Figure 4.

Increased frequency of migDCs in tumor-draining lymph nodes after IM-T9P1-ASO therapy. (A) Total number of migratory DCs (migDCs—CD11c^int MHC-II^hi cells), resident DCs (rDCs—CD11c^hi MHC-II^int), macrophages (F4/80⁺ cells), and B cells (CD19⁺ cells) in MC38 dLN and ndLN, counted by flow cytometry 2 days after IM-T9P1-ASO or Ctr-ASO treatment (n=5 mice/group). (B) Representative flow cytometry histograms of PD-L1 surface expression in migDCs and rDCs 8 days after Ctr-ASO or IM-T9P1-ASO treatment. (C) Flow cytometry analysis showing the CD80:PD-L1 expression ratio on migDCs and rDCs 8 days after Ctr-ASO or IM-T9P1-ASO therapy (n=5 mice/group). (D) Proportion of PD-1⁺ Tim3^– CD8⁺ T cells after Ctr-ASO or IM-T9P1-ASO therapy at the indicated time points (n=5 mice/group). (E) Lymphocyte trafficking was inhibited in MC38 tumor-bearing mice using FTY720 during IM-T9P1-ASO therapy as illustrated (n=11 mice/group). Kaplan-Meier survival curves with the respective number of tumor-free surviving mice are shown. Data are presented as mean±SEM. For comparisons between two groups, Student’s two-tailed t-test was used. For comparisons between multiple groups, one-way analysis of variance was used. For survival analyses, the Mantel-Cox log-rank test was used. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ASO, antisense oligonucleotides; cDC, conventional DC; CpG, cytosine-phosphate-guanine; DC, dendritic cell; dLN, draining lymph node; FMO, fluorescence minus one; MHC, major histocompatibility complex; ndLN, non-draining LN; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand 1; s.c., subcutaneously; Tim3, T cell immunoglobulin and mucin-domain containing-3