Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes
© Gullo et al; licensee BioMed Central Ltd. 2008
Received: 06 November 2007
Accepted: 28 April 2008
Published: 28 April 2008
As the eradication of tumor cells in vivo is most efficiently performed by cytolytic T lymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described.
Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity.
By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p < 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells.
Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens.
Multiple myeloma (MM), a malignancy of terminally differentiated plasma cells, is the most common hematologic cancer in the United States (US) . Currently, MM is incurable and there is an urgent need for the development of novel and curative forms of therapy, including immunotherapy. A major limitation on the treatment of all cancers is the evasion strategy developed by tumor cells to bypass immune surveillance. Thus far, specific anti-cellular therapy against MM has been difficult to develop because of the down regulation of tumor cell surface antigens (Ag) and the attenuation of host immunity by MM cells. The majority of MM cellular immunotherapies have focused on deriving anti idiotypic (Id) patient-specific CTL [2–4]. Other strategies include the use of MM-specific RNA transduced dendritic cells and MM apoptotic bodies pulsed dendritic cells [5, 6]. Unfortunately, although some of these studies were promising none of them have achieved high response rates and therefore have not been actively pursued.
As eradication of tumor cells in vivo is most efficiently performed by CD8+ and/or CD4-CD8- natural killer (NK) cytolytic T lymphocytes (CTL) [7, 8], various methods for increasing recognition by lymphocytes have been developed. Since the success of such therapies depends significantly on the efficiency of Ag presentation, tumor cells have been genetically modified to present antigens directly to tumor reactive T cells . Other potentially safer methods include the use of cytokines and/or adjuvants to increase the antigenicity of the tumor itself or to enhance the immune recognition of the tumor in vivo . Reinfusion of tumor-specific CTLs as therapy against cancer (i.e. adoptive immunotherapy) has been used in the treatment of a number of cancers  proving particularly effective in some patients with melanoma . Several groups have also recently characterized a novel class of cells that have potent innate anti-tumor properties termed Cytokine Induced Killer (CIKs)  or Interferon-γ producing Killer Dendritic Cells (IKDCs) . Those as well as newly developed anti-tumor cytolytic cells with NK cell markers  demonstrate renewed interest in finding non antigen-specific anti-tumor effector cells for cellular adoptive therapy.
Unfortunately, searches for novel tumor-associated (TAAs) for many cancers have been extremely slow and those that do exist have rarely translated into clinically effective therapies . Thus finding a source of tumor-specific antigens with adjuvant properties that can be used in an adoptive-immunotherapy setting to elicit strong T cell responses is greatly desired. In a landmark paper by Srivastava et al., tumor antigens were isolated from chemically induced sarcomas and found to be capable of mediating tumor rejection . One of these tumor antigens was identified as tumor rejection Ag-1 (TRA-1, gp96, Grp94), a heat shock protein (HSP)-90 family member , which is capable of mediating tumor rejection of various cancers. Heat shock proteins are induced when cells are exposed to stress (e.g. DNA damage) , and have now been intimately linked to antigen presentation pathways [19, 20]. In fact, HSPs Grp94, HSP-90, and Grp78 are known to carry peptides that are stimulatory to CD8+ CTL via a process known as cross-presentation  and are being currently used for immunotherapy [22–24]. Expression of HSP can be induced using ultraviolet (UV) light irradiation , which damages DNA and triggers the stress response in the cell . Hence, we embarked on this study to determine if UV-irradiation could be used as a means to concomitantly increase HSP expression and tumor cell antigenicity of MM cells, resulting in the production of effective anti-tumor CTLs. We demonstrate that UV-irradiation indeed induces total RNA, protein, as well as surface expression of Grp94 and Grp78, two potent immunogenic HSPs. Furthermore, UV-irradiated MM cells were shown to be effective in priming CD8+ CTL and resulted in efficient induction of anti-MM CTL in-vitro. Finally, recognition of the UV-irradiated tumor cells by the CTL was partially inhibited by the ansymcin antibiotic 17-AAG, an HSP-protein inhibitor [9, 16, 18].
The human RPMI 8226 MM (CCL-155) cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD) and cultured in complete media consisting of 90% RPMI 1640 with L-glutamine media, 10% fetal bovine serum (FBS), 25 IU/ml penicillin, 25 μg/ml streptomycin, and additional 5 mM L-glutamine. Normal human peripheral blood (PB) was obtained from volunteer donors with informed consent and under institutional review board (IRB) approval (Singapore General Hospital, IRB approval #103/2003). Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque (Amersham Pharmacia, Uppsala, Sweden) density gradient sedimentation and cultured in complete media. Cell cultures were maintained at 37°C with 5% CO2 in a humidified atmosphere. Cells were enumerated using standard trypan blue (Gibco BRL, Life Technologies, Carlsbad, CA) exclusion assays.
UV-irradiation of RPMI 8226 MM cell line
UV-irradiation was performed using the Stratagene Stratalinker UV Crosslinker (Stratagene, La Jolla, CA). For various experiments, UV-irradiation (6 mJ/cm2 to 240 mJ/cm2) was performed on open 10 cm diameter tissue culture dishes containing RPMI 8226 MM cells (1.5 × 106 in 3.0 mL of complete media).
Cell proliferation assays
Cell proliferation was assessed using DNA synthesis assay. Here, standard tritiated thymidine (3H-TdR, Perkin Elmer Life Sciences, Boston, MA) incorporation assays were performed. Cells (5,000 cells/mL) were first incubated with 0.25 μCi/well overnight and then treated for 3 hrs in 96-well tissue culture plates (200 μL/well) with various doses of UV-irradiation or left untreated. Next, cells were harvested onto fiberglass filters using a cell harvester (Tomtec Mach III Auto, Tomtec, Hamden, CT) and counted on a beta plate reader (Wallac 1450 MicroBeta TriLux, Turku, Finland). Each proliferation result is an average of three independent experiments. Readings are expressed as counts per minute (CPM) and derived from triplicate values for each condition. No proliferation was observed in long-term cultures for cells exposed to 120 mj/cm2 or above UV-irradiation.
Annexin V/propidium iodide (PI) staining
In order to assay for the presence of apoptosis, RPMI 8226 MM cells (1 × 105/sample) were dually stained using fluorescein isothiocynate (FITC) labeled annexin V (annexin V-FITC) and PI (BD Pharmingen, San Diego, CA) according to the protocol provided by the manufacturer. Briefly, cells were washed and suspended in 100 μL of binding buffer, then stained using 5 μL of annexin V-FITC and 2 μL of PI. Analysis was performed on the Cytomics FC500 flow cytometric analyzer (Beckman Coulter, Miami, FL). In this assay, annexin V+/PI- cells represent MM cells in early stages of apoptosis, whereas dually positive annexin V+/PI+ cells are MM cells that have undergone apoptosis. Data from each flow histogram quadrant (Annexin-V FITC detected in the first channel and PI in the second channel) is then tabulated and represented as % apoptosis in chart form.
Priming of PBMCs under 'mixed-culture' conditions
Priming of PBMCs was performed by co-culture of PBMCs (5 × 105/well) and UV-irradiated (120 mJ/cm2, 5 × 106/well) or mitomycin (100 μg/ml, 5 × 106/well) treated RPMI 8226 MM cells in 6-well tissue culture plates. Thus the responder cell to stimulator cell ratio was 1:10. Cell co-cultures were maintained in complete media with recombinant human interleukin-2 (IL-2, R & D Systems, Minneapolis, MN, 0.5 ng/mL). Weekly priming (Days 0, 7, 14, 21 and 28) was performed by adding UV-irradiated or mitomycin-treated RPMI 8226 MM cells (5 × 106/well) into the respective existing co-culture. Half-media exchanges containing IL-2 were performed every 3 days to 5 days. Cells were collected and washed weekly before each 'mixed-culture' priming was performed.
Indirect fluorescence flow cytometric analysis
T cell subset immunophenotyping and HSP expression were performed using indirect fluorescence flow cytometric analysis (Cytomics FC500 flow cytometric analyzer, Beckman Coulter). The following mouse anti-human monoclonal antibodies (mAbs) were used for effector cell immunophenotyping -anti-CD8, anti-CD45, anti-CD56, anti-granzyme A, and anti-perforin (Beckman Coulter). Antibodies were directly conjugated with fluorophores (FITC and phycoerythrin (PE)). Lymphocytes were identified as CD45bright plus low side-scatter cells. For assessment of heat shock protein expression, goat anti-Grp78 (C-20, sc-1051) and goat anti-Grp94 (C-19, sc-1794) (both from Santa Cruz Biotechnology, Santa Cruz, CA) were used. The secondary antibody used (where appropriate) was donkey anti-goat FITC mAb (Santa Cruz). Briefly, primed PBMCs or UV-irradiated RPMI 8226 MM cells (0.5 × 106 to 1.0 × 106/sample) were harvested, washed three times in phosphate buffered saline (PBS), blocked with 10% human AB serum in PBS, and stained using the appropriate mAb at 4°C for 30 mins. Next, cells were washed three times in PBS prior to analysis. Statistical analysis was performed using the Chi-squared test (Microsoft Excel, Microsoft Office 97, Microsoft Corp., Redmond, WA).
A tritiated thymidine (3H-TdR)-based cytotoxicity assay (JAM assay) was used to evaluate the killing of viable RPMI 8226 MM cells by primed and non-primed PBMCs . In this assay, 3H-TdR is first incorporated into DNA during labeling. During analysis, degraded DNA is washed through a fiberglass filter, leaving behind the intact, high molecular weight DNA. Briefly, RPMI 8226 MM cells (target cells, T) were first labeled with 3H-TdR (10 μCi/ml, PerkinElmer Life Sciences, Waltham, MA USA) for 12 hrs in 96-well tissue culture plates (2 × 104 cells/well). Primed or non-primed PBMCs (effector, E) were co-cultured with target cells at various E:T (0:1, 25:1, 50:1, 75:1 or 100:1) ratios for various times. Next, cells were harvested onto fiberglass filters using a cell harvester (Tomtec Inc. Hamden, CT USA) and counted on a beta plate reader (PerkinElmer,). The percentage of specific killing relative to medium-stimulated controls was calculated as [(Spontaneous – Experimental)/Spontaneous] × 100], where the radioactivity (cpm) in T cells exposed to control medium was defined as S and that of treated cells was defined as E .
Whole cell extracts (WCE) were obtained from non-UV-irradiated and UV-irradiated RPMI 8226 MM cells (3.0 × 106 cells/sample) using EBC1 lysis buffer, which contains 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% NP-40, 0.5 μg/mL phenylmethylsulfonyl fluoride (PMSF), 50 mM NaF, 1 mM NaVO4, and a Complete® protease inhibitor tablet (Roche Diagnostics GmbH, Mannheim, Germany) in every 50 mL. Proteins were quantified using Bradford's method (Bio-Rad, Hercules, CA), and resolved (20 μg/lane) in a 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Next, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Schleicher & Schuell, Keene, NH), and blocked for 2 hrs using Tris-buffered saline (TBS) – 20 mM Tris pH 7.6, 150 mM NaCl – containing 1.0% Tween-20 (Sigma-Aldrich, St Louis, MO). Membranes were probed using goat anti-Grp78 (C-20, sc-1051) or goat anti-Grp94 (C-19, sc-1794) mAbs 1:200 dilution (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr, then washed thrice with TBS containing 0.2% Tween-20 (TBST), and then reprobed using horseradish peroxidase (hrp)-conjugated donkey anti-goat IgG mAb 1:15,000 dilution (Santa Cruz Biotechnologies) for 2 hrs. Next, membranes were washed 6 times with TBST, and chemiluminescene detection was preformed using ChemiGlow® reagents and filmless imaging on the FluoChem Imager™ (both from Alpha Innotech, San Leandro, CA). Spot densitometry was performed using AlphaEaseFC™ software (Alpha Innotech). In some cases, to determine relative expression, the normalized values for each condition were derived by taking the integrated density values from the protein in question and dividing that by the values for Actin in that condition.
Quantitative Real Time Reverse Transcription PCR (Q-PCR)
Data was analyzed using the Relative Quantitation software (Roche Diagnostics) and is expressed as relative expression.
Results and Discussion
Effects of UV-irradiation on the proliferation and apoptosis of RPMI 8226 cell line
Since, lymphocyte priming requires long-term cell co-culture with UV-irradiated MM cells, we next compared the effect of different doses (6, 12, 24, 60, 120 or 240 mJ/cm2) of UV-irradiation on MM cell proliferation for up to 8 weeks. Importantly, a low UV dose that prevents overgrowth of cell cultures by the surviving MM cells but that still maintain tumor antigen presentation was selected, as this maneuver would facilitate antigen recognition by T lymphocytes . The minimal UV- irradiation dose that will inhibit long-term growth of RPMI 8226 MM cell line is also 120 mJ/cm2 (data not shown). Hence, this dose of UV-irradiation was used in subsequent experiments.
Expansion of CD8+CTLs by UV-irradiated RPMI 8226 MM under 'mixed-culture' priming conditions
Efficient CTL redirected lysis of fresh RPMI cells following 'mixed-culture' T cell priming conditions
Up regulation of heat shock protein expression on RPMI-8226 cells following UV-irradiation
HSP-peptide inhibitors block UV-irradiated RPMI recognition by cytolytic T cells
Successful cancer immunotherapy requires the reconstitution of host tumor immunosurveillance, and the production of durable cures through induction of immunological memory . Although both humoral and cellular vaccines have been shown to produce cures in the short term, durable long-term cures are only possible with cell-based vaccines (e.g. CTL vaccines), because immunological memory is a cellular function. Unfortunately, the use of autologous CTLs as cellular anti-tumor vaccines for the treatment of MM has been associated with a lack of clinical efficacy; and this is widely thought to be due to the deletion of critical CTL clones that specifically target the tumor [35, 41, 42]. Although this may theoretically be overcome by the use of allogeneic CTL anti-tumor vaccines, this form of immunotherapy is frequently associated with limited  to significant  graft-versus-host-disease (GVHD), and may pose real dangers to the patient.
A special form of anti-cancer vaccination strategy involves the introduction of non-cellular tumor rejection molecules or Ag into a mammalian host to induce cellular immune responses. This vaccination strategy has previously been shown to be effective in promoting tumor recognition and rejection in both autologous as well as allogeneic mice [18, 19]. The principal molecules mediating this process are the HSP molecular chaperones, especially TRA-1. Tumor rejection Ag-1 is a HSP , which has been shown to mediate tumor rejection in various cancers . These molecules act to chaperone tumor-specific peptides to sites in the body where tumor Ag is most effectively presented, thereby inducing tumor recognition and tumor-specific CTL (both CD8+ as well as NK cells) activation. Ultraviolet irradiation has also been shown to result in the presentation of cellular antigens. In fact, high doses of UV-B irradiation induces proinflammatory apoptosis and necrosis, where the production of inflammatory cytokines is accompanied by exposure and release of autoantigens and autoimmune disease .
In the present study, UV-irradiation was used to enhance the immunogenicity of MM cells while at the same time inducing apoptosis of the tumor cells. Ultraviolet light-induced Fas expression may serve to target stress-injured cells for removal by FasL-bearing cells or by FasL produced by the cells themselves in response to the stimuli, and may represent a general function of the Fas/FasL pathway in facilitating the apoptosis and elimination of undesirable or harmful cells . However, UV-irradiation also appears to engage the apoptotic axis of TNFR1 , and appears to involve initial formation of the Fas-FADD-caspase-8 death complex in an FasL-independent manner . Some types of chemotherapeutic drugs such as anthracyclins, as well as UV-C irradiation, can lead to the cell surface expression of calreticulin (CRT) which has recently been shown to confer anti-tumor immunogenicity to otherwise less immunogenic tumor cells . It will be interesting to see if UV- irradiation of MM cells, as was done in this study, also leads to the surface expression of CRT. Furthermore, the expression of costimulatory molecules on dendritic cells (DC) is upregulated after co-incubation with UV-irradiated tumor cells, and UV-irradiated tumor cells-pulsed DCs stimulated allogeneic T lymphocytes more efficiently than DCs pulsed with γ-irradiated cells . This increase in the ability of human cancer cells to induce CTLs by UV-irradiation has been found to be independent of the corresponding effect on histocompatibility locus Ag (HLA) expression . In parallel with the induction of tumor cell immunogenicity, UV-irradiation made tumor cells more sensitive to natural killer cell-mediated cytotoxicity and to lysis by TNF, suggesting that immunogenicity and TNF sensitivity are two independent UV-induced properties . It is important to note however, that the effects of mitomycin C treatment on MM cells did not alter the Grp94 or Grp78 expression significantly nor did it result in early induction of apoptosis (data not shown) and thus it served as our control in our CTL assays. The effects of 17-AAG were not tested on mitomycin treated MM cells.
When allogeneic PBMCs were exposed to these UV-irradiated MM cells, through the process of repetitive priming, CD8+ CTLs were induced. Furthermore, these CTLs demonstrated strong tumor-specific activity. These data therefore confirm that not only were cytolytic NK cells preferentially induced, but that these CTLs were functionally reactive in targeting co-cultured tumor cells as well. In addition, since CTLs are widely thought to be the principal mediators of host tumor immunosurveillance , these data further suggest that this vaccination strategy could both reconstitute host tumor immunosurveillance as well as mediate immunological memory; i.e. be the 'Holy Grail' of cancer immunotherapy.
In conclusion, our study has shown in a functional way that it is feasible to devise an autologous cell-based anti-cancer vaccine using UV-irradiated MM cells as immunogens. Primed CTLs were associated with significant eradication of co-cultured MM cells, demonstrating in a functional and efficacious method to promote tumor rejection. Finally, since UV-irradiation increases HSP expression, we therefore postulate that the mechanism for CTL priming involves the induction of tumor peptide-loaded HSP expression on the surface of UV-irradiated MM cells, which in turn preferentially induced cytolytic T cell activation. Our study, therefore, provides the framework for the development of preclinical and clinical studies using anti-tumor cytolytic T cell vaccines for treating MM, and in which UV-irradiated tumor cells are used as immunogens.
List of abbreviations
cytolytic T lymphocytes
- E to T:
effector to target
Heat Shock Proteins
tumor associated antigen
tumor rejection antigen-1
- 17 AAG:
This work was supported by grants from SingHealth (grant # SU089/2003, and SU106/2004); and the Department of Clinical Research, SGH (grant # DCR/P01/2003) to CG and GT.
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