Development of PLGA Nanoparticles with a Glycosylated Myelin Oligodendrocyte Glycoprotein Epitope (MOG_35–55) against Experimental Autoimmune Encephalomyelitis (EAE)

Multiple sclerosis (MS) is one of the most common neurodegenerative diseases in young adults, with early clinical symptoms seen in the central nervous system (CNS) myelin sheaths due to an attack caused by the patient’s immune system. Activation of the immune system is mediated by the induction of an antigen-specific immune response involving the interaction of multiple T-cell types with antigen-presenting cells (APCs), such as dendritic cells (DCs). Antigen-specific therapeutic approaches focus on immune cells and autoantigens involved in the onset of disease symptoms, which are the main components of myelin proteins. The ability of such therapeutics to bind strongly to DCs could lead to immune system tolerance to the disease. Many modern approaches are based on peptide-based research, as, in recent years, they have been of particular interest in the development of new pharmaceuticals. The characteristics of peptides, such as short lifespan in the body and rapid hydrolysis, can be overcome by their entrapment in nanospheres, providing better pharmacokinetics and bioavailability. The present study describes the development of polymeric nanoparticles with encapsulated myelin peptide analogues involved in the development of MS, along with their biological evaluation as inhibitors of MS development and progression. In particular, particles of poly(lactic-co-glycolic) acid (PLGA) loaded with peptides based on mouse/rat (rMOG) epitope 35–55 of myelin oligodendrocyte glycoprotein (MOG) conjugated with saccharide residues were developed. More specifically, the MOG35–55 peptide was conjugated with glucosamine to promote the interaction with mannose receptors (MRs) expressed by DCs. In addition, a study of slow release (dissolution) and quantification on both initially encapsulated peptide and daily release in saline in vitro was performed, followed by an evaluation of in vivo activity of the formulation on mouse experimental autoimmune encephalomyelitis (EAE), an animal model of MS, using both prophylactic and therapeutic protocols. Our results showed that the therapeutic protocol was effective in reducing EAE clinical scores and inflammation of the central nervous system and could be an alternative and promising approach against MS inducing tolerance against the disease

ICOS agonist treatment reduces the frequency of CD8⁺ T cells but does not affect the polyclonal CD4⁺ T cell compartment.

<p>IAV infected BALB/c mice were treated with ICOS agonist or hamster IgG isotype (control antibodies) as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100970#pone-0100970-g001" target="_blank">Figure 1A</a>. On day 7–9 post infection the percentage of CD8⁺ in BAL (day 7, pooled data from two independent experiments) and lung (day 8) as well as CD4⁺ T cells in lung (day 9) was determined by flow cytometry. The dots in all experiments represent data from individual mice (A). <i>In vitro</i> apoptosis assays were performed, as described in materials and methods, followed by annexin-V staining on live T cells and subsequent flow cytometric analysis. Dot plot graphs indicate percentages of apoptotic CD8⁺ and CD4⁺ T cells (mean of triplicate wells) plotted against increasing concentration of ICOS agonist antibody added to the culture. CD3 alone (mean of triplicate wells) represents that cells were stimulated by anti-CD3 treatment, in the absence of ICOS agonist; untreated (mean of duplicate wells) represents that cells were neither anti-CD3 stimulated nor treated with ICOS agonist. Column statistics were performed for the apoptosis assay (the data is expressed by mean/SEM) (B and C).</p

Increased frequency of T_regs and elevated IL-10 levels in mice treated with ICOS agonist during acute IAV infection.

<p>(A) Mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100970#pone-0100970-g001" target="_blank">Figure 1A</a>. On day 7, 8 and 9 post infection, mice were sacrificed and CD4⁺ T cells isolated from the lung (left panel: day 8 p.i.; right panel: day 9 p.i.), BLN (left panel: day 7 p.i.; right panel: day 9 p.i.) (pooled data from two independent experiments) and spleen (left panel: day 8 p.i.; right panel day 9 p.i.) (pooled data from two independent experiments) were analyzed for the intracellular expression of the T_reg-specific transcription factor Foxp3. (B) Bronchoalveolar lavage fluid of mice infected with IAV and treated with ICOS agonist or control antibody was collected on day 8 post infection and IL-10 concentration was determined by ELISA. The data obtained were pooled from two independent experiments (C) In addition, tryptophan (trp) and kynurenine (kyn) concentrations were determined in serum samples (pooled data from two independent experiments) and bronchoalveolar lavage fluid by reversed-phase HPLC technology. IDO enzymatic activity is estimated as kyn/trp ratio. Dots represent data obtained for individual mice. Statistical analysis was done by non-parametric Mann-Whitney test.</p

Attenuated immune-mediated pneumonia in ICOS agonist treated mice.

<p>BALB/c mice were IAV infected and treated with ICOS agonist and isotype control antibody as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100970#pone-0100970-g001" target="_blank">Figure 1A</a>. On day 7 and 9 post infection lungs were collected and subjected to histological examination. (A) A – ICOS agonist, day 7: bronchiointerstitial pneumonia, grade 1, lymphocytic and neutrophilic; alveolar infiltration, grade 1, with lymphocytes, neutrophils and fewer alveolar histiocytes; B – isotype control, day 7: bronchiointerstitial pneumonia, grade 2, lymphocytic, with scattered neutrophils; alveolar infiltration, grade 1, with lymphocytes, fewer neutrophils and alveolar histiocytes; C – ICOS agonist, day 9: bronchiointerstitial pneumonia, grade 2, lymphocytic, with scattered neutrophils; alveolar infiltration, grade 2, with lymphocytes, fewer neutrophils and alveolar histiocytes; D – isotype control, day 9: bronchiointerstitial pneumonia, grade 2.5, lymphocytic, with scattered neutrophils; alveolar infiltration grade 2.5, with lymphocytes, fewer neutrophils and alveolar histiocytes; bronchial epithelial necrosis, grade 2; E – ICOS agonist, day 7: bronchointerstitial pneumonia, grade 1; F – isotype control, day 7: bronchointerstitial pneumonia, grade 3; G- ICOS agonist, day 7: Alveolar infiltration, grade 1; H - isotype control, day 7: Alveolar infiltration, grade 3; I - ICOS agonist, day 7: Necrosis of bronchial epithelium, grade 1; J – isotype control, day 7: Necrosis of bronchial epithelium: grade 2. (B and C) Inflammation score for alveolar infiltration, broncho-interstitial pneumonia, affected lung tissue and bronchiola & alveolar necrosis on day 7 and 9, respectively, post IAV infection. 1 = mild; 1.5 = mild to moderate; 2 = moderate; 2.5 = moderate to severe; 3 = severe; % = area of affected tissue; n = 5 mice in the ICOS agonist treated group, n = 3 mice in the isotype treated group. (D) Bronchoalveolar lavage fluid was collected on day 8 post IAV infection and concentration of IL-6, IFN-γ (data obtained were pooled from two independent experiments) and TNF-α was determined by ELISA. Dots represent data obtained for individual mice. Statistical analysis was done by non-parametric Mann-Whitney test.</p

Schematic representation of the experimental procedure.

<p>(A) BALB/c mice were infected intranasal with a sub-lethal dose of IAV on day 0. Intraperitoneal injection of ICOS agonistic antibody or hamster IgG isotype control antibody was performed on day 1 (200 µg/mouse) and day 5 (100 µg/mouse) post infection followed by body weight monitoring for 2 weeks. For functional analyses, mice were sacrificed and analyzed between day 7 and 9 post infection, i.e. during the peak of adaptive immunity. (B) Mice were IAV infected and treated with ICOS agonist (n = 5) or PBS (n = 5) as described above and body weight was monitored over a time period of two weeks post IAV infection (non-parametric Mann-Whitney test was used to observe weight difference between two groups).</p

Data_Sheet_1.PDF

Class IA phosphatidylinositol 3-kinase (PI3K) catalytic subunits p110α and p110δ are targets in cancer therapy expressed at high levels in T lymphocytes. The role of p110δ PI3K in normal or pathological immune responses is well established, yet the importance of p110α subunits in T cell-dependent immune responses is not clear. To address this problem, mice with p110α conditionally deleted in CD4+ and CD8+ T lymphocytes (p110α−/−ΔT) were used. p110α−/−ΔT mice show normal development of T cell subsets, but slightly reduced numbers of CD4+ T cells in the spleen. “In vitro,” TCR/CD3 plus CD28 activation of naive CD4+ and CD8+ p110α−/−ΔT T cells showed enhanced effector function, particularly IFN-γ secretion, T-bet induction, and Akt, Erk, or P38 activation. Tfh derived from p110α−/−ΔT cells also have enhanced responses when compared to normal mice, and IL-2 expanded p110α−/−ΔT CD8+ T cells had enhanced levels of LAMP-1 and Granzyme B. By contrast, the expansion of p110α−/−ΔT iTreg cells was diminished. Also, p110α−/−ΔT mice had enhanced anti-keyhole limpet hemocyanin (KLH) IFN-γ, or IL-4 responses and IgG1 and IgG2b anti-KLH antibodies, using CFA or Alum as adjuvant, respectively. When compared to WT mice, p110α−/−ΔT mice inoculated with B16.F10 melanoma showed delayed tumor progression. The percentage of CD8+ T lymphocytes was higher and the percentage of Treg cells lower in the spleen of tumor-bearing p110α−/−ΔT mice. Also, IFN-γ production in tumor antigen-activated spleen cells was enhanced. Thus, PI3K p110α plays a significant role in antigen activation and differentiation of CD4+ and CD8+ T lymphocytes modulating antitumor immunity.</p

Delayed virus clearance as a consequence of ICOS agonist treatment during acute IAV infection in mice.

<p>(A) BALB/c mice were infected and treated with ICOS agonist or control antibody as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100970#pone-0100970-g001" target="_blank">Figure 1A</a>. On day 9 post infection, influenza-specific <i>in vivo</i> CTL assays were performed, as described in materials and methods. Dot plots represent percentage specific lysis of peptide-loaded APCs by IAV-specific cytotoxic T cells in ICOS agonist and isotype control treated mice. Every dot represents data obtained for an individual mouse. (B) BALB/c mice were treated as described before with ICOS agonist antibodies and the respective isotype control antibodies. On day 7, 8 and 9 post IAV infection mice were sacrificed and NP copy numbers as an indicator for viral load was determined in the lung by quantitative RT-PCR. Dots represent data obtained for individual mice. The data were pooled from two independent experiments. Statistical analysis was done by non-parametric Mann-Whitney test.</p

Co-inherited mutations of Fas and caspase-10 in development of the autoimmune lymphoproliferative syndrome-1

<p><b>Copyright information:</b></p><p>Taken from "Co-inherited mutations of Fas and caspase-10 in development of the autoimmune lymphoproliferative syndrome"</p><p>http://www.biomedcentral.com/1471-2172/8/28</p><p>BMC Immunology 2007;8():28-28.</p><p>Published online 13 Nov 2007</p><p>PMCID:PMC2211507.</p><p></p>d for 6 days in RPMI 1640 +10% FCS+rIL-2 (2 U/mL). Before activation (day 0), and at day 3 and day 6 of culture, cells were stained with a FITC-conjugated anti-Fas mAb and analyzed with a cytofluorimeter. The shows the cytofluorimetric staining of cells from Pt.1, Pt.2, and a control donor after 6 days of culture. The shows the MFI ratio calculated for each subject at different times of culture. Control data are the medians ± interquartile ranges (25–75% range) from 5 control donors; their 5percentile value at day 6 was MFI-R = 6.48. b) Caspase-10 activity was evaluated in PHA-activated T cells cultured for 12 days (see Methods) in RPMI 1640 +10% FCS+rIL-2 (10 U/mL) and then treated or not with an anti-Fas mAb for 3 hours. Results are expressed as relative caspase activity % calculated as follows: (result displayed by each subject/mean of the results displayed by the 2 controls run in the same experiment) × 100; 100% indicates the mean of the results obtained with the 2 control donors run in parallel with the patient samples in each experiment; the dotted horizontal lines indicate the 5percentile of the activity displayed by all normal controls. The color code is the same in all panels

Analysis of mixed bone marrow chimeric mice.

Analysis of spleen cells of Rag2-/-γc-/-recipient mice reconstituted with 107 donor BM cells containing a 1:1 mixture of B6.SJL.CD45.1+ (WT CD45.1+) plus ICOS−/−CD45.2+ BM cells. (A) The scheme depicts the mixed BM mouse model generated. (B) Representative dot-plot of the spleen lymphoid cells from one mixed BM chimeric mouse nine weeks after being transferred. ICOS staining for the CD45.1+ (blue) and CD45.2+ (red) populations is shown at the right; isotype staining control in grey. (C) Analysis of the frequency of positive cells or median of fluorescence intensity (MFI) of the markers NK1.1, ICOS-L and CD11b in the spleen NK cells of mixed BM chimeric mice. NK cells were selected as CD3-NK1.1+ILC. (D) Analysis of the relative number of T (CD3+NK1.1-), B (CD19+) and DCs (CD11chigh) cells, and ICOS expression in T cells in the spleen of mixed BM chimeric mice. B6.SJL.CD45.1+ (denoted as WT, white circles) and ICOS-KO CD45.2+ (KO, black circles). Data (mean±SEM) from two reconstitution experiments, each with four animals, are shown. The eight chimeric mice were analyzed individually. Wilcoxon–matched rank test was used to compare paired results of CD45.1 vs. CD45.2 cells in each mouse. Significant p value as indicated in the graphs.</p

DataSheet_1_Inducible T-Cell Costimulator Mediates Lymphocyte/Macrophage Interactions During Liver Repair.pdf

The liver capacity to recover from acute liver injury is a critical factor in the development of acute liver failure (ALF) caused by viral infections, ischemia/reperfusion or drug toxicity. Liver healing requires the switching of pro-inflammatory monocyte-derived macrophages(MoMFs) to a reparative phenotype. However, the mechanisms involved are still incompletely characterized. In this study we investigated the contribution of T-lymphocyte/macrophage interaction through the co-stimulatory molecule Inducible T-cell co-stimulator (ICOS; CD278) and its ligand (ICOSL; CD275) in modulating liver repair. The role of ICOS/ICOSL dyad was investigated during the recovery from acute liver damage induced by a single dose of carbon tetrachloride (CCl4). Flow cytometry of non-parenchymal liver cells obtained from CCl4-treated wild-type mice revealed that the recovery from acute liver injury associated with a specific up-regulation of ICOS in CD8+ T-lymphocytes and with an increase in ICOSL expression involving CD11bhigh/F4-80+ hepatic MoMFs. Although ICOS deficiency did not influence the severity of liver damage and the evolution of inflammation, CCl4-treated ICOS knockout (ICOS-/-) mice showed delayed clearance of liver necrosis and increased mortality. These animals were also characterized by a significant reduction of hepatic reparative MoMFs due to an increased rate of cell apoptosis. An impaired liver healing and loss of reparative MoMFs was similarly evident in ICOSL-deficient mice or following CD8+ T-cells ablation in wild-type mice. The loss of reparative MoMFs was prevented by supplementing CCl4-treated ICOS-/- mice with recombinant ICOS (ICOS-Fc) which also stimulated full recovery from liver injury. These data demonstrated that CD8+ T-lymphocytes play a key role in supporting the survival of reparative MoMFs during liver healing trough ICOS/ICOSL-mediated signaling. These observations open the possibility of targeting ICOS/ICOSL dyad as a novel tool for promoting efficient healing following acute liver injury.</p