CD40 stimulation of b cell chronic lymph

research paper CD40 stimulation of B-cell chronic lymphocytic leukaemia cells enhances the anti-apoptotic profile, but also Bid expression and cells remain susceptible to autologous cytotoxic T-lymphocyte attack Arnon P Kater,1 Ludo M Evers,1 Ester B M Remmerswaal,2 Annelieke Jaspers,2 Michiel F Oosterwijk,1,2 Rene´ A W van Lier,2 Marinus H J van Oers1 and Eric Eldering2 Department of Haematology, and 2Laboratory for Experimental Immunology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands Received 28 June 2004; accepted for publication 13 September 2004 Correspondence: Eric Eldering, Department of Experimental Immunology, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands E-mail e.eldering@amc.uva.nl Summary To enhance the poor antigen-presenting capacity of B-cell chronic lymphocytic leukaemia (B-CLL), CD40 triggering has been considered as an active immunotherapy However, CD40 stimulation also has an antiapoptotic effect and may further impair the dysregulated response of B-CLL to apoptotic stimuli Therefore, we measured the expression of virtually all regulators of apoptosis before and after CD40 stimulation These findings were correlated with sensitivity for chemotherapy- and death-receptorinduced apoptosis and T-cell-mediated killing CD40 stimulation enhanced the constitutive anti-apoptotic profile of B-CLL cells by upregulation of Bcl-xL and Bfl-1 and downregulation of the BH3-only protein Harakiri Unexpectedly, the BH3-only protein Bid was strongly induced Functionally, CD40-stimulated B-CLL cells became resistant to drug-induced apoptosis and, despite upregulation of CD95 and Bid, were not sensitive to CD95L In contrast, autologous T cell killing, triggered by loading CLL cells with viral (CMV) peptides, was very efficient both before and after CD40 stimulation Upon CTL interaction, CLL targets underwent mitochondrial depolarization and caspase-3 activation Thus, despite an increased anti-apoptotic profile, CD40 triggered B-CLL cells remain excellent targets for resident cytotoxic T cells These data support therapeutic exploitation of CD40 stimulation in B-CLL, provided that a strong CTL component is induced Keywords: chronic lymphocytic leukaemia, apoptosis, immunotherapy, Bid B-cell chronic lymphocytic leukaemia (B-CLL) is considered as a prime example of malignancy involving defects in cell death regulation This results in relative chemoresistance (Kitada et al, 1998), making this disease currently incurable As B-CLL is characterized by a slow accumulation rate, allowing time for the generation of an immune response against the tumour cells, it is an attractive disease for immunotherapeutic approaches However, B-CLL B cells are ineffective antigenpresenting cells (APCs) because they lack important costimulatory surface molecules and not express strong immunogenic tumour-specific peptides (Ranheim & Kipps, 1993) In addition, the death-receptor pathway seems impaired by the absence or very low levels of the death receptors CD95, DR-4 and -5 and increased levels of the surrogate caspase Flip doi:10.1111/j.1365-2141.2004.05225.x (Panayiotidis et al, 1995; Olsson et al, 2001) CD40–CD40Ligand (CD40L) interactions have been used to augment the APC capacity of B-CLL cells CD40 triggering increases the expression of a variety of immune accessory molecules such as death receptors (CD95), adhesion molecules (CD54) and co-stimulatory molecules (CD80, CD86 and CD70) (Ranheim & Kipps, 1993; Wang et al, 1997; Kato et al, 1998; Kitada et al, 1999) CD40-activated leukaemic B cells can induce proliferation of allogeneic and autologous CD4+ and CD8+ T cells (Kato et al, 1998; Buhmann et al, 1999; Krackhardt et al, 2002) Different groups have shown immune responses of primed allogeneic and autologous T cells against CD40-activated B-CLL cells in vitro (Kato et al, 1998; Buhmann et al, 1999; Chu et al, 2000; Krackhardt et al, 2002) Infusion of autologous B-CLL ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 127, 404–415 CD40-stimulated B-CLL Upregulate Bid and Remain Sensitive to CTLs cells, virally transduced to express high levels of recombinant murine CD40L, showed encouraging results in a recent phase I clinical trial (Wierda et al, 2000) A possible drawback for the clinical application of CD40stimulated B-CLL cells might be that, CD40 triggering may further impair the dysfunctional response to apoptotic stimuli Although most groups have found decreased sensitivity to spontaneous apoptosis, data on sensitivity to different deathinducing stimuli, such as cytostatic drugs, are inconsistent (Romano et al, 1998; Younes et al, 1998; Kitada et al, 1999; Grdisa, 2003; de Totero et al, 2003) However, although CD95 is upregulated by CD40 triggering, current data on sensitivity to death-receptor-induced cell death are contradictory (Wang et al, 1997; Younes et al, 1998; Kitada et al, 1999; Chu et al, 2002) Another matter of debate is the molecular mechanisms involved in the altered response to apoptotic triggers Augmented expression of anti-apoptotic Bcl-xL upon CD40 ligation was found in most (Kitada et al, 1999; Granziero et al, 2001; Pedersen et al, 2002), but not all studies (Chu et al, 2000) Similar contradictions were reported for the antiapoptotic protein Mcl-1 (Pedersen et al, 2002; de Totero et al, 2003) Moreover, most studies concentrated on a single apoptotic pathway, such as the death-receptor pathway (Chu et al, 2000) or the family of inhibitor of apoptosis proteins (IAPs) (Granziero et al, 2001) or were restricted to only a subset of apoptotic regulators (Kitada et al, 1999; Pedersen et al, 2002) Finally, no data are available regarding the contribution of the BH3-only pro-apoptotic molecules, which are currently considered for prime significance in apoptosis regulation (Puthalakath & Strasser, 2002) Thus, at present, a comprehensive understanding of the influence of CD40 stimulation on regulators of apoptosis and ensuing sensitivity to physiological and pharmacological apoptotic stimuli as well as cytotoxic T-lymphocytes (CTL) attack is lacking Yet, in order to safely implement clinical applications, such knowledge is crucial In this study, we investigated the influence of CD40 triggering of B-CLL cells on the expression of virtually all direct regulators of apoptosis We correlated these findings with the sensitivity of CD40-triggered B-CLL cells to chemotherapy- and deathreceptor-induced apoptosis and cytotoxicity, mediated by autologous cytomegalovirus (CMV)-specific T cells We found that only four apoptotic regulators were significantly altered upon CD40 triggering, among which was a pronounced increase in Bid protein Although resistant to death-receptor and chemotherapy-induced apoptosis, CD40stimulated B-CLL cells were excellent targets for autologous cytotoxic T-cell-mediated killing Materials and methods Patient samples After obtaining informed consent, blood was drawn from patients fulfilling the diagnostic criteria of B-CLL (Kipps, 1995) and from those who had not received treatment for at least months Peripheral blood mononuclear cells (PBMC), obtained via density-gradient centrifugation were either directly frozen (for T-cell experiments) in fetal calf serum (FCS) containing 10% dimethyl sulphoxide (DMSO; Sigma Chemical Co., St Louis, MO, USA) and stored in liquid nitrogen before use or were first enriched for B-CLL cells by negative depletion T cells, monocytes and granulocytes were depleted as described (Lens et al, 1999), by using a-CD3, a-CD14 and a-CD16 immunomagnetic beads and a magnetic particle concentrator (both Dynal A.S., Oslo, Norway) Purified leukaemic cells contained ‡95% of CD5+ CD19+ B cells as assessed by flow cytometry Cell culture B-cell chronic lymphocytic leukaemia cells were stimulated by co-culture with fibroblasts (NIH3T3 cells) that had been stably transfected with either a plasmid encoding human CD40L (3T40L) or negative control plasmid (3T3, both, kind gifts from D van Baarle, Sanquin, Amsterdam, The Netherlands) Fibroblast were irradiated (30 Gy) and precultured overnight at 1Ỉ5 · 105 cells/well in 24-well plates (Costar, Corning Inc., Rochester, NY, USA) in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Life Technology, Paisley, UK) supplemented with 10% (v/v) heat-inactivated FCS (ICN Biomedicals, Meckenheim, Germany), 100 U/ml penicillin and 100 lg/ml streptomycin Thawed B-CLL cells (typically >80% vital cells) were resuspended in the above described medium and ml of 2Ỉ5 · 106cells/ml were added to the adherent cells After d, B-CLL cells were gently removed and used either for total cellular RNA isolation (5 · 106 stimulated or unstimulated BCLL cells), flowcytometric analyses or studies for responsiveness to different apoptotic stimuli Flow cytometry Cells were washed and resuspended in staining media [phosphate-buffered saline (PBS) containing 0Ỉ5% (weight/volume) bovine serum albumin (BSA)] For analysis of the expression of cell surface markers, different combinations of antibodies were used CD80-phycoerythrin (PE), CD86-fluorescein isothiocyanate (FITC), CD19-PE, CD27-FITC and human leucocyte antigen (HLA)–DR–PE were purchased from Becton Dickinson (San Jose, CA, USA), CD54-PE and HLA-abc-FITC were obtained from Pharmingen (Becton Dickinson Biosciences Pharmingen, San Jose, CA, USA), CD5-FITC was obtained from Sanquin, CD95-FITC was obtained from IQ products (Groningen, The Netherlands), CD58 from Immunotech (Marseille, France) and CD70-PE from Ancell (Kordia Life Sciences, Leiden, The Netherlands) After incubation for 30 at 4°C protected from light, cells were washed twice in PBS/0Ỉ5% BSA and expression of cell surface markers was analysed using FACSCalibur flow cytometry and CellQuest software (Becton Dickinson) ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 127, 404–415 405 A P Kater et al Reversed transcriptase multiplex ligation-dependent probe amplification reagents, reactions and analysis above For drug-induced apoptosis, cells were incubated with fludarabine (80 lmol/l; Sigma) for a maximum of 48 h For the induction of CD95-mediated apoptosis, we used an agonistic mAb (5 lg/ml, FAS10, kind gift of L Aarden, Sanquin) and murine embryonic cell line (MEC clone 3), mock transfected or stably transfected with murine CD95L (kind gift of J P Medema, LUMC, Leiden, the Netherlands), cultured in the same medium as described above B-CLL cells were co-cultured with confluent MEC cells for 24 h Jurkat T cells were used as positive control for CD95-induced apoptosis Cell viability was analysed via detection of phosphatidyl serine on the outer membrane of apoptotic cells as described previously (Koopman et al, 1994) Propidium iodide (PI; Sigma) was added (5 lg/ml) to distinguish necrotic from apoptotic cells Multiplex ligation-dependent probe amplification probes consist of two oligonucleotides that anneal to adjacent sites on a target sequence and are then ligated by a heat stable ligase Each ligated probe gives rise to an amplification product of unique length and the resulting DNA mixture is analysed by capillary sequencer and standard software for identification and relative quantification of the amplicons (Schouten et al, 2002) Each MLPA probe consists of one short synthetic oligonucleotide (Biolegio, Malden, the Netherlands) and one phage M13-derived long probe oligonucleotide Preparation of the M13-derived MLPA probe oligonucleotides and all probes used in the apoptosis gene probe set, as well as detailed reaction conditions for reverse transcriptase (RT-MLPA) have been described elsewhere (Schouten et al, 2002; Eldering et al, 2003) Briefly, RNA samples (40–60 ng of total RNA) were first reverse transcribed using a gene-specific probe mix The resulting cDNA was annealed overnight at 60°C to the MLPA probes Annealed oligonucleotides were covalently linked by Ligase-65 at 54°C (MRC, Amsterdam, the Netherlands) Ligation products were amplified by polymerase chain reaction (PCR; 33 cycles, 30 s at 95°C; 30 s at 60°C and at 72°C) using one unlabelled and one 6-carboxy-fluorescein (FAM)-labelled primer (10 pmol/l) After the PCR stage, aliquots of samples were mixed with Genescan-500 ROX size standards and run on an ABI 3100 capillary sequencer (Applied Biosystems, Warrington, UK) in Genescan mode Data were analysed with genescan and genotyper software packages (Applied Biosystems), successively Category tables containing the area for each assigned peak (scored in arbitrary units) were compiled in genotyper and exported for further analysis with Microsoft excel spreadsheet software The sum of all peak data was set at 100% to correct for fluctuations in total signal between samples, and individual peaks were calculated relative to the 100% value Signals below the detection limit in medium were assigned a value corresponding to the threshold value for noise cut-off in genescan In order to identify and select patient samples with detectable CMV-specific CD8+ cells, PBMC from B-CLL patients were stained with CD8-PE (Becton Dickinson) and allophycocyanin-conjugated A2 or B7 CMV tetramers (kind gift of Sanquin) as previously described (van Leeuwen et al, 2002) Of those selected patients, thawed PBMC (2 · 105 CD8+ cells/ ml, approximately 5–10 · 106 total cells/ml, depending on the percentage of CD8+ cells) were incubated in 25 cm2 culture flasks (Costar, Cambridge, MA, USA) with CMVpp65-derived peptide (HLA-A2-binding NLVPMVATV or the HLA-B7binding TPRVTGGGAM, IHB-LUMC peptide synthesis library facility, Leiden, The Netherlands), added at a final concentration of 1Ỉ25 lg/ml For stimulation of T cells, interleukin (IL)2 (50 U/ml; Biotest Ag, Dreieich, Germany) was added After week, cells were re-stimulated for one additional week with irradiated (30 Gy) CMV peptide-loaded Epstein–Barr virus (EBV)-transformed cell lines expressing either HLA-A2 or HLA-B7 (5 · 104 cells/ml) in the presence of IL-2, as previously described (van Leeuwen et al, 2002) Western blotting Cytotoxic assays Western blotting was performed as described previously (Mackus et al, 2002), after separation of protein samples by 13% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Antisera against Bfl-1 and Bid were kind gifts of Prof Dr J Borst (NKI, the Netherlands) Rabbit antiserum to Bcl-xL was purchased from Transduction Laboratories (Erembodegem, Belgium) and the monoclonal antibody (mAb) against Tubulin was from Sigma (Sigma– Aldrich Chemie B.V., Zwijndrecht, the Netherlands) Cytotoxic T-lymphocytes activity was measured in a standard 51 Cr release assay (van Leeuwen et al, 2002) using freshly thawed or CD40-stimulated CMV peptide-loaded B-CLL cells as experimental targets As control targets, unstimulated or CD40 stimulated B-CLL cells without CMV-peptide was used Briefly, target cells were labelled with 50 ll 51Cr (30 lg/ml, 18Ỉ5 MBq/mg; Amersham, Buckinghamshire, UK) for h After washing, cells were incubated with 0Ỉ1 mg/ml peptide or medium for h at 37°C and washed again Graded numbers of autologous CMV-specific effector cells, obtained as described above, were incubated with · 103 51Cr-labelled target cells After h, released radioactivity was measured and specific lysis was calculated according to the following formula: percentage of specific release ¼ [(experimental release)spontaneous Induction of apoptosis After co-culture with 3T3 or 3T40L, B-CLL cells were washed and resuspended in IMDM plus supplements as described 406 Stimulation of virus-specific autologous cytotoxic T-lymphocytes ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 127, 404–415 CD40-stimulated B-CLL Upregulate Bid and Remain Sensitive to CTLs release)/(maximum release)spontaneous release)] · 100 Results are presented as specific lysis, adjusted for the number of tetramer-binding cells in each well, as determined by flowcytometric analysis Evaluation of mitochondrial membrane potential (DWm) and caspase-3 cleavage Prior to the cytotoxic assay, cells were preincubated for 30 at 37°C with 100 nmol/l MitoTracker Orange (CMTMRos, Molecular probes, Leiden, The Netherlands) to measure DWm, washed twice in IMDM medium and incubated with CMV peptide or medium as described above A total of 5000 B-CLL cells were used as targets in a 4-h cytotoxic assay After the assay, cells were washed in HEPES buffer, double-stained with allophycocyanin-conjugated anti-CD19 (Becton Dickinson) and analysed on a fluorescence-activated cell sorting (FACS)scan The presence of activated caspase-3 was studied by flow cytometry using apoptosis kit I (Becton Dickinson) To distinguish targets from effectors, cells were stained with APC-conjugated anti-CD19 (Becton Dickinson), as effectors contained less than 2% CD19+ cells Statistical analysis A paired sample two-sided Student’s t-test was used for analysis of differences between stimulated and unstimulated B-CLL cells and an unpaired two-sided Student’s t-test was used to compare differences between different cell types P < 0Ỉ05 were considered statistically significant For MLPA analysis, results were considered to be significant if both, at least, a 0Ỉ5-fold or two-fold induction and a statistically significant difference was found Results Profiling of apoptosis genes in CD40L stimulated B-CLL cells by RT-MLPA reveals alterations in both anti- and pro-apoptotic regulators B-CLL samples were co-cultured with fibroblasts that had been stably transfected with either a huCD40L-encoding (3T40L) or 3T3 Cells were stimulated for d, as prior studies showed maximal APC activity after this period (Krackhardt et al, 2002; Hoogendoorn et al, 2004) In agreement with previous reports, d of CD40 stimulation resulted in expression of high levels of immune accessory molecules such as CD54, CD80, CD86, CD70 and CD95, as compared with cells immediately after isolation and after d of culturing in the presence of control (3T3) fibroblasts (Fig and data not shown) The RT-MLPA procedure provides an efficient means to accurately quantify the relative expression of up to 45 transcripts in a one-tube assay The apoptosis probe set essentially targeted all direct regulators of apoptosis and three housekeeping genes (b2M, FTL and PARN; see also Fig 2) (Eldering et al, 2003) Using this novel assay, we compared expression profiles of purified B cells from eight B-CLL patients prior to and after CD40L triggering Four patients had unmutated B-CLL [2 are indicated with an asterisk Inset: difference in gene expression after 3T3 and 3T40L stimulation of the significantly altered genes is shown The y-axis represents relative expression as a percentage of total signal in the sample 3T3 stimulated samples are indicated with a filled square ( ), 3T40L stimulated samples with a filled triangle ( ) directly be activated by granzyme B (Alimonti et al, 2001) The induction of Bid protein was confirmed in samples from four patients Compared with the low levels, detectable by Western blot immediately after isolation of B-CLL cells, Bid protein was strongly increased after d of CD40 ligation (Fig 3) Increased expression of Flip (Kato et al, 1998; Granziero et al, 2001; Pedersen et al, 2002) and Survivin (Granziero et al, 408 2001) was also reported after CD40 triggering The present study confirmed that CD40 ligation increased Flip and survivin mRNA levels with respect to baseline, but these changes did not reach statistical significance (Flip 2ặ3-fold induction, P ẳ 0ặ11; survivin 6ặ5-fold induction, P ẳ 0Ỉ09) An increased expression of the p53-responsive BH3-only member Puma during cell culture has also been noted previously (unpublished ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 127, 404–415 CD40-stimulated B-CLL Upregulate Bid and Remain Sensitive to CTLs A C – – + + – B + – – + – + LC LC Bcl-Xl Bfl-1 + – + – + Tubulin Bid Fig Increased expression of Bcl-xL, Bfl-1 and Bid protein upon CD40 stimulation of B-CLL cells Protein samples from unstimulated and CD40stimulated B-CLL cells, as indicated by minus and plus symbols, respectively, were separated on 13% gels and probed for presence of (A) Bcl-xL (two patients), (B) Bfl-1 (two patients) and (C) Bid (four patients) Designated bands migrated at the expected apparent molecular weight (A) and (B) 25 lg of protein was used per sample and equal loading is visualized by the aspecific staining of background bands indicated by LC (loading control); (C) 50 lg of protein was applied and Tubulin was used as loading control observations) Importantly, no differences in gene expression profiles after CD40 stimulation between patients with unmutated and mutated IgVH genes were found CD40 stimulation reduces spontaneous and drug-induced apoptosis To study the functional consequences of anti- and proapoptotic alterations after CD40 stimulation, the sensitivity to apoptotic stimuli triggering the intrinsic mitochondrial pathway and the extrinsic death-receptor pathway were tested In contrast to the presumed longevity of B-CLL cells in vivo, they die rapidly in vitro (Collins et al, 1989) As shown in Fig A, co-culturing of B-CLLs with CD40L-expressing cells reduced this spontaneous apoptosis, consistent with earlier reports (Kitada et al, 1999; Granziero et al, 2001; Pedersen et al, 2002; Grdisa, 2003) The percentage of B-CLL cells undergoing apoptosis in d was reduced by culturing with 3T40L, from an average of 68 ± 6% to 23 ± 7% (P < 0Ỉ005) To investigate sensitivity of cytostatic drugs after CD40 stimulation, cells were cultured for another 48 h in the presence or absence of the purine analogue fludarabine Following d of co-culture with 3T3 plus another 48 h in medium, 48 ± 25% of cells were apoptotic This percentage appears lower than shown in Fig 4A (this was because of accumulating cellular debris in the 5-d cultures, which was excluded from the analyses as a result of FACS gating) Addition of fludarabine increased apoptosis to 80 ± 15% (P ¼ 0Ỉ002; Fig 4B) CD40L treatment significantly decreased sensitivity to fludarabine-mediated apoptosis compared with un-stimulated cells (27 ± 8%; P ¼ 0Ỉ00004) There was a small but significant difference in apoptotic cells in CD40L-stimulated cells after fludarabine treatment compared with medium (Fig 4B) This became apparent only after 48 h of incubation (24 h data not shown), consistent with a recent report (Grdisa, 2003) The apoptosis gene expression profile with or without CD40 stimulation in combination with fludarabine treatment was compared by RT-MLPA Data shown are only for the genes that displayed significant changes after either CD40 or fludarabine treatment (Fig 5A and B) Fludarabine treatment resulted only in upregulation of Bax and Puma, in agreement with our previous study (unpublished observations), irrespective of prior stimulation via CD40 The expression profiles in combination with the functional data shown in Fig 4B suggested that the enhanced levels of Bcl-xL and Bfl-1 via CD40 triggering counterbalanced the increase in Puma, thereby rendering cells less sensitive to fludarabinemediated apoptosis This finding indicates that, at least in this system, the CD40L pathway operates independent from the p53 pathway B-CLL cells are not sensitized to death-receptor-induced cell death upon CD40 stimulation To examine whether increased expression of CD95 by CD40 ligation (see Fig 1) sensitized B-CLL for CD95-induced apoptosis, cells were cultured for 24 h in the presence of an agonistic CD95 antibody No sensitivity to CD95-mediated apoptosis was observed in both unstimulated and CD40activated B-CLL cells, although >80% of control Jurkat cells underwent apoptosis (data not shown) As it is known that membrane-bound CD95L provides a stronger stimulus than a soluble agonistic antibody (Suda et al, 1997; Sieg et al, 1999), we next used cells expressing CD95L MEC-CD95L cells, but not untransfected MEC cells, consistently induced >80% of control Jurkat cells to undergo apoptosis within h (Fig 3C) Untreated B-CLL cells were not sensitive to CD95L and CD40 activation hardly changed this (