TLR7/8-signaling enhances DCIR-mediated secondary CD8⁺ T-cell response by mDCs. (A) Blood-derived mDCs from an HLA-A201⁺ donor were targeted with 12nM, 2nM, or 200pM of anti–DCIR.doc-coh.FluMP complex mAb, activated with either TLR3, TLR4, or TLR7/8 agonists (poly I:C, LPS, or CL075) and cocultured with autologous CD8⁺ T cells for 10 days. Graph represents the percentage of FluMP-specific CD8⁺ T cells measured with a specific HLA-A201–FluMP(58-66) tetramer for each amount of anti–DCIR.doc-coh.FluMP complex mAb and with each DC-activator tested. DCs with no activation were used as a control: no activation (—), TLR7/8 (♦), TLR3 (*), and TLR4 (○) agonists; CL075, poly I:C, and LPS, respectively. Data are representative of 4 independent experiments with 4 different donors. The graph represents mean ± SD; n = 3. (B) Blood-derived mDCs from an HLA-A201⁺ donor were targeted with 8nM anti–DCIR.doc-coh.FluMP or IgG4.doc-coh.FluMP complex mAb, activated with TLR7/8, TLR3, and TLR4 agonists (CL075, poly I:C, and LPS, respectively) and cocultured with autologous CD8⁺ T cells for 10 days. Graph represents the percentage of FluMP-specific CD8⁺ T cells as measured with a specific HLA-A201–FluMP(58-66) tetramer. Conditions indicated in the graph are as follows: no activation, CL075 1μg/mL; poly I:C, 10 μg/mL; and LPS, 50 ng/mL. The graph represents mean ± SD; n = 3. (C) Same experiment as in panel B. Graph represents the mean percentage of FluMP-specific CD8⁺ T cells as measured with a specific HLA-A201–FluMP(58-66) tetramer. Conditions indicated in the graph are as follows: no activation; CL075-0.2μg/mL and 2 μg/mL; poly I:C, 5 μg/mL and 25 μg/mL; LPS, 10 ng/mL and 100 ng/mL.