Yonsei Med J.  2014 Jul;55(4):1014-1027. 10.3349/ymj.2014.55.4.1014.

Identification of Pancreatic Cancer-Associated Tumor Antigen from HSP-Enriched Tumor Lysate-Pulsed Human Dendritic Cells

• Kim HS
• Kang D
• Moon MH
• Kim HJ

• KMID: 2130828
• DOI: http://doi.org/10.3349/ymj.2014.55.4.1014

Abstract

PURPOSE
Vaccine strategies utilizing dendritic cells (DCs) to elicit anti-tumor immunity are the subject of intense research. Although we have shown that DCs pulsed with heat-treated tumor lysate (HTL) induced more potent anti-tumor immunity than DCs pulsed with conventional tumor lysate (TL), the underlying molecular mechanism is unclear. In order to explore the molecular basis of this approach and to identify potential antigenic peptides from pancreatic cancer, we analyzed and compared the major histocompatibility complex (MHC) ligands derived from TL- and HTL-pulsed dendritic cells by mass spectrophotometry.
MATERIALS AND METHODS
Human monocyte-derived dendritic cells were pulsed with TL or HTL prior to maturation induction. To delineate differences of MHC-bound peptide repertoire eluted from DCs pulsed with TL or HTL, nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS) was employed.
RESULTS
HTL, but not TL, significantly induced DC function, assessed by phenotypic maturation, allostimulation capacity and IFN-gamma secretion by stimulated allogeneic T cells. DCs pulsed with TL or HTL displayed pancreas or pancreatic cancer-related peptides in context of MHC class I and II molecules. Some of the identified peptides had not been previously reported as expressed in pancreatic cancer or cancer of other tissue types.
CONCLUSION
Our partial lists of MHC-associated peptides revealed the differences between peptide profiles eluted from HTL-and TL-loaded DCs, implying that induced heat shock proteins in HTL chaperone tumor-derived peptides enhanced their delivery to DCs and promoted cross-presentation by DC. These findings may aid in identifying novel tumor antigens or biomarkers and in designing future vaccination strategies.

Keyword

Tumor lysate; heat shock proteins; dendritic cells; tumor antigens; nanoflow LC-MS-MS

MeSH Terms

Antigens, Neoplasm/*immunology
Cell Line, Tumor
Dendritic Cells/*immunology
Humans
Pancreatic Neoplasms/*immunology
Antigens, Neoplasm
Pancreatic Carcinoma

Figure

• 

  Fig. 1 Western blot analysis for HSP family protein expression by TL and HTL. Human gastric cancer cell line (N87) and human pancreatic ductal adenocarcinoma cell line (Panc-1) were exposed to 42℃ for 1 hr, followed by recovery for 24 hr at 37℃. Tumor cell lysates from untreated tumor lysate (TL) or tumor lysate preparation after heat treatment (HTL) were prepared as described in the Materials and Methods section and immunoblotted with indicated antibodies. HTL, heat-treated tumor lysate; TL, tumor lysate; HSP, heat shock protein.

• 

  Fig. 2 Phenotypic maturation of dendritic cells induced by HTL, HSP-enriched tumor lysate prepared from heat-treated tumor cells. Human dendritic cells (5×105/mL) were incubated with 4 mg/mL of TL or HTL for 4 hr. After washing, cells were cultured in the presence of GM-CSF and IL-4 (iDC) or in the presence of GM-CSF, IL-4 with cytokine cocktail, composed of TNF-α, IL-1β, IL-6, and PGE2 (mDC), for 48 hr. DCs were collected and stained with monoclonal antibodies specific to HLA-DR, CD40, CD86, CD80, CD1a, and CD83. Gray histograms indicate untreated DCs, fine-line histograms, TL-pulsed DCs; and thick line histograms, HTL-pulsed DCs. Histograms show the expression of CD1a, CD40, CD80, CD86, HLA-DR, and CD83, which represent one representative experiment of four performed. Numbers denote MFI values. HTL, heat-treated tumor lysate; TL, tumor lysate; HSP, heat shock protein; DC, dendritic cell; iDC, immature DC; MFI, median fluorescence intensity; mDC, mature DC.

• 

  Fig. 3 HTL-pulsed DCs enhanced T cell proliferation, IL-12 production and IFN-γ production. (A) DC exhibits enhanced allostimulatory capacity after pulsing with HTL compared to that of TL-pulsed DC. Gamma-irradiated DC were added to CD3+ T cells at the indicated DC/T cell ratio and incubated for an 5 days before the addition of [3H] thymidine to assess T cell proliferation. Data represent one of three independent experiments with similar results. The error bars are the SEM of triplicate determinations. (B) The production of IL-12p70 was slightly increased by HTL-pulsing. Mature DCs were generated by stimulating immature DCs (iDC) with cytokine cocktail for 48 hr. The error bars are the SD of triplicate determinations. (C) Measurement of IFN-γ in supernatants of allogeneic T cells stimulated by unpulsed, TL-pulsed or HTL-pulsed mature DCs. Allogeneic T cells were stimulated as described in Materials and Methods. The error bars are the SD of triplicate determinations. *p<0.05, **p<0.01. HTL, heat-treated tumor lysate; TL, tumor lysate; DC, dendritic cell.

• 

  Fig. 4 Base peak chromatograms of naturally processed MHC class I- and II-associated peptides derived from TL-DC (A) and HTL-DC (B). Peptides were eluted from DCs by treatment with citrate-phosphate buffer (pH 3.3) and peptides were separated using a homemade pulled tip capillary column by binary gradient elution at 200 nL/min (see details in Materials and Method section). TL, tumor lysate; DC, dendritic cell; HTL, heat-treated tumor lysate; MHC, major histocompatibility complex.

• 

  Fig. 5 Identification of tumor-associated peptides eluted from TL-DC or HTL-DC. The MS-MS spectra of m/z 625.94 ([M+3H]3+) from HTL-DC (A) and of m/z 828.32 ([M+2H]2+) from TL (B), which were found to originate from the same protein, human S100 calcium binding protein A4 (residues 94-110). HTL, heat-treated tumor lysate; TL, tumor lysate; DC, dendritic cell.

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