csb e08718h mouse ifn gamma granzyme b dual color elispot kit r d systems  (R&D Systems)

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

Journal: Nature Cancer

Article Title: An anti-PD-1–GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy

doi: 10.1038/s43018-022-00334-9

Figure Lengend Snippet: a – c , Growth inhibition of CT26 ( a ), EMT6 ( b ) and JC cells ( c ) in syngenic mice following indicated treatments and doses (IP frequency indicated by arrows). Each point on the curve represents the mean tumor volume for each group ( n = 7 mice for CT26 and n = 10 for EMT6 and JC). d , e , Flow cytometry analysis of draining lymph nodes collected from CT26 and JC models (24 h post second dose). ICOS + and Ki67 + are shown as a percentage of CD8 + T cells (CT26 ( d ) ( n = 3 mice) and JC ( e ) ( n = 4 mice)). f , g , CT26 tumor growth rechallenge study ( f ) ( n = 7 mice) and accumulation of MuLV gp70-antigen-specific T cells ( g ) in a fully regressed CT26 model following treatment with anti-muPD-muGITR-L bispecific ( n = 4 mice). h – k , Number of CT26-specific GZMB + CD8 + T cells (TDLNs ( h ) ( n = 3 mice)), percentage of CT26 cell killing measured by caspase-3/7 staining ( i ), percentage of CT-26 specific GZMB + CD8 + T cells (TILS ( j ) ( n = 5 mice)) and GZMB + shown as percentage of CD3 − CD49b + NK and CD8 + T cells in the tumor ( k ) ( n = 5 mice). l , Growth inhibition of EMT6 cells in syngeneic mice by anti-muPD-muGITR-L bispecific and 1:1 combination following in vivo depletion of CD8 + and CD4 + T cells ( n = 10 mice). m , n , Tumor NanoString analysis of CD8a ( m ) and GZMB genes ( n ) (CT26 model, n = 5 mice). Each point on the curve represents the mean tumor volume for each group. a – h , j – n , Data presented as mean ± s.e.m. Statistical significance was calculated by two-way ANOVA with Tukey’s correction for multiple comparisons (statistics refer to anti-PD-1–GITR-L bispecific versus combination).

Article Snippet: The flow cytometry gating strategy is described in Extended Data Fig. . CT26-specific, GZMB-positive cells were quantified in CD8 + T cells isolated from TDLNs using the mouse GZMB ELISPOT kit (catalog no. XEL1865, R&D).

Techniques: Inhibition, Flow Cytometry, Staining, In Vivo