Surface Immunoglobulin Expression Research Articles

B cell immunosenescence: different features of naive and memory B cells in elderly

Elderly people show a reduced protection against new infections and a decreased response to vaccines as a consequence of impairment of both cellular and humoral immunity. In this paper we have studied memory/naïve B cells in the elderly, evaluating surface immunoglobulin expression, production of the pro- and anti-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-10, and presence of somatic hypermutation, focusing on the IgG(+)IgD(-)CD27(-) double negative (DN) B cells that are expanded in the elderly. Our results show that naïve B cells from young donors need a sufficiently strong stimulus to be activated "in vitro", while naïve B cells from old subjects are able to produce IL-10 and TNF-α when stimulated "physiologically" (α-CD40/IL-4), suggesting that these cells might play a role in the control of the immuno-inflammatory environment in the elderly. In addition, in the elderly there is an accumulation of DN B cells with a reduced rate of somatic hypermutation. Thus, DN B lymphocytes may be exhausted cells that are expanded and accumulate as a by-product of persistent stimulation or impaired germinal center formation.

Transcriptional and epigenetic regulation of B cell development

B cell development starts in the bone marrow where hematopoietic stem cells (HSCs) progress through sequential developmental stages, as it differentiates into a naïve B cell expressing surface immunoglobulin. In the periphery, B cells that encounter antigen can further differentiate into antibody-secreting plasma cells. In this review, we focus on two factors, E47 and ELL2, which play important roles in the regulation of B cell development in the bone marrow and differentiation of mature B cells into plasma cells in the periphery, respectively. First, E47 activity is required for B cell development in the bone marrow. In addition, we have identified a cell-intrinsic role for E47 in regulating efficient self-renewal and long-term multilineage bone marrow reconstitution potential of HSCs. Second, we explored the role of transcription elongation factors in the super elongation complex (SEC), including ELL2 (eleven-nineteen lysine-rich leukemia factor) in driving poly(A) site choice and plasma cell development. We found that elongation factors impel high levels of IgH mRNA production and alternative processing at the promoter proximal, secretory-specific (sec) poly(A) site in plasma cells by enhancing RNA polymerase II modifications and downstream events. The sec poly(A) site, essentially hidden in B cells, is found by SEC factors in plasma cells.

Cytomorphologic findings of B‐cell lymphomas with concurrent IGH/BCL2 and MYC rearrangements (dual‐translocation lymphomas)

B-cell lymphomas with concurrent IGH/BCL2 and MYC gene rearrangements, termed dual-translocation or double-hit lymphomas (DTLs), rarely are identified. They usually are characterized by highly aggressive behavior, a poor prognosis, and complex karyotypes. The objective of this study was to review and describe the cytomorphologic findings in different types of cytologic preparations and clinicopathologic characteristics of patients with DTLs. Patient samples with IGH/BCL2 and MYC rearrangements that were detected by fluorescence in situ hybridization during the period from October 2003 to September 2009 were selected for morphology review. Clinical data and results from additional studies were collected from patient reports. Cytologic samples from 14 patients (5 men and 9 women) were reviewed. The most common cytomorphologic pattern was a mixed cell population consisting predominantly of large cells (88.2%), mainly centroblasts (94.1%), with dark blue cytoplasm (76.4%) accompanied by apoptotic bodies (64.7%), with marked cellular pleomorphism (94.1%). Nuclear segmentation was present in 64.7% of samples, conferring a "coffee bean" nucleus, and cytoplasmic vacuoles were observed in 46.6% of samples. Immunophenotyping revealed the expression of CD20, CD19, surface immunoglobulin, and CD10 in 13 samples. Other chromosomal aberrations were also identified. Seven patients died of their disease, and the time from progression to death ranged from 1 month to 16 months. Large cells with deeply basophilic cytoplasm, cytoplasmic vacuoles, and frequent segmented nuclei, particularly in fine needle aspirate smears and especially in patients with clinically aggressive and/or unusual clinical features, should trigger a fluorescence in situ hybridization analysis for IGH/BCL2 and MYC translocation to identify this entity.

Chronic Lymphocytic Leukemia (CLL) Signaling Profiling Identifies CLL-Specific Signaling Impairment That Discriminates CLL B-Cells From Normal Circulating B Cells

AbstractAbstract 1374CLL is the most common leukemia in the Western world. It is characterized by the clonal expansion of CD5+/CD19+ B cells, expressing surface immunoglobulins (sIg), most often of the IgM subset. Clinically, it can either manifest as an indolent disease with little impact on the lifespan of a subset of patients or as an aggressive, highly drug-resistant disease with lethal outcome but all possible scenarios in between these extremes are routinely observed. The degree of somatic mutations of the sIg has been shown to affect survival; patients expressing highly mutated sIg have a better prognosis than patients whose sIg are less than 2% different from germline sequences. Therefore, it is intuitive that signaling through the sIg, or B-cell receptor (BCR), must have a role in determining the fate of the CLL cells, veering the intracellular machinery towards proliferation or apoptosis.We have systematically investigated the downstream events caused by BCR stimulation via crosslinking of the sIg in a series of about 60 heterogeneous CLL patients and 15 normal donor peripheral blood mononuclear cells (PBMCs). Using multiplexed phosphoflow cytometry we were able to assess the intensity of multiple signaling events at specific stages of the BCR signaling pathway. By combining phosphospecific staining and classic immunophenotyping we were also able to evaluate signaling intensity at the at the single cell level as well as correlating the signal intensity of CLL-specific surface markers to that of the phosphoproteins analyzed. We used principal component analysis to combine phosphospecific staining within individual cells and generate a variable that discriminates efficiently between CLL and PBMCs B-cells.We found that signaling properties of CLL B-cells were impaired when compared to normal B cells from healthy donors, both at the level of upstream members of the BCR signaling pathway (pBLNK, pSYK, pZap70, pBTK, pPLCg2) and downstream components such as pp38 and pERK. However, within the cohort of CLL samples we identified a small subpopulation in which signaling proceeded as well or more efficiently than in normal B cells. We also used our principal component variable to quantify the heterogeneity of signaling responses within populations of CLL B-cells and correlate them with clinical parameters that are known prognostic indicators in CLL.In conclusion, multiplexed phosphoflow cytometry of individual CLL cells provides a novel and highly specific method to determine the use of signaling pathways (such as BCR-mediated) in comparison to normal B cells. Our studies indicate a significant decrease in BCR signaling of CLL B cells, which involves both upstream and downstream components. Disclosures:van den Brink:Cytheris: Research Funding.

IGH Repertoire Analysis In Multiple Myeloma (MM): Lack of Intra-Disease Homology and Occasional Clustering with Sequences of Other B-Cell Neoplasms Sharing Identical Geographical Origin

AbstractAbstract 2951 Background:The identification of stereotyped immunoglobulin (IG) receptors has improved our knowledge on the pathogenesis of several B-cell malignancies, suggesting the role of antigen-driven stimulation in chronic lymphocitic leukemia (CLL), marginal-zone lymphoma (MZL) and mantle-cell lymphoma (MCL). Multiple myeloma (MM) is a terminally-differentiated neoplasm no longer expressing surface IG; however some reports suggest the existence of early B-lymphocyte precursors which could be susceptible to antigen-driven stimulation. IG heavy chain (IGH) repertoire has not been extensively investigated in MM, with the largest available reports containing less than 80 complete sequences. Aims:To address this issue we created a database of MM IGH sequences including our institutional records (mostly derived from minimal residual disease studies) and sequences available from the literature. We planned a two-step analysis: a) first we characterized the MM repertoire and performed intra-MM clustering analysis; b) then we compared our MM series to a large public database of IGH sequences from neoplastic and non-neoplastic B-cells in search of similarities between MM sequences and other normal or neoplastic IGH repertoires. Patients and methods:131 MM IGH genes were amplified and sequenced at our Institutions and belonged to Italian patients, while 214 MM IGH sequences from non-Italian patients were derived from published databases (NCBI-EMBL-IMGT/LIGM-DB) for a total of 345 fully interpretable MM sequences (out of 396). 28590 IGH sequences from other malignant and non-malignant B-cells were retrieved from the same public databases, including approximately 4500 CLL/Non-Hodgkin lymphoma (NHL) sequences and comprising 500 sequences from Italian patients. All sequences were analyzed using the IMGT database and tools (Lefranc et al., Nucleic Acid Res. 2005; http://imgt.cines.fr/) to identify IGHV-D-J gene usage, to assess the somatic hypermutation (SHM) rate and to identify HCDR3. HCDR3 aminoacidic sequences were aligned together using the ClustalX 2.0 software (Larkin et al., Bioinformatics, 2007; http://www.clustal.org/). Subsets of stereotyped IGH receptors were defined according to Stamatopoulos et al. (Blood, 2007). Result:IGHV analysis in MM was almost in keeping with the normal B-cell repertoire, showing a less remarkably biased IGH usage compared to CLL, MCL and MZL (with seven genes accounting for 40% of cases, compared to respectively five, three and two genes). However, a modest but significant over-representation of IGHV1-69, 2–5, 2–70, 3–21, 3–30-3, 3–43, 5–51 and 6-1 genes and under-representation of the IGHV1-18, 1–8, 3–30, 3–53 and 4–34 was noticed. The rate of somatic hypermutation in MM followed a Gaussian distribution with a median value of 7.8%. Intra-MM search for HCDR3 similarities never met minimal requirements for stereotyped receptors. When MM sequences were compared to non-MM database, only a minority of MM sequences (2.6%, n=9) clustered with sequences from lymphoid tumors and normal B-cells (figure 1A). In particular two non-Italian MM sequences clustered with previously characterized, uncommon CLL subsets (n.37 and n.71 according to Murray et al., Blood 2008). Moreover, novel provisional clusters were observed including three MM-CLL subsets, one MM-NHL subset, and three MM-normal B-cell subsets. While the MM-normal B-cell clusters involved non-Italian patients, we unexpectedly noticed that the four MM-CLL/MM-NHL clusters were composed exclusively of Italian patients, as shown in figure 1B, although Italian subjects represented less than 12% of the entire CLL-NHL database. Conclusion:The analysis of the largest currently available database of MM IGH sequences indicates the following: 1) MM IGH repertoire is closer to physiological distribution than that of CLL, MCL and MZL; 2) MM specific clusters do not occur to a frequency detectable with currently available databases; 3) 98% of MM sequences are not related to other “highly-clustered” lymphoproliferative disorders; 4) Uncommon clustering phenomena may follow a geographical rather than a disease-related pattern. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Montelukast treatment (cysteinyl leukotriene receptor antagonist) in a model of food allergy: modifications in lymphatic cell population from rectal mucosa

The aim is to determine immunopathological modifications in rectal mucosa from rabbits after local challenge in ovalbumin (OVA) sensitized animals previously treated with montelukast. thirty two rabbits divided into four groups: G1: normal; G2: subcutaneously OVA sensitized; G3: sensitized, locally OVA challenged and sampled 4 hours after challenge; and G4: sensitized, locally OVA challenged and treated 4 hours before challenge with montelukast (0.15 mg/kg). Specific anti-OVA IgE levels were evaluated by passive cutaneous anaphylaxis test (PCA). In each group 200 high microscopical power fields (HPF) were counted. Results were expressed as arithmetic mean and SE. Anti -CD4, CD5, micro chain monoclonal antibodies were used. Avidin biotin horseradish peroxidase system was used. CD 4: G1: 8.3 +/- 0.06; G2: 13.4 +/- 0.08, G3: 8.25 +/- 0.06, G4: 11.8 +/- 0.02. CD 5: G1: 7.3 +/- 0.05; G2: 9.4 +/- 0.05, G3: 11.3 +/- 0.06, G4: 8.1 +/- 0.06. mu chain: G1: 10.4 +/- 0.06; G2: 3.8 +/- 0.02, G3: 6.0 +/- 0.10, G4: 2.2 +/- 0.10. In all cases, experimental groups (G3 vs. G4) presented statistical significant differences (p < 0.05). CD4+, CD5+ cells and mu chain+ decrease in experimental group (G4), probably due to lymphocyte migration inhibition to challenged mucosa. mu chain+ cell decrease could be based on B cell activation and expression of different surface immunoglobulins. Cells expressing mu chain decreased in G2 and G3 likely due to activation of B cells and subsequent expression of other immunoglobulin chains in cell surface. We conclude that obtained data are important to elucidate immunopathology of local anaphylactic reaction in rectal mucosa from systemic sensitized animals after treatment with montelukast.

Endoscopic Diagnosis of Childhood Burkitt's Lymphoma of the Colon

A 11-year-old previously healthy girl presented with abdominal pain for 8 weeks. Her physical examination was unremarkable except for a mass located in the right upper quadrant. Abdominal computed tomography scan revealed a 4-cm solid mass arising from the right colon (Fig 1). Positron emission tomography with [F]fluorodeoxyglucose scanning exhibited abnormal uptake in the right upper quadrant of the abdomen (Fig 2). Colonoscopy showed a 4 4 cm near-obstructive nonulcerated wall mass in the right colon (Fig 3). An endoscopic biopsy of the mass was performed. Histology showed a rich cellular infiltrate with medium-sized atypical lymphocytes with round to irregular nuclei, prominent nucleoli, and scant cytoplasm with a starry sky appearance (Fig 4A). Tumor cells showed positive staining for CD45, CD20, and CD10 (Fig 4B, immunostaning for CD20). A diagnosis of Burkitt’s lymphoma was confirmed by cytogenetic analysis of the tumor, with the t(8;14)(q24; q32) translocation being found. Bone marrow and CSF study did not show any malignant cells. The patient underwent four cycles of chemotherapy according to the LMB 96 protocol, with complete disappearance of the palpable abdominal mass confirmed on computed tomography scanning. The patient currently remains free of disease at a follow-up of 5 years. Burkitt’s lymphoma is a mature B-cell neoplasm characterized by the expression of surface immunoglobulins and the presence of characteristic reciprocal chromosomal translocations involving the c-myc oncogene and an immunoglobulin gene. Burkitt’s lymphoma is prevalently a childhood malignancy and represents an oncological emergency because of its high proliferation fraction and short doubling time. Burkitt’s lymphoma is highly sensitive to chemotherapy including cyclophosphamide, vincristine, cytarabine, and methotrexate. The overall cure rates are approximately 90% in children and 65% to 80% in adults with contemporary chemotherapy regimens. Most children with Burkitt’s lymphoma present with primary involvement of the abdomen or the head and neck region. Patients with primary localized disease in the abdomen may present with abdominal pain or swelling, gastrointestinal bleeding, intestinal perforation, or intestinal obstruction resulting from direct compression of the lumen by an expanding mass or by an intussusception triggered by the intraluminal projection of the tumor mass. These abdominal symptoms often lead to emergency laparotomies before a diagnosis of Burkitt’s lymphoma can be made. Surgical resection could be associated with

SELECTIVE INFLUENCES IN THE B-CELL RECEPTOR IMMUNOGLOBULIN HEAVY AND LIGHT CHAIN IN HAIRY CELL LEUKEMIA

Hairy cell leukemia (HCL) is a rare, chronic B-cell neoplasm characterized by leukemic hairy cells (HCs) present in blood, bone marrow, and splenic red pulp, with atrophy of white pulp. Lymph node involvement is infrequent. HCL is typically associated with markers of activation, that include expression of CD25, CD11c, FMC7, and CD103 at high intensity. A distinctive feature of HCL is expression of multiple surface immunoglobulin (sIg) isotypes, although its prevalence in HCL is not fully mapped.

Expression of Surface IgG Is High in Chronic Lymphocytic Leukaemia, and Co-Expression with IgM Confers a Worse Prognosis.

Abstract 4371 IntroductionThe classical immunophenotype of Chronic Lymphocytic Leukaemia (CLL) involves weak or moderate expression of surface immunoglobulin (sIg) M and D. Expression of sIgG has generally been considered to be rare (published estimates 4-28%). The B Cell Receptor is thought to be central to the pathophysiology of CLL. Patients and MethodsWe present data from an analysis of 748 patients with CLL who have had flow cytometry performed at Barts and the London NHS trust. This includes patients from several other clinical centres serving a population of 3.7 million. A cohort of cases (n=26) displaying differing patterns of sIgG and sIgM expression was investigated further by: 4-colour flow cytometry; detection of constant (C) gene mRNA transcripts including Ca, Cm, Cg, or Cd to confirm isotype status; variable heavy chain somatic mutation rate (VH status); and functionality of the sIg using calcium flux and cell proliferation assays ([H3]- thymidine incorporation) after BCR crosslinking using isotype-specific F(ab')2 anti-human Ig. We also analysed whether differing patterns of sIgG and sIgM expression conveyed prognostic impact. Results- Prevalence of sIgG expression399 of the 748 patients with CLL had complete sIgG and sIgM expression data. We found that 33% of all CLL cases express sIgG (19% with co-expression of sIgG and sIgM). 19% appeared to have neither sIgG nor sIgM expression. When tested for, sIgD appeared to be present in most of this IgG-IgM- subset. The expression pattern appeared to be stable over time in the majority of cases. Functionality of sIgGTo explore the signalling capacity of the sIg, a cohort of 26 patients with readily available cells was chosen that displayed a variety of expression patterns of sIgG and sIgM (6 IgG-IgM+, 3 IgG+IgM-, 15 IgG+IgM+, 2 IgG-IgM- ). This cohort underwent further testing:Four-colour flow cytometry confirmed the presence of both sIgG and sIgM on single CLL cells in cases of IgG+IgM+ identified in the diagnostic laboratory, as well as the sIgG and sIgM expression of other cases. In the IgG-IgM+ cases 3 of 6 were VH mutated, one had Cg mRNA transcripts detectable, with all expressing Cm. The IgG+IgM- cases were VH mutated and contained only mRNA for Cg gene transcripts. Of the IgG+IgM+ cases 10/15 were VH mutated. All had detectable Cg and Cm transcripts, with several expressing Ca and Cd. One IgG-IgM- case had no detectable C-gene transcripts but detectable VDJ-genes, the other had Cm and Cd transcripts detectable in the absence of sIgG and sIgM. Both IgG-IgM- cases were VH mutated. Crosslinking of the sIg with appropriate anti sIgG or sIgM F(ab')2 resulted in calcium flux and cell proliferation. These data confirm that the expressed sIgG and sIgM is produced by the CLL cell, and are functionally competent to signal. Prognostic impact: Survival and treatment data were available on 382 patients in our database. Of these, 244 had complete sIgG and sIgM expression determination. Analyses of the sIgG and sIgM expression subgroups suggested that while there is no difference in time to first treatment (median 4.5 years), the IgG+IgM+ group (n=52, median OS 7 years) had a significantly shorter overall survival (OS) compared to the IgG-IgM+ (n=126, median survival not reached), IgG+IgM- (n=29, median OS 11 years), or IgG-IgM- (n=37, median survival not reached) groups. [Display omitted] ConclusionsIn a large cohort of patients with CLL, we show that expression of sIgG is common, with a large number of cases co-expressing sIgG and sIgM simultaneously. When stimulated, both isotypes are capable of signal transduction resulting in cellular activation. These malignant cells appear to have deranged mechanisms of Class Switch Recombination. The IgG+IgM+ group of patients appears to have a more treatment resistant disease, conferring shortened survival. Many different groups have suggested the B Cell Receptor to be an important component of CLL pathophysiology. Our data support these models and provide further insight into the underlying biology of CLL, as well as offering another prognostic marker and possible target for treatment. Disclosures:No relevant conflicts of interest to declare.

Elevation of clonal serum free light chains in patients with HIV-negative primary effusion lymphoma (PEL) and PEL-like lymphoma

Elevation of clonal serum free light chains in patients with HIV-negative primary effusion lymphoma (PEL) and PEL-like lymphoma

Acute Lymphoblastic Leukemia With Burkitt-like Morphologic Features and High Myeloperoxidase Activity

Expression of a high level of myeloperoxidase (MPO) as a sole myeloid marker in acute leukemias that express typical lymphoblastic markers is unusual. Herein we report 5 cases of MPO+, otherwise typical acute lymphoblastic leukemia (ALL) without the expression of other myeloid markers. In most cases, MPO positivity was detected in more than 20% of blasts by immunologic (flow cytometric) and enzymatic testing. The striking feature of most of these cases is a morphologic picture reminiscent of that seen in Burkitt-like B-cell ALL with basophilic cytoplasm and vacuoles but no expression of surface immunoglobulin. All cases responded to ALL therapy and should be distinguished from myeloid leukemia and from Burkitt leukemia/lymphoma.

免疫グロブリンH鎖(Igα)を細胞表面に発現し，L鎖の発現を認めないびまん性大細胞型B細胞リンパ腫

A 59-year-old woman with goiter complained of nausea, vomiting and weight loss in April 2000. She underwent an endoscopic examination and was admitted to our hospital because gastric biopsy specimens revealed that she had diffuse large B-cell lymphoma. A thyroid biopsy also detected the diffuse infiltration of lymphoma cells, which were positive for CD19, CD20, CD38 and HLA-DR. Although the cells expressed surface immunoglobulin a chain, they lacked expressions of the kappa and lambda light chains. Chromosomal analysis of the thyroid cells showed 47, XX, t(2 ; 3)(q31 ; q13), + 3, t(8 ; 22)(q24 ; q11). After five courses of biweekly CHOP chemotherapy, she received autologous peripheral blood stem cell transplantation in October 2000. Currently, she has maintained complete remission for more than 4 years.

Immunophenotypic studies of monoclonal gammopathy of undetermined significance

BackgroundMonoclonal gammopathy of undetermined significance (MGUS) is a common plasma cell dyscrasia, comprising the most indolent form of monoclonal gammopathy. However, approximately 25% of MGUS cases ultimately progress to plasma cell myeloma (PCM) or related diseases. It is difficult to predict which subset of patients will transform. In this study, we examined the immunophenotypic differences of plasma cells in MGUS and PCM.MethodsBone marrow specimens from 32 MGUS patients and 32 PCM patients were analyzed by 4-color flow cytometry, using cluster analysis of ungated data, for the expression of several markers, including CD10, CD19, CD20, CD38, CD45, CD56 and surface and intracellular immunoglobulin light chains.ResultsAll MGUS patients had two subpopulations of plasma cells, one with a "normal" phenotype [CD19(+), CD56(-), CD38(bright +)] and one with an aberrant phenotype [either CD19(-)/CD56(+) or CD19(-)/CD56(-)]. The normal subpopulation ranged from 4.4 to 86% (mean 27%) of total plasma cells. Only 20 of 32 PCM cases showed an identifiable normal subpopulation at significantly lower frequency [range 0–32%, mean 3.3%, p << 0.001]. The plasma cells in PCM were significantly less likely to express CD19 [1/32 (3.1%) vs. 13/29 (45%), p << 0.001] and more likely to express surface immunoglobulin [21/32 (66%) vs. 3/28 (11%), p << 0.001], compared to MGUS. Those expressing CD19 did so at a significantly lower level than in MGUS, with no overlap in mean fluorescence intensities [174 ± 25 vs. 430 ± 34, p << 0.001]. There were no significant differences in CD56 expression [23/32 (72%) vs. 18/29 (62%), p = 0.29], CD45 expression [15/32 (47%) vs. 20/30 (67%), p = 0.10] or CD38 mean fluorescence intensities [6552 ± 451 vs. 6365 ± 420, p = 0.38]. Two of the six MGUS cases (33%) with >90% CD19(-) plasma cells showed progression of disease, whereas none of the cases with >10% CD19(+) plasma cells evolved to PCM.ConclusionMGUS cases with potential for disease progression appeared to lack CD19 expression on >90% of their plasma cells, displaying an immunophenotypic profile similar to PCM plasma cells. A higher relative proportion of CD19(+) plasma cells in MGUS may be associated with a lower potential for disease progression.

The POZ/BTB Domain Transcription Factor Miz-1 (Zbtb17) Is Required during Early B Cell Development for the Survival of B-Cell Progenitors and Is Essential for the Formation of Mature Follicular B Cells

B-cell development takes place in the bone marrow and is defined by a number of sequential steps that include the up-regulation of CD19, the rearrangement of immunoglobulin heavy and light chain genes (V(D)J recombination) and the expression of surface immunoglobulin. The early steps are regulated by cytokine signaling and the hierarchical expression of transcription factors, among them EBF, Pax5 and E2A and any interference with these critical elements leads to partial or total abrogation of B cell development. Here we present evidence that the POZ/BTB domain transcription factor Miz-1 (Zbtb17) represents an important novel regulator of the early development of follicular B cells. We have used gene targeting in mice to generate a non-functional allele of Miz-1 in all hematopoietic cells. In these mice, the development of adult follicular B cells is almost entirely abrogated, whereas the formation of marginal zone B-cells remain unaffected. Miz-1 deficiency correlated with the absence of CD19+ pro B-cells from the bone marrow and a block at the transition of the pre-pro-B cell to the pro-B cell stage. Although common lymphoid progenitors (CLPs) that are at the origin of B-cell development were present in Miz-1 deficient mice, they showed decreased expression of E2A, EBF and Pax5 compared to their wild type counterparts. Moreover, they were unable to differentiate in culture into more mature B cells even on stroma cells (OP9) and the presence or absence of IL-7. Interestingly, a forced expression of EBF or PAX5 in Miz-1 deficient progenitor cells did not rescue this phenotype. Furthermore, fetal B cell development, which has been shown to depend on EBF and Pax5, is not altered in Miz-1 deficient mice, suggesting that Miz-1 acts in a pathway that is independent of these critical B-cell regulators. In contrast, however, to EBF and Paxc5, the co-expression of a Bcl-2 transgene almost completely restored the development of more mature CD19+ or IgM+ B-cells in Miz-1 deficient mice. This indicated that Miz-1 is implicated in the regulation of cell survival at early stages of B cell development. Since it has been shown before that Bcl-2 is a downstream effector of Miz-1, it is conceivable that Miz-1 regulates Bcl-2 in the early B cell precursors, possibly as an element of the IL-7 signaling pathway, and thereby ensures their survival and proper development. We conclude that Miz-1 represents a novel regulator of early B cell development that exerts its function at a precise step in adult mice independently of other well-established regulators of B-cell development such as EBF or Pax5.

Concurrent Chromosomal Alterations at 3q27, 8q24 and 18q21 in An Atypical Form of Burkitt's Lymphoma

Introduction: Chromosomal translocations are common in non-Hodgkin's lymphomas (NHL), most frequently involving the genes bcl-2 in the t(14;18) of follicular lymphoma (FL), c-myc in the t(8;14) of Burkitt's lymphoma (BL) and bcl-6 in the t(3;14) of follicular or diffuse large B-cell (DLBC) lymphoma. We report the clinical features, pathology and genetic findings in an exceedingly rare case of Burkitt's lymphoma that showed concurrent involvement of these three chromosomal loci.Case Report: This is a 65 year old Caucasian female who presented with a rapidly growing right supraclavicular lymph node over a few weeks. FNA biopsy showed typical morphology of Burkitt's lymphoma. Similar morphologic features were found on the bone marrow biopsy. There was widespread disease with no CNS involvement. Flow cytometry from peripheral blood and immunohistochemistry on the cellblock showed B-cell phenotype positive for CD 10, CD19, CD20 (negative CD20 by immunohistochemistry), HLA-DR, cytoplasmic CD79a, and negative for CD34 and TdT. The interesting finding was the lack expression of surface or cytoplasmic immunoglobulin and expression of weak Bcl-6. Almost 90–95% expressed Ki67. The cytogenetic analysis reportedly demonstrated a complex karyotype t(3;8;14), and t(14;18) involving c-myc (8q24), bcl-2 (18q21), and bcl-6 (3q27). After 7 cycles of hyper CVAD-R she had bone marrow biopsy which showed residual disease. She also had a biopsy confirmed relapse as left arm nodule and left leg nodular infiltrate at 8 and 12 months form the diagnosis, respectively.Discussion: This is a complex case of high grade B-cell lymphoma with morphology suggestive of Burkitt's lymphoma. However the classification was challenging by the lack of surface immunoglobulin expression that might be expected in mature B-cell neoplasm “DLBCL, FL”, and the lack of TdT and CD34 that might be expected in precursor B-cell neoplasm “BL”. The diagnosis was highly dependent on the cytogenetic findings, which was significant for the presence of t(8;14) albeit in a three way translocation t(3;8;14), and t(14;18) involving c-myc (8q24), bcl-2 (18q21), and bcl-6 (3q27). The lymphoma was therefore described as “Burkitt's transformation”. This is a rare translocation pattern, but has been described in follicular lymphoma, grade 3; diffuse large cell lymphoma; and Burkitt's lymphoma.Conclusion: BL might lack surface immunoglobulin expression making the diagnosis of high grade B-cell lymphoma challenging if based on the morphology and immunophenotyping alone. The cytogentetic findings better delineate sub-types of lymphoma. Molecular evidence of multiple oncogene deregulations, especially when involving the c-myc gene, appears to be associated with a dire clinical outcome.

The effect of mesenchymal stem cells on the viability, proliferation and differentiation of B-lymphocytes

Mesenchymal stem cells are multilineage non-hematopoietic progenitor cells that play a key role in supporting the lymphohematopoietic system. Their distribution in bone marrow and secondary lymphoid organs allows an intimate interaction with T- and B-lymphocytes. While their effect on T-lymphocytes has been extensively analyzed, data on the effect of mesenchymal stem cells on B cells are more limited. We analyzed the effects of mesenchymal stem cells on B-lymphocytes and the pathways involved in these effects. The effect of MSC on the proliferation and viability of B cells was evaluated using MTT assays, annexin/7-amino-actinomycin D and propidium iodide staining. The B-cell maturation pattern was established using flow cytometry based on the expression of different markers related to the differentiation of B cells, such as CD38, CD138, CD19 and CCR7, and to the expression of surface and intracellular immunoglobulins. Finally, western blot assays were used to identify the pathways involved in the effects of mesenchymal stem cells on B-lymphocytes. Mesenchymal stem cells increased viability and blocked the cell cycle of B-lymphocytes in the G(0)/G(1) phase. In vitro exposure of B cells to plasmacytoid dendritic cells induced B-cell differentiation as shown by an increased number of CD38(++)/CD138(++) cells, which also displayed higher levels of cytoplasmic immunoglobulin and lower levels of CD19, CCR7 and surface immunoglobulin. Interestingly, this maturation pattern was inhibited by adding mesenchymal stem cells to the culture. Finally, mesenchymal stem cells modified the phosphorylation pattern of the extracellular response kinase 1/2 and p38 pathways which are both involved in B-cell viability, proliferation and activation. Mesenchymal stem cells increase B-cell viability while inhibiting proliferation, arresting B-lymphocytes in the G(0)/G(1) phase of the cell cycle. The presence of mesenchymal stem cells blocked B-cell differentiation as assessed by flow cytometry. Finally, mesenchymal stem cells modified the activation pattern of the extracellular response kinase and the p38 mitogen-activated protein kinase pathways in B-lymphocytes.

Targeting Lentiviral Vectors to Antigen-Specific Immunoglobulins

Gene transfer into B cells by lentivectors can provide an alternative approach to managing B lymphocyte malignancies and autoreactive B cell-mediated autoimmune diseases. These pathogenic B cell populations can be distinguished by their surface expression of monospecific immunoglobulin. Development of a novel vector system to deliver genes to these specific B cells could improve the safety and efficacy of gene therapy. We have developed an efficient method to target lentivectors to monospecific immunoglobulin-expressing cells in vitro and in vivo. We were able to incorporate a model antigen CD20 and a fusogenic protein derived from the Sindbis virus as two distinct molecules into the lentiviral surface. This engineered vector could specifically bind to cells expressing surface immunoglobulin recognizing CD20 (alphaCD20), resulting in efficient transduction of target cells in a cognate antigen-dependent manner in vitro, and in vivo in a xenografted tumor model. Tumor suppression was observed in vivo, using the engineered lentivector to deliver a suicide gene to a xenografted tumor expressing alphaCD20. These results show the feasibility of engineering lentivectors to target immunoglobulin- specific cells to deliver a therapeutic effect. Such targeting lentivectors also could potentially be used to genetically mark antigen-specific B cells in vivo to study their B cell biology.

Lymphoma immunophenotyping: “borderline” lymphomas

Immunophenotyping of B-cell lymphoproliferative disorders is indispensable, especially in disorders with CD19(+) CD5(+) B lymphocytes, where we have to make the distinction between low grade neoplasia, such as chronic lymphocytic leukemia with CD23(+) malignant lymphocytes, and aggressive neoplasia such as mantle cell lymphoma with CD23(-) malignant lymphocytes. We found some cases of CD19(+) CD5(+) lymphoproliferative disorders that do not meet all criteria for diagnosis of chronic lymphocytic leukemia or mantle cell lymphoma. For instance, we found cases with a low or no expression of CD23, asociated with absence of expression of FMC7 and surface immunoglobulins. These cases could be classified as "borderline" CD19(+) CD5(+) B cell lymphoproliferative disorders, with an intermediate neoplasic grade.

Deubiquitination of FANCD2 Is Required for DNA Crosslink Repair

SummaryMonoubiquitination of FANCD2 and PCNA promotes DNA repair. It causes chromatin accumulation of FANCD2 and facilitates PCNA's recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted USP1 in chicken cells to dissect its role in a stable genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensitivity. This defective DNA repair is associated with constitutively chromatin-bound, monoubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible impact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA damage. In DT40, USP1 autocleavage is not stimulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage.

High Frequency of Surface Immunoglobulin IgG in B-Cell Chronic Lymphocytic Leukaemia.

Background: A common diagnostic feature of chronic lymphocytic leukaemia (CLL) is the weak surface immunoglobulin (sIg) expression of the IgM/D isotypes. The occurrence of sIgG is believed to be an atypical finding in CLL.Aims: To demonstrate that sIgG expression is a real and not artefactual by molecular and functional assays.Methods: 194 unselected, consecutive cases of CLL were analysed during routine diagnostic testing for sIgG and sIgM. Flow cytometry was performed using fresh whole blood and red cell lysis (Pharmalyse solution, BD). For confirmation of these results and demonstration of functionality, we used 4-colour flow cytometry, PCR for mRNA detection, proliferation assay (3H incorporation), and calcium flux assay.Results: 23% (44/194) of all CLL cases displayed sIgG, with 39 being sIgG+ alone. PCR results showed a distinct band indicating the presence of mRNA for IgG. Four-colour flow cytometry identified single cells that co-expressed CD19/CD160/sIgG and ruled out sIgM expression. In vitro proliferation assays demonstrated that sIgG is functional when triggered by F(Ab')2 fragments specific for sIgG isotype. The strength of the signal in one case after stimulation via sIgG led to a 3.5-fold increase in cellular proliferation. Calcium flux assays also demonstrated that the IgG isotype was functional, one case showing a 50% increase in fluorescence intensity after stimulation via sIgG.Discussion: 23% of CLL cases have sIgG expression. In addition to a high frequency of sIgG expression, this study has shown sIgG to be capable of signalling. If antigen-drive is important in CLL, an IgG B-cell receptor should have higher affinity for a putative antigen, which potentially would be a poor prognostic feature. However, such cases would also be expected to show mutated VH genes, indicating a better prognosis. Further study is required to elucidate the pathological significance of sIgG expression and its role in CLL.