Granulysin-mediated cognate immune responses are hampered in TLR4 mutant mice. C3H/HeN and HeJ mice (4 mice per group) were injected intraperitoneally either with 50 μg of OVA alone or with 15-kDa GNLY (20 μg/mouse) or alum. One week later, mice were boosted with the same combination. After 2 days, mice were killed, and the ability to induce OVA-specific splenocyte proliferation (A) and cytokine production (B) was measured. Splenocytes from individual spleens from each group (5 × 10⁵/0.2 mL/well) were stimulated with 50 μg/mL OVA for 72 hours with [³H]-TdR pulse (0.5 μCi/well) for the last 18 hours. For cytokine measurement, (1 × 10⁶/0.2 mL/well) cells from individual spleen were plated with 50 μg/mL OVA, and supernatants were collected after 72 hours. Splenocyte proliferation (mean ± SD) [³H]-TdR incorporation (counts per minute) of triplicate wells and cytokine production from individual mice of 1 experiment representative of 2 are shown. *P < .01, **P < .001, ***P < .0005. ns, not significant.