scholarly journals Repression of the Pontin (RUVBL1, TIP49) Gene By BCL6: Implications for the Pathogenesis of Human B and T Cell Lymphomas

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4821-4821
Author(s):  
Beverly W Baron ◽  
Rebecca Baron ◽  
Joseph M. Baron
Keyword(s):  
T Cell ◽  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Binding Site ◽  
Germinal Center ◽  
Master Regulator ◽  
Protein Levels ◽  
T Cell Lymphomas ◽  
Evolutionarily Conserved

Abstract The human BCL6 gene encodes a transcriptional repressor that is needed for germinal center B cell development and T follicular helper cell differentiation, and it is best known for its association with certain human lymphomas, especially the diffuse large B-cell type. We devised a cell system in which the BCL6 repressive effects are inhibited with the goal of detecting the consequently upregulated BCL6 target genes. In order to do this, we converted the BCL6 zinc fingers (BCL6ZF), which can bind DNA but lack repressive effects, into a transcriptional activator and used this construct to compete with endogenous BCL6 in BJAB cells (Burkitt lymphoma cell line) which express high levels of BCL6. We used subtractive hybridization methodology to amplify differentially expressed sequences and identified the Pontin gene (also called Pontin 52, RUVBL1, TIP49, NMP238) as a potential target of BCL6 repression. We confirmed Pontin as a BCL6 target. Northern blots prepared from BJAB cells that had been transfected with the BCL6 ZF construct or the vector in which it was cloned were hybridized with the cDNA fragment of Pontin obtained from cDNA subtraction and amplification. Scanning densitometry, used to normalize relative band intensity to β -actin, indicated that differential expression of Pontin as compared with the vector control was 2.6-fold (p < 0.02). We found an exact match to 7 (out of 8) bases described as a preferential BCL6 binding site in the promoter region of the Pontin gene. ChIP assays indicated that BCL6 was bound to this site, but the appropriate controls did not enrich DNA from this region. Transfection assays showed that the BCL6 protein represses transcription from the BCL6 consensus binding site in the Pontin promoter (Fig. 1, 6.8-fold as compared with the control, p < 0.0001). Transfections of siRNAs targeting the BCL6 protein in BJAB cells led to a significant increase in endogenous Pontin (Fig. 2, 2-fold, p < 0.02). Finally, immunohistochemistry performed on 21 randomly selected B and T cell lymphomas showed an inverse relationship between BCL6 and Pontin in a majority of the tumors. Pontin is a transcriptional cofactor with ATPase activity that is part of the AAA+ (ATPases Associated with diverse cellular Activities) superfamily which includes a large group of ring-shaped complexes that are involved in multiple cellular processes. The Pontin protein is expressed virtually ubiquitously, is evolutionarily conserved, and is expressed in both the cell nucleus and cytoplasm. The BCL6 protein, also expressed ubiquitously and evolutionarily conserved, is upregulated 3 to 34 times in the nucleus of lymph node germinal center B cells as compared with resting B cells. It has been called a "master regulator" of the germinal center. It is possible, judging from the many essential functions of Pontin, that it, too, is a kind of "master regulator." Further understanding of the relationship between BCL6 and Pontin may help to provide new insights concerning the initiation and progression of B and T cell lymphomas. Figure 1. Transfection assays: The BCL6 Protein represses transcription from the Pontin Promoter Figure 1. Transfection assays: The BCL6 Protein represses transcription from the Pontin Promoter Figure 2. Knockdown of BCL6 protein levels by siRNA increases Pontin protein expression Figure 2. Knockdown of BCL6 protein levels by siRNA increases Pontin protein expression Disclosures No relevant conflicts of interest to declare.

scholarly journals Paracrine Regulation of Germinal Center B Cell Adhesion through the c-Met–Hepatocyte Growth Factor/Scatter Factor Pathway

1997 ◽  
Vol 185 (12) ◽  
pp. 2121-2131 ◽  
Author(s):  
Robbert van der Voort ◽  
Taher E.I. Taher ◽  
Robert M.J. Keehnen ◽  
Lia Smit ◽  
Martijn Groenink ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
T Cell ◽  
B Cells ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Germinal Center ◽  
Scatter Factor ◽  
Hepatocyte Growth

T cell–dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell migration and interaction with FDC critically depend on integrin-mediated adhesion. To date, the physiological regulators of this adhesion were unkown. In the present report, we have identified the c-met–encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signaling pathway regulating B cell adhesion. We observed that c-Met is predominantly expressed on CD38+CD77+ tonsillar B cells localized in the dark zone of the GC (centroblasts). On tonsil B cells, ligation of CD40 by CD40-ligand, induces a transient strong upregulation of expression of the c-Met tyrosine kinase. Stimulation of c-Met with HGF/SF leads to receptor phosphorylation and, in addition, to enhanced integrin-mediated adhesion of B cells to both VCAM-1 and fibronectin. Importantly, the c-Met ligand HGF/SF is produced at high levels by tonsillar stromal cells thus providing signals for the regulation of adhesion and migration within the lymphoid microenvironment.

scholarly journals Germinal center B cell development has distinctly regulated stages completed by disengagement from T cell help

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Ting-ting Zhang ◽  
David G Gonzalez ◽  
Christine M Cote ◽  
Steven M Kerfoot ◽  
Shaoli Deng ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
B Cell Differentiation ◽  
B Cell Maturation ◽  
Germinal Center B Cell ◽  
T Cell Help ◽  
Dose Dependent

To reconcile conflicting reports on the role of CD40 signaling in germinal center (GC) formation, we examined the earliest stages of murine GC B cell differentiation. Peri-follicular GC precursors first expressed intermediate levels of BCL6 while co-expressing the transcription factors RelB and IRF4, the latter known to repress Bcl6 transcription. Transition of GC precursors to the BCL6hi follicular state was associated with cell division, although the number of required cell divisions was immunogen dose dependent. Potentiating T cell help or CD40 signaling in these GC precursors actively repressed GC B cell maturation and diverted their fate towards plasmablast differentiation, whereas depletion of CD4+ T cells promoted this initial transition. Thus while CD40 signaling in B cells is necessary to generate the immediate precursors of GC B cells, transition to the BCL6hi follicular state is promoted by a regional and transient diminution of T cell help.

T cell-intrinsic Interferon Regulatory Factor-1 expression suppresses differentiation of CD4 + T cell populations that support chronic gammaherpesvirus infection.

2021 ◽  
Author(s):  
C. N. Jondle ◽  
K. E. Johnson ◽  
W. P. Mboko ◽  
V. L. Tarakanova
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
T Cell Subsets ◽  
Viral Reservoir ◽  
B Cell Differentiation ◽  
Follicular Helper ◽  
Interferon Regulatory Factor 1 ◽  
Latent Viral Reservoir

Gammaherpesviruses are ubiquitous pathogens that establish life-long infection and are associated with B cell lymphomas. To establish chronic infection, these viruses usurp B cell differentiation and drive a robust germinal center response to expand the latent viral reservoir and gain access to memory B cells. Germinal center B cells, while important for the establishment of latent infection, are also thought to be the target of viral transformation. The host and viral factors that impact the gammaherpesvirus-driven germinal center response are not clearly defined. We showed that global expression of the antiviral and tumor-suppressor interferon regulatory factor 1 (IRF-1) selectively attenuates the murine gammaherpesvirus 68 (MHV68)-driven germinal center response and restricts expansion of the latent viral reservoir. In this study we found that T cell intrinsic IRF-1 expression recapitulates some aspects of antiviral state imposed by IRF-1 during chronic MHV68 infection, including attenuation of the germinal center response and viral latency in the spleen. We also discovered that global and T cell-intrinsic IRF-1 deficiency leads to unhindered rise of IL-17A-expressing and follicular helper T cell populations, two CD4 + T cell subsets that support chronic MHV68 infection. Thus, this study unveils a novel aspect of antiviral activity of IRF-1 by demonstrating IRF-1-mediated suppression of specific CD4 + T cell subsets that support chronic gammaherpesvirus infection. Importance Gammaherpesviruses infect over 95% of the adult population, last the lifetime of the host, and are associated with multiple cancers. These viruses usurp the germinal center response to establish lifelong infection in memory B cells. This manipulation of B cell differentiation by the virus is thought to contribute to lymphomagenesis, though exactly how the virus precipitates malignant transformation in vivo is unclear. IRF-1, a host transcription factor and a known tumor suppressor, restricts the MHV68-driven germinal center response in a B cell-extrinsic manner. We found that T cell intrinsic IRF-1 expression attenuates the MHV68-driven germinal center response by restricting the CD4 + T follicular helper population. Further, our study identified IRF-1 as a novel negative regulator of IL-17-driven immune responses, highlighting the multifaceted role of IRF-1 in gammaherpesvirus infection.

scholarly journals Potential anti-EBV effects associated with elevated interleukin-21 levels: a case report

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Kristian Assing ◽  
Christian Nielsen ◽  
Marianne Jakobsen ◽  
Charlotte B. Andersen ◽  
Kristin Skogstrand ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Formation ◽  
Memory B Cells ◽  
Memory B Cell

Abstract Background Germinal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4+ T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8+ T cells able to contain chronic virus infections. Case presentation We present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4+ T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8+ lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pTFH, p = 0.01), reduced frequencies of peripheral CD4+ Bcl-6+ T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pTFH formation, pTFH and CD4+ IL-21+ frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman’s rho: − 0.86, p < 0.001. Conclusions To the best of our knowledge, this is the first report of elevated CD4+ IL-21+ T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.

Current targeted therapies in lymphomas

2019 ◽  
Vol 76 (22) ◽  
pp. 1825-1834
Author(s):  
Clement Chung
Keyword(s):  
T Cell ◽  
B Cells ◽  
Adverse Effects ◽  
Cell Surface ◽  
Supportive Care ◽  
B Cell ◽  
Targeted Therapies ◽  
B Cell Lymphoma ◽  
Lymphocyte Antigen ◽  
T Cell Lymphomas

Abstract Purpose This article summarizes current targeted therapies that have received regulatory approval for the treatment of B- and T-cell lymphomas. Summary Over the last 20 years, new drug therapies for lymphomas of B cells and T cells have expanded considerably. Targeted therapies for B-cell lymphomas include: (1) monoclonal antibodies directed at the CD20 lymphocyte antigen, examples of which are rituximab, ofatumumab, and obinutuzumab; (2) gene transfer therapy, an example of which is chimeric antigen receptor–modified T-cell (CAR-T) therapy directed at the CD19 antigen expressed on the cell surface of both immature and mature B cells; and (3) small-molecule inhibitors (ibrutinib, acalabrutinib, copanlisib, duvelisib, and idelalisib) that target the B-cell receptor signaling pathway. Of note, brentuximab vedotin is an antibody–drug conjugate that targets CD30, another lymphocyte antigen expressed on the cell surface of both Hodgkin lymphoma (a variant of B-cell lymphoma) and some T-cell lymphomas. Although aberrant epigenetic signaling pathways are present in both B- and T-cell lymphomas, epigenetic inhibitors (examples include belinostat, vorinostat, and romidepsin) are currently approved by the Food and Drug Administration for T-cell lymphomas only. In addition, therapies that target the tumor microenvironment have been developed. Examples include mogamulizumab, bortezomib, lenalidomide, nivolumab, and pembrolizumab. In summary, the efficacy of these agents has led to the development of supportive care to mitigate adverse effects, due to the presence of on- or off-target toxicities. Conclusion The therapeutic landscape of lymphomas has continued to evolve. In turn, the efficacy of these agents has led to the development of supportive care to mitigate adverse effects, due to the presence of on- or off-target toxicities. Further opportunities are warranted to identify patients who are most likely to achieve durable response and reduce the risk of disease progression. Ongoing trials with current and investigational agents may further elucidate their place in therapy and therapeutic benefits.

scholarly journals Human Germinal Center CD4+CD57+T Cells Act Differently On B Cells Than Do Classical T-Helper Cells

1995 ◽  
Vol 4 (3) ◽  
pp. 189-197 ◽  
Author(s):  
Farida Bouzahzah ◽  
Alain Bosseloir ◽  
Ernst Heinen ◽  
Léon J. Simar
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Functional Study ◽  
Target Cells ◽  
Helper Cells ◽  
Ig Synthesis

We have isolated two subtypes of helper T cells from human tonsils: CD4+CD57+cells, mostly located in the germinal center (GC), and CD4+CD57-cells, distributed through the interfollicular areas but also present in the GC. In a functional study, we have compared the capacities of these T-cell subtypes to stimulate B cells in cocultures. In order to block T-cell proliferation while maintaining their activation level, we pretreated isolated T cells with mitomycin C prior to culture in the presence of B cells and added polyclonal activators such as PHA and Con A, combined or not with IL-2. Contrary to CD4+CD57-cells, CD4+CD57+cells did not markedly enhance B-cell proliferation. Even when sIgD-B cells typical of germinal center cells were tested, the CD4 CD57 cells had no significant effect. This is in accordance with the location of these cells: They mainly occupy the light zones of the GC where few B cells divide. Even when added to preactivated, actively proliferating cells, CD4+CD57+cells failed to modulate B-cell multiplication. On the supernatants of B-cell-T-cell cocultures, we examined by the ELISA technique the effect of T cells on Ig synthesis. Contrary to CD57-T cells, whose effect was strong, CD57+T cells weakly stimulated Ig synthesis. More IgM than IgG was generally found. Because CD57 antigen is a typical marker of natural killer cells, we tested the cytolytic activity of tonsillar CD4+CD57+cells on K562 target cells. Unlike NK cells, neither CD4+CD57+nor CD4+CD57-cells exhibit any cytotoxicity. Thus, germinal center CD4+CD57+cells are not cytolytic and do not strongly stimulate either B-cell proliferation or Ig secretion. CD4+CD57-cells, however, enhance B-cell proliferation and differentiation, thus acting like the classical helper cells of the T-dependent areas.

Dysregulation of Pax5 Activity Contributes to the Extinction of the B-Cell Phenotype in Reed-Sternberg Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 517-517 ◽  
Author(s):  
Graham P Collins ◽  
Jennifer C Paterson ◽  
Gillian E May ◽  
Rajeev Gupta ◽  
Teresa Marafioti ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Binding Site ◽  
Cell Lines ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Methylation Status ◽  
Cell Phenotype ◽  
Transfected Cells

Abstract Hodgkin/Reed-Sternberg cells (HRS cells) are thought to be derived from post-germinal centre B-cells and yet have down-regulated the B-cell phenotype. The B-cell transcription factor Pax5 is important in the maintenance of B-cell identity and we demonstrate that it is down-regulated in HRS cells lines and in HRS cells of the majority of primary classical Hodgkin Lymphoma (cHL) cases. Specifically, 3/30 cases were negative for Pax5, 16/30 were weakly positive, 10/30 cases were moderately positive and 1/30 showed Pax5 staining of equivalent intensity to infiltrating, polyclonal B-cells. In order to functionally test the relevance of a reduced Pax5 expression level, the cHL cell lines L428 and L1236 were stably transfected with Pax5 using a lentiviral transfection system. Transfection of L1236 resulted in up-regulation of CD79a protein expression. However, CD79a was not upregulated in L428 and expression of the Pax5 target genes Cd19 and Blnk was unaffected by Pax5 transfection in both cell lines. Chromatin immunoprecipitation demonstrated that Pax5 failed to bind the high affinity binding site within the Cd19 promoter in the cHL lines despite high levels of Pax5 expression, appropriately localised to the nucleus. Pax5 could, however, bind synthetic oligonucleotide corresponding to this site (as shown by electrophoretic mobility shift assays) raising the possibility that epigenetic modification in vivo may be responsible for the failure to bind DNA. Bisulphite genome sequencing confirmed that in cHL cell lines, the region surrounding the Pax5 binding site in the Cd19 promoter was extensively methylated. Moreover, histone modification analysis also demonstrated an absence of markers of accessible, active chromatin (di- and trimethylated H3K4) and an enrichment of a marker indicating closed, repressive chromatin (trimethylated H3K27). Within the Cd79a promoter, previous studies have implicated the methylation status of a single cytosine residue within the binding site for a Pax5-Ets1 complex to be an important determinant of activation of the Cd79a gene. Interestingly, this residue was shown to be largely methylated in L428 cells but largely unmethyated in L1236 cells, providing a likely mechanism for the differential activation of this gene by transfected Pax5 protein. To investigate whether the observed epigenetic changes were responsible for preventing Pax5 binding and activity at the Cd19 and Cd79a promoters, Pax5 transfected cHL cell lines were cultured in the presence of the demethylating agent 5-aza-2-deoxycytidine. Up-regulation of Cd19 and Cd79a expression was significantly greater in Pax5 transfected cells than in control transfected cells. To conclude: our data suggests that dysregulation of Pax5 activity (at the levels of protein expression and epigenetic modification of the Pax5 binding sites) is important in mediating the extinction of the B-cell programme in HRS cells.

scholarly journals Rapid Emergence of T Follicular Helper and Germinal Center B Cells Following Antiretroviral Therapy in Advanced HIV Disease

2021 ◽  
Vol 12 ◽  
Author(s):  
Chun-Shu Wong ◽  
Clarisa M. Buckner ◽  
Silvia Lucena Lage ◽  
Luxin Pei ◽  
Felipe L. Assis ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Antiretroviral Therapy ◽  
B Cell ◽  
Germinal Center ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Cd4 T Cell ◽  
Follicular Helper ◽  
Cell Counts

Low nadir CD4 T-cell counts in HIV+ patients are associated with high morbidity and mortality and lasting immune dysfunction, even after antiretroviral therapy (ART). The early events of immune recovery of T cells and B cells in severely lymphopenic HIV+ patients have not been fully characterized. In a cohort of lymphopenic (CD4 T-cell count &lt; 100/µL) HIV+ patients, we studied mononuclear cells isolated from peripheral blood (PB) and lymph nodes (LN) pre-ART (n = 40) and 6-8 weeks post-ART (n = 30) with evaluation of cellular immunophenotypes; histology on LN sections; functionality of circulating T follicular helper (cTfh) cells; transcriptional and B-cell receptor profile on unfractionated LN and PB samples; and plasma biomarker measurements. A group of 19 healthy controls (HC, n = 19) was used as a comparator. T-cell and B-cell lymphopenia was present in PB pre-ART in HIV+ patients. CD4:CD8 and CD4 T- and B-cell PB subsets partly normalized compared to HC post-ART as viral load decreased. Strikingly in LN, ART led to a rapid decrease in interferon signaling pathways and an increase in Tfh, germinal center and IgD-CD27- B cells, consistent with histological findings of post-ART follicular hyperplasia. However, there was evidence of cTfh cells with decreased helper capacity and of limited B-cell receptor diversification post-ART. In conclusion, we found early signs of immune reconstitution, evidenced by a surge in LN germinal center cells, albeit limited in functionality, in HIV+ patients who initiate ART late in disease.

scholarly journals Bcl-x rather than Bcl-2 mediates CD40-dependent centrocyte survival in the germinal center

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1359-1364 ◽  
Author(s):  
JM Tuscano ◽  
KM Druey ◽  
A Riva ◽  
J Pena ◽  
CB Thompson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Protein Levels ◽  
Staphylococcus Aureus Cowan ◽  
A Cell ◽  
Tonsil Tissue ◽  
Cell Fractions ◽  
Selection Of

Both rapid B-cell proliferation and programmed cell death (PCD) occur during the differentiation and selection of B cells within the germinal center. To help elucidate the role of Bcl-x in B-cell antigen selection and PCD within the germinal center, we examined its expression in defined B-cell populations and by immunochemistry of tonsil tissue. Purified B-cell fractions enriched for centrocytes express high amounts of Bcl-x and relatively low amounts of Bcl-2, whereas fractions enriched for centroblasts lack significant levels of both proteins. Consistent with this observation, immunocytochemistry localized Bcl-x within cells scattered throughout the germinal center. Stimulation of tonsil B cells with either CD40 or Staphylococcus aureus Cowan increase bcl-x mRNA and protein levels. Treatment of a cell line with a germinal center phenotype (RAMOS) or the tonsillar B-cell centroblast fraction with CD40 rapidly increased Bcl-x levels and partially rescued B cells from PCD. These data suggest that Bcl-x rather than Bcl-2 may rescue centrocytes during selection in the germinal center.

Is Zap-70 Protein Detection by Flow Cytometry a Reliable Tool in CLL Prognostic Evaluation?.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4771-4771
Author(s):  
Magali Le Garff-Tavernier ◽  
Michel Ticchioni ◽  
Rémi Letestu ◽  
Martine Brissard ◽  
Frédéric Davi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Flow Cytometric Analysis ◽  
Protein Detection ◽  
Flow Cytometric ◽  
Mutational Status ◽  
Healthy Donors

Abstract Background : Expression of ZAP-70 protein has been shown to be correlated with mutational status of immunoglobulin heavy-chain variable region (IgVH) genes, a major prognostic factor in CLL. We investigated whether the detection of ZAP-70 protein by flow cytometric analysis using unconjugated and conjugated monoclonal antibodies (mAbs) could be applied securely in the workup of patients with CLL. Methods: Flow cytometric analysis of ZAP-70 protein was performed using the method described by Crespo et al (N Engl J Med2003;348:1764) with minor modifications. Both fresh and cryopreserved mononuclear cells from CLL patients and healthy donors were fixed and permeabilized using Fix and Perm kit (Caltag Laboratories), incubated with anti-ZAP-70 mAb (clone 2F3.2, Upstate Biotechnology) and then revealed with goat antimouse FITC mAb (Immunotech). Finally cells were incubated with CD3-APC, CD56-APC and CD19-PC5. We also tested 3 mAbs conjugated to various fluorochromes: 2F3.2-FITC (Upstate), 1E7.2-PE (eBioscience), 1E7.2-PE or -Alexa 488 (Caltag). ZAP-70 protein detection in B-cells was expressed either as a percentage of its expression in the T and NK-cells or as a ratio (R) of T-cell mean cell fluorescence (MCF) to B-cell MCF. Western blotting of protein lysates from purified B-cells was carried out to control results obtained by cytometry in 55 samples. Mutational status was defined using a cutoff of 98%. Results: In 13 healthy donors, the mean percentage of ZAP-70 protein expression obtained by flow cytometry with unconjugated mAb (clone 2F3.2) was 4.69% ± 1.93 [range 2–9%] and the R ratio was 6.64 ± 1.54 and &gt; 4.8. In 83 B-CLL samples, ZAP-70 expression was determined using the same method and compared to IgVH mutational status. Results in table below show a 75% concordance between gene mutations and ZAP-70 expression when considering a percentage of positive B-cells &gt; 20%. A better concordance (81%) is obtained with a threshold T-cell MCF/ B-cell MCF at 4 determined by Youden’s index. To note the high concordance (90%) between unmutated status and ZAP-70 + expression (19/21). Comparison with at least 1 of the 3 conjugated mAbs has been performed for 63 samples, with discordant results in our laboratories. 62 mutated IgVH samples 21 unmutated IgVH samples ZAP-70 B-Cells + ≤ 20 % : 43 &gt; 20% : 19 T-cell MCF/B-cell MCF ≥ 4 : 48 &lt; 4 : 19 Conclusions: Our data document the concordance between IgVH gene mutational status and ZAP-70 protein expression measured by flow cytometry, particularly in ZAP-70 negative samples. We found that the indirect method of labelling with unconjugated anti-ZAP-70 mAb remains until now, in our hands, the gold standard method compared to the available dyes conjugate mAbs.

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