Dendritic-Tumor Fusion Cells Derived Heat Shock Protein70-Peptide Complex Has Enhanced Immunogenicity

<div><p>Tumor-derived heat shock protein70-peptide complexes (HSP70.PC-Tu) have shown great promise in tumor immunotherapy due to numerous advantages. However, large-scale phase III clinical trials showed that the limited immunogenicity remained to be enhanced. In previous research, we demonstrated that heat shock protein 70-peptide complexes (HSP70.PC-Fc) derived from dendritic cell (DC)-tumor fusions exhibit enhanced immunogenicity compared with HSP70.PCs from tumor cells. However, the DCs used in our previous research were obtained from healthy donors and not from the patient population. In order to promote the clinical application of these complexes, HSP70.PC-Fc was prepared from patient-derived DC fused directly with patient-derived tumor cells in the current study. Our results showed that compared with HSP70.PC-Tu, HSP70.PC-Fc elicited much more powerful immune responses against the tumor from which the HSP70 was derived, including enhanced T cell activation, and CTL responses that were shown to be antigen specific and HLA restricted. Our results further indicated that the enhanced immunogenicity is related to the activation of CD4+ T cells and increased association with other heat shock proteins, such as HSP90. Therefore, the current study confirms the enhanced immunogenicity of HSP70.PC derived from DC-tumor fusions and may provide direct evidence promoting their future clinical use.</p></div

Activation of T cells by HSP70.PC derived from DC-tumor fusion cells.

<p>Indicated numbers of lymphocytes and DCs (10:1 ratio) were incubated with HSP70.PC-Tu (2 μg/ml) or HSP70.PC-Fc (2 μg/ml) in medium containing 10% human AB serum in the presence of 10 U/ml human IL-2. Medium alone was used as a control. After 5 days of incubation, the stimulated T cells were harvested by nylon wool separation. A, ELISPOT enumeration of IFN-γ producing cells. B, Intracellular expression of IFN-γ in CD4 and CD8 cells (data from Patient 1 shown). The statistical significance was determined by one-way ANOVA (* indicates P<0.05, HSP70.PC vs. medium, ** indicates P<0.05, HSP70.PC-Tu vs. HSP70.PC-Fc).</p

Characterization of DC-tumor fusion cells.

<p>A, Fusion cells were double-stained for the unique DC marker (HLA-DR-PE) and the tumor marker (HER2-FITC) and analyzed by 2-color flow cytometry to quantify the percentage of double-positive fusion cells. B, Identification of DC-tumor fusion cells by laser confocal microscopy. DC-tumor fusion cells (cytospin on the slides) were identified directly by observation of HER2-FITC and HLA-DR-PE under confocal microscopy. Left panel, HER2-FITC positive; middle panel, HLA-DR-PE; right panel, merged image showing double-positive fusion cells.</p

CTL responses against tumor stimulated by HSP70.PC derived from DC-tumor fusion cells.

<p>The indicated number of lymphocytes and DCs (10:1 ratio) were incubated with HSP70.PC-Tu or HSP70.PC-Fc (2 μg/ml) in medium containing 10% human AB serum in the presence of 10 U/ml human IL-2. Medium alone was used as a control. After 5 days of incubation, the stimulated T cells were harvested by nylon wool separation for use as effector cells. CTL activity against autologous breast tumor cells was analyzed by LDH release assay (A from Patient 1 [HLA-A2+/A11-], C from Patient 2 [HLA-A2+/A11-], E from Patient 3 [HLA-A2-/A11+]). DC mixed with tumor were included as control in Patient 4 (G) and Patient 5 (H). (*indicates P<0.05, HSP70.PC-Fc, HSP70.PC-Tu, DC mix tumor vs. Medium. ** indicates P<0.05, HSP70.PC-Fc vs. HSP70.PC-Tu, DC mix tumor or Medium). In order to test the tumor specificity of CTL responses, different tumor targets with different HLA phenotypes (breast tumor cell line MCF7 HLA-A2+ A11-, SKBR3 HLA-A2- A11+, BT20 HLA-A2- A11-, leukemia cell line K562 HLA-A2- A11-, autologous monocytes) were included in CTL assays (B from Patient 1, D from Patient 2, F from Patient 3). All determinations were conducted in triplicate and expressed as the mean ± SD. The statistical significance was determined by one-way ANOVA. (*indicates P<0.05, target vs. target treated with anti-MHC class antibody. ** indicates P<0.05, autologous breast tumor cells vs. MCF7 or SKBR3).</p

pH dependency of luminescence vs. fluorescence <i>in vitro</i>.

<p>(<b>A</b>) SDS-PAGE gel of purified His-tagged pHlash protein stained with Coomassie Blue dye. Leftmost lane is molecular weight standards with KDa indicated, while the other lanes are the purified pHlash protein loaded at 0.2, 1, and 2 µg per lane. (<b>B</b>) Raw data (not normalized) of luminescence emission spectra of purified pHlash protein at different pH values (pH 5.4–9.0), measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043072#pone-0043072-g001" target="_blank">Figure 1</a>. (<b>C</b>) pH dependence of total luminescence of pHlash (i.e., integration of total light emitted from 400–600 nm), (<b>D</b>) pH dependence of fluorescence emitted (510–600 nm) from pHlash during excitation at 490 nm (no substrate was presented to pHlash during the fluorescence measurements). Error bars are +/− S.D., but in some cases the error bars are so small that they are obscured by the symbols (n = 3).</p

Response of yeast pH to weak acid treatment.

<p>(<b>A</b>) pH response of BCECF-AM loaded yeast cells (red) compared with pHlash transformed yeast cells (blue). (<b>B</b>) Response of BCECF-AM loaded yeast cells to weak acid (20 µM sodium butyrate, upper panel). Yeast cells were suspended in 20 mM MES buffer (pH 5.0). After 10 min baseline recording, 20 µM final concentration of Na-butyrate (pH 5.0) was added (total added volume was 20 µl, which is the same volume as that for the pH 5.0 buffer that was used as a control in the lower panel). (<b>C</b>) Response of pHlash-expressing yeast cells to weak acid. Yeast cells were suspended in 20 mM MES buffer (pH 5.0) with 10 µM native coelenterazine. After 10 min baseline recording, 20 µM final concentration of Na-butyrate (pH 5.0) was added (upper panel). An equal volume of pH 5.0 buffer was added as control (lower panel). The change in BRET ratio of pHlash reports the intracellular acidification after treatment with Na-butyrate. (<b>D</b>) Histogram depiction of multiple replicates of the BCECF protocol illustrated in panel B. (<b>E</b>) Histogram depiction of multiple replicates of the pHlash protocol illustrated in panel C. In panels A, D, and E, error bars are +/− S.D., but in some cases the error bars are so small that they are obscured by the symbols (n = 3 for panel A, n = 5 for panels D and E). ** p<0.01.</p

pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH

<div><p>We report the development of a genetically encodable and ratiometic pH probe named “pHlash” that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor–composed of a donor luciferase that is genetically fused to a Venus fluorophore–exhibits pH dependence of its spectral emission <em>in vitro</em>. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification <em>in vivo</em>. Its spectral ratio response is H⁺ specific; neither Ca⁺⁺, Mg⁺⁺, Na⁺, nor K⁺ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H⁺ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.</p> </div

BRET imaging of pHlash-transfected HeLa cells after NaF treatment.

<p>Cells were imaged as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043072#pone-0043072-g007" target="_blank">Figure 7</a> at different times after adding an equal volume of control medium or an equal volume of medium containing 40 mM NaF to achieve a final concentration of 20 mM NaF (both media contained 2.5 µM ViviRen™). The luminescence images from the blue (∼400–505 nm) and yellow (∼505–600 nm) channels of the Dual-View™ were the median values after 7 sequential 2-sec exposures. Cells were imaged at 0, 3, 5, and 10 min after adding control or NaF-containing media (time 0 =  image before adding media). Ratio images were made by the images obtained from the yellow channel divided by the images from the blue channel (505–600 nm/400–505 nm), and the scales illustrate the pseudocolor encoding of BRET ratio. The plots under the BRET images show the time-dependent changes in the calculated BRET ratio and in the cellular pH estimated from the <i>in vivo</i> calibration curve shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043072#pone-0043072-g008" target="_blank">Figure 8C</a>; values are shown as mean ± S.D. (n = 6 cells for both the control and NaF sets).</p

Characterization of patient-derived DCs and autologous breast tumor cells.

<p>Phenotype of patient-derived auto breast tumor cells (from Patient 1, Patient 2, Patient 3). Tumor cells were stained with antibodies against tumor antigens MUC1, HER2, HLA-A2, HLA-A11 and HLA-DR, CD86 and analyzed by flow cytometry. Phenotype of patient-derived DCs (from Patient 1). DCs were stained with antibodies against MUC1, HER2, HLA-A2, HLA-A11, HLA-DR, and CD86.</p

Characterization of HSP70.PC derived from tumor cells and DC-tumor fusion cells.

<p>A, Association of HSP proteins in HSP70.PC-Tu and HSP70.PC-Fc. Lysates from patient-derived breast tumor cells and from DC-tumor fusion cells were immunoprecipitated with anti-HSP70 mAb followed by immunoblotting with mAbs against HSP110 and HSP90; HSP70 was used as a loading control (results from Patient 1 and Patient 2 are shown). B, The results are representative of three independent blots. The relative protein density was determined by densitometric analysis and normalized between different blots (The ODr (relative optical density) of each band on the blot was normalized according to the mean of these sums: normODr = (S1 + S2 + S3)/3Si.). The statistical significance for the ratio of HSP110, HSP90, to HSP70 between HSP70.PC-Tu and HSP70.PC-Fc was determined by Student’s t-test (* indicates P<0.05).</p