Fig. 10

Bisp-AFN is superior in the induction of tumor-specific CTLs. A-C Flow cytometry analysis of gp100-specific CD8⁺ T cell proliferation in draining LN of B16-bearing mice p.l. injected with PBS (grey) or Bisp-AFN (green) at days 7 and 9 after tumor inoculation. One day prior to immunization, gp100-specific CD8⁺ T cells (pMel) were adoptively transferred in B16 tumor-bearing mice. Data show percentages of gp100-specific CD8⁺ T cells that have undergone at least one division (A), stacked bar chart representing the percentage of cells per division peak (B) and representative flow cytometry proliferation profile for each treatment (C). Shown is a summary of two independent experiments (6/group/experiment). D, E B16-bearing mice were p.l. injected at days 7 and 9 after tumor inoculation with the indicated conditions. Five days after the first delivery, the killing potency of the induced tumor-specific CD8⁺ T cells was analyzed in draining LN by in vivo cytotoxicity assay (D). The correlation between tumor size at time of read out and % specific lysis was plotted (E). A summary of two independent experiments is shown (5–6/group/experiment). F Frequencies of the top-10 largest T cell clones within the top-100 most-abundant TCRB sequences in the draining LN (left) and tumor (right). Note that colors do not represent the same sequence in different bars. G, J Frequency distribution plots of TCRB V/J pairing usage within the top-100 most-abundant LN sequences (G) and tumors sequences that also appear in the LN (J). H, K Average TCRB V/J pairing usage frequencies within the top-100 most-abundant LN sequences (H) and tumors sequences that also appear in the LN (K). Corresponding circos plots for each treatment group are shown next to the heatmaps. I Amount of TCRB sequences from the draining LN that are also retrieved in the matched tumor (left) with relative frequencies of these sequences after treatment with Bisp-AFN. A summary of three mice per group was analysed. Results show bar charts of individual values with mean ± SEM (A, D). An unpaired nonparametric Mann–Whitney t-test (A) or parametric t-test (D) was performed depending on Shapiro Wilk normality test * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001