HS is a potent stimulator of alloreactive T-cell responses through the TLR4- and MyD88-dependent activation of DCs. (A) TLR and NLR agonists were assayed in allogeneic T-cell proliferation assay between purified T cells (2 × 10⁵/well) from C57BL/6 mice and bone marrow–derived BALB/c DCs (2.5 × 10⁴/well). Cells were cocultured either alone (media) or in the presence of LPS (100 ng/mL), Pam3CSK4 (2 μg/mL), hyaluronan (HA; 100 μg/mL), sonicated-HA (sHA; 100 μg/mL), fibronectin (FN; 100 μg/mL), fibrinogen (Fbn; 100 μg/mL), HS (100 μg/mL), Hsp70 (5 μg/mL), HMGB1 (1 μg/mL), C12-iE-DAP (1 μg/mL), or L18-MDP (1 μg/mL) for 72 hours and then pulsed [³H]thymidine for 16 hours. Proliferation was determined by ³H incorporation and results are expressed as cpm ± SEM. Baseline alloreactivity is indicated by the dotted line (*P < .05 compared with media alone). (B) Proliferation performed as described in panel A ± the addition of the LPS inhibitor polymyxin B (PMB; 10 μg/mL; *P < .05). (C) Proliferation assay performed as described in panel A with purified responder T cells (R) from either WT (+) or MyD88^−/− (−) C57BL/6 mice were cocultured with DC stimulators (S) from either WT (+) or MyD88^−/− (−) BALB/c mice (*P < .05 compared with media alone in S+/R+ group. (D-E) Analysis of proliferation and IFN-γ production in proliferation assays performed as described in panel A using WT, TLR4^−/−, and MyD88^−/− BALB/c DCs as stimulators and purified C57BL/6 T cells as responders (*P < .05). Results are representative of 3 independent experiments.