immune functions in liver and kidney.</p