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Induction of Cross-Priming of Naive CD8⁺ T Lymphocytes by Recombinant Bacillus Calmette-Guérin That Secretes Heat Shock Protein 70-Major Membrane Protein-II Fusion Protein¹

Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan

Because Mycobacterium bovis bacillus Calmette-Guérin (BCG) unconvincingly activates human naive CD8⁺ T cells, a rBCG (BCG-70M) that secretes a fusion protein comprising BCG-derived heat shock protein (HSP)70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed to potentiate the ability of activating naive CD8⁺ T cells through dendritic cells (DC). BCG-70M secreted HSP70-MMP-II fusion protein in vitro, which stimulated DC to produce IL-12p70 through TLR2. BCG-70M-infected DC activated not only memory and naive CD8⁺ T cells, but also CD4⁺ T cells of both types to produce IFN-. The activation of these naive T cells by BCG-70M was dependent on the MHC and CD86 molecules on BCG-70M-infected DC, and was significantly inhibited by pretreatment of DC with chloroquine. Both brefeldin A and lactacystin significantly inhibited the activation of naive CD8⁺ T cells by BCG-70M through DC. Thus, the CD8⁺ T cell activation may be induced by cross-presentation of Ags through a TAP- and proteosome-dependent cytosolic pathway. When naive CD8⁺ T cells were stimulated by BCG-70M-infected DC in the presence of naive CD4⁺ T cells, CD62L^lowCD8⁺ T cells and perforin-producing CD8⁺ T cells were efficiently produced. MMP-II-reactive CD4⁺ and CD8⁺ memory T cells were efficiently produced in C57BL/6 mice by infection with BCG-70M. These results indicate that BCG-70M activated DC, CD4⁺ T cells, and CD8⁺ T cells, and the combination of HSP70 and MMP-II may be useful for inducing better T cell activation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by a Grant-in-Aid for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour, and Welfare of Japan.

² Address correspondence and reprint requests to Dr. Masahiko Makino, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, 4-2-1 Aobacho, Higashimurayama, Tokyo 189-0002, Japan. E-mail address: mmaki{at}nih.go.jp

³ Abbreviations used in this paper: DC, dendritic cell; BCC, Mycobacterium bovis bacillus Calmette-Guérin-derived cytosolic protein; BCG, Mycobacterium bovis bacillus Calmette-Guérin; BCG-SM, rBCG that secretes major membrane protein-II; HSP, heat shock protein; MMP, major membrane protein; MOI, multiplicity of infection.