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.

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.

NFAT2 Is a Critical Regulator Of Anergy Induction In CLL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 869-869
Author(s):  
Melanie Märklin ◽  
Jonas S. Heitmann ◽  
B. Sc. ◽  
David Worbs ◽  
B. Sc. ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Gene Expression Analysis ◽  
Target Genes ◽  
Flow Cytometric Analysis ◽  
Knock Out ◽  
Flow Cytometric

Abstract NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. Expression of CD38 and ZAP70 define a subgroup of patients with enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggessive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype refering to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL and in anergy induction in CLL cells. For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eµ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eµ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 wks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). To identify novel NFAT2 target genes in CLL cells, we performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays. Mice with NFAT2 knock out exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells and a significantly reduced life expectancy (200 vs. 325 days) as compared to control animals. Flow cytometric analysis at distinct time points showed a pronounced infiltration by CD5+ B cells of the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko exhibited significantly higher rates of proliferation than control animals. To identify NFAT2 target genes resonsible for the observed alterations in the disease phenotype, we subsequently peformed a gene expression analysis with CD5+CD19+ CLL cells from TCL1+NFAT2 ko mice with CLL cells from TCL1+ mice serving as controls. Here, we detected a significantly altered expression of 22 genes associated with B cell anergy in the TCL1+NFAT2 ko cohort. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1, Hspa14 and CD166 constituting the strongest hits with up to 10fold reduced gene expression. Downregulation of the identified target genes was subsequently confirmed using RT-PCR and Western Blotting. In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells. In addition, we could show that NFAT2 controls the expression of several anergy-associated genes and that its absence prevents the acquisition of an anergic phenotype by the CLL cells correlating with a significantly more aggressive course of the disease. Taken together, our data demonstrate that NFAT2 plays an essential role in the pathogenesis of CLL and implicate this transcription factor as a potential target in its treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differentiation of normal human pre-B cells in vitro.

1990 ◽  
Vol 172 (1) ◽  
pp. 325-334 ◽  
Author(s):  
J G Villablanca ◽  
J M Anderson ◽  
M Moseley ◽  
C L Law ◽  
R L Elstrom ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Flow Cytometric Analysis ◽  
Magnetic Bead ◽  
B Cell Differentiation ◽  
Differentiated Cells ◽  
Flow Cytometric ◽  
Normal Human

The differentiation of surface Ig- pre-B cells into surface Ig+ B cells is a critical transition in mammalian B cell ontogeny. Elucidation of the growth factor requirements and differentiative potential of human pre-B cells has been hampered by the absence of a reproducible culture system that supports differentiation. Fluorescence-activated cell sorting and magnetic bead depletion were used to purify fetal bone marrow CD10+/surface mu- cells, which contain 60-70% cytoplasmic mu+ pre-B cells. CD10+/surface mu- cells cultured for 2 d were observed to differentiate into surface mu+ cells. Analysis by Southern blotting provided direct evidence that rearrangement of kappa light chain genes occurs in culture, and flow cytometric analysis revealed the appearance of surface Ig+ B cells expressing mu/kappa or mu/lambda. Unexpectedly, the kappa/lambda ratio in differentiated cells was the inverse of what is normally observed in adult peripheral blood. Differentiation occurs in the absence of exogenous growth factors or cytokines, suggesting that a stimulus-independent differentiative inertia might characterize pre-B cells in vivo. Future use of this model will facilitate our understanding of normal and abnormal human pre-B cell differentiation.

Dietary Products Induce Apoptosis in CLL B Cells and Reveal Potential as a Therapeutic Combination That Can Overcome Stromal Cell Mediated Protection.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3130-3130
Author(s):  
Asish K. Ghosh ◽  
Tait D. Shanafelt ◽  
Charla Secreto ◽  
Neil E. Kay
Keyword(s):  
B Cells ◽  
B Cell ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Combined Treatment ◽  
Flow Cytometric Analysis ◽  
Physical Contact ◽  
Flow Cytometric ◽  
Induced Apoptosis

Abstract Background: B-cell chronic lymphocytic leukemia (CLL) is characterized by defects in the level of apoptosis rather than accelerated leukemic cell proliferation. We have been interested in identification of naturally occurring substances that induce apoptosis in CLL B cells with potential for therapeutic application with a favorable toxicity profile. Two such agents, epigallocatechin-3-gallate (EGCG) in green tea and curcumin from the spice turmeric have recently been studied by our laboratory. We have shown previously that in vitro treatment of CLL B cells with EGCG induces apoptosis [Blood, 2004, 104:788] and is now being tested in a Phase I/II clinical trial. In this study, we explored the possibility of using curcumin in the induction of CLL B cell apoptosis either alone or in combination with EGCG. We also tested their ability to induce CLL B cell apoptosis in the presence of human stromal cells. Results: In vitro treatment of CLL B cells with curcumin induces apoptosis in a dose (5–20 μM) and time dependent manner. Further analysis suggested that curcumin treatment inhibited the effects of the constitutively active proteins STAT3, AKT and NF-κB and suppressed expression of XIAP in CLL B cells. Interestingly, we observed that curcumin/EGCG combination was more effective in the induction of apoptosis in CLL B cells as compared to the corresponding individual dose of the drug alone. We next evaluated the effects of curcumin when CLL B cells were co-cultured with the human stromal cell line HS-5. Curcumin-induced apoptosis was significantly inhibited when CLL B cells were co-cultured in physical contact with HS-5 cells. We also found protection against curcumin-induced cell death, albeit less, when CLL B cells were co-cultured with HS-5 separated by transwells. However, higher doses of curcumin can overcome HS-5 mediated apoptosis-protection of CLL B cells in transwells but, did not overcome the protective effect of physical contact with HS-5. Further analysis of CLL B cells co-cultured with HS-5 in transwells demonstrated elevated levels of STAT3 with increased phosphorylation at serine-727 and increased expression of Mcl-1 and XIAP. At this stage, we employed combined therapeutic approach to evaluate whether combining curcumin with EGCG could overcome stromal cell-mediated protection. Interestingly, combined treatment of CLL B cells could significantly overcome stromal cell-mediated protection against apoptotic cell death, although the effect was more pronounced when CLL B cells were separated by transwells (see figure below). Conclusion: Together, these results suggest that curcumin induces apoptosis in CLL B cells in vitro and may mediate its effects by suppressing the constitutively active signaling pathways that are known to be important in apoptosis resistance This work also demonstrates that stromal cells and their soluble products can resist drug-induced apoptosis of leukemic CLL B cells. Importantly, the apoptosis-inducing activity of curcumin and EGCG is not impaired completely in the presence of stromal cells, suggesting that the combination of curcumin/EGCG may warrant clinical testing in patients with CLL. Figure. Combined treatment of CLL B cells with curcumin/EGCG reduces stromal cell mediated protection against apoptosis. CLL B cells were cultured alone or co-cultured with HS-5 cells and treated with the indicated doses of curcumin (C) and/or EGCG (E). After 24h, cells were collected for Annexin/PI staining for flow cytometric analysis. Mean percent of annexin/PI positive cells are presented with standard error bars. Figure. Combined treatment of CLL B cells with curcumin/EGCG reduces stromal cell mediated protection against apoptosis. CLL B cells were cultured alone or co-cultured with HS-5 cells and treated with the indicated doses of curcumin (C) and/or EGCG (E). After 24h, cells were collected for Annexin/PI staining for flow cytometric analysis. Mean percent of annexin/PI positive cells are presented with standard error bars.

A NUP98-HOXD13 Fusion Gene Impairs B Lymphocyte Development and Function In a Mouse Model for Myelodysplastic Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 279-279
Author(s):  
Abdul Gafoor A. Puthiyaveetil ◽  
Bettina Heid ◽  
David L. Caudell
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
B Lymphocytes ◽  
Marginal Zone ◽  
Fusion Gene ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Cell Fractions ◽  
And Function

Abstract Abstract 279 Transgenic mice which express the fusion gene NUP98-HOXD13 (NHD13) have been shown to develop characteristic features of Myelodysplastic syndrome (MDS) including impaired hematopoietic differentiation and peripheral blood cytopenias in the presence of normocellular or hypercellular bone marrow (BM). It is evident that B-cells play a role in the progression of MDS by immune modulation or as direct targets of mutations resulting in ALL, or as cells that influence the BM microenvironment in which a neoplastic myeloid clone evolves. Choi and colleagues suggested a block in differentiation during the early development of B lymphocytes in the BM of NHD13 mice leading to lymphopenia consistent with the observation in some MDS patients. In this study, we sought to further delineate the role of NHD13 on B lymphocytes which escaped the initial differentiation block in the BM. We hypothesized that NHD13 impairs maturation and function of IgM+ B lymphocytes contributing to immunodeficiency. To study this, we performed blood smear examination, Complete Blood Counts (CBC), quantitative ELISA for antibody concentrations, and flow cytometric analysis of B cell fractions from the BM and spleen in 8–12 week-old transgenic and wild type (WT) mice. CBCs revealed significant lymphopenia and ELISA showed higher IgM concentrations (n=10, p<0.001), reduced levels of IgG1 (n=10, p<0.05) and IgE (n=10, p<0.01). The IgG2a, IgG2b, and IgG3 antibody levels were comparable to WT counterparts. Flow cytometric analysis of BM and splenic B cell fractions revealed reduced numbers of B cells in Hardy fractions D and F (n=10, p<0.01) indicative of impaired differentiation prior to these stages; splenic fractions in NHD13 mice were comparable to WT controls. Next, to assess the peripheral maturation and functional efficiency of B lymphocytes in the context of a comprehensive immune stimulation, a cohort of five WT and five preclinical transgenic mice were injected with 100 μ g dinitrophenol (DNP) followed by a booster dose on day 21. Mice were euthanized on day 28 and whole blood, spleen, lymphnodes and BM were harvested. CBC evaluation revealed significant lymphopenia in NHD13 mice (n=5, p<0.001). Quantitative ELISA for DNP specific antibodies showed comparable levels of serum IgM and significantly reduced levels of serum IgG1 (n=5, p<0.001), IgG2a (n=5, p<0.001), IgG2b (n=5, p<0.01), IgG3 (n=5, p<0.001) and IgE (n=5, p<0.01). Flow cytometric analysis of peripheral blood showed reduced numbers of B220+ IgM+ B cells (n=5, p<0.01), but comparable percentages of CD4+ and CD8+ T-cells. Detailed flow cytometric analysis of B-cell fractions in the BM and spleen of DNP-stimulated mice revealed a reduction in subpopulations of B lymphocytes. The earliest B cell lineage population, Pre-Pro B, was comparable to the WT controls. Hardy Pro B fraction B (n=5, p<0.001) and Pre B fractions E (n=5, p<0.01) and F (n=5, p<0.01) from BM of stimulated mice were significantly reduced in contrast to fractions C and C', which were higher (n=5, p<0.05 and p<0.001 respectively), indicative of cell growth arrest at these stages. Flow cytometry of splenic B-cell fractions from the DNP-stimulated mice showed significantly lower Transitional 1 (n=5, p<0.01), Follicular (n=5, p<0.05) and Marginal Zone (n=5, p<0.001) populations upon antigenic stimulation suggestive of defective clonal expansion of IgM+ cells even after escaping the block in the BM. Histopathology of the spleen revealed smaller lymphoid follicles with poorly developed mantle and marginal zone regions in the transgenic mice when compared to WT controls, consistent with the flow cytometric data. This study indicates that when NHD13 mice are immunologically challenged, B lymphocytes undergo impaired differentiation in the BM and maturation in the spleen, as well as reduced antibody class switching and subsequently lower antibody production. Analysis of B cell subsets during development and specific IgG/IgE antibody production, suggest that the NHD13 transgene might impair VDJ gene recombination and class switch recombination that are critical during these phases of B cell development. Disclosures: No relevant conflicts of interest to declare.

Genetic Loss of NFAT2 Induces Profound Acceleration of CLL in the TCL1 Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 862-862
Author(s):  
Melanie Märklin ◽  
Stefanie Bugl ◽  
Jonas S. Heitmann ◽  
Alexandra Poljak ◽  
Bettina S ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Mouse Model ◽  
Transgenic Mice ◽  
B Cell ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Knock Out ◽  
Flow Cytometric

Abstract Abstract 862 NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated and relocated to the cytoplasm by a network of several kinases. Although identified originally as a major transcriptional regulator in T cells, it is now clear that NFAT transcription factors also possess important roles in other cells of the hematopoietic system including dendritic cells, mast cells, megakaryocytes and B cells. Several recent studies have demonstrated that Calcineurin/NFAT signaling is involved in the pathogenesis of a wide array of hematological malignancies including diffuse large B cell lymphoma, CLL as well as Burkitt and Burkitt-like lymphomas. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL. For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice, in which the Cre recombinase is expressed under the control of the B cell-specific CD19 promoter. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eμ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eμ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 weeks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). Mice with NFAT2 knock out exhibited a significantly accelerated accumulation of CD5+CD19+ CLL cells as compared to control animals. Flow cytometric analysis at distinct time points showed a tremendous infiltration by CD5+ B cells in the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. At approximately 26 weeks of age, NFAT2 ko mice showed an approximately 40 fold increased lymphocyte count in the peripheral blood than their litter mate controls (1500/μL vs. 60000/μL). Splenomegaly and lymphatic adenopathy was also significantly increased in the NFAT ko population. Furthermore, NFAT2 ko mice showed a dramatically reduced median survival (200 vs. 325 days) and maximum survival (265 vs. 398 days) in comparison to regular TCL1 transgenic mice. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko at an age of approximately 7 months exhibited significantly higher rates of proliferation than control animals. In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells implicating Ca2+/NFAT signaling as a potential target for the treatment of this disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD22 Gating for Flow Cytometric Analysis in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia after CD19 CAR-T Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 13-13
Author(s):  
Xinjian Yu ◽  
Pan Li ◽  
Heyuan Feng ◽  
Jian Kang ◽  
Xichang Zheng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Hla Dr ◽  
Car T ◽  
Normal Expression ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Antibody Panel

CD19 has been as a classic marker for gating B-cells in flow cytometric analysis. However, with the application of CD19 chimeric antigen receptor (CAR) T-cell therapy for treating relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), CD19 gating is no longer adequate for B-ALL patients post CD19 CAR-T therapy owing to CD19 antigen loss, under this circumstance, cytoplasmic CD79a (cCD79a) emerged to replace CD19 as a gating marker of B-cells. Nevertheless, cCD79a was intracellular staining and time consuming, sometimes with unspecific stain or without enough cells for analysis after permeabilization and extra washing. CD22 is another pan B marker expressed on the surface of B lymphocytes at different stages, could be as alternative choice of B-cell identification after CD19 CAR-T. Here, we evaluated the possibility of CD22 as a B-cell gating marker by comparing the expression of CD22 and cCD79a in CD19 negative relapsed B-ALL patients. We routinely used cCD79a to gate B-cells in patients after CD19 CAR-T therapy, CD22 expression was detected simultaneously. The standard antibody panel consist of CD45, CD19, CD22, CD10, CD20, CD34, CD38, CD58, cCD79a, CD81, HLA-DR and CD123. CD22 antibody conjugated with phycoerythrin (PE) or allophycocyanin (APC) was used, extra antibodies could be added based on patient's immunophenotype at initial diagnosis. Bone marrow specimens were prepared by a standard stain-lyse-wash procedure, no less than 300,000 events were acquired by 8-color BD FACS Canto II flow cytometer, data were analyzed using FACS Diva 8.0.1 software. Antigen normal expression and partial expression were defined as &gt;80% and 20-80% of gated cells displaying interested antigen, respectively. Dim expression was defined as more than one log weaker than the normal counterpart's mean fluorescence intensity (MFI). From April 2018 to June 2020, a total of 40 CD19-negative relapsed B-ALL patients post CD19 CART therapy were included in this study, consisting of 14 adults and 26 children younger than 18 years old, with a median age of 12 years (range, 1-64). The simultaneous expression of cCD79a and CD22 among these patients were analyzed. All 40 patients (100%) had normal cCD79a expression on B-cells. Although CD22 was expressed on all cases as well, normal expression was seen in 33 (82.5%) patients whereas 7 (17.5%) were partial or dim (P/D) expression, the P/D expression rendered CD22 as a gating marker unsuitable. To figure out whether this P/D CD22 expression was related to CD19 CAR-T treatment, we checked back the immunophenotype of these 7 patients and found that, before CD19 CAR-T, 4 (10%) patients with P/D CD22 expression and 3 (7.5%) with normal CD22 expression. When we excluded these 4 patients with existed P/D CD22 expression, 91.7% (33/36) of patients accordantly expressed normal CD22 and cCD79a, which indicated that CD22 could be a B-cell gating marker for 91.7% of patients with normal CD22 expression before CD19 CAR-T therapy. To obtain optimal signal, CD22 antibody conjugated with bright fluorescein such as PE or APC is recommended. It is noted that CD22 also expressed on basophils, therefore, HLA-DR should be included in the antibody panel to exclude this interference since basophils do not express HLA-DR. Sometimes, there might be CD22 stain on plasmacytoid dendritic cells (pDCs) which express HLA-DR too, it is not difficult for us to differentiate B-cell from pDCs based on the facts that CD22 expression on pDCs is weaker than that on B cells, pDCs have bright CD123 expression and are located at a relatively fixed position in CD45/SSC plot. In conclusion, our data on the comparison between CD22 and cCD79a expression in CD19 negative relapsed B-ALL patients revealed that, under most circumstances (with 82.5%-91.7% chance), CD22 gating alone could efficiently identify B cells in patients after CD19 CAR-T therapy, which is also a cost-effective and labor-saving strategy for routine practice compared to cCD79a gating. In case of partial or dim CD22 expression, the cCD79a gating is needed. Disclosures No relevant conflicts of interest to declare.

Increased Stability of Bcl-2 mRNA in B-Cell Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4789-4789
Author(s):  
Ebenezer A. Kio ◽  
Mauricio Pineda-Roman ◽  
Yoko Otake ◽  
Robert K. Stuart ◽  
Daniel J. Fernandes
Keyword(s):  
B Cells ◽  
B Cell ◽  
Gradient Centrifugation ◽  
Flow Cytometric Analysis ◽  
Lymphocytic Leukemia ◽  
Potential Therapeutic Target ◽  
Protein Levels ◽  
Cell Extracts ◽  
Flow Cytometric

Abstract B-cell CLL is characterized by the accumulation of mononuclear B cells that are resistant to apoptosis as a result of bcl-2 oncogene overexpression. Nucleolin has recently been identified as a bcl-2 mRNA stabilizing protein that binds specifically to a 139 base AU-rich instability element (ARE) (Sengupta et al., J. Biol. Chem.279:10855–10863, 2004). Thus, studies were done to address the question whether the increased levels of bcl-2 mRNA in CLL are related to stabilization of bcl-2 mRNA by nucleolin. B cells were isolated from the blood of 9 patients with CLL and 5 normal volunteers by density gradient centrifugation followed by positive selection with CD19 immuno-magnetic microbeads. Flow cytometric analysis indicated that greater than 90% of the CLL and normal B cells were CD19 positive and CD3 negative. Western blotting revealed that cytoplasmic nucleolin and total cellular bcl-2 protein levels were elevated 18-fold (p&lt;0.001) and 5-fold (p&lt;0.001) respectively, in CLL compared to normal B cells. To directly examine the ability of nucleolin to stabilize bcl-2 mRNA, in vitro RNA decay assays were carried out using capped and polyadenylated bcl-2 mRNA transcripts. The average half-life of the bcl-2 mRNA transcript containing the ARE instability element was 12.5 min in S100 extracts of normal B cells, but was increased to 39.7 min in CLL cell extracts. Purified recombinant nucleolin (280 nM) stabilized bcl-2 mRNA when added to S100 extracts of normal B cells, but had no significant effect on bcl-2 mRNA stability in CLL cell extracts. The results are consistent with the hypothesis that resistance of CLL cells to apoptosis is related to overexpression of cytoplasmic nucleolin and increased stability of bcl-2 mRNA. These findings warrant further investigation of nucleolin as a potential therapeutic target in CLL.

scholarly journals Flow cytometric analysis of the binding of eleven lectins to human T- and B-cells and to human T- and B-cell lines

Cytometry ◽  
1984 ◽  
Vol 5 (2) ◽  
pp. 204-209 ◽  
Author(s):  
Jeannette Malin-Berdel ◽  
Günther Valet ◽  
Eckhart Thiel ◽  
John Anthony Forrester ◽  
Lutz Gürtler
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Flow Cytometric Analysis ◽  
T And B Cells ◽  
Flow Cytometric

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

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