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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-06-1822.
NEOPLASIA
From the Molecular Immunology Group, Tenovus Research
Laboratory, Southampton University Hospitals Trust, Southampton,
United Kingdom; Cancer Research Oncology Unit, Cancer
Sciences Division, School of Medicine, University of Southampton,
Southampton General Hospital, Southampton, United Kingdom;
and Department of Haematology, Royal Bournemouth Hospital, Bournemouth,
United Kingdom.
The mutational status of tumor immunoglobulin VH
genes is providing a powerful prognostic marker for chronic
lymphocytic leukemia (CLL), with patients having tumors expressing
unmutated VH genes being in a less favorable subset.
However, the biologic differences correlating with VH gene
status that could determine the clinical course of the disease are
unknown. Here we show that differing responses to IgM ligation are
closely associated with VH gene status. Specifically, 80%
of cases with unmutated VH genes showed increased global
tyrosine phosphorylation following IgM ligation, whereas
only 20% of samples with mutated VH genes responded
(P = .0002). There was also an association between
response to IgM ligation and expression of CD38
(P = .015). The Syk kinase, critical for transducing
B-cell receptor (BCR)- derived signals, was constitutively present
in all CLL samples, and there was a perfect association between global
phosphorylation and induction of phosphorylation/activation of Syk.
Nonresponsiveness to anti-IgM could be circumvented by ligation of IgD
(10 of 15 samples tested) or the BCR-associated molecule CD79 Chronic lymphocytic leukemia (CLL) is the most
common adult B-cell malignancy in the Western world and is
characterized by the accumulation of monoclonal CD5+ B
cells with the appearance of small mature lymphocytes (for a review,
see Caligaris-Cappio and Hamblin1). The clinical course of
CLL is heterogeneous, with some patients progressing rapidly with early
death, whereas others exhibit a more stable, possibly, nonprogressing
disease lasting many years. The clinical management of CLL is therefore
challenging and considerable effort has been directed toward the
identification of clinically useful prognostic markers to guide treatment.
Somatic mutation of the immunoglobulin VH genes in normal B
cells is a key event in increasing diversity during maturation of
immune responses. The mutation status of the VH genes
expressed in CLL cells is a powerful prognostic marker for CLL;
patients with unmutated genes have a worse prognosis.2,3
The cell surface expression of CD38 may be another potential prognostic marker in CLL. A relatively high level of CD38 surface expression by
CLL cells has been shown to be a marker of poor
prognosis.3-5 Although originally suggested to correlate
with unmutated VH gene status, this is controversial and
other studies have shown CD38 expression does not correlate with
VH gene status and can vary during disease
progression.6-8 Taken together, the studies suggest that
VH gene status and CD38 expression are independent
prognostic markers for CLL.
Although both VH gene status and CD38 may have clinical
utility as prognostic markers in CLL, the biologic differences between CLL subtypes that underlie their very different disease courses are not
known. CLL cells have heterogeneous responses to stimulation via cell
surface receptors including CD40,9 CD5,10 and
the B-cell receptor (BCR).11-13 BCR is a key molecule that
triggers signaling pathways that regulate proliferation,
differentiation, and apoptosis in B cells. BCR comprises membrane
immunoglobulin, the antigen-binding subunit, and a heterodimer of
CD79 Previous studies have found that CLL cells differ in their responses to
IgM ligation based on differences in the constitutive levels of
Syk11 or phosphorylation of Syk.12 However,
at that time no correlation with VH gene mutational status
was available. Because differences in BCR function may contribute to
differences in biologic behavior, we have analyzed the signaling
response to BCR cross-linking in CLL and its relationship to
VH gene mutational status and expression of CD38.
Patients' cells
If not known, VH gene mutation status was determined as
previously described.2 Daudi, Ramos, and Jurkat cells were
cultured in RPMI 1640 medium containing 10% (vol/vol) fetal calf serum (FCS), 2 mM glutamine, 1% (wt/vol) sodium pyruvate, and antibiotics. CLL samples were thawed and maintained for 1 hour at 37°C in this medium before the assays were performed. Cell viability was 80% to
100% following thawing and remained unchanged during the experiment.
Flow cytometry
Tyrosine phosphorylation Cells were washed in RPMI 1640 medium and resuspended at a density of 2 × 107 cells/250 µL in RPMI 1640 medium and were untreated or incubated for 2 minutes at 37°C with 20 µg/mL goat anti-IgM, goat anti-IgD, or goat immunoglobulins as a control (all from Southern Biotechnology Associates, Birmingham, AL). We used goat IgG antibody because this binds poorly to human Fc receptors17 and has been shown previously to behave similarly to F(ab')2 fragments in CLL signaling assays.13 For stimulation of CD79 , we used the mouse
monoclonal antibody (mAb) ZL7-416 and mouse
IgG1 (Southern Biotechnology Associates) as a control.
Reactions were stopped by centrifugation and the pellets resuspended in
150 µL lysis buffer (Tris [tris(hydroxymethyl)aminomethane] 20 mM,
pH 7.5, EDTA [ethylenediaminetetraacetic acid] 1 mM, NaCl 140 mM,
1:100 dilution of inhibitor cocktail for mammalian tissues [Sigma,
Poole, Dorset, United Kingdom], sodium orthovanadate 2 mM) containing
1% (vol/vol) Brij 97 detergent (polyoxythylene 10 oleyl ether
[Sigma]) for 60 minutes at 4°C. Cell debris was removed by
centrifugation at 12 000g and the supernatant boiled in
sample buffer. Proteins were separated by denaturing sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins
were transferred to nitrocellulose membranes (Schleicher and Schuell,
Dassel, Germany) and probed with mouse mAbs against phosphotyrosine
4G10 (Upstate Biotechnology, Lake Placid, NY) followed by horseradish
peroxidase-conjugated antimouse antibodies. Bound immunocomplexes were
subsequently detected using enhanced chemiluminescence (Perbio,
Tattenhall, Cheshire, United Kingdom) and visualized on a Fluor-S Max
imager (Biorad, Hemel Hempstead, Herts, United Kingdom). The fold
increase in Syk phosphorylation in cells treated with anti-IgM, IgD, or
CD79 antibodies relative to control cells was quantified using
Quantity One software (Biorad).
Constitutive levels of Syk Untreated lysed cell samples boiled in sample buffer from tyrosine phosphorylation studies (see "Tyrosine phosphorylation") were run on denaturing SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Separate gels were probed with mouse mAbs against human Syk 4D10.1 (Upstate Biotechnology) and then detected as described. Ramos and Jurkat cell lines were used as controls.Immunoprecipitation Brij-97-lysed supernatants were precleared for 30 minutes with protein G-coated Sepharose beads (Amersham Pharmacia Biotech) followed by incubation with 2 µg specific antibody for 2 hours at 4°C. Packed protein G-Sepharose beads (15 µL) blocked with 5% (wt/vol) bovine serum albumin were added to the sample, which was then incubated for a further 60 minutes at 4°C. The beads were washed 4 times with cold lysis buffer and boiled in sample buffer. The precipitated proteins were separated by SDS-PAGE and specific proteins detected by immunoblotting. To detect Syk phosphorylation, immunoprecipitations were performed using antiphosphotyrosine antibody 4G10 followed by immunoblotting with anti-Syk 4D10.1. Samples that had at least 2 times more phosphorylated Syk following anti-IgM, IgD, or CD79
stimulation were considered to be responsive. To detect Tyr525/526
phosphorylated Syk, immunoprecipitations were performed using a rabbit
polyclonal antibody against Tyr525/526 phosphorylated Syk (New England
Biolabs, Hitchin, Herts, United Kingdom) followed by immunoblotting
with mouse anti-Syk 4D10.1. To detect Lyn, immunoprecipitations
were preformed using rabbit polyclonal antibody against Lyn (sc-15;
Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit control antibodies
(ChromPure Rabbit IgG, whole molecule; Jackson Immunoresearch
Laboratories, West Grove, PA).
Statistics Statistical analysis was performed using the SPSS for Windows program version 10 (SPSS UK, Woking, Surrey, United Kingdom). Chi-squared analysis was used for comparison of mutation status or CD38 with signaling responsiveness. Values for MEF were calculated using the software program, TallyCAL (Dako). Scattergrams of CD20, surface Ig, and CD79 MEF levels were prepared using the GraphPad Prism (version
3) software (San Diego, CA), and comparisons with mutation, CD38, and
signaling status made using the Mann-Whitney test. The comparison of
Syk expression and signaling status was made using the Mann-Whitney test.
Patients' immunophenotype and VH gene status All patients with CLL studied were untreated, most were Binet stage A (Table 1). CLL cells from all patients studied expressed CD5, CD19, surface IgM (sIgM), and CD23. The levels of sIgM, CD79, and CD20, expressed as MEF values, were variable (Table 1). Of the cases studied here, 15 of 40 (38%) had unmutated VH
genes and 17 of 38 (45%) were positive for CD38 expression (Table
2). There was no significant correlation
between VH gene status or CD38 expression and cell surface
expression of IgM, CD20, or CD79 (Figure
1).
Signaling through IgM We analyzed responsiveness to IgM ligation by measuring increases in global protein tyrosine phosphorylation. Daudi cells were used as a positive control and showed a rapid increase in global tyrosine phosphorylation when treated with anti-IgM, but not with control antibody (Figure 2A), as reported.12 In contrast, the response of CLL samples was variable with only a proportion of cases responding to anti-IgM stimulation shown by a visible increase in band intensity (Figure 2B; Table 2). For example patients 4 and 7 showed increased global tyrosine phosphorylation, whereas patients 24 and 28 displayed no response. Nonresponsiveness was not simply due to delayed phosphorylation in some samples because there was no evidence for increased global tyrosine phosphorylation in any of 4 nonresponding samples, which were incubated with anti-IgM for up to 2 hours (data not shown). In addition, IgM responsiveness was repeatable in duplicate fresh and frozen samples from 6 patients studied (3 responsive to anti-IgM treatment and 3 nonresponsive, data not shown).
All CLL samples expressed 2 heavily phosphorylated proteins of
approximately 55 kDa regardless of whether the cell could signal through the BCR (Figure 2B). Human Lyn occurs as 2 isoforms with molecular weights of 53 and 56 kDa. To determine whether the proteins in CLL cells were phosphorylated Lyn, we performed immunoprecipitations using a Lyn-specific antibody (Figure 3).
The tyrosine phosphorylated proteins immunoprecipitated by the anti-Lyn
antibody were identical to the approximately 55-kDa proteins detected
by direct immunoblotting using a phosphotyrosine-specific antibody.
Therefore, Lyn appears to be constitutively phosphorylated in CLL cells
independent of anti-IgM stimulation. Lyn phosphorylation apparently did
not alter following IgM ligation and was present in responsive and
nonresponsive CLL samples.
Analysis of PTK Syk The nonresponsiveness of CLL cells to IgM ligation has been suggested previously to correlate with reduced constitutive expression of Syk11 or to a failure to induce Syk phosphorylation.12 To resolve this discrepancy, we first analyzed the constitutive expression of Syk in CLL samples. Syk was readily detected in all samples analyzed (data not shown). Although there was variability in the level of expression between samples, samples with the lowest levels retained responsiveness to anti-IgM and there was no significant difference in the mean levels between mutated and unmutated CLL (P = .3). Because constitutive Syk expression did not correlate with responsiveness, we next measured Syk phosphorylation by immunoprecipitating tyrosine phosphorylated proteins and immunoblotting for Syk. As with global tyrosine phosphorylation, increases in Syk phosphorylation were detected only in a proportion of CLL samples (Figure 4A). Further, there was a perfect correlation between responsiveness to anti-IgM measured by visible increases in global tyrosine phosphorylation and specific tyrosine phosphorylation of Syk (Table 2). Examples of patients 7 and 28 are shown in Figures 2B and 4A. Hence, specific measurement of phosphorylated Syk by densitometry enabled quantification of the signaling response in all cases, rather than the arbitrary method of a visible increase in global tyrosine phosphorylation. When 6 patients were retested for phosphorylation of Syk following anti-IgM treatment, all gave reproducible results to that shown in Table 2 (data not shown).
Syk is phosphorylated on multiple tyrosine residues and these modifications can activate or inhibit Syk function. In particular, phosphorylation of tyrosine residues 525 and 526 is required for Syk activation.18 Immunoprecipitations were performed using an antibody that specifically detects Tyr525/526 phosphorylated Syk (Figure 4B). In the 6 cases studied, we readily detected increased Tyr525/526 phosphorylation of Syk in Daudi cells and in responsive (patients 9, 11, and 18) but not nonresponsive (patients 24, 26, and 30) CLL samples. We then assessed responsiveness to IgM ligation in all cases and
compared the results with the VH gene mutational status and with expression of CD38, each important prognostic markers for CLL.
There was a statistically significant association between anti-IgM
responsiveness and unmutated VH genes
(P = .0002; Table 2); 12 of 15 (80%) of samples with
unmutated VH genes responded to anti-IgM, whereas only 5 of
25 (20%) of samples with mutated VH genes responded. In
addition, there was also an association between CD38 expression and
anti-IgM responsiveness, although this was not as strong as the
correlation with mutations status: 11 of 17 (65%) of CD38+
and 5 of 20 (25%) of CD38 Differential signaling via BCR components Although some CLL samples were resistant to stimulation with anti-IgM antibodies, it was possible that nonresponsiveness could be circumvented by stimulation of other BCR components. All samples expressed IgM, CD79, and IgD (data not shown) and we therefore determined whether stimulation of CLL samples with antibodies to these
molecules increased tyrosine phosphorylation of Syk (Figures 5-6;
results summarized in Table 3). Of the 15 samples tested that did not
respond to anti-IgM, 10 (66%) were
responsive to anti-IgD (Figure 5). Similarly, of 15 samples tested, 12 (80%) were responsive to anti-CD79 (Figure 6). The 3 samples that
did not respond to anti-CD79 were nonresponsive to either anti-IgM
or anti-IgD. Therefore, there are various patterns of differential
responsiveness to BCR ligation in CLL suggesting that multiple
molecular defects underlie failure to respond to anti-IgM. Most CLL
samples that are nonresponsive to anti-IgM retain responsiveness to
anti-IgD and CD79. A smaller proportion of CLL samples fail to
respond to anti-IgM or anti-IgD but do respond to anti-CD79, or
fail to respond to ligation of any of the BCR components tested. By contrast, CLL samples that were responsive to anti-IgM were also sensitive to anti-CD79 and anti-IgD.
CLL is a heterogeneous disease and recent evidence suggests VH gene mutation status may have prognostic utility, guiding clinical management. The biologic differences that correlate with these prognostic markers and underlie the variable clinical behavior of CLL are not known. Here we have shown that the capacity to signal through membrane IgM correlates closely with VH gene status. We used Syk phosphorylation as a measure of responsiveness to BCR
stimulation, because Syk tyrosine phosphorylation plays a key role in
downstream signaling, including activation of PLC The BCR has a profound impact on the behavior of B cells,
including induction of proliferation or apoptosis, depending on the
stage of development, and on the context and strength of stimulation (for a review, see Healy and Goodnow15). In the
CD38+ subset of CLL, ligation of sIgM has been reported to
result in accelerated apoptosis.19 In contrast, ligation
of sIgD resulted in cell survival.19 At present, our data
relate only to Syk phosphorylation and tyrosine phosphorylation,
leaving open the question of whether signaling in the unmutated cases
leads to apoptosis. What is clear in the mutated subset is that there
is a differential capacity to transduce signals from the BCR,
dependent on which component of the BCR complex is targeted. The
fact that the apparent deficit in signaling via sIgM can be
bypassed by stimulating via sIgD or CD79 A hierarchy of factors appears to be involved in failure to signal via
sIgM. Most CLL samples that fail to respond to anti-IgM retain
responsiveness to anti-IgD and anti-CD79 The features of the cases, mostly with mutated VH genes,
which were unresponsive to signaling, are reminiscent of B cells that
have undergone receptor desensitization, following chronic stimulation
by antigen.23 This anergic state has been well documented in mice where the desensitized BCR remains able to bind antigen, but
fails to transduce signals.23 As with our observations in CLL, the murine desensitized receptors remain responsive to signaling via CD79 The nature of antigen involved in mediating the proposed desensitized state is unclear, although the biased usage of the V4-34 gene in the mutated subset might point to either a viral antigen25 or an autoantigen.26,27 It appears that less malignant behavior is associated with the anergic state, perhaps suggesting that the unmutated cases with more competent BCRs are better able to receive signals for maintenance or proliferation. Clearly there are many steps between BCR signaling and cellular response that need to be uncovered in vitro, and their relevance to conditions in vivo determined, before we can connect these proximal events to tumor progression.
We thank Professor Martin Glennie and Dr Mark Cragg (Tenovus Laboratory, Southampton, United Kingdom) for supplying antibodies, particularly for CD79 analysis. We would also like to thank Zadie Davis, Jenny Orchard, and Mo Tiller for technical assistance.
Submitted June 19, 2002; accepted August 27, 2002.
Prepublished online as Blood First Edition Paper, September 26, 2002; DOI 10.1182/blood-2002-06-1822.
Supported by Tenovus United Kingdom and Cancer Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Freda Stevenson, Molecular Immunology Group, Tenovus Research Laboratory, Southampton University Hospitals Trust, Tremona Road, Southampton SO16 6YD, United Kingdom; e-mail: fs{at}soton.ac.uk.
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Top
Abstract
Introduction
) with unmutated (unmut, 
), signaling (sig, 
)
with nonsignaling (nonsig, 
), and CD38+ (38pos, 
)
with CD38
(38neg, 
) groups. None of the comparisons
shows a significant difference. Only samples for which the expression
of all surface molecules were known were included in the
analysis.
2, SLP-65, and
calcium mobilization. The constitutive levels of Syk expression do not
differ between anti-IgM responsive and nonresponsive CLL, and we found
a perfect correlation between Syk phosphorylation and increases in
global tyrosine phosphorylation following anti-IgM stimulation.
Importantly, there was a strong correlation observed between
VH gene status and anti-IgM-induced phosphorylation, with cases expressing unmutated VH genes mostly showing induced
phosphorylation of PTK Syk. CD38 expression has been reported to be
associated with a poor prognosis,