Photoinduced Electron Transfer of PAMAM Dendrimer-Zinc(II) Porphyrin Associates at Polarized Liquid|Liquid Interfaces

The heterogeneous photoinduced electron-transfer reaction of the ion associates between NH2-terminated polyamidoamine (PAMAM) dendrimers and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato zinc(II) (ZnTPPS4-) was studied at the polarized water|1,2-dichloroethane (DCE) interface. The positive photocurrent arising from the photoreduction of ZnTPPS4- by a lipophilic quencher, decamethylferrocene, in the interfacial region was significantly enhanced by the ion association with the PAMAM dendrimers. The photocurrent response of the dendrimer-ZnTPPS4- associates was dependent on the pH condition and on the generation of dendrimer. A few cationic additives such as polyallylamine and n-octyltrimethyammonium were also examined as alternatives to the PAMAM dendrimer, but the magnitude of the photocurrent enhancement was rather small. The high photoreactivity of the dendrimer-ZnTPPS4- associates was interpreted mainly as a result of the high interfacial concentration of photoreactive porphyrin units associated stably with the dendrimer which was preferably adsorbed at the polarized water|DCE interface. The photochemical data observed in the second and fourth generation PAMAM dendrimer systems demonstrated that the higher generation dendrimer which can incorporate a porphyrin molecule more completely in the interior is less efficient for the photocurrent enhancement at the interface. These results indicated that the photoreactivity of ionic reactant at a polarized liquid|liquid interface can readily be modified via ion association with the charged dendrimer. © 2015 American Chemical Society

CD62L+ NKT cells have prolonged persistence and antitumor activity in vivo

Vα24-invariant natural killer T cells (NKTs) localize to tumors and have inherent antitumor properties, making them attractive chimeric antigen receptor (CAR) carriers for redirected cancer immunotherapy. However, clinical application of CAR-NKTs has been impeded, as mechanisms responsible for NKT expansion and the in vivo persistence of these cells are unknown. Here, we demonstrated that antigen-induced expansion of primary NKTs in vitro associates with the accumulation of a CD62L+ subset and exhaustion of CD62L– cells. Only CD62L+ NKTs survived and proliferated in response to secondary stimulation. When transferred to immune-deficient NSG mice, CD62L+ NKTs persisted 5 times longer than CD62L– NKTs. Moreover, CD62L+ cells transduced with a CD19-specific CAR achieved sustained tumor regression in a B cell lymphoma model. Proliferating CD62L+ cells downregulated or maintained CD62L expression when activated via T cell receptor alone or in combination with costimulatory receptors. We generated HLAnull K562 cell clones that were engineered to express CD1d and costimulatory ligands. Clone B-8-2 (HLAnullCD1dmedCD86high4-1BBLmedOX40Lhigh) induced the highest rates of NKT expansion and CD62L expression. B-8-2–expanded CAR-NKTs exhibited prolonged in vivo persistence and superior therapeutic activities in models of lymphoma and neuroblastoma. Therefore, we have identified CD62L as a marker of a distinct NKT subset endowed with high proliferative potential and have developed artificial antigen-presenting cells that generate CD62L-enriched NKTs for effective cancer immunotherapy

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

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