Inhibition of Antibody Production in Vitro with Bispecific CD20 X CD95 Antibodies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1114-1114
Author(s):  
Kristina Nalivaiko ◽  
Martin Hofmann ◽  
Ludger Grosse-Hovest ◽  
Peter H Krammer ◽  
Hans-Georg Rammensee ◽  
...  
Keyword(s):  
B Cells ◽  
Autoimmune Disease ◽  
B Cell ◽  
Antibody Production ◽  
Conflicts Of Interest ◽  
Death Receptor ◽  
Pokeweed Mitogen ◽  
Resting Cells ◽  
Cd20 Antibodies

Abstract Abstract 1114 Antibodies directed against the B-cell associated CD20 surface antigen can target normal as well as malignant B cells. They are sucessfully used for the treatment of B-cell derived leukemia and lymphoma and antibody mediated autoimmune disease, respectively. We have previously described that bispecific antibodies with specificity for CD20 and the death receptor CD95 are capable of inducing CD95 mediated apoptosis selectively in CD20-positive lymphoma cells. We now show that CD20 X CD95 hybrid antibodies induce apoptosis in pokeweed mitogen (PWM) activated B cells expressing CD95, but not in resting cells lacking it. Antibody production induced by PWM in vitro is profoundly inhibited. These results indicate that bispecific CD20 X CD95 antibodies may be used for the treatment of antibody mediated autoimmune disease. Compared to monospecific CD20 antibodies these reagents offer a new effector principle and specificity for activated rather than resting B cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evidence for enhanced Bruton’s tyrosine kinase activity in transitional and naïve B cells of patients with granulomatosis with polyangiitis

Rheumatology ◽  
2019 ◽  
Vol 58 (12) ◽  
pp. 2230-2239
Author(s):  
Anouk von Borstel ◽  
Wayel H Abdulahad ◽  
Jan Stephan Sanders ◽  
Jasper Rip ◽  
Stefan F H Neys ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Antibody Production ◽  
Granulomatosis With Polyangiitis ◽  
Cytokine Production ◽  
B Cell Subsets ◽  
Auto Antibody ◽  
Subset Distribution ◽  
Cell Cytokine Production

Abstract Objectives To determine Bruton’s tyrosine kinase (BTK) protein and phosphorylation levels in B cell subsets of granulomatosis with polyangiitis (GPA) patients and to investigate the effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production. Methods BTK protein and phosphorylation levels were determined by flow cytometry in peripheral blood B cells of 29 untreated GPA patients [9 active and 20 remission GPA patients (10 ANCA– and 10 ANCA+)], 9 age- and sex-matched healthy controls (HCs) and 9 untreated active RA patients. The effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production was determined in the same donors in peripheral blood mononuclear cell cultures. Results BTK protein levels were significantly increased in transitional and naïve B cells of active GPA and RA patients compared with remission GPA patients and HCs. Both B cell subsets of active patients were more sensitive to B cell receptor stimulation, as BTK and phospholipase Cγ2 phosphorylation were increased in these patients. In vitro BTK blockade had profound effects on B cell cytokine production, plasma cell formation and (auto)antibody production in both GPA patients and HCs. Interestingly, the effect of BTK blockade was less pronounced in active GPA patients, possibly due to increased activation of B cells. Conclusion We show that BTK protein and phosphorylation levels are most profoundly increased in newly emerging B cells of active GPA patients compared with remission patients. BTK blockade greatly inhibits in vitro B cell effector functions in GPA patients and HCs. These promising data identify BTK as an interesting novel therapeutic target in the treatment of GPA.

Mucosal Epithelial Cells Initiate Frontline Immunoglobulin Class Switching through an SLPI-Regulated Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3898-3898
Author(s):  
Andrea Cerutti ◽  
Bing He ◽  
April Chiu ◽  
Meimei Shan ◽  
Paul Santini ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
B Cells ◽  
Epithelial Cells ◽  
B Cell ◽  
Antibody Production ◽  
Cytidine Deaminase ◽  
Dna Recombination ◽  
Class Switching ◽  
Class Switch

Abstract Introduction. Class switching from IgM to IgG and IgA is central to immunity against microbes and usually occurs in draining lymph nodes and requires activation of B cells by CD4+ T cells expressing CD40 ligand. Growing evidence indicates that B cells can mount frontline IgG and IgA responses at mucosal sites of entry through an alternative CD40-independent pathway involving B cell-activating factor of the TNF family (BAFF, also known as BLyS) and a proliferation-inducing ligand (APRIL). These innate factors are usually produced by dendritic cells and stimulate B cells through at least three distinct receptors. Together with dendritic cells, epithelial cells have a key position at the host-environment interface. Therefore, we asked whether epithelial cells play a role in frontline antibody production. Methods. Tonsillar tissue sections from healthy donors were analyzed for expression of activation-induced cytidine deaminase (AID) by immunohistochemistry and in situ hybridization. A simplified in vitro model reproducing the geometry of mucosal surfaces was used to evaluate the role of epithelial cells in class switching. Briefly, primary epithelial cells and B cells were cultured in the upper and lower chambers, respectively, of a trans-well system. Monocyte-derived dendritic cells were positioned on a filter separating the two chambers. Various microbial product analogues were used to mimic infection. RNA interference was performed to knockdown BAFF in epithelial cells. AID expression, CSR, antibody production and signaling were evaluated in B cells as reported (Litinsky et al., Nat. Immunol.2002, 3:822–829; Qiao et al., Nat. Immunol.2006, 7:302–310). Results. We found that the upper respiratory mucosa of healthy subjects comprised intraepithelial pockets filled with B cells expressing AID, a DNA-editing enzyme associated with ongoing class switch DNA recombination (CSR). Epithelial cells released innate class switch-inducing factors, including BAFF, after sensing microbial products through TLRs, thereby inducing AID expression, CSR, and ultimately IgG and IgA production in neighboring B cells. Epithelial cell-induced antibodies comprised polyreactive IgG and IgA capable of recognizing multiple microbial determinants. Intraepithelial class switching was enhanced by thymic stromal lymphopoietin (TSLP), an epithelial IL-7-like cytokine that augments the innate B cell-licensing functions of dendritic cells, and restrained by secretory leukocyte protease inhibitor (SLPI), an epithelial alarm antiprotease that suppresses AID expression in activated B cells. Conclusions. The present findings indicate that epithelial cells function as non-immune sentinels capable to autonomously orchestrate compartmentalized IgG and IgA responses at the interface between host and environment. This implies that mucosal vaccines should activate both epithelial and immune cells to elicit optimal antibody production.

Soluble Bcma in Myeloma Serum Binds Its Ligands BAFF and Prevents Normal Antibody Production in Multiple Myeloma Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1799-1799
Author(s):  
Eric Sanchez ◽  
Abigail Gillespie ◽  
Nika Manik Harutyunyan ◽  
George Tang ◽  
Jillian Gottlieb ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Antibody Production ◽  
Conflicts Of Interest ◽  
Scid Mice ◽  
Response To Treatment ◽  
Control Group ◽  
Raji Cells

Abstract Introduction: A hallmark of multiple myeloma (MM) is the low levels of uninvolved immunoglobulin (Ig) levels. B-cell maturation antigen (BCMA) is a receptor expressed in mature non-malignant and malignant B lymphocytes, including plasma cells. Its ligands are B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL). We previously demonstrated that BCMA is present in the serum of MM patients (pts) and that its levels predict survival (Sanchez et al. Br J Haematol 2012). We hypothesized that circulating BCMA binds it ligands, preventing normal plasma cell development in MM patients which may explain their reduction in uninvolved Ig levels. Methods: BCMA-Fc and control Ig were obtained and reconstituted in PBS (R&D Systems). Retro-orbital bleeds were performed on C57 Bl/6 and SCID mice implanted with the human MM xenografts LAGλ-1, LAGk-1A or LAGk-2. Human BCMA and mouse BAFF, IgM, IgA and IgG levels were measured with ELISA (R&D Systems & Bethyl Laboratories). The Raji B-cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). Human IgA and IgG levels were determined in MM patients using nephelometry (Immage 800, Beckman Coulter). Hevylite® Assays (Binding Site) were used to quantify the levels of heavy-light chain isoform pairs in MM patients. Results: We determined if mBAFF formed complexes with human BCMA (hBCMA) in the plasma from SCID mice implanted with LAGλ-1, LAGκ-2 or LAGκ-1A, and were able to identify mBAFF-hBCMA complexes in plasma samples from these mice. To determine what effect human BCMA had on Ig levels in immune competent mice, rhBCMA-Fc or control Ig-Fc (100 mg) was injected into C57 Bl/6 mice, and plasma IgA, IgM and IgG levels were measured. Decreases in IgA levels were observed following BCMA treatment when compared to baseline plasma IgA on days 4 and 6 (P = 0.0031 and P = 0.0064, respectively), and the control group (P = 0.0087 and P = 0.0221). Samples were also analyzed for mouse IgM levels with similar marked reductions when compared to the untreated (P = 0.0001) and Ig-Fc (P = 0.0088) groups. For plasma IgG levels, a marked decrease was observed on day 6 following rhBCMA-Fc administration when compared to its baseline levels (P = 0.0023), and also when compared to the Ig-Fc control protein (P = 0.0014) and the untreated control (P = 0.0129) groups. We then set out to determine if sera from MM patients contained BCMA-BAFF complexes, using ELISA plates coated with an anti-human BAFF antibody followed by exposure to a polyclonal anti-human BCMA antibody. A strong absorbance indicating the presence of BCMA-BAFF complexes was detected in serum samples from MM patients, whereas no antibody cross reactivity was observed in control samples. We also determined whether human MM serum or rhBCMA-Fc blocked BAFF from binding to Raji B-cells. Raji cells (B-cell line) were incubated with serum from a MM patient containing hBCMA (0.75μg/ml) or rhBCMA-Fc (3 μg/ml) in the presence of rhBAFF (500 ng/ml). Myeloma serum and rhBCMA-Fc decreased rhBAFF binding to Raji cells by 71% (from 96.8 to 25.6 %) and 74% (from 96.8 to 22.9 %), respectively. Next, we determined whether serum BCMA levels inversely correlated with uninvolved Ig levels in MM pts. For pts with IgA (n = 134) or IgG (n = 313) MM, higher BCMA levels (> 100 ng/ml) correlated with below normal levels of uninvolved IgG in IgA MM and uninvolved IgA in IgG MM, whereas lower BCMA levels (< 100 ng/ml) correlated with normal uninvolved levels (P < 0.0001). Using the Hevylite Assay, similar results were observed for the levels of BCMA compared to uninvolved IgG isoforms in both pts with involved IgG lambda (n = 62, P = 0.0006) and IgG kappa (n = 117, P < 0.0001) MM. Conclusions: Our laboratory previously has reported that serum levels of BCMA are increased in the serum of MM patients, and correlates with response to treatment and predicts survival. We now demonstrate 1) the formation of complexes of BCMA with its B-cell ligand BAFF in the plasma of MM xenografts and sera of MM patients, 2) show that rhBCMA and MM patient serum blocks the binding of BAFF to human B-cells, 3) administration of rhBCMA to normal mice results in marked reductions in their antibody levels, and 4) show that BCMA levels inversely correlate with uninvolved Ig levels in MM pts. Thus, the lack of normal antibody production in MM pts results, in part, from circulating BCMA binding its ligands, preventing production of normal antibody-producing cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Enhanced B Cell Expansion, Survival, and Humoral Responses by Targeting Death Receptor 6

2002 ◽  
Vol 197 (1) ◽  
pp. 51-62 ◽  
Author(s):  
Clint S. Schmidt ◽  
Jinqi Liu ◽  
Tonghai Zhang ◽  
Ho Yeong Song ◽  
George Sandusky ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Expansion ◽  
Death Receptor ◽  
Immunoglobulin M ◽  
Type I ◽  
Factor C

Targeted disruption of death receptor (DR)6 results in enhanced CD4+ T cell expansion and T helper cell type 2 differentiation after stimulation. Similar to T cells, DR6 is expressed on resting B cells but is down-regulated upon activation. We examined DR6−/− B cell responses both in vitro and in vivo. In vitro, DR6−/− B cells undergo increased proliferation in response to anti–immunoglobulin M, anti-CD40, and lipopolysaccharide. This hyperproliferative response was due, at least in part, to both increased cell division and reduced cell apoptosis when compared with wild-type B cells. Consistent with these observations, increased nuclear levels and activity of nuclear factor κB transcription factor, c-Rel, and elevated Bcl-xl expression were observed in DR6−/− B cells upon stimulation. In addition, DR6−/− B cells exhibited higher surface levels of CD86 upon activation and were more effective as antigen-presenting cells in an allogeneic T cell proliferation response. DR6−/− mice exhibited enhanced germinal center formation and increased titers of immunoglobulins to T-dependent as well as T-independent type I and II antigens. This is the first demonstration of a regulatory role of DR6 in the activation and function of B cells.

Examination of the Relative Rates of Rituximab-Mediated Loss of CD20 From B Cells Via Trogocytosis Versus Internalization. Implications for Rituximab Therapy of CLL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2404-2404
Author(s):  
Ronald P Taylor ◽  
Paul V Beum ◽  
Margaret A Lindorfer ◽  
Elizabeth M Peek
Keyword(s):  
B Cells ◽  
Cell Surface ◽  
B Cell ◽  
Transgenic Mouse ◽  
Conflicts Of Interest ◽  
Cell Clone ◽  
Signal Molecules ◽  
Human Igg ◽  
Anti Cd20

Abstract Abstract 2404 The use of the anti-CD20 monoclonal antibody (mAb) rituximab (RTX) in the immunotherapy of cancer has led to substantial advances in the treatment of B cell malignancies. However, many patients are refractory to RTX therapy and a substantial fraction of responding patients suffer relapses. In some patients the loss or lack of efficacy can be explained because levels of CD20 are substantially reduced on target B cells as a consequence of RTX therapy. This phenomenon may be due to growth and proliferation, over a period of at least several months, of a CD20-negative B cell clone. In contrast, we have reported that levels of CD20 expressed on circulating CLL cells can be reduced > 90% in less than one hour following RTX infusion. These clinical observations, as well as our results seen in vitro and in mouse models, suggest that after the body's effector mechanisms that promote mAb-mediated cell killing are exhausted or saturated, an alternative reaction predominates. RTX-CD20 complexes on targeted B cells are rapidly removed from the B cells by monocytes and macrophages through an Fc receptor-mediated endocytic process known as trogocytosis, or the shaving reaction. Beers et al. recently reported (Blood, 2010) that human CD20 transgenic mouse B cells opsonized with Rit m2a, a mouse IgG2a mAb with RTX activity, internalized both bound mAb and up to half of cell-surface CD20 after incubation for 2 hrs at 37C. They also reported that on average 40% of cell-surface CD20 was internalized when CLL cells opsonized with RTX were similarly treated. It would thus appear that as a consequence of RTX therapy, both trogocytosis by effector cells and direct internalization by B cells can promote loss of CD20 from B cells targeted by RTX. Both of these reactions will reduce the efficacy of RTX therapy, and a key question must focus on the relative rates of trogocytosis and internalization in promoting CD20 loss in vivo. To address these questions we examined human CD20 transgenic mouse 38C13 B cells (a kind gift from Dr. Josee Golay) for RTX-mediated CD20 internalization versus shaving. To test for internalization of cell-bound RTX, CD20+ 38C13 cells were reacted with Al488- or biotinylated RTX for 10 min to 2 hours at 37C, then washed and secondarily probed with the following reagents: 1, Al647 mAb HB43, specific for the Fc region of human IgG; 2, Al488-streptavidin to detect surface-bound biotinylated RTX; 3, Anti-Al488 antibodies that induce quenching of surface-bound Al488 RTX; or 4, The cells were subjected to an acid wash, re-equilibrated at neutral pH, and re-tested for Al488 RTX binding. Our flow cytometry results reveal rapid and stable binding of Al488-labeled or biotinylated RTX to the CD20+ 38C13 cells. After two hours at 37C, the amount of cell-bound RTX that had been internalized, defined by the 4 different assays, was much less than that reported by Beers, and ranged between 5 and 25%, suggesting that the degree of internalization may be model-dependent. Moreover, reaction of acceptor THP-1 cells (a monocytic line) with the RTX-opsonized cells leads to rapid shaving and removal of ~ 90% of bound RTX and CD20 from the 38C13 cells. After just 45 min at 37C in the presence of adhered THP-1 monocytes, there is a 10-fold reduction in fluorescence intensity signal (molecules of equivalent soluble fluorochrome) on the 38C13 cells attributable to either bound Al488 RTX or due to secondary development with Al647 anti-human IgG mAb HB43. Alternatively, Al488 RTX-opsonized CD20+ 38C13 cells were reacted in solution with THP-1 monocytes at varying THP-1/38C13 cell ratios. Within 45 min at 37C at a 3/1 ratio more than half of cell bound Al488 RTX and CD20 were removed from the 38C13 cells, and large amounts of Al488 RTX were found on the acceptor THP-1 cells. Our results indicate that the shaving reaction is considerably more rapid and indeed leads to the removal of much more bound anti-CD20 mAb RTX (and CD20) than the internalization reaction, suggesting that the shaving reaction is most likely primarily responsible for the rapid down-modulation of CD20 that is observed when CLL patients are treated with RTX. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeting autoantigen to B cells prevents the induction of a cell-mediated autoimmune disease in rats.

1992 ◽  
Vol 175 (3) ◽  
pp. 655-659 ◽  
Author(s):  
M J Day ◽  
A G Tse ◽  
M Puklavec ◽  
S J Simmonds ◽  
D W Mason
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Autoimmune Disease ◽  
Antibody Production ◽  
Specific Antigen ◽  
Reciprocal Relationship ◽  
Delayed Type Hypersensitivity ◽  
A Cell

Immunization protocols that induce high levels of delayed-type hypersensitivity are often associated with low levels of antibody production, whereas alternative immunization strategies can produce the opposite effect. This reciprocal relationship appears to depend, at least in part, on the fact that T cell-derived lymphokines that are predominantly involved in one type of response inhibit the development of those T cells that promote the alternative one. Such a regulatory mechanism is likely to be bistable in that whenever one form of response is established, spontaneous development of the alternative one will be inhibited. We have applied this concept to the control of a cell-mediated autoimmune disease in rats. By covalently linking the autoantigen to anti-IgD antibody, we have targeted it to B cells for presentation to antigen-specific T cells. This form of presentation favors antibody production and may be expected to antagonize the cell-mediated disease-inducing response to the same antigen. To test this hypothesis, use was made of the fact that experimental allergic encephalomyelitis (EAE), when induced with the encephalitogenic peptide of guinea pig myelin basic protein, is purely a cell-mediated disease. The experiments show that Lewis rats, immunized with the peptide in its encephalitogenic form, were protected from disease when simultaneously injected with the peptide coupled to anti-IgD monoclonal antibodies. Control experiments showed that neither peptide nor anti-IgD alone were protective, and the peptide covalently coupled to irrelevant antibodies also failed to protect. Spleen cells from animals protected from disease by the anti-IgD-peptide conjugate, when activated in vitro with the encephalitogen, were able to transfer EAE to naive recipients. The results demonstrate that a cell-mediated immune response can be controlled by appropriate targeting of the specific antigen without inducing T cell anergy and suggest a potential strategy for preventing autoimmune diseases that are essentially cell-mediated in type.

The MiR-23a MicroRNA Cluster Inhibits B Cell Development.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1465-1465
Author(s):  
Jason Mullenix ◽  
Kimi Y Kong ◽  
Kristin Severns Owens ◽  
Jason Rogers ◽  
Shannon FitzPatrick ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Mature Mirnas ◽  
Cell Development ◽  
B Cell Development

Abstract Abstract 1465 Poster Board I-488 The miR-23a microRNA (miRNAs) cluster inhibits both [ITALIC]in vitro[/ITALIC] and [ITALIC]in vivo[/ITALIC] B cell development. When murine hematopoietic progenitor cells expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed over a five-fold decrease in the generation of CD19+ B cells compared to control cultures. Conversely, we observed over a five-fold increase in CD11b+ myeloid cells. When irradiated mice were transplanted with bone marrow expressing the miR-23a cluster we observed a two-fold decrease in bone marrow and splenic B cells, 8 weeks post-transplant compared to control mice. The miR-23a cluster codes for a single pri-transcript, which when processed yields three mature miRNAs: miR-23a, miR-27a, and miR-24-2. All three mature miRNAs are more abundant in myeloid cells compared to other hematopoietic cells. In vitro miR-24 alone is necessary and sufficient to inhibit B cell development. The promoter for the cluster contains conserved binding sites for the essential myeloid transcription factors PU.1 and C/EBP alpha. Chromatin immunoprecipitations demonstrated that PU.1 and C/EBP alpha are associated with the promoter in myeloid cells. In addition, C/EBP alpha is bound to several highly conserved regions upstream of the promoter. Both PU.1 and C/EBP alpha promote myeloid development at the expense of lymphopoiesis. Our work suggests that the miR-23a cluster may be a critical downstream target of PU.1 and C/EBP alpha in the specification of myeloid cell fate. Although miRNAs have been identified downstream of PU.1 and C/EBP alpha in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in the regulating lymphoid cell fate acquisition. We are currently identifying targets of miR-24 that may mediate the inhibitory effect on B lymphopoiesis. Disclosures No relevant conflicts of interest to declare.

ROR1 Can Interact With TCL1 and Enhance Leukemogenesis In Eµ-TCL1 Transgenic Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 868-868
Author(s):  
George F. Widhopf ◽  
Bing Cui ◽  
Emanuela M. Ghia ◽  
Liguang Chen ◽  
Karen Messer ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Ki 67 ◽  
Pathway Network ◽  
Intravenous Injections ◽  
Z Scores

Abstract ROR1 is an onco-embryonic antigen found on chronic lymphocytic leukemia (CLL) B cells, but not on normal adult tissues. We generated B6 transgenic (Tg) mice with human ROR1 regulated by the murine Ig promoter/enhancer (ROR1 Tg). Such animals had B-cell-restricted expression of ROR1 and infrequently developed ROR1+/CD5+/B220low leukemia resembling human CLL at ≥15 months of age. The leukemia cells of these animals had phenotypic features in common with those of the leukemia that originates in B6 Eµ-TCL1-Tg (TCL1) mice, which develop a CLL-like disease at ≥ 9 months of age. However, in contrast to human CLL, the leukemia that develops in TCL1 Tg mice does not express ROR1, indicating that expression of this antigen is not necessary for leukemogenesis. However, in immune-precipitation and mass spectrometry studies we found that ROR1 could complex with TCL1 in human CLL cells. TCL1 is a proto-oncogene product that can serve as a co-activator of AKT. To investigate whether expression of ROR1 could affect the biology of the leukemia that develops in TCL1 Tg mice, we crossed our ROR1 Tg animals with TCL1-Tg mice. Progeny with both transgenes (ROR1xTCL1) developed CD5+/B220low B-cell leukemia at a significantly younger median age than did littermates with either transgene alone. ROR1xTCL1 leukemia B-cells had higher levels of pAKT than TCL1 leukemia-cells and expressed high-levels of human ROR1, which we also found could complex with TCL1. Exploratory subnetwork analyses of transcriptome microarray data on isolated leukemia cells using Ingenuity Pathway network tools revealed 51 subnetworks that were expressed at different levels between the two types of leukemia, 21 of which had z scores in excess of 0.8, and an associated functional annotation with a false-discovery-rate (FDR) of less than 0.05. This analysis implied that ROR1xTCL1 leukemia cells had higher expression of subnetworks implicated in embryonic and tumor-cell proliferation, but lower expression of subnetworks involved in cell-cell adhesion or cell-death, than did TCL1 leukemia-cells. ROR1xTCL1 leukemia-cells also had higher proportions of Ki-67-positive cells, lower proportions of cells undergoing spontaneous apoptosis, and produced more aggressive disease upon adoptive transfer than TCL1 leukemia-cells. We examined the activity of two different mouse anti-human ROR1 mAbs, D10 and 4A5, which bind to distinct non-overlapping epitopes of ROR1, as assessed in cross-blocking studies. Treatment of ROR1xTCL1 leukemia cells with D10 in vitro resulted in reduced expression of pAKT within 1 hour after addition of the antibody to the leukemia cells, an effect that was not apparent in control Ig or 4A5-treated cells. We treated ROR1 Tg mice engrafted with CD5+/B220low/ROR1+ leukemia cells with intravenous injections of D10, 4A5, or control mouse IgG (mIgG), at 10 mg/kg. At five weeks, mice given D10 in one representative experiment had significantly fewer CD5+/B220low leukemia cells (2.4 ± 1.0 x106, n=3) in the blood than mice that received mIgG (2.0 ± 0.3 x107, n=3, p=0.032), whereas the number of leukemia cells in the blood of mice given 4A5 (1.6 ± 0.3 x107, n=3, p>0.05) was not significantly different than that of mice treated with mIgG. Furthermore, mice that received CD5+/B220low/ROR1+ B cells and that were treated with D10 had significantly smaller spleens than mice that were treated with mIgG or 4A5. Although D10 was effective in inhibiting the engraftment of ROR1xTCL1 leukemia cells, administration of either anti-ROR1 mAb had no effect on the endogenous non-leukemia (CD5-/B220Hi/ROR1+) B cells or T cells, or on engraftment of leukemia cells from TCL1 Tg mice, which do not express ROR1. Our data demonstrate that ROR1 accelerates progression of TCL1 leukemia and may be a target for therapy of patients with CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human immune response to group A streptococcal carbohydrate (A-CHO). I. Quantitative and qualitative analysis of the A-CHO-specific B cell population responding in vitro to polyclonal and specific activation.

1985 ◽  
Vol 161 (3) ◽  
pp. 547-562 ◽  
Author(s):  
F Emmrich ◽  
B Schilling ◽  
K Eichmann
Keyword(s):  
Immune Response ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Pokeweed Mitogen ◽  
Specific Antibodies ◽  
Group A ◽  
Precursor B Cells

The immune response to the group-specific carbohydrate of group A streptococci (A-CHO) provides an informative in vitro model for the investigation of several aspects of human anticarbohydrate immune responses. A-CHO-specific B cells can be polyclonally activated by pokeweed mitogen (PWM), and, specifically, by in vitro immunization with streptococcal vaccine. High levels of A-CHO-specific antibodies, mainly directed to the immunodominant side chain N-acetyl-D-glucosamine (GlcNAc), occur in healthy adult individuals. Serum antibody levels are reflected in high frequencies of precursor B cells among peripheral blood lymphocytes. In one particular case, greater than 15% of all B cells activated by PWM for IgM production were found to produce IgM anti-A-CHO antibodies, as determined in limiting dilution experiments, as well as by analyzing Ig concentrations in bulk culture experiments. The case with the lowest proportion observed had 0.3% A-CHO-specific B cells among IgM-producing B cells. Preferential PWM activation of anti-A-CHO-producing B cells could be excluded. The comparison of the proportions of anti-A-CHO IgM produced in vivo, and of B cells producing antibodies of this specificity in peripheral blood, suggests a similar distribution of specific precursor B cells in the antibody-producing lymphoid tissue compartments and in peripheral blood. However, nearly all specific antibodies produced in vitro belong to the IgM isotype, whereas IgG anti-A-CHO in high amounts, mostly exceeding the specific IgM, was found only among anti-A-CHO antibodies produced in vivo. Low anti-A-CHO IgG production was seen in polyclonally activated as well as in antigen-activated cultures, whereas, in contrast, total IgG was produced in considerable amounts after polyclonal activation. This suggests a different distribution pattern, and/or diverse differentiation requirements for anti-A-CHO-producing B cells, compared with other B cell species.

scholarly journals Cell-to-cell interaction controlled by immunoglobulin genes. Role of Thy-1-, Lyt-1+, Ig+ (B') cell in allotype-restricted antibody production.

1982 ◽  
Vol 156 (2) ◽  
pp. 443-453 ◽  
Author(s):  
K Okumura ◽  
K Hayakawa ◽  
T Tada
Keyword(s):  
T Cells ◽  
B Cells ◽  
Antibody Response ◽  
B Cell ◽  
Antibody Production ◽  
Helper T Cells ◽  
Secondary Antibody ◽  
T And B Cells ◽  
Athymic Nude Mice

A novel lymphocyte subpopulation, designated "B' cell" because of its characteristic dual expression of Ig and Lyt-1 antigen, is described in relation to its ability to augment the in vitro secondary antibody response. The cells are found in the spleens of normal unprimed mice as well as those of athymic nude mice and represent a small of normal unprimed mice as well as those of athymic nude mice and represent a small number (2-3%) of immunoglobulin-positive cells. No other distinguishing surface markers of conventional T and B cells, such as Thy-1, Lyt-2, Ia, and ThB antigens, are detected on the B' cell. In the in vitro anti-hapten secondary antibody response, the addition of a small number of B' cells from unprimed mice to the mixture of T and B cells greatly augmented the anti-hapten antibody formation when the number of carrier-specific helper T cells was limited. This augmentation was observed only when B and B' cells shared the same set of IgVH genes. The identity of the immunoglobulin gene between T cells and B or B' cells was not necessary for optimum antibody production. The results indicate that the presence of B' cells is necessary for the induction of an optimum antibody response when helper T cells are limited. It is suggested that B' cells deliver an additional signal to the B cell network to magnify the antibody response.

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