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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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Related in: MedlinePlus

Memory CD8+ T cells activated by cross-presentation of heat shock protein 90 (Hsp90)–peptide complex became functional peptide-specific CTLs. CD8+ T cells separated from PBMCs (5 × 103 cells/well) from patient 1 (Table1) were stimulated with human monocyte-derived dendritic cells loaded with survivin-2B80-88 (400 μg/mL), Hsp90 (400 μg/mL), precursor peptide survivin-2B75-93 (400 μg/mL), and Hsp90 (100 or 400 μg/mL)–survivin-2B75-93 (100 or 400 μg/mL) complex, were added to each well along with HLA-A24-transfected T2 (T2-A24) cells (5 × 104 cells/well) that had been preincubated with survivin-2B80-88 (10 μg/mL) or HIV with an HIV peptide as a negative control (−). After incubation in a 5% CO2 humidified chamber at 37°C for 24 h, the wells were washed vigorously five times with PBS and incubated with a biotinylated anti-human γ-interferon (IFN-γ) antibody and HRP-conjugated avidin. Spots were visualized and analyzed using KS ELISPOT. Data are shown as means + SEM of three independent experiments. *P < 0.01.
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fig03: Memory CD8+ T cells activated by cross-presentation of heat shock protein 90 (Hsp90)–peptide complex became functional peptide-specific CTLs. CD8+ T cells separated from PBMCs (5 × 103 cells/well) from patient 1 (Table1) were stimulated with human monocyte-derived dendritic cells loaded with survivin-2B80-88 (400 μg/mL), Hsp90 (400 μg/mL), precursor peptide survivin-2B75-93 (400 μg/mL), and Hsp90 (100 or 400 μg/mL)–survivin-2B75-93 (100 or 400 μg/mL) complex, were added to each well along with HLA-A24-transfected T2 (T2-A24) cells (5 × 104 cells/well) that had been preincubated with survivin-2B80-88 (10 μg/mL) or HIV with an HIV peptide as a negative control (−). After incubation in a 5% CO2 humidified chamber at 37°C for 24 h, the wells were washed vigorously five times with PBS and incubated with a biotinylated anti-human γ-interferon (IFN-γ) antibody and HRP-conjugated avidin. Spots were visualized and analyzed using KS ELISPOT. Data are shown as means + SEM of three independent experiments. *P < 0.01.

Mentions: To further confirm whether survivin-2B-specific CD8+ T cells activated by Hsp90-mediated cross-presentation were functional or not, we carried out an ELSPOT assay using CD8+ T cells from a patient who had been vaccinated with survivin-2B peptide with incomplete Freund's adjuvant. Figure3 shows that stimulation of CD8+ T cells from the patient with Hsp90–survivin-2B75-93 precursor peptide complex clearly increased functionally positive survivin-2B-specific CD8+ T cells compared with stimulation with survivin-2B75-93 precursor peptide or survivin-2B80-88 peptide. When CD8+ T cells from the patient were stimulated with Hsp90 (400 μg/mL)–precursor peptide (400 μg/mL) complex, the number of IFN-γ-positive spots was less than that of CD8+ T cells stimulated with Hsp90 (100 μg/mL)–precursor peptide (100 μg/mL) complex. These results were due to the formation of fused large spots that were observed when stimulated with Hsp90 (400 μg/mL)–precursor peptide (400 μg/mL) complex and therefore the number of ELISPOT counted became smaller than that of Hsp90 (100 μg/mL)–precursor peptide (100 μg/mL) complex. These findings indicated that Hsp90–peptide complex is efficiently cross-presented by human Mo-DCs and is capable of stimulating peptide-specific CTLs.


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)

Memory CD8+ T cells activated by cross-presentation of heat shock protein 90 (Hsp90)–peptide complex became functional peptide-specific CTLs. CD8+ T cells separated from PBMCs (5 × 103 cells/well) from patient 1 (Table1) were stimulated with human monocyte-derived dendritic cells loaded with survivin-2B80-88 (400 μg/mL), Hsp90 (400 μg/mL), precursor peptide survivin-2B75-93 (400 μg/mL), and Hsp90 (100 or 400 μg/mL)–survivin-2B75-93 (100 or 400 μg/mL) complex, were added to each well along with HLA-A24-transfected T2 (T2-A24) cells (5 × 104 cells/well) that had been preincubated with survivin-2B80-88 (10 μg/mL) or HIV with an HIV peptide as a negative control (−). After incubation in a 5% CO2 humidified chamber at 37°C for 24 h, the wells were washed vigorously five times with PBS and incubated with a biotinylated anti-human γ-interferon (IFN-γ) antibody and HRP-conjugated avidin. Spots were visualized and analyzed using KS ELISPOT. Data are shown as means + SEM of three independent experiments. *P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4317773&req=5

fig03: Memory CD8+ T cells activated by cross-presentation of heat shock protein 90 (Hsp90)–peptide complex became functional peptide-specific CTLs. CD8+ T cells separated from PBMCs (5 × 103 cells/well) from patient 1 (Table1) were stimulated with human monocyte-derived dendritic cells loaded with survivin-2B80-88 (400 μg/mL), Hsp90 (400 μg/mL), precursor peptide survivin-2B75-93 (400 μg/mL), and Hsp90 (100 or 400 μg/mL)–survivin-2B75-93 (100 or 400 μg/mL) complex, were added to each well along with HLA-A24-transfected T2 (T2-A24) cells (5 × 104 cells/well) that had been preincubated with survivin-2B80-88 (10 μg/mL) or HIV with an HIV peptide as a negative control (−). After incubation in a 5% CO2 humidified chamber at 37°C for 24 h, the wells were washed vigorously five times with PBS and incubated with a biotinylated anti-human γ-interferon (IFN-γ) antibody and HRP-conjugated avidin. Spots were visualized and analyzed using KS ELISPOT. Data are shown as means + SEM of three independent experiments. *P < 0.01.
Mentions: To further confirm whether survivin-2B-specific CD8+ T cells activated by Hsp90-mediated cross-presentation were functional or not, we carried out an ELSPOT assay using CD8+ T cells from a patient who had been vaccinated with survivin-2B peptide with incomplete Freund's adjuvant. Figure3 shows that stimulation of CD8+ T cells from the patient with Hsp90–survivin-2B75-93 precursor peptide complex clearly increased functionally positive survivin-2B-specific CD8+ T cells compared with stimulation with survivin-2B75-93 precursor peptide or survivin-2B80-88 peptide. When CD8+ T cells from the patient were stimulated with Hsp90 (400 μg/mL)–precursor peptide (400 μg/mL) complex, the number of IFN-γ-positive spots was less than that of CD8+ T cells stimulated with Hsp90 (100 μg/mL)–precursor peptide (100 μg/mL) complex. These results were due to the formation of fused large spots that were observed when stimulated with Hsp90 (400 μg/mL)–precursor peptide (400 μg/mL) complex and therefore the number of ELISPOT counted became smaller than that of Hsp90 (100 μg/mL)–precursor peptide (100 μg/mL) complex. These findings indicated that Hsp90–peptide complex is efficiently cross-presented by human Mo-DCs and is capable of stimulating peptide-specific CTLs.

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