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[Cancer Research 65, 10041-10049, November 1, 2005]
© 2005 American Association for Cancer Research

Immunology

Protein Transduction of Dendritic Cells for NY-ESO-1-Based Immunotherapy of Myeloma

Ramesh B. Batchu1, Amberly M. Moreno1, Susann M. Szmania1, Grant Bennett1, Guilio C. Spagnoli3, Selvarangan Ponnazhagan2, Bart Barlogie1, Guido Tricot1 and Frits van Rhee1

1 Myeloma Institute for Research and Therapy, Section for Gene and Immunotherapy, University of Arkansas for Medical Sciences, Little Rock, Arkansas; 2 Department of Pathology, The University of Alabama at Birmingham, Birmingham, Alabama; and 3 Department of Surgery, University of Basel, Switzerland

Requests for reprints: Frits van Rhee, Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 776, Little Rock, AR 72205. Phone: 501-296-1503, ext. 1547; Fax: 501-686-6442; E-mail: vanrheefrits{at}uams.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Myeloma vaccines, based on dendritic cells pulsed with idiotype or tumor lysate, have been met with limited success, probably in part due to insufficient cross-priming of myeloma antigens. A powerful method to introduce myeloma-associated antigens into the cytosol of dendritic cells is protein transduction, a process by which proteins fused with a protein transduction domain (PTD) freely traverse membrane barriers. NY-ESO-1, an immunogenic antigen by itself highly expressed in 60% of high-risk myeloma patients, was purified to near homogeneity both alone and as a recombinant fusion protein with a PTD, derived from HIV-Tat. Efficient entry of PTD-NY-ESO-1 into dendritic cells, confirmed by microscopy, Western blotting, and intracellular flow cytometry, was achieved without affecting dendritic cell phenotype. Experiments with amiloride, which inhibits endocytosis, and N-acetyl-L-leucinyl-L-norleucinal, a proteasome inhibitor, confirmed that PTD-NY-ESO-1 entered dendritic cells by protein transduction and was degraded by the proteasome. Tetramer analysis indicated superior generation of HLA-A2.1, CD8+ T lymphocytes specific for NY-ESO-1157-165 with PTD-NY-ESO-1 compared with NY-ESO-1 control protein (44% versus 2%, respectively). NY-ESO-1-specific T lymphocytes generated with PTD-NY-ESO-1 secreted IFN-{gamma} indicative of a Tc1-type cytokine response. Thus, PTD-NY-ESO-1 accesses the cytoplasm by protein transduction, is processed by the proteasome, and NY-ESO-1 peptides presented by HLA class I elicit NY-ESO-1-specific T lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Autologous blood stem cell–supported high-dose melphalan therapy has emerged as the treatment of choice for the primary care of younger patients with multiple myeloma (1, 2). Further benefit from "tandem" autotransplants, suggested in Arkansas' Total Therapy studies (3), has now been confirmed in a randomized trial (IFM 94) by the French Myeloma Cooperative Group (1). Systematic investigations of prognostic factors as part of Total Therapy phases I and II have identified the presence of metaphase cytogenetic abnormalities as the dominant feature associated with short survival, present in one third of newly diagnosed patients (4). Recent gene expression profiling studies of purified multiple myeloma cells revealed high levels of expression of a number of cancer/testis (C/T) antigens, especially among patients deemed at high risk (i.e., with CKS1B overexpression and with translocations involving MAF, MAFB, and MMSET; ref. 5). These results were confirmed by immunohistochemical studies of bone marrow biopsy specimens, showing frequent C/T antigen expression among patients with cytogenetic abnormalities and almost universally at relapse (6). Given the relatively minor progress in the treatment of such high-risk patients, we sought to exploit this feature of frequent C/T antigen expression therapeutically by developing a vaccine-based approach.

C/T antigens are either not expressed or expressed at very low levels in normal tissues but are overexpressed in a variety of tumors including myeloma (7–11). The C/T antigen, NY-ESO-1, a 22-kDa protein (12), is one of the most immunogenic C/T antigens described thus far and stimulates both spontaneous and vaccine-induced cellular and humoral antitumor responses in humans (6, 10, 13–16). We have recently reported that NY-ESO-1 protein is expressed in 60% of myeloma patients with abnormal metaphase cytogenetics. We have also provided evidence of preexistent cellular and humoral immunity to NY-ESO-1 in myeloma (6). Furthermore, after expansion, spontaneously present NY-ESO-1-specific T cells were able to kill primary myeloma cells. NY-ESO-1 immunotherapy therefore may confer specific antitumor activity without targeting normal tissues.

Immunogenic viral and tumor peptide epitopes recognized by CD8+ cytotoxic T cell (CTL) are typically derived from intracellular proteins, which have been processed in the cytoplasm, transported to the endoplasmic reticulum, and loaded onto HLA class I molecules before egress to the cell surface. This complex process is referred to as the HLA class I antigen-processing pathway (17). In contrast, exogenous antigens pulsed onto dendritic cells are internalized by macropinocytosis, degraded into peptides inside vesicular intracellular compartments, and loaded onto HLA class II molecules for presentation at the cell surface to induce CD4+ T-helper cell responses (18). This separation of HLA class I and II pathways is not absolute. Professional antigen-presenting cells (APC) exogenously pulsed with tumor lysates (19), myeloma idiotype, or tumor proteins (20, 21) rely on a relatively inefficient process called "cross-priming", in which the immunoproteasome plays a critical role (22) to access the HLA class I pathway of antigen processing and presentation. Although murine (23–25) and human (19, 26–29) studies indicated that dendritic cells pulsed with polypeptides, tumor lysates, or proteins can indeed stimulate tumor-specific CTLs, CTL induction with exogenous antigen is often suboptimal (30–32).

We have previously shown that NY-ESO-1 protein can be expressed in the cytoplasm of monocyte-derived dendritic cells by gene modification with NY-ESO-1-containing viral vectors (33). However, recent concerns regarding the safety of viral vectors, especially the immunogenicity of viral capsid proteins and insertional mutagenesis, may ultimately limit clinical application.

HIV-1 TAT protein has the ability to freely traverse membrane bilayers and enter directly into the cytoplasm (34, 35), a process known as protein transduction. A nonimmunogenic 11-amino-acid motif known as the protein transduction domain (PTD: YGRKKRRQRRR) is responsible for this phenomenon. Fusion proteins generated with this 11-amino-acid TAT (PTD) sequence traverse membrane barriers efficiently, enter the cytosol (36, 37), and have immediate access to the HLA class I pathway for highly efficient antitumor CTL induction (38–40). We therefore explored NY-ESO-1 protein transduction as a safe and effective alternative to virus-mediated gene delivery. We report cloning of the PTD-NY-ESO-1 gene and purification of the PTD-NY-ESO-1 fusion protein and show that the PTD-NY-ESO-1 fusion protein efficiently enters the dendritic cell cytosol without altering dendritic cell phenotype or functional activity. In addition, we show that monocyte-derived dendritic cells transduced with the PTD-NY-ESO-1 protein can induce CD8+ cellular antitumor immunity superior to that achieved with NY-ESO-1 protein alone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid vector construction. Bacterial protein expression vectors for NY-ESO-1 and PTD-NY-ESO-1 were constructed as follows. NY-ESO-1 was PCR amplified (forward primer, 5'-GCATGCAGGCCGAAGGCCGGGGCACAGGG-3'; reverse primer, 5'-AGATCTGCGCCTCTGCCCTGAGGGAGGC-3') from pET-NY-ESO-1 and subcloned into a pDRIVE T vector (Qiagen, Inc., Valencia, CA). NY-ESO-1 was excised from the pDRIVE vector with SphI and BglII and cloned into the SphI-BglII sites in the pQE-70 expression vector (Qiagen) in frame with the downstream 6Xhis-tag sequence to generate pQE-NY-ESO-1. PTD-NY-ESO-1 was generated by excising NY-ESO-1 from pET-NY-ESO-1 with KpnI and XhoI and ligated into a pTAT vector (a gift from Dr. S. Dowdy, Washington University, St. Louis, MO) in frame and upstream of the PTD and 6Xhis-tag.

Expression of PTD-NY-ESO-1 and NY-ESO-1. Escherichia coli BL21 Star (DE3-pLysS)–competent cells (Invitrogen, Carlsbad, CA) were transformed with PTD-NY-ESO-1 and NY-ESO-1 plasmids. Conditions for protein expression were optimized by first analyzing clones grown in various bacterial growth media, such as Turbo Broth, Superior Broth, Power Broth, Hyper Broth, M9Y (glucose) Broth, LB Broth (Miller; pEX Protein Expression Media optimization kit, U.S. Biologicals, Swampscott, MA), and 2XYT (Sigma Chemicals, St. Louis, MO), then second by analyzing protein expression at various induction periods. Protein expression was induced by adding 1 mmol/L isopropylthio-ß-D-galactoside (IPTG, Sigma Chemicals) after allowing starter cultures to reach an optimal absorbance, which varied for each protein (see Results).

Affinity purification and gel extraction of PTD-NY-ESO-1 and NY-ESO-1 proteins. Bacterial cell pellets were lysed by adding a denaturation buffer [8 mol/L Urea, 100 mmol/L NaCl, 20 mmol/L HEPES (pH 8.0)] followed by sonication (50 Sonic dismembrator, Fisher Scientific, Pittsburgh, PA). Lysates were applied to a preequilibrated nickel-charged resin (Ni-NTA agarose, Qiagen) and allowed to incubate on a rotary shaker for 1 hour at room temperature. The equilibration/washing buffer for column purification was the denaturation buffer containing both 5 mmol/L imidazole and 10% glycerol for NY-ESO-1 or 20 mmol/L imidazole for PTD-NY-ESO-1. The elution of NY-ESO-1 and PTD-NY-ESO-1 proteins from the columns was optimized by adding increasing amounts of imidazole in a step-gradient fashion. To achieve a high level of purity, the affinity-purified proteins were further separated and visualized on preparatory PAGE gels using an Insite kit (National Diagnostics, Atlanta, GA). Protein bands were excised from the polyacrylamide gels and then purified by electroelution against a 0.1% SDS-Tris-glycine buffer. Buffer exchange with PBS was carried out using 10,000 MWCO Amicon Ultra centrifugal filter devices (Millipore, Bedford, MA). Purity of the proteins was further analyzed by high-pressure liquid chromatography (Beckman Instruments System Gold high-pressure liquid chromatography, Iowa State University of Science and Technology, Protein Facility, Ames, IA). Molecular mass of the proteins was identified by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF, ThermoBioanalysis Dynamo, Amphotech Ltd. Beverly, MA) and SDS-PAGE electrophoresis. The presence of bacterial endotoxin in recombinant protein preparations was estimated by a quantitative chromogenic Limulus Amebocyte Lysate test (QCL-1000 kit, Bio Whittaker, Walkersville, MD) as per manufacturer's instructions.

Electrophoresis and Western blotting. For electrophoresis and blotting experiments, lysates of monocyte-derived dendritic cells, pulsed with NY-ESO-1 or PTD-NY-ESO-1, were prepared as previously described with minor modifications (33). A PTD-specific polyclonal antibody was custom made (Biocarta, San Diego, CA) using the TAT-PTD sequence with two additional amino acids (CGGRKKRRQRRR). The NY-ESO-1 monoclonal antibody clone B9.8 was used to identify both NY-ESO-1 and PTD-NY-ESO-1 (41).

Generation of monocyte-derived dendritic cells. Peripheral blood mononuclear cells (PBMC) were collected from normal individuals after informed consent. Monocyte-derived dendritic cells were generated as previously described (42).

Inhibition of endocytosis and the immunoproteasome in monocyte-derived dendritic cells. Approximately 3 µmol/L of NY-ESO-1 or PTD-NY-ESO-1 protein were added to the dendritic cell culture at the indicated time intervals to access the dendritic cell cytosol via direct translocation of the PTD-NY-ESO-1 protein by protein transduction. Normal human fibroblasts were similarly pulsed with 3 µmol/L of each recombinant protein. Amiloride (50 µmol/L; ALEXIS Biochemicals, San Diego, CA) was used in experiments designed to block macropinocytosis. N-acetyl-L-leucinyl-L-norleucinal (LLNL, 50 µmol/L, Sigma Chemicals) was added to maturing monocyte-derived dendritic cells at the indicated time intervals to inhibit immunoproteasome activity and show that PTD-NY-ESO-1 was degraded by the proteasome.

Flow cytometry of monocyte-derived dendritic cells. Monocyte-derived dendritic cells were stained with FITC- or phycoerythrin-conjugated antibodies, including HLA-DR, CD1a, CD83, CD11c, CD80, CD86, mannose receptor, CD3, CD14, and CD19 (R&D Systems, Minneapolis, MN) as previously described (42). Stained monocyte-derived dendritic cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Diego, CA). For intracellular staining, 2 x 105 NY-ESO-1- or PTD-NY-ESO-1-pulsed monocyte-derived dendritic cell were stained with either the custom-made PTD-specific antibody (recognizing CGGRKKRRQRRR) or the anti-NY-ESO-1 antibody using as secondary label goat anti-mouse FITC (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Isotype-specific negative controls designed for both extracellular and intracellular staining were used in all experiments.

Immunofluorescence and deconvolution/confocal microscopy. Cytospin slides of ~2 x 105 antigen exposed monocyte-derived dendritic cells or fibroblasts were prepared as reported previously (33). Slides were incubated with NY-ESO-1 mouse anti-human antibody (41) at room temperature for 1 hour followed by two washes with PBS (pH 7.2) containing 0.05% IGPAL CA-630 (NP40; Sigma, St. Louis, MO). The cells were further incubated with goat anti-mouse Alexa 488 (Molecular Probes, Eugene, OR), washed, fixed, and analyzed using either a Zeiss 410 confocal laser-scanning microscope or a Zeiss deconvolution microscope Axioskop2 mot plus (Microscopy Laboratory, University of Arkansas for Medical Sciences, Little Rock, AR). Data were analyzed using Image J software (http://rsb.info.nih.gov/ij/index.html).

Induction of NY-ESO-1-specific T lymphocytes and tetramer analysis. Approximately 2 x 105 monocyte-derived dendritic cells were coincubated with 3 µmol/L of NY-ESO-1 and PTD-NY-ESO-1 overnight. Monocyte-derived dendritic cells were irradiated to 2,500 cGy and subsequently cocultured with 2 x 106 autologous CD14-depleted PBMCs. Cultures were replenished every 3 days with one half fresh media [RPMI and 10% autologous plasma with interleukin 2 (IL-2), 10 IU/mL]. Cultures were restimulated weekly with NY-ESO-1- or PTD-NY-ESO-1-pulsed, irradiated PBMC or monocyte-derived dendritic cells for 4 to 6 weeks. Generation of NY-ESO-1-specific CD8+ NY-ESO-1-specific T lymphocytes was ascertained by tetramer staining with NY-ESO-1157-165 tetramer phycoerythrin (Beckman Coulter, Immunomics Operations, San Diego, CA) as previously reported (6).

ELISPOT assays. ELISPOT assays were done with an IFN-{gamma} or IL-4 ELISPOT kit (BD Biosciences PharMingen, San Diego, CA) according to the manufacturer's recommendations. NY-ESO-1-specific T lymphocytes (1 x 105) and 1 x 104 mock or protein pulsed monocyte-derived dendritic cells were plated per well and incubated for 16 hours at 37°C in a CO2 incubator. As controls, 1 x 105 unstimulated NY-ESO-1-specific T lymphocytes, unstimulated PBMCs, and nonspecifically stimulated PBMCs were included. The spots were counted using an ELISPOT plate reader and software (Cellular Technologies, Inc., Cleveland, OH). At least four wells per sample were counted and presented as a mean value.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of NY-ESO-1 and PTD-NY-ESO-1 bacterial expression vectors and optimization of NY-ESO-1 and PTD-NY-ESO-1 protein expression. We constructed PTD-NY-ESO-1 and control NY-ESO-1 bacterial expression vectors as described in the Materials and Methods. Restriction digestion analysis confirmed the construction of the vectors (data not shown). Vector identity was also determined by DNA sequencing (DNA Sequencing Core Facility, University of Arkansas for Medical Sciences). Between the 6Xhis-tag and PTD sequence in pPTD vector, there is an anchor of 80 bp to achieve proper folding of the PTD sequence. Because this portion is also translated into the fusion protein of PTD-NY-ESO-1, we ran the HLA-Peptide Binding algorithms SYFPEITHI (http://www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm) and BIMAS (http://bimas.dcrt.nih.gov/molbio/hla_bind/) with the inserted sequence and confirmed that there are no peptides in this sequence predicted to bind to HLA class I molecules (data not shown; ref. 43). Therefore, it is likely that the additional translated region does not contain immunogenic epitopes capable of inducing nonspecific T lymphocytes, which may interfere with NY-ESO-1-specific T-lymphocyte induction.

Several problems with precipitation were experienced during the initial purifications of both proteins, which required further optimization of both expression and purification for each protein individually. We observed optimal expression in Turbo Broth for NY-ESO-1 and 2XYT medium for PTD-NY-ESO-1 (data not shown). Optimal conditions for NY-ESO-1 protein and PTD-NY-ESO-1 expression comprised incubating overnight starter cultures until the absorbency at A600 nm reached 0.5 and 0.7, respectively. To induce protein expression, 1 mmol/L IPTG was added to a 1:20 dilution of NY-ESO-1 and a 1:10 dilution of PTD-NY-ESO-1 starter cultures, which were then incubated for a further 3 and 4 hours, respectively, before harvesting.

High-grade purification of NY-ESO-1 and PTD-NY-ESO-1 proteins. A high level of protein purity is a necessary requirement for dendritic cell pulsing, because it cannot be excluded that peptides derived from bacterial-contaminant proteins may have a higher affinity for HLA class I and result in undesirable immune responses. Hence, a three-step procedure was devised to obtain highly purified NY-ESO-1 and PTD-NY-ESO-1 protein from the bacterial lysates. First, the 6Xhis-tag in both protein sequences allowed purification from cell lysates by his-tag affinity chromatography using nickel-charged resin affinity columns (Qiagen). NY-ESO-1 and PTD-NY-ESO-1 proteins were effectively eluted at imidazole concentrations of 100 and 500 mmol/L, respectively, although there was minor contamination in both cases (Fig. 1A; NY-ESO-1 purification profile not shown). Next, the his-tag affinity-purified NY-ESO-1 and PTD-NY-ESO-1 proteins were further separated on preparatory polyacrylamide gels (Fig. 1B) and excised to eliminate any possible contaminating proteins from the his-tag preparations. Lastly, the proteins were extracted from excised bands by electroelution. SDS-PAGE analysis identified a homogeneous preparation of the NY-ESO-1 and PTD-NY-ESO-1 proteins with expected molecular sizes of ~22 and 32 kDa, respectively (Fig. 1C, lanes 4 and 7).



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  		Figure 1. Stepwise isolation of >95% pure NY-ESO-1- and PTD-NY-ESO-1-recombinant proteins. A, purification profile of PTD-NY-ESO-1 analysis by SDS-PAGE before and after column purification. Lane 1, protein marker; lane 2, noninduced culture; lane 3, induced culture; lane 4, 0.2 mol/L imidazole wash; lane 5, 0.5 mol/L imidazole eluate; lane 6, 1 mol/L imidazole eluate. B, preparative SDS-PAGE using Insite Dye (National Diagnostics) to visualize concentrated PTD-NY-ESO-1 eluate from column purification. The large fluorescent band at 32 kDa was excised for further purification by electroelution. C, SDS-PAGE analysis of PTD-NY-ESO-1 after electroelution. Lane 1, marker protein; lanes 2 and 3, control and induced cell lysates of NY-ESO-1, respectively; lane 4, purified NY-ESO-1 protein; lanes 5 and 6, control and induced PTD-NY-ESO-1, respectively; lane 7, purified PTD-NY-ESO-1 protein. D, MALDI-TOF analysis of PTD-NY-ESO-1 displays a highly purified, large peak at the expected size of the protein. Western blot analysis with NY-ESO-1 antibody (E) and with PTD antibody (F) show highly purified preparations of both proteins. E and F, control (lane 1) and induced cell lysates of NY-ESO-1 (lane 2), respectively; purified NY-ESO-1 protein (lane 3); control (lane 4) and induced PTD-NY-ESO-1 (lane 5); purified PTD-NY-ESO-1 protein (lane 6).

 
MALDI-TOF mass spectrometry confirmed the presence of >99% pure preparation of the PTD-NY-ESO-1 (Fig. 1D). Western blot analysis with the B9.8 NY-ESO-1 antibody confirmed the identity of the NY-ESO-1 and PTD-NY-ESO-1 proteins in induced and purified samples (Fig. 1E). We further confirmed presence of the PTD motif in the PTD-NY-ESO-1 protein and not in NY-ESO-1 with the PTD antibody (Fig. 1F, lanes 5 and 6). Endotoxin levels in the protein preparations were <0.1 EU/mL (data not shown), indicating that the proteins will not be toxic to mammalian cells.

Efficient transduction of human fibroblasts and monocyte-derived dendritic cells by PTD-NY-ESO-1. Unlike monocyte-derived dendritic cells, fibroblasts have minimal endocytotic activity and thus take up little external protein. Hence, the entry of substantial amounts of external proteins into fibroblasts must depend on protein transduction.

We therefore compared the entry efficiency of PTD-NY-ESO-1 versus NY-ESO-1 protein into human fibroblasts after pulsing with 3 µmol/L of either protein and immunofluorescent staining with NY-ESO-1 antibody. NY-ESO-1 protein was readily detected in the cytoplasm of fibroblasts pulsed with PTD-NY-ESO-1 protein but not in fibroblasts, which were either mock pulsed or pulsed with NY-ESO-1 protein (Fig. 2A).



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  		Figure 2. Highly efficient protein transduction of human fibroblasts and monocyte-derived dendritic cell by PTD-NY-ESO-1. A, fibroblasts were mock-pulsed (i) or pulsed-chased for 30 minutes at 37°C with 3 µmol/L of the NY-ESO-1 (ii) and PTD-NY-ESO-1 (iii) proteins and stained with NY-ESO-1 antibody (clone B9.8) followed by visualization with Alexa 488 (magnification, 100x) using a Zeiss deconvolution microscope Axioskop2 mot plus. Immature monocyte-derived dendritic cells were pulsed-chased for the indicated time intervals at 37°C with 3 µmol/L of the NY-ESO-1 (B) and PTD-NY-ESO-1 (C) proteins and then stained with NY-ESO-1 antibody (clone B9.8) followed by visualization with Alexa 488 (magnification, 100x). Time intervals for both (B) and (C) included (i) 2 minutes, (ii) 15 minutes, (iii) 30 minutes, and (iv) 2 hours.

 
Next, we compared the ability of the PTD-NY-ESO-1 and NY-ESO-1 protein to access the cytoplasm of monocyte-derived dendritic cells by pulsing on day 6 with 3 µmol/L of PTD-NY-ESO-1 protein or NY-ESO-1 control protein. We observed very little fluorescent staining in NY-ESO-1-pulsed monocyte-derived dendritic cells after 2 hours (Fig. 2B). In contrast, PTD-NY-ESO-1 was present in monocyte-derived dendritic cells within 2 minutes of pulsing, indicating that protein transduction is a highly efficient and rapid way to introduce NY-ESO-1 into monocyte-derived dendritic cell cytoplasm (Fig. 2C). Studies using deconvolution and confocal microscopy confirmed that PTD-NY-ESO-1 was localized to the dendritic cell cytosol (Fig. 3D).



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  		Figure 3. Entry of PTD-NY-ESO-1 into monocyte-derived dendritic cell occurs by protein transduction rather than macropinocytosis and is localized to the dendritic cell cytosol. Dendritic cells were treated with amiloride (50 µmol/L), which inhibits dendritic cell macropinocytosis, for 30 minutes at 37°C before pulsing with NY-ESO-1 (B) or PTD-NY-ESO-1. NY-ESO-1 is only present in dendritic cells pulsed with PTD-NY-ESO-1 protein (C). Mock-pulsed control (A). PTD-NY-ESO-1 cytoplasm was localized to the dendritic cell cytosol as shown by confocal microscopy (magnification, 60x) and 4x zoom (D). D, left, PTD-NY-ESO-1-pulsed dendritic cell after 14 hours of incubation. Bottom arrow, location of the nucleus surrounded by PTD-NY-ESO-1 protein (top arrow) in the cytosol. Right, bright-field image of the same field of view as left.

 
Semiquantitative analysis of monocyte-derived dendritic cells shows that large amounts of PTD-NY-ESO-1 enter the cytoplasm. Monocyte-derived dendritic cells were also processed for flow cytometry and Western blotting to allow for semiquantitative analysis of the amount of PTD-NY-ESO-1 or NY-ESO-1 in monocyte-derived dendritic cells. We observed >65% of transduced cells with PTD-NY-ESO-1, whereas <10% of the monocyte-derived dendritic cells contained NY-ESO-1; unpulsed cells exhibited only background fluorescence (data not shown). We also detected protein in monocyte-derived dendritic cells pulsed with PTD-NY-ESO-1, but not with NY-ESO-1 (data not shown), by Western blot analysis thus corroborating flow cytometry results that indicate efficient entry of PTD-NY-ESO-1 into monocyte-derived dendritic cells.

Inhibition of macropinocytosis by amiloride does not abrogate protein transduction of monocyte-derived dendritic cells by PTD-NY-ESO-1. Macropinocytosis is significantly blocked by adding amiloride (50 µmol/L), which inhibits membrane ruffling and endocytosis; however, protein transduction occurs independent of endocytosis and is not affected by amiloride (30). Monocyte-derived dendritic cells were therefore treated with amiloride to further substantiate that the entry of PTD-NY-ESO-1 into monocyte-derived dendritic cells is indeed mediated through protein transduction (30) and not by macropinocytosis. Limited entry of NY-ESO-1 protein into monocyte-derived dendritic cells occurred in the presence of amiloride (Fig. 3B), whereas efficient entry of PTD-NY-ESO-1 was still observed (Fig. 3C).

Immunoproteasome processes PTD-NY-ESO-1 in monocyte-derived dendritic cells. Peptides destined to access the HLA class I pathway via cross-priming are mostly processed by the immunoproteasome in monocyte-derived dendritic cells (44). LLNL is a potent immunoproteasome inhibitor. To examine if the PTD-NY-ESO-1 peptides are processed by the immunoproteasome, we incubated mature monocyte-derived dendritic cells with and without LLNL before pulsing with PTD-NY-ESO-1. A time course experiment showed that PTD-NY-ESO-1 was maximally present at 14 hours after pulsing in the cytosol of dendritic cells incubated without LLNL. However, after 14 hours, the PTD-NY-ESO-1 protein was rapidly degraded in dendritic cells not treated with LLNL (Fig. 4A). In contrast, PTD-NY-ESO-1 was still readily detectable in dendritic cells treated with LLNL at 20 hours, the last time point tested (Fig. 4B). This experiment clearly indicates that PTD-NY-ESO-1 protein is processed by the immunoproteasome, which is required for accessing the HLA class I pathway for antigen processing and presentation and essential to the generation of NY-ESO-1-specific T cells.



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  		Figure 4. Degradation of PTD-NY-ESO-1 is proteasome-mediated. Mature monocyte-derived dendritic cells were either untreated (A) or treated (B) with the proteasome inhibitor LLNL (50 µmol/L) and pulsed chased with PTD-NY-ESO-1 (3 µmol/L) for the indicated time intervals followed by staining with NY-ESO-1 antibody (clone B9.8). Time intervals for both (A) and (B) included (i) 5 hours, (ii) 12 hours, (iii) 14 hours, (iv) 16 hours, (v) 18 hours, and (vi) 20 hours. PTD-NY-ESO-1 persisted after 14 hours in dendritic cells treated with LLNL but was rapidly degraded in untreated dendritic cells. Maximum accumulation of PTD-NY-ESO-1 was observed after 14 hours, indicating that this time point may be the most advantageous incubation period for optimal presentation of NY-ESO-1 peptides by MHC class I molecules.

 
Protein transduction did not alter the phenotypic characteristics of monocyte-derived dendritic cells. Analysis of monocyte-derived dendritic cell surface expression levels of CD1a, CD11c, CD86 (B7.2), HLA-DR, CD83, and CD80 (B7.1) are consistent with a mature dendritic cell phenotype, indicating that pulsing with PTD-NY-ESO-1 did not interfere with the expression of critical monocyte-derived dendritic cell surface molecules (Fig. 5i-vi).



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  		Figure 5. Protein transduction of monocyte-derived dendritic cells with PTD-NY-ESO-1 does not alter immunophenotype. Fluorescence-activated cell sorting analysis of PTD-NY-ESO-1-treated monocyte-derived dendritic cells is compatible with a mature dendritic cell immunophenotype. Percentage (%) indicates the level of expression of the respective surface dendritic cell molecule compared with isotype control. The left peak is the isotype control. The grey line is the staining for the molecule of interest. Dendritic cells stained (i) for CD11c, (ii) for CD1a, (iii) for CD86 (B7.2), (iv) for HLA-DR, (v) for CD83, and (vi) for CD80 (B7.1).

 
Efficient generation of NY-ESO-1-specific T cell lines with monocyte-derived dendritic cells transduced with PTD-NY-ESO-1. The efficacy of NY-ESO-1-specific T-cell induction using protein transduction was evaluated by stimulating purified CD3+ T cells from an HLA-A*0201+ NY-ESO-1-expressing myeloma patient with autologous monocyte-derived dendritic cell or PBMC pulsed with 50 µg/mL of the PTD-NY-ESO-1 or control NY-ESO-1 at weekly intervals. After four restimulations, the number of NY-ESO-1157-165 peptide-specific T cells were analyzed with phycoerythrin-labeled NY-ESO-1157-165/HLA-A*0201 tetramers by flow cytometry. We have previously shown that the NY-ESO-1157-165 peptide epitope is highly immunogenic and presented in the context of HLA-A*0201 (6). The fraction of NY-ESO-1157-165 peptide-specific T cells was 44% in lines generated with PTD-NY-ESO-1-pulsed monocyte-derived dendritic cell compared with only 2% in the NY-ESO-1-pulsed monocyte-derived dendritic cell (Fig. 6A). Analysis by ELISPOT showed that the CTLs generated with PTD-NY-ESO-1 secreted substantially higher levels of IFN-{gamma}, but not IL-4, indicating polarization of the cytokine response towards Tc1 type (Fig. 6B and C). This data also shows that the process of protein transduction does not render the dendritic cells functionally inactive.



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  		Figure 6. CTL lines generated with PTD-NY-ESO-1 contain a high number of NY-ESO-1-specific T cells by tetramer analysis (A) and are of Tc1 type as shown by ELISPOT assays (B and C). A, highly specific NY-ESO-1 CD8+ T cells were obtained after stimulation with mature monocyte-derived dendritic cells pulsed with PTD-NY-ESO-1 protein on a weekly basis for 5 weeks. i, CD8+ CTLs generated with PTD-NY-ESO-1-pulsed mature monocyte-derived dendritic cell after three restimulations (right) and four restimulations (left). ii, marked reduction in NY-ESO-1-specific CTLs generated with NY-ESO-1 protein pulsed mature monocyte-derived dendritic cell after three and four stimulations. ELISPOT assay (B and C). Top, 105 PTD-NY-ESO-1-specific CTLs were cocultured with 104 monocyte-derived dendritic cell mock pulsed with E711-20 peptide, or pulsed with NY-ESO-1157-165 peptide, NY-ESO-1 protein, or PTD-NY-ESO-1 protein. B, columns, average number of spots per 106 T cells from quadruplicate wells. C, images of IL-4 and INF-{gamma} secretion from representative wells of PTD-NY-ESO-1-pulsed monocyte-derived dendritic cell targets and PTD-NY-ESO-1-specific CTLs as effectors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Induction of potent CTL responses is critical to achieve effective therapeutic anticancer vaccination. A number of new vaccination strategies have been developed, which often use whole antigen to stimulate CD4+ T-cell responses. This facilitates CD8+-mediated tumor rejection and generates effective long-lasting antitumor T-cell memory. In addition, use of proteins rather than peptides does not restrict the use of a vaccine to specific HLA types and allows for the natural selection of (multiple) T-cell epitopes. A variety of different methods have been used to deliver whole antigen into dendritic cells, including the use of immune complexes (44, 45), mannosylated antigens (46), liposomal delivery (47), particulate antigen (48), apoptotic tumor cells (49, 50), tumor lysates (51), toxins fused to CTL epitopes (52), pulsing with tumor-derived RNA (53), and the creation of dendritic cell tumor hybrids (54). The mere existence of this myriad of delivery methods reflects the inherent difficulty in introducing tumor antigen into the cytosol of dendritic cells.

Dendritic cell vaccination trials in myeloma have mostly relied on exogenous pulsing of dendritic cell with idiotype or alternatively myeloma lysates, which results in suboptimal access of tumor peptides via cross-priming to HLA class I at the dendritic cell surface and thus poor CTL induction. In addition, idiotype is probably a weak tumor rejection antigen. We addressed both issues by (a) selecting the highly immunogenic tumor-specific antigen, NY-ESO-1, which is frequently expressed in high-risk myeloma and (b) modifying this antigen with a PTD to allow for abundant access to the cytosol and the HLA class I pathway antigen processing and presentation. The PTD abrogates the need for cross-priming of protein antigen, which is usually inefficient.

Protein transduction technology has been used to introduce many therapeutic proteins inside mammalian cells by allowing free trafficking through the membrane barrier (55). In the present study, we constructed a NY-ESO-1 protein expression vector in frame with a PTD motif, derived from the HIV-1 TAT protein. PTD-NY-ESO-1 and control NY-ESO-1 proteins were highly purified. Rapid uptake of PTD-NY-ESO-1 into monocyte-derived dendritic cells was confirmed by immunohistochemistry, flow cytometry, Western blotting, and staining with a PTD antibody. Further confirmation of protein transduction was obtained by inhibiting the macropinocytotic activity of monocyte-derived dendritic cells with amiloride, which did not affect the entry of PTD-NY-ESO-1 into the dendritic cell cytosol. We showed that PTD-NY-ESO-1 was processed by the proteasome, because blocking of the proteasome substantially prolonged the cytoplasmic presence of PTD-NY-ESO-1. Presentation of tumor peptides on the surface of dendritic cells may be limited by the quick turnover of MHC class I molecules, and the prolonged presence of PTD-NY-ESO-1 in monocyte-derived dendritic cells may augment the presentation of MHC/NY-ESO-1 peptide complexes.

Tetramer analysis indicated superior generation of antigen-specific T lymphocytes with PTD-NY-ESO-1 compared with NY-ESO-1 control protein (44% versus 2%, respectively). Efficient IFN-{gamma} but not IL-4 secretion by NY-ESO-1-specific T lymphocytes generated with PTD-NY-ESO-1 indicated Tc1-type cytokine responses that are needed for the maintenance and memory of CTLs (56). Together, these findings suggest that PTD-NY-ESO-1 accesses the cytoplasm by protein transduction, is processed by the proteasome, and that NY-ESO-1 peptides presented by HLA-class I elicit NY-ESO-1-specific T lymphocytes. It is likely that these T cells will be cytotoxic to NY-ESO-1-expressing targets, as we have previously shown (6). It is important to point out that the CTL data were obtained with the cells from a single myeloma patient and that the T-cell response obtained with PTD-NY-ESO-1 require further study in other myeloma patients to ensure reproducibility. Lastly, characterization of quantitative differences in the efficiency of antigen presentation when using the PTD-NY-ESO-1 protein internalized by the endocytic pathway is a further point of interest, which needs to be addressed in future studies.

Other investigators have also reported potent immune responses using Tat-PTD as a vehicle to deliver tumor and viral antigens into dendritic cells (38). Vaccination of mice with melanoma-specific antigen-transduced monocyte-derived dendritic cells by TAT-mediated protein has been shown to generate durable and robust CTL responses, which regresses tumors (39). Tanaka et al. observed that a TAT fusion protein with HER-2/neu generated efficient CTL responses in contrast to the poor CTL generation observed without the presence of a TAT PTD (38). Furthermore, TAT-fusion proteins do not only induce specific CTLs but also generate potent T-helper cell responses due to simultaneous loading of the HLA class II pathway of antigen processing and presentation (40).

Other proteins with transduction capability have been described including the herpes simplex virus transcription factor, VP22 (57), and the Drosophila homeotic transcription protein Antennapedia (AntpHD; ref. 58). AntpHD-linked antigenic epitopes are able to prime antigen-specific CTL in vivo in mice and a VP-22 has been shown to enhance cross-presentation of MART epitopes by human dendritic cells (58, 59). PTD-mediated transduction of proteins seems independent of receptor, transport, and endocytotic mechanisms and does not require energy, because it also occurs at 4°C. The mechanism of PTD is not fully understood, but the various PTD domains share highly charged, basic arginine residues, and the transduction efficiency seems related to the number and location of arginine residues in the PTD sequence. It has been argued that the internalization of PTDs is mediated by an interaction between arginine residues and charged membrane constituents (60).

In a recent study, recombinant NY-ESO-1 protein that was formulated in a special adjuvant called ISCOMATRIX induced broad humoral and cellular immune responses in patients with NY-ESO-1-positive melanomas. Patients who received such vaccinations relapsed less frequently than patients receiving NY-ESO-1 alone or placebo (61). ISCOMATRIX contains a mixture of saponin, cholesterol, and phospholipids and delivers NY-ESO-1 to the dendritic cell cytosol. This study supports our use of whole length recombinant PTD-NY-ESO-1 protein as potent immunogen and emphasizes the need for accessing the cytosol of APCs directly.

We have already established, in a pilot trial with tumor lysate–pulsed dendritic cells, a clinical model for vaccinating myeloma patients who receive autologous transplantation. This approach entails early vaccination before transplantation, collection of primed vaccine-induced T cells, and reinfusion of these cells immediately after autotransplantation. Multiple booster vaccines are then given immediately after reinfusion of primed cells in an effort to circumvent the immune paralysis observed after transplantation and boost the T-cell response in the setting of a relatively empty lymphoid system to achieve preferential expansion of vaccine induced T cells (62). A similar approach has recently been published by Dudley et al. using adoptively transferred melanoma-specific T-cell clones (63). A recombinant PTD-NY-ESO-1 protein could be employed in a similar fashion and may result in clinically effective immunotherapy for a group of myeloma patients who have a poor prognosis and are unlikely to have durable responses to PBSCT alone.


   Acknowledgments
 
Grant support: Multiple Myeloma Research Foundation Senior Research grant G1-11186-01E (F. van Rhee), Multiple Myeloma Research Foundation Junior Research grant G1-11052-01-E (R.B. Batchu), NIH program project G1-10507-05-03E (R.B. Batchu), Lymphoma and Leukemia Society Translational research grant G1-10676-03E (F. van Rhee), and NIH grant R01CA90850 (S. Ponnazhagan).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/ 5/05. Revised 7/ 6/05. Accepted 8/17/05.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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