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

Article Title: Antitumor in situ vaccination effect of TNFα and IL-12 plasmid DNA electrotransfer in a murine melanoma model

doi: 10.1007/s00262-018-2133-0

Figure Lengend Snippet: Experimental design. The tested therapeutic groups were as follows: TNF, gene electrotransfer (GET) of the TNFα plasmid; IL-12 GET, GET of the IL-12 plasmid repeated twice with an interval of 6 days; and TNF + IL-12, concomitant GET of the TNFα and IL-12 plasmids, followed 6 days later by GET of the IL-12 plasmid. Additional control groups were as follows: CTRL complete control, EP electroporation only, pControl GET of a control plasmid. On days 2 and 8 tumors were harvested for determination of TNFα and IL-12 expression by RT-PCR. Tumor growth was monitored until the tumor reached a volume of 300 mm 3 , which was also used as the endpoint event for plotting the Kaplan–Meier survival curve. Mice that were tumor free for 90 days were subjected to secondary challenge with an injection of tumor cells. Tumors, blood and lymph nodes were collected on days 4 and 10. Tumors were used for histological determination of immune cell infiltration (H&E, hematoxylin and eosin staining) and immunohistochemical (IHC) determination of granzyme B-positive cells. Granzyme B ELISpot was performed on the PBMC and single-cell suspensions isolated from the lymph nodes

Article Snippet: A Mouse Granzyme B ELISpot kit (R&D Systems, Minneapolis, MN, USA) was used to detect granzyme B-positive cells in blood and lymph node samples.

Techniques: Electrotransfer, Plasmid Preparation, Control, Electroporation, Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Staining, Immunohistochemical staining, Enzyme-linked Immunospot, Isolation

Journal: Cancer Immunology, Immunotherapy

Article Title: Antitumor in situ vaccination effect of TNFα and IL-12 plasmid DNA electrotransfer in a murine melanoma model

doi: 10.1007/s00262-018-2133-0

Figure Lengend Snippet: Results of the granzyme B ELISpot performed on single-cell suspensions from the lymph nodes and PBMC 4 days after treatment. a Number of cells isolated from the lymph nodes. b Number of PBMC isolated from blood. c Number of granzyme B-positive cells in the lymph nodes after exposure to tumor cells, normalized to cells that were not stimulated with tumor cells. d Number of granzyme B-positive PBMC after exposure to tumor cells, normalized to cells that were not stimulated with tumor cells. Circles represent values for individual samples; boxes show the variance with the median values for three samples per experimental group. CTRL control group, EP application of electroporation only, TNF TNFα GET, IL-12 IL-12 GET, TNF + IL-12 TNFα and IL-12 GET

Article Snippet: A Mouse Granzyme B ELISpot kit (R&D Systems, Minneapolis, MN, USA) was used to detect granzyme B-positive cells in blood and lymph node samples.

Techniques: Enzyme-linked Immunospot, Isolation, Control, Electroporation

Journal: American Journal of Cancer Research

Article Title: Immune response evoked by tumor-associated NADH oxidase (tNOX) confers potential inhibitory effect on lung carcinoma in a mouse model

doi:

Figure Lengend Snippet: The protein expression and immune responses of the human tNOX recombinant proteins in mice. Recombinant human tNOX was expressed in E. coli and purified with His-tag purification resin. A. The expressed, purified and dialyzed proteins were resolved by SDS-PAGE and detected by western blot analysis with anti-His tag and anti-tNOX antibodies, and are shown in Lanes 1, 2 and 3, respectively. The molecular weight (KDa) marker (M) is shown at the left. Arrow indicates the tNOX protein band. B. Blood samples were collected at 0, 14, 28 and 35 days after the first vaccination and antibody titers were measured by ELISA. The specific anti-tNOX antibody response was significantly higher in the tNOX vaccine group compared with unvaccinated controls (*P < 0.05). C. Splenocytes of mice were stimulated with human tNOX protein and IFN-γ-secreting T cells were evaluated by ELISpot assay. Representative images show proliferative spots of splenocytes from mice vaccinated with tNOX (tNOX) or without tNOX (NC). D. The frequencies of tNOX-specific IFN-γ-secreting T cells was significantly higher in the tNOX vaccine group compared to the NC group (*P < 0.05). The average spot counts of the NC and tNOX vaccine groups were 67 ± 13 and 155 ± 42, respectively.

Article Snippet: ELISpot assays were performed using a Mouse IFN-γ ELISpot Development Module (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Recombinant, Purification, SDS Page, Western Blot, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Cancers

Article Title: A TLR4 Agonist Induces Osteosarcoma Regression by Inducing an Antitumor Immune Response and Reprogramming M2 Macrophages to M1 Macrophages

doi: 10.3390/cancers15184635

Figure Lengend Snippet: LipoMP-LPS induced systemic adaptive immune responses. ( A ) Flow cytometry analysis of the immune cell populations in the spleen of OsA tumor bearing rats injected with high- or low-dose LipoMP-LPS, or with PBS as controls. For each group, the modification in cells density is expressed as fold change in comparison to control group (set up arbitrary at 1). Data are presented as mean ± SEM ratio of mean percentages of positive cells among CD45+ cells, normalized to mean spleen weights. ** p < 0.005; *** p < 0.001; ( B ) Mean spleen weights in groups. Data are presented as mean ratio ± SEM of spleen weights. *** p < 0.001; ( C ) Analysis of IFN-γ secretion by total splenocytes in each groups using ELISpot assays. Data are mean ± SEM of IFN-γ spots (4 × 10 4 cells/wells in triplicate). IFN-g production (i.e., number of spots per 40,000 cell/well) was increased in LipoMP-LPS treated group compared with control; ( D ) Plasmatic levels of IFN-γ in each group. Data are mean ± SEM of plasma concentrations of IFN-γ evaluated for 4 animals/treatment group. * p < 0.05.

Article Snippet: The Rat IFN-γ ELISpot kit (R&D, Minneapolis, MN, USA) was used to measure IFN-γ released by splenocytes of 3 animals from the different treatment groups.

Techniques: Flow Cytometry, Injection, Modification, Comparison, Control, Enzyme-linked Immunospot, Clinical Proteomics