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. 2023 Apr 14;14:1104711. doi: 10.3389/fimmu.2023.1104711

Figure 6.

Figure 6

Role of type 1 IFN in remodulation of Ti-DCs in the MIP treated TME: (A) Activation and maturation markers on Ti-DCs including CD80, CD86, MHC-II and CD40 were analyzed by flow cytometry in wild type and IFNR1-/- mice. Representative histogram plots indicate MFI of each activation marker (n = 5). (B) Ex-vivo DC suppression assay: CFSE labeled tumor-specific CD3+ T cells were obtained as described earlier. These were added to culture wells containing tumor pulsed BMDCs (1:1). Sorted Ti-DCs either from Control or MIP treated tumor of wild type or IFNR1-/- mice were co-incubated with these cultures in a 1:1:1 ratio (T cell/pulsed BMDC/sorted DC). The histogram plot is a representative of CFSE dye dilution which indicates tumor specific T cell proliferation of one mouse per group (proliferation induced by tumor pulsed BMDCs in the presence of WT or IFNR1-/- Ti-DCs). The line graph represents percentage of tumor specific T cells in each generation of cell division (n = 5 mice/group). For the line graph, statistical significance was determined by two-way ANOVA. (***:p=0.0001 ; **:p<0.01) (C) Ti-DC mediated T cell proliferation assay: CFSE labeled tumor-specific CD3+ T cells were co-cultured with Ti-DCs sorted from tumor bearing wild type or IFNR1-/- mice treated with or without MIP (T cell: TI-DC ~ 10: 1) (n = 4 mice/group). (D) The importance of type 1 IFN in inducing the function of Ti-DCs after MIP immunotherapy is demonstrated by the production of IL-12 and IL-6 from Ti-DCs. (E) After MIP treatment, mRNA levels of Ccl22was determined from the tumor infiltrating DCs of WT and IFNR1-/- mice (n = 4); Intratumoral levels of CCL22 was also analyzed by ELISA in all the four groups (n = 5). All the bar graphs represent results as means ± SEM of one experiment. Statistical analysis was performed using unpaired Student’s t test (*:p<0.05, **:p<0.01, ns, non-significant). Two independent experimental repeats were generated.