scholarly journals An improved clonal excess assay using flow cytometry and B-cell gating

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1178-1185
Author(s):  
BW Letwin ◽  
PK Wallace ◽  
KA Muirhead ◽  
GL Hensler ◽  
WH Kashatus ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Fluorescence Intensity ◽  
Light Scatter ◽  
B Cell Malignancies ◽  
Clonal Proliferation ◽  
Minimal Disease ◽  
Low Levels

In humans with B-cell malignancies, the presence of monoclonal B lymphocytes (clonal proliferation) can be detected by comparing the fluorescence intensity distributions of lymphocytes stained with anti- kappa and anti-lambda reagents. The sensitivity of previously described single-color immunofluorescence techniques to low levels of clonal excess is limited by background from cytophilic immunoglobulins on non- B cells and by the low proportion of circulating B cells in individuals with minimal disease. We have used two-color immunofluorescence and B- cell gating to develop an improved assay that avoids false positives due to non-B cells, without requiring restrictive light scatter gates that may exclude true positives. This method is sensitive to 0.2% monoclonal B cells admixed with fresh normal lymphocytes, to 0.6% monoclonal B cells admixed with normal lymphocytes that have been stored for up to 72 hours, and readily detects 1% monoclonal cells in patient specimens. The two color B-cell gated assay offers sensitivity equivalent to the single-color assay and improved specificity for detection of low levels of clonal excess.

scholarly journals An improved clonal excess assay using flow cytometry and B-cell gating

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1178-1185 ◽  
Author(s):  
BW Letwin ◽  
PK Wallace ◽  
KA Muirhead ◽  
GL Hensler ◽  
WH Kashatus ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Fluorescence Intensity ◽  
Light Scatter ◽  
B Cell Malignancies ◽  
Clonal Proliferation ◽  
Minimal Disease ◽  
Low Levels

Abstract In humans with B-cell malignancies, the presence of monoclonal B lymphocytes (clonal proliferation) can be detected by comparing the fluorescence intensity distributions of lymphocytes stained with anti- kappa and anti-lambda reagents. The sensitivity of previously described single-color immunofluorescence techniques to low levels of clonal excess is limited by background from cytophilic immunoglobulins on non- B cells and by the low proportion of circulating B cells in individuals with minimal disease. We have used two-color immunofluorescence and B- cell gating to develop an improved assay that avoids false positives due to non-B cells, without requiring restrictive light scatter gates that may exclude true positives. This method is sensitive to 0.2% monoclonal B cells admixed with fresh normal lymphocytes, to 0.6% monoclonal B cells admixed with normal lymphocytes that have been stored for up to 72 hours, and readily detects 1% monoclonal cells in patient specimens. The two color B-cell gated assay offers sensitivity equivalent to the single-color assay and improved specificity for detection of low levels of clonal excess.

scholarly journals Expression of FAK and Its Involvement in the Progression of B-Cell Chronic Lymphocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3309-3309
Author(s):  
Cristina Gattazzo ◽  
Andrea Visentin ◽  
Alberto Pavan ◽  
Veronica Martini ◽  
Federica Frezzato ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocytes ◽  
Focal Adhesion ◽  
Poor Prognosis ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling ◽  
Cell Chronic Lymphocytic Leukemia

Abstract INTRODUCTION B-cell chronic lymphocytic leukemia (B-CLL) is a disorder characterized by the accumulation of clonal CD5+ B lymphocytes, due to uncontrolled growth and resistance to apoptosis. Although the prognosis and clinical outcome has dramatically improved by recent innovative therapies, B-CLL still remains an incurable disease. Since signaling events downstream the BCR engagement are important for the progression of B cells, BCR signaling has been investigated in B-CLL in order to design new agents to specifically treat this disease. We demonstrated that Lyn, one of the first kinases involved in BCR signaling pathway, is overexpressed, constitutively active and anomalously distributed in malignant B cells, as compared to normal B lymphocytes. The Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, is the primary enzyme involved in the engagement of integrins and assembly of Focal Adhesion. FAK is regulated primarily through tyrosine phosphorylation by Lyn after BCR engagement and was found to be overexpressed in many kinds of human cancers. However, a downmodulation of FAK expression and its association to poor prognosis have also been reported. The aim of this study was to investigate the role of FAK in CLL patients. METHODS Blood samples were collected from 5 controls and 50 B-CLL patients. Informed consent was obtained according to the Declaration of Helsinki. Untouched peripheral blood B cells were purified using the RosetteSep for human B cells isolation kit. The samples that were used had at least 95% of normal CD19+ or neoplastic CD5+/CD19+ cells, as assessed by flow-cytometry. Level of FAK protein was evaluated by Western blotting (Wb) and Flow Cytometry assay (FC). Levels of FAK were correlated to clinical parameters of patients. RESULTS We observed that FAK was downmodulated in 56% of analyzed patients with respect to healthy subjects (respectively, Wb: 0.28±0.25 vs 0.85±0.32, p<0.001; FC: 35%±29 vs 60%±16, p<0.05). We also identified that lower levels of FAK expression were related to the prognostic markers of poor outcome (the expression of ZAP70, CD38 and an unmutated-IGHV genes status, p<0.05) and to a shorter Treatment Free Survival (p<0.05). Moreover, patients (n=6) who had an indolent course and were responsive to the standard treatment, showed normal expression of this kinase already at diagnosis. In contrast, patients (n=6) with a more aggressive disease, had a lower expression of FAK, that was further downmodulated during the progression of disease, irrespective of how the patients were treated. CONCLUSIONS From the data presented in this report we propose that FAK downmodulation could be considered as a new marker of poor prognosis and as a putative predictor for high-risk subgroups of CLL, even in early-stage disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals B Cells of the Proliferative Fraction of CLL Clones Exhibit Activated B-Cell and Myeloid-Cell Signatures Suggesting Enhanced Antigen-Presentation, Integrin Responsiveness, and IL-4 Receptiveness: Additional Targets for CLL Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 302-302
Author(s):  
Xiao-Jie Yan ◽  
Florencia Palacios ◽  
Wentian Li ◽  
Sophia Yancopoulos ◽  
Carlo Calissano ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Antigen Presentation ◽  
B Lymphocytes ◽  
Research Funding ◽  
Myeloid Cell ◽  
Integrin Signaling ◽  
Gene Sets

Abstract CLL results from the accumulation of monoclonal B lymphocytes that derive from a small fraction of cells with proliferative activity. Because expression of the DNA mutator, activation-induced cytidine deaminase (AID) is restricted to these dividing cells, they can develop new DNA abnormalities leading to more lethal disease. Hence, such cells are important targets for therapy. Our previous study indicated that the B-cell subset with low levels of CXCR4 and high levels of CD5 (CXCR4DimCD5Bright) is enriched in these cells ("proliferative fraction", PF), whereas the less vital, resting cells exhibit a CXCR4BrightCD5Dim phenotype ("resting fraction", RF). In this study, we focused on analyzing the significantly differentially expressed genes (DEGs) between PF and RF. PF and RF were isolated from 26 CLL (13 U-CLL and 13 M-CLL) and microarrays (llumina HT12) were performed. Selected DEGs between PF and RF were confirmed by rtQ-PCR and/or by flow cytometry. Array data were interpreted using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). First, focusing on the Immunologic Signature of B lymphocytes with GSEA, we found the PF was enriched in gene sets shared with IgM memory cells and pre-germinal center B cells, whereas RF was enriched in gene sets in common with naïve, IgD+ B cells. Notably, the PF also shared gene sets with myeloid dendritic cells and monocytes. Protein expression of 10 myeloid markers (CD68, CD1c, CD11a, CD11b, CD11c, CD13, CD31, CD205, CXCR3 and CLECL1) was documented in PF B cells from 11 U-CLL and 11 M-CLL patients, and each was more highly expressed in the PF than RF. No difference in myeloid markers was observed between U-CLL and M-CLL, suggesting that this expression is independent of IGHV mutation status. DEGs were also determined based on expression ratios for PF and RF for each patient; t tests were performed using R. With a fold change cutoff of > 1.5 or < -1.5 and a level of significance of P < 0.01, we identified 198 genes significantly upregulated in PF and 88 in RF. The top biological-function categories identified using IPA indicated that these genes related to cellular development (n=37), cellular growth and proliferation (n=49), cellular movement (n=42) and cell survival and death (n=50). In addition, these DEGs mapped to 8 canonical pathways with Z-scores ≥2, the highest being the integrin signaling pathway (Z-score = 2.887). Twelve of 13 genes were upregulated in the PF and correlated significantly with the integrin pathway: ACTG, ARPC5, ARPC1B, BCAR3, CAPNS1, ITGAX, ITGB1, ITGB2, ITGB7, PFN1, RAC2 and RHOC. Upregulation of integrin subunits was confirmed by Q-PCR and cell surface staining by flow cytometry. Preliminary cellular adhesion experiments suggest PF bind to fibronectin coated plates, and those cells that bind survive better. Next, comparing PF and RF expression ratios for U-CLL vs. M-CLL revealed 502 DEGs in U-CLL and 179 in M-CLL; 144 genes were shared by both U-CLL and M-CLL. IPA analysis of the latter genes correlated best with integrin signaling, and the potential upstream regulators of these were IFNg, IL1, F2 and IFNa. The Rho Family GTPases signaling pathway was significant (10 genes) for DEGs unique to U-CLL. No significant pathway or bio-function relationship was observed in DEGs only in M-CLL. Finally, IPA analysis showed IL4 as an upstream regulator of DEGs unique to the PF of U-CLL based on upregulation of 12 IL4 target genes (APRT, CCL5, CDKN1A, DECR1, IL17RB, ITGAL, ITGB1, LTA, MAPKAPK3, PIGR, SAMSN1, TIMP1). Three other IL-4 targeted genes were paradoxically under-represented in the U-CLL PF (BCL2, CCR7 and VIPR1). The upregulated findings are consistent with PF B cells inducing T cells to produce IL-4 via co-receptors on B/myeloid cells that foster a Th2 response (e.g., CLECL1). In conclusion, gene expression profiling indicates that cells of the PF display a dual activated B cell/myeloid cell phenotype suggesting enhanced antigen-presentation capacities. Integrin signaling appears to be a key pathway for these cells which could foster cell proliferation, survival, and migration, especially in U-CLL clones where integrin activation can lead to Rho GTPases activation. Finally, genes regulated by IL-4 in the PF could be induced by interactions of autologous T cells with CLL B cells. These findings suggest antigen-presentation and integrin and IL-4 signaling pathways as therapeutic targets in CLL, particularly for U-CLL. Disclosures Barrientos: AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Janssen: Consultancy.

A Phase I Clinical Trial of Treatment of B-Cell Malignancies with Autologous Anti-CD19-CAR-Transduced T Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2865-2865 ◽  
Author(s):  
James N. Kochenderfer ◽  
Mark E. Dudley ◽  
Maryalice Stetler-Stevenson ◽  
Wyndham H. Wilson ◽  
John E. Janik ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Adoptive Transfer ◽  
B Cell Malignancies ◽  
B Lineage

Abstract Abstract 2865 T cells can be genetically modified to express chimeric antigen receptors (CARs) that specifically recognize the B-cell antigen CD19. Adoptive transfer of autologous T cells expressing anti-CD19 CARs is an attractive new approach for treating B-cell malignancies. We have constructed a CAR that consists of the variable regions of a mouse-anti-human-CD19 antibody coupled to the signaling domains of CD28 and CD3-zeta. We have treated 5 patients with 2 doses of 60 mg/kg of cyclophosphamide and 5 doses of 25 mg/m2 of fludarabine followed by infusions of anti-CD19-CAR-transduced T cells and administration of high-dose IL-2. All of the patients received infusions of cells that produced cytokines in a CD19-specific manner. The percentage of the infused cells that expressed the anti-CD19 CAR as measured by flow cytometry ranged from 45% to 65%. The first patient enrolled on our trial has follicular lymphoma. He was treated twice. The patient obtained a partial remission (PR) from his first course of chemotherapy, 0.4×109 anti-CD19-CAR-transduced T cells, and IL-2 (reported in Kochenderfer et al. Blood First Edition); however, he subsequently developed progressive disease, and 40 weeks after his first CAR-transduced T cell infusion he received a second course of chemotherapy followed by 2×109 CAR-transduced T cells and IL-2. The second course of treatment resulted in an additional PR and was not associated with any toxicity that could be attributed to the CAR-transduced T cells. At last follow-up, a small amount residual disease detected only by positron emission tomography remained. In this first patient, the initial treatment course resulted in eradication of blood and bone marrow B-lineage cells for 39 weeks. In contrast to the prolonged eradication of B-lineage cells after the initial treatment course, the number of polyclonal blood B cells normalized 9 weeks after the second CAR-transduced T cell infusion. CAR-transduced T cells were present at a level of 0.1% of total peripheral blood mononuclear cells (PBMCs) one month after the first CAR-transduced T cell infusion. Despite the five-fold higher dose of CAR-transduced T cells administered with the second treatment, CAR-transduced T cells were not detected in the blood one month after the second CAR-transduced T cell infusion. The second patient treated on our protocol had follicular lymphoma and had received extensive prior therapy including autologous stem cell transplantation. After an initially uncomplicated course, this patient developed pneumonia caused by culture-proven influenza A virus and died 18 days after CAR-transduced T cell infusion. Quantitative PCR was used to measure the level of CAR-transduced cells in multiple tissues obtained from this patient at autopsy. CAR-transduced cells were widely distributed with the highest levels in the spleen and bone marrow. The third patient treated on our trial obtained a complete remission of advanced chronic lymphocytic leukemia (CLL) after treatment with chemotherapy, infusion of 2×109 anti-CD19-CAR-transduced T cells, and IL-2. At the time of last follow-up, three months after treatment, adenopathy had resolved, CLL cells were not detected by flow cytometry analysis of the blood and bone marrow, and the number of normal polyclonal B cells in the blood was below normal levels. This patient had a period of fever and hypotension 7 days after cell infusion that was associated with an elevated serum interferon-gamma level of 1532 pg/mL. At the time of the hypotensive episode 7 days after cell infusion, anti-CD19-CAR-transduced cells made up 2.1% of PBMCs. The fourth patient treated on our study obtained a PR of splenic marginal zone lymphoma that continues 2 months after treatment with chemotherapy, 2×109 CAR-transduced T cells, and IL-2. This patient did not have prolonged depletion of normal B cells after treatment, and he did not have any toxicity that could be attributed to the anti-CD19 CAR-transduced T cells. We recently treated a fifth patient who has CLL. Follow-up on this patient is too short to evaluate toxicity or response. In conclusion, we have shown that adoptive transfer of anti-CD19-CAR-transduced T cells with in vivo activity is feasible. The promising results obtained on this trial raise important questions for future research aimed at optimizing therapy with anti-CD19-CAR-transduced T cells. Disclosures: No relevant conflicts of interest to declare.

Detecting Cryptic Neoplastic B-Cells: Flow Cytometric Analysis of B-Cell Lymphoma Concealed in Polyclonal B-Cells and Other Intense Reactive Components, with Emphasis on Large B-Cell Lymphoma in Fine Needle Aspirate (FNA)/ Body Fluid (BF).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4560-4560
Author(s):  
Endi Wang ◽  
Joan E. Etzell
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
Flow Cytometric Analysis ◽  
B Cell Lymphoma ◽  
Light Scatter ◽  
Color Flow ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Flow cytometry is widely used in the diagnosis of lymphoma. In B-cell lymphoma, light chain restriction (LCR) is especially useful for distinguishing B-cell malignancy from a reactive lymphoid proliferation. Usually the neoplastic B-cell population predominates, and monoclonality is conspicuous as demonstrated by distinct LCR. However, if the specimen contains a large reactive component, especially polyclonal B-cells, a small number of neoplastic B-cells will be buried in the background, thus creating a diagnostic challenge. We retrospectively analyzed three/four-color flow cytometry by examining 12 cases of B-cell lymphoma with a small proportion of neoplastic B-cells hidden in polyclonal B-cells and other intense reactive components. These 12 cases (9 FNA, 1 BF, 2 tissues) comprised 7% of specimens diagnostic or suspicious for lymphoma by flow cytometry at our institution. Eight cases were diffuse large B-cell lymphoma (DLBCL) (18.6% of DLBCL and 27.6% of DLBCL in FNA/BF) while four were lymphoma of other types (3% of non-DLBCL). In all the cases, LCR was obscured by polyclonal B-cells (8/12) or absence of surface immunoglobulin (sIg) in neoplastic B-cells (4/12), and morphology resembled a reactive picture. The clues by flow cytometry prompting further analysis included increased forward angle light scatter(FSC)/side angle light scatter(SSC) (12/12), brighter CD20 (6/12), dim CD20 (1/12), absence of sIg (4/12), and a distinct population of B-cells co-expressing CD10 (2/12), CD5 (1/12), CD2 (1/12), CD23 (4/12) or CD38 (1/12). By backgating/regating suspected subpopulations, the concealed neoplastic B-cells were demonstrated by enriched LCR (5 to10 fold) or tight clustering on FSC/SSC. In conclusion, cryptic neoplastic B-cells, especially diffuse large B-cell lymphoma in FNA/BF, can be extracted, according to their altered immunophenotypic features, from background polyclonal B-cells and reactive T-cells via manipulation of gating strategies.

The Shaving Reaction Is Facilitated through Trogocytosis: Concerted Transfer of Both Rituximab (RTX) and the Membrane Dye PKH26 from B Lymphocytes to Macrophages Is Demonstrable by Flow Cytometry, Fluorescence Microscopy, and High Resolution Digital Imaging in a Flow Cytometric Environment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2096-2096 ◽  
Author(s):  
David A. Mack ◽  
Paul V. Beum ◽  
Margaret A. Lindorfer ◽  
Andrew W. Pawluczkowycz ◽  
Ronald P. Taylor
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
High Resolution ◽  
Fluorescence Microscopy ◽  
B Cell ◽  
B Lymphocytes ◽  
Digital Imaging ◽  
Antigenic Modulation ◽  
Raji Cells ◽  
Flow Cytometric

Abstract Opsonization of CD20+ B lymphocytes with the anti-CD20 mAb RTX can promote substantial loss of B cell-bound RTX and CD20 in the presence of acceptor monocytes (Beum et al., J Immunol, 2006). This process of antigenic modulation, or shaving, occurs when patients with chronic lymphocytic leukemia are treated with the standard RTX dose of 375 mg/m2, and represents an escape mechanism for mAb-targeted malignant cells. Shaving in vitro, and likely in vivo, is mediated by mononuclear phagocytic cells: Fc gamma receptors on these cells bind and take up CD20-RTX immune complexes. This process may occur by trogocytosis, in which donor and acceptor cells form immunologic synapses, allowing membrane fragments and cell-surface proteins to be removed from donor cells and transferred to and internalized by acceptor cells. To investigate this hypothesis, Raji cells were stained with the lipophilic fluorescent reporter membrane dye PKH26, and opsonized with RTX or Alexa (Al) 488-conjugated RTX. The cells were incubated with retinoic acid-treated THP-1 cells, a human monocyte/macrophage cell line. THP-1 cells in the mixtures were then stained for CD11b and CD14 for identification. Quantitative analysis of cells by flow cytometry and high resolution digital imaging in a flow cytometric environment (ImageStream, AMNIS Corp.) demonstrated time-dependent transfer of PKH26 from RTX-opsonized Raji cells to THP-1 cells, as illustrated in a representative kinetics experiment in the table. Fluorescence intensity values are given for THP-1 cells after incubation with naïve (control), or RTX-opsonized Raji cells, both of which were PKH26 labeled. Molecules of equivalent soluble fluorochrome (MESF) units or geometric mean fluorescence (GMF) units are used, based on flow cytometry or ImageStream analysis, respectively. PKH26 Fluorescence Intensities Acquired on THP-1 Cells after Incubation with Control or RTX-Opsonized PKH26-Labeled Raji Cells Time of Incubation of Raji and THP-1 Cells, min <1 5 15 45 Control (no RTX on Raji cells) MESF 170 240 260 630 GMF 900 900 1300 3300 Experimental (RTX-opsonized Raji Cells) MESF 700 1200 1700 4300 GMF 3500 6100 8300 19000 Based on the PKH26 signal, a small but readily demonstrable portion of Raji cell membrane is transferred from RTX-opsonized cells to THP-1 cells. Transfer corresponds to only ~5–10% of total PKH26 on Raji cells, which is reasonable as B cells are left intact after shaving. However, we observed >65% transfer of Al488 RTX from B cells to THP-1 cells. Results were confirmed by fluorescence microscopy and by inspection of ImageStream digital images of THP-1 and Raji cells. We believe that transfer of RTX and CD20 from RTX-opsonized B cells to acceptor monocytes/macrophages proceeds via trogocytosis, and plays an important role in the resistance of some B-cell malignancies to RTX therapy. Based on reports of antigenic modulation of targets for other immunotherapeutic mAbs used in cancer treatment, the trogocytic mechanism we document for RTX is likely to underlie antigenic modulation promoted by these mAbs as well.

Junctional Adhesion Molecule C (JAM-C) Constitutes a New Marker for the Differential Diagnosis of B Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3940-3940
Author(s):  
Thomas Matthes ◽  
Christiane Ody ◽  
Beat Imhof ◽  
Carmen Donate ◽  
Dominique Cossali ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Marginal Zone ◽  
Plasma Cells ◽  
B Cell Differentiation ◽  
B Cell Lymphomas

Abstract Abstract 3940 Poster Board III-876 Introduction Differentiation of naïve B cells into plasma cells or memory cells occurs in the germinal centres (GC) of lymph follicles or alternatively in the marginal zone via a GC- and T cell independent pathway. It is currently assumed that B cell lymphomas correspond to normal B cell differentiation stages, but the precise correlation of several B cell lymphomas to these two pathways remains controversial. We have previously shown that junctional adhesion molecule C (JAM-C) originally identified at the cell-cell border of endothelial cells, constitutes also a marker of B lymphocytes with a tightly regulated expression during B cell differentiation: immature B cells, GC-B cells and plasma cells stain negatively, whereas mature, memory and marginal zone derived B cells stain strongly positive. Here we test the expression of JAM-C on a series of patients with B cell lymphomas. Methods B lymphocytes from the peripheral blood of 158 untreated patients were analyzed using flow cytometry with standard antibody panels (CD5, CD10, CD11c, CD22, CD23, CD25, CD38, CD103, FMC7, sIg). Diagnosis of a B cell lymphoma was established according to WHO guidelines, using additionally RT-PCR, karyotyping, or FISH, if necessary. Expression of JAM-C was studied by flow cytometry with a polyclonal antibody obtained from a rabbit immunized with the soluble JAM-C molecule. Results MCL, HCL and MZBL with a supposed origin in the marginal zone stained mostly positive, whereas CLL and FL with a supposed origin in the germinal centre showed mostly a negative staining. No correlation was found in CLL between JAM-C expression and staining for ZAP70 or CD38. In 12 cases routine work-up was not able to precisely establish a diagnosis of CLL or MZBL, and CLL or MCL. In these cases the presence of JAM-C was considered a strong argument against a GC-origin of the malignant B cells. Addition of JAM-C to antibodies used in the Matutes score increased the sensitivity and specificity of this score for the diagnosis of CLL. Furthermore, it may help differentiating MZBL from LPL which otherwise display overlapping immunophenotypes. Conclusion JAM-C constitutes a new diagnostic marker for the differential diagnosis of B cell lymphomas, and is particularly useful for the distinction between CLL and LPL (negative staining) on the one hand and mantle cell and marginal zone B cell lymphomas (positive staining) on the other hand. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Synergistic cooperation and crosstalk between MYD88L265P and mutations that dysregulate CD79B and surface IgM

2017 ◽  
Vol 214 (9) ◽  
pp. 2759-2776 ◽  
Author(s):  
James Q. Wang ◽  
Yogesh S. Jeelall ◽  
Peter Humburg ◽  
Emma L. Batchelor ◽  
Sarp M. Kaya ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
B Lymphocytes ◽  
B Cell Malignancies ◽  
B Cell Survival ◽  
Rate Limiting ◽  
Self Antigen ◽  
Endoglycosidase H

CD79B and MYD88 mutations are frequently and simultaneously detected in B cell malignancies. It is not known if these mutations cooperate or how crosstalk occurs. Here we analyze the consequences of CD79B and MYD88L265P mutations individually and combined in normal activated mouse B lymphocytes. CD79B mutations alone increased surface IgM but did not enhance B cell survival, proliferation, or altered NF-κB responsive markers. Conversely, B cells expressing MYD88L265P decreased surface IgM coupled with accumulation of endoglycosidase H–sensitive IgM intracellularly, resembling the trafficking block in anergic B cells repeatedly stimulated by self-antigen. Mutation or overexpression of CD79B counteracted the effect of MYD88L265P. In B cells chronically stimulated by self-antigen, CD79B and MYD88L265P mutations in combination, but not individually, blocked peripheral deletion and triggered differentiation into autoantibody secreting plasmablasts. These results reveal that CD79B and surface IgM constitute a rate-limiting checkpoint against B cell dysregulation by MYD88L265P and provide an explanation for the co-occurrence of MYD88 and CD79B mutations in lymphomas.

Immunological Shift On Day 15 of Remission Induction in Children with CD10-Positive B-Cell Precursor ALL Treated by ALL-MB 2008 Protocol.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2650-2650
Author(s):  
Alexander Popov ◽  
Tatiana Verzhbitskaya ◽  
Grigory Tsaur ◽  
Egor Shorikov ◽  
Leonid Saveliev ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Fluorescence Intensity ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Remission Induction ◽  
Cell Precursor ◽  
Childhood All

Abstract Abstract 2650 Poster Board II-626 Statement. Flow cytometric (FC) minimal residual disease (MRD) monitoring during remission induction is a strong tool for prediction of outcome in children with acute lymphoblastic leukemia (ALL) of B-cell precursor (BCP) origin. Antigens' expression changes during treatment. Immunological shift can significantly complicate MRD detection. Immunophenotypical changes could vary during antileukemic treatment. Aim. To compare CD19, CD10, CD34, CD20, CD45 and CD58 expression at diagnosis and on day 15 of remission induction of currently applied ALL-MB 2008 treatment protocol and to evaluate significance of this changes for FC MRD monitoring. Patients and methods. Since November 2008 till July 2009 38 cases of childhood ALL were enrolled onto ALL-MB 2008 trial in Pediatric Oncology and Hematology Center in Ekaterinburg. Among them 22 patients were selected for present study as they fulfilled following criteria: had CD10-positive BCP-ALL and were MRD-positive ≥ 10−4 on day 15. MRD detection in bone marrow was performed by 9-color FC. We compared mean fluorescence intensity (MFI) values at diagnosis and on day 15. We also analyzed changes in CD19, CD20, CD45 and CD58 expression by normal B-lymphocytes on day 15 because at this time-point B-lineage regeneration is absent in bone marrow and MRD has to be discriminated only from mature B-cells. Coefficient of variation (CV) of fluorescence intensity and percentage of positive cells were used for evaluation of cells' distribution changes. Results. At diagnosis we found significant differences in antigen expression between tumor cells and B-lymphocytes. CD10, CD34, CD58 overexpression and CD19, CD20, CD45 underexpression by leukemic blasts were noted. On day 15 CD10, CD34 and CD58 were downmodulated (p = 0,0030, p = 0,0007 and p = 0,0017 respectively) while CD19, CD20 and CD45 were upmodulated (p = 0,0033, p = 0,0273 and p = 0,0001 respectively). At the same time CD20, CD45 and CD58 expression by mature B-lymphocytes decreased (p = 0,0013, p = 0,0130 and p = 0,0067 respectively) while CD19 was stable (p = 0,1060). Hence malignant and normal cells became closer on dot plots. On day 15 significant differences in CD10, CD34, CD20, CD45 and CD58 between blasts and lymphocytes were still presented. CD19 expression by leukemic cells on day 15 became higher than by mature B-cells (median MFI = 20284, range1587-34505 and median MFI = 15823, range 811–30882, p = 0,0196). Cells' distribution by CD10 and CD34 became more heterogeneous (p = 0,0364 and p = 0,0008 respectively), CD45 expression became more homogeneous (p = 0,0003) while CD20 and CD58 expression CV didn't change significantly (p = 0,1913 and p = 0,3443 respectively). CD20- and CD45-positive cells' percentage increased (p = 0,0015 and p = 0,0002 respectively) and CD34-positive cells' percentages decreased (p = 0,0003). Discussion. Changes in antigens' expression could complicate MRD detection so far as they decrease immunophenotypical differences between tumor blasts and mature B-lymphocytes. Interestingly, disparity of our results and previously shown data was found. Differences in immunological shift could be partially explained by differences in induction intensity of ALL-MB 2008 and international protocols. Conclusion. Significant immunological shift occurred on day 15 of ALL-MB 2008 remission induction. These antigens' expression changes have to be taken into account by the researcher for the successful FC MRD monitoring. Disclosures: No relevant conflicts of interest to declare.

scholarly journals AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1046.1-1046
Author(s):  
L. Schlicher ◽  
P. Kulig ◽  
M. Murphy ◽  
M. Keller
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Activation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
B Cell Activation ◽  
Human B Cells ◽  
Circulating Lymphocytes

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

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