Fig. 6: Macrophages and NK cells are required for trained immunity to enhance vaccine efficacy.
A Dosing scheme for macrophage and NK cell depletion in CT26-bearing mice (Created in BioRender. Hamdan, F. (https://BioRender.com/sckv9it). B Tumor sizes from n = 8 mice per group (“****” p < 0.0001). C UMAP projection of CD8⁺ splenocytes at day 23 (n = 3 per group). D Heat maps showing CD8⁺ T-cell memory subsets stimulated with PBS or Syl peptide (n = 6 per group). E Dosing scheme for macrophage and NK adoptive transfer (Created in BioRender. Hamdan, F. (https://BioRender.com/knbij2y). F Tumor sizes from n = 5 mice per group. G Heat maps of CD8⁺ T-cell memory subsets stimulated with Syl peptide (n = 3 per group). H Quantification of IFN-γ⁺ CD8⁺ splenocytes after Syl stimulation at day 23 (n = 3 per group) (PC + Trained Macrophages vs. Mock + Macrophages p = 0.0001; vs. Mock + Trained Macrophages p = 0.0003; vs. Mock + NK p = 0.0001; vs. Mock + Trained NK p = 0.0001; vs. PC + Macrophages p = 0.0021; vs. PC + NK p = 0.0003; vs. PC + Trained NK p = 0.0003). I Quantification of TNF-α⁺ CD8⁺ splenocytes after Syl stimulation at day 23 (n = 3 per group) (PC + Trained Macrophages vs. Mock + Macrophages p = 0.0003; vs. Mock + Trained Macrophages p = 0.0006; vs. Mock + NK p = 0.0031; vs. Mock + Trained NK p = 0.0014; vs. PC + Macrophages p < 0.0001; vs. PC + NK p = 0.0015; vs. PC + Trained NK p = 0.0190). J Schematic of NK chemotaxis transwell assay (Created in BioRender. Hamdan, F. (https://BioRender.com/8yxqt1n): CFSE-labeled NK cells placed in the upper chamber migrated toward macrophages stimulated with Ad5/3; NK cells were treated with 10 µg/mL anti-CXCR3 antibody (n = 3 technical replicates). K Confocal images of CFSE-labeled NK cells migrating toward macrophages. L DC chemotaxis assay in which NK cells stimulated with Ad5/3 were treated with 10 µg/mL anti-CCL5 antibody (n = 3 technical replicates). M Confocal images of CFSE-labeled DCs migrating toward NK cells. Statistical significance was assessed with two-way ANOVA for tumor growth along with a Šídák’s multiple comparisons test. A one-way ANOVA for flow cytometry and cell migration analyses along with a Tukey’s multiple comparisons test. All data are presented as the mean ± SD. Source data are provided as a Source Data file.
