Fig. 1. Selection of CU-T12-9 as a TLR2 signaling agonist.

(A) Role of TLRs in the innate and adaptive immunity responses. Recognition of PAMPs by TLRs expressed on APCs, such as dendritic cells, up-regulates cell surface expression of costimulatory molecules (CD80 and CD86), major histocompatibility complex class II (MHC II), and T cell receptor (TCR). Induction of CD80/86 on APCs by TLRs leads to the activation of T cells specific to pathogens. TLRs also induce expression of cytokines, such as IL-12, IL-6, and TNF-α, as well as chemokines and their receptors, triggering many other events associated with dendritic cell maturation. The above cytokines will contribute to the differentiation of activated T cells into T helper effector cells, building long-term protective immunity (51). (B) Chemical structure of CU-T12-9. (C) CU-T12-9 activates SEAP signaling in a dose-dependent manner. HEK-Blue hTLR2 cells were incubated with CU-T12-9 or GA for 24 hours, and activation was evaluated by SEAP secretion in the culture supernatants by the luminescence assay. (D) Human TLR2, TLR3, TLR4, TLR5, TLR7, and TLR8 HEK-Blue cells were incubated with CU-T12-9 (0 to 20 μM) or TLR-specific agonist for 24 hours, and activation was evaluated by the luminescence assay. (E to I) As positive control, agonists that selectively activate a specific TLR were used: TLR1/TLR2, Pam₃CSK₄ (0 to 66 nM or 0 to 100 ng/ml); (E) TLR3, polyinosinic-polycytidylic acid [poly(I:C)] (0 to 10.9 μg/ml); (F) TLR4, lipopolysaccharide (LPS) (0 to 36.5 ng/ml); (G) TLR5, FLA-BS (0 to 10 μg/ml); (H) TLR7 and (I) TLR8, R848 (0 to 6 μg/ml). Data are means ± SD of triplicate and representative of three independent experiments.