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Heat shock protein 90 targets a chaperoned peptide to the static early endosome for efficient cross-presentation by human dendritic cells.

Tanaka T, Okuya K, Kutomi G, Takaya A, Kajiwara T, Kanaseki T, Tsukahara T, Hirohashi Y, Torigoe T, Hirata K, Okamoto Y, Sato N, Tamura Y - Cancer Sci. (2014)

Bottom Line: In this study, we found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived DCs and induced peptide-specific CTLs.Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5(+), EEA1(+) static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through an endosome-recycling pathway.Our data indicate that targeting of the antigen to a "static" early endosome by Hsp90 is essential for efficient cross-presentation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan; United Graduate School of Veterinary Sciences, Yamaguchi University, Yamaguchi, Japan.

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Cross-presentation of heat shock protein 90 (Hsp90)-chaperoned peptides by human monocyte-derived dendritic cells (Mo-DCs). (a) Human Mo-DCs (1 × 105) were pulsed with Hsp90 (400 μg/mL), precursor peptide survivin-2B75-93 (400 μg/mL) alone, a complex of Hsp90 (100 or 400 μg/mL) and survivin-2B75-93 (100 or 400 μg/mL), a simple mixture of both, or survivin-2B80-88 peptide (for positive control) for 2 h at 37°C and then fixed with 0.01% glutaraldehyde, washed, and cultured with survivin-2B80-88-specific CTL clone (1 × 105/well). Activation of CTLs was measured as γ-interferon (IFN-γ) production using ELISA. (b) Mo-DCs (1 × 105) were loaded with various doses of survivin-2B80-88 peptide (6, 25, 100, and 400 μg/mL) or Hsp90–survivin-2B75-93 precursor peptide complex (6/6, 25/25, 100/100, and 400/400 μg/mL) for 2 h in 100 μL Opti-MEM and fixed with 0.01% glutaraldehyde. The cells were washed and cultured overnight with 1 × 105 survivin-2B80-88-specific CTL clone. Activation of CTLs was measured as IFN-γ production using ELISA. Data are shown as means + SEM of three independent experiments. *P < 0.01.
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fig01: Cross-presentation of heat shock protein 90 (Hsp90)-chaperoned peptides by human monocyte-derived dendritic cells (Mo-DCs). (a) Human Mo-DCs (1 × 105) were pulsed with Hsp90 (400 μg/mL), precursor peptide survivin-2B75-93 (400 μg/mL) alone, a complex of Hsp90 (100 or 400 μg/mL) and survivin-2B75-93 (100 or 400 μg/mL), a simple mixture of both, or survivin-2B80-88 peptide (for positive control) for 2 h at 37°C and then fixed with 0.01% glutaraldehyde, washed, and cultured with survivin-2B80-88-specific CTL clone (1 × 105/well). Activation of CTLs was measured as γ-interferon (IFN-γ) production using ELISA. (b) Mo-DCs (1 × 105) were loaded with various doses of survivin-2B80-88 peptide (6, 25, 100, and 400 μg/mL) or Hsp90–survivin-2B75-93 precursor peptide complex (6/6, 25/25, 100/100, and 400/400 μg/mL) for 2 h in 100 μL Opti-MEM and fixed with 0.01% glutaraldehyde. The cells were washed and cultured overnight with 1 × 105 survivin-2B80-88-specific CTL clone. Activation of CTLs was measured as IFN-γ production using ELISA. Data are shown as means + SEM of three independent experiments. *P < 0.01.

Mentions: We first examined whether human Hsp90 facilitated cross-presentation of the chaperoned precursor peptide by human Mo-DCs. The Mo-DCs were pulsed with Hsp90 alone, the survivin-2B75-93 precursor peptide alone, a simple mixture of both, a complex of them generated in vitro at double concentration, or survivin-2B80-88 peptide (for positive control) for 2 h at 37°C and then fixed, washed, and cultured with survivin-2B80-88-specific CTL clone. The Hsp90–survivin-2B75-93 precursor peptide complex elicited a significant amount of IFN-γ production both at 100 and 100 μg/mL, whereas Hsp90 alone, survivin-2B75-93 precursor peptide alone, or a simple mixture of both did not induce IFN-γ production by CTLs (Fig.1a). Strikingly, IFN-γ production induced by Hsp90–survivin-2B precursor peptide complex was much greater than that induced by survivin-2B peptide. These results indicated that cross-presentation of survivin-2B-derived peptide was enhanced when an exogenous precursor peptide was complexed to Hsp90. To confirm these observations, we compared the efficacy of CTL activation between survivin-2B80-88 peptide and Hsp90–survivin-2B75-93 precursor peptide complex in a dose titration assay (Fig.1b). We observed that stimulation of the survivin-2B80-88-specific CTL clone with Hsp90–survivin-2B75-93 precursor peptide complex was more effective than stimulation with survivin-2B80-88 peptide at any dose.


Heat shock protein 90 targets a chaperoned peptide to the static early endosome for efficient cross-presentation by human dendritic cells.

Tanaka T, Okuya K, Kutomi G, Takaya A, Kajiwara T, Kanaseki T, Tsukahara T, Hirohashi Y, Torigoe T, Hirata K, Okamoto Y, Sato N, Tamura Y - Cancer Sci. (2014)

Cross-presentation of heat shock protein 90 (Hsp90)-chaperoned peptides by human monocyte-derived dendritic cells (Mo-DCs). (a) Human Mo-DCs (1 × 105) were pulsed with Hsp90 (400 μg/mL), precursor peptide survivin-2B75-93 (400 μg/mL) alone, a complex of Hsp90 (100 or 400 μg/mL) and survivin-2B75-93 (100 or 400 μg/mL), a simple mixture of both, or survivin-2B80-88 peptide (for positive control) for 2 h at 37°C and then fixed with 0.01% glutaraldehyde, washed, and cultured with survivin-2B80-88-specific CTL clone (1 × 105/well). Activation of CTLs was measured as γ-interferon (IFN-γ) production using ELISA. (b) Mo-DCs (1 × 105) were loaded with various doses of survivin-2B80-88 peptide (6, 25, 100, and 400 μg/mL) or Hsp90–survivin-2B75-93 precursor peptide complex (6/6, 25/25, 100/100, and 400/400 μg/mL) for 2 h in 100 μL Opti-MEM and fixed with 0.01% glutaraldehyde. The cells were washed and cultured overnight with 1 × 105 survivin-2B80-88-specific CTL clone. Activation of CTLs was measured as IFN-γ production using ELISA. Data are shown as means + SEM of three independent experiments. *P < 0.01.
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Related In: Results  -  Collection

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fig01: Cross-presentation of heat shock protein 90 (Hsp90)-chaperoned peptides by human monocyte-derived dendritic cells (Mo-DCs). (a) Human Mo-DCs (1 × 105) were pulsed with Hsp90 (400 μg/mL), precursor peptide survivin-2B75-93 (400 μg/mL) alone, a complex of Hsp90 (100 or 400 μg/mL) and survivin-2B75-93 (100 or 400 μg/mL), a simple mixture of both, or survivin-2B80-88 peptide (for positive control) for 2 h at 37°C and then fixed with 0.01% glutaraldehyde, washed, and cultured with survivin-2B80-88-specific CTL clone (1 × 105/well). Activation of CTLs was measured as γ-interferon (IFN-γ) production using ELISA. (b) Mo-DCs (1 × 105) were loaded with various doses of survivin-2B80-88 peptide (6, 25, 100, and 400 μg/mL) or Hsp90–survivin-2B75-93 precursor peptide complex (6/6, 25/25, 100/100, and 400/400 μg/mL) for 2 h in 100 μL Opti-MEM and fixed with 0.01% glutaraldehyde. The cells were washed and cultured overnight with 1 × 105 survivin-2B80-88-specific CTL clone. Activation of CTLs was measured as IFN-γ production using ELISA. Data are shown as means + SEM of three independent experiments. *P < 0.01.
Mentions: We first examined whether human Hsp90 facilitated cross-presentation of the chaperoned precursor peptide by human Mo-DCs. The Mo-DCs were pulsed with Hsp90 alone, the survivin-2B75-93 precursor peptide alone, a simple mixture of both, a complex of them generated in vitro at double concentration, or survivin-2B80-88 peptide (for positive control) for 2 h at 37°C and then fixed, washed, and cultured with survivin-2B80-88-specific CTL clone. The Hsp90–survivin-2B75-93 precursor peptide complex elicited a significant amount of IFN-γ production both at 100 and 100 μg/mL, whereas Hsp90 alone, survivin-2B75-93 precursor peptide alone, or a simple mixture of both did not induce IFN-γ production by CTLs (Fig.1a). Strikingly, IFN-γ production induced by Hsp90–survivin-2B precursor peptide complex was much greater than that induced by survivin-2B peptide. These results indicated that cross-presentation of survivin-2B-derived peptide was enhanced when an exogenous precursor peptide was complexed to Hsp90. To confirm these observations, we compared the efficacy of CTL activation between survivin-2B80-88 peptide and Hsp90–survivin-2B75-93 precursor peptide complex in a dose titration assay (Fig.1b). We observed that stimulation of the survivin-2B80-88-specific CTL clone with Hsp90–survivin-2B75-93 precursor peptide complex was more effective than stimulation with survivin-2B80-88 peptide at any dose.

Bottom Line: In this study, we found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived DCs and induced peptide-specific CTLs.Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5(+), EEA1(+) static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through an endosome-recycling pathway.Our data indicate that targeting of the antigen to a "static" early endosome by Hsp90 is essential for efficient cross-presentation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan; United Graduate School of Veterinary Sciences, Yamaguchi University, Yamaguchi, Japan.

Show MeSH
Related in: MedlinePlus