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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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Peptide-specific precursor CTLs were activated by cross-presentation of heat shock protein 90 (Hsp90)–peptide complex. PBMCs were isolated from patient 1 suffering from colon cancer (Table1) who had been vaccinated with survivin-2B80-88 peptide in our clinical study. The patient's PBMCs were shown to contain the survivin-2B-specific CD8+ T cells. PBMCs were stimulated with human monocyte-derived dendritic cells loaded with survivin-2B80-88 (400 μg/mL), Hsp90 (400 μg/mL), survivin-2B75-93 precursor peptide (400 μg/mL), and Hsp90 (400 μg/mL)–survivin-2B75-93 precursor peptide (400 μg/mL) complex in AIM V medium containing 10% human serum and interleukin-2 (50 U/mL) for 7 days. The stimulated PBMCs were stained with HIV tetramer (tet) or survivin-2B tetramer at 37°C for 20 min. Then a phycoerythrin-Cy5-conjugated anti-CD8 antibody was added at 4°C for 30 min. Cells were washed twice with PBS. After washing, cells were fixed with 0.5% paraformaldehyde and analyzed by flow cytometry using FACSCalibur and CellQuest software. CD8+ living cells were gated, and cells labeled with survivin-2B tetramer were referred to as tetramer-positive cells. The frequency of CTL precursors was calculated as the number of tetramer-positive cells divided by the number of CD8+ cells. Data are shown as means + SEM of three independent experiments. *P < 0.01.
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fig02: Peptide-specific precursor CTLs were activated by cross-presentation of heat shock protein 90 (Hsp90)–peptide complex. PBMCs were isolated from patient 1 suffering from colon cancer (Table1) who had been vaccinated with survivin-2B80-88 peptide in our clinical study. The patient's PBMCs were shown to contain the survivin-2B-specific CD8+ T cells. PBMCs were stimulated with human monocyte-derived dendritic cells loaded with survivin-2B80-88 (400 μg/mL), Hsp90 (400 μg/mL), survivin-2B75-93 precursor peptide (400 μg/mL), and Hsp90 (400 μg/mL)–survivin-2B75-93 precursor peptide (400 μg/mL) complex in AIM V medium containing 10% human serum and interleukin-2 (50 U/mL) for 7 days. The stimulated PBMCs were stained with HIV tetramer (tet) or survivin-2B tetramer at 37°C for 20 min. Then a phycoerythrin-Cy5-conjugated anti-CD8 antibody was added at 4°C for 30 min. Cells were washed twice with PBS. After washing, cells were fixed with 0.5% paraformaldehyde and analyzed by flow cytometry using FACSCalibur and CellQuest software. CD8+ living cells were gated, and cells labeled with survivin-2B tetramer were referred to as tetramer-positive cells. The frequency of CTL precursors was calculated as the number of tetramer-positive cells divided by the number of CD8+ cells. Data are shown as means + SEM of three independent experiments. *P < 0.01.

Mentions: As we had shown that the Hsp90–survivin-2B75-93 precursor peptide complex was efficiently cross-presented, we next examined whether cross-presentation of Hsp90–peptide complex could activate and expand peptide-specific memory CD8+ T cells from patients who had been vaccinated with survivin-2B peptide with incomplete Freund's adjuvant. Activated and expanded survivin-2B-specific CD8+ T cells were detected by tetramer staining. As shown in Figure2, the survivin-2B75-93 precursor peptide chaperoned by Hsp90 was able to activate and expand survivin-2B-specific memory CD8+ T cells more vigorously than was the precursor peptide alone. Interestingly, peptide-specific T-cell frequency was higher when stimulated with Hsp90–survivin-2B75-93 precursor peptide complex than that with survivin-2B80-88 peptide, indicating that a long peptide chaperoned by Hsp90 was efficiently cross-presented and was able to stimulate peptide-specific CD8+ T cells. To confirm these observations, we compared the efficacy of activation of survivin-2B-specific memory CD8+ T cells by stimulation with survivin-2B80-88, survivin-2B75-93 precursor peptide, or Hsp90–survivin-2B75-93 precursor peptide complex in eight patients. As shown in Table1, stimulation with Hsp90–survivin-2B75-93 complex could expand survivin-2B-specific memory CD8+ T cells from seven out of eight patients compared with stimulation with survivin-2B75-93. More importantly, in six out of eight patients, stimulation with Hsp90–survivin-2B75-93 complex expanded survivin-2B-specific memory CD8+ T cells more efficiently compared with stimulation with survivin-2B80-88.


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)

Peptide-specific precursor CTLs were activated by cross-presentation of heat shock protein 90 (Hsp90)–peptide complex. PBMCs were isolated from patient 1 suffering from colon cancer (Table1) who had been vaccinated with survivin-2B80-88 peptide in our clinical study. The patient's PBMCs were shown to contain the survivin-2B-specific CD8+ T cells. PBMCs were stimulated with human monocyte-derived dendritic cells loaded with survivin-2B80-88 (400 μg/mL), Hsp90 (400 μg/mL), survivin-2B75-93 precursor peptide (400 μg/mL), and Hsp90 (400 μg/mL)–survivin-2B75-93 precursor peptide (400 μg/mL) complex in AIM V medium containing 10% human serum and interleukin-2 (50 U/mL) for 7 days. The stimulated PBMCs were stained with HIV tetramer (tet) or survivin-2B tetramer at 37°C for 20 min. Then a phycoerythrin-Cy5-conjugated anti-CD8 antibody was added at 4°C for 30 min. Cells were washed twice with PBS. After washing, cells were fixed with 0.5% paraformaldehyde and analyzed by flow cytometry using FACSCalibur and CellQuest software. CD8+ living cells were gated, and cells labeled with survivin-2B tetramer were referred to as tetramer-positive cells. The frequency of CTL precursors was calculated as the number of tetramer-positive cells divided by the number of CD8+ cells. 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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fig02: Peptide-specific precursor CTLs were activated by cross-presentation of heat shock protein 90 (Hsp90)–peptide complex. PBMCs were isolated from patient 1 suffering from colon cancer (Table1) who had been vaccinated with survivin-2B80-88 peptide in our clinical study. The patient's PBMCs were shown to contain the survivin-2B-specific CD8+ T cells. PBMCs were stimulated with human monocyte-derived dendritic cells loaded with survivin-2B80-88 (400 μg/mL), Hsp90 (400 μg/mL), survivin-2B75-93 precursor peptide (400 μg/mL), and Hsp90 (400 μg/mL)–survivin-2B75-93 precursor peptide (400 μg/mL) complex in AIM V medium containing 10% human serum and interleukin-2 (50 U/mL) for 7 days. The stimulated PBMCs were stained with HIV tetramer (tet) or survivin-2B tetramer at 37°C for 20 min. Then a phycoerythrin-Cy5-conjugated anti-CD8 antibody was added at 4°C for 30 min. Cells were washed twice with PBS. After washing, cells were fixed with 0.5% paraformaldehyde and analyzed by flow cytometry using FACSCalibur and CellQuest software. CD8+ living cells were gated, and cells labeled with survivin-2B tetramer were referred to as tetramer-positive cells. The frequency of CTL precursors was calculated as the number of tetramer-positive cells divided by the number of CD8+ cells. Data are shown as means + SEM of three independent experiments. *P < 0.01.
Mentions: As we had shown that the Hsp90–survivin-2B75-93 precursor peptide complex was efficiently cross-presented, we next examined whether cross-presentation of Hsp90–peptide complex could activate and expand peptide-specific memory CD8+ T cells from patients who had been vaccinated with survivin-2B peptide with incomplete Freund's adjuvant. Activated and expanded survivin-2B-specific CD8+ T cells were detected by tetramer staining. As shown in Figure2, the survivin-2B75-93 precursor peptide chaperoned by Hsp90 was able to activate and expand survivin-2B-specific memory CD8+ T cells more vigorously than was the precursor peptide alone. Interestingly, peptide-specific T-cell frequency was higher when stimulated with Hsp90–survivin-2B75-93 precursor peptide complex than that with survivin-2B80-88 peptide, indicating that a long peptide chaperoned by Hsp90 was efficiently cross-presented and was able to stimulate peptide-specific CD8+ T cells. To confirm these observations, we compared the efficacy of activation of survivin-2B-specific memory CD8+ T cells by stimulation with survivin-2B80-88, survivin-2B75-93 precursor peptide, or Hsp90–survivin-2B75-93 precursor peptide complex in eight patients. As shown in Table1, stimulation with Hsp90–survivin-2B75-93 complex could expand survivin-2B-specific memory CD8+ T cells from seven out of eight patients compared with stimulation with survivin-2B75-93. More importantly, in six out of eight patients, stimulation with Hsp90–survivin-2B75-93 complex expanded survivin-2B-specific memory CD8+ T cells more efficiently compared with stimulation with survivin-2B80-88.

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