Downregulatory Molecule Research Articles

IFN-γ production by brain-resident cells activates cerebral mRNA expression of a wide spectrum of molecules critical for both innate and T cell-mediated protective immunity to control reactivation of chronic infection with Toxoplasma gondii.

We previously demonstrated that brain-resident cells produce IFN-γ in response to reactivation of cerebral infection with Toxoplasma gondii. To obtain an overall landscape view of the effects of IFN-γ from brain-resident cells on the cerebral protective immunity, in the present study we employed NanoString nCounter assay and quantified mRNA levels for 734 genes in myeloid immunity in the brains of T and B cell-deficient, bone marrow chimeric mice with and without IFN-γ production by brain-resident cells in response to reactivation of cerebral T. gondii infection. Our study revealed that IFN-γ produced by brain-resident cells amplified mRNA expression for the molecules to activate the protective innate immunity including 1) chemokines for recruitment of microglia and macrophages (CCL8 and CXCL12) and 2) the molecules for activating those phagocytes (IL-18, TLRs, NOD1, and CD40) for killing tachyzoites. Importantly, IFN-γ produced by brain-resident cells also upregulated cerebral expression of molecules for facilitating the protective T cell immunity, which include the molecules for 1) recruiting effector T cells (CXCL9, CXCL10, and CXCL11), 2) antigen processing (PA28αβ, LMP2, and LMP7), transporting the processed peptides (TAP1 and TAP2), assembling the transported peptides to the MHC class I molecules (Tapasin), and the MHC class I (H2-K1 and H2-D1) and Ib molecules (H2-Q1, H-2Q2, and H2-M3) for presenting antigens to activate the recruited CD8+ T cells, 3) MHC class II molecules (H2-Aa, H2-Ab1, H2-Eb1, H2-Ea-ps, H2-DMa, H2-Ob, and CD74) to present antigens for CD4+ T cell activation, 4) co-stimulatory molecules (ICOSL) for T cell activation, and 5) cytokines (IL-12, IL-15, and IL-18) facilitating IFN-γ production by NK and T cells. Notably, the present study also revealed that IFN-γ production by brain-resident cells also upregulates cerebral expressions of mRNA for the downregulatory molecules (IL-10, STAT3, SOCS1, CD274 [PD-L1], IL-27, and CD36), which can prevent overly stimulated IFN-γ-mediated pro-inflammatory responses and tissue damages. Thus, the present study uncovered the previously unrecognized the capability of IFN-γ production by brain-resident cells to upregulate expressions of a wide spectrum of molecules for coordinating both innate and T cell-mediated protective immunity with a fine-tuning regulation system to effectively control cerebral infection with T. gondii.

Increased expression of regulatory T cells and down-regulatory molecules in lepromatous leprosy.

T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-γ (IFN-γ) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-β (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-β. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth.

Parasitic Helminth Cystatin Inhibits DSS-Induced Intestinal Inflammation Via IL-10+F4/80+Macrophage Recruitment

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-γ and TNF-α in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-α in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.

The modular architecture of meningococcal factor H-binding protein

Meningococcal factor H binding protein (fHbp) is a promising vaccine antigen that binds the human complement downregulatory molecule factor H (fH), and this binding enhances the survival of the organism in serum. Based on sequence variability of the entire protein, fHbp has been divided into three variant groups or two subfamilies. Here, we present evidence based on phylogenetic analysis of 70 unique fHbp amino acid sequences that the molecular architecture is modular. From sequences of natural chimeras we identified blocks of two to five invariant residues that flanked five modular variable segments. Although overall, 46 % of the fHbp amino acids were invariant, based on a crystal structure, the invariant blocks that flanked the modular variable segments clustered on the membrane surface containing the amino-terminal lipid anchor, while the remaining invariant residues were located throughout the protein. Each of the five modular variable segments could be classified into one of two types, designated alpha or beta, based on homology with segments encoded by variant 1 or 3 fHbp genes, respectively. Forty of the fHbps (57 %) comprised only alpha (n=33) or beta (n=7) type segments. The remaining 30 proteins (43 %) were chimeras and could be classified into one of four modular groups. These included all 15 proteins assigned to the previously described variant 2 in subfamily A. The modular segments of one chimeric modular group had 96 % amino acid identity with those of fHbp orthologs in Neisseria gonorrhoeae. Collectively, the data suggest that recombination between Neisseria meningitidis and N. gonorrhoeae progenitors generated a family of modular, antigenically diverse meningococcal fHbps.

Lack of association between sCTLA-4 levels in human plasma and common CTLA-4 polymorphisms

BackgroundCytotoxic T lymphocyte antigen-4 (CTLA-4) is an important downregulatory molecule expressed on both T and B lymphocytes. Numerous population genetics studies have documented significant associations between autoimmune diseases and single nucleotide polymorphisms (SNPs) within and around the CTLA-4 region of chromosome 2 in man. Furthermore, circulating levels of a soluble form of CTLA-4 (sCTLA-4) have been reported in a variety of autoimmune mediated diseases. Despite these findings, the relationship between levels of sCTLA-4 protein, mRNA transcript levels, and SNPs within the CTLA-4 region have not been clearly defined. In order to further clarify this relationship, we have tested four different SNPs within the CTLA-4 region among subjects whom are negative (n = 53) versus positive (n = 28) for sCTLA-4.ResultsOur data do not support a clear association between sCTLA-4 levels and any of the four SNPs tested.ConclusionThe variation in the SNPs tested does not appear to effect sCTLA-4 protein levels, despite reports that they affect sCTLA-4 mRNA.

Erratum to Experimental frontiers for clinical applications: Novel approaches to understanding mechanisms of lymph node metastases in melanoma

Sentinel lymph nodes are the first nodes to receive lymph from primary tumors and are the preferential site of initial metastases. Sentinel nodes show morphology changes that suggests immune modulation with reduced antigen-presenting dendritic cells, activated T lymphocytes, high endothelial venules and transvenular migration of T lymphocytes. Tumor cells generate down-regulatory molecules. We postulate that tumor-induced immune dysfunction facilitates establishment of nodal metastases. Nodal immune modulation can be reversed by granulocyte macrophage colony-stimulating factor (GMCSF), a finding with implications for future therapy to prevent or reverse these nodal metastases.

Association of T Cell Costimulatory and Downregulatory Gene Polymorphisms and Susceptibility to Multiple Myeloma.

Multiple myeloma (MM) is a B-lineage malignancy characterized by an accumulation of isotype-switched, immunoglobulin-producing monoclonal plasma cells. Additionaly phenotype and functional abnormalities of T cell are observed. The implication of T cells in the development of plasmocytoma has been shown in murine model. The study was undertaken to evaluate the association between polymorphisms of the genes encoding major T cell costimulatory (CD28, ICOS) and downregulatory (CTLA-4) molecules and susceptibility to MM in a Polish population. One hundred patients with MM and 202 healthy subjects were studied. Genomic DNA was isolated from whole frozen blood using the NucleoSpinR Blood kit. Allele identification was achieved by PCR amplification. The amplified product for SNP loci was purified and minisequenced using the commercial kit SNapShot (PE Applied Biosystems). The dinucleotide repeat polymorphism was studied by PCR and fluorescence based technique. The products were analyzed on the ABI PRISM 310 Genetic Analyzer (ABI PRISM 310 capillary electrophoresis system).We found an association of 49G in exon 1 and (AT)82 alleles in 3′untranslated region in the CTLA-4 gene with MM [p=0.018, OR=1.57, 95% CI=1.08–2.29 and p=0.0013, OR=1.85, 95% CI=1.27–2.72, respectively] while no association between T/C substitution in intron 3 of the CD28 gene, the dinucleotide (GT)n repeat polymorphism in intron 4 in ICOS gene and C/T base exchange in the promoter (position −318) in the CTLA-4 gene and MM was observed. Our results indicate that the A/G substitution in exon 1 and microsatelite polymorphism in the 3′untranslated region in the CTLA-4 gene might be a susceptibility locus for MM.The present study is the largest and comprehensive to date to evaluate the association between genetic polymorphisms of genes encoding T cell costimulatory and downregulatory molecules and susceptibility to MM.

Experimental frontiers for clinical applications: Novel approaches to understanding mechanisms of lymph Node metastases in melanoma

Sentinel lymph nodes are the first nodes to receive lymph from primary tumors and are the preferential site of initial metastases. Sentinel nodes show morphology changes that suggests immune modulation with reduced antigen-presenting dendritic cells, activated T lymphocytes, high endothelial venules and transvenular migration of T lymphocytes. Tumor cells generate down-regulatory molecules. We postulate that tumor-induced immune dysfunction facilitates establishment of nodal metastases. Nodal immune modulation can be reversed by granulocyte macrophage colony-stimulating factor (GMCSF), a finding with implications for future therapy to prevent or reverse these nodal metastases.

Major peptide autoepitopes for nucleosome-centered T and B cell interaction in human and murine lupus.

The potential cross-reactivity of normal T and B cells to nuclear antigens is vast, probably due to their "education" by apoptotic cell antigens in generative lymphoid organs. Despite this "nucleocentric repertoire," as we call it, the peripheral immune system normally remains tolerant or ignorant of the products of apoptosis. However, the T helper (Th) cells, and also B cells of lupus, have a regulatory defect in the expression of CD40 ligand (CD40L). A sustained hyper-expression of CD40L by lupus T cells can be triggered by sub-threshold stimuli, and is associated with impaired phosphorylation of Cbl-b, a critical downregulatory molecule in T cell signal transduction. This CD40L hyper-expression abnormally prolongs co-stimulatory signals to autoimmune B cells, and it probably instigates APC (dendritic cells, resting anti-DNA B cells, and macrophages) to present apoptotic cell autoantigens in an immunogenic fashion. We have identified the dominant nucleosomal epitopes that are critical for cognate interactions between autoimmune Th cells and anti-DNA B cells in lupus. By scanning of overlapping synthetic peptides, and by mass spectrometry of naturally processed peptides, five major epitopes in nucleosomal histones were localized, namely H1'(22-42), H2B(10-33), H3(85-105), H4(16-39), and H4(71-94). The autoimmune T cells as well as B cells of lupus recognize these epitopes, and with age, autoantibodies against the peptide epitopes cross-react with nuclear autoantigens. Moreover, the peptide autoepitopes can be promiscuously presented and recognized by lupus T cells in the context of diverse MHC alleles. This cross-reactivity opens up the possibility of developing "universally" tolerogenic peptides for therapy of lupus in humans despite their MHC diversity. Indeed, tolerogenic therapy with a single histone peptide epitope can halt the progression of established glomerulonephritis in lupus-prone mice by "tolerance spreading" that inactivates a broad spectrum of autoimmune T and B cells in concert.

CTLA-4 up-regulation plays a role in tolerance mediated by CD45.

Cytolytic T lymphocyte-associated antigen 4 (CTLA-4) is a critical down-regulatory molecule in T cells that plays a major role in peripheral tolerance. Although the CD45 protein tyrosine phosphatase is a potent immunomodulatory target, the mechanisms by which antibody against CD45RB isoforms (anti-CD45RB) induces allograft tolerance remain unclear. We show here that anti-CD45RB treatment alters CD45 isoform expression on T cells, which is associated with rapid up-regulation of CTLA-4 expression. These effects appear specific and occur without up-regulation of other activation markers. Administration of a blocking monoclonal antibody to CTLA-4 at the time of transplantation prevents anti-CD45RB therapy from prolonging islet allograft survival. In addition, treatment with cyclosporin A blocks anti-CD45RB-induced CTLA-4 expression and promotes acute rejection. These data suggest that anti-CD45RB acts through mechanisms that include CTLA-4 up-regulation and demonstrate a link between CD45 and CTLA-4 that depends on calcineurin-mediated signaling. They demonstrate also that CTLA-4 expression may be specifically targeted to enhance allograft acceptance.

Expression of Fas ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer

BackgroundDespite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis...

Epithelioid cells from foreign-body granuloma selectively express the calcium-binding protein MRP-14, a novel down-regulatory molecule of macrophage activation.

The S-100 proteins MRP-8 and MRP-14 are expressed by cells of the myelomonocytic lineage, either alone or simultaneously during certain stages of cellular differentiation. We demonstrate that MRP-14 but not MRP-8 was detected by immunostaining in the cytoplasm of epithelioid cells on the surface of round coverslips implanted for 14 days into the subcutaneous tissue of mice. Using this experimental model, our laboratory has previously shown that epithelioid macrophages are poor phagocytic cells that release a macrophage-deactivating factor (MDF) in short-term cultures. The full chemical characterization of MDF has not been achieved so far. We provide evidence that the calcium-binding protein MRP-14 was also released by epithelioid macrophages in short-term cultures and that its neutralization from the culture medium after addition of monoclonal antibody anti-MRP-14 abolished the MDF activity of the conditioned medium. Purified or recombinant human MRP-14 but not MRP-8 inhibits the respiratory burst of BCG-activated macrophages. Recombinant mouse MRP-14 also down-regulate macrophage activation in vitro, and PMA does not revert the inhibitory effect induced by MRP-14. It is thus concluded that epithelioid cells from foreign-body granuloma express and release MRP-14 in short-term cultures and that this molecule is endowed with MDF activity.