scholarly journals A monoclonal antibody that recognizes B cells and B cell precursors in mice.

1981 ◽  
Vol 153 (2) ◽  
pp. 269-279 ◽  
Author(s):  
R L Coffman ◽  
I L Weissman
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Cell Lineage ◽  
Hematopoietic Stem ◽  
B Cell Precursors

The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

scholarly journals Autoantibodies inhibit interleukin-7-mediated proliferation and are associated with the age-dependent loss of pre-B cells in autoimmune New Zealand Black Mice

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3289-3296 ◽  
Author(s):  
MS Merchant ◽  
BA Garvy ◽  
RL Riley
Keyword(s):  
Bone Marrow ◽  
New Zealand ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Igm Antibodies ◽  
Interleukin 7 ◽  
Heat Stable Antigen ◽  
B Cell Precursors

Surface IgM+B220+ B cell precursors can be categorized as either leukosialin (CD43/S7) negative (late stage pre-B cells) or positive (pro-B/early pre-B cells). In autoimmune New Zealand Black (NZB) mice, bone marrow small pre-B cells (IgM-CD43-B220+) and pro-B/early pre-B cells (IgM-CD43+B220+) declined significantly with age. In particular, subpopulations of pro-B/early pre-B cells expressing the heat stable antigen (HSA) were found in lower proportions with age. Significant decreases in interleukin-7 (IL-7) colony forming units (CFU) were also seen in NZB mice by 6 to 8 months of age and accompanied alterations in the numbers of pro-B and pre-B cells in bone marrow. Concomitant with reduced numbers of B lineage precursor cells and IL-7 CFU in vivo, NZB mice produced serum IgM antibodies that strongly inhibited IL-7 CFU responses in vitro. Two monoclonal IgM antibodies (5G9, 2F5) derived from LPS stimulated 10-month-old NZB splenocytes recognized pre-B cell surface antigens on both pre-B cell lines and on IL-7 stimulated bone marrow pro-B/pre-B cells. However, these monoclonal antibodies (MoAb) failed to significantly stain ex vivo bone marrow cells. The 5G9 and 2F5 MoAbs also partially inhibited IL-7 CFU in vitro. These results suggest that NZB bone marrow becomes increasingly deficient in B cell precursors and especially in IL-7 responsive pre-B cells with age. IgM serum antibodies and monoclonal IgM antibodies derived from older NZB mice inhibit pre-B cell growth to IL-7. The production of such autoantibodies may interfere with B cell development in aging NZB mice by preventing IL-7-mediated proliferation.

BRAFV600E Mutations Occur In The Hematopoietic Stem Cell Compartment In Hairy Cell Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 816-816
Author(s):  
Stephen S. Chung ◽  
Jae H. Park ◽  
Eunhee Kim ◽  
Young Rock Chung ◽  
Wenhuo Hu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Hematopoietic Stem ◽  
Mutant Braf

Abstract Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder recently found to be characterized by somatic BRAFV600E mutations. The malignant cell in HCL exhibits features consistent with a mature B-lymphocyte, including cell-surface expression of the pan-B-cell marker CD19 and monotypic surface immunoglobulins with clonal rearrangements of immunoglobulin heavy and light chains. Despite possessing these stereotypic features, the cell of origin of HCL has been long debated, and no cell type along the continuum of developing B-lymphocytes has been definitively identified as the normal counterpart of HCL cells. We hypothesized that HCL may originate from immature hematopoietic cells, and therefore investigated the hematopoietic-stem/progenitor cell (HSPC) compartment in HCL patients. We found that HCL patients exhibited a significantly increased frequency of immunophenotypically defined long-term hematopoietic stem cells (LT-HSCs; lineage-negative (Lin-neg) CD34+CD38-CD90+CD45RA- cells), pro-B cells (Lin-neg CD10+ cells), and CD34-CD38+ CD10+CD19+ hematogones, as well as a decreased frequency of granulocyte-macrophage progenitor cells (Lin-neg CD34+CD38+CD45RA+CD123+) relative to age-matched normal controls. Sequencing of cDNA from highly pure FACS-sorted cell populations from the bone marrow of HCL patients revealed the presence of the BRAFV600E allele in LT-HSCs and in pro-B cells (Figure). Transplantation of LT-HSCs from the pretreatment bone marrow of HCL patients into NOD/SCID/IL2r-gnull mice resulted in stable human grafts characterized by an expanded B-progenitor population and development of a clonal population of hCD19+hCD103+hCD25+ B cells characteristic of HCL 6 months after transplantation. Together, these data suggest that HCL arises from HSCs that then differentiate into committed B-cells which ultimately give rise to the characteristic clonal B-cell proliferation of HCL. Given the human HSC genetic and functional cell data, we conditionally expressed BRafV600E from its endogenous locus at different stages of hematopoiesis, including in HSPCs and committed B cells. Mice with conditional expression of BRafV600E in Mx1Cre+ BRafV600E knock-in mice died of a lethal hematopoietic malignancy characterized by features of human HCL including splenomegaly, anemia, thrombocytopenia, increased circulating sCD25, and increased clonogenic capacity of B-lineage cells (evidenced by infinite serial replating in the presence of IL-7) (Figure). This disorder was transplantable into lethally-irradiated recipient mice. In contrast, mice with expression of BRafV600E restricted to the B-cell lineage with Cd19 Cre manifested no overt malignant phenotype up to one year of age. Stimulation of these mice with alloantigen through injections of sheep red blood cells resulted in germinal center B-cell hyperplasia, but still did not result in development of a clonal B-cell proliferation. Recent case reports have noted that refractory HCL patients respond to mutant BRAF inhibition with vemurafenib. We investigated the effect of vemurafenib on HSPCs and hematopoiesis in patients treated on a phase II study of the mutant BRAF inhibitor vemurafenib for relapsed/refractory HCL as well as in our in vivo murine models. Flow cytometric analysis of bone marrow cells from vemurafenib treated HCL patients revealed normalization of HSPC frequencies within three months of starting therapy, concomitant with an improvement in peripheral blood counts. Consistent with this, evaluation of the in vitro clonogenic capacity of sorted LT-HSC's from the bone marrow of HCL patients revealed a significant increase in myeloid/erythroid colony formation in HCL patients treated for 3 months with vemurafenib compared to their pretreatment marrows. Likewise, treatment of wildtype mice transplanted with Mx1Cre+ BRafV600E mutant bone marrow cells revealed improvement in anemia and hepatosplenomegaly with in vivo therapy. Overall, these findings link the pathogenesis of HCL to a specific somatic genetic abnormality present in HSCs and provide evidence that mature B-cell malignancies can initiate in the HSC compartment. Moreover, these data suggest that the use of therapies targeting MAP kinase signaling in HCL may lead to durable remissions not only by eliminating the mature leukemic cells but also through targeted inhibition of signaling and survival in HCL initiating cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Epstein-Barr virus transformation of human pre-B cells.

1983 ◽  
Vol 158 (2) ◽  
pp. 616-622 ◽  
Author(s):  
M Hansson ◽  
K Falk ◽  
I Ernberg
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Bone Marrow Cells ◽  
Fetal Bone ◽  
Barr Virus ◽  
Epstein Barr

In vitro infection of human B lymphocytes with Epstein-Barr virus (EBV) results in establishment of B lymphoblastoid cell lines that reflect normal B cell phenotypes. In this study we have investigated whether immature B cells from fetal bone marrow and liver can serve as targets for EBV. The fetal bone marrow cells were readily transformed by EBV. Among the resulting cell lines, five were surface Ig (sIg)-negative. Three B cell-associated antigens defined by monoclonal antibodies were expressed to the same extent on the fetal cell lines, whether they belonged to the sIg- or sIg+ group. The various differentiation stages that these cell lines may represent are discussed.

Competition Between (Mono)Clonal Plasma Cells and Normal Cells for Potentially Overlapping Bone Marrow Niches Is Associated with a Progressively Altered Cellular Distribution In MGUS Vs. Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 617-617
Author(s):  
Bruno Paiva ◽  
Martin Perez-Andres ◽  
María-Belén Vidriales ◽  
Julia Almeida ◽  
Natalia de las Heras ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Plasma Levels ◽  
Plasma Cells ◽  
Cell Populations ◽  
High Dose ◽  
Normal Cells ◽  
Hematopoietic Stem ◽  
Migration Potential ◽  
B Cell Precursors

Abstract Abstract 617 Disappearance of normal bone marrow (BM) plasma cells (N-PC) predicts malignant transformation of monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) into symptomatic myeloma (MM). The homing, behavior and survival of N-PC, but also CD34+ hematopoietic stem/precursor cells (HPC), B-cell precursors, and (mono)clonal/aberrant PC (M-PC) largely depends on their interaction with SDF-1 expressing BM stromal cell niches. Accordingly, it can be hypothesized that a certain degree of competition among BM B-cell precursors, CD34+ HPC, and PC exist for the same BM niches. Thus, progressive replacement of normal cells by M-PC could help to explain the occurrence of cytopenias and hypogammaglobulinemia in MM patients. However, this hypothesis has not been investigated in depth neither in MM nor in SMM and MGUS. In this study we analyze by 8-color multiparameter flow cytometry the distribution and competitive migration capacity of B-cell precursors, CD34+ HPC, N-PC and M-PC in the BM and PB of patients with MGUS (n=60), SMM (n=47) and MM (n=87) at diagnosis plus 12 MM cases studied after high-dose therapy/autologous stem cell transplantation (MM POST-HDT/ASCT) vs. healthy adults aged >60 years (HA; n=26). The percentage of BM M-PC as well as the number of M-PC from all BMPC found at diagnosis significantly (p<.001) increased from MGUS to SMM and MM patients. Circulating M-PC were also detected at diagnosis in the PB of MGUS (21%) vs. SMM (69%) and MM (75%) at increasing M-PC counts (p≤.002). Interestingly, PB vs. BM M-PC showed significantly lower amounts of sideward light scatter (p<.001), together with lower levels of CD38 (p=.001), CD40 (p=.006), CD56 (p=.03) and CD138 (p=.02) expression. Focusing on the CXCR4-SDF1 axis, we found that the proportion of CXCR4+ PC slightly decreased (p>.05) from N-PC from HA (42%) to M-PC from MGUS (39%), SMM (37%) and MM (35%) patients at diagnosis, while MM POST-HDT/ASCT cases showed the lowest values (28%). In contrast, plasma levels of SDF-1 increased from HA (1150 pg/mL) to MGUS (1352 pg/mL), SMM (1656 pg/mL) and MM (1778 pg/mL; p=.04 vs. HA) patients, returning to almost normal levels in MM POST-HDT/ASCT (1331 pg/mL). Thus, an inverse correlation trend (r2=.12; p=.05) between the proportion of CXCR4+ PC and SDF-1 plasma levels was found. Concerning the distribution of the normal cell populations, BM pro-B and pre-B cell precursors were significantly decreased in MM (p=.001) patients vs. HA, while it was normal in MGUS (p>.05) and SMM (p>.05). Despite the number of N-PC in the BM was significantly lower among MM and SMM vs. MGUS cases (p<.001), the distribution of circulating N-PC in PB was normal in all three groups of patients (p>.05 vs. HA). Interestingly however, the proportion of CXCR4+ PB N-PC progressively increased from HA (11%) to MGUS (14%), SMM (15%) and MM (21%; p=.05 vs. HA), while MM POST-HDT/ASCT cases showed the lowest median percentage of CXCR4+ PB N-PC (5%). CD34+ HPC were found to be depleted in the BM of MM (0.3%; p=.001) and SMM (0.4%; p=.002) patients vs. HA (0.9%) while MGUS (0.8%) and MM POST-HDT/ASCT (1.1%) patients showed normal CD34+ HPC numbers. Conversely, PB CD34+ HPC progressively increase from HA to MGUS, SMM and MM cases (p=.008 and p=.06 vs. HA, respectively). By contrast, in MM POST-HDT/ASCT patients the number of PB CD34+ HPC returned to normal/lower levels. Ex-vivo competition assays between BM B-cells, PC and CD34+ HPC for SDF-1 induced migration showed that in HA, CD34+ HPC displayed the highest migration potential in the presence of SDF-1, followed by pre-B cell precursors; conversely N-PC barely migrated. No significant differences were found for the migration of all cell populations analyzed between MGUS and SMM patients vs. HA, except for M-PC that showed an impaired migration in the presence of SDF-1. In turn, the migration potential of CD34+ HPC (p=.04), and pre-B cell precursors (p=.02) was markedly reduced in symptomatic MM, particularly at lower SDF-1 concentrations (30nM). Most interestingly, the migration of M-PC from symptomatic MM was markedly increased at both concentrations of SDF-1 used: median of 2.9% and 1.0% for SDF-1 concentrations of 30nM and 70nM, respectively. Overall, these findings provide evidence about the role of progressive competition and replacement of normal BM cells by M-PC in determining transformation of pre-malignant MGUS and SMM into symptomatic MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro tolerance induction of neonatal murine B cells as a probe for the study of B-cell diversification.

1977 ◽  
Vol 145 (5) ◽  
pp. 1382-1386 ◽  
Author(s):  
E S Metcalf ◽  
N H Sigal ◽  
N R Klinman
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Tolerance Induction ◽  
Adult Bone Marrow ◽  
Late Expression ◽  
Adult Bone ◽  
Marrow Cells

The susceptibility to in vitro tolerance induction has been implicated as a characteristic of B cells early in their development, since DNP-reactive B cells are tolerizable only during the first days after birth, and 25% of adult bone marrow cells are tolerizable. In the present study, a modification of the in vitro splenic focus technique was utilized to determine if PC-specific B cells, by virtue of their late expression (approximately 1 wk post-parturition), also display susceptibility to tolerance induction. The results demonstrate that at 7-10 days after birth, when over 90% of the DNP-specific splenic B cells are resistant to tolerance induction, the majority of PC-specific B cells are tolerizable. These results re-emphasize tolerance susceptibility as a characteristic of developing clones, confirm the late acquisition of PC-specific B cells, and support the contention that the acquisition of the specificity repertoire is a highly ordered, specifically predetermined process which is independent of antigen-driven events.

scholarly journals IRF8 regulates B-cell lineage specification, commitment, and differentiation

Blood ◽  
2008 ◽  
Vol 112 (10) ◽  
pp. 4028-4038 ◽  
Author(s):  
Hongsheng Wang ◽  
Chang Hoon Lee ◽  
Chenfeng Qi ◽  
Prafullakumar Tailor ◽  
Jianxun Feng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Cell Lineage ◽  
Transcriptional Network ◽  
Wild Type ◽  
Lineage Specification ◽  
Hematopoietic Stem

Abstract PU.1, IKAROS, E2A, EBF, and PAX5 comprise a transcriptional network that orchestrates B-cell lineage specification, commitment, and differentiation. Here we identify interferon regulatory factor 8 (IRF8) as another component of this complex, and show that it also modulates lineage choice by hematopoietic stem cells (HSCs). IRF8 binds directly to an IRF8/Ets consensus sequence located in promoter regions of Sfpi1 and Ebf1, which encode PU.1 and EBF, respectively, and is associated with transcriptional repression of Sfpi1 and transcriptional activation of Ebf1. Bone marrows of IRF8 knockout mice (IRF8−/−) had significantly reduced numbers of pre-pro-B cells and increased numbers of myeloid cells. Although HSCs of IRF8−/− mice failed to differentiate to B220+ B-lineage cells in vitro, the defect could be rescued by transfecting HSCs with wild-type but not with a signaling-deficient IRF8 mutant. In contrast, overexpression of IRF8 in HSC-differentiated progenitor cells resulted in growth inhibition and apoptosis. We also found that IRF8 was expressed at higher levels in pre-pro-B cells than more mature B cells in wild-type mice. Together, these results indicate that IRF8 modulates lineage choice by HSCs and is part of the transcriptional network governing B-cell lineage specification, commitment, and differentiation.

scholarly journals Abnormalities of Bone Marrow B Cells and Plasma Cells in Primary Immune Thrombocytopenia

2021 ◽  
Author(s):  
Tianshu Yu ◽  
Haoyi Wang ◽  
Yajing Zhao ◽  
Yafei Yu ◽  
Yu Hou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Immune Thrombocytopenia ◽  
Plasma Cells ◽  
Plasma Concentrations ◽  
Mrna Levels ◽  
Primary Immune Thrombocytopenia ◽  
B Cell Subsets

Primary immune thrombocytopenia (ITP) is an autoantibody-mediated hemorrhagic disorder where B cells play an essential role. Previous studies have focused on peripheral blood (PB), but B cells in bone marrow (BM) have not been well characterized. We aimed to explore the profile of B cell subsets and their cytokine environments in BM of ITP patients to further clarify the pathogenesis of the disease. B cell subpopulations and their cytokine/chemokine receptors were detected by flow cytometry. Plasma concentrations of cytokines/chemokines were measured by ELISA. mRNA levels of B cell-related transcription factors were determined by qPCR. Regulatory B cell (Breg) function was assessed by quantifying their inhibitory effects on monocytes and T cells in vitro. Decreased proportions of total B cells, naïve B cells and defective Bregs were observed in ITP patients compared with healthy controls (HCs), whereas elevated frequency of long-lived plasma cells was found in BM of autoantibody-positive patients. No statistical difference was observed in plasmablasts or in short-lived plasma cells between ITP patients and HCs. The immunosuppressive capacity of BM Bregs from ITP patients was considerably weaker than that from HCs. In vivo study using an active ITP murine model revealed that Breg transfusion could significantly alleviate thrombocytopenia. Moreover, over-activation of CXCL13-CXCR5 and BAFF/APRIL systems were found in ITP patient BM. Taken together, B cell subsets in BM were skewed toward a proinflammatory profile in ITP patients, suggesting the involvement of dysregulated BM B cells in the development of the disease.

CD52 Expression Patterns in Normal and Neoplastic B-Cells: Myeloma Is an Unlikely Target for Single Agent Alemtuzumab Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4883-4883
Author(s):  
A. C. Rawstron ◽  
G. Laycock-Brown ◽  
F. E. Davies ◽  
R. G. Owen ◽  
P. Hillmen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Single Agent ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Normal Controls

Abstract Alemtuzumab is a highly effective monoclonal antibody therapy for some B-cell disorders, and has been suggested as a possible therapeutic agent for treatment of myeloma. Monoclonal antibody therapeutic efficacy is closely associated with the expression level of the therapeutic target, as demonstrated by the lack of efficacy of single-agent rituximab in CLL. However, there are conflicting reports about the expression levels of CD52, the target for alemtuzumab, in plasma cell disorders. The aim of this study is to assess a large series of cases of plasma cell and B-cell disorders, utilising a standard approach to allow comparison of the target molecule. Plasma cells were assessed from patients with myeloma at presentation or relapse (n=106), monoclonal gammopathy of undetermined significance (MGUS, n=34), and from normal controls (n=19). In addition, B-cells were assessed from patients with chronic lymphocytic leukaemia (CLL, n=87), diffuse large B-cell lymphoma (DLBCL, n=10), follicular lymphoma (FCL, n=9), Waldenstroms macroglobulinaemia (WM, n=20), and also from normal bone marrow (n=37). Normal and neoplastic B-cells showed expression of CD52 (&gt;20% of cells above the CD3-control levels) in all patients except for 1/10 DLBL. B-CLL and WM are known to show responses to single-agent alemtuzumab therapy, and these two disorders had the highest levels of expression. In contrast, B-progenitor cells in normal bone marrow are unaffected by alemtuzumab, and proliferate during alemtuzumab treatment in CLL patients. The levels of CD52 expression by normal B-progenitors were 3-fold lower than CLL/WM. In DLBL and FCL, the B-cells showed very similar levels of CD52 expression to normal B-progenitors, on average 2.8-fold lower than CLL. All plasma cells, whether neoplastic (CD19− or CD19+56+) or normal (CD19+56−), showed much lower levels of expression than normal and neoplastic B-cells. Plasma cell CD52 expression was detectable in 68% of normal controls (13/19), 50% of MGUS patients (17/34), and only 43% of myeloma patients (46/106). Expression was uni-modal in all cases. There was significantly lower expression of CD52 by myeloma plasma cells than by their normal counterparts (median 2.4-fold decrease, P=0.03). Neoplastic plasma cell CD52 expression showed a high degree of inter-patient variation, but fewer than 10% of myeloma patients (7/106) had CD52 expression at a similar level to CLL cells. Neoplastic plasma cell CD52 expression was approximately 6-fold lower than that of normal B-progenitors, and nearly 20-fold lower than that of CLL cells. In summary, CD52 expression is not detectable above control levels in a significant proportion of myeloma patients. In cases with detectable CD52 expression, the antigen is at a much lower level than is present on normal B-progenitors, which actively proliferate during alemtuzumab therapy. The risk of immunosuppression due to depletion of residual normal B/T-cells must also be considered. As alemtuzumab efficacy appears to correlate with CD52 expression levels, myeloma is highly unlikely to respond to alemtuzumab as a single agent except in rare cases. However, alemtuzumab is more likely to be effective in the IgM immunosecretory disorders which show strong CD52 expression.

Rituximab Infusion Two Months after HCT Decreases Alloreactive B Cell Responses While Recipient Plasma Cells Persist.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2234-2234 ◽  
Author(s):  
Bi ta Sahaf ◽  
Julie R. Boiko ◽  
George Chen ◽  
Kartoosh Heydari ◽  
Sally Arai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Hematopoietic Cell Transplantation ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Chronic Gvhd ◽  
Lymphocytic Leukemia ◽  
Facs Analysis ◽  
Peripheral B Cells

Abstract B cells and their effector molecules, antibodies, are implicated in pathophysiology of the chronic graft-vs-host disease (cGVHD) and rituximab is effective cGVHD therapy. Here we investigate B cell reconstitution in bone marrow aspirates collected from 14 mantle cell lymphoma and 22 chronic lymphocytic leukemia patients receiving rituximab infusion 375mg/m2 weekly x 4 beginning 56 days after allogeneic hematopoietic cell transplantation (HCT) following total lymphoid irradiation 80cGy x10 daily fractions and anti-thymoglobulin (1.5 mg/Kg/day x 5). Primary GVHD prophylaxis was mycophenolic acid and cyclosporine tapered off by 6 months. We hypothesized rituximab would deplete alloreactive na•ve and memory B cells and result in less chronic GVHD. Here we present multi-parameter B cell FACS analysis characterizing extent of B cell depletion and developmental stage analysis and subsequent reconstitution kinetics. We collected bone marrow aspirates prior to rituximab, and then days 90, 180, and 365 following HCT. Peripheral B cells were detected in 17 of 34 HCT patients prior to rituximab infusion day 56. Following rituximab, peripheral blood CD19+ B cells were detected in 4 by one year, 18 by 1.5 years, and 9 by 2 years post HCT. Multi-parameter (12 colors-14 parameters) FACS analysis of bone marrow B cells using 2 different cocktails on the same bone marrow cells distinguished: common lymphoid progenitor (CD34+CD117+CD7+), Pro B cells (CD34+CD20−CD10−), pre B cells (CD34−CD20−, CD10+), immature B cell (CD20−CD38−IgM+ IgD low/neg), mature (CD20+CD38+IgD+, IgM+) B cells, and CD38+ CD138+ plasma cells. Despite only modest reconstitution of PERIPHERAL B cells 2 months after HCT (17/32), bone marrow B cells expressing CD19 were present in 9 out of 9 patients at 56 days post HCT and were depleted to less than 0.05% of total lymphocytes after 4 rituximab infusions when measured 90 days post-HCT (below table). Following rituximab, CD19+ B cells were first detected in the bone marrow 180 days after HCT. The mature CD19+ B cells accounted for 2–5% by 365 days post HCT. While rituximab depleted mature B cells, plasma cells remained unchanged. Furthermore, CD138+CD38+ plasma cells were FACS sorted shown by STR DNA polymorphism testing to be recipient derived (n=5). Consistent with observed stable plasma cell frequency, total plasma IgG showed no significant change. Inherited polymorphisms in IgG heavy chain constant regions can be recognized by allotype-specific monoclonal antibodies and thereby distinguish donor and recipient antibodies. Such allotype detection of antimicrobial IgG confirmed stable anti-VZV and EBV as well as recipient origin of these plasma IgG up to 2 years post HCT. In support of our hypothesis, alloreactive IgG responses against 5 minor histocompability antigens (mHA) encoded on Y chromosome (DBY, UTY, ZFY, RPS4Y, and EIF1AY) were decreased in TLI/ATG/rituximab treated patients. None of the 11 male patients with female donors treated with rituximab developed antibodies against H-Y proteins while 12 out of 24 (50%) F̂M undergoing TLI/ATG without rituximab developed allo-antibodies against H-Y proteins (p=0.09). In summary, multi-parameter (12 colors-14 parameters) immunophenotyping of bone marrow shows rituximab treatment two months after allo-HCT causes delayed donor derived B cell reconstitution, persistent antimicrobial IgG from persistent recipient plasma cells, and undetectable allogeneic H-Y antibodies. Summary table. Days after HCT LYMPHOID PROGENITORS CD34+ CD117+ CD7+ PRO B CELLS CD34+ CD20− CD10− PRE B CELLS CD34+ CD20− CD10− MATURE B CELLS CD20+ Ig D+ Ig M+ PLASMA CELLS CD38+ CD138+ TOTAL IgG μg/dl(pre) 56 pre = ritux n = 9 20–25% 2–6% 0.1–4% 0.2–1% 0.7–1% 655 81% 90 n = 25 20–40% 2–9% 0.5–2% ND** 0.5–3% 910 101% 180 n = 28 13–20% 5–12% 0–0.7% ND** 0.5–2% 507 60% 365 n = 16 3–8% 2–10% 0–0.5% 1–5% 0.5–3.7% 642 78%

scholarly journals Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to C mu sequence in immunoglobulin (IgG)- and IgA-secreting multiple myelomas.

1993 ◽  
Vol 178 (3) ◽  
pp. 1091-1096 ◽  
Author(s):  
P Corradini ◽  
M Boccadoro ◽  
C Voena ◽  
A Pileri
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Indirect Evidence ◽  
Specific Marker

Multiple myeloma is a B cell malignancy characterized by the expansion of plasma cells producing monoclonal immunoglobulins (Ig). It has been regarded as a tumor arising at the B, pre-B lymphocyte, or even stem cell level. Precursor cells are presumed to proliferate and differentiate giving rise to the plasma cell clonal expansion. Antigenic features and specific Ig gene rearrangement shared by B lymphocytes and myeloma cells have supported this hypothesis. However, the existence of such a precursor is based upon indirect evidence and is still an open question. During differentiation, B cells rearrange variable (V) regions of Ig heavy chain genes, providing a specific marker of clonality. Using an anchor polymerase chain reaction assay, these rearranged regions from five patients with multiple myeloma were cloned and sequenced. The switch of the Ig constant (C) region was used to define the B cell differentiation stage: V regions are linked to C mu genes in pre-B and B lymphocytes (pre-switch B cells), but to C gamma or C alpha in post-switch B lymphocytes and plasma cells (post-switch B cells). Analysis of bone marrow cells at diagnosis revealed the presence of pre-switch B cells bearing plasma cell V regions still joined to the C mu gene. These cells were not identified in peripheral blood, where tumor post-switch B cells were detected. These pre-switch B cells may be regarded as potential myeloma cell precursors.

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