MHC-dependent perforin/granzyme A–mediated killing of cognate CD8⁺ T cells in MLR is associated with triggering of TLR7/8 and TREM-1. (A) CD8⁺ 2C cells stained with calcein AM green were mixed with L-NAME–pretreated CB6/F1 imDCs. imDCs were also pretreated with blocking peptides for indicated TLRs, competitive peptide for TREM-1 or TREM-2 (50 ng/mL), or neutralizing Ab for TREM-3 (100 ng/mL). After an incubation of 5 hours, the total number of stained 2C CD8⁺ T cells in the absence (gray) or presence of imDCs at cell ratio of 1:4 (black) was evaluated by FACS analysis. (B) After incubation as described in panel A, the percentage of specific BLT esterase release was calculated as described in the Methods. In parallel, imDCs were cultured with TREM-1 agonist Ab for 5 hours without 2C CD8⁺ cells, after which time the supernatant was measured for specific BLT esterase release (TREM-1 agonist). (C) FACS evaluation of total numbers of live OT-I CD8⁺ T cells in the absence (white) or presence of untreated imDCs (light gray) or imDCs treated with SIINFEKL (black) or SIYRYYGL (gray) at the indicated cell ratios (1:2 and 1:4). (D) Killing ability of imDCs from C57BL/6 wild-type or TLR7^−/− mice loaded with SIINFEKL. (E) Killing ability of imDCs from C57BL/6 wild-type or Dap12^−/− mice loaded with SIINFEKL. Shown are averages ± SD (n ≥ 3).