Journal: iScience

Article Title: Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy

doi: 10.1016/j.isci.2024.108836

Figure Lengend Snippet: Overview of experimental design Flow cytometric analysis revealed modulation of myeloid-lineage immune cells with IL-12 treatment. (A) The resulting systemic immune response from various IL-12 treatment strategies in healthy 7–8-week male/female BALB/c mice was analyzed via seven serial blood sampling timepoints. Timepoints included baseline (T1), 12-h (T2), day 4 (T3), day 8 (T4), day 11 (T5), day 15 (T6), and day 18 (T7), at which time mice were humanely euthanized for full necropsy following cardiac puncture. Experimental mice received either weekly (IL12ns) or daily (MTD) injections at indicated timepoints. At day 18 (T7, euthanasia), peripheral blood was collected via cardiac puncture. This was followed by necropsy to harvest heart, liver, spleen, lungs, and kidneys for histopathological analysis. NanoString nCounter analysis with a custom pro-inflammatory panel was also performed on RNA isolated from the formalin-fixed, paraffin embedded liver, spleen, and lung specimens. (B) At each sampling timepoint, peripheral blood was collected via cheek bleed (∼80 μL) for analysis via an immune diagnostic platform (IDP) consisting of PBMC spectral flow cytometric analysis, plasma cytokine/chemokine analysis, and bulk PBMC RNA-sequencing [Figure by BioRender.com]. (C–P) PBMCs isolated into single cell suspension at serial blood sampling timepoints [baseline (T1), 12-h (T2), day 4 (T3, n = 7 for saline male), day 8 (T4, n = 7 for saline male), day 11 (T5, n = 7 for saline male and 10 mg female), day 15 (T6, n = 7 for saline male and 10 mg female), and day 18 (T7, n = 7 for saline male and 10 mg female)] were analyzed by spectral flow cytometry using the appended gating strategy ( Figure S7 ). Neutrophils (C) were analyzed for expression of iNOS (D), Arg-1 (E), PD-L1 (F), CD66b (G), CD80 (H), and MHC-II (I). Polymorphonuclear myeloid derived suppressor cells (PMN-MDSC) (J) were also analyzed for expression of iNOS (K), Arg-1 (L), PD-L1 (M), CD66b (N), CD80 (O), and MHC-II (P) (male mice – left, female mice – right). Each data point represents the average percent (%) of all (live) cells for each cellular population. Unless indicated above, an n = 8 mice per group per sex were included in the analysis. Where visible, error bars represent standard error of the mean (SE). For visual clarity, findings from statistical analyses can be found in . The x axis represents the timepoint of blood collection while the y axis indicates the % of all (live) cells.

Article Snippet: CD80 Antibody, anti-mouse, PE-Vio® 770, REAfinityTM, Clone REA983 , Miltenyi Biotech , 130-116-398; RRID: AB_2727516.

Techniques: Sampling, Isolation, Formalin-fixed Paraffin-Embedded, Diagnostic Assay, Clinical Proteomics, RNA Sequencing, Suspension, Saline, Flow Cytometry, Expressing, Derivative Assay

Journal: iScience

Article Title: Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy

doi: 10.1016/j.isci.2024.108836

Figure Lengend Snippet: List of immunophenotypic markers or gene IDs, alternative names, and their descriptions

Article Snippet: CD80 Antibody, anti-mouse, PE-Vio® 770, REAfinityTM, Clone REA983 , Miltenyi Biotech , 130-116-398; RRID: AB_2727516.

Techniques: Virus, Marker, Immunopeptidomics, Binding Assay

Journal: iScience

Article Title: Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy

doi: 10.1016/j.isci.2024.108836

Figure Lengend Snippet:

Article Snippet: CD80 Antibody, anti-mouse, PE-Vio® 770, REAfinityTM, Clone REA983 , Miltenyi Biotech , 130-116-398; RRID: AB_2727516.

Techniques: Recombinant, Staining, Cell Culture, Red Blood Cell Lysis, Enzyme-linked Immunosorbent Assay, Isolation, Multiplex Assay, Software, Sterility