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: FASEB bioAdvances

Article Title: AJP001, a novel helper T‐cell epitope, induces a humoral immune response with activation of innate immunity when included in a peptide vaccine

doi: 10.1096/fba.2019-00056

Figure Lengend Snippet: Vaccination with AJP001 induces antibody production and T‐cell activation in mice. A. Ang II, a mixture of Ang II and AJP001 or the AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA three times at 2‐week intervals. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). B, C. The AJP001‐Ang II conjugate vaccine (AJP001‐Ang II 100 μg) was administered intracutaneously to 7‐week‐old female BALB/cA (B) or 7‐week‐old male C57BL/6 (C) mice three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum sample collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). D. The AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA mice at a dose of 20, 100, or 500 μg per mouse three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was measured by ELISA. The titers are expressed as the dilution fold of the serum giving half‐maximal absorbance at 450 nm. All data are expressed as the mean ± SD (n = 3). * P < .05, ** P < .01 and *** P < .001 vs the saline group. E, F. Antigen‐specific activation of T cells in AJP001‐Ang II‐immunized mice was evaluated by an ELISpot assay. Splenocytes were isolated from AJP001‐Ang II‐immunized mice and stimulated with Angiotensin II or AJP001 at a concentration of 10 μg/mL. PMA and ionomycin (100 ng/m each) were added to positive control wells, and medium was added as a negative control. The number of IFN‐γ‐ (E) or IL‐4‐producing (F) cells was detected by counting spots using a stereomicroscope. The number of spots was quantified in the duplicate or triplicate wells of each mouse. The data represent the mean ± SD (n = 3). * P < .05 and ** P < .01 vs the saline group

Article Snippet: Mouse IFN‐γ ELISpot Development Module, Mouse IL‐4 ELISpot Development Module and ELISpot Blue Color Module were obtained from R&D Systems, Inc.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Adjuvant, Saline, Enzyme-linked Immunospot, Isolation, Concentration Assay, Positive Control, Negative Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Notch1 blockade by a novel, selective anti-Notch1 neutralizing antibody improves immunotherapy efficacy in melanoma by promoting an inflamed TME.

doi: 10.1186/s13046-024-03214-5

Figure Lengend Snippet: Fig. 5 Anti-N1 promotes CD8+ T cytotoxicity. A, B) Flow cytometry for markers of cell growth (Ki67), activity (CD44/CD69), exhaustion (PD-1), degranula tion (CD107a), IFNγ and TNFα in CD8+ T cells unstimulated or stimulated with PMA (50ng/ml) + ionomycin (500ng/ml) for 4 h in vitro. C) Granzyme B+ CD8 T cells treated as in A-B. Dots were counted by ELISpot. D) Expression of active Notch1 (NIC), the Notch1 selective target SNAP23 and the Notch2 selective target BCAT2 in CD8+ T cells. Data are the mean of 3 independent experiments. E, F) IFNγ and GZB ELISpot data from treated tumors. Data are the mean of two independent experiments each containing 5 tumors. P values were calculated by the Student’s t test. G) Naïve YUMM2.1 melanoma cells were co-cultured with TILs extracted from YUMM2.1 tumors treated with IgG or anti-N1, and APCs at a 5:1 ratio, then treated with 10ug/ml anti-N1 for an additional 12 h. Cells were then harvested for flow cytometry. Data are the % of alive cells in the anti-N1 group normalized to control (IgG), which was set at 1 for all treatments. Data are the mean of three independent experiments. Combo = melanoma + TILs + APCs

Article Snippet: IFNγ and GrzB expression in co-cultures were assessed using Mouse ELISpot Development Module (R&D Systems).

Techniques: Flow Cytometry, Activity Assay, In Vitro, Enzyme-linked Immunospot, Expressing, Cell Culture, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting

doi: 10.1016/j.omtm.2017.01.003

Figure Lengend Snippet: Differential Impacts of Rap + Pred on Bone Marrow Antibody-Secreting Cells (A–E) WT mice were immunized with an i.p. injection of rAAV9 vector, and then they were treated with rapamycin (R, 2 mg/kg, every other day) and prednisolone (P, 0.75 mg/kg, daily) via i.p. injection (R + P), beginning at 4 weeks post-immunization. Controls were matched naive and AAV9-immunized mice without IS treatment. Bone marrow (BM) cells were assayed at 8 weeks of IS treatment by ELISpot for (A, B, and D) IgG-secreting and (C and E) AAV9-Ab-secreting plasma cells (PCs). (B and C) p = 0.04 and p = 0.13 R + P versus non-IS. Naive, non-immunized WT mice; non-IS, non-IS-treated AAV9-immunized mice; R + P, AAV9-immunized mice treated with R + P.

Article Snippet: To assess a frequency of antibody-secreting cells (ASCs) among total bone marrow cells, IgG-ELISpot assays were performed using Mouse IgG B cell ELISpot kits (SELB004 and SEL002, R&D Systems), following the procedures provided by the manufacturer.

Techniques: Injection, Plasmid Preparation, Enzyme-linked Immunospot, Clinical Proteomics