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: Lupus science & medicine

Article Title: Impaired regulatory function of granzyme B-producing B cells against T cell inflammatory responses in lupus mice.

doi: 10.1136/lupus-2023-000974

Figure Lengend Snippet: Figure 1 B cells from the mice spleen produced granzyme B (GrB) spontaneously. (A) Spleen single-cell suspensions were isolated from B6 mice and incubated with brefeldin A (BFA) (10 µg/mL), ionomycin (1 µg/mL) and phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) for 5 hours. The expression of GrB in CD19+ B cells was detected by staining with anti-CD19, anti- CD3ε, anti-CD49b and anti-GrB. FACS gating strategy for identifying the expression of GrB on CD19+ B cells was shown. (B) Flow cytometry-sorted CD19+ B cells (1×106) from the spleen of B6 mice were set to detect the mRNA expression of GrB by PCR. (C) Freshly purified CD19+ B cells (2 × 105; middle) from B6 spleen were cultured with CpG (10 µg/mL) stimulation on mice GrB-specific ELISpot plates for 24 hours. Medium (left) and CD8a+ T cells (right) were used as blank control and positive control, respectively. Dots were counted and the representative of independent data from five different B6 mice was shown (p<0.001). ***p<0.001 (Student’s t-test C).

Article Snippet: Mouse GrB Antibody (Cat# AF1865), Normal Goat IgG Control (Cat# AB- 108- C) and the Mouse GrB ELISpot Development Module (Cat# SEL1865) were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Produced, Isolation, Incubation, Expressing, Staining, Flow Cytometry, Purification, Cell Culture, Enzyme-linked Immunospot, Control, Positive Control

Journal: Lupus science & medicine

Article Title: Impaired regulatory function of granzyme B-producing B cells against T cell inflammatory responses in lupus mice.

doi: 10.1136/lupus-2023-000974

Figure Lengend Snippet: Figure 4 Reduced granzyme B (GrB)-producing Breg cells in lupus mice. Bm12 mice spleen lymphocytes (1.2×108 cells) were injected intravenously into indicated animals (aged 6–8 weeks). Representative anti-ANAs (p<0.001) (A) staining and ELISA analysis of anti-double-stranded DNA (anti-dsDNA) (p=0.002) (B) of serum from mice described in A–B at 14 days. (C) The frequencies of GrB-producing Breg cells were assayed by flow cytometry in lupus (n=10), and naïve mice (n=10), the representative dots (left) and statistical results were shown (right) (p=0.001). Purified CD19+ B cells from lupus (n=5) and naïve mice (n=5) were subjected to detection of mRNA expression of GrB by PCR (left) (D) and quantitative PCR (right) (p=0.037) (E). CD19+ B cells (2.5×105 cells/well) from lupus (n=5) and naïve mice (n=5) were cultured with CpG stimulation (10 µg/mL) on specific mice GrB ELISpot plates for 24 hours. The representative figures (left) and statistical results (right) were shown (p<0.001) (F). *p<0.05, **p<0.01, ***p<0.001 (Student’s t-test C, E, F and Mann-Whitney U test A, B).

Article Snippet: Mouse GrB Antibody (Cat# AF1865), Normal Goat IgG Control (Cat# AB- 108- C) and the Mouse GrB ELISpot Development Module (Cat# SEL1865) were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Injection, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Purification, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunospot, MANN-WHITNEY

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