Extended Data Fig. 8: Impact of MDK on immune-based therapies.
(a) Growth curves of B16.F10-OVA inoculated in mice vaccinated with IgG-OVA (black), vaccinated with αDEC-205-OVA (green), or vaccinated with αDEC-205-OVA + rMDK (purple). Samples sizes: IgG-OVA control, n = 3; αDEC-205-OVA vaccination, n = 7; αDEC-205-OVA vaccination + rMDK, n = 7. Representative experiment from two repetitions. (b) Immune staining intratumoral CD8+ cells (pink) in B16.F10-OVA inoculated in mice, vaccinated with αDEC-205-OVA or vaccinated with αDEC-205-OVA + rMDK. Scale bars = 50 μm. Shown are representative images from 6 lesions per condition. (c) Individual fold change (Log2 scale) in tumor volume after treatment (day 24 post-treatment vs day of initiation of the treatment) with CD40 agonist or IgG2a (5 mg kg−1) in mice bearing B16.R2L-shCtrl or shMdk. Samples sizes are: shCtrl + αCD40, n = 7; shCtrl + IgG2a, n = 6; shMdk + αCD40, n = 9; and shMdk + IgG2a, n = 6. (d) Percentage of PD-1+ and CD44+ CD8+ T cells in blood from mice inoculated with B16.R2L-shCtrl or shMdk cells and treated with the CD40 agonist antibody or IgGa (5 mg kg−1). Statistical significance was defined by unpaired two-tailed t-test. Sample size: shCtrl + CD40, n = 5; shCtrl + IgG2a, n = 5; shMdk + CD40, n = 6; and shMdk + IgG2a, n = 5. Violin plots show median values (center dash-line), quartiles (dotted-lines) and minimum and maximum values (edges of the violin), with individual values plotted. (e) Individual Log2 fold change in tumor volume after treatment with IgG2a isotype control (5 mg kg−1) or with αPD-1 antibody (clone RMP1-14; 5 mg kg−1) in mice bearing B16.R2L-shCtrl or -shMdk. Data at day 20. Sample size was: shCtrl + IgG2a (n = 5); shCtrl + αPD-1 (n = 6); shMdk + IgG2a (n = 6); and (shMdk + αPD-1; n = 6). (f) Quantification by flow cytometry of circulating cDC1s (CD3-CD19-F4/80-CD11c+MHC-II+CD24+Sirp1a-) in blood in wild-type (WT) and Batf3-/- mice implanted with B16.R2L-shCtrl or B16R2L-shMdk treated with IgG2a control (5 mg kg−1) or with αPD-1 antibody (clone RMP1-14; 5 mg kg−1). Samples sizes are WT + shCtrl + IgG2a, n = 4; WT + shMdk + IgG2a, n = 5; Batf3-/- + shCtrl + IgG2a, n = 3; Batf3-/- + shMdk + IgG2a, n = 6; WT + shCtrl + αPD-1, n = 6; WT + shMdk + αPD-1, n = 6; Batf3-/- + shCtrl + αPD-1, n = 5; and Batf3-/- + shMdk + aPD-1, n = 6. (g) Survival curves and (h) individual tumor growth curves of wild-type (WT) and Batf3-/- mice implanted with B16.R2L-shCtrl or B16R2L-shMdk treated with IgG2a control (5 mg kg−1) or with αPD-1 antibody (clone RMP1-14; 5 mg kg−1). Log-rank (Mantel-Cox) test was performed to compare survival among groups. Samples sizes are WT + shCtrl + IgG2a, n = 11; WT + shMdk + IgG2a, n = 16; Batf3-/- + shCtrl + IgG2a, n = 13; Batf3-/- + shMdk + IgG2a, n = 10; WT + shCtrl + αPD-1, n = 8; WT + shMdk + αPD-1, n = 7; Batf3-/- + shCtrl + αPD-1, n = 5; and Batf3-/- + shMdk + aPD-1, n = 6. Treatments were performed every three days. Data summarizes results from two independent experiments.
