To analyze the influence of mTORi on antigen presentation ability, the BMMCs were treated with different mTORi (rapamycin: 10 nM; everolimus: 10 nM; temsirolimus: 2 µM) or DMSO (control group) to generate immature BMM-derived DCs. 1 μg/mL FITC-conjugated OVA short peptides (OVA257-264 [SIINFEKL]) (Invitrogen, Thermo Fisher Scientific, San Diego, CA, USA) were co-cultured with these cells on day 6 for 3 h as previously described [42 (link)].
To investigate whether the mTORi could restore the antigen presentation ability of BMM-derived DCs inhibited by tumor cells, BMM-derived DCs were co-cultured with TC-1 tumor cells and then pulsed with FITC-conjugated OVA short peptide. In brief, the BMMCs were treated with different mTORi (rapamycin: 10 nM; everolimus: 10 nM; temsirolimus: 2 µM) or DMSO (control group) to generate immature BMM-derived DCs. These cells were co-cultured with TC-1 tumor cells for 6 h and then pulsed with 1 μg/mL FITC-conjugated OVA short peptides for 3 h. All cells were stained with PE-conjugated anti-CD11c Ab (eBioscience) and evaluated by flow cytometry.
To investigate whether the mTORi could restore the antigen presentation ability of BMM-derived DCs inhibited by tumor cells, BMM-derived DCs were co-cultured with TC-1 tumor cells and then pulsed with FITC-conjugated OVA short peptide. In brief, the BMMCs were treated with different mTORi (rapamycin: 10 nM; everolimus: 10 nM; temsirolimus: 2 µM) or DMSO (control group) to generate immature BMM-derived DCs. These cells were co-cultured with TC-1 tumor cells for 6 h and then pulsed with 1 μg/mL FITC-conjugated OVA short peptides for 3 h. All cells were stained with PE-conjugated anti-CD11c Ab (eBioscience) and evaluated by flow cytometry.