Journal: Genome Medicine

Article Title: Cut or bind? Antigen-specific processing mechanisms define CD4 + T cell immunodominant epitopes for SARS-CoV-2 S and N proteins

doi: 10.1186/s13073-025-01577-8

Figure Lengend Snippet: Immunodominance and molecular insights on the selection of allotype-specific peptide pools. a Frequency of responder cells to each dual peptide combination measured via ELISpot (IFNg + IL-10) from PC donors. The number of spots is converted to responder cells per million of PBMCs and the frequencies determined for every individual (measured in duplicates) are shown according to the color code depicted in the legend. Dashed lines represent thresholds allowing the classification of the measured response. The first line at “10” is the maximum of responder cells detected in any negative control (HD), the second line is the minimum observed response for any combination, and the third line represents a 2-fold increase of Line 2. The dual peptide combinations are indicated on the left side and the color code of the bars indicate the distinct immunodominant responses measured (Dark gray and those immunogenic (light gray). Potential restrictions defined by IC50 determination over the two DRB1* allotypes present in these donors. Each dot represents the affinity of each peptide for either allotype as depicted by the size and color (see legend), Note that affinity differences lower than 1.5-fold are considered as possibly restricted by both allotypes (shown in green). b Antigen-intrinsic and -processing related features defining mechanistic models for peptide selection depicted as scheme. Proteolytic digestion of the two antigens tested reveals peptides resistant to proteases under the tested conditions (Res_Prot) and regions sensitive to proteases that point out at the different mechanistic models. Residues found through more than 3 peptides within series of nested peptides longer than 7 residues are considered indicative of the “First Cut” model. Disruption of series of nested peptides in more than 3 peptides are indicative of the “First Bind” model. Remaining regions with represented in more than 3 peptides with a full coverage of an antigen are considered “Privileged”. c Antigen processing mechanism and antigen-intrinsic features of every peptide tested in the dual combinations from the two model antigens. Antigen sources for every peptide are indicated as: Nu-, Sp-, o3- and Me- for Nucleocapsid, Spike, orf3a and Membrane, respectively. The first three residues of the peptide and the positions are also indicated. Res_Prot and SASA values for each candidate are compared to those of a random selection of peptides excluding all known epitopes (IEDB accession Sept. 2022) and represented according to the scale shown in the right of the panel. “+” indicates higher than median, “-“ refers to values lower than the median and “ns” stands for not significant (significance tested through a Wilcoxon Rank test)

Article Snippet: Dual secretion of IFNγ and IL-10 was determined using the enzymatic Human IFNγ/IL-10 Double-Color ELISPot Kit (Cellular Technology, Shaker Hieghts, OH, USA) and using pre-isolated CD14 + monocytes and CD4 + T cells.

Techniques: Selection, Enzyme-linked Immunospot, Negative Control, Disruption, Membrane

Journal: Mediators of Inflammation

Article Title: Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1155/2014/898630

Figure Lengend Snippet: CMV blocks IDO activity and IDO-mediated antimicrobial effects in human MSC. (a) PBL, stimulated with OKT3, were cocultured with MSC in the presence or absence of CMV. As controls UV-inactivated CMV (uvCMV), the IDO-specific inhibitor 1-L-methyl-tryptophan (1-MT; 1.5 mM), or a neutralising anti-IFN- γ antibody ( α IFN- γ ; 10 ng/mL) was used. After three days IDO activity was determined and is presented as mean kynurenine content ± SEM of three independent experiments, each done in triplicate. (b) PBL (1 × 10 5 /well), stimulated with CD3-directed mAB OKT3, were cocultured with MSC (3 × 10 4 /well) in the absence or presence of CMV (MOI 5). After three days cultures were infected with S. aureus (10–100 cfu/well) and bacterial growth was determined photometrically. As a control, cultures were supplemented with L-tryptophan (Trp; 0.6 mM) at the time point of bacterial infection. Data are given as mean OD (620 nm) ± SEM of three experiments, each done in triplicate. Significant differences ( P < 0.05) as compared to the positive control are marked by asterisks.

Article Snippet: Recombinant human IFN- γ was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Activity Assay, Infection, Control, Positive Control

Journal: Mediators of Inflammation

Article Title: Cytomegalovirus Infection Impairs Immunosuppressive and Antimicrobial Effector Functions of Human Multipotent Mesenchymal Stromal Cells

doi: 10.1155/2014/898630

Figure Lengend Snippet: CMV inhibits IDO induction by recombinant IFN- γ . (a) MSC (2 × 10 4 /well) which were infected with various amounts of CMV (MOI 0.1–10) were stimulated with IFN- γ (300 U/mL). After three days IDO activity was determined and is presented as mean kynurenine content ± SEM of three independent experiments, each done in triplicate. (b) MSC (1.5 × 10 6 /flask) were stimulated with IFN- γ (600 U/mL) in the absence or presence of CMV (MOI 5). Cells were harvested after 24 h and IDO protein was detected in Western blot analysis. β -Actin was utilized as a protein loading control, while the viral pp72 protein served as an infection control.

Article Snippet: Recombinant human IFN- γ was purchased from R&D Systems (Wiesbaden, Germany).

Techniques: Recombinant, Infection, Activity Assay, Western Blot, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: IFN? regulates retinal pigment epithelial fluid transport

doi: 10.1152/ajpcell.00255.2009

Figure Lengend Snippet: Expression and localization of IFNγ receptors. A: immunoblots of IFNγ receptor subunits 1 and 2 (IFNGR1 and IFNGR2) in human fetal and adult retinal pigment epithelium (RPE) and fetal choroidal (hfCHC) cells. M, molecular weight marker lanes; lane 1, primary hfRPE cell culture; lane 2, native human adult RPE; lane 3, primary hfCHC cells. B: immunofluorescence localization of IFNγ receptor in hfRPE cells. For IFNGR1 and IFNGR2, cross section through the z plane is shown at top. In each case, x-y plane is shown as an en face view of the apical membrane (maximum-intensity projection through the z-axis). ZO-1 (green) stains tight junctions; 4,6-diamidino-2-phenylindole (DAPI, blue) labels nuclei. Inset at higher gain shows a z-section above each panel; note that IFNGR1 and IFNGR2 are mainly located on the basolateral membrane (Ba). Ap, apical membrane.

Article Snippet: The blotted membrane was incubated with antibodies against human IFNGR1 (catalog no. MAB6731, R & D Systems, Minneapolis, MN), IFNGR2 (catalog no. sc-970, Santa Cruz Biotechnology, Santa Cruz, CA), IFNα receptor subunits 1 and 2 (IFNAR1 and IFNAR2, Abcam, Cambridge, MA), IRF-1, IRF-2, and ICSBP (Santa Cruz Biotechnology), and CFTR (CFTR antibody 217, Cystic Fibrosis Foundation Therapeutics, Bethesda, MD).

Techniques: Expressing, Western Blot, Molecular Weight, Marker, Cell Culture, Immunofluorescence, Membrane

Journal: American Journal of Physiology - Cell Physiology

Article Title: IFN? regulates retinal pigment epithelial fluid transport

doi: 10.1152/ajpcell.00255.2009

Figure Lengend Snippet: IFNγ activates JAK-STAT signaling pathway in hfRPE cells. A: IFNγ stimulated tyrosine phosphorylation of STAT1 in hfRPE cells (15 min) and can be blocked by anti-IFNGR1 blocking antibody. GAPDH was used as loading control. B: regulation of interferon regulatory factor (IRF) gene expression in hfRPE cells. Cells were treated with serum-free medium [SFM (Ctrl)], IFNγ, or cycloheximide (CHX) + IFNγ for 4 h. ICSBP, IFN consensus sequence-binding protein.

Article Snippet: The blotted membrane was incubated with antibodies against human IFNGR1 (catalog no. MAB6731, R & D Systems, Minneapolis, MN), IFNGR2 (catalog no. sc-970, Santa Cruz Biotechnology, Santa Cruz, CA), IFNα receptor subunits 1 and 2 (IFNAR1 and IFNAR2, Abcam, Cambridge, MA), IRF-1, IRF-2, and ICSBP (Santa Cruz Biotechnology), and CFTR (CFTR antibody 217, Cystic Fibrosis Foundation Therapeutics, Bethesda, MD).

Techniques: Phospho-proteomics, Blocking Assay, Control, Gene Expression, Sequencing, Binding Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: IFN? regulates retinal pigment epithelial fluid transport

doi: 10.1152/ajpcell.00255.2009

Figure Lengend Snippet: IFNγ-induced alterations in gene expression by quantitative RT-PCR

Article Snippet: The blotted membrane was incubated with antibodies against human IFNGR1 (catalog no. MAB6731, R & D Systems, Minneapolis, MN), IFNGR2 (catalog no. sc-970, Santa Cruz Biotechnology, Santa Cruz, CA), IFNα receptor subunits 1 and 2 (IFNAR1 and IFNAR2, Abcam, Cambridge, MA), IRF-1, IRF-2, and ICSBP (Santa Cruz Biotechnology), and CFTR (CFTR antibody 217, Cystic Fibrosis Foundation Therapeutics, Bethesda, MD).

Techniques: Gene Expression, Quantitative RT-PCR