Human CD4+ iNKT cell adoptive immunotherapy induces anti-tumour responses against CD1d-negative EBV-driven B lymphoma.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that are uniquely suitable as off-the-shelf cellular immunotherapies due to their lack of alloreactivity. Two major subpopulations of human iNKT cells have been delineated, a CD4- subset that has a TH1/cytolytic profile, and a CD4+ subset that appears polyfunctional and can produce both regulatory and immunostimulatory cytokines. Whether these two subsets differ in anti-tumour effects is not known. Using live cell imaging, we found that CD4- iNKT cells limited growth of CD1d+ Epstein-Barr virus (EBV)-infected B-lymphoblastoid spheroids in vitro, whereas CD4+ iNKT cells showed little or no direct anti-tumour activity. However, the effects of the two subsets were reversed when we tested them as adoptive immunotherapies in vivo using a xenograft model of EBV-driven human B cell lymphoma. We found that EBV-infected B cells down-regulated CD1d in vivo, and administering CD4- iNKT cells had no discernable impact on tumour mass. In contrast, xenotransplanted mice bearing lymphomas showed rapid reduction in tumour mass after administering CD4+ iNKT cells. Immunotherapeutic CD4+ iNKT cells trafficked to both spleen and tumour and were associated with subsequently enhanced responses of xenotransplanted human T cells against EBV. CD4+ iNKT cells also had adjuvant-like effects on monocyte-derived DCs and promoted antigen-dependent responses of human T cells in vitro. These results show that allogeneic CD4+ iNKT cellular immunotherapy leads to marked anti-tumour activity through indirect pathways that do not require tumour cell CD1d expression and that are associated with enhanced activity of antigen-specific T cells.

Ox-LDL-induced CD80+ macrophages expand pro-atherosclerotic NKT cells via CD1d in atherosclerotic mice and hyperlipidemic patients.

Atherosclerosis is an inflammatory disease of blood vessels involving the immune system. Natural killer T (NKT) cells, as crucial components of the innate and acquired immune systems, play critical roles in the development of atherosclerosis. However, the mechanism and clinical relevance of NKT cells in early atherosclerosis are largely unclear. The study investigated the mechanism influencing NKT cell function in apoE deficiency-induced early atherosclerosis. Our findings demonstrated that there were higher populations of NKT cells and interferon-gamma (IFN-γ)-producing NKT cells in the peripheral blood of patients with hyperlipidemia and in the aorta, blood, spleen, and bone marrow of early atherosclerotic mice compared with the control groups. Moreover, we discovered that the infiltration of CD80+ macrophages and CD1d expression on CD80+ macrophages in atherosclerotic mice climbed remarkably. CD1d expression increased in CD80+ macrophages stimulated by oxidized low-density lipoprotein (ox-LDL) ex vivo and in vitro. Ex vivo coculture of macrophages with NKT cells revealed that ox-LDL-induced CD80+ macrophages presented lipid antigen α-Galcer (alpha-galactosylceramide) to NKT cells via CD1d, enabling NKT cells to express more IFN-γ. Furthermore, a greater proportion of CD1d+ monocytes and CD1d+CD80+ monocytes were found in peripheral blood of hyperlipidemic patients compared with that of healthy donors. Positive correlations were found between CD1d+CD80+ monocytes and NKT cells or IFN-γ+ NKT cells in hyperlipidemic patients. Our findings illustrated that CD80+ macrophages stimulated NKT cells to secrete IFN-γ via CD1d-presenting α-Galcer, which may accelerate the progression of early atherosclerosis. Inhibiting lipid antigen presentation by CD80+ macrophages to NKT cells may be a promising immune target for the treatment of early atherosclerosis.NEW & NOTEWORTHY This work proposed the ox-LDL-CD80+ monocyte/macrophage-CD1d-NKT cell-IFN-γ axis in the progression of atherosclerosis. The proinflammatory IFN-γ+ NKT cells are closely related to CD1d+CD80+ monocytes in hyperlipidemic patients. Inhibiting CD80+ macrophages to present lipid antigens to NKT cells through CD1d blocking may be a new therapeutic target for atherosclerosis.

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.

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.

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.

Modulation of TCR signalling components occurs prior to positive selection and lineage commitment in iNKT cells.

iNKT cells play a critical role in controlling the strength and character of adaptive and innate immune responses. Their unique functional characteristics are induced by a transcriptional program initiated by positive selection mediated by CD1d expressed by CD4+CD8+ (double positive, DP) thymocytes. Here, using a novel Vα14 TCR transgenic strain bearing greatly expanded numbers of CD24hiCD44loNKT cells, we examined transcriptional events in four immature thymic iNKT cell subsets. A transcriptional regulatory network approach identified transcriptional changes in proximal components of the TCR signalling cascade in DP NKT cells. Subsequently, positive and negative selection, and lineage commitment, occurred at the transition from DP NKT to CD4 NKT. Thus, this study introduces previously unrecognised steps in early NKT cell development, and separates the events associated with modulation of the T cell signalling cascade prior to changes associated with positive selection and lineage commitment.

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.

Activation of iNKT Cells Facilitates Liver Repair After Hepatic Ischemia Reperfusion Injury Through Acceleration of Macrophage Polarization.

Macrophage polarization is critical for liver tissue repair following acute liver injury. However, the underlying mechanisms of macrophage phenotype switching are not well defined. Invariant natural killer T (iNKT) cells orchestrate tissue inflammation and tissue repair by regulating cytokine production. Herein, we examined whether iNKT cells played an important role in liver repair after hepatic ischemia-reperfusion (I/R) injury by affecting macrophage polarization. To this end, we subjected male C57BL/6 mice to hepatic I/R injury, and mice received an intraperitoneal (ip) injection of α-galactosylceramide (α-GalCer) or vehicle. Compared with that of the vehicle, α-GalCer administration resulted in the promotion of liver repair accompanied by acceleration of macrophage differentiation and by increases in the numbers of Ly6Chigh pro-inflammatory macrophages and Ly6Clow reparative macrophages. iNKT cells activated with α-GalCer produced interleukin (IL)-4 and interferon (IFN)-γ. Treatment with anti-IL-4 antibodies delayed liver repair, which was associated with an increased number of Ly6Chigh macrophages and a decreased number of Ly6Clow macrophages. Treatment with anti-IFN-γ antibodies promoted liver repair, associated with reduced the number of Ly6Chigh macrophages, but did not change the number of Ly6Clow macrophages. Bone marrow-derived macrophages up-regulated the expression of genes related to both a pro-inflammatory and a reparative phenotype when co-cultured with activated iNKT cells. Anti-IL-4 antibodies increased the levels of pro-inflammatory macrophage-related genes and decreased those of reparative macrophage-related genes in cultured macrophages, while anti-IFN-γ antibodies reversed the polarization of macrophages. Cd1d-deficient mice showed delayed liver repair and suppressed macrophage switching, compared with that in wild-type mice. These results suggest that the activation of iNKT cells by α-GalCer facilitated liver repair after hepatic I/R injury by both IL-4-and IFN-γ-mediated acceleration of macrophage polarization. Therefore, the activation of iNKT cells may represent a therapeutic tool for liver repair after hepatic I/R injury.

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.

Protective effect of trichostatin A on CD19+CD5+CD1dhigh regulatory B cells in heart transplantation.

Heart transplantation is widely used for the treatment of several heart diseases. Regulatory B cells (Breg cells) serve a critical role in immune tolerance. However, the role of Breg cells in immune tolerance in the context of allogeneic heart transplantation remains poorly understood. The present study aimed to explore the effect of histone deacetylase (HDAC) inhibitor trichostatin A (TSA)­regulated Breg on the regulation of immune tolerance in heart transplantation. By constructing anallogeneic heart transplantation mouse model, and performing flow cytometry, reverse transcription­quantitative PCR, western blotting and carboxyfluorescein succinimidyl esterstaining assays, TSA­regulated Breg cells and their effects on immune tolerance in heart transplantation were evaluated. The results demonstrated that TSA increased the frequency of CD19+CD5+CD1dhigh Breg cells both in vitro and in vivo. Moreover, TSA treatment increased the frequency of IL­10 and TGF­ß­producing CD19+CD5+CD1dhigh Breg cells, and IL­10 and TGF­ß levels in vitro and in vivo. TSA administration significantly prolonged the survival rate in a heart transplant experiment model. In addition, the IL­10 inhibitor ammonium trichloro(dioxoethylene­o,o')tellurate partially reduced the survival rate and the percentages of CD19+CD5+CD1dhigh Breg cells in mice receiving heart allografts. In contrast, anti­CD20 treatment significantly decreased the survival rate in these mice. Collectively, the present findings suggested that TSA may induce immune tolerance following heart transplantation by regulating CD19+CD5+CD1dhigh Breg cells. These results provide a theoretical basis for the prevention of immunological rejection in cardiac transplantation.

Host-derived lipids orchestrate pulmonary γδ T cell response to provide early protection against influenza virus infection.

Innate immunity is important for host defense by eliciting rapid anti-viral responses and bridging adaptive immunity. Here, we show that endogenous lipids released from virus-infected host cells activate lung γδ T cells to produce interleukin 17 A (IL-17A) for early protection against H1N1 influenza infection. During infection, the lung γδ T cell pool is constantly supplemented by thymic output, with recent emigrants infiltrating into the lung parenchyma and airway to acquire tissue-resident feature. Single-cell studies identify IL-17A-producing γδ T (Tγδ17) cells with a phenotype of TCRγδhiCD3hiAQP3hiCXCR6hi in both infected mice and patients with pneumonia. Mechanistically, host cell-released lipids during viral infection are presented by lung infiltrating CD1d+ B-1a cells to activate IL-17A production in γδ T cells via γδTCR-mediated IRF4-dependent transcription. Reduced IL-17A production in γδ T cells is detected in mice either lacking B-1a cells or with ablated CD1d in B cells. Our findings identify a local host-immune crosstalk and define important cellular and molecular mediators for early innate defense against lung viral infection.

CD1 and MR1 recognition by human γδ T cells.

The two main T cell lineages, αß and γδ T cells, play a central role in immunity. Unlike αß T cells that recognize antigens bound to the Major Histocompatibility Complex (MHC) or MHC class I-like antigen-presenting molecules, the ligands for γδ T cell receptors (TCRs) are much more diverse. However, it is now clear that γδ TCRs can also recognize MHC class I-like molecules, including CD1b, CD1c, CD1d and the MHC class I-related protein 1 (MR1). Yet, our understanding at the molecular level of γδ T cell immunity to CD1 and MR1 is still very limited. Here, we discuss new molecular paradigms underpinning γδ TCRs recognition of antigens, antigen-presenting molecules or both. The recent discovery of recognition of MR1 by a γδ TCR at a position located underneath the antigen display platform reinforces the view that γδ TCRs can approach their ligands from many directions, unlike αß TCRs that bind MHC, CD1 and MR1 targets in an aligned, end to end fashion.

Serial Stimulation of Invariant Natural Killer T Cells with Covalently Stabilized Bispecific T-cell Engagers Generates Antitumor Immunity While Avoiding Anergy.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

CD1d deficiency limits tolerogenic properties of peritoneal macrophages.

Invariant natural killer T (iNKT) cells are involved in various autoimmune diseases. Although iNKT cells are arthritogenic, transforming growth factor beta (TGFß)-treated tolerogenic peritoneal macrophages (Tol-pMφ) from wild-type (WT) mice are more tolerogenic than those from CD1d knock-out iNKT cell-deficient mice in a collagen-induced arthritis (CIA) model. The underlying mechanism by which pMφ can act as tolerogenic antigen presenting cells (APCs) is currently unclear. To determine cellular mechanisms underlying CD1d-dependent tolerogenicity of pMφ, in vitro and in vivo characteristics of pMφ were investigated. Unlike dendritic cells or splenic Mφ, pMφ from CD1d+/- mice showed lower expression levels of costimulatory molecule CD86 and produced lower amounts of inflammatory cytokines upon lipopolysaccharide (LPS) stimulation compared to pMφ from CD1d-deficient mice. In a CIA model of CD1d-deficient mice, adoptively transferred pMφ from WT mice reduced the severity of arthritis. However, pMφ from CD1d-deficient mice were unable to reduce the severity of arthritis. Hence, the tolerogenicity of pMφ is a cell-intrinsic property that is probably conferred by iNKT cells during pMφ development rather than by interactions of pMφ with iNKT cells during antigen presentation to cognate T cells. [BMB Reports 2021; 54(4): 209-214].

CD1dhiPD-L1hiCD27+ Regulatory Natural Killer Subset Suppresses Atopic Dermatitis.

Effector and regulatory functions of various leukocytes in allergic diseases have been well reported. Although the role of conventional natural killer (NK) cells has been established, information on its regulatory phenotype and function are very limited. Therefore, the objective of this study was to investigate the phenotype and inhibitory functions of transforming growth factor (TGF)-ß-producing regulatory NK (NKreg) subset in mice with MC903-induced atopic dermatitis (AD). Interestingly, the population of TGF-ß-producing NK cells in peripheral blood monocytes (PBMCs) was decreased in AD patients than in healthy subjects. The number of TGF-ß+ NK subsets was decreased in the spleen or cervical lymph node (cLN), but increased in ear tissues of mice with AD induced by MC903 than those of normal mice. We further observed that TGF-ß+ NK subsets were largely included in CD1dhiPD-L1hiCD27+ NK cell subset. We also found that numbers of ILC2s and TH2 cells were significantly decreased by adoptive transfer of CD1dhiPD-L1hiCD27+ NK subsets. Notably, the ratio of splenic Treg per TH2 was increased by the adoptive transfer of CD1dhiPD-L1hiCD27+ NK cells in mice. Taken together, our findings demonstrate that the TGF-ß-producing CD1dhiPD-L1hiCD27+ NK subset has a previously unrecognized role in suppressing TH2 immunity and ILC2 activation in AD mice, suggesting that the function of TGF-ß-producing NK subset is closely associated with the severity of AD in humans.

Lipid antigens in bile from patients with chronic liver diseases activate natural killer T cells.

Natural killer T (NKT) cells are an abundant subset of liver lymphocytes activated by lipid antigens presented on CD1d molecules that are expressed by cholangiocytes. We aimed to determine if bile from patients with chronic liver diseases contains antigenic lipids that can activate NKT cells. Using murine invariant (24.7, 24.8 and DN32.D3) and non-invariant (14S.6, 14S.7 and 14S.10) NKT hybridomas we investigated the presence of lipid antigens in bile collected from the gallbladder of patients undergoing liver transplantation due to end-stage liver disease. Biliary microbiota profiles were generated using 16S rRNA amplicon sequencing. We found that the patient bile samples contain antigens that activate both invariant and non-invariant NKT hybridomas (24.7, 24.8, DN32.D3, 14S.6, 14S.7 and 14S.10), as demonstrated by activation of at least one hybridoma by eight of 10 bile samples. Activation at high dilutions suggests that some antigens are highly potent. We used the non-invariant NKT hybridoma 14S.6 to screen 21 additional patient bile samples for NKT-reactivity and demonstrated that 12 of 21 bile samples resulted in activation, three of which gave a strong activation. Four of 12 activating bile samples contained microbial DNA. Our results reveal an immunological pathway that could be of critical importance in biliary immunology.

Bacteroides fragilis alleviates the symptoms of lupus nephritis via regulating CD1d and CD86 expressions in B cells.

Emerging evidences indicated that the dysbiosis of microbiota was related to the onset of systemic lupus erythematosus (SLE). Bacteroides fragilis (B. fragilis) ATCC 25285, a human commensal, was discovered to improve inflammatory diseases. However, whether B. fragilis (ATCC 25285) has the beneficial effects on the treatment of lupus nephritis has still remained elusive. In the present study, oral treatment with B. fragilis (ATCC 25285) ameliorated the activity of MRL/lpr mice, including decreased levels of autoantibodies and symptoms of lupus nephritis. Furthermore, we demonstrated that treatment with B. fragilis (ATCC 25285) could promote CD1d expression in B cells by Est-1 pathway, while inhibit CD86 expression via SHP-2 signaling pathway to repair the immune response of B cells in MRL/lpr mice. In addition, our findings revealed a possible role of treatment with B. fragilis (ATCC 25285) in relieving intestinal inflammation in MRL/lpr mice. Meanwhile, it was uncovered that B. fragilis (ATCC 25285) restored the Th17/Treg balance in MRL/lpr mice that was consistent with the role of B. fragilis in other autoimmune diseases. Overall, the current study may highlight the potential application of B. fragilis (ATCC 25285) to treat manifestations of SLE in high-risk individuals.

The assembly of glycosphingolipid determines their immunomodulatory effect: A novel method for structure-based design of immunotherapy.

Structure-activity relationships provide insight into the binding interactions of beta-glycosphingolipids (GSLs) with both the TCR and the CD1d molecules, as well as the subsequent immunologic response of regulatory NKT cells. AIM: To determine the effects of synthetic GSL structures on their immune modulatory functions. METHODS: GSLs of various structures were tested in vitro and in an animal model of Concanavalin A (ConA) immune-mediated hepatitis. RESULTS: In vitro, using SV40 binding to live monkey CV1 cells, the l-threo stereoisomer of C8-ß-LacCer inhibits caveolar internalization, reducing viral binding to the cell surface. In vivo, in the ConA model, LR172, which has a saturated C8 chain, and LR178, which has a trans double bond at C-2 in the C8 chain, suppressed the immune-mediated liver inflammation and reduced IFNγ levels in a dose dependent manner. The beneficial effects of LR172 and of LR178 are associated with suppression of liver apoptosis, increased phosphorylated STAT3 expression in the liver, and an increase in the NKT liver/spleen ratio. SUMMARY: The assembly of GSLs determines their immunomodulatory effect and can serve as a method for structure-based design of immunotherapy.

Combination of NKT14m and Low Dose IL-12 Promotes Invariant Natural Killer T Cell IFN-γ Production and Tumor Control.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes characterized by the expression of an invariant T cell receptor (iTCR) that recognizes glycolipid antigens presented by the MHC I-like CD1d molecule. Following antigenic stimulation, iNKT cells rapidly produce large amounts of cytokines that can trans-activate dendritic cells (DC) and promote the anti-tumor functions of cytotoxic lymphocytes, such as natural killer (NK) and CD8 T cells. Additionally, iNKT cells can mediate robust and direct cytotoxicity against CD1d+ tumor targets. However, many tumors down-regulate CD1d and evade iNKT cell attack. To circumvent this critical barrier to iNKT cell anti-tumor activity, a novel monoclonal antibody (mAb), NKT14 has been recently developed. This agonistic antibody binds directly and specifically to the iTCR of murine iNKT cells. In the current study, we demonstrate that NKT14m mediates robust activation, cytokine production and degranulation of murine iNKT cells, in vitro. Consistently, NKT14m also promoted iNKT cell activation and immunomodulatory functions, in vivo. Finally, administration of NKT14m with low dose interleukin (IL)-12 further augmented iNKT cell IFN-γ production in vivo, and this combination conferred superior suppression of tumor cell growth compared to NKT14m or IL-12 alone. Together, these data demonstrate that a combination treatment consisting of low dose IL-12 and iTCR-specific mAb may be an attractive alternative to activate iNKT cell anti-tumor functions.