Recognition of CD1d-sulfatide mediated by a type II natural killer T cell antigen receptor.

Natural killer T cells (NKT cells) are divided into type I and type II subsets on the basis of differences in their T cell antigen receptor (TCR) repertoire and CD1d-antigen specificity. Although the mode by which type I NKT cell TCRs recognize CD1d-antigen has been established, how type II NKT cell TCRs engage CD1d-antigen is unknown. Here we provide a basis for how a type II NKT cell TCR, XV19, recognized CD1d-sulfatide. The XV19 TCR bound orthogonally above the A' pocket of CD1d, in contrast to the parallel docking of type I NKT cell TCRs over the F' pocket of CD1d. At the XV19 TCR-CD1d-sulfatide interface, the TCRα and TCRß chains sat centrally on CD1d, where the malleable CDR3 loops dominated interactions with CD1d-sulfatide. Accordingly, we highlight the diverse mechanisms by which NKT cell TCRs can bind CD1d and account for the distinct antigen specificity of type II NKT cells.

Innate and adaptive stimulation of murine diverse NKT cells result in distinct cellular responses.

Natural killer T (NKT) cells recognize glycolipids presented on CD1d. They share features of adaptive T lymphocytes and innate NK cells, and mediate immunoregulatory functions via rapid production of cytokines. Invariant (iNKT) and diverse (dNKT) NKT cell subsets are defined by their TCR. The immunological role of dNKT cells, that do not express the invariant TCRα-chain used by iNKT cells, is less well explored than that of iNKT cells. Here, we investigated signals driving Toll-like receptor (TLR) ligand activation of TCR-transgenic murine dNKT cells. IFN-γ production by dNKT cells required dendritic cells (DC), cell-to-cell contact and presence of TLR ligands. TLR-stimulated DC activated dNKT cells to secrete IFN-γ in a CD1d-, CD80/86- and type I IFN-independent manner. In contrast, a requirement for IL-12p40, and a TLR ligand-selective dependence on IL-18 or IL-15 was observed. TLR ligand/DC stimulation provoked early secretion of pro-inflammatory cytokines by both CD62L+ and CD62L- dNKT cells. However, proliferation was limited. In contrast, TCR/co-receptor-mediated activation resulted in proliferation and delayed production of a broader cytokine spectrum preferentially in CD62L- dNKT cells. Thus, innate (TLR ligand/DC) and adaptive (TCR/co-receptor) stimulation of dNKT cells resulted in distinct cellular responses that may contribute differently to the formation of immune memory.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Ultrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers.

BACKGROUND: Immune repertoire sequencing of the T-cell receptor can identify clonotypes that have expanded as a result of antigen recognition or hematological malignancies. However, current sequencing protocols display limitations with nonuniform amplification and polymerase-induced errors during sequencing. Here, we developed a sequencing method that overcame these issues and applied it to γδ T cells, a cell type that plays a unique role in immunity, autoimmunity, homeostasis of intestine, skin, adipose tissue, and cancer biology. METHODS: The ultrasensitive immune repertoire sequencing method used PCR-introduced unique molecular identifiers. We constructed a 32-panel assay that captured the full diversity of the recombined T-cell receptor delta loci in γδ T cells. The protocol was validated on synthetic reference molecules and blood samples of healthy individuals. RESULTS: The 32-panel assay displayed wide dynamic range, high reproducibility, and analytical sensitivity with single-nucleotide resolution. The method corrected for sequencing-depended quantification bias and polymerase-induced errors and could be applied to both enriched and nonenriched cells. Healthy donors displayed oligoclonal expansion of γδ T cells and similar frequencies of clonotypes were detected in both enrichment and nonenriched samples. CONCLUSIONS: Ultrasensitive immune repertoire sequencing strategy enables quantification of individual and specific clonotypes in a background that can be applied to clinical as well as basic application areas. Our approach is simple, flexible, and can easily be implemented in any molecular laboratory.

γδ T Cells Contribute to Injury in the Developing Brain.

Brain injury in premature infants, especially periventricular leukomalacia, is an important cause of neurologic disabilities. Inflammation contributes to perinatal brain injury development, but the essential mediators that lead to early-life brain injury remain largely unknown. Neonates have reduced capacity for mounting conventional αßT-cell responses. However, γδT cells are already functionally competent during early development and are important in early-life immunity. We investigated the potential contribution of γδT cells to preterm brain injury using postmortem brains from human preterm infants with periventricular leukomalacia and two animal models of preterm brain injury-the hypoxic-ischemic mouse model and a fetal sheep asphyxia model. Large numbers of γδT cells were observed in the brains of mice, sheep, and postmortem preterm infants after injury, and depletion of γδT cells provided protection in the mouse model. The common γδT-cell-associated cytokines interferon-γ and IL-17A were not detectable in the brain. Although there were increased mRNA levels of Il17f and Il22 in the mouse brains after injury, neither IL-17F nor IL-22 cytokines contributed to preterm brain injury. These findings highlight unique features of injury in the developing brain, where, unlike injury in the mature brain, γδT cells function as initiators of injury independently of common γδT-cell-associated cytokines. This finding will help to identify therapeutic targets for preventing or treating preterm infants with brain injury.

Defining a novel subset of CD1d-dependent type II natural killer T cells using natural killer cell-associated markers.

Natural killer T (NKT) cells are αß T cell receptor (TCR) expressing innate-like T cells that display natural killer (NK) cell markers. Based on TCR characteristics, they are divided into two groups restricted to the MHC class I-like molecule CD1d. Type I NKT cells, most extensively studied, are identified by a semi-invariant Vα14-Jα18 (mouse, Vα24-Jα18 in humans) TCR reactive to the prototypic ligand α-galactosylceramide presented on CD1d. In contrast, type II NKT cells display diverse TCR reacting to different CD1d-presented ligands. There are no reagents that identify all type II NKT cells, limiting their exploration. Here, we searched for novel type II NKT cells by comparing Jα18-/- MHCII-/- mice that harbour type II but not type I NKT cells, and CD1d-/- MHCII-/- mice, lacking all NKT cells. We identified significantly larger populations of CD4+ and CD4- CD8- (double negative, DN) TCRß+ cells expressing NKG2D or NKG2A/C/E in Jα18-/- MHCII-/- mice compared with CD1d-/- MHCII-/- mice, suggesting that 30%-50% of these cells were type II NKT cells. They expressed CD122, NK1.1, CXCR3 and intermediate/low levels of CD45RB. Further, the CD4+ subset was CD69+ , while the DN cells were CD49b+ and CD62L+ . Both subsets expressed the NKT cell-associated promyelocytic leukaemia zinc finger (PLZF) transcription factor and Tbet, while fewer cells expressed RORγt. NKG2D+ CD4+ and DN populations were producers of IFN-γ, but rarely IL-4 and IL-17. Taken together, we identify a novel subset of primary CD4+ and DN type II NKT cells that expresses NKG2 receptors have typical NKT cell phenotypes and a TH1-like cytokine production.

Sulfatide isoform pattern in cerebrospinal fluid discriminates progressive MS from relapsing-remitting MS.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Several biomarkers including proteins and lipids have been reported in MS cerebrospinal fluid (CSF), reflecting different aspects of the pathophysiology particularly of relapsing-remitting MS (RRMS). Sulfatide, abundant in the myelin sheath and a proposed target for autoimmune attack in MS, has been reported altered in MS CSF. Here, we investigated the potential of CSF sulfatide and its isoforms as biomarkers in MS. A highly sensitive and quantitative mass spectrometry method was employed to determine levels of sulfatide isoforms in CSF from RRMS and progressive MS (PMS) patients, and healthy donors (HD). We demonstrate that levels of total CSF sulfatide and C24:1, C26:1, and C26:1-OH isoforms were significantly increased in PMS compared with RRMS patients and HD, while C23:0-OH was significantly decreased in CSF from PMS patients compared to the other two groups. Multivariate discriminant analysis showed that CSF sulfatide isoform pattern in PMS patients was distinct and non-overlapping with that of RRMS patients and HD. Sulfatide levels did not correlate with tested biomarkers or clinical parameters. The results suggest that CSF sulfatide isoform levels may be used to discriminate the phenotype of MS and might play a role in the progression of the disease.

Extracellular matrix of secondary lymphoid organs impacts on B-cell fate and survival.

We describe a unique extracellular matrix (ECM) niche in the spleen, the marginal zone (MZ), characterized by the basement membrane glycoproteins, laminin α5 and agrin, that promotes formation of a specialized population of MZ B lymphocytes that respond rapidly to blood-borne antigens. Mice with reduced laminin α5 expression show reduced MZ B cells and increased numbers of newly formed (NF) transitional B cells that migrate from the bone marrow, without changes in other immune or stromal cell compartments. Transient integrin α6ß1-mediated interaction of NF B cells with laminin α5 in the MZ supports the MZ B-cell population, their long-term survival, and antibody response. Data suggest that the unique 3D structure and biochemical composition of the ECM of lymphoid organs impacts on immune cell fate.

Recognition of microbial and mammalian phospholipid antigens by NKT cells with diverse TCRs.

CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18(+) invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and ß-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.

The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αß transgenic T cells (24αß T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αß T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.

The immune response after hypoxia-ischemia in a mouse model of preterm brain injury.

BACKGROUND: Preterm brain injury consists primarily of periventricular leukomalacia accompanied by elements of gray-matter injury, and these injuries are associated with cerebral palsy and cognitive impairments. Inflammation is believed to be an important contributing factor to these injuries. The aim of this study was to examine the immune response in a postnatal day (PND) 5 mouse model of preterm brain injury induced by hypoxia-ischemia (HI) that is characterized by focal white and gray-matter injury. METHODS: C57Bl/6 mice at PND 5 were subjected to unilateral HI induced by left carotid artery ligation and subsequent exposure to 10% O2 for 50 minutes, 70 minutes, or 80 minutes. At seven days post-HI, the white/gray-matter injury was examined. The immune responses in the brain after HI were examined at different time points after HI using RT-PCR and immunohistochemical staining. RESULTS: HI for 70 minutes in PND 5 mice induced local white-matter injury with focal cortical injury and hippocampal atrophy, features that are similar to those seen in preterm brain injury in human infants. HI for 50 minutes resulted in a small percentage of animals being injured, and HI for 80 minutes produced extensive infarction in multiple brain areas. Various immune responses, including changes in transcription factors and cytokines that are associated with a T-helper (Th)1/Th17-type response, an increased number of CD4+ T-cells, and elevated levels of triggering receptor expressed on myeloid cells 2 (TREM-2) and its adaptor protein DNAX activation protein of 12 kDa (DAP12) were observed using the HI 70 minute preterm brain injury model. CONCLUSIONS: We have established a reproducible model of HI in PND 5 mice that produces consistent local white/gray-matter brain damage that is relevant to preterm brain injury in human infants. This model provides a useful tool for studying preterm brain injury. Both innate and adaptive immune responses are observed after HI, and these show a strong pro-inflammatory Th1/Th17-type bias. Such findings provide a critical foundation for future studies on the mechanism of preterm brain injury and suggest that blocking the Th1/Th17-type immune response might provide neuroprotection after preterm brain injury.

CD4(+) type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic ß cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry diverse TCRs, prevented T1D in the NOD mouse model for the human disease. In this study, we show that CD4(+) 24αß type II NKT cells, but not CD4/CD8 double-negative NKT cells, were sufficient to downregulate diabetogenic CD4(+) BDC2.5 NOD T cells in adoptive transfer experiments. CD4(+) 24αß NKT cells exhibited a memory phenotype including high ICOS expression, increased cytokine production, and limited display of NK cell markers, compared with double-negative 24αß NKT cells. Blocking of ICOS or the programmed death-1/programmed death ligand 1 pathway was shown to abolish the regulation that occurred in the pancreas draining lymph nodes. To our knowledge, these results provide for the first time cellular and molecular information on how type II CD1d-restricted NKT cells regulate T1D.

Sulfatide attenuates experimental Staphylococcus aureus sepsis through a CD1d-dependent pathway.

Natural killer T (NKT) lymphocytes are implicated in the early response to microbial infection. Further, sulfatide, a myelin self-glycosphingolipid, activates a type II NKT cell subset and can modulate disease in murine models. We examined the role of NKT cells and the effect of sulfatide treatment in a murine model of Staphylococcus aureus sepsis. The lack of CD1d-restricted NKT cells did not alter survival after a lethal inoculum of S. aureus. In contrast, sulfatide treatment significantly improved the survival rate of mice with S. aureus sepsis, accompanied by decreased levels of tumor necrosis factor alpha and interleukin-6 in the blood. The protective effect of sulfatide treatment depended on CD1d but not on type I NKT cells, suggesting that activation of type II NKT cells by sulfatide has beneficial effects on the outcome of S. aureus sepsis in this model.

dysregulation of CD1d-restricted type ii natural killer T cells leads to spontaneous development of colitis in mice.

BACKGROUND & AIMS: CD1d-restricted natural killer (NK) T cells are a subset of immunoregulatory T cells that comprise type I (express the semi-invariant T-cell receptor [TCR] and can be detected using the α-galactosylceramide/CD1d tetramer) and type II (express diverse TCRs and cannot be directly identified). Studies in mouse models of inflammatory bowel disease revealed a complex role for type I NKT cells in the development of colitis. Type II NKT cells have been associated with intestinal inflammation in patients with ulcerative colitis. METHODS: To investigate whether dysregulation of type II NKT cells, caused by increased expression of CD1d, can contribute to colitis, we generated transgenic mice that express high levels of CD1d and a TCR from an autoreactive, type II NKT cell (CD1dTg/24αßTg mice). RESULTS: CD1dTg/24αßTg mice had reduced numbers of 24αß T cells compared with 24αßTg mice, indicating that negative selection increases among type II NKT cells engaged by abundant self-antigen. The residual 24αß T cells in CD1dTg/24αßTg mice had an altered surface phenotype and acquired a cytokine profile distinct from that of equivalent cells in 24αßTg mice. Interestingly, CD1dTg/24αßTg mice spontaneously developed colitis; adoptive transfer experiments confirmed that type II NKT cells that develop in the context of increased CD1d expression are pathogenic. CONCLUSIONS: Aberrant type II NKT cell responses directly contribute to intestinal inflammation in mice, indicating the importance of CD1d expression levels in the development and regulation of type II NKT cells.

Identification of novel glycolipid ligands activating a sulfatide-reactive, CD1d-restricted, type II natural killer T lymphocyte.

Sulfatide-reactive CD1d-restricted natural killer T (NKT) lymphocytes belong to the type II NKT cell subset with diverse TCRs, and have been found to regulate experimental auto-immune encephalomyelitis, tumor immunity, and experimental hepatitis in murine models. NKT cells can be activated by self-lipids presented by CD1d, manifested as autoreactivity. The identity of most of these self-lipids remains unknown. By isolating lipids from a CD1d-expressing, highly stimulatory antigen presenting cell, we identified isoforms of ß-glucosylceramide (GlcCer), with sphingosine and fatty acid chain lengths of C24:0 and C16:0, that activated a sulfatide-reactive type II NKT cell hybridoma. A screen of structurally related glycosphingolipids demonstrated ß-galactosylceramide (GalCer) as another ligand, and further, that the lysoforms were the most potent isoform of the glycosphingo-lipid ligands, followed by isoforms with a long fatty acid chain of C24. Thus, the same type II NKT cell was activated by several ligands, namely sulfatide, GlcCer, and GalCer. However, CD1d-dependent reactivity to antigen presenting cells lacking all GlcCer-based glycosphingolipids, or all glycosphingolipids, was maintained. This suggests that other endogenous, nonglycosphingolipid, lipid ligands contribute to steady-state autoreactivity by type II NKT cells.

The extracellular matrix of lymph node reticular fibers modulates follicle border interactions and germinal center formation.

Germinal center (GC) formation and antibody production in lymph node follicles require coordinated interactions between B-cells, T-cells and dendritic cells (DCs), orchestrated by the extracellular matrix-rich reticular fiber (RF) network. We describe a unique laminin 523-containing RF network around and between follicles that associates with PDGFrecßhighCCL19lowgp38low fibroblastic reticular cells (FRC). In the absence of FRC expression of laminin α5 (pdgfrb-cre:Lama5fl/fl), pre-Tfh-cells, B-cells and DCs are displaced from follicle borders, correlating with fewer Tfh-cells and GC B-cells. Total DCs are not altered in pdgfrb-cre:Lama5fl/fl mice, but cDC2s, which localize to laminin α5 in RFs at follicle borders, are reduced. In addition, PDGFrecßhighCCL19lowgp38low FRCs show lower Ch25h expression, required for 7α,25-dihydroxycholesterol synthesis that attracts pre-Tfh-cells, B-cells and DCs to follicle borders. We propose that RF basement membrane components represent a type of tissue memory that guides the localization and differentiation of both specialized FRC and DC populations, required for normal lymph node function.

Cerebrospinal fluid sulfatide isoforms lack diagnostic utility in separating progressive from relapsing-remitting multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system. The glycosphingolipid sulfatide, a lipid particularly enriched in the myelin sheath, has been shown to be involved the maintenance of this specific membrane structure. Sulfatide in cerebrospinal fluid (CSF) may reflect demyelination, a dominating feature of MS. We investigated the diagnostic utility of CSF sulfatide isoform levels to separate different courses or phenotypes of MS disease. MATERIAL AND METHODS: This was a mono-center, cross-sectional study of relapsing-remitting MS (RRMS) (n = 45) and progressive MS (PMS) (n = 42) patients (consisting of primary PMS (n = 17) and secondary PMS (n = 25)) and healthy controls (n = 19). In total, 20 sulfatide isoforms were measured in CSF by liquid chromatography-mass spectrometry. RESULTS: CSF total sulfatide concentrations, as well as CSF sulfatide isoform distribution, did not differ across the study groups, and their levels were independent of disease course/phenotype, disease duration, time to conversion to secondary PMS, age, and disability in MS patients. CONCLUSION: CSF sulfatide isoforms lack diagnostic and prognostic utility as a biomarker for progressive MS.

Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer.

In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding ß-galactosylceramide (ßGalCer) without sulfate. C24:2 induced IFN-γ-dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid's function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.

C1QTNF3 is Upregulated During Subcutaneous Adipose Tissue Remodeling and Stimulates Macrophage Chemotaxis and M1-Like Polarization.

The adipose tissue undergoes substantial tissue remodeling during weight gain-induced expansion as well as in response to the mechanical and immunological stresses from a growing tumor. We identified the C1q/TNF-related protein family member C1qtnf3 as one of the most upregulated genes that encode secreted proteins in tumor-associated inguinal adipose tissue - especially in high fat diet-induced obese mice that displayed 3-fold larger tumors than their lean controls. Interestingly, inguinal adipose tissue C1qtnf3 was co-regulated with several macrophage markers and chemokines and was primarily expressed in fibroblasts while only low levels were detected in adipocytes and macrophages. Administration of C1QTNF3 neutralizing antibodies inhibited macrophage accumulation in tumor-associated inguinal adipose tissue while tumor growth was unaffected. In line with this finding, C1QTNF3 exerted chemotactic actions on both M1- and M2-polarized macrophages in vitro. Moreover, C1QTNF3 treatment of M2-type macrophages stimulated the ERK and Akt pathway associated with increased M1-like polarization as judged by increased expression of M1-macrophage markers, increased production of nitric oxide, reduced oxygen consumption and increased glycolysis. Based on these results, we propose that macrophages are recruited to adipose tissue sites with increased C1QTNF3 production. However, the impact of the immunomodulatory effects of C1QTNF3 in adipose tissue remodeling warrants future investigations.

Type II NKT Cell Agonist, Sulfatide, Is an Effective Adjuvant for Oral Heat-Killed Cholera Vaccines.

Oral vaccination has the potential to offer a safer and more efficacious approach for protection against enteric pathogens than injection-based approaches, especially in developing countries. One key advantage is the potential to induce intestinal immune responses in addition to systemic immunity. In general, antigen delivery via the oral route triggers weak immune responses or immunological tolerance. The effectiveness of oral vaccination can be improved by co-administering adjuvants. However, a major challenge is the absence of potent and safe oral adjuvants for clinical application. Here, the Type II NKT cell activator sulfatide is shown for the first time to be an effective oral adjuvant for Vibrio cholerae vaccine antigens in a mouse model. Specifically, administration of sulfatide with the oral cholera vaccine Dukoral® resulted in enhancement of intestinal antigen-specific IgA in addition to Th1 and Th17 immune responses. In summary, sulfatide is a promising adjuvant for inclusion in an oral cholera vaccine and our data further support the potential of adjuvants targeting NKT cells in new vaccine strategies.