Published online before print December 6, 2004
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,1
* The Holden Comprehensive Cancer Center and Departments of
Internal Medicine and
Urology, University of Iowa, Iowa City
1 Correspondence: Holden Cancer Center at The University of Iowa, 5970Z JPP, University of Iowa, Iowa City, IA 52242. E-mail: george-weiner{at}uiowa.edu
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Key Words: B-CLL • cytogenetic status • CpG ODN • caspase
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In recent years, an increasing interest in immunotherapeutic strategies for the treatment of B cell malignancies such as B-CLL has developed. B cell malignancies, including B-CLL, are poorly antigenic [3 , 4 ]. Different approaches have begun to explore whether it is possible to overcome this obstacle by up-regulating costimulatory and antigen-presenting molecules. Strategies tested include the application of cytokines, CD40 ligation, and the use of immunostimulatory oligodeoxynucleotides (IS ODN) including CpG-containing ODN (CpG ODN) [3 , 5 6 7 8 9 10 11 12 ]. Previous data from our group and others have demonstrated that IS ODN are capable of inducing a change in phenotype of normal and malignant B cells, which would be expected to increase their immunogenicity [9 , 12 , 13 ]. IS ODN are also able to induce proliferation and protection from apoptosis in healthy B cells, thus providing a potential explanation for the expansion of B cells in response to invading pathogens in vivo [13 , 14 ].
We recently showed that the B-CLL cell-proliferative response to IS ODN is low compared with normal B cells [9 ]. In the studies outlined below, we demonstrate that various IS ODN can actually induce B-CLL apoptosis in most but not all samples and explore the potential mechanisms responsible for this effect.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ODN
Nuclease-resistant phosphorothioate (PS)-modified ODN were provided by the Coley Pharmaceutical Group (Wellesley, MA). Endotoxin levels in all ODN were <0.075 EU/ml by limulus amebocyte lysate assay. As control ODN, we used a non-IS ODN (ODN 2243). Specific sequences were as follows: IS ODN with CpG motif and PS backbone: ODN 2006-5'-TCG TCG TTT TGT CGT TTT GTC GTT-3'; IS ODN with methylated CpG motif and PS backbone: ODN 2117-5'-TCG TCG TTT TGT CGT TTT GTC GTT-3'; IS ODN without CpG motif and PS backbone: ODN 2137-5'-TGC TGC TTT TGT GCT TTT GTG CTT-3'GC; IS ODN with CpG motif and phophodiester backbone: ODN 2059-5'-TCG TCG TTT TGT CGT TTT GTC GTT-3'; control ODN with mixed PS/phosphodiester (PO) backbone: ODN 2243-5'-GGG GGA GCA TGC TGG GGG GG-3'.
Underlined nucleotides have PS linkages at their 3'-ends; all others have PO linkages. ODN were diluted in 10 mM Tris-HCl, 1 mM EDTA, pH 8, using pyrogen-free reagents. If not otherwise stated, the IS ODN used in this study was ODN 2006. The controls included medium only or ODN 2243.
Phenotypic determination
At the time-points indicated, cells were washed in ice-cold phosphate-buffered saline (PBS) and stained for expression of various antigens. For surface staining, 2 x 105 cells per sample were stained with fluorescently labeled monoclonal antibodies (mAb), which against CD5 [phycoerythrin (PE), UCHT2], CD19 (Cy-Chrome, HIB19), Fas (PE, DX2), Fas ligand (biotin, NOK-1), CD54 (PE, HA58), CD80 (PE, L307.4), and CD86 (PE, FUN-1) as well as appropriate isotype controls were purchased from BD Biosciences. For intracellular staining of 
-associated protein-70 (ZAP-70), 2 x 105 cells were fixed and permeabilized per the manufacturer’s protocol (Fix and Perm, Caltag, South San Francisco, CA). Briefly, cells were washed with PBS (400 g, 4°C, 5 min), incubated for 15 min with fixation buffer A, washed again, and then incubated with permeabilization buffer B, together with 10 µl PE-labeled anti-ZAP-70 mAb (1E7.2, eBioscience, San Diego, CA) or the corresponding isotype control antibody. Cells were then washed twice (400 g, 4°C, 5 min), resuspended in PBS, and immediately analyzed by flow cytometry using a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). Spectral overlap was corrected by appropriate compensation. For surface-staining experiments, viable cells were gated according to their typical forward-/side-scatter (FSC/SSC) characteristics and propidium iodide (PI) staining. Data were analyzed with the fluorescein-activated cell sorter (FACS) evaluation program FlowJo (version 4.5, Tree Star, Inc., Stanford, CA). Median fluorescence intensity (MFI) values were used for statistical analysis of ZAP-70 and surface marker expression.
Apoptosis and cell survival assays
Cells were harvested at the indicated time-points by staining all the cells from a given well with mAb for a lineage-specific antigen and fluorescein isothiocyanate (FITC)-labeled Annexin V (BD Biosciences) or incubated with a cell-permeable fluorogenic caspase 6 (CyToxiLuxTM, OncoImmunin, Gaithersburg, MD) or caspase 8 substrate (Caspase 8 FLICA, Immunochemistry Technologies LLC, Bloomington, MN) for 1 h, according to the manufacturer’s instructions. A predetermined number of calibration beads (CaliBRITETM Beads, BD Biosciences) were then added to each sample to allow for normalization of cell counts in different samples at different time-points. PI at 1 µg/ml was added just prior to flow cytometric detection. The count of viable cells rather than the count of apoptotic cells was used for the calculation of B-CLL cell survival because of concerns that some nonviable cells may have undergone lysis and not have been available for counting. Absolute cell survival is expressed as percentage of viable cell counts relative to initial plating counts. The absolute cell survival in IS ODN-stimulated samples divided by the cell survival in samples incubated in medium alone is referred to as relative IS ODN survival.
Fluorescence in situ hybridization (FISH)
Unstimulated PBMC from subjects with B-CLL were cultured for 24 h in RPMI-media containing 15% FCS and antibiotics. Slides were made after the cells had been processed and fixed in 3:1 methanol and acetic acid fixative. The FISH studies were performed using a CLL probe panel (VYSIS, Downers Grove, IL), which includes two cocktail probes. One set contains ATM (11q22.3)/17p13.1 (p53), and the second set has 12cen (CEP12)/13q14.3 (D13S319)/13q34. These studies were carried out using standard methods as specified by the manufacturer. Three hundred interphase nuclei were analyzed for each probe set. The cutoff for positive values was 2.2% for trisomy 12 and 2.7%, 6.4%, 6.0%, and 4.4% for ATM, p53, D13S319, and 13q34, respectively.
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for TLR9
For the analysis of Toll-like receptor 9 (TLR9) mRNA expression, B-CLL cells were isolated (>95% CD19+ CD5+) from peripheral blood, and RNA from 10 x 106 cells per sample was prepared using the RNeasy mini kit from Qiagen (Valencia, CA). Total RNA (500 ng) was reverse-transcribed using SuperScript III RNase H-RT (Invitrogen, Carlsbad, CA) in a 20-µl reaction volume. The generated cDNA (3 µl) was used per 25 µl real-time PCR carried out with the Platinum Quantitative PCR SuperMix-UDG kit (Invitrogen). Primers and probes were purchased from Integrated DNA Technologies (Coralville, IA): TLR9 forward: 5'-GCC AGA CCC TCT GGA GAA-3'; TLR9 reverse: 5'-AGA CTT CAG GAA CAG CCA GTT G-3'; TLR9 probe: 5'-/56-FAM/TAC CTT GCC TGC CTT CCT ACC CTG TGA/3BHQ-1/-3'; ß-actin forward: 5'-CAC ACC TTC TAC AAT GAG CTG CGT-3'; ß-actin reverse: 5'-ACA GCC TGG ATA GCA ACG TAC A-3'; ß-actin probe: 5'-FAM/AAC CGC GAG AAG ATG ACC CAG ATC AT/BHQ-3'. The amplification efficiency of the primers was 2.0 ± 10%. The copy number of TLR9 was calculated from a standard curve generated by plotting the log dilutions of known concentrations of a plasmid containing the amplicon versus the cycle threshold values and was normalized by the housekeeping gene ß-actin. TLR9 copy number is presented as number of transcripts per 103 copies of ß-actin.
Statistical analysis
Data are expressed as means ± SEM. To determine statistical differences in the mean of two data columns, the two-sample t-test was used. A P value of <0.05 was considered to be significant; a P value of <0.005 was considered to be highly significant. Statistical analysis was performed using the SAS System for Windows (SAS, version 8.2. 2001, SAS Institute, Cary, NC).
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Table 1. Clinical Data of B-CLL Patients Included in the Study
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Figure 1. In vitro B-CLL cell survival in response to ODN. PBMC from subjects with B-CLL were suspended in complete medium and cultured on 96-well plates at 37°C in the presence of different ODN at concentrations as indicated. On day 4, cells were harvested, and the number of viable B-CLL cells was determined by Annexin V/PI staining. (A) Bead counts were used to normalize viable B-CLL cell counts. The top plot shows the gating on the calibration beads and total mononuclear cells and the middle plot, gating on the CD5+, CD19+ cell subset. (B) Survival of IS ODN-sensitive B-CLL cells in response to different ODN at increasing concentrations. Data are from one representative experiment out of three with similar results.
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A total of 20 different subjects with B-CLL were tested. All B-CLL samples exhibited 
90% viable cells at day 0. Incubation with IS ODN resulted in lower absolute B-CLL cell survival after 4 and 7 days incubation when compared with samples cultured with control ODN or media (Fig. 2A
; IS ODNday 4, 38±6%; control ODNday 4, 50±7%, *P<0.03; IS ODNday 7, 35±5%; control ODNday 7, 52±7%, **P<0.001). However, as seen in earlier studies with 4-day cultures, B-CLL cell viability in the absence and the presence of IS ODN was heterogeneous. B-CLL cells bearing 13q deletions as a single aberration had a relative IS ODN survival <1 in all nine cases studied; i.e., the B-CLL viability was lower in the presence than in the absence of IS ODN (Fig. 2B
, left bar and Table 1
). In contrast, B-CLL cells with other chromosomal aberrations including 17p deletion, trisomy 12, or 11q deletion responded heterogeneously to IS ODN (Fig. 2B
, middle bar and Table 1
). The difference in relative IS ODN survival between the 13q and the non-13q groups was statistically significant (Fig. 2B
; 13q deletion cells: 0.61±0.09; non-13q deletion cells: 1.18±0.22, *P<0.02). As to be expected, B cells from normal, healthy donors showed proliferation in response to IS ODN and therefore had a relative IS ODN survival >1 (Fig. 2B
, right bar).
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Figure 2. Effect of IS ODN on B-CLL cell survival. PBMC from 20 subjects with B-CLL and four healthy volunteers were suspended in complete medium and cultured on 96-well plates at 37°C in the presence or absence of 5 µg/ml IS ODN or control ODN. On days 0, 4, and 7, cells were harvested, and the number of viable cells was determined by Annexin V/PI staining. (A) Average survival of B-CLL cells from 20 individuals in the presence or absence of IS ODN or control ODN is compared over time. Error bars indicate SEM. (B) The ratio of the cell count in IS ODN-treated samples to the cell count in nontreated samples is referred to as relative IS ODN survival. Average relative IS ODN survival of B-CLL samples from two cytogenetic groups as well as normal B cells at day 4 is compared. Error bars indicate SEM. Diamonds represent individual data points. (C) Comparison of IS ODN effects on unsorted versus enriched B-CLL cells. PBMC from subjects with IS ODN-sensitive B-CLL cells were isolated as described and magnetically separated from non-B-CLL cells. The frequency of plasmacytoid DC [natural interferon (IFN)-producing cells], as defined by BDCA-2 and lineage 1 staining, was below 0.006% in the enriched samples. Unsorted and enriched samples were suspended in complete medium and cultured on 96-well plates at 37°C in the presence or absence of ODN 2006 at 5 µg/ml for 4 and 7 days. Cells were harvested, and survival was determined based on Annexin V/PI staining and the use of calibration beads as described above. Similar results were obtained in two further experiments with B-CLL cells from different subjects.
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Degree of IS ODN-mediated apoptosis is not a result of level of TLR9 message or prior therapy
One potential explanation for the observed differences in response to IS ODN between the cytogenetic groups is differential expression of the IS ODN receptor TLR9. Real-time RT-PCR was performed on nine samples to assess for quantitative differences in TLR9 expression between 13q deletion B-CLL cells and other B-CLL cells. No significant differences were identified in TLR9 expression based on cytogenetic status, and there was no correlation between TLR9 message and sensitivity to IS ODN (Fig. 3
). There was no evidence for correlation between sensitivity of IS ODN-mediated apoptosis and prior therapy (Table 1)
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Figure 3. TLR9 expression in B-CLL cells with different cytogenetic status. PBMC from nine subjects with B-CLL were isolated from peripheral blood. All subjects had >95% CD19+ CD5+ cells. RNA from 106 cells per sample was prepared, and real-time RT-PCR for TLR9 was performed as described in Materials and Methods. TLR9 copy numbers for two prognostically distinct B-CLL groups are presented as number of transcripts per 103 copies of ß-actin.
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Figure 4. B-CLL cell apoptosis involves caspase activation. PBMC from five subjects with B-CLL were obtained and cultured in the presence or absence of 5 µg/ml IS ODN (ODN 2006) or control ODN (ODN 2243) at 37°C. Caspase activation on days 0, 4, and 7 was determined using cell-permeable, fluorogenic caspase substrates for caspases 8 and 6. (A) Histograms of two representative experiments. The percentages of cells positive for activated caspases are shown. Similar results were observed for five individual subjects with B-CLL cells sensitive to IS ODN-mediated apoptosis. (B) Average percentage of caspase 6-negative B-CLL cells for five individual samples on day 4. (C) PBMC from three subjects with B-CLL were obtained and cultured in the presence or absence of 5 µg/ml IS ODN and 50 µM pan-caspase inhibitor Z-Val-Ala-Asp (zVAD). Survival on day 4 was determined by Annexin V/PI staining. The y-axis represents the percentage of viable (Annexin V/PI-negative) B-CLL cells. Error bars indicate SEM.
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Figure 5. Expression of death receptors and their ligands on B-CLL cells is up-regulated by IS ODN. PBMC from subjects with B-CLL or healthy volunteers were obtained and cultured in the presence or absence of 5 µg/ml IS ODN (ODN 2006) or control ODN (ODN 2243) for 4 and 7 days. (A) The expression of Fas ligand (n=5), TRAIL (n=15), CD95/Fas (n=12), TRAIL receptors 1 and 2 (R1 and R2; n=11), and TRAIL receptors 3 and 4 (R3 and R4; n=6) was determined on B-CLL cells by FACS analysis. Relative MFI values as compared with expression on day 0 are shown. No up-regulation of markers was found with control ODN. Error bars indicate SEM. (B) Up-regulation of CD95/Fas on day 4 in response to IS ODN in normal B cells as well as B-CLL cells sensitive to IS ODN-mediated apoptosis versus those that do not undergo apoptosis in response to IS ODN.
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To test our hypothesis that death receptors might be involved in IS ODN-mediated apoptosis, we used soluble Fas-Fc or TRAIL receptor 2-Fc fusion proteins to block interaction of Fas with Fas ligand or TRAIL with TRAIL receptors on B-CLL cells, which were sensitive to IS ODN-induced apoptosis and were incubated in the presence or absence of IS ODN at 5 µg/ml and different fusion proteins at 5 µg/ml for 4 days. As illustrated in Figure 6 , IS ODN-induced apoptosis was not blocked by these fusion proteins (Fig. 6) .
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Figure 6. Effect of soluble Fas and TRAIL receptor 2 on IS ODN-induced apoptosis of B-CLL cells, which were sensitive to IS ODN-mediated apoptosis from three different subjects and were isolated and cultured in complete medium in the presence or absence of IS ODN (ODN 2006) and fusion proteins consisting of the Fc portion of human Ig and Fas, TRAIL receptor 2, or a control protein at 5 µg/ml. B-CLL cell survival after 4 days incubation was determined as described. Shown are the average percentages of three independent experiments. Error bars indicate SEM.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chromosomal aberrations are among a number of prognostic features (such as serum thymidine kinase, lactate dehydrogenase level, mutational status, ZAP-70 expression, and CD38 expression) known to correlate with B-CLL patient survival, disease progression, and treatment-free interval [2 ]. The most frequent aberration is the 13q14.3 deletion, which is found in up to 50% of patients and is generally associated with a better prognosis than the presence of other aberrations such as 17p deletion, 11q deletion, or trisomy 12. We found samples with 13q deletion, as single aberrations were sensitive to IS ODN-induced apoptosis, and B-CLL cells with other chromosomal aberrations including 17p deletion, 11q deletion, or trisomy 12 showed a more heterogeneous response. In contrast, we found no correlation between IS ODN-induced apoptosis and ZAP-70 expression in B-CLL cells. Given that there is a high concordance between ZAP-70 protein expression and Ig heavy-chain variable region (IgVH) gene mutations in B-CLL [19 ], it is unlikely that mutational status of the IgVH gene plays a role in the IS ODN-mediated effects described above.
In the data outlined above, we found that not only CpG ODN (ODN 2006, ODN 2059) but also ODN lacking the classical CpG motif (ODN 2137 and to a lesser degree, ODN 2243) have effects on B-CLL survival. These results suggest that induction of B-CLL cell apoptosis may depend not only on the TLR9 pathway activated by CpG motifs but also on other factors such as the ODN backbone (PS modification). Consistent with this conclusion is our observation that the level of B-CLL TLR9 expression did not correlate with sensitivity to IS ODN-induced apoptosis, suggesting variations in the expression of this receptor were not (or not alone) responsible for differences in response to IS ODN between B-CLL samples. In this context, it is important to mention that benign B cells also respond to some ODN lacking the CpG motif with changes in phenotype and proliferation.
We do not yet know what factors are responsible for the variable sensitivity of various cytogenetic groups to IS ODN-induced apoptosis. Potential candidates include pathways known to be directly involved in B-CLL growth, survival, and apoptosis, such as phosphatidylinositol-3-kinase (PI-3K), AKT, caspases, or members of the bcl-2 family. A report from Kienle et al. [20 ] suggests that mRNA expression of molecules such as bax, bcl-2, and PI-3K in B-CLL cells varies based on the cytogenetic or the mutational status of B-CLL cells. Ongoing studies are assessing the potential significance of these molecules in IS ODN-induced apoptosis.
Our data demonstrate that caspase activation is involved in the apoptosis-inducing effects of IS ODN in B-CLL cells. The activation of caspase 8 points to a possible involvement of death receptors. Indeed, we found IS ODN induced up-regulation of different members of the TNF receptor family, particularly CD95/Fas and its ligand, suggesting a potential mechanism by which IS ODN increases sensitivity of B-CLL cells to apoptosis. The up-regulation of CD95/Fas by IS ODN was significantly higher in B-CLL cells sensitive to IS ODN-mediated apoptosis. However, we found benign B cells also up-regulate CD95/Fas in response to IS ODN, yet such treatment leads to proliferation and resistance to apoptosis. In our B-CLL samples, IS ODN-mediated apoptosis was not inhibited by blocking Fas ligand or TRAIL using soluble CD95/Fas or TRAIL receptor 2. These data suggest other receptors or heterogeneity in downstream signaling are involved in determining the different response to IS ODN of benign B cells and B-CLL cells of different subgroups.
Additional studies are needed to explore the role death receptor family members play in the ability of IS ODN to induce apoptosis of B-CLL cells. In addition, it is unclear whether the apoptosis-inducing effect of IS ODN in B-CLL cells will have therapeutic value. A clinical trial of ODN 2006 as a single agent in subjects with B-CLL has begun recently, and information related to changes in phenotype, as well as any clinical responses, should provide valuable information in this regard.
In conclusion, a majority of B-CLL samples, particularly those with 13q14.3 deletion as a single aberration, undergoes apoptosis in response to various ODN. This is seen with ODN containing the classic CpG motif but other ODN as well. This is in contrast to the ability of ODN to stimulate proliferation and inhibit apoptosis of benign B cells, which is limited to IS ODN. IS ODN-mediated B-CLL apoptosis depends in part on caspase activation and is accompanied by up-regulation of Fas and Fas ligand. Clinical studies exploring the effect of IS ODN on B-CLL have just begun. Although the possible therapeutic value of these findings remains to be determined, our results provide further evidence for pursuing IS ODN as a potential therapeutic agent in B-CLL and may provide a valuable tool for the investigation of apoptosis pathways in B-CLL.
Received June 29, 2004; revised November 8, 2004; accepted November 15, 2004.
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