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.

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.

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.

Natural Killer T-Cell Agonist α-Galactosylceramide and PD-1 Blockade Synergize to Reduce Tumor Development in a Preclinical Model of Colon Cancer.

Murine and human invariant natural killer T (iNKT) lymphocytes are activated by α-galactosylceramide (α-GalCer) presented on CD1d. α-GalCer was first described as a lipid that had strong anti-metastatic effects in a mouse melanoma model, and it has subsequently been shown to induce efficient iNKT cell dependent tumor immunity in several tumor models. We have shown that α-GalCer treatment leads to a weak reduction of polyp burden in the autochthonous ApcMin/+ mouse model for human colon cancer, however this treatment resulted in upregulation of the inhibitory receptor PD-1 on iNKT cells. While anti-PD-1 treatment can prevent immune-suppression in other cancer types, human colon cancer is generally resistant to this treatment. Here we have used the ApcMin/+ model to investigate whether a combined treatment with α-GalCer and PD-1 blockade results in improved effects on polyp development. We find that PD-1 expression was high on T cells in polyps and lamina propria (LP) of ApcMin/+ mice compared to polyp free Apc+/+ littermates. Anti-PD-1 treatment alone promoted Tbet expression in iNKT cells and CD4 T cells, but did not significantly reduce polyp numbers. However, the combined treatment with anti-PD-1 and α-GalCer had synergistic effects, resulting in highly significant reduction of polyp numbers in the small and large intestine. Addition of PD-1 blockade to α-GalCer treatment prevented loss of iNKT cells that were skewed towards a TH1-like iNKT1 phenotype specifically in polyps. It also resulted in TH1 skewing and increased granzyme B expression of CD4 T cells. Taken together this demonstrates that a combination of immune stimulation targeting iNKT cells and checkpoint blockade may be a promising approach to develop for improved tumor immunotherapy.

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.

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.

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.

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.

Dissecting Integrin Expression and Function on Memory B Cells in Mice and Humans in Autoimmunity.

Immunological memory ensures life-long protection against previously encountered pathogens, and in mice and humans the spleen is an important reservoir for long-lived memory B cells (MBCs). It is well-established that integrins play several crucial roles in lymphocyte survival and trafficking, but their involvement in the retention of MBCs in secondary lymphoid organs, and differences between B cell subsets in their adhesion capacity to ICAM-1 and/or VCAM-1 have not yet been confirmed. Here, we use an autoimmune mouse model, where MBCs are abundant, to show that the highest levels of LFA-1 and VLA-4 amongst B cells are found on MBCs. In vivo blockade of VLA-4 alone or in combination with LFA-1, but not LFA-1 alone, causes a release of MBCs from the spleen into the blood stream. In humans, we find that in peripheral blood, spleens, and tonsils from healthy donors the highest expression levels of the integrins LFA-1 and VLA-4 are also found on MBCs. Consistent with this, we found MBCs to have a higher capacity to adhere to ICAM-1 and VCAM-1 than naïve B cells. In patients with the autoimmune disease rheumatoid arthritis, it is the MBCs that have the highest levels of LFA-1 and VLA-4; moreover, compared with healthy donors, naïve B and MBCs of patients receiving anti-TNF medication have enhanced levels of the active form of LFA-1. Commensurate levels of the active αL subunit can be induced on B cells from healthy donors by exposure to the integrin ligands. Thus, our findings establish the selective use of the integrins LFA-1 and VLA-4 in the localization and adhesion of MBCs in both mice and humans.

Promotion or Suppression of Murine Intestinal Polyp Development by iNKT Cell Directed Immunotherapy.

The glycosphingolipid α-galactosylceramide (α-GalCer) is a well-described immune activator with strong anti-tumor properties in animal models. It is presented on CD1d and acts by stimulating the invariant, type I, natural killer T (iNKT) lymphocytes to rapidly secrete TH1 and TH2 associated cytokines. This in turn promotes activation of a diversity of immune cells including natural killer (NK) cells with anti-tumor functions. Prior to tumor development, iNKT cells can also perform tumor surveillance and naturally protect from emergence of cancer. In contrast, we have recently demonstrated that iNKT cells naturally promote polyps in the spontaneous murine adenomatous polyposis coli (Apc) ApcMin/+ model for colon cancer, associated with suppressed TH1 immunity and enhanced immunoregulation. Here we investigated whether iNKT cell directed immunotherapy could subvert the polyp promoting function of iNKT cells and reduce polyp growth in this model. We treated ApcMin/+ mice with α-GalCer, or synthetic derivatives of this ligand (C-glycoside and C20:2) that have enhanced immunoregulatory properties. Treatment with iNKT cell ligands led to increased iNKT cell division, but reduced iNKT cell frequencies, lower NK1.1 expression and elevation of PD-1. ApcMin/+ mice that had been treated either long-term (5-15 weeks of age), or short-term (12-15 weeks of age) with α-GalCer demonstrated a significant decrease in polyp burden. Surprisingly, long-term treatment with the TH1 biasing ligand C-glycoside did not have significant effects on polyps, while long-term treatment with the TH2 biasing ligand C20:2 enhanced polyp growth. In stark contrast, short-term treatment with C20:2 led to reduction in polyp numbers and size. Reduced polyp burden after long-term treatment was associated with increased expression of genes indicating a pro-inflammatory polyp microenvironment. Polyp-reducing short-term treatment led to CD8 T cell activation specifically in polyps, and decreased tumor infiltrating and splenic macrophages, and a switch toward a pro-inflammatory phenotype. Thus, iNKT cell directed therapy could subvert the natural polyp enhancing function of iNKT cells, overcome immunosuppression, and reduce polyps. However, different iNKT cell activating ligands had opposite effects, and the timing of treatment had a major influence on outcomes.

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.

Type II NKT Cells: An Elusive Population With Immunoregulatory Properties.

Natural killer T (NKT) cells are unique unconventional T cells that are reactive to lipid antigens presented on the non-polymorphic major histocompatibility class (MHC) I-like molecule CD1d. They have characteristics of both innate and adaptive immune cells, and have potent immunoregulatory roles in tumor immunity, autoimmunity, and infectious diseases. Based on their T cell receptor (TCR) expression, NKT cells are divided into two subsets, type I NKT cells with an invariant TCRα-chain (Vα24 in humans, Vα14 in mice) and type II NKT cells with diverse TCRs. While type I NKT cells are well-studied, knowledge about type II NKT cells is still limited, and it is to date only possible to identify subsets of this population. However, recent advances have shown that both type I and type II NKT cells play important roles in many inflammatory situations, and can sometimes regulate the functions of each other. Type II NKT cells can be both protective and pathogenic. Here, we review current knowledge on type II NKT cells and their functions in different disease settings and how these cells can influence immunological outcomes.

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.

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.

High Interferon-γ Uniquely in Vδ1 T Cells Correlates with Markers of Inflammation and Axonal Damage in Early Multiple Sclerosis.

We have identified a population of T lymphocytes in peripheral blood, Vδ1 TCRγδ T lymphocytes, which unexpectedly was uniquely expressing high production of interferon-γ in newly diagnosed, untreated multiple sclerosis (MS) patients. IFN-γ production in this population distinctly correlated to parameters of clinical disease activity, inflammation, and neuronal damage. These Vδ1 T lymphocytes belong to a population of innate T lymphocytes that recognize antigen in the context of CD1d/CD1c and which include reactivity to the myelin glycosphingolipid sulfatide. Importantly, patients treated with natalizumab, blocking leukocyte transmigration to central nervous system, had completely normalized levels of interferon-γ-producing Vδ1 T lymphocytes. A biomarker and early sign of demyelinating disease in MS is much warranted and would help identify immunopathogenesis and prognosis of disease as well as monitor success with adequate treatment. The present study identifies the Vδ1 T lymphocytes as an early marker of MS and a possible link to understanding the disease etiology.

The Yin and Yang of Invariant Natural Killer T Cells in Tumor Immunity-Suppression of Tumor Immunity in the Intestine.

CD1d-restricted invariant natural killer T (iNKT) cells are known as early responding, potent regulatory cells of immune responses. Besides their established role in the regulation of inflammation and autoimmune disease, numerous studies have shown that iNKT cells have important functions in tumor immunosurveillance and control of tumor metastasis. Tumor-infiltrating T helper 1 (TH1)/cytotoxic T lymphocytes have been associated with a positive prognosis. However, inflammation has a dual role in cancer and chronic inflammation is believed to be a driving force in many cancers as exemplified in patients with inflammatory bowel disease that have an increased risk of colorectal cancer. Indeed, NKT cells promote intestinal inflammation in human ulcerative colitis, and the associated animal model, indicating that NKT cells may favor tumor development in intestinal tissue. In contrast to other cancers, recent data from animal models suggest that iNKT cells promote tumor formation in the intestine by supporting an immunoregulatory tumor microenvironment and suppressing TH1 antitumor immunity. Here, we review the role of iNKT cells in suppression of tumor immunity in light of iNKT-cell regulation of intestinal inflammation. We also discuss suppression of immunity in other situations as well as factors that may influence whether iNKT cells have a protective or an immunosuppressive and tumor-promoting role in tumor immunity.

Altered expression of Butyrophilin (BTN) and BTN-like (BTNL) genes in intestinal inflammation and colon cancer.

Several Butyrophilin (BTN) and Btn-like (BTNL) molecules control T lymphocyte responses, and are genetically associated with inflammatory disorders and cancer. In this study, we present a comprehensive expression analysis of human and murine BTN and BTNL genes in conditions associated with intestinal inflammation and cancer. Using real-time PCR, expression of human BTN and BTNL genes was analyzed in samples from patients with ulcerative colitis, irritable bowel syndrome, and colon tumors. Expression of murine Btn and Btnl genes was examined in mouse models of spontaneous colitis (Muc2-/-) and intestinal tumorigenesis (ApcMin/+). Our analysis indicates a strong association of several of the human genes with ulcerative colitis and colon cancer; while especially BTN1A1, BTN2A2, BTN3A3, and BTNL8 were significantly altered in inflammation, colonic tumors exhibited significantly decreased levels of BTNL2, BTNL3, BTNL8, and BTNL9 as compared to unaffected tissue. Colonic inflammation in Muc2-/- mice significantly down-regulated the expression of particularly Btnl1, Btnl4, and Btnl6 mRNA, and intestinal polyps derived from ApcMin/+ mice displayed altered levels of Btn1a1, Btn2a2, and Btnl1 transcripts. Thus, our data present an association of BTN and BTNL genes with intestinal inflammation and cancer and represent a valuable resource for further studies of this gene family.

A New Mouse Model That Spontaneously Develops Chronic Liver Inflammation and Fibrosis.

Here we characterize a new animal model that spontaneously develops chronic inflammation and fibrosis in multiple organs, the non-obese diabetic inflammation and fibrosis (N-IF) mouse. In the liver, the N-IF mouse displays inflammation and fibrosis particularly evident around portal tracts and central veins and accompanied with evidence of abnormal intrahepatic bile ducts. The extensive cellular infiltration consists mainly of macrophages, granulocytes, particularly eosinophils, and mast cells. This inflammatory syndrome is mediated by a transgenic population of natural killer T cells (NKT) induced in an immunodeficient NOD genetic background. The disease is transferrable to immunodeficient recipients, while polyclonal T cells from unaffected syngeneic donors can inhibit the disease phenotype. Because of the fibrotic component, early on-set, spontaneous nature and reproducibility, this novel mouse model provides a unique tool to gain further insight into the underlying mechanisms mediating transformation of chronic inflammation into fibrosis and to evaluate intervention protocols for treating conditions of fibrotic disorders.