Autophagy Creates a CTL Epitope That Mimics Tumor-Associated Antigens

<div><p>The detailed mechanisms responsible for processing tumor-associated antigens and presenting them to CTLs remain to be fully elucidated. In this study, we demonstrate a unique CTL epitope generated from the ubiquitous protein puromycin-sensitive aminopeptidase, which is presented via HLA-A24 on leukemic and pancreatic cancer cells but not on normal fibroblasts or EBV-transformed B lymphoblastoid cells. The generation of this epitope requires proteasomal digestion and transportation from the endoplasmic reticulum to the Golgi apparatus and is sensitive to chloroquine-induced inhibition of acidification inside the endosome/lysosome. Epitope liberation depends on constitutively active autophagy, as confirmed with immunocytochemistry for the autophagosome marker LC3 as well as RNA interference targeting two different autophagy-related genes. Therefore, ubiquitously expressed proteins may be sources of specific tumor-associated antigens when processed through a unique mechanism involving autophagy.</p> </div

Induced autophagy does not result in PSA epitope presentation in fibrobast cells.

<p>A. Immunofluorescence assays for endogenous LC3 in fibroblast cells after low nutrient culture or rapamycin treatment. In low nutrient conditions, fibroblast cells were cultured in medium supplemented with 10%, 5%, 0,5% FCS or in Hank’s Balanced Salt Solution (starved). B, CTL response to autophagy-induced fibroblast cells treated with low nutrient culture conditions or rapamycin. Target cells were treated with low nutrient culture conditions or rapamycin for 4h, washed twice and cultured with CTL overnight. Next day, supernatants were harvested and IFN-γ measured by ELISA. K562-A24 cells and K562-A2 cells were used as positive and negative control, respectively. The results show means ± SD of triplicates.</p

The epitope is presented and processed through a vacuolar pathway.

<p>A, K562 cells expressing both HLA-A2 or A24 and CMV pp65 (A2-pp65-K562 or A24-pp65-K562) were acid-stripped and incubated at 37°C for 9 h in the presence or absence of BFA. Then, the cells were co-cultured with either 16F3 or an HLA-A24-restricted CMV pp65-specific CTL clone for an additional 5 h. BFA was also added during the co-culture. After fixation and permeabilization, the cells were stained for CD3, CD8, CD69 and IFN-γ. CD3⁺ and CD8⁺ T cells were gated and analyzed using a flow cytometer. The frequency of IFN-γ producing cells is shown as the percentage of the total CD3⁺ CD8⁺ T cells. B-C, IFN-γ secretions of clones for 4 h after stimulation with A24-pp65-K562 cells (B) or KP-3 cells (C) treated with acid buffer for peptide stripping and/or inhibitors for 14 h was detected using an IFN-γ catch assay. 7-AAD⁻ alive CD8⁺ T cells were gated and analyzed using a flow cytometer. The frequency of IFN-γ secreting cells is shown as the percentage of the total alive CD8⁺ T cells. Whereas irreversibly acting lactacystin was removed during co-culture period (B), CQ and Baf A were retained in the media (B, C) because of their reversible nature. To exclude the possibility that CQ and Baf A could be inhibitory for 16F3 to produce IFN-γ, cognate or irrelevant peptides were added at concentrations of 1 µg/ml (B, C) and the T-cell response was examined.</p

Autophagy is involved in the 16F3 epitope processing in cancer cells.

<p>A, K562 cells expressing both HLA-A2 or A24 and CMV pp65 were treated with an acid buffer for peptide stripping and incubated for 14 h in the presence or absence of 3-MA. Next, the cells were co-cultured with each clone for 4 h for IFN-γ secretion detected via the IFN-γ catch assay. The frequency of IFN-γ-secreting cells is shown as the percentage of the total living CD8⁺ T cells. 3-MA was not added during the co-culture period. In the lower panels, histograms of the IFN-γ signal and their mean fluorescence intensity are shown. B, RT-PCR analysis of scrambled, atg5- or atg7-specific siRNA-treated cells performed 70 h after transfection. The intensities of the bands were calculated using the ImageJ software. C, The CTL response against KP-3 and MIA PaCa-2 cells transfected with scrambled, atg5- or atg7-specific siRNA for 70 h was examined using an IFN-γ ELISA. Three different siRNA targets were chosen for each autophagy-associated gene. The results are the means ± SD of triplicates. Similar results were obtained in three separate experiments.</p

Characterization of a CTL clone, designated as 16F3, from in vitro culture with aAPCs.

<p>A, Surface expression of HLA-A24, CD86 and 4-1BBL molecules on the K562 cells used for stimulation. The shaded and solid area show non-transduced and lentiviral-transduced cells, respectively. B–D, IFN-γ secretion of 16F3 upon incubation with various cells. The 16F3 cells were incubated with K562 cells expressing either HLA-A2 or HLA-A24 (B) for 4 h, and the IFN-γ secreting cells were detected and analyzed. The frequency is shown as the percentage of the total living CD8⁺ T cells. HLA-A24-positive fibroblast cells, normal human bronchial epithelial cells and B-LCLs were used as representative non-cancerous cells (C). Five HLA-A24-positive pancreatic carcinoma cell lines were also used as stimulator cells (D). The data are representative of three independent experiments.</p

Degradation of full-length PSA protein is inhibited by CQ but not by lactacystin.

<p>A, Cells were cultured with or without CQ (50 µM) overnight, and then the cell lysates were subjected to Western blot analysis for PSA and p62 as a positive control for autophagic digestion. B, Cells were cultured with or without lactacystin (5 µM) overnight, and the cell lysates were subjected to Western blot analysis for PSA and HIF-2α as a positive control for proteasomal digestion. C, The intensities of the bands were calculated using the ImageJ software.</p

Constitutively active autophagy is involved in the 16F3 epitope processing of cancer cells.

<p>A, An immunofluorescence assay was performed to examine the expression of endogenous LC3. B, Double staining for endogenous LC3 and PSA was performed. Cells were cultured with BafA for 4 h, and then immunocytochemistry was performed. A yellow signal indicates colocalization. C, KP-3 and MIA PaCa-2 cells were transfected with plasmids expressing an mRFP-GFP-LC3 tandem-tagged fluorescent protein. Forty hours after transfection, the cells were fixed and analyzed via microscopy. A white signal indicates colocalization. The bars indicate 10 µm (A–C). D, The status of autophagic flux was measured via the LC3-II expression level. Cells were cultured with or without CQ for 2 h, and then the cell lysates were subjected to Western blot analysis for LC3.</p

Ubiquitous expression of the PSA mRNA and protein within various cells irrespective of clone recognition.

<p>A, Two RT-PCR primer sets are designed to discriminate between wild type and variant PSA (PSAv), indicated by horizontal arrows (top). RT-PCR analysis was performed using the PSA primer sets and those for β-actin (bottom). B, The expression of the PSA and β-actin proteins was analyzed using Western blot analysis. C, KP-3 and MIA PaCa-2 cells were transfected for 70 h with scrambled or PSA-specific siRNA. Three different siRNA targets were chosen for the PSA gene, and Western blot analysis of the PSA protein in siRNA-treated cells was performed. The intensities of the bands were calculated using the ImageJ software. D, CTL responses against siRNA-treated cells were examined using an IFN-γ ELISA. The results are expressed as the means ± SD of triplicate values. Similar results were obtained in three separate experiments.</p