A Glycolipid-Peptide-Hapten Tricomponent Conjugate Vaccine Generates Durable Antihapten Antibody Responses in Mice.

Eliciting an antihapten antibody response to vaccination typically requires the use of constructs where multiple copies of the hapten are covalently attached to a larger carrier molecule. The carrier is required to elicit T cell help via presentation of peptide epitopes on major histocompatibility complex (MHC) class II molecules; as such, attachment to full-sized proteins, alone or in a complex, is generally used to account for the significant MHC diversity in humans. While such carrier-based vaccines have proven extremely successful, particularly in protecting against bacterial diseases, they can be challenging to manufacture, and repeated use can be compromised by pre-existing immunity against the carrier. One approach to reducing these complications is to recruit help from type I natural killer T (NKT) cells, which exhibit limited diversity in their antigen receptors and respond to glycolipid antigens presented by the highly conserved presenting molecule CD1d. Synthetic vaccines for universal use can, therefore, be prepared by conjugating haptens to an NKT cell agonist such as α-galactosylceramide (αGalCer, KRN7000). An additional advantage is that the quality of NKT cell help is sufficient to overcome the need for an extra immune adjuvant. However, while initial studies with αGalCer-hapten conjugate vaccines report strong and rapid antihapten antibody responses, they can fail to generate lasting memory. Here, we show that antibody responses to the hapten 4-hydoxy-3-nitrophenyl acetyl (NP) can be improved through additional attachment of a fusion peptide containing a promiscuous helper T cell epitope (Pan DR epitope, PADRE) that binds diverse MHC class II molecules. Such αGalCer-hapten-peptide tricomponent vaccines generate strong and sustained anti-NP antibody titers with increased hapten affinity compared to vaccines without the helper epitope. The tricomponent vaccine platform is therefore suitable for further exploration in the pursuit of efficacious antihapten immunotherapies.

Self-Adjuvanting Lipoprotein Conjugate αGalCer-RBD Induces Potent Immunity against SARS-CoV-2 and its Variants of Concern.

Safe and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are the best approach to successfully combat the COVID-19 pandemic. The receptor-binding domain (RBD) of the viral spike protein is a major target to develop candidate vaccines. α-Galactosylceramide (αGalCer), a potent invariant natural killer T cell (iNKT) agonist, was site-specifically conjugated to the N-terminus of the RBD to form an adjuvant-protein conjugate, which was anchored on the liposome surface. This is the first time that an iNKT cell agonist was conjugated to the protein antigen. Compared to the unconjugated RBD/αGalCer mixture, the αGalCer-RBD conjugate induced significantly stronger humoral and cellular responses. The conjugate vaccine also showed effective cross-neutralization to all variants of concern (B.1.1.7/alpha, B.1.351/beta, P.1/gamma, B.1.617.2/delta, and B.1.1.529/omicron). These results suggest that the self-adjuvanting αGalCer-RBD has great potential to be an effective COVID-19 vaccine candidate, and this strategy might be useful for designing various subunit vaccines.

Imaging α-GalCer-Activated iNKT Cells in a Hepatic Metastatic Environment.

Patients with colorectal cancer frequently develop liver metastases after, and perhaps as a consequence of, lifesaving surgical resection of the primary tumor. This creates a potential opportunity for prophylactic metastatic treatment with novel immunostimulatory molecules. Here, we used state-of-the-art intravital imaging of an experimental liver metastasis model to visualize the early behavior and function of invariant natural killer T (iNKT) cells stimulated with α-galactosylceramide (α-GalCer). Intravenous α-GalCer prior to tumor cell seeding in the liver significantly inhibited tumor growth. However, some seeding tumor cells survived. A multiple dosing regimen reduced tumor burden and prolonged the life of mice, whereas tumors returned within 5 days after a single dose of α-GalCer. With multiple doses of α-GalCer, iNKT cells increased in number and granularity (as did NK cells). As a result, the total number of contacts and time in contact with tumors increased substantially. In the absence of iNKT cells, the beneficial effect of α-GalCer was lost. Robust cytokine production dissipated over time. Repeated therapy, even after cytokine dissipation, led to reduced tumor burden and prolonged survival. Serial transplantation of tumors exposed to α-GalCer-activated iNKT cells did not induce greater resistance, suggesting no obvious epigenetic or genetic immunoediting in tumors exposed to activated iNKT cells. Very few tumor cells expressed CD1d in this model, and as such, adding monomers of CD1d-α-GalCer further reduced tumor growth. The data suggest early and repeated stimulation of iNKT cells with α-GalCer could have direct therapeutic benefit for patients with colorectal cancer who develop metastatic liver disease.

Expansion of invariant natural killer T cells from systemic lupus erythematosus patients by alpha-Galactosylceramide and IL-15.

CD1d-restricted invariant natural killer T cells (iNKT cells) may play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Interleukin (IL)-15 is a pro-inflammatory cytokine which is over-expressed in SLE patients. In the present study, we investigated the iNKT cell expansion of mononuclear cells (MNCs) from SLE patients following 10 days' culture with α-galactosylceramide (α-Galcer) and /or IL-15. We sought to determine the phenotypic and functional characteristics of the expanded iNKT cells compared to healthy controls and correlated with disease activity. We observed that 1. The percentages of Vα24+/Vß11+ iNKT cells following 10-day incubation was lower in SLE groups compared to controls; 2. The percentages and absolute numbers of Vα24+/Vß11+ iNKT cells were expanded by α-galactosylceramide (α-Galcer), and further enhanced with IL-15 in SLE patient, but the effect of IL-15 was much lower than controls; 3.IL-15 +α-Galcer expanded CD3+/CD56+ NKT-like cells from SLE patients, especially with active disease 4. The CD161+ Vα24+/Vß11+ iNKT cells in SLE were more responsive to α-Galcer stimulation than the CD161- counterpart; 5. IL-15 decreased apoptosis of α-Galcer activated SLE iNKT cells; 6. IL-15 enhanced CD69, CD1d and CD11a expression on α-Galcer treated iNKT cells; 7. The IL-4 production of iNKT cells was decreased in SLE patients compared to controls; 8. IL-15 increased IFN-γ and IL-4 production of SLE iNKT cells; 8. IL-15 failed to augment the ability of iNKT cells to aid NK-mediated K562 cytolysis in SLE patients; 9. CD161 positivity, granzyme B and perforin expression of α-Galcer+IL-15 expanded iNKT cells correlated with C3 levels in SLE patients. Taken together, our results demonstrated numeric and functional deficiency of iNKT cells and their response to IL-15 in SLE patients. Our finding may provide insight for using adoptive iNKT cell therapy in autoimmune diseases.

Host immunomodulatory lipids created by symbionts from dietary amino acids.

Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation1. However, little is known about the molecular mechanisms that control immune development in the host-microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d-BfaGC-NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system.

α-Galactosylceramide-Reactive NKT Cells Increase IgG1 Class Switch against a Clostridioides difficile Polysaccharide Antigen and Enhance Immunity against a Live Pathogen Challenge.

All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and, when conjugated to a protein carrier, induces a protective antibody response in animal models. Given that CD1d-restricted natural killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG titers were evident in sera from immunized mice. The inclusion of α-GC had a modest influence on isotype switch but increased the IgG1/IgG2c ratio. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18-/- mice were not protected by the PSII/α-GC immunization modality. Absence of NKT cells similarly had a modest effect on isotype switch, but ratios of IgG1/IgG2c decreased. These results indicate that α-GC-driven NKT cells move the humoral immune response against C. difficile PSII antigen toward Th2-driven IgG1 and may contribute to augmented protection. This study suggests that NKT activation represents a pathway for additional B-cell help that could be used to supplement existing efforts to develop vaccines against polysaccharides derived from C. difficile and other pathogens.

Inbred Strain Characteristics Impact the NKT Cell Repertoire.

NKT cells are primed lymphocytes that rapidly secrete cytokines and can directly kill cancerous cells. Given the critical role NKT cells play in cancer immune surveillance, we sought to investigate the effect of mutations in Brca1, specifically a conditional deletion of exon 11, on type I invariant NKT cell development. We observed a significant reduction in invariant NKT cells in both primary lymphoid and peripheral organs in Brca1 mutant mice compared with wild-type C57BL/6. However, the original Brca1 mutant strain was on a mixed background containing FVB/N. We determined that strain differences, rather than mutations in Brca1, led to the observed loss in NKT cells. Importantly, we found that whereas FVB/N mice lack Vß8, there was a striking increase in the total number of thymic type I CD1d-α-galactosylceramide tetramer positive NKT cells and skewing of the NKT cell population to NKT2 compared with C57BL/6 mice. Collectively, our data demonstrate the profound effect genetics can have on NKT cell subset differentiation.

Glycolipid-Containing Nanoparticle Vaccine Engages Invariant NKT Cells to Enhance Humoral Protection against Systemic Bacterial Infection but Abrogates T-Independent Vaccine Responses.

CD4+ T cells enable the critical B cell humoral immune protection afforded by most effective vaccines. We and others have recently identified an alternative source of help for B cells in mice, invariant NK T (iNKT) cells. iNKT cells are innate glycolipid-specific T cells restricted to the nonpolymorphic Ag-presenting molecule CD1d. As such, iNKT cells respond to glycolipids equally well in all people, making them an appealing adjuvant for universal vaccines. We tested the potential for the iNKT glycolipid agonist, α-galactosylceramide (αGC), to serve as an adjuvant for a known human protective epitope by creating a nanoparticle that delivers αGC plus antigenic polysaccharides from Streptococcus pneumoniae αGC-embedded nanoparticles activate murine iNKT cells and B cells in vitro and in vivo, facilitate significant dose sparing, and avoid iNKT anergy. Nanoparticles containing αGC plus S. pneumoniae polysaccharides elicits robust IgM and IgG in vivo and protect mice against lethal systemic S. pneumoniae However, codelivery of αGC via nanoparticles actually eliminated Ab protection elicited by a T-independent S. pneumoniae vaccine. This is consistent with previous studies demonstrating iNKT cell help for B cells following acute activation, but negative regulation of B cells during chronic inflammation. αGC-containing nanoparticles represent a viable platform for broadly efficacious vaccines against deadly human pathogens, but their potential for eliminating B cells under certain conditions suggests further clarity on iNKT cell interactions with B cells is warranted.

Synthesis and Evaluation of Liposomal Anti-GM3 Cancer Vaccine Candidates Covalently and Noncovalently Adjuvanted by αGalCer.

GM3, a typical tumor-associated carbohydrate antigen, is considered as an important target for cancer vaccine development, but its low immunogenicity limits its application. αGalCer, an iNKT cell agonist, has been employed as an adjuvant via a unique immune mode. Herein, we prepared and investigated two types of antitumor vaccine candidates: (a) self-adjuvanting vaccine GM3-αGalCer by conjugating GM3 with αGalCer and (b) noncovalent vaccine GM3-lipid/αGalCer, in which GM3 is linked with lipid anchor and coassembled with αGalCer. This demonstrated that ßGalCer is an exceptionally optimized lipid anchor, which enables the noncovalent vaccine candidate GM3-ßGalCer/αGalCer to evoke a comparable antibody level to GM3-αGalCer. However, the antibodies induced by GM3-αGalCer are better at recognition B16F10 cancer cells and more effectively activate the complement system. Our study highlights the importance of vaccine constructs utilizing covalent or noncovalent assembly between αGalCer with carbohydrate antigens and choosing an appropriate lipid anchor for use in noncovalent vaccine formulation.

Harnessing the Versatility of Invariant NKT Cells in a Stepwise Approach to Sepsis Immunotherapy.

Sepsis results from a heavy-handed response to infection that may culminate in organ failure and death. Many patients who survive acute sepsis become immunosuppressed and succumb to opportunistic infections. Therefore, to be successful, sepsis immunotherapies must target both the initial and the protracted phase of the syndrome to relieve early immunopathology and late immunosuppression, respectively. Invariant NKT (iNKT) cells are attractive therapeutic targets in sepsis. However, repeated treatments with α-galactosylceramide, the prototypic glycolipid ligand of iNKT cells, result in anergy. We designed a double-hit treatment that allows iNKT cells to escape anergy and exert beneficial effects in biphasic sepsis. We tested the efficacy of this approach in the sublethal cecal ligation and puncture mouse model, which mirrors polymicrobial sepsis with progression to an immunosuppressed state. Septic mice were treated with [(C2S, 3S, 4R)-1-O-(α-d-galactopyranosyl)-N-tetracosanoyl-2-amino-1,3,4-nonanetriol] (OCH), a TH2-polarizing iNKT cell agonist, before they received α-galactosylceramide. This regimen reduced the morbidity and mortality of cecal ligation and puncture, induced a transient but robust IFN-γ burst within a proinflammatory cytokine/chemokine landscape, transactivated NK cells, increased MHC class II expression on macrophages, and restored delayed-type hypersensitivity to a model hapten, consistent with recovery of immunocompetence in protracted sepsis. Structurally distinct TH2-polarizing agonists varied in their ability to replace OCH as the initial hit, with their lipid chain length being a determinant of efficacy. The proposed approach effectively exploits iNKT cells' versatility in biphasic sepsis and may have translational potentials in the development of new therapies.

Acute invariant NKT cell activation triggers an immune response that drives prominent changes in iron homeostasis.

Iron homeostasis is an essential biological process that ensures the tissue distribution of iron for various cellular processes. As the major producer of hepcidin, the liver is central to the regulation of iron metabolism. The liver is also home to many immune cells, which upon activation may greatly impact iron metabolism. Here, we focus on the role of invariant natural killer T (iNKT) cells, a subset of T lymphocytes that, in mice, is most abundant in the liver. Activation of iNKT cells with the prototypical glycosphingolipid antigen, α-galactosylceramide, resulted in immune cell proliferation and biphasic changes in iron metabolism. This involved an early phase characterized by hypoferremia, hepcidin induction and ferroportin suppression, and a second phase associated with strong suppression of hepcidin despite elevated levels of circulating and tissue iron. We further show that these changes in iron metabolism are fully dependent on iNKT cell activation. Finally, we demonstrate that the biphasic regulation of hepcidin is independent of NK and Kupffer cells, and is initially driven by the STAT3 inflammatory pathway, whereas the second phase is regulated by repression of the BMP/SMAD signaling pathway. These findings indicate that iNKT activation and the resulting cell proliferation influence iron homeostasis.

Lipo-Based Vaccines as an Approach to Target Dendritic Cells for Induction of T- and iNKT Cell Responses.

In this study we developed a liposome-based vaccine containing palmitoylated synthetic long peptides (SLP) and alpha galactosylceramide (αGC) to specifically target dendritic cells (DC) for activation of both innate (invariant natural killer T-cells [iNKT]) and adaptive (CD8+ T-cells) players of the immune system. Combination of model tumor specific antigens (gp100/MART-1) formulated as a SLP and αGC in one liposome results in strong activation of CD8+ and iNKT, as measured by IFNγ secretion. Moreover, addition of lipo-Lewis Y (LeY) to the liposomes for C-type lectin targeting increased not only uptake by monocyte-derived dendritic cells (moDC), dermal dendritic cells and Langerhans cells but also enhanced gp100-specific CD8+ T- and iNKT cell activation by human skin-emigrated antigen presenting cells in an ex vivo explant model. Loading of moDC with liposomes containing LeY also showed priming of MART-126-35L specific CD8+ T-cells. In conclusion, chemically linking a lipid tail to a glycan-based targeting moiety and SLP combined with αGC in one liposome allows for easy generation of vaccine formulations that target multiple skin DC subsets and induce tumor antigen specific CD8+ T- and iNKT cells. These liposomes present a new vaccination strategy against tumors.

A phase I study of loco-regional immunotherapy by transbronchial injection of α-galactosylceramide-pulsed antigen presenting cells in patients with lung cancer.

We conducted a phase I study of the trans-bronchial injection of α-galactosylceramide (αGalCer)-pulsed antigen presenting cells (APCs) to evaluate their safety, immune responses, and anti-tumor activities. Patients with advanced or recurrent non-small cell lung cancer (NSCLC) refractory to standard treatments were eligible. αGalCer-pulsed APCs were administered intratumorally or intranodally by bronchoscopy. Twenty-one patients were enrolled in this study. No severe adverse events related to the cell therapy were observed during this study in any patient. After αGalCer-pulsed APCs were administrated, increased iNKT cell numbers were observed in PBMCs from eight cases, and IFN-γ producing cells were increased in the peripheral blood of 10 cases. Regarding clinical responses, one case exhibited a partial response and eight were classified as stable disease. In the tumor microenvironment, IFN-γ expression was upregulated after treatment in partial response or stable disease cases and TGF-ß was upregulated in progressive disease cases.

Vaccines adjuvanted with an NKT cell agonist induce effective T-cell responses in models of CNS lymphoma.

Aim: The efficacy of anti-lymphoma vaccines that exploit the cellular adjuvant properties of activated natural killer T (NKT) cells were examined in mouse models of CNS lymphoma. Materials & methods: Vaccines were prepared by either loading the NKT cell agonist, α-galactosylceramide onto irradiated and heat-shocked B- and T-lymphoma cells, or chemically conjugating α-galactosylceramide to MHC-binding peptides from a lymphoma-associated antigen. Vaccine efficacy was analyzed in mice bearing intracranial tumors. Results: Both forms of vaccine proved to be effective in preventing lymphoma engraftment through activity of T cells that accessed the CNS. Established lymphoma was harder to treat with responses constrained by Tregs, but this could be overcome by depleting Tregs prior to vaccination. Conclusion: Simply designed NKT cell-activating vaccines enhance T-cell responses and have the potential to protect against CNS lymphoma development or prevent CNS relapse. To be effective against established CNS lymphoma, vaccines need to be combined with Treg suppression.

Combined proinflammatory cytokine and cognate activation of invariant natural killer T cells enhances anti-DNA antibody responses.

Invariant natural killer T (iNKT) cells serve as early rapid responders in the innate immune response to self-derived autoantigens and pathogen-derived danger signals and antigens. iNKT cells can serve both as helpers for effector B cells and negatively regulate autoreactive B cells. Specifically, iNKT cells drive B cell proliferation, class switch, and antibody production to induce primary antigen-specific immune responses. On the other hand, inflammasome-mediated activation drives accumulation of neutrophils, which license iNKT cells to negatively regulate autoreactive B cells via Fas ligand (FasL). This positions iNKT cells at an apex to support or inhibit B cell responses in inflammation. However, it is unknown which effector mechanism dominates in the face of cognate glycolipid activation during chronic inflammation, as might result from glycolipid vaccination or infection during chronic autoimmune disease. We stimulated iNKT cells by cognate glycolipid antigen α-galactosylceramide (αGalCer) and measured B cell activation during interleukin 18 (IL-18)-induced chronic inflammation. Moreover, glycolipid-activated iNKT cells increased the serum concentration of autoantibodies, frequency of germinal center (GC) B cells, and antigen-specific plasma cells induced during chronic IL-18-mediated inflammation, as compared with IL-18 alone. Further, activation of iNKT cells via cognate glycolipid during IL-18-mediated inflammation overrides the licensing function of neutrophils, instead inducing iNKT follicular helper (iNKTfh) cells that in turn promote autoimmunity. Thus, our data demonstrate that glycolipids which engage iNKT cells support antigen-specific B cell help during inflammasome-mediated inflammation.

Activation of Invariant Natural Killer T Cell Subsets in C57BL/6J Mice by Different Injection Modes of α-galactosylceramide.

Whether different injection modes of α-galactosylceramide (α-GalCer) affect the activation of different subsets of invariant natural killer T (iNKT) cells in different tissues and organs of mice is unclear. This study included healthy control, subcutaneous injection, and intraperitoneal injection groups (n=10 in each group). The subcutaneous and intraperitoneal injection groups were injected with α-Galcer (0.1 mg/kg weight), and then the changes in thymus, spleen, and liver iNKT cell frequencies and subsets were observed. The intraperitoneal injection of α-GalCer could increase the frequency of splenic iNKT cells, but the subcutaneous injection did not affect the frequency. Neither injection had any effect on the frequency of iNKT cells in the thymus and liver. The subcutaneous injection of α-GalCer increased the rate of iNKT2 subsets in the thymus but did not affect the rate of iNKT1 subsets. However, the intraperitoneal injection of α-GalCer did not affect thymus iNKT1 and iNKT2 subsets. Interestingly, the subcutaneous injection of α-GalCer significantly increased the proportion of iNKT1 in the spleen and liver but did not significantly change the proportion of iNKT2. The intraperitoneal injection of α-GalCer significantly increased the rate of iNKT2 in spleen and liver but decreased the rate of iNKT1. Subsets of iNKT1 or iNKT2 cells in the spleen and liver were selectively activated by the subcutaneous or intraperitoneal injection of α-GalCer. It provides a valuable means for treating tumors and certain autoimmune diseases. Further exploration of the activation mechanism may provide new ideas about the development of related vaccines.

Interleukin-7 protects against bacterial respiratory infection by promoting IL-17A-producing innate T-cell response.

Interleukin-7 (IL-7) is a critical cytokine in B- and T-lymphocyte development and maturation. Recent evidence suggests that IL-7 is a preferential homeostatic and survival factor for RORγt+ innate T cells such as natural killer T (NKT) cells, γδT cells, and mucosal-associated invariant T (MAIT) cells in the periphery. Given the important contribution of these populations in antibacterial immunity at barrier sites, we questioned whether IL-7 could be instrumental in boosting the local host immune response against respiratory bacterial infection. By using a cytokine-monoclonal antibody approach, we illustrated a role for topical IL-7 delivery in increasing the pool of RORγt+ IL-17A-producing innate T cells. Prophylactic IL-7 treatment prior to Streptococcus pneumoniae infection led to better bacterial containment, a process associated with increased neutrophilia and that depended on γδT cells and IL-17A. Last, combined delivery of IL-7 and α-galactosylceramide (α-GalCer), a potent agonist for invariant NKT (iNKT) cells, conferred an almost total protection in terms of survival, an effect associated with enhanced IL-17 production by innate T cells and neutrophilia. Collectively, we provide a proof of concept that IL-7 enables fine-tuning of innate T- cell functions. This might pave the way for considering IL-7 as an innovative biotherapeutic against bacterial infection.

Distinct CD1d docking strategies exhibited by diverse Type II NKT cell receptors.

Type I and type II natural killer T (NKT) cells are restricted to the lipid antigen-presenting molecule CD1d. While we have an understanding of the antigen reactivity and function of type I NKT cells, our knowledge of type II NKT cells in health and disease remains unclear. Here we describe a population of type II NKT cells that recognise and respond to the microbial antigen, α-glucuronosyl-diacylglycerol (α-GlcADAG) presented by CD1d, but not the prototypical type I NKT cell agonist, α-galactosylceramide. Surprisingly, the crystal structure of a type II NKT TCR-CD1d-α-GlcADAG complex reveals a CD1d F'-pocket-docking mode that contrasts sharply with the previously determined A'-roof positioning of a sulfatide-reactive type II NKT TCR. Our data also suggest that diverse type II NKT TCRs directed against distinct microbial or mammalian lipid antigens adopt multiple recognition strategies on CD1d, thereby maximising the potential for type II NKT cells to detect different lipid antigens.

B Cell-Based Vaccine Transduced With ESAT6-Expressing Vaccinia Virus and Presenting α-Galactosylceramide Is a Novel Vaccine Candidate Against ESAT6-Expressing Mycobacterial Diseases.

Early secretory antigenic target-6 (ESAT6) is a potent immunogenic antigen expressed in Mycobacterium tuberculosis as well as in some non-tuberculous mycobacteria (NTM), such as M. kansasii. M. kansasii is one of the most clinically relevant species of NTM that causes mycobacterial lung disease, which is clinically indistinguishable from tuberculosis. In the current study, we designed a novel cell-based vaccine using B cells that were transduced with vaccinia virus expressing ESAT6 (vacESAT6), and presenting α-galactosylceramide (αGC), a ligand of invariant NKT cells. We found that B cells loaded with αGC had increased levels of CD80 and CD86 after in vitro stimulation with NKT cells. Immunization of mice with B/αGC/vacESAT6 induced CD4+ T cells producing TNF-α and IFN-γ in response to heat-killed M. tuberculosis. Immunization of mice with B/αGC/vacESAT6 ameliorated severe lung inflammation caused by M. kansasii infection. We also confirmed that immunization with B/αGC/vacESAT6 reduced M. kansasii bacterial burden in the lungs. In addition, therapeutic administration of B/αGC/vacESAT6 increased IFN-γ+ CD4+ T cells and inhibited the progression of lung pathology caused by M. kansasii infection. Thus, B/αGC/vacESAT6 could be a potent vaccine candidate for the prevention and treatment of ESAT6-expressing mycobacterial infection caused by M. kansasii.

Structure-Function Implications of the Ability of Monoclonal Antibodies Against α-Galactosylceramide-CD1d Complex to Recognize ß-Mannosylceramide Presentation by CD1d.

iNKT cells are CD1d-restricted T cells recognizing lipid antigens. The prototypic iNKT cell-agonist α-galactosylceramide (α-GalCer) alongside compounds with similar structures induces robust proliferation and cytokine production of iNKT cells and protects against cancer in vivo. Monoclonal antibodies (mAbs) that detect CD1d-α-GalCer complexes have provided critical information for understanding of antigen presentation of iNKT cell agonists. Although most iNKT cell agonists with antitumor properties are α-linked glycosphingolipids that can be detected by anti-CD1d-α-GalCer mAbs, ß-ManCer, a glycolipid with a ß-linkage, induces strong antitumor immunity via mechanisms distinct from those of α-GalCer. In this study, we unexpectedly discovered that anti-CD1d-α-GalCer mAbs directly recognized ß-ManCer-CD1d complexes and could inhibit ß-ManCer stimulation of iNKT cells. The binding of anti-CD1d-α-GalCer mAb with ß-ManCer-CD1d complexes was also confirmed by plasmon resonance and could not be explained by α-anomer contamination. The binding of anti-CD1d-α-GalCer mAb was also observed with CD1d loaded with another ß-linked glycosylceramide, ß-GalCer (C26:0). Detection with anti-CD1d-α-GalCer mAbs indicates that the interface of the ß-ManCer-CD1d complex exposed to the iNKT cell TCR can assume a structure like that of CD1d-α-GalCer, despite its disparate carbohydrate structure. These results suggest that certain ß-linked monoglycosylceramides can assume a structural display similar to that of CD1d-α-GalCer and that the data based on anti-CD1d-α-GalCer binding should be interpreted with caution.