Chronic Lymphocytic Leukemia, Normal B and T Cells

ZAP-70 is expressed by normal and malignant human B-cell subsets of different maturational stage

Abstract

ZAP-70 tyrosine kinase is involved in signalling pathways following T-cell receptor stimulation and was originally described only in T cells and natural killer cells. ZAP-70 expression has been reported in normal mouse B lineage cells and in human malignant B lymphocytes, mainly in chronic lymphocytic leukemia (CLL) where it correlates with clinical outcome. We analyzed several B-cell lines and ex vivo malignant B cells, ranging from acute lymphoblastic leukemia to multiple myeloma and reflecting different stages of B-cell differentiation, and they showed ZAP-70 expression regardless their maturation stage. We then analyzed by Western blot and flow cytometry different human normal B-lymphocyte subpopulations: naïve, germinal center and memory B cells from tonsils, CD19+ CD5+ cells from cord blood and CD19+ lymphocytes from peripheral blood. All expressed ZAP-70 protein, though at different levels depending on their differentiation, activation and tissue localization. In addition, ZAP-70 expression levels could be modulated following stimulation via the B-cell receptor. These findings implicate a potential role of ZAP-70 in the signalling pathway of B lymphocytes at different maturational stages, indicate that ZAP-70 expression is not a CLL-specific feature among B-cell malignancies and suggest that the absence of ZAP-70 rather than its presence should be considered abnormal for malignant B lymphocytes.

Introduction

ZAP-70 is a 70-kDa ζ-chain CD3-receptor-associated protein tyrosine kinase (PTK)1 that operates in the T-lymphocyte signalling pathways triggered by the stimulation of the T-cell receptor. It is generally considered the T-lymphocyte counterpart of Syk, a B-cell receptor (BCR)-associated kinase that belongs to the same PTK family and plays a similar role in the antigen receptor signalling in the B-lineage cells.2

Interestingly, ZAP-70 was originally described to be exclusively present in T cells and natural killer (NK) cells,1 while Syk, though originally defined as a B-cell-specific tyrosine kinase, was soon reported to be expressed also in murine and human thymocytes and, three- to fourfold less, in peripheral T cells.3

Evidence is gathering that the pattern of expression of ZAP-70 might not be as lineage specific as previously thought. The first hint came from the gene transcript analysis of B-chronic lymphocytic leukemia (CLL) cells. Although homogeneous in terms of surface phenotype, CLL is heterogeneous in terms of clinical outcome and biological features, including the mutational status of the immunoglobulin variable heavy chain (IGHV) genes. The expression of ZAP-70 gene in CLL cases grouped according to the presence or the absence of somatic mutations in the IGHV genes revealed that ZAP-70 was significantly associated with the unmutated CLL subset.4 High levels of ZAP-70 protein are detectable in the majority of cases of unmutated CLL and ZAP-70+ CLL B cells express levels of ZAP-70 protein that are comparable to those expressed by normal blood T cells.4, 5, 6, 7 On functional grounds it has become evident that CLL cells expressing ZAP-70 show an enhanced signal transduction via the BCR5 and the forced expression of ZAP-70 in the negative cases allows for a more effective IgM signalling.8 Taken together these data, which indicate that ZAP-70 might be involved in the natural history of the disease and possibly contribute to the more aggressive clinical behavior generally associated with unmutated CLL, led to the idea that ZAP-70 expression might be a CLL-related feature. In a way this hypothesis was corroborated by the finding that only few other B-lymphoma cell lines (LILA, LK6, OCI-Ly2) were found positive for ZAP-70.9 Nevertheless, doubts on its real pattern of expression in B-lineage cells have arisen when the protein was clearly demonstrated in bone marrow (BM) immature and splenic mature mouse B cells.10 Recently, ZAP-70 has been demonstrated also in normal human B cells, though only in a subset showing an activated phenotype.11 In addition, ZAP-70 has been reported to be expressed in a variable number of other mature and immature human B-lymphoid malignancies.12, 13, 14, 15

In this work, we have studied by Western blot (WB), flow cytometry (FC) and immunohistochemistry normal and malignant human B-cell subpopulations. We demonstrate that different subsets of human mature B lymphocytes express ZAP-70, regardless their tissue of origin or activation status, and with differences in terms of intensity of expression. ZAP-70 expression levels could be modulated in peripheral blood (PB) B lymphocytes following stimulation through the surface immunoglobulin (Ig). In addition, several B-cell lines and fresh malignant B cells, from acute leukemia to multiple myeloma (MM), purified ex vivo, show expression of the kinase regardless their maturation stage.

These findings imply a role for ZAP-70 in the normal B-cell function and suggest that, as for the natural history of CLL, it is the absence of ZAP-70 expression rather than its presence that might be considered abnormal.

Patients and methods

Tissue samples and cell lines

Normal conventional B cells were obtained from donors’ buffy coats while normal CD5+ B cells (B1 cells)16 were purified from cord blood.

Tonsils were obtained, as discarded tissues, from children undergoing tonsillectomy. Organs were cut with a scalpel blade and passed through a fine wire mesh to prepare a single-cell suspension.17 All normal samples were processed and cells used for experiments immediately after collection.

Leukemic lymphocytes were obtained from the PB of 40 CLL patients, diagnosed according to the National Cancer Institute-Working Group (NCI-WG).18 Peripheral blood, BM or infiltrated lymph nodes were obtained from patients with different B-cell malignancies. These included two pediatric common acute lymphoblastic leukemias (ALL), 15 follicular lymphomas (FL), three hairy cell leukemias (HCL), four mantle cell lymphomas (MCL), six diffuse large B-cell lymphomas (DLCL), 27 marginal zone lymphomas (MZL). All tissue samples were obtained following institutional guidelines at our institutions. Mononuclear cells from all different tissues were isolated by Ficoll–Hypaque gradient (density, 1.077 g/ml; Pharmacia, Uppsala, Sweden). When not used directly for experiments at the time of preparation, they were frozen in liquid nitrogen. All patients were untreated or off-therapy since at least 6 months. The following cell lines have been used and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/l L-glutamine and 15 μg/ml gentamicin (Life Technologies, San Giuliano, Milanese, Milan, Italy): REH, 207, Nalm-6 (ALL), MEC-1 (CLL), Namalwa, Daudi, Raji (Burkitt lymphomas), RL, H2, DHL-16, DHL6 (DLCL), SKMM-1, U266, Karpas 422 (MM), Jurkat (acute T-cell leukemia), 16 EBV transformed lymphoblastoid cell lines (12 obtained from PB and four from tonsils) from healthy individuals.

Flow cytometry analysis and purification

To analyze ZAP-70 expression by FC, PB and buffy coats cells were stained with a mixture of CD19PECy5.5 (Caltag, Burlingame, CA, USA), CD56PE, CD16PE (Beckman-Coulter, Miami, FL, USA) and CD5APC (BD Biosciences, San Josè, CA, USA) antibodies, fixed and permeabilized using the Fix-and-Perm kit (Caltag, Burlingame, CA, USA) and incubated with AlexaFluor488-conjugated anti-ZAP-70 monoclonal antibody (clone 1E7.2) (Caltag, Burlingame, CA, USA) or irrelevant isotypic control.19 All cells were analyzed on properly compensated FACSCalibur (BD-Biosciences).

To purify the different B-cell subsets, properly stained blood and tonsils mononuclear cells were fluorescein-activated cell sorting (FACS) sorted on a Coulter Altra (Beckman-Coulter).17 In detail, donors’ buffy coats and cord blood were first depleted of CD3+ and CD14+ cells using MACS-microbeads (Miltenyi Biotec, Srl, Bologna, Italy), then stained with anti CD19PECy5.5, (Caltag, Burlingame, CA, USA) and CD5APC (BD Biosciences, San Josè, CA, USA) antibodies in order to separate CD19+CD5 and CD19+CD5+ B cells. Normal CD15+ granulocytes, CD14+ monocytes and CD3+ T lymphocytes were positively purified using MACS microbeads.

Tonsillar B cells were stained with anti-IgDFITC (Southern Biotechnology Associates), anti-CD19PE and anti-CD38PC5 (Beckman-Coulter) and then FACS-purified in order to obtain Naive B cells (CD19+, IgD+, CD38), Germinal Center B cells (GC-B – CD19+, IgD, CD38+) and Memory B cells (CD19+, IGD, CD38).17

Chronic lymphocytic leukemia leukemic cells were purified using a B–lymphocyte enrichment kit (RosetteSep™ StemCell Technologies), following manufacturer's instructions.

Purity of both normal and leukemic B lymphocytes was always above 99%, as checked on a Coulter FC500 (Beckman-Coulter). All preparations were virtually devoid of NK and T lymphocytes.

B-cell culture and stimulation

B cells isolated from three donor's buffy coats were cultured for up to 72 h in the presence of either goat anti-human IgM (10 μg/ml) or a mixture of soluble recombinant human CD40L (sCD40L) (100 ng/ml) (Alexis Corporation, San Diego, CA, USA), IL-4 (50 ng/ml), IL-6 (30 ng/ml) and IL-10 (50 ng/ml) (ImmunoTools, Friesoythe; Germany).11 Soluble recombinant human CD40L was used together with 1 μg/ml of enhancer, according to manufacturer's instructions (Alexis Corporation).

Immunoprecipitation, Western blot and densitometry analysis

Purified leukocyte populations or cell lines were lysed, as previously described.20 Total cell lysates obtained from MEC-1,SKMM-1, NALM-6 and Jurkat21 cell lines were incubated overnight at 4°C with antiphosphotyrosine antibody 4G10 (Upstate Biotechnology, Charlottesville, VI, USA) immobilized on protein G-coated Sepharose Beads (Amersham Biosciences, Cologno Monzese, Italy). After a quick spin, the left over was collected and the beads were washed three times with lysis buffer. Immunocomplexes were recovered by incubation with Laemmli buffer (5′ at 95°C).

Immunoprecipitated or whole proteins were resolved on a 10% acrylamide sodium dodecyl sulphate–polyacrylamide gel electrophoresis, after loading 10 μg of protein per lane. Proteins were transferred to nitrocellulose followed by incubation with murine anti-ZAP-70 (clone 29, BD-Biosciences, San Josè, CA, USA) and anti-β-actin (Sigma-Aldrich, Gallarate, MI, USA) antibodies. Immunoreactivity was revealed by incubation with a goat anti-mouse IgG horseradish peroxidase conjugated (Upstate Biotechnology, Charlottesville, VI, USA) followed by ECL (Amersham Biosciences) reaction and film exposures. Densitometry analysis was performed with Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA, USA). ZAP-70 expression levels were evaluated as optical density (OD) ratio with β-actin.

Immunohistochemistry analysis

Formalin-fixed tissue sections (5 μm) of 20 BM trephine biopsies from patients with MM with variable infiltration of plasma cells were cut on slides covered with adhesive. Sections were deparaffinized and endogenous peroxidase was quenched with 1.5% hydrogen peroxidase in methanol (10 min). A series of antibodies were applied to characterize malignant plasma cells (CD38, CD138). All reagents were purchased from DakoCytomation, (Glostrup, Denmark). Mouse monoclonal antibody to ZAP-70 (clone 2F3.2, diluition 1/200, Upstate, Lake Placid, NY, USA) and an isotypic control antibody were utilized, with a polymeric labelling two-step method (Super sensitivetm ihc detection system, Biogenex, San Ramon, CA, USA) as revelation system, according to the manufacturer's instructions, using 3,3′-diaminobenzidine as the chromogen. After immunostaining, the slides were lightly counterstained with hematoxilin and coverslipped.

Statistical analysis

Statistical analysis of ZAP-70/actin ratio data has been performed using one-tailed signed-rank Wilcoxon test. Results were considered statistically significant when the P-value was <0.05. Statistical analysis was performed with the software STATA 8.2 (StataCorp, College Station, TX, USA).

Results

ZAP-70 expression in different B-cell lines representing different stages of differentiation

We first performed a WB analysis on different B-cell lines commonly used in the laboratory settings and resembling different stages of B-cell differentiation: B-ALL (REH, 207, NALM-6) as precursors B cells, BL (Namalwa, Daudi, RAJI) and DLBL (H2, RL, DHL-6) as germinal center B cells and MM (SKMM-1, U-266, KARPAS-422) as plasma cells. In addition, we also analyzed a CLL cell line (MEC-1) given the notion that CLL cases may be ZAP-70 positive. Sixteen different EBV-transformed B-cell lines were also analyzed to study activated non-neoplastic B cells.

The expression of ZAP-70 was heterogeneous as only NALM-6 among three ALL cell lines, DAUDI among three BL cell lines, SKMM-1 among three MM cell lines and the CLL cell line MEC-1 were positive for ZAP-70, though at variable levels. None of the four DLCL cell lines expressed the protein. Among EBV-transformed B-cell lines, 4/12 cell lines obtained from PB and 1/4 obtained from tonsils expressed readily detectable levels of the protein. ZAP-70 levels were consistently similar in different determinations and negative cases were always found negative. Of note, in none of the cell lines analysed (namely MEC-1, SKMM-1 and Nalm-6) ZAP-70 was found phosphorylated, after immunoprecipitation in contrast to Jurkat cells used as controls21(data not shown).

ZAP-70 is expressed in several human B-cell malignancies other than chronic lymphocytic leukemia

By immunoblot we examined CD19+CD5+ PB leukemic cells from 40 CLL patients. ZAP-70 expression was very variable among all cases, ranging from virtually negative to strongly positive (Figure 1a). In details, nine cases showed no detectable levels of the protein (Figure 1a, lanes 1 and 2) while 11 showed a faint band on the WB analysis (Figure 1a, lanes 3 and 4). All negative/low cases had a ZAP-70/β-actin OD ratio ranging between 0 and 0.3. In contrast, the remaining 18 cases (Figure 1a, lanes 5 and 6) showed significant levels of the protein (ZAP-70/β-actin OD ratio >0.3). The intensity of expression appeared to correlate with the mutational status of the IGHV genes in each CLL case studied, with negative or low cases preferentially carrying mutated IGHV genes (17/20 cases), while strongly positive samples being unmutated (12/18 cases).

Figure 1
figure1

ZAP-70 expression in purified leukemic CD19+CD5+ chronic lymphocytic leukemia (CLL) cells. (a) Leukemic cells from representative CLL cases were analyzed for ZAP-70 expression by immunoblot. The corresponding ZAP-70/β-actin optical density (OD) ratio is shown beneath each lane. Cases 1 and 2: negative; cases 3 and 4: low; cases 5 and 6: positive. (b) Unpurified peripheral blood (PB) cells from three selected CLL cases (cases 1, 3 and 5) were analyzed by flow cytometry (FC) after staining with anti-CD19PECy5.5 and AlexaFluor 488-conjugated isotype control (left panels) or anti-ZAP-70 antibodies (right panels). The upper panels were obtained by gating only lymphocytes as identified on SSC and FSC profiles and show ZAP-70 expression (right upper panel) on both CD19+ and CD19 cell fractions; the lower panels were obtained from the same files by gating CD19+ lymphocytes, allowing to calculate the percentage of positivity on CD19+ cells. (c) Data of 27 CLL patients are plotted according to ZAP-70 expression detected by FC analysis (x axis: percentage of positive cells) and by WB analysis (y axis: ZAP-70/β-actin OD ratio). A trendline is shown. In addition, a vertical line divides ZAP-70 (<20%) from ZAP-70+ (>20%) CLL cases, as detected by FC. For clarity, ZAP-70 negative/low cases, as detected by WB analysis, are divided from positive patients by a horizontal line, arbitrarily placed at OD ratio 0.3.

We then analyzed 27 of these samples for ZAP-70 expression by FC, using an alexafluor488-labelled anti-ZAP-70 antibody in order to test the reproducibility of the results using a different, though simpler, methodology (Figure 1b). The 20% cutoff value3 has been used to define positive and negative results. Twenty-one out of 27 (77.8%) cases analyzed with both techniques were concordant in terms of expression of the protein (Figure 1c), with the WB methodology being more sensitive than FC, as expected. Five remaining cases scored positive in WB while they were negative by FC (Figure 1c – upper left quadrant). Only one additional case was found to be positive (39%) by FC and showed a faint band by WB (ZAP-70/β-actin OD ratio: 0.29) (Figure 1c – lower right quadrant). Interestingly, this case was also the only one of the present series carrying 97% similarity on the IGHV genes. The overall analysis of the concordance between ZAP-70 expression, regardless the methodology used, and the IGHV mutational status showed very similar results. Twenty out of 25 cases (80%) were concordant based on FC results and, seemingly, 19/25 (76%) were concordant when analyzed by WB.

Given the good correlation between FC and WB detection of ZAP-70 expression (Figure 1c), we proceeded to analyze by FC a wider spectrum of B-cell malignancies. ZAP-70 expression was variable among all different lymphoma subtypes ranging from negative to positive and was considered positive, according to the 20% cutoff value,3 in 2/2 CD10+ leukemic samples from pediatric ALL (FC value: 78±5%), 2/4 MCL (FC value: 45±7%), 5/15 FL (FC value: 37±11%), 2/6 DLCL (FC value: 37±2%), 5/27 MZL (FC value: 34±18%) and 1/3 HCL (FC value: 79%).

Our data paralleled and confirmed with a different technique, the recent finding12, 13, 14 showing, by immunohistochemistry, that several different human B-cell lymphomas are expressing ZAP-70. Among these works, no definitive data on the expression in MM plasma cells have been reported. As our analysis on cell lines suggested that ZAP-70 could be also expressed in malignant plasma cells, we next analyzed 20 different BM samples infiltrated with MM cells by immunohistochemistry. In 4/20 cases (20%) malignant plasma cells appeared strongly ZAP-70 positive (Figure 2).

Figure 2
figure2

ZAP-70 expression in malignant bone marrow (BM)-derived plasma cells. ZAP-70 expression was analyzed in BM trephine biopsies infiltrated with multiple myeloma (MM) plasma cells, by immunohistochemistry and found positive (a) in 4/20 cases analyzed, while the remaining were scored negative (b).

ZAP-70 is expressed in all normal human B-cell subsets regardless the stage of maturation

As the above-reported findings indicate that in human B-lineage cells ZAP-70 is expressed in different stages of differentiation and maturation, we investigated by immunoblot the expression of ZAP-70 in different B-cell subsets purified from three human tonsils, based on the expression of CD19, CD38 and IgD, whose differential expression define specific stages of B-cell maturation. As shown in Figure 3a, naïve B cells (CD19+CD38IgD+), GC-B cells (CD19+CD38+IgD) and memory B cells (CD19+CD38IgD)17 expressed ZAP-70 at similar intensity, being about three times less than CD3+ T lymphocytes (Figure 3a).

Figure 3
figure3

ZAP-70 expression in normal B-cell subsets and normal leukocyte subsets. (a) ZAP-70 expression analyzed by immunoblot analysis in naïve, GC-B, memory, CD19+CD5+ and CD19+ B cells sorted from one representative tonsil, one cord blood and one donor buffy coat, respectively. The corresponding ZAP-70/β-actin optical density (OD) ratio is shown beneath each lane. (b) As control, ZAP-70 expression was also performed on purified CD15+ granulocytes, CD14+ monocytes and CD3+ T lymphocytes. The data shown here are representative of three different sets of experiments. (c) Unpurified cells from buffy coats were analyzed by flow cytometry (FC) after staining with anti-CD19PECy5.5 and AlexaFluor 488-conjugated isotype control (left panels) or anti-ZAP-70 antibodies (right panels). The upper panels were obtained by gating only lymphocytes as identified on SSC and FSC profiles and show ZAP-70 expression (right upper panel) on both CD19+ and CD19 cell fractions; the lower panels were obtained from the same files by gating CD19+ lymphocytes, allowing to calculate the percentage of positivity on CD19+ cells. The data here shown are representative of five different experiments.

We next analyzed ZAP-70 expression in circulating B lymphocytes including normal CD19+ B cells purified from three different donor buffy coats and normal CD19+CD5+ B cells sorted from three different cord bloods (Figure 3a). The analysis revealed the expression of ZAP-70 in both subsets, though cord blood B cells showed higher expression as compared to peripheral B cells (Figure 3a). This finding was somehow expected as cord blood-derived B cells are considered to be activated. Peripheral blood-derived B lymphocytes were consistently less positive than any other B-cell subset (about five times less than tonsillar B cells) (Figure 3a). As controls, we analyzed ZAP-70 expression in purified peripheral CD15+ granulocytes and CD14+ monocytes that were negative (Figure 3b).

ZAP-70 expression in normal B cells is confirmed by flow cytometry analysis

We then tested ZAP-70 expression in normal B cells by using FC assay (Figure 3c) on unpurified PB leukocytes. CD19+ B cells from 5/6 healthy donors showed a dim positivity, still higher than background (range of positive cells 19.1–27.3%), When B lymphocytes were distinguished on the basis of CD5 expression, no particular differences in terms of ZAP-70 positivity was evident (data not shown).

ZAP-70 expression may be modulated after stimulation via the BCR

We aimed at defining whether ZAP-70 expression in B cells could be modulated by exogenous stimulation. As PB-derived B lymphocytes showed consistently lower levels of the kinase than any other B-cell subset, we cultured CD19+ B cells purified from three different buffy coats in the presence of anti-IgM antibodies or of a mixture of sCD40L, IL-4, IL-6 and IL-10, for up to 3 days. Interestingly, the levels of ZAP-70 expression (Figure 4a) increased in a statistical significant manner as compared to the unstimulated B lymphocytes (Figure 4b) in both experimental situations.

Figure 4
figure4

ZAP-70 expression in normal PB-derived B cells is upregulated following in vitro stimulation. (a) ZAP-70 expression has been analyzed by immunoblot in B lymphocytes purified from buffy coats and cultured for up to 72 h in media only (US: unstimulated), in the presence of soluble recombinant human CD40L (sCD40L), IL-4, IL-6 and IL-10 (sCD40L+cyt) or in the presence of anti-IgM antibodies (α-IgM). The corresponding ZAP-70/β-actin optical density (OD) ratio is shown beneath each lane. The data shown here are representative of three different sets of experiments. (b) Mean ZAP-70 expression values (±s.d.) of each culture condition from all three different experiments performed, defined as ZAP-70/β-actin OD ratio. *Indicates a significant difference (P<0.05) between both stimulating conditions (sCD40L+cyt and α-IgM) as compared to the unstimulated control (US).

Discussion

Since its cloning, ZAP-70 has been considered a T-cell-specific tyrosine kinase, whose expression was strictly limited to T-cell lineage and NK cells.1, 3 This assumption was somehow mirrored by the existence of Syk protein, which is a tyrosine kinase closely related to ZAP-70 that was originally described as selectively expressed by B lymphocytes. Syk was subsequently demonstrated to be expressed in murine and human thymocytes and, three- to fourfold less, in mature peripheral T lymphocytes.3

A first exception to the assumption of the T-cell-restricted expression of ZAP-70 was reported9 and confirmed4, 5, 6, 7, 19, 22 in malignant B lymphocytes of CLL where it identifies a subset.

Bone marrow B-lineage progenitors and splenic mature B cells in the mouse have been subsequently shown to express this kinase10 that appears to play an indispensable role during B-cell development. The mouse model finding was not paralleled by the human system, where ZAP-70 was recently demonstrated to be only expressed in one subset of normal B cells, showing an activated phenotype.11 In contrast, it has been reported that, besides CLL, several other human B-cell malignancies express detectable levels of ZAP-70 when analyzed by immunohistochemistry.12, 13, 14

Here we present evidence that ZAP-70 is actually expressed in all mature B-cell subpopulations tested, regardless the tissue of origin and the level of in vivo activation. Both conventional CD19+ B cells as well CD5+ B lymphocytes express ZAP-70. In addition, naive, GC-B and memory B cells highly purified from tonsils were found to express the molecule. Interestingly, the intensity of expression appeared to increase with the level of potential in vivo activation status, that is B cells in the inflamed tonsil were expressing higher levels as compared to the circulating cells, being the B lymphocytes of the PB the lower expressors. In accordance to these in vivo evidences, in vitro stimulation through the surface Ig of PB-derived B lymphocytes led to a significant increase of ZAP-70 expression. These experiments suggest that the activation following an antigen-like encounter may be involved in triggering the expression of the kinase also in vivo.

Given the consistent expression of ZAP-70 in all normal mature B cells subsets here tested – CD5+ and CD5, circulating in the blood or located in the tonsils – even though at low levels, it is striking to note that a sizeable fraction of CLL cases is virtually negative for this protein.4 This evidence suggests that, in contrast to the current hypothesis, it is the absence of ZAP-70 expression rather than its presence that has to be considered abnormal. Hence understanding the mechanisms responsible for ZAP-70 downmodulation may be useful to understand the biological basis of the clinical differences that are observed among the different CLL subsets. A recent work suggests that ZAP-70 methylation status is significantly related to the expression of the protein with the majority of ZAP-70+ cases lacking methylation of a highly conserved intronic region of the gene.23 Further studies are clearly needed to unravel the mechanisms controlling ZAP-70 expression in both normal and malignant B lymphocytes.

To this end, the evidence that, within normal B cells, the highest levels of ZAP-70 are present in tonsillar B cells, which have been activated following antigenic stimulation, may somehow help to interpret the observation that the strongest expression is detected in unmutated CLL. A possible explanation for these findings is that ZAP-70 levels may reflect the in vivo activation status. The progressive decline of the kinase expression from activated B cells involved in immune responses to resting B lymphocytes circulating in the blood is consistent with a scenario where mutated CLL cases, though previously stimulated, have progressively become resting elements, even if they retain the expression of some activation molecules.24 In contrast, unmutated cases which show not only an activated phenotype24 but also an increased responsiveness to IgM ligation25 might be blocked in an abortive activation status which does not allow them to proceed further in the maturation process. Following our in vitro experiments of stimulation through the BCR, the possibility exists that a persistent antigenic or antigen-like stimulation26 may be responsible for maintaining elevated levels of the tyrosine kinase, as it may happen during a normal immune reaction (e.g. in the inflamed tonsil). However, it has to be underscored that activation per se seems not to be sufficient to induce ZAP-70 expression as witnessed by the heterogeneous expression detected in EBV-transformed B lymphocytes.

In addition, our analysis of ZAP-70 expression in CLL by WB also revealed a continuum of expression rather than a positive–negative phenomenon4 as previously suggested in a study based on FC.19 This confirms the complexity of establishing a clear cutoff value to define positive and negative cases, in terms of clinical prognosis. Regarding this point, it is of potential interest the observation here reported that normal circulating B cells express low but detectable levels of ZAP-70 as shown by FC (Figure 3d). The percentage of expression (between 19 and 27%) is intriguingly close to the cutoff value currently used to define prognosis in CLL (20%), with cases >20% having a poor outcome.6, 7, 19, 22 These evidences might give a biological reason for such threshold that has been defined only on a statistical basis. They might indicate that certain level of the protein may be physiological for circulating (and resting) B cells, though a stronger expression might become a hallmark of the cellular involvement in an active immune response as suggested by the strong expression in the tonsil. On the other hand our findings question the possibility to use normal B lymphocytes as ‘negative’ control to define CLL cells positivity as suggested.19

The analysis of several cell lines and of several lymphoproliferative disorders purified ex vivo showed ZAP-70 expression throughout all stages of B-cell differentiation, from acute leukemia to MM. These evidences indicate that ZAP-70 expression cannot be considered a CLL-specific feature among B cells malignancies.

Interestingly, MCL and MMs showed consistent levels of expression. The former are considered to derive from antigen-unexperienced B cells while the latter are thought to be unresponsive to antigen as normal plasma cells are, given the absence of Ig expression on the cellular surface. Accordingly, the presence of ZAP-70 in malignant plasma cells is rather puzzling and cast some doubt on the whole hypothesis of ZAP-70 expression following antigenic stimulation. Given the fact that only a fraction of MM cases are expressing the kinase further analysis on a larger series of cases, are warranted in order to elucidate potential differences in terms of clinical behavior or biological features between negative and positive cases.

In conclusion, we report the unexpected expression of ZAP-70 in all normal mature human B-cell subsets tested, regardless their stage of maturation and tissue localization. Nevertheless, the different B-cell subpopulations show different level of expression, possibly depending on their activation status. Increased expression of ZAP-70 appears to correlate with recent/persistent cell activation, while the expression of the molecule progressively disappears along with the acquisition of a resting state. Accordingly, in vitro stimulation through the surface Ig induces upregulation of the kinase expression. These findings open a new area of investigation with the obvious need to understand the role of this kinase in the normal B-cell signalling pathway. The ‘physiological’ presence of ZAP-70 in all normal B cells is also reflected by its expression in several B-cell malignancies ranging from B-cell precursors to plasma cells. It remains to be elucidated which mechanisms are responsible for downmodulating the kinase in both normal and neoplastic B cells.

This might help understanding some elements of the pathogenesis of B-lymphoproliferative disorders and especially the biological basis of the clinical differences that characterize the different CLL subsets.

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Acknowledgements

CS is a recipient of a scholarship from FIRC, Milano. We are grateful to Ms Rose Mary Carletti for her precious technical assistance and to Dr Michela Frenquelli for her continuous support in the lab work. We are indebted with Alessandro Ambrosi, Eliana La Ferrara e Giuseppe Guida for their precious advice on statistical analysis. This work was supported in part by Associazione Italiana per la Ricerca sul Cancro (AIRC), Progetto Oncologia CNR-MIUR, MURST 40%, the CLL Global Research Foundation, Ministero della Sanità (R.F.2002, no. 183), Fondazione CARIPLO and MIUR-Firb (RBNE01LNX7-006).

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Scielzo, C., Camporeale, A., Geuna, M. et al. ZAP-70 is expressed by normal and malignant human B-cell subsets of different maturational stage. Leukemia 20, 689–695 (2006). https://doi.org/10.1038/sj.leu.2404138

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Keywords

  • ZAP-70
  • B lymphocytes
  • B-lymphoid malignancies
  • chronic lymphocytic leukemia

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