granzyme b  (R&D Systems)

Journal: Annals of neurology

Article Title: Pathogenic Potential of Eomesodermin-Expressing T-Helper Cells in Neurodegenerative Diseases.

doi: 10.1002/ana.26920

Figure Lengend Snippet: FIGURE 2: Cytotoxic characteristics of eomesodermin (Eomes)-expressing T helper (Th) cells, and the relationship between Eomes expression and clinical information of neurodegenerative disease. (A) Representative flow cytometry plots showing the sorting protocol of CCR5+CX3CR1+ cells and CCR5CX3CR1 cells, and the pattern of CD107a expression in those cells after stimulation. (B) Representative flow cytometry plots showing patterns of granzyme B and Eomes expression in CD3+CD4+CD8 Th cells from the peripheral blood of an amyotrophic lateral sclerosis (ALS) patient and a healthy control participant (HC). (C) Quantification of the proportion of granzyme B+ cells in CD3+CD4+ cells from ALS (left, n = 28), Alzheimer’s disease (AD; right, n = 20), and age-matched HC participants for each group (for ALS, n = 25; for AD, n = 19); ***p < 0.001, *p < 0.05; Mann–Whitney U test. (D) Correlation analysis between the proportion of granzyme B expression in Eomes+ Th cells and the frequency of Eomes+ Th cells; p < 0.0001, r2 = 0.36, Spearman’s rank correlation coefficient. (E) Comparison of granzyme B spot counts in the ELISpot assay of CD3+CD4+ cells from the peripheral blood after stimulation for 3 days; ALS, n = 5; AD, n = 5; HC, n = 5; **p < 0.01; Kruskal–Wallis test with Dunn’s multiple comparison test. (F) Relationship between disease duration and Eomes expression in ALS patients; p = 0.49, r2 = 0.02, Spearman’s rank correlation coefficient. (G) Quantification of Eomes expression in Th cells in ALS patients with tracheostomy and invasive ventilation (TIV; n = 4), non-invasive ventilation (NIV; n = 10), and without any ventilatory support (non-NIV, n = 14); p = 0.97; Kruskal–Wallis test. (H) Relationship between disease duration and Eomes expression in AD patients; p = 0.30, r2 = 0.06, Spearman’s rank correlation coefficient. (I) Relationship between Eomes expression and scores of the Hasegawa Dementia Scale-Revised (HDS-R, left) or Mini-Mental State Examination (MMSE, right), both of which are screening tests for cognitive function with a maximum score of 30; p = 0.036, r2 = 0.22 (left), p = 0.11, r2 = 0.14 (right); Spearman’s rank correlation coefficient. n.s. = not significant.

Article Snippet: CD4+ T cells were isolated with a MACSxpress whole blood CD4 T cell isolation kit (Miltenyi Biotec) and incubated for 3 days using Human Granzyme ELISpot Kit (Diaclone, Besançon, France).

Techniques: Expressing, Cytometry, Control, MANN-WHITNEY, Comparison, Enzyme-linked Immunospot

Journal: Journal for Immunotherapy of Cancer

Article Title: Nanoparticles targeting immune checkpoint protein VISTA induce potent antitumor immunity

doi: 10.1136/jitc-2024-008977

Figure Lengend Snippet: Survival study in the YUMM1.7 melanoma model in mice. Mice-bearing YUMM1.7 tumors were treated when the tumors were ~60 mm 3 . The dual-LNPs were injected intratumorally (IT) weekly for a total of 4 weeks at a dose containing 4 µg of CPG and 10 µg of VISTA siRNA. Mice were IT injected with VISTA-siRNA-only LNPs at a dose of a 10 µg siRNA 3 days after a dual-LNP treatment. Similar schedule/dose were used for the control treatments. ( A ) Tumor growth curves for groups treated with dual LNP (n=21 mice), control LNP (VISTA siRNA+non-stimulatory GPC; n=5 mice), control LNP (CPG+non-targeting siRNA; n=5 mice), vehicle LNP (n=5 mice), CPG and VISTA mAb (n=5 mice), or PBS (n=13 mice). ( B ) Kaplan-Meier survival. ( C ) Tumor growth curves of the complete responders and non-complete responders in the dual-LNP-treated group. ( D ) Kaplan-Meier survival of complete and non-complete responders in the dual-LNP-treated group. ( E ) A subset of complete responders (n=5) was rechallenged with YUMM1.7 cells on their opposite flank 30 days after completion of the dual-LNP treatments. The tumor growth was compared with naïve controls (n=7). ( F ) Kaplan-Meier survival of rechallenged mice. A subset of complete responders (n=5) was used for ELISPOT assays. CD4 and CD8 T cells were isolated 12 days following rechallenge. T cells were stimulated with irradiated YUMM1.7 cells (100 Gy). ( G ) ELISPOT results for Granzyme B from lymphatic CD8 + T cells following rechallenge. ( H ) ELISPOT results for IFN-γ from splenic CD8 + T cells following rechallenge. Statistics were analyzed by Student’s t-test. LNP, lipid nanoparticle; VISTA, V-domain immunoglobulin suppressor of T cell activation; PBS, phosphate-buffered saline.

Article Snippet: Cells were seeded in ELISPOT kits for IFN-γ (CTL) or Granzyme B ELISPOT (R&D Systems) at a density of 300,000 effector cells (from spleen or lymph node) and 10,000 stimulant cells (irradiated YUMM1.7 cells).

Techniques: Injection, Control, Enzyme-linked Immunospot, Isolation, Irradiation, Activation Assay, Saline