D. Spaner, Division of Molecular and Cellular Biology, Research Institute, S-116A, Research Building, Sunnybrook Health Sciences Center, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5. E-mail: email@example.com
Cytotoxic chemotherapies do not usually mediate the expression of an immunogenic gene programme in tumours, despite activating many of the signalling pathways employed by highly immunogenic cells. Concomitant use of agents that modulate and complement stress-signalling pathways activated by chemotherapeutic agents may then enhance the immunogenicity of cancer cells, increase their susceptibility to T cell-mediated controls and lead to higher clinical remission rates. Consistent with this hypothesis, the microtubule inhibitor, vincristine, caused chronic lymphocytic leukaemia (CLL) cells to die rapidly, without increasing their immunogenicity. Protein kinase C (PKC) agonists (such as bryostatin) delayed the death of vincristine-treated CLL cells and made them highly immunogenic, with increased stimulatory abilities in mixed lymphocyte responses, production of proinflammatory cytokines, expression of co-stimulatory molecules and activation of c-Jun N-terminal kinase (JNK), p38 and nuclear factor kappa B (NF-κB) signalling pathways. This phenotype was similar to the result of activating CLL cells through Toll-like receptors (TLRs), which communicate ‘danger’ signals from infectious pathogens. Use of PKC agonists and microtubule inhibitors to mimic TLR-signalling, and increase the immunogenicity of CLL cells, has implications for the design of chemo-immunotherapeutic strategies.
Co-operation between cytotoxic drugs and anti-tumour immune responses may improve the outcome of cancer patients, compared to either modality alone . Cytotoxic agents can decrease the tumour burden but, at best, kill only a portion of target cells . T cells may eliminate specifically the remaining tumour cells , but the latter are required to be immunogenic, or able to support type 1 immune responses through expression of co-stimulatory molecules, such as CD80 and CD86, adhesion molecules such as CD54 and other molecules such as CD83, along with production of inflammatory cytokines and chemokines . However, the immunogenicity of tumour cells is intrinsically weak, in part because of the oncogenic signalling events that drive cancer progression . If chemotherapeutic drugs could increase the immunogenicity of the tumour cells they fail to kill, this might facilitate susceptibility to T cells.
Leukaemia cells become highly immunogenic in vitro when treated with Toll-like receptor (TLR) and protein kinase C (PKC) agonists and cytokines . Interestingly, chemotherapeutic drugs activate many of the same signalling pathways as these immunomodulatory agents . Why, then, do chemotherapeutic drugs not normally increase the immunogenicity of tumour cells? One reason might be the kinetics of signalling induced by cytotoxic drugs that mediate apoptosis rather than immunogenicity. Another might be failure to activate all the signalling pathways required for an immunogenic gene programme . Accordingly, co-treatment with agents that modulate and complement the stress-signalling pathways activated by chemotherapeutic agents may enhance the immunogenicity of cancer cells.
To model this possibility, we studied the effects of vincristine and PKC agonists on chronic lymphocytic leukaemia (CLL) B cells, as a recent trial of a PKC agonist, bryostatin-1, and vincristine in advanced stage lymphoma patients (which included patients with CLL) had shown surprising efficacy  even though vincristine-based regimens are not usually effective in the treatment of CLL . Because we and others had found that PKC agonists modulate cytokine and TLR-signalling in CLL cells [3,7,8], experiments were designed to address the possibility that the signalling properties of vincristine and PKC-agonists complemented each other to enhance lymphoma immunogenicity in vivo.
Materials and methods
Blood was from consenting CLL patients (with persistent increases of clonal CD19+CD5+ immunoglobulin (Ig)Mlo cells  and characterized in Table 1) who had not been treated for at least 3 months. Normal B cells were from healthy volunteers. Protocols were approved by the Local Review Board.
Phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-labelled CD3, CD28, CD80, CD86, CD54, CD83, CD19 and tumour necrosis factor (TNF)-α antibodies were from Pharmingen (San Francisco, CA, USA). 7-Aminoactinomycin D (7-AAD) was from Sigma (St Louis, MO, USA). Stock solutions of phorbol dibutyrate (PDB) (Sigma) (5 mg/ml), picolog [7,10] (1·8 mg/ml) and U0126, SB203580, SP600125, G06976 and rottlerin (from Calbiochem, San Diego, CA, USA) (25 mg/ml) were made in dimethylsulphoxide (DMSO). Dexamethasone (Pharmascience Inc., Montreal, Quebec, Canada), vincristine, taxotere and interleukin (IL)-2 (Chiron Corp., San Francisco, CA, USA) were from the hospital pharmacy. Staphylococcal enterotoxin A (SEA) was from Toxin Technology, Inc. (Madison, WI, USA). The TLR-7/8 agonist, S28690 (from 3M Pharmaceuticals, St Paul, MN, USA) , was dissolved in serum-free AIM-V™ media (Gibco BRL, Grand Island, NY, USA) (with 33% DMSO) at 1·3 mg/ml and stored in the dark at 4°C. Antibodies to c-Jun N-terminal kinase (JNK), p38, p42/p44 extracellular regulated kinase (ERK), inhibitor kappa B (IκB) and β-actin and the serine/threonine-phosphorylated forms of JNK, p38, ERK, IκB and myristoylated alanine-rich protein kinase C substrate (MARCKS) were from Cell Signaling Technology (Beverly, MA, USA). The TNF-α converting enzyme (TACE) inhibitor, TAPI , was from Peptides International (Louisville, KY, USA). 5,6-Carboxyfluorescein diacetate succinimidyl ester (CFSE) was from Molecular Probes (Eugene, OR, USA).
CLL and T cells were isolated as described previously  using the RosetteSep-negative selection technique (StemCell Technologies, Vancouver, British Columbia, Canada), according to the manufacturer's instructions (with minor modifications). Briefly, 75% of the plasma volume was removed from heparinized whole blood samples to concentrate the peripheral blood mononuclear cells, increase the yield of isolated cells and minimize the required antibodies. Total T cells were obtained with antibodies against CD16, CD19, CD36, CD56 and glycophorin A with > 96% purity, respectively. CLL-B cells were isolated with antibodies against CD2, CD3, CD14, CD16, CD56 and glycophorin A yielding > 98% CD19+/CD5+ cells.
Activation of CLL cells
Purified CLL cells (1·0 × 106 cells/ml) were cultured in AIM-V plus 2-mercaptoethanol (2-ME) (Sigma) (5 × 10−5 M) for 3–4 days at 37°C in 5% CO2. S28690 was used at a previously optimized final concentration of 0·1 µg/ml . PDB, vincristine, signal transduction and PKC isozyme inhibitors were added at the concentrations indicated in the figure legends. Viable cells that excluded Trypan blue were counted in a haemocytometer.
Mixed lymphocyte responses (MLRs)
Normal T cells were adjusted to 5 × 105 cells/ml in AIM-V. Activated CLL cells were washed at least four times (to remove residual immunomodulators), irradiated (2500 cGy) and suspended at 5 × 105 cells/ml, or less. Responders and stimulators were then mixed in a 1:1 (vol : vol) ratio and cultured in 96-well round-bottomed plates (Becton Dickinson Labware, San Jose, CA, USA) without additional cytokines or serum. Proliferation was measured 4–6 days later by a colorimetric assay .
Flow cytometric analysis was performed as described previously . Cells were incubated with pre-optimized volumes of CD80-PE and CD83-FITC, CD54-PE and CD86-FITC or CD19-PE and CD5-FITC antibodies for 20 min, washed once in phosphate-buffered saline (PBS) and then resuspended in PBS plus 1% albumin prior to flow cytometric analysis. Negative controls were isotype-matched irrelevant antibodies (such as CD3-PE and CD28-FITC for purified B cell populations). Staining of nucleated cells was determined by gating on forward- and side-scatter properties. Ten thousand viable counts were analysed with a fluorescence activated cell sorter (FACS)can flow cytometer and Cellquest software (Becton Dickinson). Standardization of the flow cytometer was performed before each experiment using SpheroParticles (Spherotech Inc., Chicago, IL, USA).
Detection of membrane TNF-α (mTNF-α)
CLL cells, 1–2 × 106, were cultured alone or activated with PDB and/or vincristine or S28690 in 5 ml polystyrene tubes (Becton Dickinson Labware). TAPI (100 µM) was added to each tube and CD19 or CD83-FITC and TNF-α-PE antibodies were added 4 h later. Subsequent steps were the same as for conventional immunophenotyping .
For microscopic analysis, 4 × 106 purified CLL cells were fixed with 4% paraformaldehyde (Canemco, Lakefield, Quebec, Canada) for 30 min at room temperature and permeabilized with 0·1% Triton X-100 for 20 min at room temperature. Cells were then incubated with a monoclonal FITC-conjugated anti-β tubulin (TUB 2·1; Sigma) in 5% bovine serum albumin (BSA) for 1 h. Cell nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes) at a 1:600 dilution for 10 min at room temperature. Tubulin images were obtained using a Zeiss LSM510 confocal microscope equipped with a Plan-Neofluar 63 × 1·0W objective, an argon laser illumination source, and imaged with a 505–530 nm bandpass filter. Images were captured and analysed using Zeiss LSM software. DAPI imaging was performed using a Zeiss Axiovert 200M epifluorescence microscope equipped with a Hamamatsu ORCA-1394 camera and a 100× Zeiss Apochromat DIC 1·4 oil objective. Image capture and analysis was performed using Zeiss AxioVision software.
Protein extracts were made from activated CLL cells as described previously . The proteins were resolved in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobilon-P transfer membranes (Millipore Corp., Billerica, MA, USA). Western blot analysis was then performed according to the manufacturer's protocols for the specific antibodies. Supersignal West Pico Luminal Enhancer and Stable Peroxide Solution (Pierce, Rockford, IL, USA) created the chemiluminescent signal that was detected using a Syngene InGenius system (Syngene, Cambridge, UK).
Cytokine levels in culture supernatants (from activated CLL cells after 48 h) were determined by a multi-analyte fluorescent bead assay with a Luminex-100TM system (Luminex Corp., Austin, TX, USA). A kit for human IL-5, IL-6, granulocyte–macrophage colony-stimulating factor (GM–CSF), IL-10 and TNF-α measurement was used according to the manufacturer's instructions (Licoplex/Millipore, Billerica, MA, USA). Individual cytokine concentrations were determined from standard curves using Bio-Plex Manager V3·0TM software (Bio-Rad, Mississauga, Ontario, Canada). The assay was linear between 30 and 1000 pg/m for each cytokine.
Isolation of total RNA and synthesis of cDNA
Total RNA was extracted using the RNeasy kit (Qiagen, Mississauga, Ontario, Canada), according to the manufacturer's instructions. To remove contaminating genomic DNA, 10 µg of the total RNA preparation was incubated with 10 units of RNase-free Dnase I (Promega, Madison, WI, USA) for 30 min at 37°C. Total RNA concentration was determined in a spectrophotometer at 260 nm. cDNA was made with the Superscript First Strand Synthesis System for reverse transcription–polymerase chain reaction (RT–PCR) (Invitrogen, Carlsbad, CA, USA) in a 20-µl reaction containing 3 µg of DNase I-treated total RNA, 20 mmol/l Tris-HCl (pH 8·4), 50 mmol/l KCl, 2·5 mmol/l MgCl2, 10 mmol/l dithiothreitol (DTT), 0·5 µg oligodeoxythymidylic acid 18 (oligodT18), 0·5 mmol/l each of desoxiadenosine-5′-triphosphate (dATP), deoxyguanosine triphosphate (dGTP), desoxicitidin-5′-triphosphate (dCTP) and 2′-deoxythymidine 5′-triphosphate (dTTP), and 200 units Superscript II reverse transcriptase. The priming oligonucleotide was annealed to total RNA by incubating at 70°C for 5 min and then cooling to 4°C. Reverse transcription was performed at 42°C for 2 h, and cDNA was stored at –20°C until PCR analysis.
Real-time PCR amplification
The following primers were used to amplify human TNF-α and hypoxanthine phosphoribosyltransferase (HPRT) transcripts: TNF-α forward, 5′-ACCTCTCTCTAATCAGCCC-3′, reverse, 5′-AGGAGCACATGGGTGGAG-3′; HPRT forward, 5′-GAGGATTTGGAAAGGGTGTT-3′, reverse, 5′-ACAATAGCTCTTCAGTCTGA-3′.
PCR was performed on a DNA engine Opticon System (MJ Research Inc., Waltham, MA, USA) using SYBR Green I as a double-stranded DNA-specific binding dye. PCR reactions were cycled 40 times after initial denaturation (95°C, 15 min) with the following parameters: denaturation at 95°C for 15 s, annealing of primers at 57°C (TNF), 54°C (TLR-7) and 52°C (HPRT) for 20 s, and extension at 72°C for 20 s. Fluorescent data were acquired during each extension phase. After each PCR reaction, a melting curve analysis of amplification products was performed by cooling the samples to 4°C and then increasing the temperature to 95°C at 0·2°C/s. Fast loss of fluorescence is observed uniquely at the denaturing/melting temperature of the amplified DNA fragment. Standard curves were generated with serial 10-fold dilutions of cDNAs obtained using the same primers as for real-time PCR.
SEA-directed cytotoxicity assay
Sensitivity of activated CLL cells to cytotoxic effectors was evaluated by flow cytometry as described previously . Briefly, lymphokine activated killer (LAK) cells were made from normal peripheral blood mononuclear cells (PBMCs) cultured in AIM-V with IL-2 (500 U/ml) for 7–10 days and fed every 3 days. SEA-reactive T cells were made from PBMCs stimulated with SEA (1 ng/ml) in AIM-V plus 10% fetal calf serum (FCS) for 10–14 days. Effectors were collected by density gradient centrifugation and suspended in AIM-V.
Target CLL cells were suspended at 1 × 107/ml in phosphate-buffered saline (PBS) plus 5% FCS, pelleted and then incubated with 100 µl of CFSE (50 µM in PBS) for 5 min at room temperature in the dark before being washed and resuspended in AIM-V. For the assay, effectors (at 1 and 3 × 106/ml) and targets (at 1 × 106/ml) were mixed (1:1) in a final volume of 200 µl in 5 ml polystyrene tubes (BD Biosciences, Bedford, MA, USA) and cultured for 4 h at 37°C. SEA (1 ng/ml) was added to some tubes to activate effector cells and bind them to targets . The samples were then washed twice and 7-AAD and annexin V-PE wereadded, according to the manufacturer's instructions (BD-Pharmingen).
For analysis of cellular cytotoxicity, gates were set on CFSE-stained CLL cells using an FL1-histogram. The percentages of target cells that bound annexin V-PE and/or 7AAD were determined by further gating in an FL2/FL3 dot-plot.
Student's t-test was used for P-values of differences between sample means and paired t-tests were used to compare responses of the same cells to different treatments.
Effect of vincristine on CLL cell survival and co-stimulatory molecule expression
Consistent with previous reports of their vincristine-sensitivity , CLL cells [regardless of clinical stage or other prognostic factors (Table 1)] died rapidly in response to vincristine doses as low as 100 ng/ml (Fig. 1a). The effects on co-stimulatory molecules (expressed to varying degrees by CLL cells [7,13]) were measured on cells that remained viable after 24–48 h. As exemplified in Fig. 4a (compare the first and second panels on the left) and summarized in Fig. 1b, CD80, CD83, CD86 and CD54 expression decreased markedly on these cells.
Whether vincristine decreased the expression of these molecules directly, or selected simply for a subpopulation of CLL cells with low expression of these molecules, is not clear from these data. Regardless, the results suggested that treatment with an effective cytotoxic agent killed CLL cells in vitro, leaving behind a population of tumour cells with weak expression of co-stimulatory molecules and presumably poorly equipped to engage in productive anti-tumour T cell responses.
Protection from vincristine-mediated killing by PKC agonists
In contrast to the effects of vincristine alone (and consistent with previous reports that PKC agonists prevent death of CLL cells by microtubule disruption ), addition of PDB before (not shown) or with vincristine protected cells from death (Fig. 1a and c). CLL cells continued to die with ongoing exposure to vincristine, but the protective effect of PDB was apparent even after 5 days in culture (Fig. 1d). The effect was not due to PDB-induced proliferation of vincristine-resistant cells, as PDB does not cause CLL cells to proliferate in these culture conditions (Fig. 1d) [3,7]. Other PKC agonists were studied to determine if protection was specific for phorbol esters. In particular, picolog (a synthetic bryostatin analogue ) also protected CLL cells from vincristine, at similar doses as PDB (Fig. 1c, solid bars).
Mechanism of PDB-mediated survival of vincristine-treated CLL cells
Because some PKC substrates stabilize microtubules , PDB may have simply reversed the microtubule disruption caused by vincristine. Accordingly, the state of the microtubules was assessed by confocal microscopy. In untreated CLL cells (Fig. 2a, left upper panel) and in cells treated with PDB alone (Fig. 2a, right upper panel), microtubules were seen to emerge from microtubular organizing centres (MTOCs) as shown by staining with tubulin antibodies. After treatment with vincristine alone, the microtubule system collapsed completely (Fig. 2a, left lower panel). In CLL cells treated with both vincristine and PDB, the MTOCs remained but microtubules were no longer seen (Fig. 2a, right lower panel). These findings were consistent with ongoing microtubule disruption in cells treated with both vincristine and PDB.
CLL cells treated with vincristine alone were smaller than untreated cells, suggesting that they were undergoing apoptosis, while CLL cells activated with PDB were larger, regardless of concomitant treatment with vincristine (Fig. 2a and b). Increased nuclear staining with DAPI in the cells treated with vincristine (Fig. 2b, middle row) was consistent with apoptosis. The nuclear DAPI patterns of the other cells confirmed their viability.
To gain insight into the signalling pathways responsible for survival or immunogenicity, small molecule inhibitors were used. U0126 , SP600125 , SB203580  and dexamethasone  are inhibitors of p42/p44 ERK, JNK, p38 and nuclear factor kappa-B (NF-κB), respectively. The combination of U0126 and dexamethasone abrogated almost completely the survival effects of PDB on CLL cells treated with vincristine at 3 µg/ml, with U0126 being the major contributor, while SP600125 and SB203580 did not decrease significantly the survival of CLL cells treated with both PDB and vincristine (Fig. 3a). These results suggested that PDB protected cells from vincristine by activating ERK and NF-κB. JNK and p38 signalling did not appear to affect survival, but presumably mediated some of the changes in immunogenic properties (see below).
Phorbol esters, bryostatins and bryologues act through both PKC isozymes and PKC-independent pathways . Phosphorylation of the classical PKC isozyme substrate, MARCKS , was measured to support that PDB acted through PKC. PDB caused MARCKS phosphorylation, which was unchanged by vincristine (Fig. 3c, columns 3 and 4, first row). To determine if PDB protected CLL cells from vincristine by activating PKC, specific isozyme inhibitors were used. Go6976 inhibits classical PKC isozymes (such as PKC-α and -β) and rottlerin inhibits the novel isozyme, PKC-δ, but not the classical isozymes . These inhibitory activities were confirmed in CLL cells (Fig. 3c, columns 5–7), which express PKC-α, -β and -δ. However, neither Go6976 or rottlerin prevented the survival effects of phorbol esters on CLL cells treated with vincristine at 3 µg/ml (Fig. 3b). The combination of Go6976 and rottlerin partially reversed the effects of phorbol esters on survival (Fig. 3b), but not to the same extent as U0126 and dexamethasone (Fig. 3a). Accordingly, the mechanism of PDB-mediated survival of vincristine-treated CLL cells appeared to be relatively independent of PKC.
Despite being able to block phosphorylation of MARCKS by PDB (Fig. 3c, column 5), Go6976 did not block PDB-induced phosphorylation of ERK (Fig. 3c, column 5, second and third rows) or phosphorylation of IκB and its subsequent degradation (Fig. 3c, column 5, fourth and fifth rows), which allows NF-κb to traffic into the nucleus and mediate gene transcription . Similarly, phosphorylation of ERK and degradation of IκB in CLL cells activated with PDB and vincristine were not prevented by rottlerin (Fig. 3c, column 6, rows 2–5). However, the combination of rottlerin and Go6976, which partially reversed the survival effects of PDB (Fig. 3b), was able to prevent phosphorylation and degradation of IκB (Fig. 3c, column 7, fourth and fifth rows), but not phosphorylation of ERK (Fig. 3c, column 7, second and third rows). These findings support the importance of ERK and NF-κB activation as the factors provided by phorbol esters which allow CLL cells to survive in the face of vincristine, as suggested by the results in Fig. 3a.
Effect of vincristine and PKC agonists on co-stimulatory molecule expression by CLL cells
Expression of CD80, CD86, CD54 and CD83 were measured by flow cytometry to determine if co-stimulatory molecule expression on CLL cells changed after exposure to vincristine and PDB. CD80 and CD86 increased on most CLL cells treated with both PDB and vincristine, compared to PDB alone (Fig. 4a and b). No obvious relationship of these changes to the clinical characteristics of the patient samples (Table 1) were apparent. While CD54 decreased, increases in CD83 expression were most significant (Fig. 4). CD83 is a marker of strong antigen-presenting cells (APCs) and was found previously to correlate directly with increased CLL cell immunogenicity [7,13]. These findings suggested that vincristine-treated CLL cells, whose survival had been prolonged by PDB, were more immunogenic.
Effect of vincristine and phorbol esters on TNF-α production by CLL cells
We have found previously that increased production of the proinflammatory cytokine, TNF-α, is associated with highly immunogenic CLL cells [3,8]. Accordingly, the effect of vincristine on TNF-α production by CLL cells was studied at the transcriptional and translational levels and compared to the effects of imidazoquinoline, S28690, a conventional TLR-7 agonist [3,8].
Expression of TNF-α mRNA transcript numbers (relative to HPRT transcripts) increased 50–200-fold within 2 h after stimulation by S28690 (Fig. 5a). Vincristine increased TNF-α transcripts minimally (1·5-fold) while PDB alone increased them by only 25–50-fold. However, the addition of vincristine to PDB caused TNF-α transcripts to increase to the levels reached in response to the bona fide TLR-agonist (Fig. 5a).
Protein expression was then studied by flow cytometry, using a TACE inhibitor to prevent solubilization of membrane TNF-α. Vincristine increased mTNF-α production by CLL cells treated concomitantly with PDB, sometimes to the same level as S28690 (Fig. 5b and c). Vincristine, alone, did not induce TNF-α production by CLL cells. Increased production of soluble TNF-α was confirmed by enzyme-linked immunosorbent assays (ELISAs) (Fig. 5d).
Effect of vincristine and phorbol esters on immunogenic properties and lytic sensitivity of CLL cells
The ability to stimulate allogeneic T cell proliferation in MLRs is a property of immunogenic cells. The stimulatory abilities of CLL cells treated with both PDB and vincristine were compared to CLL cells alone, CLL cells treated with vincristine alone, and also to CLL cells treated with PDB and the TLR-7/8 agonist, S28690 (which was shown previously to make CLL cells highly immunogenic [3,8]) (Fig. 6a). These latter cells represent a ‘yardstick’ against which to measure the immunogenicity of CLL cells treated with both vincristine and PDB. Untreated CLL cells, or after treatment with vincristine, had little stimulatory activity (Fig. 6a). CLL cells treated with PDB alone were better able to stimulate T cell proliferation, as reported previously . Concomitant treatment with vincristine increased this ability (albeit to a somewhat lesser extent than cells treated with PDB S28690) (Fig. 6a).
Given that PDB could prevent direct killing of CLL cells by vincristine, it was possible that it could also prevent them from being killed by cytotoxic effector lymphocytes. To check this, a previously described flow cytometric assay  was used to compare the killing of CLL cells cultured for 62 h in AIM-V media alone, with PDB or with PDB and vincristine (Fig. 6b and c). Note that by this time cells treated with vincristine alone were mainly dead. The CLL targets were labelled with CFSE and then incubated with effector T cells. Annexin-V (to label apoptotic cells) and 7-AAD (to identify necrotic cells) were then added and the percentages of dead and dying CLL targets were determined by gating on FITC+ cells. CLL cells treated with both PDB and vincristine were significantly more sensitive to cytotoxic T cells than CLL cells alone, or treated with only PDB (Fig. 6b and c), as would be expected for more immunogenic cells.
Taken together, the results suggested that PDB extended the life of vincristine-treated CLL cells, and augmented a number of their immunogenic properties.
Signalling profile of CLL cells treated with PDB and vincristine
Both vincristine and PDB activate mitogen-activated protein kinase (MAPK) and NF-κB signalling pathways [3,23], which couple stressful biological situations to the immune system through APCs . Assessment of the activation state of the JNK, ERK and p38 pathways was performed by immunoblotting, with antibodies against phosphorylated forms of these MAPKs. NF-κB activity was indicated with antibodies against the phosphorylated form of the inhibitor, IκB, which allows NF-κB dimers to traffic to the nucleus and engage in gene transcription .
Two JNK isoforms arise by differential mRNA splicing . Vincristine caused p54 isoform phosphorylation preferentially in CLL cells, while PDB phosphorylated mainly the p46 isoform (Fig. 7a, third row, upper panels). Treatment with both PDB and vincristine caused phosphorylation of both isoforms, also seen in CLL cells treated with PDB and S28690. CLL cells treated with both PDB and vincristine expressed phosphorylated p42/p44 MAPK (ERK1 and ERK2) (Fig. 7a, second row, upper panels). Activation of ERK was due mainly to PDB, as vincristine alone did not cause phosphorylation of these kinases. PDB and vincristine individually caused phosphorylation of p38 MAPK and IκB with additive effects when CLL cells were treated with both agents (Fig. 7a, first and second rows, bottom panels).
In summary, both JNK isoforms, along with p42/p44 ERK, p38 and IκB became phosphorylated (suggesting activation of the respective pathways) in CLL cells treated with both PDB and vincristine. Strikingly, this signalling pattern was also found in cells treated with a bona fide TLR agonist (Fig. 7a) that had been shown previously to increase the immunogenicity of CLL cells [3,8].
The observations in this paper suggest that vincristine can enhance the immunogenicity of CLL cells under certain conditions. On its own, vincristine killed CLL cells rapidly, and the remaining cells had decreased co-stimulatory molecule expression (Figs 1 and 4) and weak T cell stimulatory properties (Fig. 6). However, in combination with PKC agonists, vincristine increased the immunogenicity of CLL cells, as measured by greater co-stimulatory molecule expression (Fig. 4), inflammatory cytokine production (Fig. 5) and stimulation of T cell proliferation (Fig. 6). This state of enhanced immunogenicity was marked by augmented ERK, JNK, p38 and NF-κB signalling (Fig. 7).
CD54 is considered to be a good surrogate marker for activated APCs . Despite their increased immunogenicity, expression of CD54 was found to be lower on CLL cells treated with both vincristine and PDB (Fig. 4). Microtubule inhibitors have been noted previously to lower CD54 expression and thereby increase the resistance of tumour cells to cytotoxic effectors . However, we have found that CD83 correlates most closely with strong immunogenic capabilities of CLL cells . The fact that CD83 expression increased significantly following treatment with both PDB and vincristine (Fig. 4) may help to explain the stronger immunogenicity of the tumour cells despite lower CD54 levels. Methods to enhance the expression of CD54 (perhaps with cytokines such as IL-2 [3,13]) might enhance further the immunogenic effects of phorbol esters and vincristine.
Consistent with previous observations of the association of unrestrained activation of JNK and apoptotic death , vincristine activated both JNK isoforms and caused tumour cells to undergo apoptosis (Figs 2 and 7). However, in the context of signalling through immunoreceptors, such as TLRs, both JNK and p38 are associated with the production of inflammatory cytokines, chemokines and co-stimulatory molecules . Phorbol esters allowed CLL cells to survive for several days longer in the presence of vincristine through pathways that were independent of JNK and p38 (Fig. 3a). While phorbol esters induced some aspects of immunogenicity on their own during this period of time, vincristine contributed increased CD83 expression (Fig. 4), TNF-α mRNA and protein expression (Fig. 5), T cell stimulatory ability and lytic sensitivity (Fig. 6). We suggest that these contributions of vincristine were mediated partly through enhanced JNK and p38 signalling (Fig. 7).
Accordingly, increased immunogenicity of CLL cells appeared to result from co-operative signalling between PKC agonists and vincristine, as shown by the diagram in Fig. 7b. PDB caused the phosphorylation of p42/p44 ERK and p46 JNK, while vincristine caused phosphorylation of p54 JNK and IκB. The combination of PDB and vincristine led to increased phosphorylation of p38 and IκB (Fig. 7a). Increased survival of vincristine-treated CLL cells in the presence of PDB appeared to be mediated mainly by enhanced ERK and NF-κB activity (indicated by the changes in phosphorylation status of p42/p44 ERK proteins and IκB) as it was prevented mainly by the combination of a mitogen-activated protein extracellular signal-related kinase (MEK) inhibitor (U0126) and an NF-κB inhibitor (dexamethasone) (Fig. 3a). The results are consistent with a model whereby PKC activation (through ERK and NF-κB) increased the strength and duration of vincristine-induced signalling (through JNK, p38 and NF-κB) for sufficient time to allow the changes in immunogenic properties (Fig. 7b). For example, CD83 was the most significantly increased co-stimulatory molecule on CLL cells treated with both PDB and vincristine (Fig. 4). Because the CD83 promoter includes only SP1 and NF-κB sites , increased expression of CD83 caused by vincristine may be explained partly by the enhanced NF-κB activity (Fig. 7).
The changes in immunogenicity and signal transduction caused by PDB and vincristine were strikingly similar to those seen with PDB and the TLR-7/8 agonist, S28690 (Figs 5–7) . S28690 did not appear to affect microtubules (not shown), but the TLR-4 agonist, endotoxin, has been reported to do so . TLRs couple innate to adaptive immunity, in part, by increasing the immunogenicity of APCs and are activated usually by molecular patterns that reflect biological stress from infections . However, endogenous, non-infectious markers of stress (such as heat shock proteins) can also activate TLRs . The studies reported here suggest that persistent loss of cytoskeletal integrity (Fig. 2) activates the immune system in a similar fashion to TLRs.
Not all microtubule inhibitors appear capable of increasing the immunogenicity of CLL cells. For example, cytotoxic doses of taxotere (which prevents tubulin polymer dissociation ) were not overcome by PDB and did not increase the expression of co-stimulatory molecules on PDB-treated CLL cells (not shown). These observations suggest that microtubule disruption (rather than stabilization) was involved in the signalling events that caused enhanced immunogenicity of CLL cells.
The modality by which tumour cells are killed by chemotherapeutic agents may profoundly affect the clinical outcome . Apoptosis is associated with normal developmental processes and may obviate, or even subvert, an effective anti-tumour immune response. Necrosis may cause inflammation but not necessarily an appropriate immune response. The term ‘immunogenic death’ has been coined to describe a cell death modality that is associated with molecular events that include cell surface changes and production of factors that mobilize effective anti-tumour immune responses . The results described in this paper suggest that PKC agonists switch the effects of vincristine from rapid apoptosis/necrosis to a slower, immunogenic death process. Given that advanced stage lymphoma patients often have profound T cell defects, it would follow that the effects of vincristine and bryostatin (or bryostatin analogues, such as picolog) may potentially be improved by incorporating strategies to improve anti-tumour T cell responses, such as vaccination or adoptive T cell transfer .
This study was supported by grants from the Ontario Institute of Cancer Research (OICR) (#07Nov – 61), Canadian Institutes of Health Research (CIHR) (MOP-79389), the National Cancer Institute of Canada (NCIC) with funds from the Terry Fox Foundation (#018005), and the Leukemia and Lymphoma Society of Canada (to D. E. S.), the CIHR (to J. W. B.), and the National Institute of Health (CA31845 to P. A. W.). J. T. was supported by a graduate studentship and a Harold E. Johns studentship award from the NCIC.
Dr Wender is the inventor of Picolog. Otherwise the authors have no conflicts of interest to declare.