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 > 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 > 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 > 20% : 19 T-cell MCF/B-cell MCF ≥ 4 : 48 < 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.

Analysis of PRAME Protein Expression in Normal Lymphoid Tissues and during B Ontogenesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3924-3924
Author(s):  
Lorena L. de Figueiredo-Pontes ◽  
Fabio M. do Nascimento ◽  
Rodrigo S. de Abreu e Lima ◽  
Rodrigo Proto-Siqueira ◽  
Aglair B. Garcia ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Mononuclear Cells ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Healthy Donors ◽  
Chronic Lymphoproliferative Diseases

Abstract PRAME (Preferentially Expressed Antigen in Melanoma) gene was originally isolated in melanoma. A significant increase in the number of PRAME transcripts has been demonstrated in hematologic malignancies such as acute myeloid and lymphoid leukemias, multiple myeloma and chronic lymphoproliferative diseases. Furthermore, our group generated an anti-PRAME monoclonal antibody (MoAb) and by quantitative flow cytometry has demonstrated that PRAME protein was aberrantly expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma. However, the expression of this antigen in normal lymphoid tissues and during B cells ontogeneis has not been characterized. To address this question, PRAME protein expression was studied by flow cytometry in peripheral blood (PB, n=15) and bone marrow (BM, n=6) from healthy donors, lymphonodes (n=4) and spleen (n=4) from patients submitted to lymphonode excision or splenectomy for non malignant diseases. First, we determined in which hematopoietic lineage PRAME was expressed by concomitantly staining PB, BM, lymphonode and spleen mononuclear cells (MCs) with anti-PRAME and a panel of MoAbs specific to B(CD19)/ T(CD3)/ NK(CD16/56), monocytic(CD14) and granulocytic(CD33) markers. PRAME was detected exclusively in CD19+ cells. The median percenatge of PRAME positive cells was 5,31% (2,55–12,34%), 13,01% (8,47–38,15%), 12,79% (3,15–23,06%) and 17,5% (12,67–27,43%) in PB, BM, lymphonode and spleen MCs, respectively. Amongst CD19+ cells, we have observed that PRAME was expressed by 42,39% (16,16–75,72%), 16% (13–69,5%), 15,16% (5,49–41,20%) and 48,82%(12,67–58,89%) in PB, BM, lymphonode and spleen, respectively. To establish in which stage of B ontogenesis PRAME was expressed on, cell suspensions stained with anti-CD19 were submitted to positive magnetic separation and labeled with anti-PRAME, CD5, CD27, CD38, CD34, CD10 and IgD MoAbs. PRAME+/CD19+ cells were CD5−, CD27+, CD38+, CD34−, CD10− and IgD+, thus suggesting that PRAME is expressed by the memory B cell compartment of the normal lymphoid tissues. This study defines PRAME as a B cell antigen that may accompany the neoplastic clone proliferation of mature B cell neoplasms. Although PRAME is mainly an embryonic antigen, expressed by carcinomas of immature phenotype, it is expressed by mature B cells in normal and pathological lymphoid tissues. Our findings suggest that maturational events occurring at the germinal center of lymphoid follicles affects PRAME expression.

scholarly journals POS0387 ACPA STATUS CORRELATES WITH DIFFERENTIAL IMMUNE PROFILE OF RHEUMATOID ARTHRITIS PATIENTS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 423.2-424
Author(s):  
A. Floudas ◽  
M. Canavan ◽  
T. McGarry ◽  
V. Krishna ◽  
S. Nagpal ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Synovial Tissue ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Algorithm Analysis ◽  
Treat To Target ◽  
Tnf Α ◽  
Acpa Status

Background:Rheumatoid arthritis (RA) is a progressive erosive autoimmune disease that affects 1% of the world population. Anti-citrullinated protein autoantibodies (ACPA) are routinely used for the diagnosis of RA, however 20-30% of patients are ACPA negative. ACPA status is a delineator of RA disease endotypes with similar clinical manifestation but potentially different pathophysiology. Elucidating the underlying mechanisms of disease pathogenesis could inform a treat to target approach for both ACPA-positive and ACPA-negative RA patients.Objectives:To identify peripheral blood and synovial tissue immune population differences that associate with RA disease endotype.To identify unique RA patient synovial tissue gene signatures and enriched pathways that correlate with ACPA status.Methods:Detailed high dimensionality flow cytometric analysis with supervised and unsupervised algorithm analysis of ACPApos and ACPAneg RA patient peripheral blood and synovial tissue single cell suspensions. Ex vivo peripheral blood and synovial tissue T cell stimulation and cytokine production characterisation. RNAseq analysis with specific pathway enrichment analysis of APCApos and ACPAneg RA patient synovial tissue biopsies.Results:Detailed profiling based on high dimensionality flow cytometric analysis of key peripheral blood and synovial tissue immune populations including B cells, T follicular helper (Tfh) cells, T peripheral helper cells (Tph) and CD4 T cell proinflammatory cytokine responses with supervised and unsupervised algorithm analysis revealed unique RA patient peripheral blood B cell and Tfh cell profiles. ACPApos RA patients were characterised by significantly (*P=0.03) increased frequency of Tfh (CXCR5+CD4+) cells and distinct clustering influenced by increased switched (IgD-CD27+) and DN (IgD-CD27-) memory B cells compared to APCAneg RA patients. Surprisingly synovial tissue B cell subpopulation distribution was similar between ACPAneg and ACPApos RA patients, with significant accumulation of switched and double negative memory B cells, highlighting a key role for specific B cell subsets in both disease endotypes. Interestingly, synovial tissue CD4 T cell proinflammatory cytokine (TNF-α, IFN-γ, IL-2, GM-CSF, IL-17A, IL-22, IL-4) production was markedly different between ACPAneg and APCApos RA patients with hierarchical clustering and PCA analysis revealing endotype specific cytokine profiles with ACPAneg RA patient synovial T cells showing increased TNF-α (P=0.01) expression. RNAseq analysis of RA patient synovial tissue revealed significant disease endotype specific gene signatures with specific enrichment for B cell receptor signalling and T cell specific pathways in ACPApos compared to ACPAneg RA patients. Additionally, significantly different chemokine receptor expression based on RA patient ACPA status was observed with increased CXCR3 (P<0.001), CCR7 (P=0.002), and CCR2 (P=0.004) but decreased CXCR7 (P=0.007) expression in APCApos compared to ACPAneg RA patient synovial biopsies.Conclusion:ACPA status associates with unique synovial tissue immune cell and gene profile signatures highlighting differences in the underlying immunological mechanisms involved, therefore reinforcing the need for a treat to target approach for both endotypes of RA.Figure 1.RNAseq analysis of synovial tissue biopsies revealed specific T cell related pathway enrichment in ACPA positive compared to ACPA negative RA patients (n=50, analysis performed with the DESq2 and pathfindeR pipelines in R).Disclosure of Interests:Achilleas Floudas: None declared, Mary Canavan: None declared, Trudy McGarry Employee of: Novartis, Vinod Krishna Employee of: Janssen, Sunil Nagpal Employee of: Janssen, GSK, Douglas Veale Speakers bureau: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Consultant of: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Grant/research support from: Janssen, Abbvie, Pfizer, UCB, Ursula Fearon Speakers bureau: Abbvie, Grant/research support from: Janssen, Abbvie, Pfizer, UCB

AB0144 STRATIFICATION OF PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME BY MEASURING B-CELL HEALTH

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1372-1373
Author(s):  
G. M. Verstappen ◽  
J. C. Tempany ◽  
H. Cheon ◽  
A. Farchione ◽  
S. Downie-Doyle ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Cell Differentiation ◽  
Research Support ◽  
Differentiation Capacity ◽  
Cell Responses ◽  
Healthy Donors

Background:Primary Sjögren’s syndrome (pSS) is a heterogeneous immune disorder with broad clinical phenotypes that can arise from a large number of genetic, hormonal, and environmental causes. B-cell hyperactivity is considered to be a pathogenic hallmark of pSS. However, whether B-cell hyperactivity in pSS patients is a result of polygenic, B cell-intrinsic factors, extrinsic factors, or both, is unclear. Despite controversies about the efficacy of rituximab, new B-cell targeting therapies are under investigation with promising early results. However, for such therapies to be successful, the etiology of B-cell hyperactivity in pSS needs to be clarified at the individual patient level.Objectives:To measure naïve B-cell function in pSS patients and healthy donors using quantitative immunology.Methods:We have developed standardised, quantitative functional assays of B-cell responses that measure division, death, differentiation and isotype switching, to reveal the innate programming of B cells in response to T-independent and dependent stimuli. This novel pipeline to measure B-cell health was developed to reveal the sum total of polygenic defects and underlying B-cell dysfunction at an individual level. For the current study, 25 pSS patients, fulfilling 2016 ACR-EULAR criteria, and 15 age-and gender-matched healthy donors were recruited. Standardized quantitative assays were used to directly measure B cell division, death and differentiation in response to T cell-independent (anti-Ig + CpG) and T-cell dependent (CD40L + IL-21) stimuli. Naïve B cells (IgD+CD27-) were sorted from peripheral blood mononuclear cells and were labeled with Cell Trace Violet at day 0 to track cell division until day 6. B cell differentiation was measured at day 5.Results:Application of our standardized assays, and accompanying parametric models, allowed us to study B cell-intrinsic defects in pSS patients to a range of stimuli. Strikingly, we demonstrated a hyperresponse of naïve B cells to combined B cell receptor (BCR) and Toll-like receptor (TLR)-9 stimulation in pSS patients. This hyperresponse was revealed by an increased mean division number (MDN) at day 5 in pSS patients compared with healthy donors (p=0.021). A higher MDN in pSS patients was observed at the cohort level and was likely attributed to an increased division burst (division destiny) time. The MDN upon BCR/TLR-9 stimulation correlated with serum IgG levels (rs=0.52; p=0.011). No difference in MDN of naïve B cells after T cell-dependent stimulation was observed between pSS patients and healthy donors. B cell differentiation capacity (e.g., plasmablast formation and isotype switching) after T cell-dependent stimulation was also assessed. At the cohort level, no difference in differentiation capacity between groups was observed, although some pSS patients showed higher plasmablast frequencies than healthy donors.Conclusion:Here, we demonstrate defects in B-cell responses both at the cohort level, as well as individual signatures of defective responses. Personalized profiles of B cell health in pSS patients reveal a group of hyperresponsive patients, specifically to combined BCR/TLR stimulation. These patients may benefit most from B-cell targeted therapies. Future studies will address whether profiles of B cell health might serve additional roles, such as prediction of disease trajectories, and thus accelerate early intervention and access to precision therapies.Disclosure of Interests:Gwenny M. Verstappen: None declared, Jessica Catherine Tempany: None declared, HoChan Cheon: None declared, Anthony Farchione: None declared, Sarah Downie-Doyle: None declared, Maureen Rischmueller Consultant of: Abbvie, Bristol-Meyer-Squibb, Celgene, Glaxo Smith Kline, Hospira, Janssen Cilag, MSD, Novartis, Pfizer, Roche, Sanofi, UCB, Ken R. Duffy: None declared, Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant of: Consultant for Bristol-Myers Squibb, Speakers bureau: Speaker for Bristol-Myers Squibb, Roche and Janssen-Cilag, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant of: Consultant for Bristol-Myers Squibb, Roche, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Speaker for Bristol-Myers Squibb and Novartis., Philip D. Hodgkin Grant/research support from: Medimmune, Vanessa L. Bryant Grant/research support from: CSL

Roles of Endogenous IL-10 and IL-10-Competent and CD5+ B Cells in Autoimmune Thyroiditis in NOD.H-2h4 Mice

Endocrinology ◽  
2020 ◽  
Vol 161 (4) ◽  
Author(s):  
Jing Qin ◽  
Na Zhao ◽  
Shuo Wang ◽  
Shanshan Liu ◽  
Yongping Liu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Autoimmune Thyroiditis ◽  
Flow Cytometric Analysis ◽  
Cell Group ◽  
Autoantibody Production ◽  
Flow Cytometric ◽  
Cell Groups ◽  
Regulatory Capacity ◽  
Anti Inflammatory Cytokine

Abstract Interleukin (IL)-10 is a highly important anti-inflammatory cytokine in the immune system. CD1dhi and CD5+ B cells are both traditionally defined IL-10-secreting B cells. In recent years, a B cell group with combined markers of CD1dhi and CD5+ has been widely studied as it has been reported to suppress autoimmunity in mouse models of autoimmune diseases through IL-10 mechanisms. From the perspective of origination, CD1dhi and CD5+ B cells are developed from different B cell lineages. Whether the regulatory capacity of these 2 B cell groups is consistent with their ability to secrete IL-10 has not been determined. In this study, we generated IL-10 knockout NOD.H-2h4 mice to investigate the function of endogenous IL-10 in autoimmune thyroiditis and conducted adoptive transfer experiments to explore the respective roles of CD5+ and CD1dhi B cells. In our results, the IL-10–/– NOD.H-2h4 mice developed thyroiditis, similar to wild-type NOD.H-2h4 mice. The CD5+ B cells were more capable of secreting IL-10 than CD1dhi B cells in flow cytometric analysis, but the CD1dhi B cells showed more suppressive effects on thyroiditis development and autoantibody production, as well as Th17 cell response. In conclusion, endogenous IL-10 does not play an important role in autoimmune thyroiditis. CD1dhi B cells may play regulatory roles through mechanisms other than secreting IL-10.

Immune Defects of B- and T-Cell Compartments in Natalizumab-Induced Progressive Multifocal Leukoencephalopathy,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3228-3228
Author(s):  
Alessandra Sottini ◽  
Ruggero Capra ◽  
Cinzia Zanotti ◽  
Marco Chiarini ◽  
Federico Serana ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
B Cells ◽  
Progressive Multifocal Leukoencephalopathy ◽  
B Cell ◽  
B Lymphocyte ◽  
Jc Virus ◽  
Excision Circles ◽  
The Moment

Abstract Abstract 3228 Objective: Progressive multifocal leukoencephalopathy (PML) is a rare, but often fatal demyelinating brain disease caused by the JC virus, which usually occurs in immunosuppressed patients, including those with hematological malignancies or receiving monoclonal antibodies-based immunotherapies. PML is likely the result of a complex combination of several pathogenic mechanisms, such as alterations of peripheral cell-mediated immunity and mobilization of JC virus-carrying CD34+-hematopoetic stem cells and pre-B-cells. Taking advantage of the availability of samples from a multiple sclerosis (MS) patient treated with the anti-α4β1 monoclonal antibody natalizumab who developed PML, which was monitored for 35 months since before therapy initiation, we investigated the role of B and T lymphocytes in PML onset. Methods: Real-Time PCR was used to measure the release of T and B cells from the production sites by means of T-cell receptor excision circles (TRECs) and K-deleting excision circles (KRECs) analysis and to quantify transcripts for immature hematopoietic cells such as terminal deoxynucleotidyl transferase, CD34, and pre-B lymphocyte gene 1. Naïve and mature T- and B-cell subsets were identified by flow cytometry, T-cell heterogeneity was quantified by spectratyping and IgA, IgG and IgM by turbidimetric assay. Data were compared to those of untreated and natalizumab-treated MS patients and healthy donors. Results: After 34 months of natalizumab therapy, a 42 years old female developed PML, diagnosed on the basis of magnetic resonance imaging and JC virus positivity in cerebrospinal fluid. Before therapy, her thymus and bone marrow produced a significant low number of TRECs+ and KRECs+ cells. While TRECs remained low during all therapy period, KRECs and transcripts for pre-B lymphocyte gene 1, which is selectively expressed in pre-B cells, peaked after 6 months of therapy, remained high at 12 and 15 months of treatment, and then decreased at the moment of PML onset. Flow cytometry confirmed a deficient production of CD4+CD45RA+CCR7+CD31+ recent T emigrants, counterbalanced by an increased number of CD8+CCR7–CD45RA+ TEMRA cells for all observation period, but showed a modification of peripheral CD4 and CD8 cell number only at the moment of PML. While the percentage of naïve B cells increased by about 70% after 6 months of therapy, the number of B lymphocytes within each B-cell subpopulations remained low for the entire treatment period. T-cell repertoire and immunoglobulin production were altered. Interpretation: Although performed in a single patient, data all agree in demonstrating that a deficient production of new TRECs+ T lymphocytes, together with an increase of newly produced KRECs+ B cells just few months after therapy beginning may predispose to PML. These findings encourage further researches on the utility of the TRECs/KRECs assay as a potential tool for the identification of patients at risk of developing PML after monoclonal antibodies-based therapies. Disclosures: No relevant conflicts of interest to declare.

IL-21 and IL-6 Mediate Interactions Between T Cells and Malignant B Cells in the Bone Marrow Microenvironment in Waldenstrom's Macroglobulinemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1554-1554
Author(s):  
Lucy S. Hodge ◽  
Steve Ziesmer ◽  
Frank J Secreto ◽  
Zhi-Zhang Yang ◽  
Anne Novak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Tumor Microenvironment ◽  
B Cell ◽  
Bone Marrow Microenvironment ◽  
Waldenström’S Macroglobulinemia ◽  
Waldenström's Macroglobulinemia

Abstract Abstract 1554 T cells in the tumor microenvironment influence the biology of malignant cells in many hematologic malignancies, often through cytokine-mediated interactions. Recent studies involving healthy B cells and CD4+T cells identified an interplay between IL-6 and IL-21, whereby IL-6 increased IL-21 production by T cells, driving the differentiation and IL-6 secretion of nearby B cells. In addition to their known effects on healthy B cell function, IL-6 and IL-21 have also been implicated in the pathology of various lymphomas. In Waldenstrom's macroglobulinemia (WM), IL-6 is elevated in the bone marrow and is associated with increased IgM production. However, the function of IL-21 in the WM tumor microenvironment and its relationship to IL-6 is poorly understood. Our objective in this study was to characterize IL-21 production and function in WM and to examine the role of IL-6 and IL-21 in regulating interactions between malignant B cells and T cells in the tumor microenvironment. Immunohistochemistry revealed significant IL-21 staining in bone marrows of patients with WM (n=5), but the areas of infiltration by WM in the bone marrow sections appeared negative for IL-21 staining. To better understand the origin of IL-21 in in the tumor microenvironment, IL-21 expression was assessed by PCR in the CD19−CD138− fraction of cells remaining in patient bone marrow aspirates after positive selection for malignant B cells (n=5). IL-21 transcript was detected in 4/5 samples. CD19−CD138− cells activated with anti-CD3 and anti-CD28 antibodies expressed higher levels of IL-21 transcript and secreted significantly higher levels of IL-21 protein compared to unstimulated cells, suggesting that IL-21 in the WM bone marrow is derived from activated T cells. Intracellular expression of IL-21 protein was confirmed in CD4+ and CD8+ cells within the CD19−CD138− population using flow cytometry. Furthermore, dual staining of WM bone marrow sections with antibodies against IL-21 and CD3 or CD20 revealed co-staining of IL-21 with CD3+ T cells but not with CD20+ B cells. The response of WM B cells to T-cell derived IL-21 was then assessed in positively selected CD19+CD138+ WM B cells (n=5) and in the MWCL-1 cell line. Using flow cytometry, both the IL-21 receptor and the required common gamma chain subunit were detected on all patient samples as well as on MWCL-1 cells. Treatment of MWCL-1 cells with IL-21 (100 ng/mL) for 72 h increased proliferation by 35% (p<0.05) and IgM secretion by 80% (p<0.005). Similarly, in primary CD19+CD138+ WM cells (n=5), proliferation increased on average by 38% and IgM secretion by 71%. No apoptotic effects were associated with IL-21 in WM. Characterization of STAT activation in response to IL-21 revealed significant phosphorylation of STAT3 in both CD19+CD138+ WM cells and MWCL-1 cells and was associated with increases in BLIMP-1 and XBP-1 protein and decreases in PAX5. As STAT3 activation is known to regulate IL-6, we assessed the effect of IL-21 on B cell-mediated IL-6 secretion using ELISA. IL-21 significantly increased IL-6 secretion by both primary CD19+CD138+ WM cells (n=4) and MWCL-1 cells (87.9 +/− 10.9 ng/mL vs. 297.8 +/− 129.2 ng/mL, p<0.05). Treatment with IL-6 and IL-21 together had no additional effect over IL-21 alone on proliferation or IgM secretion in MWCL-1 cells, but culturing anti-CD3/anti-CD28-activated CD19−CD138−cells from WM bone marrows with IL-6 significantly increased IL-21 secretion (n=3). Overall, these data indicate that T-cell derived IL-21 significantly promotes growth and immunoglobulin production by malignant WM B cells and that subsequent IL-6 secretion by malignant B cells may enhance the secretion of IL-21 by T cells within the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Flow Cytometric Analysis Revealed a Significant Accumulation of Terminally Differentiated T cell Subtypes in the Circulating Lymphocytes of Cytomegalovirus (CMV) Positive Follicular Lymphoma Patients

2021 ◽  
pp. 1-9
Author(s):  
Lugos MD ◽  
◽  
Dangana A ◽  
Ntuhun BD ◽  
Oluwatayo BO ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Flow Cytometric Analysis ◽  
B Cell Lymphoma ◽  
T Cell Subsets ◽  
Cmv Infection ◽  
Flow Cytometric ◽  
The Impact

Follicular lymphoma (FL), a non-Hodgkin lymphoma, is an indolent cancer of the B cell lineage that runs a chronic deterioration course that can result in multiple treatment episodes leading to resistance and possible transformation to diffuse large B cell lymphoma. Cytomegalovirus (CMV) reactivation during chemotherapy or after an organ or hematopoietic stem cell transplantation is a major cause of morbidity and mortality. This study tests the hypothesis that some of the heterogeneity of FL might result from chronic infection with Cytomegalovirus (CMV). This research was intended to appraise the impact of CMV infection on the subtypes of T cells in follicular lymphoma patients. We accessed stored peripheral blood mononuclear cells (PMBCs) from patients of known CMV serostatus recruited into an FL clinical trial. We undertook a multicolour flow cytometric analysis of the PBMCs and compared the number of lymphocyte subtypes of CMV-positive and CMV-negative FL patients. Data showed a significant increase in the quantity of terminally differentiated (TEMRA) T cell subsets, including EM3-CD8 (P=0.005), EM3-CD4 (P=0.018), E-CD4 (P=0.029), E-CD8 (P=0.033) and pE2-CD4 (P=0.046) phenotypes, as well as increased NKT cells (P=0.031) among CMV-positive patients compared to the negative group. Our findings support the hypothesis that recurrent infections characterise CMV infection in FL due to accelerated immune senescence and the accumulation of exhausted T cells. Based on the data, a case could be argued for the routine application of CMV screening in FL before treatment with chemo-immunotherapy to implement enhanced infection surveillance in CMV-positive patients. These discoveries can eventually help improve the treatment approaches in the management of FL toward a combinatorial viewpoint for direct cytotoxic and indirect immunomodulatory outlook

scholarly journals Flow Cytometric Detection of the Double-Positive (CD4+CD8+)/PD-1bright T-Cell Subset Is Useful in Diagnosing Nodular Lymphocyte-Predominant Hodgkin Lymphoma

2021 ◽  
Author(s):  
Zhongchuan Will Chen ◽  
Juanita Wizniak ◽  
Chuquan Shang ◽  
Raymond Lai
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
B Cell Lymphoma ◽  
The Other ◽  
Non Hodgkin Lymphoma ◽  
Flow Cytometric ◽  
Large B Cell

Context.— Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is characterized by neoplastic lymphocyte-predominant cells frequently rimmed by CD3+/CD57+/programmed death receptor-1 (PD-1)+ T cells. Because of the rarity of lymphocyte-predominant cells in most cases, flow cytometric studies on NLPHL often fail to show evidence of malignancy. Objective.— To evaluate the diagnostic utility of PD-1 in detecting NLPHL by flow cytometry, in conjunction with the CD4:CD8 ratio and the percentage of T cells doubly positive for CD4 and CD8. Design.— Flow cytometric data obtained from cases of NLPHL (n = 10), classical Hodgkin lymphoma (n = 20), B-cell non-Hodgkin lymphoma (n = 22), T-cell non-Hodgkin lymphoma (n = 5), benign lymphoid lesions (n = 20), angioimmunoblastic T-cell lymphomas (n = 6) and T-cell/histiocyte–rich large B-cell lymphomas (n = 2) were analyzed and compared. Results.— Compared with the other groups, NLPHL showed significantly higher values in the following parameters: CD4:CD8 ratio, percentage of T cells doubly positive for CD4 and CD8, percentage of PD-1–positive T cells, and median fluorescence intensity of PD-1 expression in the doubly positive for CD4 and CD8 subset. Using a scoring system (0–4) based on arbitrary cutoffs for these 4 parameters, all 10 NLPHL cases scored 3 or higher, as compared with only 3 cases from the other groups, producing an overall sensitivity of 100% and a specificity of 96% (72 of 75). Two of the 3 outliers were non-Hodgkin lymphoma, and both showed definitive immunophenotypic abnormalities leading to the correct diagnosis. The remaining outlier was a case of T-cell/histiocyte–rich large B-cell lymphoma. Conclusions.— The inclusion of anti–PD-1 in flow cytometry is useful for detecting NLPHL in fresh tissue samples, most of which would have otherwise been labeled as nondiagnostic or reactive lymphoid processes.

scholarly journals Circulating Exosomes Inhibit B Cell Proliferation and Activity

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2110
Author(s):  
Jan C. Schroeder ◽  
Lisa Puntigam ◽  
Linda Hofmann ◽  
Sandra S. Jeske ◽  
Inga J. Beccard ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Surface Expression ◽  
Receptor Expression ◽  
Healthy Individuals ◽  
Inhibitory Effects ◽  
Healthy Donors ◽  
Checkpoint Receptors

(1) Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by a distinctive suppression of the anti-tumor immunity, both locally in the tumor microenvironment (TME) and the periphery. Tumor-derived exosomes mediate this immune suppression by directly suppressing T effector function and by inducing differentiation of regulatory T cells. However, little is known about the effects of exosomes on B cells. (2) Methods: Peripheral B cells from healthy donors and HNSCC patients were isolated and checkpoint receptor expression was analyzed by flow cytometry. Circulating exosomes were isolated from the plasma of HNSCC patients (n = 21) and healthy individuals (n = 10) by mini size-exclusion chromatography. B cells from healthy individuals were co-cultured with isolated exosomes for up to 4 days. Proliferation, viability, surface expression of checkpoint receptors, and intracellular signaling were analyzed in B cells by flow cytometry. (3) Results: Expression of the checkpoint receptors PD-1 and LAG3 was increased on B cells from HNSCC patients. The protein concentration of circulating exosomes was increased in HNSCC patients as compared to healthy donors. Both exosomes from healthy individuals and HNSCC patients inhibited B cell proliferation and survival, in vitro. Surface expression of inhibitory and stimulatory checkpoint receptors on B cells was modulated in co-culture with exosomes. In addition, an inhibitory effect of exosomes on B cell receptor (BCR) signaling was demonstrated in B cells. (4) Conclusions: Plasma-derived exosomes show inhibitory effects on the function of healthy B cells. Interestingly, these inhibitory effects are similar between exosomes from healthy individuals and HNSCC patients, suggesting a physiological B cell inhibitory role of circulating exosomes.

Flow Cytometric Analysis of Acute Leukemias

2003 ◽  
Vol 127 (1) ◽  
pp. 42-48 ◽  
Author(s):  
Zahid Kaleem ◽  
Eric Crawford ◽  
M. Hanif Pathan ◽  
Leah Jasper ◽  
Michael A. Covinsky ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
B Cell ◽  
Flow Cytometric Analysis ◽  
Multiparameter Flow Cytometry ◽  
Correct Identification ◽  
Acute Leukemias ◽  
Cytochemical Analysis ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—Acute leukemia displays characteristic patterns of surface antigen expression (CD antigens), which facilitate their identification and proper classification and hence play an important role in instituting proper treatment plans. In addition to enzyme cytochemical analysis, multiparameter flow cytometric analysis has become commonplace in most laboratories for that purpose. The essential role and caveats of flow cytometry in that regard, however, have received little scrutiny. Objective.—To evaluate the expression of commonly used immunomarkers and patterns in various acute leukemias to help define the best use and role of multiparameter flow cytometry in the diagnosis and proper classification of acute leukemias. Design.—We have retrospectively analyzed the immunophenotypic data from 508 de novo adult and pediatric acute leukemia patients, as studied using multiparameter flow cytometry in addition to routine morphologic and enzyme cytochemical analysis. Cytogenetic and/or molecular data were correlated in all 41 cases of acute promyelocytic leukemia (APL) and in 203 other cases of acute leukemia where those data were available. We have also determined the positive and negative predictive values of a combined CD34 and HLA-DR expression pattern for the differentiation of APL from other myeloid leukemias. Results.—In acute myeloid leukemia (AML) other than APL, expression of CD34 was seen in 62% and expression of HLA-DR in 86% of all cases. Twenty-six (10%) of 259 cases of non-APL AML were negative for both CD34 and HLA-DR as opposed to 33 (80%) of 41 cases of APL. None of the cases of APL were positive for both CD34 and HLA-DR in contrast to 149 (58%) of 259 cases of non-APL AML. Fifty-three cases were found to be examples of minimally differentiated AML (AML M0) based on the lack of expression of cytoplasmic CD3 and cytoplasmic CD79a and expression of one or more myelomonocytic-associated antigens and/or myeloperoxidase. Expression of CD20 was seen in 40 (24%) of 168 cases of precursor B-cell acute lymphoblastic leukemia (pB-ALL) and 52 (29%) lacked CD34 expression. Five of 180 cases of pB-ALL and 2 cases of precursor T-cell ALL (pT-ALL) were negative for terminal deoxynucleotidyl transferase (TdT). Aside from cytoplasmic CD3, CD5 and CD7 were the most sensitive antigens present in all 21 cases of pT-ALL. CD33 was more sensitive but less specific than CD13 for myeloid lineage. Conclusion.—Aside from identification of blasts, flow cytometry was found to be especially useful in the correct identification of AML M0, differentiation of APL from AML M1/M2, and correct identification of TdT-negative ALL and unusual variants, such as transitional B-cell ALL and undifferentiated and biphenotypic acute leukemias.

Sign in / Sign up

Export Citation Format

Share Document