ZAP-70 Expression in Normal Pro/Pre B Cells, Mature B Cells, and in B-Cell Acute Lymphoblastic Leukemia

Materials and Methods

Samples. Normal lymphocytes were isolated on a Ficoll/Hypaque (Seromed, Berlin, Germany) gradient from peripheral blood lymphocytes (PBL; n = 2) and bone marrow (n = 8) obtained from adults and tonsil (n = 4) obtained from children. In addition, tumoral cells from 29 B-ALL cases, classified according to their maturation status (12), were included in this study: status B-I (CD19⁺, CD10⁻, cIgM⁻), four cases; B-II (CD19⁺, CD10⁺, cIgM⁻), 18 cases; B-III (CD19⁺, CD10⁺, cIgM⁺), one case; and B-IV/Burkitt (CD19⁺, CD10⁺, sIgM⁺), six cases (Table 1). Moreover, tumoral lymphocytes obtained from 10 patients diagnosed with CLL were also analyzed for ZAP-70 expression. The cell lines Jurkat (T-ALL), NALM-6 (B-ALL), Ramos, Daudi, Namalwa, Raji (Burkitt), JVM-2 (prolymphocytic leukemia), and NC-NC (lymphoblastoid) were used as controls. The main clinical (age, response to treatment, duration of response, and survival) and biological variables of the cases diagnosed with B-ALL were recorded and correlated with ZAP-70 expression. Samples were obtained after informed consent and approval from the local Ethical Committee.

In normal PBLs, CD19⁺ lymphocytes were negatively selected using the B-cell Isolation kit II (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) followed by a further negative selection using anti-CD56, anti-CD2, and anti-CD3-FITC labeled (Becton Dickinson, San Jose, CA). In normal bone marrow, CD19⁺/CD10⁺ lymphocytes were positive selected using anti-CD10-phycoerythrin (PE) followed by anti-CD19-FITC (Becton Dickinson). In tonsils, the subpopulations CD19⁺/CD27⁻, CD19⁺/CD27⁺, and CD19⁻ were isolated by separation of CD19⁺ cells with anti-CD19-FITC followed by further purification of CD27⁺ cells with anti-CD27-PE (Becton Dickinson). In tumoral samples (B-ALLs and CLL cases), isolation of CD19⁺ cells was done by separation of CD3⁺ cells with anti-CD3-FITC. Labeled cells were collected correspondingly using anti-FITC, anti-PE, or anti-biotin Microbeads (Miltenyi Biotec). Isolated samples were analyzed for ZAP-70 expression when the presence of T and natural killer cells was <1%.

Flow cytometry analysis of ZAP-70. Flow cytometry analysis of ZAP-70 expression was done as previously described (7). Several surface markers combinations were used to assess ZAP-70 expression on the different cell subsets: (a) for B-cell subsets in normal bone marrow, to analyze simultaneously T cells and different subsets of B cells, ZAP-70-FITC/CD20-PE+CD3−PE/CD19-peridin chlorophyll protein cychrome 5.5 (PerCP Cy5.5)/CD10-allophycocyanine (APC)+CD3-APC or CD34-APC+CD3-APC; (b) for CLL, ZAP-70-FITC/CD3-PE+CD56-PE/CD19-PerCP Cy5.5/CD5-APC; and (c) for B-ALL, ZAP-70-FITC/CD3-PE+CD56-PE/CD19-PerCP Cy5.5/CD10-APC or CD34-APC. ZAP-70 monoclonal antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and the surface markers were from Becton Dickinson (CD56-PE, CD19 PerCP Cy5.5, CD5-APC, CD3-APC) and Immunotech (Marseilles, France; CD20-PE, CD10-APC, CD34-APC, CD3-PE). Mean fluorescence channel intensity was obtained for each lymphoid subpopulation, and the ratios between T and pro/pre B cells or mature B cells were compared using the Mann-Whitney test.

Double immunofluorescence analysis of ZAP-70. Five-micrometer-thick paraffin-embedded tissue sections of reactive tonsil, tonsil cell suspensions, or mononucleated cells from peripheral blood were dewaxed and rehydrated when necessary and then submitted to antigen retrieval by heating in 0.01 mol/L Tris-EDTA (pH 9.0) for 2 minutes in a conventional pressure cooker. Double immunofluorescence labeling for ZAP-70 and CD20 was done following the instructions previously described (13, 14).

The staining was examined using an E800 Eclipse fluorescence microscope (Nikon, Kingston-upon-Thames, United Kingdom). Images were captured using an Axiocam CCD camera and Axiovision software (Imaging Associates, Bicester, United Kingdom) and then opened using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA).

Quantitative real-time RT-PCR of ZAP-70. Total RNA was isolated from 20 B-ALL cases (in eight cases from isolated CD19⁺ cells), purified CD19⁺/CD27⁻, CD19⁺/CD27⁺, and CD19⁻ tonsil cells, normal PBLs, 10 CLL cases, and from cell lines using the guanidinium thiocyanate method (Ultraspec RNA; Biotecx Laboratories, Houston, TX) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of RNA using 260 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA), 26 units of RNAse inhibitor (Promega, Madison, WI), and 4.7 μg of random hexanucleotides (Pharmacia, Uppsala, Sweden).

Quantitative real-time RT-PCR (QRT-PCR) was done with Taqman technology and the ABI PRISM 7700 Sequence Detector System (Applied Biosystems, Foster City, CA). ZAP-70 primers and Taqman probe (FAM labeled at the 5′ end) were used at 300 and 200 nmol/L, respectively. Sequences of primers and the probe can be found at http://www.idibaps.ub.edu/images/noticias/ZAP70SEQ.pdf. The Jurkat T-cell line was used as a positive control for expression of ZAP-70, and GUS (β-glucoronidase) expression was used as endogenous control. The cycle threshold values of each sample were used to analyze the data, as the cycle threshold is inversely proportional to the amount of target cDNA. All samples were analyzed in triplicates. The PCR arbitrary units (AU) were defined as the mRNA levels of ZAP-70 normalized to the GUS expression. RNA from Jurkat T cells was used as reference control to normalize ZAP-70/GUS amplification ratios.

ZAP-70 expression was analyzed by QRT-PCR and flow cytometry in 7 B-ALLs and 10 CLLs. Values obtained by both methods were correlated using a regression test. Finally, to assess the influence in ZAP-70 expression of T cells present in B-ALL samples, serial dilutions of Jurkat and JVM-2 (a cell line that did not express ZAP-70; ref. 7) were done and analyzed by QRT-PCR.

Western blotting. Cryopreserved B-ALL cells were thawed and maintained at 37°C and 5% CO₂ in RPMI/10% FCS for 1 hour. Then, cells were washed with RPMI serum-free culture medium and resuspended in 400 μL of RPMI serum-free. For pervadanate treatment, equal volumes of solutions containing 300 mmol/L H₂O₂ and 100 mmol/L NaVO₄ were mixed immediately before use and added to B-ALL cells at a final concentration of 1.5 mmol/L H₂O₂ and 0.5 mmol/L NaVO₄. Cells were incubated for 4 minutes at 37°C, pelleted, and lysed on ice for 30 minutes in lysis buffer containing 20 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, 140 mmol/L NaCl, 1% Triton X-100, 2 mmol/L NaVO₄, and 1× proteases inhibitor cocktail (Sigma, St Louis, MO). After centrifugation (5 minutes at 13.2 × 10³ rpm), supernatants were collected and used for Western blot analysis.

Isolated populations from normal tonsils, bone marrow, and PBLs were pelleted and lysed on ice for 30 minutes in lysis buffer, and supernatants were collected by centrifugation.

Cell lysates were subjected to electrophoresis in 10% polyacrylamide gels and transferred to Immobilon-phosphate membranes. Membranes were incubated with the following primary antibodies: anti-phosphotyrosine, anti-phospho-ZAP-70 (Tyr³¹⁹)/phospho-Syk (Tyr³⁵²; Cell Signaling Technology, Beverly, MA); anti-ZAP-70, clone 2F3.2; anti-PLCγ2; anti-LAT, anti-phospho-LAT (Tyr²²⁶); anti-Lck, clone 3A5; and anti-α-tubulin (all purchased from Upstate). Chemiluminescent images were detected with the LAS-3000 system and analyzed with Image Gauge V4.0 software (Fuji Photo Film Co., Carrollton, TX).

ZAP-70 mutational analysis in B-ALLs. Genomic DNA was isolated from tumoral lymphocytes from four B-ALL patients (two of them with high ZAP-70 expression) using a sodium chloride extraction method (15). The coding region of the ZAP-70 gene (starting at the position −697 bp upstream of the transcription start site and including intronic flanking regions) was amplified by PCR (see http://www.idibaps.ub.edu/images/noticias/ZAP70SEQ.pdf). PCR products were purified and directly sequenced from both strands using the Big Dye Terminator Cycle sequencing V3.1 Ready Reaction. Sequence analysis and alignments were done using the Blast 2 sequences tool (16).

Results

ZAP-70 expression in normal B-cell subsets. ZAP-70 protein was analyzed by flow cytometry in pro/pre B cells (CD19⁺/CD10⁺/CD20⁻ or CD19⁺/CD34⁺) and in CD19⁺/CD20⁺/CD34⁻/CD10⁻ (mature) B-cell subpopulations from eight normal bone marrows. Among the different B-cell subsets analyzed, expression of ZAP-70 was found to be higher in pro/pre B cells (CD19⁺/CD10⁺ or CD19⁺/CD34⁺) than in mature B cells (CD19⁺/CD34⁻/CD10⁻; Fig. 1A and B). The analysis of mean fluorescence intensity showed that ZAP-70 ratios between T cells and pro/pre B cells were lower than ratios between T cells and mature B cells (3.72 versus 5.51, respectively; P = 0.002; data not shown). To confirm these results and to exclude the possibility of a nonspecific staining of ZAP-70 in the different B-cell subsets, CD19⁺/CD10⁺ cells from bone marrow were isolated and ZAP-70 expression analyzed by Western blotting. With this method, expression of ZAP-70 was confirmed in CD19⁺/CD10⁺ purified cells. (Fig. 1C). Unfortunately, analysis of ZAP-70 in the CD19⁺/CD34⁺ subset was not done due to the low number of recovered cells.

ZAP-70 expression was then analyzed in mature B cells derived from normal tonsil. In CD19⁺/CD27⁻ and CD19⁺/CD27⁺ cells from tonsil, levels of ZAP-70 expression obtained by QRT-PCR were as low as the negative controls, whereas in CD19⁻ cells, the expression was similar to PBLs (Table 1). Furthermore, tonsil slides and cell suspension were analyzed using immunofluorescence with anti-ZAP-70 and anti-CD20 double staining. Again, ZAP-70 was found expressed in T-cell areas, whereas no mature B cells in germinal center, mantle zone or marginal zone, were positively stained with ZAP-70 (Fig. 2). In addition, immunoblot analysis done on protein extracts obtained from these subsets did not detect a significant presence of ZAP-70 protein (Fig. 3). Moreover, double staining of tonsil cell suspensions detected only one double-positive cell for CD20 and ZAP-70 among >1,000 cells analyzed (Fig. 3).

ZAP-70 expression was analyzed in normal PBLs by immunofluorescence and Western blot. Immunofluorescence analysis did not detect cells with double-positive staining of ZAP-70 and CD20. These results were confirmed by Western blot analysis of CD19⁺ purified cells (Fig. 4).

ZAP-70 expression in B-ALLs. B-ALLs are aggressive neoplasms that derive from B cells with pro/pre B phenotype (B-I, B-II, B-III) or from mature germinal center B cells (B-IV and/or Burkitt/ALL). Because we found ZAP-70 expression in normal CD34⁺ or CD10⁺ B cells, the analysis of its expression was then done in a series of 29 B-ALL cases at different status of maturation.

ZAP-70 expression was ascertained by FC in 16 cases, by QRT-PCR in 20 cases, and by both methods in seven cases. The flow cytometry analysis of primary B-ALL cells disclosed a high expression of ZAP-70 in 12 of 16 cases (average expression, 80%; range, 56-99%; Table 1; Fig. 5), whereas in the remaining cases, the expression was considered low (average expression, 7%; range, 1-14%).

Expression of ZAP-70 was also analyzed by QRT-PCR in 20 B-ALLs cases. To rule out the influence of contaminating T cells in non–CD19⁺-purified cases, two different strategies were done: ZAP-70 expression analysis in serial dilutions of T cells and a ZAP-70 nonexpressing cell line (Jurkat and JVM-2), and comparison of the values obtained before and after B-cell purification in eight B-ALL cases. Using these approaches, we found that the presence of <10% of T cells did not significantly influence the values of ZAP-70 expression (data not shown). Consequently, when CD19⁺ purification was not possible, only the B-ALL cases with a total number of T and natural killer cells <10% were included in the analysis.

Normal B cells and ZAP-70 nonexpressing cell lines exhibited a mean relative expression of 0.042 AU (SD, ±0.052 AU). Therefore, values of QRT-PCR above 0.20 AU (average ± 3 SDs) were considered as having high relative levels of ZAP-70 expression. With this cutoff, 9 of 20 B-ALLs showed high levels of ZAP-70 expression (range, 0.34-0.81 AU), whereas in the remaining cases, these levels were similar to normal B cells (range, 0.01-0.19 AU; Table 1; Fig. 5). Of note, in 7 B-ALL cases and in 10 CLL cases in which ZAP-70 expression was analyzed by QRT-PCR and flow cytometry, there was a logarithmic correlation between both methods (R² = 0.7; P < 0.0001; Fig. 5).

Overall, we found a high expression of ZAP-70 in 13 of 23 (56%) B-ALLs with pro/pre B phenotype. Moreover, four of six Burkitt/ALL disclosed increased expression of ZAP-70. The levels of ZAP-70 in these Burkitt/ALL cases were found in the higher range of the series.

ZAP-70 expression and clinicobiological variables in B-ALLs. No relationship was observed between ZAP-70 expression levels and the maturation status of the B-ALLs or the detected genetic abnormalities, including the presence of bcr/abl rearrangement (Table 1). Of note, all the cases with high ZAP-70 expression showed increased expression of CD38, whereas the three cases with low CD38 disclosed low levels of ZAP-70 (Fisher's exact test, P = 0.05).

Twenty patients diagnosed with B-ALL were evaluable for prognosis. Cases with a lymphoid blast crisis of a chronic myelogenous leukemia, Burkitt's lymphoma, or without enough clinical data were not included in the analysis. There was no correlation between ZAP-70 expression and response to the induction treatment (data not shown). As per the duration of response and survival, the series was too heterogeneous to perform a meaningful analysis, because the great majority of patients diagnosed with B-ALL bcr/abl⁺ underwent allogeneic transplant, for this reason being not comparable with the rest of the patients from this series.

Mutational analysis of ZAP-70 in B-ALLs. Different point mutations in the exons 3, 12, and 13 of the ZAP-70 gene have been shown to cause deficient ZAP-70 protein expression in human T cells, leading to absence of peripheral CD8⁺ T cells and/or different forms of severe combined immunodeficiency (2, 3, 17–19). To rule out the presence of mutations in the coding portion of the ZAP-70 gene in B-ALLs not expressing ZAP-70, sequencing of the 5′-untranslated region, coding, and adjoining intronic regions was done, and the sequences were compared with the wild-type sequence (Genbank accession no. L05148.1). The mutational analysis did not reveal the presence of mutations, deletions, or insertions in B-ALL cases with low ZAP-70 expression. Three polymorphisms already described (see http://www.idibaps.ub.edu/images/noticias/ZAP70SEQ.pdf) were found in both ZAP-70 expressing and nonexpressing B-ALLs.

ZAP-70 phosphorylation and pre-TCR/TCR pathway in B-ALL cases. To determine whether ZAP-70 protein was phosphorylated in B-ALL cells, Western blot analysis was done using an anti–phospho-ZAP-70/phospho-Syk antibody that detects a phosphorylated-active ZAP-70 protein (P-ZAP-70) at the Tyr³¹⁹ residue, and the orthologous Tyr³⁵² residue in Syk (P-Syk). These residues are required for the assembly of a signaling complex that leads to the activation of the PLCγ1-dependent and Ras-dependent signaling cascades (20, 21). Syk protein is the other member of the Syk/ZAP-70 family normally expressed in cells of B-cell lineage that is required for the pre-BCR/BCR signaling (22–24). To rule out an alternate expression between Syk and ZAP-70 in B-ALL, the expression and the phosphorylation status of Syk protein was also analyzed in B-ALL cells. Western blot of total cell lysates revealed that Syk was expressed in all the samples analyzed, including cases with low ZAP-70 expression (data not shown). In untreated B-ALL cells, Western blot analysis revealed that neither ZAP-70 nor Syk were constitutively activated. Treatment of these cells with pervadanate, a broad phosphatase inhibitor (25), significantly increased the intensity of ZAP-70 and Syk phosphorylation (Fig. 6). Interestingly, Lck protein, a Src-kinase required for ZAP-70 kinase phosphorylation activity (4, 26, 27), was detected in all B-ALL cases (Fig. 6).

Different signaling elements belonging to the pre-TCR/TCR signal pathway have been described in human and mice pro/pre B cells and B-ALL cells (28–30). One of these elements is the linker for activation of T cells (LAT) protein (27–29, 31–33), a transmembrane adapter molecule expressed in T cells but not in normal mature B cells. In CD19⁺-purified B-ALL cells, we found that LAT was expressed in all the cases, confirming the results previously reported in normal pro/pre B cells (28, 29). Tyr²²⁶ residue of LAT becomes phosphorylated in T cells following TCR engagement (32, 34, 35). Using an anti-P-LAT antibody, this residue proved to be phosphorylated upon pervanadate stimulation (Fig. 6). These results showed the presence of other pre-TCR/TCR signaling elements in B-ALLs apart from ZAP-70.

Discussion

ZAP-70, a tyrosine kinase of the Syk/ZAP-70 family that plays a critical role in the signal transduction from the TCR, has been reported to be expressed in T and natural killer derived cells (1–3). Recently, ZAP-70 expression has been shown to be expressed in mice pro/pre B cells, its presence being important for B-cell development (11). Surprisingly, ZAP-70 expression has also been found in a mature B-cell–derived neoplasm as CLL (6), particularly in cases with unmutated IgV_H genes (7–9), where it seems to contribute to enhancing the signal from the BCR (10). These results pose the question whether ZAP-70 expression in CLL is an aberrant phenomenon related to the neoplasm or is a reflection of a normal B-cell counterpart of CLL expressing ZAP-70. The abovementioned results prompted us to analyze ZAP-70 expression in different human B-cell subsets and B-ALLs. Among normal B cells, ZAP-70 was only found expressed in CD34⁺ or CD10⁺ B cells, whereas no expression was observed in mature B-cell subsets from bone marrow, tonsil, and peripheral blood. Moreover, the expression of ZAP-70 protein found in CD34⁺ or CD10⁺ B cells obtained by flow cytometry and Western blot was inferior to that in T cells, in accordance to the results found in mice pro/pre B cells (11). Although expected, ZAP-70 expression in human pro/pre B cells has not been previously reported. Thus, recent gene expression profile done on pro/pre B cells did not clarify whether ZAP-70 was expressed (30).

Our results reasonably rule out the possibility of the existence of a significant proportion of mature B cells expressing ZAP-70. In this sense, expression of ZAP-70 in normal mature B cells has not been reported because this gene was cloned and found to be expressed only in cell lines of T or natural killer lineage (1). Thus, we and others showed that ZAP-70 is expressed in pro/pre B cells from mice and human but not in mature B cells. The expression found in mice splenic cells was attributed to mature B cells, but these cells were not phenotypically characterized and would correspond to immature B cells transiting to the spleen. In addition to that, we previously found the lack of ZAP-70 expression in normal CD5⁺ or CD5⁻ B cells from peripheral blood (7). Furthermore, in a recently published report (36), it was found ZAP-70 expression in a very low percentage of mature B cells from different tissues. Although an overinterpretation of ZAP-70 intensity and/or T-cell contamination could not be completely excluded in that article, it is also possible that in our report, even with the use of several and sensitive methods, a small percentage of B cells expressing ZAP-70 was not detected, as we observed a <0.1% B cells expressing ZAP-70 in tonsil.

ZAP-70 expression was found in 56% of B-ALL cases analyzed with pro/pre B phenotype. This is probably a consequence of the cell of origin of these neoplasms, as we found ZAP-70 expressed in normal pro/pre B cells. ZAP-70 expression was also analyzed in a small series of patients recently analyzed (37). In this report, a expression of ZAP-70 was found in all the cases analyzed, opposite to our results and to those recently published, in which ZAP-70 expression was analyzed by oligonucleotide arrays (38). In the later report, it was found an important correlation between ZAP-70 expression and the relapse rate (38). This analysis, however, was done in a series of cases without relevant genetic abnormalities (38). The series of patients herein reported was too heterogeneous in terms of genetic abnormalities and given therapy to analyze the clinical effect of ZAP-70 expression.

There is no clear explanation for the lack of ZAP-70 expression, found at both mRNA and protein levels, in some cases of pro/pre B-ALLs. In this sense, we did not observe mutations in the coding or 5′-untranslated region regions of the gene, as has been described in severe combined immunodeficiencies (2, 3, 17–19). In this sense, the lack of expression of ZAP-70 found in some B-ALL cases could be related to other pre-BCR signaling defects also described in this leukemia, similar to the lack of expression of SLP-65 or Syk reported in some B-ALL cases (39, 40).

Expression of other components of the pre-TCR/TCR signaling cascade, like LAT and SLP-76, has been found in mice and human pro/pre B cells. These proteins are probably acting as functionally redundant signaling molecules to the pre-BCR components (28, 29) to ensure maximum efficiency of its signal transduction. In our study, in B-ALLs ZAP-70 and Syk were found phosphorylated at the Tyr³¹⁹ residue and at the Tyr³⁵² residue, respectively. In addition, LAT, P-LAT, and the Src-kinase Lck were also found in all the B-ALL cases analyzed. These findings point out that ZAP-70, when expressed, is functionally active in B-ALL cells. In this setting, our finding of pre-TCR/TCR signaling elements in pro/pre B-ALL cells is in agreement with the hypothesis of the usage of pre-TCR/TCR signaling elements by the pre-BCR (28).

Interestingly, ZAP-70 expression was observed in Burkitt/ALL cases with the t(8;14)(q24;q32) at the highest levels observed among B-ALLs and CLLs (7). In accordance to this, we and others recently described ZAP-70 expression in a variable percentage of Burkitt's lymphomas (41–43), suggesting that this gene could play a role in the pathogenesis of this disease. The expression of ZAP-70 found in Burkitt and in 50% of CLL cases (7) suggests that this gene is aberrantly expressed in mature B-cell–derived neoplasms, as we did not observe ZAP-70 expression in normal mature B cells. Increased expression of ZAP-70 in a mature neoplasm like CLL correlates with an adverse behavior of the disease (7). Further investigations are warranted to determine whether ZAP-70 expression in Burkitt's lymphoma is associated with particular clinical and biological features.

In conclusion, this study shows that ZAP-70 was expressed in normal human pro/pre B cells but not in normal mature B cells. ZAP-70 expression was also found in one half of the B-ALLs analyzed, probably reflecting the lineage origin of such cases. In addition, other elements of the pre-TCR/TCR signaling cascade were found to be both expressed and phosphorylated in B-ALLs. Finally, the absence of ZAP-70 in normal mature B cells suggests that the expression observed in mature B-cell–derived neoplasms with different cellular origin, such as Burkitt/ALL and CLL reflects an aberrant phenomenon. Further investigations focused on the mechanisms regulating ZAP-70 expression in mature B-cell neoplasms are warranted.