Extended Data Fig. 2: Characterization of vaccine-induced immune responses.
From: A vaccine targeting resistant tumours by dual T cell plus NK cell attack
a, b, Inhibition of MICA/B shedding and surface stabilization by vaccine-induced Abs. Flow cytometric analysis of cell surface MICA/B levels (a) and shed MICA/B in supernatants (b) for human A375 melanoma, A549 lung carcinoma, HCT116 colon carcinoma and K562 myelogenous leukemia cell lines 24 h following incubation with 10 µl of sera from Ctrl-vax (blue) or MICB-vax (red) immunized mice; isotype control Ab staining shown in grey (a). c, d, Inhibition of MICB shedding and surface stabilization by vaccine-induced Abs on mouse tumor cell lines. Flow cytometric analysis of cell surface MICB levels (c) and shed MICB in supernatants (d) for mouse B16F10 (MICB) melanoma, EL4 (MICB) lymphoma and 4T1 (MICB) triple negative breast cancer cell lines 24 h following incubation with 10 µl of sera from Ctrl-vax (blue) or MICB-vax (red) immunized mice. Isotype control Ab staining shown in grey (c). e–g. Vaccine efficacy at different MICB expression levels induced in tumor cells using a doxycycline (dox) inducible promoter. Representative flow cytometry histograms showing MICB surface expression levels on B16F10 (MICB-dox) tumors in vivo in mice treated with PBS (blue histogram) or different concentrations of doxycycline (dox): low dox (2.5mg/kg, orange), medium dox (5mg/kg, red) or high dox (10mg/kg, green) or control B16F10 (Ctrl-dox) tumors treated with high dox (10mg/kg, grey) (e). Analysis of B16F10 (MICB-dox) tumor growth kinetics at different MICB expression levels by tumor cells. Mice received Ctrl-vax (grey, blue, black) or MICB-vax (orange, red, green) on day 0 and a boost on day 14. B16F10 (MICB-dox) tumor cells were implanted on day 21, and MICB expression was induced on tumor cells on day 25 when tumors were palpable by treating mice with different concentrations of doxycycline as indicated (f) (n=7 mice/group). Quantification of serum levels of shed MICB in mice immunized with Ctrl-vax (grey, blue, black) or MICB-vax (orange, red, green) 96 h post dox-mediated induction of MICB on B16F10 (MICB-dox) tumor cells. Serum levels of shed MICB were analyzed in 5 randomly selected mice in each group (g). h–i, Representative flow cytometry histogram showing surface MICB levels on B16F10 (MICB) clone G12 (red) or indicated pooled clones (gradient of turquoise). Grey histogram represents isotype antibody staining of B16F10 (MICB) clone G12 (h). Assessment of therapeutic efficacy of MICB-vax (red, green) or Ctrl-vax (blue, grey) in mice with tumors established with B16F10 (MICB) clone G12 or pooled clones (B3, A3, C6, B1, G1) (n=8 mice/group) (i). j–n, Assessment of vaccine efficacy targeting MICA or MICB α3 domains. The MSR scaffold was formulated with antigens, GM-CSF and CpG. Mice received one or two doses of Ctrl-vax, MICB-vax (j–m) or MICA-vax (n) and were then challenged with B16F10 tumor cells expressing MICB (allele 005) (j, k) or MICA (allele 009) (m, n) or EL4 tumor cells expressing MICB (allele 005) (l). Vaccination and tumor challenge schedule is illustrated above each experiment; n = 7 mice/group (j–l), n = 6 mice in Ctrl-vax, n = 8 mice in MICB-vax (m) and n = 6 mice/group (n). For experiments shown in (j), tumor-free mice were rechallenged on day 120 using the same dose of B16F10 (MICB) tumor cells as in the initial inoculation. Representative data from two experiments (a–d, j–n). Data from single experiment (e–g, h–i). Two-tailed unpaired Student’s t-test (b, d); one-way ANOVA with Tukey’s multiple comparison test (g); two-way ANOVA with Bonferroni’s post hoc test (f, i (left), j (left), k. l, m, n); log-rank (Mantel-Cox) test (i (right), j (right). Data depict mean +/− SD (b, d) or mean +/− SEM (f, g, i–n).
