Immune-system-dependent anti-tumor activity of a plant-derived polyphenol rich fraction in a melanoma mouse model.

Recent findings suggest that part of the anti-tumor effects of several chemotherapeutic agents require an intact immune system. This is in part due to the induction of immunogenic cell death. We have identified a gallotannin-rich fraction, obtained from Caesalpinia spinosa (P2Et) as an anti-tumor agent in both breast carcinoma and melanoma. Here, we report that P2Et treatment results in activation of caspase 3 and 9, mobilization of cytochrome c and externalization of annexin V in tumor cells, thus suggesting the induction of apoptosis. This was preceded by the onset of autophagy and the expression of immunogenic cell death markers. We further demonstrate that P2Et-treated tumor cells are highly immunogenic in vaccinated mice and induce immune system activation, clearly shown by the generation of interferon gamma (IFN-γ) producing tyrosine-related protein 2 antigen-specific CD8+ T cells. Moreover, the tumor protective effects of P2Et treatment were abolished in immunodeficient mice, and partially lost after CD4 and CD8 depletion, indicating that P2Et's anti-tumor activity is highly dependent on immune system and at least in part of T cells. Altogether, these results support the hypothesis that the gallotannin-rich fraction P2Et's anti-tumor effects are mediated to a great extent by the endogenous immune response following to the exposure to immunogenic dying tumor cells.

Estrogen receptor regulates MyoD gene expression by preventing AP-1-mediated repression.

Cell growth and differentiation are opposite events in the myogenic lineage. Growth factors block the muscle differentiation program by inducing the expression of transcription factors that negatively regulate the expression of muscle regulatory genes like MyoD. In contrast, extracellular clues that induce cell cycle arrest promote MyoD expression and muscle differentiation. Thus, the regulation of MyoD expression is critical for muscle differentiation. Here we show that estrogen induces MyoD expression in mouse skeletal muscle in vivo and in dividing myoblasts in vitro by relieving the MyoD promoter from AP-1 negative regulation through a mechanism involving estrogen receptor/AP-1 protein-protein interactions but independent of the estrogen receptor DNA binding activity.

A new aspect in glycolipid biology: glycosphingolipids as antigens recognized by T lymphocytes.

T cells may recognize a large variety of ligands with different chemical structures. Recently, glycosphingolipids have also been shown to stimulate human T lymphocytes. Recognition of glycosphingolipids is restricted by the nonpolymorphic CD1 molecules, expressed by professional antigen-presenting cells and by macrophages infiltrating inflammatory sites. CD1 molecules have a structure resembling that of classical MHC class I molecules, with the terminal extracellular domains characterized by two antiparallel alpha helices placed on two hydrophobic pockets. The glycosphingolipids bound to CD1 insert the lipid tails in the two pockets and position the hydrophilic head on the external part of CD1. The TCR interacts with aminoacids present in the two alpha helices and with residues provided by the carbohydrate moiety of glycosphingolipids and discriminates their structural variations. T cells recognizing self-glycosphingolipids release proinflammatory cytokines and may have a pathogenetic role in autoimmune diseases such as multiple sclerosis.

Presentation of the same glycolipid by different CD1 molecules.

Five CD1 molecules are expressed in humans and it is unclear whether they have specialized or redundant functions. We found that sulfatide is a promiscuous CD1-binding ligand and have isolated T cell clones that are specific for sulfatide and restricted by distinct CD1 molecules. These clones have been used to compare the capacity of different CD1 to present the same glycolipid, to induce effector functions, and to form persistent immunogenic complexes. CD1a, CD1b, and CD1c molecules similarly load sulfatide on the cell surface without processing, and prime Th1 and Th2 responses. Stimulation by sulfatide-loaded CD1a persists much longer than that by CD1b and CD1c in living cells. Use of recombinant soluble CD1a confirmed the prolonged capacity to stimulate T cells. Moreover, other glycosphingolipids bind to all CD1, which suggests the presence of additional promiscuous ligands. Thus, group I CD1 molecules present an overlapping set of self-glycolipids, even though they are quite divergent from an evolutionary point of view.

Immunity to glycolipid antigens in microbial infections.

T cells recognize ligands of different chemical structures. Recently, it has become clear that also self glycosphingolipids and bacterial lipoglycans may act as T cell stimulatory ligands. This type of antigen recognition is restricted by the non-polymorphic CD1 molecules, which have a structure resembling that of classical MHC molecules. Glycolipids insert their hydrophobic lipid tails in two pockets below the antigen-binding groove and position their hydrophilic heads on the external part of CD1 molecules. TCR interacts with these carbohydrates and discriminates their structural variations. Glycolipid-specific T cells may provide protection during bacterial and parasite infection probably with different mechanisms: by secreting pro-inflammatory lymphokines, by the direct killing of infected target cells, and by helping specific B cells in Ig production. Lipoglycans represent excellent candidates for new anti-microbial vaccines due to their wide distribution in the microbial world and their structural composition which does not change and thus cannot give rise to escape mutants. Moreover, these vaccines might induce anti-microbial protective T cell responses in the whole population due to the non-polymorphic nature of CD1 presenting molecules.

Ligand-independent assembly of recombinant human CD1 by using oxidative refolding chromatography.

CD1 is an MHC class I-like antigen-presenting molecule consisting of a heavy chain and beta(2)-microglobulin light chain. The in vitro refolding of synthetic MHC class I molecules has always required the presence of ligand. We report here the use of a folding method using an immobilized chaperone fragment, a protein disulphide isomerase, and a peptidyl-prolyl cis-trans isomerase (oxidative refolding chromatography) for the fast and efficient assembly of ligand-free and ligand-associated CD1a and CD1b, starting with material synthesized in Escherichia coli. The results suggest that "empty" MHC class I-like molecules can assemble and remain stable at physiological temperatures in the absence of ligand. The use of oxidative refolding chromatography thus is extended to encompass complex multisubunit proteins and specifically to members of the extensive, functionally diverse and important immunoglobulin supergene family of proteins, including those for which a ligand has yet to be identified.

Locally inducible CD66a (CEACAM1) as an amplifier of the human intestinal T cell response.

CD66a is an adhesion molecule member of the carcinoembryonic antigen immunoglobulin-like family present on the surface of epithelial cells, granulocytes and IL-2 activated T cells. We studied whether CD66a is expressed in vivo by T lymphocytes and whether it affects TCR-mediated activation. CD66a was detected by histochemistry, flow cytometry analysis, reverse transcription PCR and Western blot on fresh colon biopsies and T cell clones. A fraction of T cells in the lamina propria express CD66a, which is induced by IL-7 and IL-15 cytokines. T cells express four different CD66a splice variants and at least two forms of the protein are glycosylated in a cell type-specific manner. Triggering of CD66a on T cells with physiological ligands or with specific mAb increases TCR-mediated lymphokine release, in an antigen dose-independent manner. This effect requires the presence of the CD66a intracytoplasmic domain, which contains two immunoglobulin receptor family tyrosine-based inhibitory motif-like domains, as shown by stimulation of Jurkat cells transfected with different CD66a isoforms and is associated with increased induction of AP1 and NFkappaB transcription factors. These data indicate that CD66a amplifies T cell activation and thus could facilitate crosstalk between epithelial cells and T lymphocytes in intestinal immune response.

The alphabeta T cell response to self-glycolipids shows a novel mechanism of CD1b loading and a requirement for complex oligosaccharides.

The structural basis for the T cell recognition of lipoglycans remains to be elucidated. We have described autoreactive T cells responsive to GM1 ganglioside presented by CD1b. We show that glycosphingolipids bind to CD1b on the cell surface at neutral pH and are recognized without internalization or processing. Furthermore, soluble GM-CD1b complexes stimulate specific T cells. Oligosaccharide groups containing five or more sugars are required to build a minimal epitope for TCR recognition. This suggests a mechanism for T cell recognition of glycosphingolipids in which much of the CD1b-bound ligand is exposed. Binding to CD1b is a highly reversible process and other ceramide-containing glycosphingolipids displace GM1. These nonantigenic compounds act as blockers and may prevent harmful autoreactivity in vivo.

Self glycolipids as T-cell autoantigens.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by discrete areas of demyelination. An autoimmune response against components of myelin is thought to contribute to disease pathogenesis. Here we identify glycolipids as new targets recognized by T cells in multiple sclerosis patients. Circulating T cells reactive with glycolipids are more frequent in MS patients than in control donors as shown by enzyme-linked immunospot assay. They specifically recognize different types of glycolipids, such as gangliosides, sulfatide and galactosylceramide and release IFN-gamma and TNF-alpha. T cells specific for gangliosides were found to be CD8+, TCR alphabeta+, restricted by the MHC-like CD1b molecule and specific for epitopes residing in the carbohydrate moiety of gangliosides. Our findings suggest that in addition to self proteins, self glycolipids may represent potential source of autoantigens recognized by T cells in autoimmune diseases.

TTF-2 does not appear to be a key mediator of the effect of cyclic AMP on thyroglobulin gene transcription in primary cultured dog thyrocytes.

TTF-2 is a thyroid-specific winged-helix transcription factor which has been proposed to play a key role in the hormonal control of thyroglobulin and thyroperoxidase genes transcription in FRTL-5 cells. We have analyzed TTF-2 DNA-binding activity in primary cultures of dog thyrocytes maintained in control condition or in the presence of the cAMP agonist forskolin. Binding of 35S-labelled nuclear proteins to the TTF-2 recognition sequence identified the presence of two molecular species of 41.5 and 42.5 kDa. TTF-2 DNA-binding activity was clearly detectable in nuclear extracts from unstimulated cells and appeared increased in forskolin-treated cells. Thus, the presence of TTF-2 DNA-binding activity does not correlate with the cAMP-dependent activity of thyroglobulin and thyroperoxidase genes in this cell system. In addition, the mutation of the TTF-2 binding site in the thyroglobulin promoter resulted in a very reduced but still clearly cAMP-dependent promoter activity when assayed by transient expression in the same cells. These results do not support a dominant role for TTF-2 in the cAMP-dependent control of thyroglobulin gene transcription in primary cultured thyrocytes.

Major histocompatibility complex class I molecules modulate activation threshold and early signaling of T cell antigen receptor-gamma/delta stimulated by nonpeptidic ligands.

Killer cell inhibitory receptors and CD94-NKG2-A/B heterodimers are major histocompatibility complex class I-specific inhibitory receptors expressed by natural killer cells, T cell antigen receptor (TCR)-gamma/delta cells, and a subset of TCR-alpha/beta cells. We studied the functional interaction between TCR-gamma/delta and CD94, this inhibitory receptor being expressed on the majority of gamma/delta T cells. When engaged by human histocompatibility leukocyte antigen class I molecules, CD94 downmodulates activation of human TCR-gamma/delta by phosphorylated ligands. CD94-mediated inhibition is more effective at low than at high doses of TCR ligand, which may focus T cell responses towards antigen-presenting cells presenting high amounts of antigen. CD94 engagement has major effects on TCR signaling cascade. It facilitates recruitment of SHP-1 phosphatase to TCR-CD3 complex and affects phosphorylation of Lck and ZAP-70 kinase, but not of CD3 zeta chain upon TCR triggering. These events may cause abortion of proximal TCR-mediated signaling and set a higher TCR activation threshold.

Thymocytes control the CD4 gene differently from mature T lymphocytes.

We analyzed the activity of the enhancer, the promoter and the silencer of the human CD4 gene during T cell development using transgenic mice. Immunofluorescence studies on thymic populations of mice carrying transgenes in various combinations of these regulatory DNA elements revealed that thymocytes control the CD4 gene in a different manner than mature peripheral T lymphocytes. The 5'-positive regulatory unit, consisting of the promoter and the 5' enhancer, is already active at the CD4-CD8-double-negative (DN) stage of development. However, its activity becomes lower in the double-positive and a fraction of the CD4+ CD8int/- cell population, indicating that an additional enhancer, located in either the first or the third intron of the CD4 gene, is required for CD4 gene expression in this population. The other studied regulatory element is the minimal CD4 silencer which inhibits CD4 gene expression in peripheral CD8 T lymphocytes. This silencer is inactive in the most immature DN thymocytes, which probably use a distinct silencer mechanism to down-regulate CD4 gene expression. Unexpectedly, the CD4 silencer is also active in CD4+ CD8int/- cells of the thymus, implying that an anti-silencer may be required to resume CD4 expression in this cell population. Altogether, the CD4 gene is regulated by several positive and negative regulatory mechanisms which come into play in a developmentally coordinated manner.

Functional inactivation in the whole population of human V gamma 9/V delta 2 T lymphocytes induced by a nonpeptidic antagonist.

Nonpeptidic compounds stimulate human T cells bearing the TCR-gamma delta in the absence of major histocompatibility complex restriction. We report that one of these ligands, 2,3-diphosphoglyceric acid (DPG), which induces expansion of V gamma 9/V delta T cells ex vivo, antagonizes the same cell population after repetitive activation. Stimulation with DPG results in partial early protein tyrosine phosphorylation and a prolonged, but reversible, state of unresponsiveness to agonist ligands in V gamma 9/V delta 2, but not in other T cells. These findings show that TCR antagonism is a general phenomenon of T cells. However, in contrast to the clonal specificity of altered peptides antagonizing alpha beta T cells, all the tested V gamma 9/V delta 2 polyclonal cell lines and clones become unresponsive, a fact that may be relevant for the regulation of their response in vivo.

Identification and characterization of a human CD4 silencer.

Using transgenic mice, we have identified a human CD4 silencer contained within a 484-bp fragment in the first intron of the CD4 gene. Further experiments have mapped a lineage-specific silencing activity to a region of 190 bp. This region contains two protein-binding sites detected by deoxyribonuclease I footprinting analyses. Tested in transient transfection assays, these two DNA elements showed significant silencing activity restricted to the CD8 phenotype. In CD4 cells, either no clear effect (FP I) or strong enhancing activity (FP II) was observed by transient transfection assays. Despite the lineage-specific activity of these two elements, electrophoretic mobility shift assays (EMSA) showed similar levels of protein binding to the silencer element FP I in CD4 and CD8 T cells. Base substitutions in the FP I fragment abolished the silencing activity in transfected CD8 cells as well as the protein binding in EMSA, suggesting an important role of this protein-DNA interaction in CD4 gene regulation.

Human, bovine, canine and rat thyroglobulin promoter sequences display species-specific differences in an in vitro study.

The proximal promoter regions of the thyroglobulin gene from man, beef, dog and rat were compared by transient expression in primary cultured dog thyrocytes. All four promoter regions were able to control properly the expression of a reporter gene in response to cyclic AMP stimulation. Surprisingly, despite extensive sequence conservation, the transcriptional activities of these four mammalian thyroglobulin promoters were differently affected by equivalent mutations. Homologous sequence elements from these promoter regions also exhibited distinct binding characteristics in mobility-shift experiments conducted in the presence of nuclear proteins from bovine thyroids. Our observations show that the highly conserved thyroglobulin promoters may exhibit unexpected functional differences in a specific assay and indicate that some of the molecular mechanisms involved in the control of thyroglobulin gene expression have evolved differently within mammals.

Identification of a transcriptional enhancer upstream from the bovine thyroglobulin gene.

The DNA sequences corresponding to a DNaseI-hypersensitive region identified previously in bovine thyroglobulin gene chromatin (Hansen et al. (1988) Eur. J. Biochem. 178, 387-393) exhibited the properties of a transcriptional enhancer in a transient assay in primary cultured dog thyrocytes, but did not so in transfected HeLa cells. By contrast to the thyroglobulin proximal promoter, the enhancer element did not require cyclic AMP stimulation of the thyrocytes to be active. Using a bi-directional deletion approach, the minimal region displaying enhancer activity has been localized between positions -1906 and -1744 relative to the thyroglobulin gene transcription start. DNA-footprinting experiments revealed the presence of several binding sites for the thyroid-specific transcription factor TTF-1 within the enhancer sequence.

Functional role of TTF-1 binding sites in bovine thyroglobulin promoter.

We have studied the binding of purified TTF-1 on the bovine thyroglobulin gene promoter. DNase I footprinting experiments revealed three binding sites which corresponded in location to the A, B and C sites found in the rat thyroglobulin promoter. Mutants in the A and C regions showing reduced binding of TTF-1, also exhibited largely decreased promoter activity in transient expression experiments in primary-cultured dog thyrocytes. Two mutants in the B site that exhibited a reduced capacity to bind TTF-1 also displayed a drastically affected transcriptional activity in transient assays. As in the rat, sites A and C only are critical for promoter activity, these results suggest that full occupancy of the B site is required for thyroglobulin promoter activity in the cow only.

Isolation and characterization of the canine thyroglobulin gene promoter region.

The 5' flanking sequences from the canine thyroglobulin gene were isolated by homology screening with the evolutionary conserved sequence from the bovine thyroglobulin promoter and sequenced. Transient expression in primary cultured dog thyrocytes demonstrated that the canine clone contains a functional promoter inducible by cAMP. DNAse I footprinting assays showed that the thyroid-specific transcription factor TTF-1, purified from bovine thyroid, also recognizes the canine thyroglobulin promoter. Similar footprints were obtained with crude nuclear extracts from primary cultured dog thyrocytes.

Cloning and sequence analysis of TFE, a helix-loop-helix transcription factor able to recognize the thyroglobulin gene promoter in vitro.

A cDNA that encodes a transcription factor able to recognize the thyroglobulin gene promoter in vitro was isolated from a dog thyroid cDNA expression library in lambda gt11. The library was screened with a multimerized 20 bp-oligonucleotide probe corresponding to the -126 to -107 bp region of the bovine thyroglobulin gene promoter. The specificity of DNA sequence recognition was demonstrated by DNA binding experiments realized with beta-galactosidase-fusion protein immobilized on nitrocellulose filters and various unlabelled multimerized competing DNA fragments. The encoded protein, TFE, appears to be the canine counterpart of a recently cloned human transcription factor, ITF-2, that binds to the mu E5 kappa E2 motif found in both immunoglobulin heavy and light chains genes enhancers and belongs to the basic-Helix-Loop-Helix family of transcription factors. When TFE protein was produced in a rabbit reticulocyte lysate, it displayed the same specificity of DNA sequence recognition as the beta-galactosidase fusion protein and immobilization of the translation product on nitrocellulose still appeared to be essential for detecting in vitro DNA binding activity. Functional data failed to assign a role for TFE in the control of thyroglobulin gene transcription in vitro, suggesting that the selection of TFE clone resulted from the fortuitous presence of a high affinity binding site in the probe used for screening the expression library.

Sex steroids modulate the pituitary parameters involved in the regulation of TSH secretion in the rat.

The sex-related differences observed in the regulation of TSH secretion was further investigated by determination of the densities of T3 nuclear and TRH membrane receptors as well as the activity of 5'-deiodinase (5'D) in the anterior pituitary gland of adult male and female rats. The respective modulatory roles of androgens and estrogens on these parameters were evaluated by similar determinations carried out in castrated and in estrogen-treated male rats. The density of pituitary T3 and TRH receptors and the activity of 5'D type II were significantly greater in the female than in the male rats. The E2-treated male rats disclosed a female profile, viz. also greater densities of T3 and TRH receptors when compared with control male rats (2.3 +/- 0.2 vs 1.8 +/- 0.2 fmol T3/mg gland and 9.4 +/- 0.8 vs 6.0 +/- 0.8 fmol TRH/mg gland, mean +/- SEM), whereas no changes were found in the castrated rats. The E2-treated rats and the castrated rats exhibited an increased pituitary activity of 5'D, type II (0.87 +/- 0.10 and 0.66 +/- 0.05, respectively, vs control 0.34 +/- 0.07 pmol rT3.h-1.(mg protein)-1), suggestive of a stimulatory effect of E2 and of an inhibitory effect of androgens on this parameter. In contrast, no differences in hepatic 5'D were found between all groups, illustrating the well-known tissue-specific regulation of 5'D. These results demonstrate that the sex difference in the density of pituitary T3 and TRH receptors and the activity of 5'D in the adult rat is mainly due to a modulatory effect of estrogens, which may be responsible for the sex-dependent regulation of TSH secretion.