Figure 6.

Ovalbumin–heat shock protein 70 (OVA‐HSP70) can induce specific CTLs without Toll‐like receptors (TLR)/MyD88 and CD40 signaling. On day 0, recipient mice were transferred with OT‐I CD8⁺ T cells and simultaneously vaccinated with 10 μg OVA‐HSP70. On day 3, mice were injected i.v. with two kinds of target cells. One was labeled with a high concentration of carboxyfluorescein succinimidyl ester (CFSE) and pulsed with 1 μM OVA _257–264, the other was labeled with a low concentration of CFSE and not pulsed with peptide. Twenty hours later, recipient mice were killed and the frequency of the two types of target cells remaining in the spleen was analyzed by flow cytometry (a). Bone marrow dendritic cells from C57BL/6 or MyD88 KO mice were incubated with OVA‐HSP70 for 3 h then fixed. The bone marrow dendritic cells and OT‐I CD8⁺ T cells were then co‐cultured in 96‐well round‐bottom plates. Supernatants were collected and the amount of γ‐interferon (IFN‐γ) was measured by ELISA assay (b). C57BL/6 and MyD88 KO mice were vaccinated three times in a 1‐week interval with 10 μg OVA‐HSP70. Seven days after the final immunization, recipient mice were killed and the spleen cells were stimulated with OVA _257–264 in vitro. Induction of antigen‐specific CD8⁺ T cells was detected with H‐2K ^b/OVA _257–264‐specific tetramers by flow cytometry (c), and CTL activity of the splenocytes was measured by ⁵¹ Cr release assay (d). E.G7, OVA cDNA transfected derivative of the EL4 cell line; EL4, methylchoranthlene‐induced thymoma of C57BL/6 (H‐2^b) origin; E/T, effector/target.