Extended Data Fig. 1: Characterization of vaccine targeting MICA/B vaccine α3 domain.
From: A vaccine targeting resistant tumours by dual T cell plus NK cell attack
a, HPLC gel filtration analysis of affinity-purified ferritin (immunogen for Ctrl-vax) and MICB-ferritin (immunogen for MICB-vax) proteins. The proteins formed nanoparticles of ~957 kDa (MICB-ferritin) and ~468 kDa (ferritin); molecular weight standards are indicated. b, SDS-PAGE analysis of purified ferritin and MICB-ferritin proteins under reducing conditions. c, Electron microscopy image showing MICB-ferritin protein assembled into nanoparticles (98,000x magnification). d, Identification of MICB transgenic (Tg) mice by PCR amplification using genomic DNA extracted from tail biopsies (lanes 2,6, MICB Tg; lane 7, positive control amplification from plasmid with the transgene cassette; lane 8, negative control reaction). e, Quantitative RT-PCR analysis of luciferase and MICB mRNA in the prostate of WT (grey) and MICB-tg (orange) mice (n = 2 WT, n = 3 MICB-Tg mice) normalized to mouse Hprt mRNA. f, Quantification of day 14 MICB-specific serum Ab titers (fluorescence-based immunoassay, DELFIA) in WT and MICB-tg mice immunized with Ctrl-vax (n = 4 mice/group) (grey) (120μg ferritin protein, 100µg CpG, 1µg GM-CSF) or MICB-vax (n = 5 mice/group) (200μg MICB-ferritin, 100µg CpG, 1µg GM-CSF) without MSR (left) or with MSR (right) scaffold. g, Quantification of MICB-specific serum Ab titers (fluorescence-based immunoassay, DELFIA) in mice immunized with Ctrl-vax (grey) or MICB-vax using differing protein doses, 50μg (yellow), 100μg (blue) or 200μg (red) (n = 2 mice/group). h, Representative flow cytometry plots showing binding of polyclonal serum Abs from mice immunized with Ctrl-vax (blue) or MICB-vax (red) to cell surface MICB on B16F10 (MICB) tumor cells; serum dilutions are indicated for each condition. i, Titers of MICB Ab isotypes assessed by ELISA (n = 5 mice/group) in MICB transgenic mice immunized with Ctrl-vax (blue) or MICB-vax (red). j, Analysis of MICB-specific CD8 T-cell responses in the spleen of mice immunized with Ctrl-vax (blue) or MICB-vax (red). Intracellular cytokine staining (IFNγ) and CFSE dilution is shown in representative flow cytometry plots (left); data are quantified for T-cells from both vaccine groups (3 mice/group, right). k, Analysis of human NKG2D dimer (left) and mouse NKG2D dimer (right) binding to cell surface MICB on B16F10 (MICB-ZsGreen) tumor cells pre-incubated with sera (5μl) from Ctrl-vax or MICB-vax mice. l, Representative flow cytometry plots showing binding of anti-human MICA/B antibody (6D4, specific for MICA/B α1-α2 domains) to cell surface MICB on B16F10 (MICB) tumor cells pre-incubated with sera from mice immunized with Ctrl-vax (blue) or MICB-vax (red). Representative data from >3 independent experiments (a, b). Data from a single experiment with technical replicates (c). Representative data from three experiments (d, e, g, h, i). Representative data from two experiments (j, k, l). Two-tailed unpaired Student’s t-test (e); Two-way ANOVA with Tukey’s multiple comparison test (f, g); Two-way ANOVA with Sidak’s multiple comparison test (i, j). Data depict mean +/− SD (e,g) or mean +/− SEM (f, i, j).
