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Find video protocols related to scientific articles indexed in Pubmed.
Effective functional maturation of invariant natural killer T cells is constrained by negative selection and T-cell antigen receptor affinity.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-16-2013
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The self-reactivity of their T-cell antigen receptor (TCR) is thought to contribute to the development of immune regulatory cells, such as invariant NK T cells (iNKT). In the mouse, iNKT cells express TCRs composed of a unique V?14-J?18 rearrangement and recognize lipid antigens presented by CD1d molecules. We created mice expressing a transgenic TCR-? chain that confers high affinity for self-lipid/CD1d complexes when randomly paired with the mouse iNKT V?14-J?18 rearrangement to study their development. We show that although iNKT cells undergo agonist selection, their development is also shaped by negative selection in vivo. In addition, iNKT cells that avoid negative selection in these mice express natural sequence variants of the canonical TCR-? and decreased affinity for self/CD1d. However, limiting the affinity of the iNKT TCRs for "self" leads to inefficient Egr2 induction, poor expression of the iNKT lineage-specific zinc-finger transcription factor PLZF, inadequate proliferation of iNKT cell precursors, defects in trafficking, and impaired effector functions. Thus, proper development of fully functional iNKT cells is constrained by a limited range of TCR affinity that plays a key role in triggering the iNKT cell-differentiation pathway. These results provide a direct link between the affinity of the TCR expressed by T-cell precursors for self-antigens and the proper development of a unique population of lymphocytes essential to immune responses.
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Nod1 and Nod2 enhance TLR-mediated invariant NKT cell activation during bacterial infection.
J. Immunol.
PUBLISHED: 10-25-2013
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Invariant NKT (iNKT) cells act at the crossroad between innate and adaptive immunity and are important players in the defense against microbial pathogens. iNKT cells can detect pathogens that trigger innate receptors (e.g., TLRs, Rig-I, Dectin-1) within APCs, with the consequential induction of CD1d-mediated Ag presentation and release of proinflammatory cytokines. We show that the cytosolic peptidoglycan-sensing receptors Nod1 and Nod2 are necessary for optimal IFN-? production by iNKT cells, as well as NK cells. In the absence of Nod1 and Nod2, iNKT cells had a blunted IFN-? response following infection by Salmonella enterica serovar Typhimurium and Listeria monocytogenes. For Gram-negative bacteria, we reveal a synergy between Nod1/2 and TLR4 in dendritic cells that potentiates IL-12 production and, ultimately, activates iNKT cells. These findings suggest that multiple innate pathways can cooperate to regulate iNKT cell activation during bacterial infection.
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MAIT cell recognition of MR1 on bacterially infected and uninfected cells.
PLoS ONE
PUBLISHED: 01-14-2013
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Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant ?? TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.
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Colonic inflammation in mice is improved by cigarette smoke through iNKT cells recruitment.
PLoS ONE
PUBLISHED: 01-01-2013
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Cigarette smoke (CS) protects against intestinal inflammation during ulcerative colitis. Immunoregulatory mechanisms sustaining this effect remain unknown. The aim of this study was to assess the effects of CS on experimental colitis and to characterize the intestinal inflammatory response at the cellular and molecular levels. Using the InExpose® System, a smoking device accurately reproducing human smoking habit, we pre-exposed C57BL/6 mice for 2 weeks to CS, and then we induced colitis by administration of dextran sodium sulfate (DSS). This system allowed us to demonstrate that CS exposure improved colonic inflammation (significant decrease in clinical score, body weight loss and weight/length colonic ratio). This improvement was associated with a significant decrease in colonic proinflammatory Th1/Th17 cytokine expression, as compared to unexposed mice (TNF (p=0.0169), IFN? (p<0.0001), and IL-17 (p=0.0008)). Smoke exposure also induced an increased expression of IL-10 mRNA (p=0.0035) and a marked recruitment of iNKT (invariant Natural Killer T; CD45+ TCR?+ CD1d tetramer+) cells in the colon of DSS-untreated mice. Demonstration of the role of iNKT cells in CS-dependent colitis improvement was performed using two different strains of NKT cells deficient mice. Indeed, in J?18KO and CD1dKO animals, CS exposure failed to induce significant regulation of DSS-induced colitis both at the clinical and molecular levels. Thus, our study demonstrates that iNKT cells are pivotal actors in the CS-dependent protection of the colon. These results highlight the role of intestinal iNKT lymphocytes and their responsiveness to environmental stimuli. Targeting iNKT cells would represent a new therapeutic way for inflammatory bowel diseases.
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V?2 natural killer T cell antigen receptor-mediated recognition of CD1d-glycolipid antigen.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-07-2011
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Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR ?-chain is invariant, NKT TCR V? exhibits greater diversity, with one (V?11) and three (V?8, V?7, and V?2) V? chains in humans and mice, respectively. With the exception of the V?2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2? that are critical for CD1d binding. Thus, how V?2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2?-encoded tyrosine residues, we show that the V?2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the V?8.2 and V?7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR ?-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the V?2 NKT TCR and the V?8.2 and V?7 NKT TCRs, with the V?2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the V?2 NKT TCR-CD1d-?GalCer complex, the CDR2? loop mediated fewer contacts with CD1d, whereas the CDR1? and CDR3? loops contacted CD1d to a much greater extent compared with most V?11, V?8.2, and V?7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR ?-chain of the V?2 NKT TCR that enables CD1d-Ag ligation.
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Recognition of ?-linked self glycolipids mediated by natural killer T cell antigen receptors.
Nat. Immunol.
PUBLISHED: 04-20-2011
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The most potent foreign antigens for natural killer T cells (NKT cells) are ?-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct ?-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated ?-linked agonists ?-galactosylceramide (?-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the ?-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the ?-linked self ligands to resemble the conformation of foreign ?-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to ?-linked antigens.
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A molecular basis for NKT cell recognition of CD1d-self-antigen.
Immunity
PUBLISHED: 01-25-2011
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The antigen receptor for natural killer T cells (NKT TCR) binds CD1d-restricted microbial and self-lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags) and report the 2.3 Å resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self- and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline-encoded CDR3? loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d-restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.
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T cell receptor CDR2 beta and CDR3 beta loops collaborate functionally to shape the iNKT cell repertoire.
Immunity
PUBLISHED: 04-21-2009
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Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.
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Differential recognition of CD1d-alpha-galactosyl ceramide by the V beta 8.2 and V beta 7 semi-invariant NKT T cell receptors.
Immunity
PUBLISHED: 04-01-2009
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The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR alpha chain is typically invariant, the beta chain expression is more diverse, where three V beta chains are commonly expressed in mice. We report the structures of V alpha 14-V beta 8.2 and V alpha 14-V beta 7 NKT TCRs in complex with CD1d-alpha-galactosylceramide (alpha-GalCer) and the 2.5 A structure of the human NKT TCR-CD1d-alpha-GalCer complex. Both V beta 8.2 and V beta 7 NKT TCRs and the human NKT TCR ligated CD1d-alpha-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V beta domains of the V beta 8.2 and V beta 7 NKT TCR-CD1d complexes resulted in altered TCR beta-CD1d-mediated contacts and modulated recognition mediated by the invariant alpha chain. Mutagenesis studies revealed the differing contributions of V beta 8.2 and V beta 7 residues within the CDR2 beta loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V beta usage in NKT cells.
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Role of invariant NK T lymphocytes in immune responses to CpG oligodeoxynucleotides.
J. Immunol.
PUBLISHED: 02-10-2009
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Unmethylated CpG oligodeoxynucleotides (ODNs), by activating cells of the innate immune system, such as dendritic cells and NK cells, are potent adjuvants for type 1 immune responses. In the present study, we aimed to investigate the role of invariant NKT (iNKT) cells, a subset of lipid-reactive innate lymphocytes, in CpG ODN-induced innate and acquired type 1 responses. Our data show that, in response to the CpG ODN type B 1826, splenic and hepatic iNKT cells become activated and produce IFN-gamma, but not IL-4, both in vitro and in vivo. This Th1 bias is independent from the Ag-presenting molecule CD1d and strongly requires IL-12, at least in vitro. We also report that iNKT cell activation, in response to CpG ODN type B, results in the transactivation of NK cells. To address the potential role of iNKT cells in type 1 innate immunity induced by CpG ODN, a murine model of malignant melanoma was used. We show that CpG ODN type B protects mice against B16F10-induced lung metastasis in wild-type mice, but in a less efficient manner in iNKT cell-deficient animals. Finally, we report that immunization of wild-type mice with CpG ODN type B plus keyhole limpet hemocyanin biases the immune response toward a Th1 direction, an effect strongly mediated by iNKT cells. We conclude that iNKT cells amplify the innate and acquired response to CpG ODN type B, with potentially important consequences for the regulation of immune responses.
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Recognition of CD1d-sulfatide mediated by a type II natural killer T cell antigen receptor.
Nat. Immunol.
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Natural killer T cells (NKT cells) are divided into type I and type II subsets on the basis of differences in their T cell antigen receptor (TCR) repertoire and CD1d-antigen specificity. Although the mode by which type I NKT cell TCRs recognize CD1d-antigen has been established, how type II NKT cell TCRs engage CD1d-antigen is unknown. Here we provide a basis for how a type II NKT cell TCR, XV19, recognized CD1d-sulfatide. The XV19 TCR bound orthogonally above the A pocket of CD1d, in contrast to the parallel docking of type I NKT cell TCRs over the F pocket of CD1d. At the XV19 TCR-CD1d-sulfatide interface, the TCR? and TCR? chains sat centrally on CD1d, where the malleable CDR3 loops dominated interactions with CD1d-sulfatide. Accordingly, we highlight the diverse mechanisms by which NKT cell TCRs can bind CD1d and account for the distinct antigen specificity of type II NKT cells.
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Strategy of lipid recognition by invariant natural killer T cells: one for all and all for one.
Immunology
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Invariant natural killer T (iNKT) cells are evolutionarily conserved lipid-reactive T cells that bridge innate and adaptive immune responses. Despite a relatively restricted T-cell receptor (TCR) diversity, these cells respond to a variety of structurally distinct foreign (i.e. microbial or synthetic) as well as host-derived (self-) lipid antigens presented by the CD1d molecule. These multi-tasking lymphocytes are among the first responders in immunity, and produce an impressive array of cytokines and chemokines that can tailor the ensuing immune response. Accordingly, iNKT cells play important functions in autoimmune diseases, cancer, infection and inflammation. These properties make iNKT cells appealing targets in immune-based therapies. Yet, much has to be learned on the mechanisms that allow iNKT cells to produce polarized responses. Responses of iNKT cells are influenced by the direct signals perceived by the cells through their TCRs, as well as by indirect co-stimulatory (and potentially co-inhibitory) cues that they receive from antigen-presenting cells or the local milieu. A decade ago, biochemists and immunologists have started to describe synthetic lipid agonists with cytokine skewing potential, paving a new research avenue in the iNKT cell field. Yet how iNKT cells translate various antigenic signals into distinct functional responses has remained obscure. Recent findings have revealed a unique and innate mode of lipid recognition by iNKT cells, and suggest that both the lipid antigen presented and the diversity of the TCR modulate the strength of CD1d-iNKT TCR interactions. In this review, we focus on novel discoveries on lipid recognition by iNKT cells, and how these findings may help us to design effective strategies to steer iNKT cell responses for immune intervention.
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T cells interact with T cells via CD40-CD154 to promote autoimmunity in type 1 diabetes.
Eur. J. Immunol.
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We have investigated the role of CD40 signaling in islet-reactive, diabetogenic CD4(+) Th1 T-cell clones. Using multispectral flow cytometry, we showed that CD40 and CD154 are co-expressed and form complexes on the surface of activated T cells. We also demonstrate that activated Tcells can transactivate CD4(+) CD40(+) T cells through the CD40-CD154 pathway. To investigate the role of CD40 signaling on Th1 cells, we used the diabetogenic clone BDC-5.2.9 retrovirally transduced with a truncated form of the CD40 molecule to produce a CD40 dominant-negative T-cell clone. Upon challenge with antigen in vitro, the production of IFN-#x003B3; by BDC-5.2.9 CD40DN was greatly reduced and, in vivo, the dominant-negative variant was unable to induce diabetes. Transduction with the CD40DN vector was also effective in preventing transfer of disease by primary NOD CD4(+) T cells. Ex vivo analysis of pancreatic infiltrates after transfer of BDC-5.2.9 CD40DN cells revealed an overall reduction of cell numbers and cytokine production by both T cells and macrophages. These data indicate that CD40 is an important signaling molecule on autoreactive CD4(+) T cells and contributes to their pathogenic effector function.
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