Starting Over: The Search for Endogenous NKT Cell Ligands

Research Highlights: Immunotherapy

Natural killer T cells are important in the pathogenesis of asthma: The many pathways to asthma

Natural Killer T (NKT) cells play an important role in the immune system as demonstrated by their involvement in tumour surveillance, infection and inflammation. Unlike conventional T cells that recognises peptide antigens when presented by the Major Histocompatibility Complex (pMHC), NKT cells recognise glycolipids, presented by a MHC class I-like molecule (CD1d-α-GalCer) to invoke an immune response. Human semi-invariant NKT cells are unique as the majority of them express a T cell receptor (TCR) bearing an invariant α-chain and restricted β-chain repertoire (Vα24-Jα18;Vβ 11). The mouse orthologue also expresses an invariant α-chain (Vα14-Jα18) but a slightly more diverse β-chain repertoire (Vβ2, Vβ7 and Vβ8.2), where Vβ8.2 is most commonly expressed. The crystal structures of the human NKT TCR-CD1d-α-GalCer and mouse orthologue complexes have provided structural insights into how the NKT TCR can recognise a lipid antigen. While it is clear that the NKT TCR docks CD1d-α-GalCer in a different conformation compared to the TCR-pMHC complexes, there was no clear information on the energetic footprint of the NKT TCR's recognition of CD1d-α-GalCer and also how the NKT TCR can differentiate between closely related α-GalCer analogues to induce a biased cytokine response. In this study, an alanine scanning mutagenesis experiment carried out on the human NKT TCR (Vα24-Jα18;Vβ11) and CD1d, as well as the use of α-GalCer analogues, demonstrated that the Jα18-encoded CDR3α loop and Vβ11-encoded CDR2β loop of the NKT TCR play a crucial role in maintaining its interactions with CD1d-α-GalCer. The minimal usage of only six residues, which are also evolutionary conserved in the mouse NKT TCR (Vα14-Jα18;Vβ8.2), explains the semi-invariant nature of the NKT TCR as well as the basis of NKT cell cross-species reactivity. Furthermore, the interactions these residues made are localised directly above the F′ pocket of CD1d, distal from the galactosyl head group of α-GalCer. The use of α-GalCer analogues that contains glycosyl head group modifications, further demonstrated the lesser energetic contribution the NKT TCR CDR1α loop plays in its interactions with the sugar head group of α-GalCer. In contrast, the NKT TCR CDR3α loop, which makes interactions with α-GalCer as well as CD1d, was determined to be the key CDR loop that is energetically important in glycolipid recognition. NKT cells can differentiate between α-GalCer analogues with small modifications on their glycosyl head group to stimulate a biased T helper (Th) cytokine response. Therefore, the ability of how the mouse Vβ8.2 NKT TCR is able to distinguish between different α-GalCer analogues was further investigated through a combination of biophysical, structural and functional experiments. These data had provided further insight into how NKT cells can recognise and differentiate between structurally similar variants of α-GalCer, also referred to as Altered Glycolipid Ligands (AGLs). The crystal structures of all five NKT TCR-CD1d-AGL complexes revealed minimal structural differences. Variations in terms of affinity and kinetics of the NKT TCR engagement onto CD1d-AGLs as well as differences in cellular responses between AGLs were observed. Modifications on the glycosyl head group of the AGLs, directly impacted NKT cell activation as well as the affinity and t1/2 of the NKT TCR recognition. Furthermore, for these glycosyl head group modified AGLs, ligand potency, as determined by the amount of cytokines produced by the NKT cells, was directly affected by the t1/2 of the NKT TCR-CD1d-AGL interaction. In addition, modifications on the acyl chain of the AGLs do not affect the NKT TCR interaction but reduced NKT cell proliferation. This indicated an alternative antigen processing and presentation pathway for these AGLs by CD1d. On the other hand, truncation of the sphingosine chain resulted in a reduction of NKT TCR affinity resulting in an induced-fit mechanism by the NKT TCR. Collectively, the minimal binding requirements of CD1d restriction as well as the molecular basis of NKT fine specificity in CD1d-AGLs recognition were elucidated.

β-Glycoglycosphingolipid-Induced Alterations of the STAT Signaling Pathways Are Dependent on CD1d and the Lipid Raft Protein Flotillin-2

Function and Therapeutic Potential of In Vitro-Expanded Natural Killer T (NKT) Cells from Patients with Hematopoietic Malignancies.

BackgroundGlioblastoma is an aggressive primary brain tumor typically resistant to currently available immunotherapies. A deeper understanding of the underlying immunoregulatory mechanisms in glioblastoma is needed to enhance future immunotherapeutic approaches....

A subset of natural killer T (NKT) cells with a specialized form of the T cell receptor (TCR) containing an invariant TCR α chain (in mice Vα14i, in humans Vα24i) that recognizes antigens presented by the major histocompatibility complex (MHC)-like molecule CD1d is involved in the immune response to microbial pathogens. Presentation of lipids or lipopeptides by the CD1 class of MHC-like molecules displayed on dendritic cells is now a recognized mechanism for the activation of T cells. However, the microbial and possibly endogenous lipids that activate these CD1d-restricted NKT cells have remained elusive. Kinjo et al . and Mattner et al . report the identification of microbial glycosphingolipid ligands that activate NKT cells. Both groups reported that Gram-negative bacteria that do not possess lipopolysaccharide (LPS), which is a Toll-like receptor (TLR) agonist, activated CD1d-restricted NKT cells both in vivo and in vitro. Both groups also identified specific glycosphingolipids from the Gram-negative bacteria that activated the NKT cells. CD1d-restricted NKT cells are unusual in that they are abundant even in the absence of prior stimulation and do not require clonal expansion. Thus, their response is most similar to an innate immune response in kinetics and in magnitude. Mattner et al . reported that the response to Gram-positive bacteria, such as Salmonella , required TLR signaling and presentation of an endogenous glycosphingolipid, isoglobotrihexosylceramide (iGb3), by dendritic cells. Thus, CD1d-restricted NKT cells appear to have two mechanisms for activation in response to microbial pathogens: an indirect mechanism that involves TLR recognition of LPS by dendritic cells, leading to the presentation of an endogenous lipid for Gram-positive bacteria, and a direct mechanism whereby the NKT cells are directly activated by microbial glycosphingolipids presented by CD1d molecules. Y. Kinjo, D. Wu, G. Kim, G.-W. Xing, M. A. Poles, D. D. Ho, M. Tsuji, K. Kawahara, C.-H. Wong, M. Kronenberg, Recognition of bacterial glycosphingolipids by natural killer T cells. Nature 434 , 520-525 (2005). [PubMed] J. Mattner, K. L. DeBord, N. Ismail, R. D. Goff, C. Cantu, III, D. Zhou, P. Saint-Mezard, V. Wang, Y. Gao, N. Yin, K. Hoebe, O. Schneewind, D. Walker, B. Beutler, L. Teyton, P. B. Savage, A. Bendelac, Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434 , 525-529 (2005). [PubMed]

Natural killer T (NKT) cells are a novel subset of T cells that express both T-cell and natural killer (NK)-cell markers. NKT cells recognize non-classical MHC class I molecule CD1d in conjunction with glycolipid antigens. Among the reported glycolipids presented by human or mouse CD1d, α-galactosylceramide (α-GalCer) shows the highest immune-stimulatory activities. α-GalCer is derived from a marine sponge natural product and has been shown to possess antitumor activity. Further studies indicate that α-GalCer can bind to CD1d and activate NKT cells through T cell receptor (TCR) recognition to produce T helper 1 (TH1) and T helper 2 (TH2) cytokines, such as interferon-γ (IFN-γ) and interleukin-4 (IL-4), respectively. Modification of α-GalCer may alter the level of stimulatory activity and/or switch the cytokine pattern released by NKT cells. Several reports have described the structural requirement for α-GalCer recognition by CD1d-restricted NKT cells, but few of them studied the effect of sugar conformation. Herein we designed and synthesized conformationally constrained α-GalCer analogues with different fatty acyl chains. The key step employs the glycosylation of exo-glycals, offering the advantage of exclusive stereoselectivity. We also evaluated their ability to activate mouse NKT cells by measuring cytokine release profiles.

Enhancement of Dendritic Cell Therapy for Malignancy by Co-Activation of CD1d Restricted NKT Cells, NK Cells, and Antigen CTL.

Natural Killer T (NKT) cells are a lipid-antigen reactive T cell subset that is restricted to the antigen presenting molecule CD1d. They possess diverse functional properties that contribute to inflammatory and regulatory immune responses. The most studied lipid antigen target for these T cells is α-galactosylceramide (αGC). The commensal organism Bacteroides fragilis (B. fragilis) produces several forms of αGC, but conflicting information exists about the influence of these lipids on NKT cells. Herein, we report the total synthesis of a major form of αGC from B. fragilis (Bf αGC), and several analogues thereof. We confirm the T cell receptor (TCR)-mediated recognition of these glycolipids by mouse and human NKT cells. Despite the natural structure of Bf αGC containing lipid branching that limits potency, we demonstrate that Bf αGC drives mouse NKT cells to proliferate and differentiate into producers of the immunoregulatory cytokine, interleukin-10 (IL-10). These Bf αGC-experienced NKT cells display regulatory function by inhibiting the expansion of naïve NKT cells upon subsequent exposure to this antigen. Moreover, this regulatory activity impacts more than just NKT cells, as demonstrated by the NKT cell-mediated inhibition of antigen-stimulated mucosal-associated invariant T (MAIT) cells (a T cell subset restricted to a different antigen presenting molecule, MR1). These findings reveal that B. fragilis-derived NKT cell agonists may have broad immunoregulatory activity, providing insight into the mechanisms influencing immune tolerance to commensal bacteria and highlighting a potential means to manipulate NKT cell function for therapeutic benefit.

Natural killer T cells and rheumatoid arthritis: friend or foe?

Are NKT cells a useful predictor of COVID-19 severity?

Current tumor therapies, including immunotherapies, focus on passive eradication or at least reduction of the tumor mass. However, cancer patients quite often suffer from tumor relapse or metastasis after such treatments. To overcome these problems, we have developed a natural killer T (NKT) cell-targeted immunotherapy focusing on active engagement of the patient’s immune system, but not directly targeting the tumor cells themselves. NKT cells express an invariant antigen receptor α chain encoded by Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans and recognize glycolipid ligand in conjunction with a monomorphic CD1d molecule. The NKT cells play a pivotal role in the orchestration of antitumor immune responses by mediating adjuvant effects that activate various antitumor effector cells of both innate and adaptive immune systems and also aid in establishing a long-term memory response. Here, we established NKT cell-targeted therapy using a newly discovered NKT cell glycolipid ligand, RK, which has a stronger capacity to stimulate both human and mouse NKT cells compared to previous NKT cell ligand. Moreover, RK mediates strong adjuvant effects in activating various effector cell types and establishes long-term memory responses, resulting in the continuous attack on the tumor that confers long-lasting and potent antitumor effects. Since the NKT cell ligand presented by the monomorphic CD1d can be used for all humans irrespective of HLA types, and also because NKT cell-targeted therapy does not directly target tumor cells, this therapy can potentially be applied to all cancer patients and any tumor types.

Author SummaryA central tenet of immunology is that cellular responses that protect us from pathogens result from molecular recognition of foreign compounds (antigens). The role of self-antigens in immune activation is less clear. We show here that an endogenous lipid called lyso-phosphatidylcholine (LPC) is recognized as an antigen by a subpopulation of human T lymphocytes, called natural killer T (NKT) cells, and specifically by the best-studied subgroup of these cells known as invariant NKT (iNKT) cells. NKT cells have attracted the interest of immunologists because they can potently influence the outcome of diverse immune responses; for example, they can promote bacterial clearance and tumor rejection, and they can also quell autoimmune disease pathology. Previous studies indicated that NKT cells are activated by self-antigens, but the identity of the relevant compounds remained unclear. Our finding that LPC is a self-antigen for iNKT cells suggests that these lymphocytes are attuned to highly conserved lipid signaling pathways that are fundamental to normal physiological processes and are markedly up-regulated during inflammation. Thus, these results provide a new molecular basis for understanding how iNKT cells contribute to a wide variety of immune responses.

Natural killer T (NKT) cells rapidly respond to antigenic stimulation with cytokine production and direct cytotoxicity. These innate-like characteristics arise from their differentiation into mature effector cells during thymic development. A subset of mature NKT cells remain thymic resident, but their activation and function remain poorly understood. We examined the roles of CD28 and CTLA-4 in driving the activation of thymic resident NKT cells. In contrast to studies with peripheral NKT cells, the proliferation of thymic NKT cells was significantly impaired when CD28 engagement was blocked, but unaffected by CTLA-4 activation or blockade. Within NKT subsets, however, stage 3 NKT cells, marked by higher NK1.1 expression, were significantly more sensitive to the loss of CD28 signals compared to NK1.1− stage 2 NKT cells. In good agreement, CD28 blockade suppressed NKT cell cytokine secretion, lowering the ratio of IFN-γ:IL-4 production by NK1.1+ NKT cells. Intriguingly, the activation-dependent upregulation of the master transcription factor PLZF did not require CD28-costimulation in either of the thymic NKT subsets, underlining a dichotomy between requirements for early activation vs subsequent proliferation and effector function by these cells. Collectively, our studies demonstrate the ability of CD28 co-stimulation to fine tune subset-specific responses by thymic resident NKT cells and contextually shape the milieu in this primary lymphoid organ.