Rapid construction of a dendritic cell vaccine through physical perturbation and apoptotic malignant T cell loading
© Salskov-Iversen et al; licensee BioMed Central Ltd. 2005
Received: 04 April 2005
Accepted: 19 July 2005
Published: 19 July 2005
We have demonstrated that adherence and release of monocytes from a plastic surface drives their differentiation into immature dendritic cells (DC,) that can mature further during overnight incubation in the presence of apoptotic malignant T cells. Based on these results, we sought to develop a clinically, practical, rapid means for producing DC loaded with malignant cells.
A leukapheresis harvest containing the clonal, leukemic expansion of malignant CD4+ T cells was obtained from the blood of patients with cutaneous T cell lymphoma (CTCL). CTCL cells were purified with a CD3-magnetic bead column where CD3 engagement rendered the malignant T cells apoptotic. The monocyte fraction was simultaneously activated by column passage, re-added to the apoptotic CTCL cells and co-cultured overnight. CTCL cell apoptosis, DC differentiation and apoptotic malignant T cell ingestion were measured by immunostaining.
The results demonstrate that as monocytes passed through the column matrix, they became activated and differentiated into semi-mature DC expressing significantly increased levels of class II, CD83 and CD86 (markers associated with maturing DC) and reduced expression of the monocyte markers CD14 and CD36. Apoptotic malignant T cells were avidly engulfed by the phagocytic transitioning DC. The addition of supportive cytokines further enhanced the number of DC that contained apoptotic malignant T cells.
Functional studies confirmed that column passaged DC increased class II expression as shown by significantly enhanced stimulation in mixed leukocyte culture compared to control monocytes. In addition, DC loaded with apoptotic CTCL cells stimulated an increase in the percentage and absolute number of CD8 T cells compared to co-cultivation with non-loaded DC. After CD8 T cells were stimulated by DC loaded with malignant cells, they mediated increased apoptosis of residual CTCL cells and TNF-α secretion indicating development of enhanced cytolytic function.
We report a simple one-step procedure where maturing DC containing apoptotic malignant T cells can be prepared rapidly for potential use in vaccine immunotherapy. Ready access to both the DC and apoptotic cells provided by this system will allow extension to other malignancies through the addition of a variety of apoptotic tumor cells and maturation stimuli.
Cutaneous T cell lymphoma (CTCL) is a malignant expansion of mature, clonal CD4 T cells with an affinity for epidermal localization . The tumor cells proliferate in the epidermis around a central Langerhans cell (LC) and previous studies have demonstrated that immature DC play a crucial role in the life cycle of the malignancy . The final stages of CTCL are characterized by systemic spread, immunosuppression and a poor prognosis. Despite the malignancy's dependence on immature DC for proliferative support, DC immunotherapy has been of benefit in this disease [3, 4].
Two strategies for the treatment of CTCL, extracorporeal photopheresis (ECP) and transimmunization, have been used to successfully treat this aggressive malignancy [4, 5]. The underlying principle of these treatments is extracorporeal establishment and re-infusion of malignant T cell-loaded DC . In both therapies, a leukapheresis product is treated with the drug 8-methoxypsoralen (8-MOP) and passed through a plastic ultraviolet light (UVA) exposure plate. The 8-MOP intercalates in the DNA of nucleated cells and is cross-linked to adjacent pyrimidine bases by UVA light activation. The cross-link formation is a lethal defect and replicating cells are rendered apoptotic. At the same time, monocytes are activated by adherence and release from the plastic exposure plate surface and begin to transition into immature DC . In the ECP treatment, both apoptotic CTCL cells and transitioning DC are re-infused into the patient immediately and association of the DC and apoptotic tumor cells occurs inefficiently in vivo.
The transimmunization procedure was devised as a more effective modification of ECP and named to designate the transfer of tumor antigens to competent antigen presenting cells (APC) that could display the full complement of tumor antigens in the context of co-stimulatory and adhesion molecules. In the transimmunization procedure, the apoptotic malignant T cells and the transitioning DC are co-cultured overnight enabling the up-take of the apoptotic cells by the avidly phagocytic immature DC . The activated monocytes produce cytokines that comprise the constituents of monocyte conditioned media thereby, potentiating the maturation of the malignant T cell-loaded DC . The differentiating DC are re-infused the next day into the patient where they can further mature and have the potential to migrate to lymph nodes and induce anti-tumor immunity.
In the current studies, we sought to explore the role of physical perturbation in the monocyte to DC transition by examining whether passage through a separation column that contains a porous matrix is sufficient to induce overnight DC differentiation from monocytes. Studies  suggest that trans-migrating monocytes passing through the small spaces of an endothelial cell layer become activated and assume the phenotype of immature DC. This monocyte-to-DC transition can be preserved by phagocytosis of particulate material such as zymosan . We have also previously demonstrated that CD3-binding renders antigen-experienced proliferating CTCL cells apoptotic . We therefore sought to take advantage of the dual observations of the role of physical stimulation in DC maturation and the rapid apoptotic cell death mediated by CD3-binding to develop in one day a clinically practical vaccine. We demonstrate that a simple one-step procedure using CD3-magnetic beads to render the malignant T cells apoptotic and the separation column matrix to simultaneously activate monocytes results in overnight production of apoptotic cell-loaded DC. These immature DC generated in the absence of cytokines could be driven to differentiate further when exogenous cytokines were added. Functional evaluation of the malignant T cell loaded DC, developed by this methodology, demonstrated a significantly enhanced stimulatory capacity in mixed leukocyte culture and the ability to promote CD8 T cell expansion and cytolytic capacity.
Therefore, this approach yields malignant cell loaded DC in a rapid time-frame without extensive cell culture, exogenous factors or cell isolation and manipulation. This method may provide a clinically practical means for the production of immunogenic DC for cancer vaccine therapy.
Materials and methods
Therapeutic leukapheresis specimens were obtained from 7 CTCL patients (in accordance with the guidelines of the Yale human investigation committee). All patients had advanced disease with clonal CD4+ T cell populations present in the peripheral circulation as determined by immunophenotyping with antibodies to the clonotypic variable region of family-specific T cell receptor (TCR) or polymerase chain reaction to detect rearrangements of the beta or gamma chain of the TCR. All patients were undergoing treatment with standard ECP.
Mononuclear cells (MNC) were isolated by centrifugation over a ficoll-hypaque gradient followed by two washes in RPMI 1640 (Gibco, Gaithersburg, MD) containing 10% AB serum and 2 mM EDTA. MNC (2 × 107) were incubated with 40 μl Macs α-human CD3 microBeads (Miltenyi Bioteck, Auburn CA) following the manufacturer's directions. The cells were separated by passage through a Macs Separation Column (Miltenyi Bioteck) consisting of a magnetized iron matrix. CD3 positive and negative cells were counted, re-mixed together and incubated overnight. As a control, MNC (2 × 107) were also incubated with 40 μl Macs α-human CD4 microBeads. After treatment, the cells were incubated in 3 ml RPMI 1640 containing 15% AB serum and 15% autologous plasma in one well of a 12 well tissue culture plate (Falcon). In some experiments half of the recombined cells obtained after CD3 column passage were incubated overnight in RPMI containing 10% FCS (Gibco) in the presence of the cytokines GM-CSF 800 U/ml and IL4 1000 U/ml (R & D Systems, Minneapolis, MN). Day 0 baseline cells were immediately removed for immunostaining while Day 1 cells were incubated overnight.
In order to monitor DC differentiation, the cells were stained by two-color immunofluorescence with a panel of antibodies to monocytes, DC and apoptotic cells. Cells (1 × 106) were incubated with 10–20 μl of fluorocrome conjugated monoclonal antibody for 30 minutes in the dark at 4°C. The antibodies were directly conjugated to fluorescein (FITC) or phycoerythrin (PE) and included: CD14-FITC (monocytes) + CD86-PE (co-stimulatory molecule highly expressed on DC); HLA-DR-FITC (anti-class II MHC molecule) and CD83-PE (DC maturation marker); and their isotype matched controls (Beckman Coulter Immuno-Tech, Hialeah, FL). Cells were washed once and suspended in PBS and read on a XL flow cytometer (Beckman Coulter) within 24 hours.
Combined membrane and cytoplasmic staining was performed following manufacturers instructions (Intraprep kit, Beckman Coulter). Antibody combinations included: membrane CD36-FITC (receptor for apoptotic cells) + cytoplasmic CD83 PE; DR-FITC + cytoplasmic CD83-PE; and isotype controls (Beckman Coulter). To detect apoptotic cells, lymphocytes were stained with: membrane HLA-DR-FITC (class II MHC) + cytoplasmic Apo2.7-PE (apoptotic cells); and isotype controls. Data was analyzed using the CXP software (Beckman Coulter).
Cells were double-stained for membrane HLA-DR-FITC + cytoplasmic Apo2.7-PE following the manufacturer's instructions for combined membrane and cytoplasmic staining (see immunophenotyping). In addition, cells were double stained for cytoplasmic LAMP-2 FITC (lysosomal marker, Research Diagnostics) and HLA-DR-PE. Cells were prepared for microscopy following the instructions for Molecular Probes "Slow Fade Light" anti-fade kit (Molecular Probes Inc, Eugene, OR). Specimens were kept in the dark at 4° until microscopy was performed on a Zeiss confocal microscope.
Mixed leukocyte culture assay
The mixed leukocyte culture assay was performed by isolating control leukocytes from two normal donors. Control T cells were purified with CD4 magnetic beads and the column effluent containing monocytes and B cells was γ-irradiated to prevent differentiation and used as a source of stimulators. Transitioning DC from CTCL patients were obtained one day prior to the normal control cells and cultured overnight without cytokines, γ-irradiated and used as stimulators for the control lymphocytes. The cells were adjusted to 4 × 106/ml and 50 μl of responding cells and 50 μl of stimulating cells co-cultured in round bottom microtiter wells with the addition of 100 μl of RPMI 1640 containing 15% AB serum and 15% autologous plasma for 6 days at 37°C under a 5% CO2 atmosphere. The wells were pulsed with 1 μCi/well 3[H]-thymidine 16 hours prior to harvest (PhD harvester, Cambridge Tech., Cambridge, MA). The incorporation of the isotope was evaluated in a liquid scintillation counter.
CD8 T cell purification and expansion
CD8 T cells were purified with CD8-magnetic beads (≥96% purity) and suspended in RPMI 1640/15% autologous serum and IL2 and added to DC that had been column eluted from the same CTCL patient. The cells were co-cultured overnight with 1.1 × 106 CD8 T cells/well added to CD3-bead rendered apoptotic CTCL cells or viable CTCL cells (4 × 106/well). After overnight culture, the cells were harvested, counted, and immunophenotyped for markers of T cells (CD3, CD4, CD8) and apoptosis (Apo2.7).
Tumor necrosis-α(TNF-α) ELISA
The production of TNF-α was measured in an ELISA assay (R&D Systems, Minneapolis, MN) essentially as described by the manufacturer.
The expression of DC markers and the MLC response was evaluated statistically by the student's t test or if the data was not normally distributed the Mann-Whitney Rank Sum Test using the Sigma Stat analysis program.
Passage of monocytes through a separation column induces monocyte to DC transition
Transitioning DC increase their expression of the maturation marker, CD83
The increase in cytoplasmic CD83 expression is shown in Fig. 2B. As expected only a small percentage of cells express the DC differentiation marker, CD83 on primary isolation (0.5%, Fig. 2B-a). Overnight incubation of the leukapheresis (Fig 2B-b) increases CD83 expression to an equivalent degree as CD83 expression detected after passage through a CD4-magnetic bead column (Fig. 2B-c). More than one third of the monocytes transitioned into semi-mature DC as shown by the increased expression of cytoplasmic CD83 (Fig. 2B-d) found when CD3-separated apoptotic CTCL cells were added to the column activated monocytes.
Induction of simultaneous DC differentiation and CTCL cell apoptosis and engulfment
Differentiation of the DC population was also demonstrated by the increase in expression of membrane class II MHC molecules. Physical manipulation did not increase class II expression from the primary value obtained on initial isolation (Fig. 3B-a), when leukapheresis cells were cultured overnight (Fig. 3B-b). No enhancement of class II expression was noted even when the column activated monocytes were co-cultured overnight with CD4-bead separated CTCL cells (Fig. 3B-c). However, the overnight addition of apoptotic CTCL cells, obtained after CD3-binding, to transitioning DC increased class II expression from 55% (Day 0, Fig. 3B-a) to 72% (Fig. 3B-d).
Statistical evaluation of the enhanced expression of DC differentiation markers
Demonstration of DC loading with apoptotic cells by confocal microscopy
To confirm that class II molecules co-localized in lysosomal compartments in a pattern found in semi-mature DC , cells were stained with a lysosomal marker LAMP2 and an antibody to class II MHC molecules (Fig. 5B). In Fig. 5B-a, a cell that has been activated by passage through the separation column and co-cultivated overnight with CTCL cells rendered apoptotic by CD3-magnetic bead binding was stained with an anti-class II antibody (red). In Fig. 4B-b lysosomal compartments were visualized with an antibody that binds to the lysosomal membrane (LAMP2, green). Merging of the 2 fluorochromes (Fig. 5B-c, yellow) demonstrates colocalization of class II MHC molecules in lysosomal compartments. When class II staining was monitored on column activated transitional cells that had been co-incubated with control CTCL cells selected by CD4-magnetic bead separation (Fig. 5B-d, red), strong membrane staining was found. Weak lysosomal staining was localized beneath the plasma membrane (Fig 5B-e, green). When the pictures were merged, class II MHC molecules did not exhibit entry into the lysosomal compartment (Fig. 5B-f). The presence of class II MHC molecules in lysosomes is consistent with differentiation into semi-mature DC , and suggests that class II molecules have migrated to lysosomal compartments where they would have the opportunity for loading with peptides derived from processed apoptotic material.
The addition of supportive cytokines enhances monocyte to DC differentiation
We sought to maximize induction of maturing DC loaded with apoptotic malignant T cells through the addition of exogenous cytokines known to be important for DC differentiation . To study the effect of supportive cytokines on the phenotype of the developing DC, we divided the column separated cells in half and co-incubated them overnight with CD3-bead rendered apoptotic CTCL cells with and without GM-CSF and IL-4.
Cytokines enhance DC maturation
Class II expression and up-take of apoptotic material is enhanced in the presence of cytokines
The baseline expression of class II MHC molecules on the cell membrane of monocytes on primary isolation is shown in Fig. 8a. Freshly isolated monocytes express a reduced intensity of class II expression and contain a small percentage of cytoplasmic apoptotic material. After column separation and co-incubation with CD3-magnetic bead treated apoptotic cells, membrane class II expression is enhanced (Fig. 8b) and large amounts of apoptotic material can be detected in the cytoplasm of the transitioning DC. Exogenous cytokines further increase the percentage of class II-positive cells that contain apoptotic material (Fig. 8c). Therefore, the addition of exogenous cytokines enhances both the differentiation of immature DC and the ingestion of apoptotic material improving the overnight yield of maturing apoptotic T cell loaded DC.
Functional analysis of the differentiating DC obtained after column passage
The level of apoptosis found when CD8 T cells were cultured overnight with column activated-DC loaded with CTCL cells doubled (56%, Fig. 10B-a) in comparison to the level of apoptosis present when CD8 T cells were added to non-loaded DC that had been cultivated with viable CTCL cells (27%, Fig. 10B-b). The baseline level of apoptosis was 24% when malignant T cell loaded DC (Fig. 10B-c), or non-loaded DC (Fig. 10B-d) were cultured in the absence of CD8 T cells. These results indicate that residual CTCL cells may be lysed in the presence of CD8 T cells stimulated with DC that have ingested apoptotic malignant T cells.
Finally, further support for the contention that functional CD8 T cells were expanded by overnight exposure to column-activated DC loaded with malignant T cells was obtained by evaluation of the levels of TNF-α found in the culture supernatants. In Figure 10B-e, supernatants from CD8 T cells cultured overnight alone contained minimal levels of TNF-α. CD8 T cells stimulated with column differentiated DC loaded with CD3-bead rendered apoptotic malignant T cells or not loaded both significantly (P ≤ 0.001 & P ≤ 0.014) stimulated release of TNF-α. However, DC that had engulfed apoptotic cells caused the release of three fold more TNF-α than non-loaded DC, indicating that CD8 T cell activation had occurred and the release of a molecule that promotes tumor cytolysis was present.
Development of effective DC based cancer vaccine technology has been limited by the extensive manipulation and extended period of in vitro culture required for generation of mature DC loaded with the appropriate tumor antigens. We have circumvented some of these limitations through modification of a successful technology that permits both DC differentiation from peripheral monocytes and simultaneous loading of DC with apoptotic malignant T cells containing the full complement of potential tumor antigens . DC are the most potent APC displaying when mature high levels of co-stimulatory, adhesion and MHC molecules which can present peptides derived from apoptotic cells to the immune system . Therefore, the development of a simple rapid means of generating malignant cell-loaded DC could advance the immunotherapy of CTCL and perhaps other malignancies.
Immunotherapy has played a major role in the treatment of CTCL since the introduction of ECP by Edelson and colleagues in 1987 . The mechanism underlying the success of ECP treatment was defined by the demonstration that the simultaneous introduction of apoptotic malignant T cells and the differentiation of monocytes into DC resulted in patients receiving CTCL cell-loaded DC that have the capacity to present antigen, derived from the CTCL cells, to cytotoxic lymphocytes and initiate an immune response towards the malignant CD4 T cells. Previous studies had demonstrated that despite the clonal expansion of CD4+ malignant T cells in the peripheral blood of CTCL patients, circulating populations of CD8 T cells that retained the capacity to lyse autologous malignant T cells  could be identified. One antigen that served as an immunogen recognized by cytotoxic T cells in CTCL was determined to be peptides derived from the beta chain of the TCR that was clonotypically displayed on the malignant T cells [12, 13]. Therefore, the potential for development of an anti-malignant T cell immune response exists in CTCL patients and immunotherapeutic approaches designed to expand anti-tumor CD8 T cells could be effective in this disease.
We sought to exploit our understanding of the mechanism of ECP to develop more efficient, rapid, clinically practical means for producing malignant T cell-loaded DC. In the current study, we demonstrate that DC loaded with apoptotic cells can be produced in one day without extensive manipulation or the use of exogenous cytokines. The use of CD3-antibody to render CTCL cells apoptotic and passage of the treated MNC through the small pores of the iron matrix of a separation column followed by overnight co-incubation resulted in the generation of DC containing material derived from apoptotic CTCL cells. DC differentiation was demonstrated by both the reduction in monocyte markers and the significant increase in class II MHC molecules and co-stimulatory molecules, as well as the increase in CD83, a marker of maturing DC. The internalization of apoptotic blebs was confirmed by localization of the apoptotic material in the cytoplasm, indicating that processing of the apoptotic CTCL-derived material could make peptides available for MHC loading and transport to the cell membrane . The ability to increase the number of maturing CTCL cell-loaded DC by the addition of exogenous cytokines demonstrates that this technique can produce cell populations that can be manipulated to maximize the production of DC that contain apoptotic material thereby providing access to a spectrum of CTCL cell-derived epitopes, without the requirement for identification or isolation of individual peptides that may be relevant for induction of an anti-CTCL cell immune response.
Furthermore, we show that DC produced in this fashion are effective stimulators of alloproliferation in MLC confirming the significant up-regulation of class II MHC molecules. The malignant T cell loaded DC stimulated CD8 T cell expansion and an increase in apoptotic cell death and the significantly enhanced release of TNF-α. These results indicate that CD8 T cells that have been stimulated by malignant T cell loaded DC, produced by this methodology, may develop the ability to mediate tumor cell cytolysis.
The current studies support our previous results demonstrating that monocyte differentiation into DC could be driven by increasing levels of physical perturbation . We confirm that leukapheresis alone generates modest monocyte activation and conversion into immature DC that can be enhanced by further manipulation and the addition of apoptotic cells. We also demonstrate that CD3-binding is a potent means of rendering CTCL cells apoptotic  even when the CTCL cells are not cultured but directly isolated from the patients. The current study combines and extends these two previous observations into a format for simple, rapid, clinically practical DC vaccine generation.
Current approaches to DC vaccine technology include peptide pulsing , one week or longer of culture with cytokines , cell fusion with tumor cell partners , and the use of a variety of vectors designed to introduce tumor antigens into the DC . These methods are generally cumbersome, require extensive in vitro manipulation, and are limited to a small set of known tumor epitopes that may be lost from the patient's tumor, due to immuoselective pressures. Clinical results with these techniques have been variable and seldom provide long-term responses . In contradistinction, treatment of CTCL patients with ECP has demonstrated an excellent safety profile and in multiple studies in the literature an overall response rate for all stages of the disease of 55.7% and a complete response rate of 17.6% . Pilot studies using transimmunization to enhance the interaction of apoptotic tumor cells and differentiating DC through simple overnight incubation has demonstrated encouraging results in some patients , that suggest that the therapy retains the safety profile of ECP but may be more potent and effective in a shorter time course.
The technology proposed in this study is likely to be as safe as transimmunization and ECP since it retains the same features of limited cellular manipulation and culture. The replacement of 8-MOP with CD3 antibody should not lead to significant apoptotic cell death and potential tumor lysis syndrome since CD3-binding renders only 30% of the CTCL cell population apoptotic . Since the CD3 antibody is conjugated to the magnetic beads any free antibody could be removed by a second passage through the magnet prior to re-infusion, thereby, limiting the induction of anti-CD3 antibodies. However, presentation of portions of the CD3 antibody after DC ingestion may provoke an immune response that could prevent further therapy. These potential safety issues will require careful monitoring in future clinical trials.
The current results demonstrate that further development of this technology through passage over a column that permits the one-step apoptotic cell death of CTCL cells, sparing of normal cells and activation of monocytes into the DC pathway may further improve the immunogenicity of the reinfusate. Since only proliferating tumor cells are rendered apoptotic by the CD3 antibody, normal resting lymphocytes will not be impacted which is in contrast to the use of 8-MOP/UVA that targets the DNA of all nucleated cells. This preservation of normal T cells may serve to improve the induction of anti-CTCL immune responses to the re-infused apoptotic cell-loaded DC by preventing damage to by-stander normal cells and precluding their uptake that could lead to tolerance induction .
Using a peristaltic pump it should be possible to rapidly flow a leukapheresis product through a magnetic separation column. Due to the concentration of MNC obtained with the leukapheresis procedure, high yields of monocytes approaching 108 cells could be obtained and activated by this procedure . Since CTCL patients have large populations of circulating malignant T cells (approaching >90% of the lymphocyte population), CD3-treatment would provide substantial apoptotic tumor cells for DC loading. Because both activated monocytes and apoptotic malignant T cells are obtained individually and can be re-added after treatment, the optimal conditions for apoptotic T cell and DC co-cultivation can be determined empirically. This access to both cell populations would permit the opportunity for loading DC with other tumor antigens, including solid tumors rendered apoptotic by irradiation or other methods.
Other studies have determined that physical separation of DC clusters by simple pipetting  or cell transfer [8, 23] is among the most potent means of inducing DC maturation. Furthermore, even semi-mature DC are effective at cross-priming peptide  derived from exogenous material into the class I pathway for presentation to CD8 T cells. Our simple approach to rapid DC vaccine construction takes advantage of both physical stimulation and production of apoptotic material providing access to a broad spectrum of CTCL antigens for cross-priming into the class I pathway.
Further studies to determine the functional ability of the CTCL cell-loaded DC produced by this methodology will be required to confirm the immunogenicity of the proposed vaccine components. We have already demonstrated that DC loaded with apoptotic malignant T cells are potent immunostimulators in mixed leukocyte culture , can provoke positive clinical results in treated patients  and that responsive patients treated by standard ECP develop increased levels of circulating CD8 T cells . The current results indicate that the development of DC loaded with apoptotic cells for use in immunotherapy can be performed in a rapid, simple, clinically practical manner that provides ready access to the major cell types so that additional strategies to optimize the vaccine components can be implemented and monitored prior to re-infusion.
The authors wish to acknowledge research support from: The Danish Cancer Society, M. S.-I. and the NY Cardiac Association, C.L.B. & R.L.E
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