Journal: Biomedicines

Article Title: Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19

doi: 10.3390/biomedicines10030630

Figure Lengend Snippet: List of fluorochrome-labeled antibodies used in flow cytometry studies. FITC, fluorescein isothiocyanate; PE, phycoerythrin; PE/Cy7, tandem comprising phycoerythrin and cyanine 7; APC, allophycocyanin; APC/Cy7, tandem comprising allophycocyanin and cyanine 7.

Article Snippet: INF-γ , 45-15 , PE , Miltenyi Biotec , 130-113-493.

Techniques: Flow Cytometry

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: Cancer Immunology, Immunotherapy : CII

Article Title: Immunological synapse formation as a key mechanism in T cell-dependent bispecific antibody-mediated immune activation and cytotoxicity

doi: 10.1007/s00262-025-04036-w

Figure Lengend Snippet: Cytotoxic activity and inflammatory cytokine release in co-culture systems. a–c After co-culturing WT or KO, T cells, and TDB, the color reaction with WST-8 reagent in an hour was measured. The co-culture times were 24 h, 48 h, and 72 h of co-culture, respectively. Red: WT + T cell + TDB, blue: KO + T cell + TDB. Error bars represent the mean ± SD ( n = 6), ** p < 0.005, *** p < 0.0005, n.s. not significant. d Measurement of IFN-γ release by ELISA after 24 h of co-culture time. Co-culture supernatants were obtained and used for assay using the ELISA Kit. Red: WT + T cell + TDB, blue: KO + T cell + TDB, gray: negative control (T cell + TDB). Red letters are the results of the WT and N/C statistical tests, and blue letters are the results of the KO and N/C statistical tests. Error bars represent the mean ± SD ( n = 3), *** p < 0.0005, n.s. not significant. e IFN-γ release at TDB: 10 ng/mL for each co-culture time. Error bars represent the mean ± SD ( n ≧ 3), *** p < 0.0005, n.s. not significant. f Cancer cell death when the IFN-γ release measured in e is added to the standard. From left to right, IFN-γ release at 6 h, 24 h, 48 h, and 72 h was added. Error bars represent the mean ± SD ( n ≧ 3), *** p < 0.0005, n.s. not significant. g-i TNF-α version of the same experiment as d – f

Article Snippet: T cells (E:T = 5:1, 1.0 × 10 6 cells/50 μL/well) were added and co-cultured. hEx3 was added at a final concentration of 0.1–100 ng/mL in 100 μL of medium and cultured for 24 h. The culture supernatant was then collected and assayed using human IFN-gamma ELISA Kit, human TNF-alpha ELISA Kit, human CXCL10 ELISA Kit (Proteintech, Rosemont, IL), and human HMGB1 ELISA Kit (Chondrex, Woodinville, WA) according to the protocols provided.

Techniques: Activity Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Negative Control