scholarly journals Toll-like receptor 7 (TLR7)–driven accumulation of a novel CD11c+ B-cell population is important for the development of autoimmunity

Blood ◽  
2011 ◽  
Vol 118 (5) ◽  
pp. 1305-1315 ◽  
Author(s):  
Anatoly V. Rubtsov ◽  
Kira Rubtsova ◽  
Aryeh Fischer ◽  
Richard T. Meehan ◽  
Joann Z. Gillis ◽  
...  
Keyword(s):  
B Cells ◽  
Autoimmune Disease ◽  
B Cell ◽  
Cell Population ◽  
Peripheral Blood ◽  
Elderly Women ◽  
Toll Like Receptor ◽  
Direct Role ◽  
Autoreactive Antibodies

Abstract Females are more susceptible than males to many autoimmune diseases. The processes causing this phenomenon are incompletely understood. Here, we demonstrate that aged female mice acquire a previously uncharacterized population of B cells that we call age-associated B cells (ABCs) and that these cells express integrin αX chain (CD11c). This unexpected population also appears in young lupus-prone mice. On stimulation, CD11c+ B cells, both from autoimmune-prone and healthy strains of mice, secrete autoantibodies, and depletion of these cells in vivo leads to reduction of autoreactive antibodies, suggesting that the cells might have a direct role in the development of autoimmunity. We have explored factors that contribute to appearance of ABCs and demonstrated that signaling through Toll-like receptor 7 is crucial for development of this B cell population. We were able to detect a similar population of B cells in the peripheral blood of some elderly women with autoimmune disease, suggesting that there may be parallels between the creation of ABC-like cells between mice and humans.

scholarly journals Posttransplant Cyclophosphamide Promote Naïve-Subset Dominant B Cell Recovery Coordinated with Early Treg Expansion; Implication of Reduced Risk of Pathogenesis into Chronic Gvhd

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4566-4566
Author(s):  
Miki Iwamoto ◽  
Yusuke Meguri ◽  
Takumi Kondo ◽  
Hiroyuki Sugiura ◽  
Shuntaro Ikegawa ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Chronic Gvhd ◽  
Lymphocyte Subset ◽  
Cell Recovery ◽  
Haplo Group

Abstract Posttransplant cyclophosphamide (PTCy) is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). We recently studied the immune reconstitution dynamics of each lymphocyte subset after PTCy-based transplant using murine haploidentical BMT model and reported that PTCy strongly promoted Treg-dominant T-cell reconstitution and stem cell-derived mature B-cell generation with broad BCR-diversity. We also found that the early reconstitution of Treg could contribute to promote naïve B cell emergence from bone marrow, indicating the T and B cell recovery might be mutually coordinated after PTCy-based transplant (Iwamoto et al, ASH2017). However, the detailed process of immune reconstitution in patients after haploidentical HSCT with PTCy has not been well studied. To address this issue, we here investigated the early dynamics of donor-lymphocyte subset chimerisms in patient after clinical PTCy-based haploidentical HSCT with comparing those in patients after low-dose ATG-based haploidentical HSCT and patients after cord blood transplantation. Laboratory studies were undertaken in 13 adult patients who received HLA-mismatched allogeneic graft; unrelated cord blood (n=5), and haploidentical related peripheral blood after ATG-based conditioning (n=5) and haploidentical related peripheral blood after PTCy-based conditioning (n=5). Blood samples were obtained before and at 1, 2, 4, 6 and 8 weeks after HSCT. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density gradient centrifugation and cryopreserved before being analyzed. After thawing, to analyze the subset-specific chimerism, PBMCs were stained with anti-HLA monoclonal antibodies and other subset-specific antibodies as follows: Pacific Blue conjugated anti-CD4, eFluor450 conjugated anti-CD3, PE-Cy7 conjugated anti-CD25, anti-CD14, APC conjugated anti-CD127, anti-CD56, and APC-eFluor780 conjugated anti-CD8a, anti-CD19. Gated lymphotes (CD4+Tcons, CD4+Tregs, CD8+T cells, B cells, NK cells, Monocytes) were analyzed their chimerism by flowcytometry. To examine the detailed phenotype of B cells, the expression of CD27, CD24, CD38 and IgD were tested. Flowcytometry-based method enables us to analyze the lymphocyte subset chemerism in the very early phase after HSCT. At 2 weeks after HSCT, our analysis revealed that CD4+Tcons, CD4+Tregs and CD8+T cells had already achieved complete donor chimerisms (>95% in all subsets) in patients after ATG-based SCT and had been approaching complete donor chimerisms (85.8%, 75.4% and 87.2%, respectively) in patients after CBT. In contrast, percentage of donor chimerisms of CD4+Tcons, CD4+Tregs and CD8+T cells after PTCy-based haplo-SCT was 73.5%, 59.6% and 59.2%, respectively, and those remained to be in the lower levels than other 2 groups. However, at 4 weeks after HSCT, all examined patients achieved complete donor chimerism of T cells, NK cells and Monocytes (>90%). At 8 weeks after HSCT, the number of B cells in PTCy-based haplo-group was higher than in ATG-based haplo-group (3494 vs 1901/mm3). Of note, B cell population in PTCy-based haplo-group at 8 weeks contained the significantly higher percentage of CD24+CD27-IgD+CD38+ transitional/naïve subset and the significantly lower percentage of CD24+CD27+IgD-CD38neg/dim activated/switched-memory subset when compared to B cell population in ATG-based haplo-group (59.9% vs 10.2%, 2.6% vs 21.5%, P<0.02 respectively), suggesting PTCy treatment might be associated with the favorable B cell reconstitution with naïve-subset dominant composition. Moreover, in patients after PTCy-based haplo-group, the percentage of activated/switched-memory subsets in B cell population at 8 weeks was inversely correlated with percentage of Treg in CD4 T cells at 4 weeks (P<0.05, r2=0.77). Taken together, consistently with our murine study, the current data from clinical samples again suggest that PTCy-based immune-modulation lead to coordinated T and B cell recovery, especially promoting naïve-subset dominant B cell recovery with help of the early expansion of Treg, which might reduce the risk of subsequent chronic GVHD. These data provide the important information for understanding the immunological reconstitution after PTCy-based haploidentical HSCT. Disclosures No relevant conflicts of interest to declare.

In Vivo Depletion of Cynomolgus Monkey B Cells Targeted by a Novel Monoclonal Antibody (Xi-20H5) Specific for a Shared Portion of the Human Immunoglobulin Variable Light Chain Lambda 1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2354-2354
Author(s):  
Thierry Sornasse ◽  
Keri Tate ◽  
Kimberly Milner ◽  
Thomas Theriault ◽  
Dan W. Denney ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Peripheral Blood ◽  
Cynomolgus Monkey ◽  
Patient Specific ◽  
B Cell Malignancies ◽  
Variable Regions ◽  
Variable Light

Abstract Genitope is developing a novel, personalized treatment for surface immunoglobulin (Ig) positive B cell malignancies. This treatment is based on a panel of monoclonal antibodies (Mabs) directed against shared epitopes expressed on different subsets of the variable regions of human Ig. The concept of targeting surface tumor Ig was explored in a series of clinical trials performed by Dr. Ronald Levy and his colleagues at Stanford. In these studies, Mabs directed against patient specific (idiotypic) determinants of the surface tumor Ig produced significant clinical benefit in relapsed follicular non-Hodgkin’s lymphoma patients. A number of these patients have had long term remissions. In Genitope’s planned clinical use, each patient will receive a single Mab from the panel selected based on its reactivity with the patient’s tumor. The selected Mab will react with the patient’s tumor and a minority of normal B cells, leaving the majority of the normal B cell repertoire intact. The ability of the panel members to provide therapeutic effect requires binding to tumor surface Ig in the presence of serum containing soluble Ig molecules. Mab Xi-20H5, a member of this panel of antibodies, is specific for a shared determinant on the human Ig variable light chain lambda 1. It binds to 25 to 35% of normal human B cells from peripheral blood and to 20 to 30% of normal cynomolgus monkey B cells from peripheral blood. In this study, we sought to demonstrate that, despite the presence of serum Ig, Mab Xi-20H5 would bind to surface Ig expressed on monkey B cells in vivo, resulting in specific depletion of target B cells. Six naïve cynomolgus monkeys received 8 intravenous infusions of the Mab Xi-20H5 at a dose of 40 mg/kg on days 1, 2, 3, 4, 7, 10, 14 & 17. Two naïve control animals received 8 infusions of vehicle only following the same schedule. The frequencies of lymphocyte sub-populations and of target B cells were monitored by flow cytometry on plasma-depleted whole blood samples. Samples were collected 23 hours after each infusion. In addition, two baseline samples were collected prior to treatment. The frequencies of lymphocyte sub-populations and of target B cells were compared to the average of the two baseline measurements. Frequencies of target B cells bound by Mab Xi-20H5 decreased in all treated animals while no significant change was detectable in the control animals. The bulk of the reduction in target B cell frequencies was observed 23 hours after the first infusion (range: 22% – 62%, average 41%). Frequencies of target B cells continued to decrease moderately with additional daily infusions (days 2 – 4), resulting in maximum reduction in target B cell frequency at 23 h post infusion 4 (range: 39% – 78%, average 54%). The frequencies of total B and T lymphocytes did not significantly change during the treatment. In vivo administration of Mab Xi-20H5 results in depletion of target B cells in a manner consistent with the expectation of an immunotherapeutic Mab aimed at treating surface Ig expressing B cell malignancies.

Differential effects of epratuzumab on peripheral blood B cells of patients with systemic lupus erythematosus versus normal controls

2007 ◽  
Vol 67 (4) ◽  
pp. 450-457 ◽  
Author(s):  
A M Jacobi ◽  
D M Goldenberg ◽  
F Hiepe ◽  
A Radbruch ◽  
G R Burmester ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
B Cells ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Peripheral Blood ◽  
Therapeutic Option ◽  
Normal Subjects ◽  
Systemic Lupus

Objective:B lymphocytes have been implicated in the pathogenesis of lupus and other autoimmune diseases, resulting in the introduction of B cell-directed therapies. Epratuzumab, a humanised anti-CD22 monoclonal antibody, is currently in clinical trials, although its effects on patients’ B cells are not completely understood.Methods:This study analysed the in vivo effect of epratuzumab on peripheral B cell subsets in 12 patients with systemic lupus erythematosus, and also addressed the in vitro effects of the drug by analysing anti-immunoglobulin-induced proliferation of isolated B cells obtained from the peripheral blood of 11 additional patients with lupus and seven normal subjects.Results:Upon treatment, a pronounced reduction of CD27– B cells and CD22 surface expression on CD27– B cells was observed, suggesting that these cells, which mainly comprise naïve and transitional B cells, are preferentially targeted by epratuzumab in vivo. The results of in vitro studies indicate additional regulatory effects of the drug by reducing the enhanced activation and proliferation of anti-immunoglobulin-stimulated lupus B cells after co-incubation with CD40L or CpG. Epratuzumab inhibited the proliferation of B cells from patients with systemic lupus erythematosus but not normal B cells under all culture conditions.Conclusions:Epratuzumab preferentially modulates the exaggerated activation and proliferation of B cells from patients with lupus in contrast to normal subjects, thus suggesting that epratuzumab might offer a new therapeutic option for patients with systemic lupus erythematosus, as enhanced B cell activation is a hallmark of this disease.

VCAM-1 Expression in B-Lymphopoiesis and Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3247-3247
Author(s):  
Faith M. Young ◽  
Raymond E. Felgar ◽  
Antonia P. Eyssallenne ◽  
Andrea Bottaro ◽  
Timothy P. Bushnell
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Stromal Cell ◽  
Ex Vivo ◽  
Surface Expression ◽  
Lymphoid Cells ◽  
B Lymphoid

Abstract Vascular Cell Adhesion Molecule-1 (VCAM; CD106), a member of the Ig Superfamily of molecules, binds to the β-1 integrin, Very Late Antigen-4 (VLA-4; CD49d); this interaction plays an integral role in leukocyte trafficking as well as lymphocyte-stromal cell interactions. VCAM can be shed from the surface of cells, and, in humans, serum levels of soluble VCAM (sVCAM) parallel activity and remission states in acute lymphocytic leukemia (ALL) and inflammation. Although widely investigated as a stromal-cell associated molecule, our lab and others have recently identified VCAM expression on normal bone-marrow derived B-lymphoid cells. Using FACS technology, we found that surface expression of VCAM is closely modulated at specific stages of B cell development, with relatively high levels on the pro-B cell population, down-modulation in pre-B cells at the onset of immunoglobulin (Ig) gene rearrangement, and subsequent re-expression at variable levels in immature and mature peripheral B cell subsets. We have verified VCAM transcripts by cDNA PCR in highly purified populations of murine precursor B cells. Normal human bone marrow precursor B-lymphoid populations (hematogones) also demonstrate VCAM surface protein expression. Finally, in an animal model of BCR/ABL+ ALL, we found that VCAM expression is dramatically increased on lymphoblasts when compared to normal reference populations in bone marrow and spleen. VCAM expression in human lymphoid malignancies is currently under investigation. Antibody-mediated VCAM cross-linking on primary B-cell precursors ex-vivo generates intracellular reactive oxygen species, demonstrating that signaling through this molecule has functional consequences. Intriguingly, in-vivo, VCAM expression is limited to B-lymphoid cells harvested from tissues such as bone marrow, spleen and lymph node; since, in the same animal, peripheral blood lymphocytes and peritoneal cells do not express readily detectable levels of the surface antigen. VCAM-expressing B-lymphoid cells cultured ex-vivo gradually lose surface expression over 24 hours. The tissue-associated modulation of VCAM expression is preserved in the murine Ph+ lymphoblasts; leukemia cells isolated from the peripheral blood express very low levels of surface VCAM compared to those harvested from bone marrow or spleen. Our data suggests that VCAM expression is dependent on tissue-specific microenvironmental signals in-vivo. B-lymphoid expression of both VCAM and its ligand VLA-4 is a surprising finding that has broad implications regarding leukemic cell interaction with endothelial cells, the bone marrow retention and trafficking of precursor- and leukemic-B cell populations, and the interpretation of an extensive experimental database predicated on the stromal-cell specificity of VCAM expression and function.

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 Production of colony-stimulating activity by normal and neoplastic human B lymphocytes

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1340-1347 ◽  
Author(s):  
V Pistoia ◽  
R Ghio ◽  
S Roncella ◽  
F Cozzolino ◽  
S Zupo ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Target Cells ◽  
Sephadex Column ◽  
Colony Stimulating Activity

Abstract Normal human B cells were purified from peripheral blood or tonsils and tested for their ability to release colony-stimulating activity (CSA) in short-term cultures. The target cells used in the CSA assays were from peripheral blood or bone marrow. Unstimulated B cells produced CSA in amounts similar to those present in the GCT-conditioned medium used as a positive control. The B cell-derived CSA predominantly promoted the growth of colonies that contained macrophages alone or macrophages and granulocytes. CSA eluted in a single peak from a G-75 Sephadex column with an approximate molecular weight (mw) of 65 to 70 kilodaltons (kd). Fractionation of tonsil B lymphocytes on Percoll density gradients showed that large B cells, probably already activated in vivo, were the main source of CSA. By contrast, small, resting B cells recovered from a different fraction of the Percoll gradient released minimum amounts or no CSA. However, these B cells became CSA producers following stimulation with Staphylococcus aureus Cowan (SAC) in vitro. B cells purified from the peripheral blood of nine out of 12 patients with B-cell chronic lymphocytic leukemia (B-CLL) also released CSA in vitro in the absence of stimuli. These findings suggest that by releasing CSA, B cells may have a role in the regulation of hematopoiesis and in the control of the inflammatory process.

B-cell kinetics in humans: rapid turnover of peripheral blood memory cells

Blood ◽  
2005 ◽  
Vol 105 (9) ◽  
pp. 3633-3640 ◽  
Author(s):  
Derek C. Macallan ◽  
Diana L. Wallace ◽  
Yan Zhang ◽  
Hala Ghattas ◽  
Becca Asquith ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Average Rate ◽  
The Elderly ◽  
Gas Chromatography Mass Spectrometry ◽  
Memory Cells ◽  
Total Blood ◽  
Deuterium Enrichment

Abstract Information about the kinetic behavior and lifespan of lymphocytes is crucial to understanding the mechanisms that regulate processes such as immunologic memory. We have used in vivo labeling of dividing cells with 6,6-2H2-glucose, combined with cell sorting and gas-chromatography-mass spectrometry for deuterium enrichment, in order to analyze the kinetics of human total, naive, or memory B lymphocytes, separated from peripheral blood using monoclonal antibodies. We show that total blood B cells of young adults divide at an average rate of 1.9% (±1.0%) per day and at a similar though slightly slower rate, 1.5% (±1.3%) per day, in the elderly. Separation of naive and memory B cells according to expression of CD27 indicates that naive peripheral blood B cells divide slowly (0.46% per day), while memory cells proliferate more rapidly (2.66% per day). These data are compatible with the view that B-cell memory may be maintained by clones of proliferating B cells.

Rescue of B Cells Development in an Animal Model of X-Linked Agammaglobulinemia(XLA) Via B Lineage-Specific Lentiviral Gene Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1287-1287
Author(s):  
S. Humblet-Baron ◽  
W. Zhang ◽  
K. Kipp ◽  
S. Khim ◽  
J. Jarjour ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Lentiviral Vector ◽  
Cell Development ◽  
B Cell Development ◽  
Gfp Expression ◽  
B Lineage

Abstract X-linked agammaglobulinemia (XLA) is a human immunodeficiency caused by mutations in Bruton’s tyrosine kinase (Btk) and characterized by an arrest in early B-cell development, absence of serum immunoglobulin, and recurrent bacterial infections. Using Btk and Tec double deficient (Btk/Tec−/ −) mice as a model for XLA, we recently showed that onco-retroviral-mediated Btk gene transfer into hematopoietic stem cells (HSC) reconstituted in vivo Btk-dependent B-cell development and function (Yu et al. Blood 104(5):1281–90). In order to increase the safety of this approach, we developed a SIN-lentiviral vector with a B cell specific enhancer/promoter element, Eμ B29. Using SIN-lentiviral vectors expressing GFP, we observed that Eμ B29 consistently promoted 3–5 fold higher GFP expression in human B lineage cells derived from transduced HSC in vitro and in vivo (ASGT 2002 abstract #1302). We also evaluated this vector, CSOM-Eμ B29-GFP-WPRE, in lentiviral transgenic mice where it exhibited the highest GFP expression in peripheral B cells compared with all other hematopoietic lineages. Specifically, in more than 8 independent founder strains the MFI for GFP expression in B cells was > 3 fold higher than that in T cells (p=0.0002). Based upon these findings we developed Eμ B29-huBtk SIN-lentiviral vectors with or without the insulator element derived from the chicken β-globulin insulator (HS4). Using both vectors to transduce Btk −/ − DT40 B cells, followed by cloning by limiting dilution, we demonstrate Btk protein expression by intracellular staining and western blotting and full rescue of Btk-dependent, B cell receptor (BCR)-mediated Ca2+ signaling in all clones evaluated including those exhibiting a single viral integration. Next we tested the capacity of these vectors to reconstitute Btk-dependent B-cell development and function in a cohort of Btk/Tec−/ − mice. Marrow from 5-FU treated Btk/Tec −/ − mice was harvested, cultured on fibronectin coated plates with growth factors (mIL-3,mIL-6, mSCF, mTPO and mFLT3ligand) and concentrated lentivirus (2.3x107pg/106 cells measured by p24 level). After 48h of in vitro culture, cells were transplanted into lethally irradiated animals and transplanted animals were serially evaluated for presence of B cells in the peripheral blood. B-cell numbers progressively increased with a significant difference as early as within 6 weeks in mice receiving transduced (16–18% B220+ cells) vs. control marrow (8–9%; mock transduced). Further, mature B cells (B220+IgMlowIgDhi) represented 14–20% of total B cells in treated compared to <5% in control mice. Finally, mice receiving transduced cells exhibited a rescue of total serum IgM and IgG3 levels and responses to TI-II dependent immunization. Results of two additional animal cohorts will be presented. In summary, our data demonstrate that Eμ B29-Btk SIN-lentiviral vector specifically promotes Btk expression in B lineage cells, and correction of the Btk-deficient phenotype in vitro and in vivo. Peripheral blood B cells were analyzed for relative IgM and IgD expression at 6 weeks post reconstitution. Representative data from animals receiving mock-vs/ EμB29-Btk transduced marrow are shown. Upper left quadrant shows percentage of circulating mature B cells. Peripheral blood B cells were analyzed for relative IgM and IgD expression at 6 weeks post reconstitution. Representative data from animals receiving mock-vs/ EμB29-Btk transduced marrow are shown. Upper left quadrant shows percentage of circulating mature B cells.

Hspa9 Haploinsufficiency Induces a Cell-Intrinsic Defect in Mouse B-Cell Progenitors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1714-1714
Author(s):  
Kilannin Krysiak ◽  
Justin Tibbitts ◽  
Tim H Chen ◽  
Matthew J. Walter
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Cell Maturation ◽  
Chromosome 5 ◽  
B Cell Maturation

Abstract Abstract 1714 Patients with myelodysplastic syndromes (MDS) have a clonal hematopoietic stem cell disorder that results in dysplastic hematopoietic cells in their bone marrow as well as peripheral blood cytopenias. In addition to the commonly described erythroid and myeloid differentiation defects associated with MDS, a reduction in bone marrow B-cell progenitors exists in patients. The genetic events contributing to the reduction in B-cell progenitors remain poorly understood. The most common cytogentic abnormality identified in patients with MDS, occurring in approximately 35% of patients, is heterozygous interstitial deletion or loss of the long arm of chromosome 5 (5q). The interstitial deletions on chromosome 5 are single copy losses, and no biallelic disruptions of genes in deleted regions have been identified, implicating haploinsufficiency as the underlying genetic mechanism. We, and others, have shown that the levels of HSPA9 mRNA expression are reduced ∼50% in patients with del(5q) when compared to MDS patients without del(5q), consistent with a haploinsufficient phenotype. To model haploinsufficiency, we used shRNA to achieve ∼50% knockdown of Hspa9 in a murine bone marrow transplant model. This model showed a significant reduction in mature B-cells in the bone marrow, spleen, and peripheral blood of recipient mice, implicating HSPA9 haploinsufficiency may contribute to the B-cell alterations observed in MDS patients with del(5q). To further evaluate HSPA9 haploinsufficiency in vivo, we created a mouse model with a heterozygous deletion of Hspa9 (Hspa9+/−) and confirmed a 50% reduction in Hspa9 protein levels in bone marrow and spleen of these mice by Western blot. Hspa9+/− mice are born at normal Mendelian frequencies (N>100), however, breeding heterozygous mice suggests Hspa9−/− mice are embryonic lethal (24 Hspa9+/+:38 Hspa9+/−:0 Hspa9−/−). No significant differences in mature lineage markers, complete blood counts, and hematopoietic organ cellularity, have been identified up to 12 months of age. However, as early as 2 months of age, the numbers of bone marrow CFU-preB colonies as assessed by methylcellulose assay, are significantly reduced in Hspa9+/− mice compared to Hspa9+/+ littermates (14 vs 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p<0.0001). We performed noncompetitive bone marrow transplants of Hspa9+/− or Hspa9+/+ donor cells into Hspa9+/+ recipient mice and confirmed that the reduction of B-cell progenitors is a hematopoietic cell intrinsic phenotype (N=7–9 mice/genotype, p=0.002). We also confirmed that the Hspa9+/− bone marrow microenvironment did not contribute to the phenotype as transplantation of Hspa9+/+ donor bone marrow cells into Hspa9+/− recipients did not alter the number of CFU-preB colonies (N=5). Total frequencies of common lymphoid progenitors and B-cell precursors (Hardy fractions A, B/C, D, E and F) as assessed by flow cytometry are no different in Hspa9+/− and Hspa9+/+ mice. Therefore, we hypothesize that early Hspa9+/− B-cells may have an intrinsic signaling defect which can be compensated for in vivo. Early B-cell maturation is dependent on intracellular signaling mediated through cell surface receptors in response to environmental cytokines. Consistent with our hypothesis, we showed that Hspa9+/− CFU-preB in vitro colony formation is partially rescued by increasing concentrations of IL7 while Hspa9+/+ colony numbers remain unchanged (fold change in colony formation from 10ng/mL to 50ng/mL IL7 was 1.80 for Hspa9+/− vs. 0.80 for Hspa9+/+, p=0.03, N=6 mice/genotype). Supplementation of the media with another cytokine that contributes to early B-cell maturation, Flt3 ligand, does not alter Hspa9+/− or Hspa9+/+ CFU-preB colony formation, further implicating altered IL7 signaling. We are currently investigating the downstream responses to IL7 stimulation in B-cell progenitors from Hspa9+/− mice. Collectively, these data implicate loss of HSPA9 as a contributing factor in the reduction of B-cell progenitors observed in patients with del(5q) associated MDS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Selective expression of CD45 isoforms defines CALLA+ monoclonal B- lineage cells in peripheral blood from myeloma patients as late stage B cells

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 711-719 ◽  
Author(s):  
GS Jensen ◽  
MJ Mant ◽  
AJ Belch ◽  
JR Berenson ◽  
BA Ruether ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Plasma Cell ◽  
Late Stage ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Lower Proportion ◽  
Large Subset ◽  
B Lineage

Abstract The peripheral blood lymphocytes from 42 patients with multiple myeloma (MM) and 13 patients with monoclonal gammopathy of undetermined significance (MGUS) were studied by three-color immunofluorescence (IF) using antibodies directed to a broad range of B-cell markers (CD19, CD20, CD21, CD24), CALLA (CD10), PCA-1 (a plasma cell marker), and to the high and low molecular weight isoforms of the leukocyte common antigen, CD45RA (p205/220) and CD45RO (p 180). CD45RA is expressed on pre-B and B cells, and a transition from CD45RA to CD45RO defines differentiation towards plasma cells. Peripheral blood mononuclear cells (PBMC) from patients with myeloma included a large subset of B- lineage cells (mean of 39% to 45%) that were CALLA+ and PCA-1+ in all patients studied, including newly diagnosed patients and patients undergoing chemotherapy. Southern blot analysis indicated the presence of monoclonal Ig rearrangements in PBMC and a substantial reduction in the germ-line bands consistent with the presence of a large monoclonal B-cell subset. Avoidance of purification methods involving depletion of adherent cells was essential for detection of the abnormal B cells. Phenotypically, this abnormal B-cell population corresponded to late B or early pre-plasma cells (20% to 80% of PBMC), as defined by the concomitant expression of low densities of CD19 and CD20, moderate densities of CALLA and PCA-1, and strong expression of CD45RO on all B cells, with weakly coexpressed CD45RA on a small proportion. Heterogeneity in the expression of CD45RA and CD45RO within the abnormal B-cell population from any given patient suggested multiple differentiation stages. Abnormal B cells similar to those in MM were also detected in MGUS, although as a lower proportion of PBMC (26%). Abnormal B cells from patients with MGUS expressed predominantly the CD45RO isoform, but had a lower proportion of CALLA+ and PCA-1+ cells than were found on B cells from MM. This work indicates that the large subset of circulating monoclonal B lymphocytes from myeloma patients are at a late stage in B-cell differentiation, continuously progressing towards the plasma cell stage.

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