Motility and tumor infiltration are key aspects of invariant natural killer T cell anti-tumor function.

Dysfunction of invariant natural killer T (iNKT) cells contributes to immune resistance of tumors. Most mechanistic studies focus on their static functional status before or after activation, not considering motility as an important characteristic for antigen scanning and thus anti-tumor capability. Here we show via intravital imaging, that impaired motility of iNKT cells and their exclusion from tumors both contribute to the diminished anti-tumor iNKT cell response. Mechanistically, CD1d, expressed on macrophages, interferes with tumor infiltration of iNKT cells and iNKT-DC interactions but does not influence their intratumoral motility. VCAM1, expressed by cancer cells, restricts iNKT cell motility and inhibits their antigen scanning and activation by DCs via reducing CDC42 expression. Blocking VCAM1-CD49d signaling improves motility and activation of intratumoral iNKT cells, and consequently augments their anti-tumor function. Interference with macrophage-iNKT cell interactions further enhances the anti-tumor capability of iNKT cells. Thus, our findings provide a direction to enhance the efficacy of iNKT cell-based immunotherapy via motility regulation.

Intrinsic factors and CD1d1 but not CD1d2 expression levels control invariant natural killer T cell subset differentiation.

Invariant natural killer T (NKT) cell subsets are defined based on their cytokine-production profiles and transcription factors. Their distribution is different in C57BL/6 (B6) and BALB/c mice, with a bias for NKT1 and NKT2/NKT17 subsets, respectively. Here, we show that the non-classical class I-like major histocompatibility complex CD1 molecules CD1d2, expressed in BALB/c and not in B6 mice, could not account for this difference. We find however that NKT cell subset distribution is intrinsic to bone marrow derived NKT cells, regardless of syngeneic CD1d-ligand recognition, and that multiple intrinsic factors are likely involved. Finally, we find that CD1d expression levels in combination with T cell antigen receptor signal strength could also influence NKT cell distribution and function. Overall, this study indicates that CD1d-mediated TCR signals and other intrinsic signals integrate to influence strain-specific NKT cell differentiation programs and subset distributions.

Do antigen-presenting CD1a, CD1b, CD1c, and CD1d molecules bind different self-lipids?

Humans express four different lipid antigen-presenting molecules, CD1a, CD1b, CD1c, and CD1d, that are differentially expressed on antigen-presenting cells and which recycle through different endosomal compartments. Huang et al. now answer the question on whether the four CD1 isoforms selectively bind certain lipids.

Lipids regulate peripheral serotonin release via gut CD1d.

The crosstalk between the immune and neuroendocrine systems is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones and neurotransmitters release in response to environmental cues, such as self-lipids and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promoted peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. We also demonstrated that inhibitory sphingolipids from symbiotic microbe Bacteroides fragilis represses 5-HT release. Mechanistically, CD1d ligation on EC cells transduced a signal and restrained potassium conductance through activation of protein tyrosine kinase Pyk2, leading to calcium influx and 5-HT secretion. Together, our data reveal that by engaging with iNKT cells, gut chemosensory cells selectively perceive lipid antigens via CD1d to control 5-HT release, modulating intestinal and systemic homeostasis.

Advanced omics techniques shed light on CD1d-mediated lipid antigen presentation to iNKT cells.

Invariant natural killer T cells (iNKT cells) can be activated through binding antigenic lipid/CD1d complexes to their TCR. Antigenic lipids are processed, loaded, and displayed in complex with CD1d by lipid antigen presenting cells (LAPCs). The mechanism of lipid antigen presentation via CD1d is highly conserved with recent work showing adipocytes are LAPCs that, besides having a role in lipid storage, can activate iNKT cells and play an important role in systemic metabolic disease. Recent studies shed light on parameters potentially dictating cytokine output and how obesity-associated metabolic disease may affect such parameters. By following a lipid antigen's journey, we identify five key areas which may dictate cytokine skew: co-stimulation, structural properties of the lipid antigen, stability of lipid antigen/CD1d complexes, intracellular and extracellular pH, and intracellular and extracellular lipid environment. Recent publications indicate that the combination of advanced omics-type approaches and machine learning may be a fruitful way to interconnect these 5 areas, with the ultimate goal to provide new insights for therapeutic exploration.

Chemical Synthesis and Molecular Interaction Analysis of α-Galactosyl Ceramide Derivatives as CD1d Ligands.

CD1d is a non-classical major histocompatibility complex (MHC) protein, responsible for lipid antigen presentation, which presents lipids to natural killer T (NKT) cells. Various CD1d lipid ligands have been reported, including microbial and endogenous glycolipids/phospholipids. Among them, an α-galactosylceramide (α-GalCer), a representative CD1d ligand, is one of the most potent ligands and its derivatives have been developed. In this chapter, the chemistry of α-GalCer and its derivatives are described with an emphasis on their chemical syntheses and molecular interaction analysis with CD1d are described.

CD1d-dependent rewiring of lipid metabolism in macrophages regulates innate immune responses.

Alterations in cellular metabolism underpin macrophage activation, yet little is known regarding how key immunological molecules regulate metabolic programs in macrophages. Here we uncover a function for the antigen presenting molecule CD1d in the control of lipid metabolism. We show that CD1d-deficient macrophages exhibit a metabolic reprogramming, with a downregulation of lipid metabolic pathways and an increase in exogenous lipid import. This metabolic rewiring primes macrophages for enhanced responses to innate signals, as CD1d-KO cells show higher signalling and cytokine secretion upon Toll-like receptor stimulation. Mechanistically, CD1d modulates lipid import by controlling the internalization of the lipid transporter CD36, while blocking lipid uptake through CD36 restores metabolic and immune responses in macrophages. Thus, our data reveal CD1d as a key regulator of an inflammatory-metabolic circuit in macrophages, independent of its function in the control of T cell responses.

Harnessing invariant natural killer T cells to control pathological inflammation.

Invariant natural killer T (iNKT) cells are innate T cells that are recognized for their potent immune modulatory functions. Over the last three decades, research in murine models and human observational studies have revealed that iNKT cells can act to limit inflammatory pathology in a variety of settings. Since iNKT cells are multi-functional and can promote inflammation in some contexts, understanding the mechanistic basis for their anti-inflammatory effects is critical for effectively harnessing them for clinical use. Two contrasting mechanisms have emerged to explain the anti-inflammatory activity of iNKT cells: that they drive suppressive pathways mediated by other regulatory cells, and that they may cytolytically eliminate antigen presenting cells that promote excessive inflammatory responses. How these activities are controlled and separated from their pro-inflammatory functions remains a central question. Murine iNKT cells can be divided into four functional lineages that have either pro-inflammatory (NKT1, NKT17) or anti-inflammatory (NKT2, NKT10) cytokine profiles. However, in humans these subsets are not clearly evident, and instead most iNKT cells that are CD4+ appear oriented towards polyfunctional (TH0) cytokine production, while CD4- iNKT cells appear more predisposed towards cytolytic activity. Additionally, structurally distinct antigens have been shown to induce TH1- or TH2-biased responses by iNKT cells in murine models, but human iNKT cells may respond to differing levels of TCR stimulation in a way that does not neatly separate TH1 and TH2 cytokine production. We discuss the implications of these differences for translational efforts focused on the anti-inflammatory activity of iNKT cells.

Sortase A-Cleavable CD1d Identifies Sphingomyelins as Major Class of CD1d-Associated Lipids.

CD1d is an atypical MHC class I molecule which binds endogenous and exogenous lipids and can activate natural killer T (NKT) cells through the presentation of lipid antigens. CD1d surveys different cellular compartments including the secretory and the endolysosomal pathway and broadly binds lipids through its two hydrophobic pockets. Purification of the transmembrane protein CD1d for the analysis of bound lipids is technically challenging as the use of detergents releases CD1d-bound lipids. To address these challenges, we have developed a novel approach based on Sortase A-dependent enzymatic release of CD1d at the cell surface of live mammalian cells, which allows for single step release and affinity tagging of CD1d for shotgun lipidomics. Using this system, we demonstrate that CD1d carrying the Sortase A recognition motif shows unimpaired subcellular trafficking through the secretory and endolysosomal pathway and is able to load lipids in these compartments and present them to NKT cells. Comprehensive shotgun lipidomics demonstrated that the spectrum and abundance of CD1d-associated lipids is not representative of the total cellular lipidome but rather characterized by preferential binding to long chain sphingolipids and glycerophospholipids. As such, sphingomyelin species recently identified as critical negative regulators of NKT cell activation, represented the vast majority of endogenous CD1d-associated lipids. Moreover, we observed that inhibition of endolysosomal trafficking of CD1d surprisingly did not affect the spectrum of CD1d-bound lipids, suggesting that the majority of endogenous CD1d-associated lipids load onto CD1d in the secretory rather than the endolysosomal pathway. In conclusion, we present a novel system for the analysis of CD1d-bound lipids in mammalian cells and provide new insight into the spectrum of CD1d-associated lipids, with important functional implications for NKT cell activation.

Re-programming mouse liver-resident invariant natural killer T cells for suppressing hepatic and diabetogenic autoimmunity.

Invariant NKT (iNKT) cells comprise a heterogeneous group of non-circulating, tissue-resident T lymphocytes that recognize glycolipids, including alpha-galactosylceramide (αGalCer), in the context of CD1d, but whether peripheral iNKT cell subsets are terminally differentiated remains unclear. Here we show that mouse and human liver-resident αGalCer/CD1d-binding iNKTs largely correspond to a novel Zbtb16+Tbx21+Gata3+MaflowRorc- subset that exhibits profound transcriptional, phenotypic and functional plasticity. Repetitive in vivo encounters of these liver iNKT (LiNKT) cells with intravenously delivered αGalCer/CD1d-coated nanoparticles (NP) trigger their differentiation into immunoregulatory, IL-10+IL-21-producing Zbtb16highMafhighTbx21+Gata3+Rorc- cells, termed LiNKTR1, expressing a T regulatory type 1 (TR1)-like transcriptional signature. This response is LiNKT-specific, since neither lung nor splenic tissue-resident iNKT cells from αGalCer/CD1d-NP-treated mice produce IL-10 or IL-21. Additionally, these LiNKTR1 cells suppress autoantigen presentation, and recognize CD1d expressed on conventional B cells to induce IL-10+IL-35-producing regulatory B (Breg) cells, leading to the suppression of liver and pancreas autoimmunity. Our results thus suggest that LiNKT cells are plastic for further functional diversification, with such plasticity potentially targetable for suppressing tissue-specific inflammatory phenomena.

CD1d expression demarcates CDX4+ hemogenic mesoderm with definitive hematopoietic potential.

To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4hi expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications.

From the banal to the bizarre: unravelling immune recognition and response to microbial lipids.

Microbes produce a rich array of lipidic species that through their location in the cell wall and ability to mingle with host lipids represent a privileged class of immune-active molecules. Lipid-sensing immunity recognizes microbial lipids from pathogens and commensals causing immune responses. Yet microbial lipids are often heterogeneous, in limited supply and in some cases their structures are incompletely defined. Total synthesis can assist in structural determination, overcome supply issues, and provide access to high-purity, homogeneous samples and analogues. This account highlights synthetic approaches to lipidic species from pathogenic and commensal bacteria and fungi that have supported immunological studies involving lipid sensing through the pattern recognition receptor Mincle and cell-mediated immunity through the CD1-T cell axis.

Cancer immunotherapy using artificial adjuvant vector cells to deliver NY-ESO-1 antigen to dendritic cells in situ.

NY-ESO-1 is a cancer/testis antigen expressed in various cancer types. However, the induction of NY-ESO-1-specific CTLs through vaccines is somewhat difficult. Thus, we developed a new type of artificial adjuvant vector cell (aAVC-NY-ESO-1) expressing a CD1d-NKT cell ligand complex and a tumor-associated antigen, NY-ESO-1. First, we determined the activation of invariant natural killer T (iNKT) and natural killer (NK) cell responses by aAVC-NY-ESO-1. We then showed that the NY-ESO-1-specific CTL response was successfully elicited through aAVC-NY-ESO-1 therapy. After injection of aAVC-NY-ESO-1, we found that dendritic cells (DCs) in situ expressed high levels of costimulatory molecules and produced interleukn-12 (IL-12), indicating that DCs undergo maturation in vivo. Furthermore, the NY-ESO-1 antigen from aAVC-NY-ESO-1 was delivered to the DCs in vivo, and it was presented on MHC class I molecules. The cross-presentation of the NY-ESO-1 antigen was absent in conventional DC-deficient mice, suggesting a host DC-mediated CTL response. Thus, this strategy helps generate sufficient CD8+ NY-ESO-1-specific CTLs along with iNKT and NK cell activation, resulting in a strong antitumor effect. Furthermore, we established a human DC-transferred NOD/Shi-scid/IL-2γcnull immunodeficient mouse model and showed that the NY-ESO-1 antigen from aAVC-NY-ESO-1 was cross-presented to antigen-specific CTLs through human DCs. Taken together, these data suggest that aAVC-NY-ESO-1 has potential for harnessing innate and adaptive immunity against NY-ESO-1-expressing malignancies.

Identification of liver-specific CD24+ invariant NK T cells with low granzyme B production and high proliferative capacity.

Invariant NK T (iNKT) cells are innate-like lymphocytes that can recognize the lipid Ag presented by MHC I like molecule CD1d. Distinct tissue distribution of iNKT cells subsets implies a contribution of these subsets to their related tissue regional immunity. iNKT cells are enriched in liver, an organ with unique immunological properties. Whether liver-specific iNKT cells exist and dedicate to the liver immunity remains elusive. Here, a liver-specific CD24+ iNKT subset is shown. Hepatic CD24+ iNKT cells show higher levels of proliferation, glucose metabolism, and mTOR activity comparing to CD24- iNKT cells. Although CD24+ iNKT cells and CD24- iNKT cells in the liver produce similar amounts of cytokines, the hepatic CD24+ iNKT cells exhibit lower granzyme B production. These liver-specific CD24+ iNKT cells are derived from thymus and differentiate into CD24+ iNKT in the liver microenvironment. Moreover, liver microenvironment induces the formation of CD24+ conventional T cells as well, and these cells exhibit higher proliferation ability but lower granzyme B production in comparison with CD24- T cells. The results propose that liver microenvironment might induce the generation of liver-specific iNKT subset that might play an important role in maintaining liver homeostasis.

Parity-induced changes to mammary epithelial cells control NKT cell expansion and mammary oncogenesis.

Pregnancy reprograms mammary epithelial cells (MECs) to control their responses to pregnancy hormone re-exposure and carcinoma progression. However, the influence of pregnancy on the mammary microenvironment is less clear. Here, we used single-cell RNA sequencing to profile the composition of epithelial and non-epithelial cells in mammary tissue from nulliparous and parous female mice. Our analysis indicates an expansion of γδ natural killer T-like immune cells (NKTs) following pregnancy and upregulation of immune signaling molecules in post-pregnancy MECs. We show that expansion of NKTs following pregnancy is due to elevated expression of the antigen-presenting molecule CD1d on MECs. Loss of CD1d expression on post-pregnancy MECs, or overall lack of activated NKTs, results in mammary oncogenesis. Collectively, our findings illustrate how pregnancy-induced changes modulate the communication between MECs and the immune microenvironment and establish a causal link between pregnancy, the immune microenvironment, and mammary oncogenesis.

Distinct CD4-CD8- (Double-Negative) Memory T-Cell Subpopulations Are Associated With Indeterminate and Cardiac Clinical Forms of Chagas Disease.

CD4-CD8- (double-negative, DN) T cells are critical orchestrators of the cytokine network associated with the pathogenic inflammatory response in one of the deadliest cardiomyopathies known, Chagas heart disease, which is caused by Trypanosoma cruzi infection. Here, studying the distribution, activation status, and cytokine expression of memory DN T-cell subpopulations in Chagas disease patients without cardiac involvement (indeterminate form-IND) or with Chagas cardiomyopathy (CARD), we report that while IND patients displayed a higher frequency of central memory, CARD had a high frequency of effector memory DN T cells. In addition, central memory DN T cells from IND displayed a balanced cytokine profile, characterized by the concomitant expression of IFN-γ and IL-10, which was not observed in effector memory DN T cells from CARD. Supporting potential clinical relevance, we found that the frequency of central memory DN T cells was associated with indicators of better ventricular function, while the frequency of effector memory DN T cells was not. Importantly, decreasing CD1d-mediated activation of DN T cells led to an increase in IL-10 expression by effector memory DN T cells from CARD, restoring a balanced profile similar to that observed in the protective central memory DN T cells. Targeting the activation of effector memory DN T cells may emerge as a strategy to control inflammation in Chagas cardiomyopathy and potentially in other inflammatory diseases where these cells play a key role.

Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d.

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αß T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αßTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

The lysophospholipid-binding molecule CD1D is not required for the alloimmunization response to fresh or stored RBCs in mice despite RBC storage driving alterations in lysophospholipids.

BACKGROUND: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses. STUDY DESIGN AND METHODS: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization. RESULTS: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG. CONCLUSION: Although storage of RBCs leads to alteration of several lysophospholipids known to be capable of binding CD1D, storage-induced RBC alloimmunization responses are not impacted by recipient CD1D deficiency.

Targeting Natural Killer T Cells in Solid Malignancies.

Natural killer T (NKT) cells are a unique subset of lymphocytes that recognize lipid antigens in the context of the non-classical class I MHC molecule, CD1d, and serve as a link between the innate and adaptive immune system through their expeditious release of cytokines. Whereas NKT have well-established roles in mitigating a number of human diseases, herein, we focus on their role in cancer. NKT cells have been shown to directly and indirectly mediate anti-tumor immunity and manipulating their effector functions can have therapeutic significances in treatment of cancer. In this review, we highlight several therapeutic strategies that have been used to harness the effector functions of NKT cells to target different types of solid tumors. We also discuss several barriers to the successful utilization of NKT cells and summarize effective strategies being developed to harness the unique strengths of this potent population of T cells. Collectively, studies investigating the therapeutic potential of NKT cells serve not only to advance our understanding of this powerful immune cell subset, but also pave the way for future treatments focused on the modulation of NKT cell responses to enhance cancer immunotherapy.