Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Differential Effects of Reactive Oxygen Species on IgG versus IgM Levels in TLR-Stimulated B Cells.

doi: 10.4049/jimmunol.1901131

Figure Lengend Snippet: FIGURE 3. Differential effects of ROS scavenging by NAC and VAS2870. B cells were stimulated with or without CpG-ODN and anti-RP105 as described in the legend of Fig. 1. When indicated, the cells were pretreated for 30 min with NAC (20 mM) or VAS (2.5 mM) prior to stimulation. (A and B) Supernatants were collected after 96 h of stimulation, and IgG (A) and IgM (B) levels were determined by ELISA. Each dot represents the value from one donor, and the horizontal lines represent median values. (A) n = 12 or 6 (VAS2870) and (B) n = 16 or 6 (VAS2870). *p # 0.03. (C and D) The fraction of IgG+ and CD27+ cells was determined by flow cytometry using Brilliant Violet–conjugated Abs directed against IgG and CD27 after 96 h of stimulation. Where indicated, cells were pretreated for 30 min with 20 mM NAC (n = 4, mean 6 SEM). (E and F) Activation of the cells was determined by analyses of CD80 expression (E) or by activation of NF-kB as revealed by the phosphorylation of IkB (F). (E) The fraction of CD80+ cells was determined by flow cytometry using FITC-conjugated Ab directed against CD80 after 24 h of stimulation. Upper panel, Graphs from one representative of six flow cytometry analyses. Lower panel, The horizontal lines represent median values, the boxes represent 50% of measured values, and the whiskers represent 6 1.5 interquartile range (n = 6). *p # 0.05 (Wilcoxon matched-pairs signed rank test). (F) Western blot analyses of p-IkB were performed after 30 min of stimulation. One of two reproducible blots are shown, using calnexin as a loading control. (G) At the indicated times after stimulation, proliferation was measured by the CellTiter-Glo assay. The luminescence values obtained were normalized against the values at time 0 and are presented as mean values (n = 4). (H–J) Ninety-six hours after stimulation, ELISPOT analyses of IgG and IgM were performed on equal numbers of cells. (H) The numbers of IgG- or IgM-producing cells are indicated as the number of spots per 1000 cells. (I) The amount of Ig produced per cell is indicated by the average spot sizes. (H and J) Each dot represents the value from one blood donor, and the horizontal lines represent median values (n = 9). *p # 0.03 (Wilcoxon matched-pairs signed rank test). (J) One representative plate from nine ELISPOT experiments.

Article Snippet: ELISPOTwas performed according to the manufacturer’s recommendations using kits from ImmunoSpot/Cellular Technology Limited: human IgG and IgM singlecolor ELISPOT (hIgG-SCE-1M/2 and hIgM-SCE-1M/2).

Techniques: Enzyme-linked Immunosorbent Assay, Cytometry, Activation Assay, Expressing, Phospho-proteomics, Western Blot, Control, Glo Assay, Enzyme-linked Immunospot, Produced

Journal: Cancer immunology research

Article Title: Identification of Immunogenic MHC Class II Human HER3 Peptides that Mediate Anti-HER3 CD4 + Th1 Responses and Potential Use as a Cancer Vaccine.

doi: 10.1158/2326-6066.CIR-21-0454

Figure Lengend Snippet: Figure 1. HER3 expression in cancer, and HER3 peptide screening of ECD and ICD class II peptide libraries. A, Expression of HER3 mRNA in normal (N) and tumor (T) tissues obtained from RNA-seq data from the GDC across cancer types (see Materials and Methods). B, Correlation between percentage of HER3 expression and overall patient survival (in months) in breast cancer. Samples were sorted in the descending order of HER3 expression and put into two groups: high HER3 (red) and low HER3 (blue). C, Correlation of overall patient survival with high HER3 (red) versus low HER3 (blue) expression in melanoma. P value indicated in individual graphs. D and E, IFN-g production at each screening step for sample 1 [normal donor (ND) 8; D] and sample 2 (ND 9; E) when stimulated with ECD peptides. F and G, IFN-g production at each screening step for sample 3 (ND 3; F) and sample 4 (ND 5; G) upon stimulation with ICD peptides. D–G, IFN-g response to negative peptide control (black) compared with HER3 peptides (red) with an immunogenic response threshold of ≥1.5-fold increase. Data represented as mean SEM with statistical significance determined using a multiple t test without correction for multiple comparisons. Each row was analyzed individually, without assuming consistent SD. , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: Whole HER3 ECD/ICD protein restimulation Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4þ T cells with HER3-DC1 as described above, followed by restimulation with iDCs pulsed with the corresponding HER3 class II peptide, a negative peptide control (BRAF class II p8), native HER3 ECD (cat. #NBP2-52128-0.05 mg, Novus Biologicals) or ICD (cat. #10201-H20B1, SinoBiological) whole protein sequence, or a negative whole protein control (HemocyaninKeyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: Expressing, RNA Sequencing, Control

Journal: Cancer immunology research

Article Title: Identification of Immunogenic MHC Class II Human HER3 Peptides that Mediate Anti-HER3 CD4 + Th1 Responses and Potential Use as a Cancer Vaccine.

doi: 10.1158/2326-6066.CIR-21-0454

Figure Lengend Snippet: Figure 4. Intratumoral HER3-DC1 administration elicits peptide-specific immune responses and delays tumor growth. A, Tumor growth in the 4T1 murine mammary carcinoma model. BALB/c mice bearing subcutaneous 4T1 tumors received either intratumoral PBS (black), unpulsed mature DC1 (blue), or HER3 peptide–pulsed DC1 (red; n ¼ 10 mice/group), starting on day 7 when tumors were palpable. Tumor growth was monitored until endpoint and was compared between control and HER3-DC1, as well as between unpulsed DC1 and HER3-DC1. , control versus HER3-DC1; #, unpulsed DC1 versus HER3-DC1. B, Individual tumor growth for each mouse from control (black)-, unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated groups. C, Percent survival in the 4T1 mouse model. Control, black; unpulsed DC1, blue; HER3-DC1, red. D, Intratumoral CD3þCD4þ and CD3þCD8þ T-cell infiltration per milligram of tumor in control (black)-, unpulsed DC1 (blue)–, and HER3-DC1 (red)–treated mice. Absolute number of immune cells was compared between control and HER3-DC1 groups. E, Frequency of CD62LþCD44þ central memory (CM), CD62LCD44þ

Article Snippet: Whole HER3 ECD/ICD protein restimulation Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4þ T cells with HER3-DC1 as described above, followed by restimulation with iDCs pulsed with the corresponding HER3 class II peptide, a negative peptide control (BRAF class II p8), native HER3 ECD (cat. #NBP2-52128-0.05 mg, Novus Biologicals) or ICD (cat. #10201-H20B1, SinoBiological) whole protein sequence, or a negative whole protein control (HemocyaninKeyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: Control

Journal: Cancer immunology research

Article Title: Identification of Immunogenic MHC Class II Human HER3 Peptides that Mediate Anti-HER3 CD4 + Th1 Responses and Potential Use as a Cancer Vaccine.

doi: 10.1158/2326-6066.CIR-21-0454

Figure Lengend Snippet: Figure 5. Intratumoral HER3-DC1 delays tumor growth and enhances immune infiltration in a HER2pos TUBO therapeutic model in a CD4-dependent manner. A, Tumor growth in the TUBO murine mammary carcinoma model. BALB/c mice were injected with TUBO tumor cells, and on day 7, mice received either PBS control (black), unpulsed mature DC1 (blue), or HER3-DC1 (red) intratumorally once weekly for six doses (n ¼ 10 mice/group). Tumor growth was monitored until endpoint and compared in control versus HER3-DC1 (*) and unpulsed DC1 versus HER3-DC1 (#) mice. B, Percent survival in TUBO mouse model. Control: black; unpulsed DC1: blue; HER3-DC1: red. C, CD3þCD4þ and CD3þCD8þ T cells per milligram of tumors from mice (A) after intratumoral DC injectionwas compared between control (black) and HER3-DC1 (red) groups. No statistical analyses were performed for the unpulsed DC1 (blue) mice (n ¼ 3/group). D, Abundance of CD4þ central memory (CD62LþCD44þ CM), effector memory (CD62LCD44þ EM), and effector (CD62LCD44 Eff) T cells in control (black) versus HER3-DC1 mice (red) per milligram of tumor tissue. Data shown are the representative from three independent experiments. E, Tumor growth of TUBO tumors after CD4 depletion. BALB/c mice were injected with anti-CD4 antibodies 3 days before subcutaneous TUBO tumor injection. When tumors were palpable, mice received either PBS control (black), intratumoral HER3-DC1 once weekly (red) for six doses, CD4 depletion antibody alone (blue; continued twice weekly until endpoint), or HER3-DC1 (green) with CD4 depletion. Tumor growth was monitored until endpoint. F and G, Percentage of CD4þIFN-gþ (F) and CD8þIFN-gþ (G) TILs in the tumors from control (black) versus HER3-DC1 (red) mice from E. H, Coculture of the lymph node immune cells with HER3 peptide–pulsed DC1 for 72 hours to detect IFN-g via ELISA. Control: black bar; unpulsed DC1: blue bar; HER3-DC1: red bar. I, Western blot for HER3, phosphorylated AKT (phAKT), and cleaved caspase-3 (clCasp-3) with total protein isolated from control- and HER3-DC1–treated TUBO tumors. b-Actin: loading control. J, Western blot for HER3 and phosphorylated p44/42 MAPK (ph-p44/42 MAPK) from control-, unpulsed DC1–, and HER3- DC1–treated TUBO tumors. b-Actin: loading control. Data represented as mean SEM with statistical significance determined using multiple t test without correction for multiple comparisons. Each row was analyzed individually, without assuming a consistent SD. A log-rank (Mantel–Cox) test was used to determine differences between the survival curves. , P ≤0.05; , P ≤0.01; , P ≤0.001; #, P ≤0.01.

Article Snippet: Whole HER3 ECD/ICD protein restimulation Immunogenicity of the identified HER3 class II epitopes was confirmed through sensitization of CD4þ T cells with HER3-DC1 as described above, followed by restimulation with iDCs pulsed with the corresponding HER3 class II peptide, a negative peptide control (BRAF class II p8), native HER3 ECD (cat. #NBP2-52128-0.05 mg, Novus Biologicals) or ICD (cat. #10201-H20B1, SinoBiological) whole protein sequence, or a negative whole protein control (HemocyaninKeyhole Limpet Native protein; cat. #SRP6195, Sigma).

Techniques: Injection, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Isolation

Journal: bioRxiv

Article Title: Modified E2 glycoprotein of hepatitis C virus enhances pro-inflammatory cytokines and protective immune response in mice

doi: 10.1101/2022.02.07.479452

Figure Lengend Snippet: Amino acid positions of interest for mutations in a linear map of sE2 are indicated (panel A). Comparison of human CD81 binding with modified E2 expressed transiently in HEK293T transfected cell culture supernatant with increasing volumes (panel B) and purified histidine tag recombinant proteins in a dose dependent concentration (panel C) are shown. Culture medium from mock transfected HEK293T cells or PBS were used as reagent control. The significance levels are expressed as *=p<0.05 ** = p < 0.005, *** = p < 0.001.

Article Snippet: Recombinant human CD81 protein (R &D Systems) was coated (4μg/ml) on ELISA plate (Thermo Fisher Scientific) and incubated overnight at 4 0 C. Next day, the plate was washed with PBS containing 0.05%Tween-20.

Techniques: Comparison, Binding Assay, Modification, Transfection, Cell Culture, Purification, Recombinant, Concentration Assay, Control

Journal: Brain, Behavior, & Immunity - Health

Article Title: Therapeutic nanoliposome vaccine targeting multiple Aβ and tau epitopes reduces AD-like brain pathologies and rescues cognitive deficits in 3xTg-AD mice

doi: 10.1016/j.bbih.2025.101167

Figure Lengend Snippet: SNAP-AD 5 immunization elicited antigen-specific B cell response without high antigen-specific T cell response in mice. (A) Representative ELISPOT wells showing IFN-γ–secreting splenocytes from SNAP-AD 5 immunized, individual antigen immunized, or untreated control mice following stimulation with medium, individual antigens, or a 5-plex antigen mix. (B) Quantification of IFN-γ spots from (A) were counted and analyzed by a two-way ANOVA followed by Bonferroni's multiple comparison test, comparing each experimental group to the control, ∗p < 0.05. (C) Representative ELISPOT wells showed B-cell producing IgG in blood from mice (n = 5) immunized with SNAP-AD 5 or untreated mice. Blood cells after lysis were stimulated with medium alone, 10 μg/mL of Igκ/Igλ antibodies, or 10 μg/mL 5-plex mixed antigen. (D) Quantification of B-cell producing IgG spots from (C) were analyzed by a one-way ANOVA with multiple comparison followed by Tukey's test, ∗ P<0.05, ∗∗∗∗P<0.0001 .

Article Snippet: Mouse IFN-γ Single-Color ELISpot kit (ImmunoSpot, Cat# mIFNgp-2M/10) and Mouse IgG Single-Color ELISpot kit (ImmunoSpot, Cat# mIgG-SCE-2M/10) were purchased for ELISpot assays.

Techniques: Enzyme-linked Immunospot, Control, Comparison, Lysis

Journal:

Article Title: Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase ▿

doi: 10.1128/JVI.00211-09

Figure Lengend Snippet: Soluble NKp44 and NKp46 receptors bind to various NDV-infected tumor cell lines. (A) PANC-1 pancreatic carcinoma, HeLa cervical carcinoma, A549 lung carcinoma, and Ma-Mel-8a melanoma cells were infected for 20 h with the nonlytic NDV strain Ulster (100 HU/106 cells) (black lines) or left uninfected (gray lines). Cells were stained with NKp30-, NKp44-, and NKp46-IgG1 Fc fusion proteins in complexes with goat anti-hIgG-PE secondary antibodies as indicated. Results for Ma-Mel-8a cells stained with a secondary antibody alone are represented by filled curves. To monitor infection efficiencies, tumor cells were stained with anti-NDV MAb HN.B, recognizing HN (top panels, black lines). For a control, uninfected cells were stained with HN.B (top panels, gray lines). (B) HeLa cells were infected for 18 h with the NDV strain Ulster (black lines) or left uninfected (gray lines). Cells were stained with the NKG2D-Fc fusion protein or with MAbs recognizing NDV HN, to control for infection, or the NKG2D ligands MICA, MICB, and ULBP1 to ULBP4, as indicated. Results for secondary antibody controls are represented by filled curves.

Article Snippet: Complexes of NCR-Fc fusion proteins (1 to 2 μg per staining) and PE-labeled goat anti-hIgG Fc (Dianova; 1:100 in FACS buffer) were allowed to form for 30 min before being added to cells for 60 min on ice.

Techniques: Infection, Staining

Journal:

Article Title: Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase ▿

doi: 10.1128/JVI.00211-09

Figure Lengend Snippet: NKp44-Fc and NKp46-Fc bind to NDV particle-coated plates. (A) Standard ELISA plates were coated with purified NDV (strain Ulster and MTH-68) particles, and the particles were allowed to react with NKp44-Fc and NKp46-Fc fusion proteins, followed by peroxidase-labeled anti-hIgG secondary antibody (anti-hIgG-POX). The assay results were developed using the peroxidase substrate o-phenylenediamine dihydrochloride and evaluated in an ELISA reader as the absorption (Abs.) at 450 nm. The binding of soluble NCR can be blocked by preincubation of the virus-coated plate with the anti-HN MAb HN.B but not the anti-F MAb Icii. Plate coating was checked by using anti-HN or anti-F0 MAb, followed by peroxidase-labeled anti-mouse IgG secondary antibody (anti-mIgG-POX). Wells without virus show low levels of nonspecific binding of NCR-Fc, anti-HN, anti-F, and secondary antibodies. Means and SEM for triplicate samples are shown. (B to F) Activation of NKp44-CD3ζ and NKp46-CD3ζ reporter cells by NDV-infected cells. (B, left) Schematic diagram showing NCR-CD3ζ reporter proteins. The ectodomains of NKp46, NKp44, and NKp30 were fused to the transmembrane domain (TMD) and cytoplasmic domain (cyt) of mouse CD3ζ (mCD3ζ) by using cDNA constructs. Residues of the respective proteins contained in the chimeras are indicated. (Right) BWZ.36 mouse thymoma cells harboring the lacZ gene under the control of the NFAT promoter were transfected with the cDNA constructs. Stainings with MAbs specific for NKp46 (αNKp46), NKp44 (αNKp44), and NKp30 (αNKp30) demonstrated surface expression of the indicated NCR-CD3ζ fusion proteins. (C) The indicated NCR-CD3ζ reporter lines were cocultured with uninfected HeLa cells or NDV Ulster-infected HeLa cells for 18 h, after which the lacZ response was measured with the colorigenic substrate CPRG in an ELISA reader. Absorption at 595 nm, expressed as means of triplicate values ± SEM, is shown. ***, P < 0.001; n.s., not significant. (D) Untransfected HeLa cells as well as HeLa cells transfected with HN cDNA were analyzed as described in the legend to panel C. Reporter cell stimulation by NDV-infected cells transfected with HN cDNA (HeLa/HN) and that by untreated HeLa cells were compared by Student's t test. **, P < 0.01; n.s., not significant. (E) The indicated NCR-CD3ζ reporter cells were cultured for 20 h in wells of an ELISA plate treated with coating buffer (no virus) for a control or coated with purified NDV Ulster particles. Other wells were coated with NKp30, NKp44, and NKp46 antibodies (1 μg/well) as indicated to monitor reporter cell activation following antibody-mediated NCR cross-linking.

Article Snippet: Complexes of NCR-Fc fusion proteins (1 to 2 μg per staining) and PE-labeled goat anti-hIgG Fc (Dianova; 1:100 in FACS buffer) were allowed to form for 30 min before being added to cells for 60 min on ice.

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Labeling, Binding Assay, Activation Assay, Infection, Construct, Transfection, Expressing, Cell Stimulation, Cell Culture