The Antidepressant Mirtazapine Rapidly Shifts Hepatic B Cell Populations and Functional Cytokine Signatures in the Mouse

IntroductionB cells are important regulators of both adaptive and innate immunity. The normal liver contains significant numbers of B cells, and their numbers increase dramatically in immune-mediated liver diseases. Our previous observations suggest a hepatoprotective effect of the antidepressant mirtazapine in human and experimental immune-mediated liver disease. Therefore, we performed a series of experiments to determine the impact of mirtazapine treatment on hepatic B cell homeostasis, as reflected by B cell number, trafficking and phenotype using flow cytometry (FCM) and intravital microscopy (IVM) analysis. Mirtazapine treatment rapidly induced a significant reduction in total hepatic B cell numbers, paralleled by a compositional shift in the predominant hepatic B cell subtype from B2 to B1. This shift in hepatic B cells induced by mirtazapine treatment was associated with a striking increase in total hepatic levels of the chemokine CXCL10, and increased production of CXCL10 by hepatic macrophages and dendritic cells. Furthermore, mirtazapine treatment led to an upregulation of CXCR3, the cognate chemokine receptor for CXCL10, on hepatic B cells that remained in the liver post-mirtazapine. A significant role for CXCR3 in the hepatic retention of B cells post-mirtazapine was confirmed using CXCR3 receptor blockade. In addition, B cells remaining in the liver post-mirtazapine produced lower amounts of the proinflammatory Th1-like cytokines IFNγ, TNFα, and IL-6, and increased amounts of the Th2-like cytokine IL-4, after stimulation in vitro.ConclusionMirtazapine treatment rapidly alters hepatic B cell populations, enhancing hepatic retention of CXCR3-expressing innate-like B cells that generate a more anti-inflammatory cytokine profile. Mirtazapine-induced hepatic B cell shifts could potentially represent a novel therapeutic approach to immune-mediated liver diseases characterized by B cell driven pathology.

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The autoantigen-binding B cell repertoires of normal and of chronically graft-versus-host-diseased mice.

Journal of Experimental Medicine ◽

10.1084/jem.165.6.1675 ◽

1987 ◽

Vol 165 (6) ◽

pp. 1675-1687 ◽

Cited By ~ 54

Author(s):

A G Rolink ◽

T Radaszkiewicz ◽

F Melchers

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

The Other ◽

Cell Populations ◽

Foreign Antigen ◽

Alloreactive T Cells ◽

Polyclonal Activator ◽

Activated B Cells

A quantitative analysis of the frequencies of autoantibody-producing B cells in GVHD and in normal mice has been undertaken by generating collections of hybridomas of activated B cells. These hybridomas secreted sufficient quantities of Ig to allow binding analyses on a panel of autoantigens. B cells have been activated in a variety of ways. In vivo they were activated by injection of alloreactive T cells of one parent, leading to GVHD by a foreign antigen, sheep erythrocytes, in a secondary response, or by the polyclonal activator LPS. B cells from an experimentally unstimulated animal were used for an analysis of the normal background. In vitro B cells were activated by alloreactive T cells or by LPS. The frequencies of hybridomas and, therefore, of activated B cells producing autoantibodies to DNA or to kidney were not significantly different in mice activated by a graft-vs.-host T cell response as compared with B cell populations activated by any of the other procedures. They were found to compose 7.1-17.1% of the total repertoire of activated B cells. Moreover, the frequencies of autoantibody-producing activated B cells does not change with time after induction of the graft-vs.-host reaction. The pattern and frequencies of autoantigen-binding specificities to cytoskeleton, smooth muscle, nuclei, mitochondria, and DNA were not found to be different in any of the groups of hybridomas. The single notable exception, found in GVHD mice, were hybridomas producing autoantibodies to kidney proximal tubular brush border. These results allow the conclusion that autoantigen-binding B cells exist in an activated state in GVHD mice, as well as in mice activated by a foreign antigen or by a polyclonal activator, in B cell populations activated in vitro either by alloreactive T cells or by a polyclonal activator, and even in the background of experimentally unstimulated animals. T cell-mediated graft-vs.-host activation, in large part, does not lead to a selective expansion of autoantigen-binding B cells. The main difference between the graft-vs.-host-activated B cell repertoire and all others is that approximately 90% of teh autoantibodies were of the IgG class, whereas al autoantibodies found in the other groups were IgM.

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The Role of B Cells in Adult and Paediatric Liver Injury

Frontiers in Immunology ◽

10.3389/fimmu.2021.729143 ◽

2021 ◽

Vol 12 ◽

Author(s):

Arzoo M. Patel ◽

Yuxin S. Liu ◽

Scott P. Davies ◽

Rachel M. Brown ◽

Deirdre A. Kelly ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

T Cell Activation ◽

Cell Biology ◽

Liver Diseases ◽

Cell Activation ◽

Persistent Inflammation ◽

Stage Disease ◽

End Stage ◽

The Impact

B lymphocytes are multitasking cells that direct the immune response by producing pro- or anti-inflammatory cytokines, by presenting processed antigen for T cell activation and co-stimulation, and by turning into antibody-secreting cells. These functions are important to control infection in the liver but can also exacerbate tissue damage and fibrosis as part of persistent inflammation that can lead to end stage disease requiring a transplant. In transplantation, immunosuppression increases the incidence of lymphoma and often this is of B cell origin. In this review we bring together information on liver B cell biology from different liver diseases, including alcohol-related and metabolic fatty liver disease, autoimmune hepatitis, primary biliary and primary sclerosing cholangitis, viral hepatitis and, in infants, biliary atresia. We also discuss the impact of B cell depletion therapy in the liver setting. Taken together, our analysis shows that B cells are important in the pathogenesis of liver diseases and that further research is necessary to fully characterise the human liver B cell compartment.

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Tumor Cells Resistant to Alemtuzumab Complement Mediated Cytotoxicity in Patients with High Risk Previously Untreated Early Stage CLL: A Possible Mechanism of Treatment Failure.

Blood ◽

10.1182/blood.v106.11.2973.2973 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2973-2973

Author(s):

Clive S. Zent ◽

Nancy D. Bone ◽

Susan M. Geyer ◽

Neil E. Kay

Keyword(s):

Flow Cytometry ◽

B Cells ◽

High Risk ◽

B Cell ◽

Cell Clone ◽

Early Stage ◽

Cell Membrane Permeability ◽

Viable Cells ◽

The Impact

Abstract The monoclonal antibodies (MoAb) alemtuzumab and rituximab have proven efficacy in the treatment of CLL. In addition, alemtuzumab is effective in patients with defective p53 function responding poorly to purine analogue therapy. The action of both MoAb is not completely understood. Proposed mechanisms include complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and direct induction of apoptosis of CLL B cells. We have done correlative studies on CLL B cells from patients enrolled in a trial of alemtuzumab and rituximab in “high risk” early stage previously untreated CLL to determine: 1. Role of apoptosis induction and CDC in each MoAb and 2. If the addition of rituximab to alemtuzumab increases their in vitro cytotoxicity. Patients and Methods: Patients with early stage, previously untreated, high risk CLL are treated with subcutaneous alemtuzumab (dose escalation over 3 days then 30 mg Mon-Wed-Fri for 4 weeks) and rituximab (375 mg/m2/dose weekly from day 8 x 4 doses). High risk disease was defined as one or more of the following features of the CLL B cell clone: (1) 17p13−; (2) 11q22−; (3) unmutated IgVH (< 2%) and either CD38+ or ZAP-70+. Blood B lymphocytes collected prior to the start of therapy were tested for response to MoAb in vitro. Cells were cultured at 2 x 106/ml in AIM-V medium using standard conditions. Alemtuzumab and rituximab were used at 20 μg/ml and complement as 10% of 40 CH50 units/ml human serum. The impact of the MoAb was measured by counting viable cells (trypan blue negative) and measuring early apoptosis (annexin V) and cell death (cell membrane permeability to propidium iodide) using flow cytometry at 1 hour, and then daily for 3 days. Results: Treatment caused rapid resolution of lymphocytosis in all 7 patients and 3 patients were negative for circulating CLL cells using a highly sensitive 3 color flow cytometry (CD5+/CD19+/CD79b-) after therapy. All patients had a clinical response (2 CR, 5 PR). Alemtuzumab and complement were rapidly cytotoxic to most CLL cells. Mean cell viability was 39% (sd: 8%) after 1 hour of incubation. Cytotoxicity was similar in all samples irrespective of FISH defects, IgVH mutation status, and in vitro resistance to F-ara-A (n = 3). Alemtuzumab was inactive in the absence of complement for all samples. Rituximab alone and together with complement did not induce cytotoxicity or apoptosis. However, the addition of rituximab to alemtuzumab and complement did increase CDC where the number of viable cells was significantly lower at 1, 24, 48, and 72 hours incubation (p = 0.075, 0.047, 0.031, 0.027, respectively, for pairwise comparisons). CLL cells surviving alemtuzumab CDC subsequently had a lower level of apoptosis than control cells, implying a selection for resistant cells. Alemtuzumab CDC on this residual population was not increased at higher concentrations of alemtuzumab or complement. This mechanism of CDC resistance is currently under investigation. Conclusion: These data suggest that alemtuzumab CDC is an important mechanism of action in patients with CLL. However, alemtuzumab CDC kills only about 61% of CLL cells in vitro, and the surviving cells are more resistant to spontaneous apoptosis. This suggests that cells that survive alemtuzimab CDC contribute to disease progression or relapse. We intend to elucidate the mechanism of this resistance using our in vitro model with the hope that treatment strategies can be deployed to remove this residual CLL B cell clone.

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CD40 Ligand Activation Alters B Cell Migration to Secondary Lymphoid Organs.

Blood ◽

10.1182/blood.v108.11.935.935 ◽

2006 ◽

Vol 108 (11) ◽

pp. 935-935

Author(s):

Yvonne A. Efebera ◽

Tahamtan Ahmadi ◽

Amanda Flies ◽

David H. Sherr

Keyword(s):

B Cells ◽

Lymph Nodes ◽

B Cell ◽

Cd40 Ligand ◽

Lymphoid Organs ◽

Cell Populations ◽

Secondary Lymphoid Organs ◽

Activated B Cells

Abstract Background: An increased understanding of the requirements for antigen presentation has encouraged development of cell-based cancer vaccines. Trials using dendritic cells (DC) as antigen presenting cells (APC) for immunotherapy of several malignancies have shown considerable success. However, the difficulty in generating large numbers of DC required for these immunizations has led to the search for alternative APC. One such candidate is the CD40 ligand (CD40L)-activated B cell, populations of which can readily be expanded in vitro. To be an effective vehicle for antigen presentation to T cells, CD40L-activated B cells must be capable of migrating to secondary lymphoid organs. Therefore, CD40L-activated B cell migration following subcutaneous or intravenous injection was evaluated. Methods: Splenic B cells from GFP transgenic mice were activated with CD40L + IL-4 and expanded in vitro prior to i.v. or s.c. injection of 3–4 x 107 into C57BL/6 mice. Recipient mice were sacrificed 2 hrs or 1–14 days thereafter and the percentage of GFP+/B220+ B cells quantified in spleens and lymph nodes by flow cytometry. Localization of these cells within lymphoid organs was determined by immunohistochemistry. In some experiments, activated C57BL/6 B cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) to evaluate cell growth in vivo. Results: Murine B cell populations were readily expanded by culture on CD40L-transfected L cells in the presence of IL-4. CD40L-activated B cells expressed high levels of CD80, CD86, and LFA-1 but decreased levels of L-selectin relative to naive cells. Following i.v. injection, activated B cells were detected in spleens and lymph nodes within 1 day. Peak concentrations of activated B cells were noted in spleens and lymph nodes on days 7 (4.8% of injected cells) and 10 (1.25% of injected cells) respectively, suggesting expansion of the activated B cell population in vivo. Naive B cells injected i.v. were detected within 1 day but their number declined precipitously thereafter. Following s.c. injection, peak levels of CD40L-activated B cells were noted on day 5 (spleens) and day 7 (lymph nodes). As determined by immunohistochemistry, both CD40L-activated and naïve B cells injected i.v. appeared in B cell regions of spleens and lymph nodes. While the kinetics of accumulation of CD40L-activated B cells injected s.c. or i.v. were similar, s.c. injected CD40L-activated B cells homed to the T cell regions of spleens and lymph nodes. CFSE experiments indicated that these activated B cells continue to grow in vivo. In contrast, naïve B cells injected s.c. only appeared in B cell regions. Conclusion: CD40L-activated B cell populations can readily be expanded in vitro, CD40L-activated B cells migrate to secondary lymphoid organs even when injected s.c., activated B cell populations expand in vivo, and s.c. injected, CD40L-activated B cells preferentially home to T cell regions of secondary lymphoid organs. These results suggest that this effective APC may serve as an important vehicle for delivery and presentation of exogenous (e.g. tumor) antigens to T cells in vivo.

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The Role of B Cells and B Cell Therapies in Immune-Mediated Liver Diseases

Frontiers in Immunology ◽

10.3389/fimmu.2021.661196 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tamsin Cargill ◽

Emma L. Culver

Keyword(s):

B Cells ◽

B Cell ◽

Liver Diseases ◽

Current Knowledge ◽

Adaptive Immune System ◽

Immune Defense ◽

Future Research ◽

Primary Biliary Cholangitis ◽

Cell Therapies ◽

Immune Mediated

B cells form a branch of the adaptive immune system, essential for the body’s immune defense against pathogens. B cell dysfunction has been implicated in the pathogenesis of immune mediated liver diseases including autoimmune hepatitis, IgG4-related hepatobiliary disease, primary biliary cholangitis and primary sclerosing cholangitis. B cells may initiate and maintain immune related liver diseases in several ways including the production of autoantibodies and the activation of T cells via antigen presentation or cytokine production. Here we comprehensively review current knowledge on B cell mechanisms in immune mediated liver diseases, exploring disease pathogenesis, B cell therapies, and novel treatment targets. We identify key areas where future research should focus to enable the development of targeted B cell therapies.

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BAFF Promotes Heightened BCR Responsiveness and Manifestations of Chronic GVHD after Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.2020008040 ◽

2021 ◽

Author(s):

Wei Jia ◽

Jonathan C. Poe ◽

Hsuan Su ◽

Sarah Anand ◽

Glenn K. Matsushima ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Chronic Gvhd ◽

Allogeneic Bone Marrow Transplantation ◽

Cell Receptor ◽

Cell Number ◽

Allogeneic Bone ◽

Donor Cells

Chronic graft versus host disease (cGVHD) patients have increased B cell-activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major MHC-mismatched model with cGVHD-like manifestations we first examined B-lymphopenic mMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B cell number. Mice that later developed cGVHD, had significantly increased numbers of recipient fibroblastic reticular cells (FRCs) with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD since BAFF transcript in CD4+ T cells from diseased mice and patients was increased. Chronic GVHD manifestations in mice associated with high BAFF/B cell ratios and persistence of B Cell Receptor (BCR)-activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR-responsiveness to surrogate antigen and NOTCH ligand. BAFF-Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR-activation or when alloantigen was present in vivo. Using T-cell depleted (BM only) BAFF-Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells and alloantibody production. We demonstrate that pathological production of BAFF promotes an altered B-cell compartment and augments BCR-responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in cGVHD patients.

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The collaborative phenotype of secondary B cells is determined by T lymphocytes during in vivo immunization.

Journal of Experimental Medicine ◽

10.1084/jem.155.2.574 ◽

1982 ◽

Vol 155 (2) ◽

pp. 574-586 ◽

Cited By ~ 4

Author(s):

N A Speck ◽

S K Pierce

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Cell Populations ◽

Antibody Synthesis ◽

Humoral Immune ◽

Igg1 Antibody ◽

Humoral Immune Responses

Previous studies have demonstrated that the B cells in immune and nonimmune mice manifest different major histocompatibility complex (MHC) collaborative phenotypes with antigen-specific T cells. Immune, or secondary B cells require syngeneic-like MHC recognition by collaborating T cells, and in its absence fail to be stimulated. Primary B cells manifest a much less stringent requisite for MHC recognition by T cells, and under conditions in which secondary B cells fail to be stimulated, primary B cells are stimulated to secrete IgM antibody. Experiments were conducted to determine whether the acquisition of the secondary B cells' MHC collaborative phenotype was dependent on the presence of T cells during in vivo immunization. B cell populations from T dependently and T independently immunized conventional BALB/c and athymic BALB/c nu/nu mice were compared in their ability to collaborate with allogeneic T cells. Although antigen alone promotes the differentiation of several secondary B cell characteristics, including an increase in the frequency of antigen-specific B cells and a preference for IgG1 antibody synthesis in vitro, the acquisition of the secondary B cells' MHC collaborative phenotype was dependent on the presence of T cells during in vivo immunization. B cell populations from T dependently and T independently immunized conventional BALB/c and athymic BALB/c nu/nu mice were compared in their ability to collaborate with allogeneic T cells. Although antigen alone promotes the differentiation of several secondary B cell characteristics, including an increase in the frequency of antigen-specific B cells and a preference of IgG1 antibody synthesis in vitro, the acquisition of the secondary B cells' MHC collaborative phenotype was found to be dependent on the presence of T cells during in vivo immunization. Thus, the restriction imposed on T cell-B-cell-collaborative interactions in secondary humoral immune responses appears to be the result of T dependent antigen-driven events.

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Recovery of B-cell homeostasis after rituximab in chronic graft-versus-host disease

Blood ◽

10.1182/blood-2010-10-307819 ◽

2011 ◽

Vol 117 (7) ◽

pp. 2275-2283 ◽

Cited By ~ 83

Author(s):

Stefanie Sarantopoulos ◽

Kristen E. Stevenson ◽

Haesook T. Kim ◽

Whitney S. Washel ◽

Nazmim S. Bhuiya ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Graft Versus Host Disease ◽

Cell Number ◽

Cell Recovery ◽

Cell Homeostasis ◽

Host Disease ◽

Graft Versus Host ◽

Cell Numbers ◽

B Cell Homeostasis

Abstract Investigation of the effects of rituximab (anti-CD20) on B-cell-activating factor of the tumor necrosis factor family (BAFF) and B cells would better define the significance of B-cell homeostasis in chronic graft-versus-host disease (cGVHD) pathophysiology. We studied 20 cGVHD patients at a median of 25 months after rituximab treatment when most patients had recovered total B-cell numbers. A total of 55% of patients had stable/improved cGVHD, and total B-cell numbers in these patients were significantly higher compared with rituximab-unresponsive patients. Although total B-cell number did not differ significantly between cGVHD groups before rituximab, there was a proportional increase in B-cell precursors in patients who later had stable/improved cGVHD. After rituximab, BAFF levels increased in all patients. Coincident with B-cell recovery in the stable/improved group, BAFF/B-cell ratios and CD27+ B-cell frequencies decreased significantly. The peripheral B-cell pool in stable/improved cGVHD patients was largely composed of naive IgD+ B cells. By contrast, rituximab-unresponsive cGVHD patients had persistent elevation of BAFF and a predominance of circulating B cells possessing an activated BAFF-RLoCD20Lo cell surface phenotype. Thus, naive B-cell reconstitution and decreased BAFF/B-cell ratios were associated with clinical response after rituximab in cGVHD. Our findings begin to delineate B-cell homeostatic mechanisms important for human immune tolerance.

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T Cell Pathway Deficiencies in Chronic Lymphocityc Leukemia: Partial Restoration with OKT3/IL-2 Activation.

Blood ◽

10.1182/blood.v110.11.4692.4692 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4692-4692

Author(s):

Mauro Di Ianni ◽

Lorenzo Moretti ◽

Beatrice Del Papa ◽

Maria De Ioanni ◽

Adelmo Terenzi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

Activated T Cells ◽

Mutational Status ◽

The Impact

Abstract As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p<0.05) in CD3+ cells from CLL patients compared with healthy donors. Panther software analysis identified 34/237 upregulated genes and 26/221 downregulated genes that were involved in specific pathways, mainly cell differentiation and proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T cell activation. The 26 dowregulated genes included Zap70, a member of the syk family protein tyrosine kinase, which is involved in T-cell activation. Zap-70 results were validated by mRNA quantification by RT-PCR (−1.77 fold in comparison with healthy controls) and by flow-cytometric analysis (Mean Intensity Fluorescence=33±12 vs 80±23.62 in controls, p<0.05). To test the hypothesis that activation with OKT3 /IL-2 could bypass these T cell deficiencies, activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity (using the 51chromium release assay) against mutated and unmutated (according to IgVH mutational status) autologous B cells, DAUDI, K562 and P815 cell lines. After 10 days’ culture, the T cell count remained unchanged; CD8 cells expanded more than CD4; TCR spectratyping analysis indicated no differences in TCR repertoires. Activation restored the ZAP-70 mRNA (+1.67 fold). The 51chromium release cytotoxicity assay showed an index > 30% in 5/20 patients. The other 15 were partially cytotoxic against P815, K562 and Daudi. Cell line analysis in all 20 confirmed prevalently T cell-mediated cytotoxicity and poor NK/LAK activity. Cytotoxicity did not correlate with B cell mutational status. We tested the cytotoxic activity of autologous activated T cells in NOD/SCID mice co-transplanted with leukaemic B cells. Only activated T cells exerting cytotoxicity vs autologous B-cell CLL prevent CLL in human-mouse chimera, as confirmed by PCR and FACS analysis which visualised only CD3+ cells. In conclusion, in patients with CLL, activating autologous T cells with OKT3 /IL-2 bypasses, at least in part, the T cell immunological deficiencies. These in vitro and in vivo findings might serve to throw light on new mechanisms that could be exploited in immunotherapy designed to exert disease control.

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Patients With DLBCL Commonly Have B-Cell Lymphopenia and Circulating Clonal B-Cell Populations With a Phenotype Concordant With The Underlying Tumour

Blood ◽

10.1182/blood.v122.21.3013.3013 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3013-3013

Author(s):

Ruth M de Tute ◽

Sharon Barrans ◽

Andy C. Rawstron ◽

Peter W.M. Johnson ◽

Andrew J Davies ◽

...

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Gene Expression Profiling ◽

Peripheral Blood ◽

Expression Profiling ◽

Common Finding ◽

Cell Populations ◽

The Impact

Abstract Clonal B-cell populations with either a CLL or a non-CLL phenotype are a common finding in normal individuals but uncertainty remains about how this relates to the development of clinically significant disease. The aim of this study was to investigate the frequency of peripheral blood clonal B-cell populations and B-cell subset abnormalities in newly presenting DLBCL patients and to determine whether the incidence of these abnormalities differed between the GCB and ABC subtypes, which are regarded as having distinct pathogenesis. The study was carried out using peripheral blood samples collected from patients entered in the UK-REMoDL-B trial. This trial is testing the hypothesis that the ABC subtype of DLBCL responds preferentially to R-CHOP- Bortezomib. Gene expression profiling is performed on the diagnostic tissue biopsy (FFPE) using the Illumina WG-DASL assay prior to randomisation classified as GCB, ABC or unclassified (UN). The availability of GEP data allows meaningful comparison with the phenotype of clonal populations detected by flow cytometry. Peripheral blood taken prior to first treatment was analysed using multi-colour flow cytometry. Following red cell lysis with ammonium chloride, samples were incubated with a panel of antibodies comprising of a CD19 and CD20 backbone, with Kappa, Lambda, CD5, CD45, CD49d, LAIR-1, CXCR5, CD31, CD95, CD38 and CD10, supplemented in some cases by CD81, CD79b, and CD43. A minimum of 500,000 events were acquired on a FacsCanto II flow cytometer (Becton Dickinson). B-cells were enumerated and any monoclonal populations identified were classified as CLL, germinal centre (GC), non-GC or not otherwise specified (NOS) where the phenotype was indeterminate. 358 samples were eligible for inclusion from patients with an average age of 62.2years (range 22.9-86.1). Abnormalities were detected in 52% of cases (B-lymphopenia ((<0.06 x 109/l) 33%, B-lymphocytosis (>1 x 109/l) 2.8%, CLL clone 3.6%, GC clone 9.8%, non-GC clone 9.8%, clonal population NOS 2.2%). Gene expression profiling results were available for 278 individuals; 51% GCB, 32% ABC and 17% unclassified. The relationship between peripheral blood B-cell findings and the GEP determined phenotype of the tumour is shown in the table:TableB-lymphopeniaCLL CloneMonoclonal GC typeMonoclonalNon-GC typeMonoclonal NOSNormalB-cellGCB n=14241/142 (29%)5/142 (3.5%)21/142 (15%)8/142 (5.6%)2/142 (1%)72/142 (51%)ABC n=8927/89 (30%)2/89 (2%)2/89 (2%)12/89 (13.5%)2/89 (2%)49/89 (55%)Unclassified n=4726/47 (55%)0/50 (0%)2/47 (4%)6/47 (12%)6/47 (5%)14/47 (30%) In patients where clonal populations were detected in the peripheral blood there was striking concordance between the phenotype of the clone and the GEP of the underlying tumour. Presence of a GC-population by flow was highly predictive of GCB GEP (84% GC–type populations detected were in GCB cases). The number of discordant cases and the number of CLL clones detected approximate to the numbers that would be expected in a normal population of a similar age. It is, therefore, likely that in most cases circulating tumour cells or a closely related precursor clone are being detected. The similarity between the results of the ABC and unclassified GEP groups suggest that these are biologically related. An unexpected finding in this study was the high incidence of B-lymphopenia at a level that might be expected to be associated with increased risk of infection. This may reflect suppression of normal B-cells by the neoplastic clone or be a marker of underlying immune dysfunction that may predispose to the development of the tumour. Immuosuppression has a role in the pathogenesis of DLBCL in the elderly and this study suggests that this may also be a factor in the wider patient population. These results may have implications for prognostic assessment and may offer opportunities for early diagnosis and possibly response assessment in some patients. The impact on outcome will be assessed in the course of the trial. Disclosures: Jack: Roche /Genentech: Research Funding.

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