Memory B-cell Populations Research Articles

Envelope-specific B-cell populations in African green monkeys chronically infected with simian immunodeficiency virus

African green monkeys (AGMs) are natural primate hosts of simian immunodeficiency virus (SIV). Interestingly, features of the envelope-specific antibody responses in SIV-infected AGMs are distinct from that of HIV-infected humans and SIV-infected rhesus monkeys, including gp120-focused responses and rapid development of autologous neutralization. Yet, the lack of genetic tools to evaluate B-cell lineages hinders potential use of this unique non-human primate model for HIV vaccine development. Here we define features of the AGM Ig loci and compare the proportion of Env-specific memory B-cell populations to that of HIV-infected humans and SIV-infected rhesus monkeys. AGMs appear to have a higher proportion of Env-specific memory B cells that are mainly gp120 directed. Furthermore, AGM gp120-specific monoclonal antibodies display robust antibody-dependent cellular cytotoxicity and CD4-dependent virion capture activity. Our results support the use of AGMs to model induction of functional gp120-specific antibodies by HIV vaccine strategies.

Ageing of the B-cell repertoire.

Older people are more susceptible to infection, less responsive to vaccination and have a more inflammatory immune environment. Using spectratype analysis, we have previously shown that the B-cell repertoire of older people shows evidence of inappropriate clonal expansions in the absence of challenge, and that this loss of B-cell diversity correlates with poor health. Studies on response to vaccination, using both spectratyping and high-throughput sequencing of the repertoire, indicate that older responses to challenge are lacking in magnitude and/or delayed significantly. Also that some of the biologically significant differences may be in different classes of antibody. We have also previously shown that normal young B-cell repertoires can vary between different phenotypic subsets of B cells. In this paper, we present an analysis of immunoglobulin repertoire in different subclasses of antibody in five different populations of B cell, and show how the repertoire in these different groups changes with age. Although some age-related repertoire differences occur in naive cells, before exogenous antigen exposure, we see indications that there is a general dysregulation of the selective forces that shape memory B-cell populations in older people.

Regulation of germinal center responses and B-cell memory by the chromatin modifier MOZ.

Memory B cells and long-lived bone marrow-resident plasma cells maintain humoral immunity. Little is known about the intrinsic mechanisms that are essential for forming memory B cells or endowing them with the ability to rapidly differentiate upon reexposure while maintaining the population over time. Histone modifications have been shown to regulate lymphocyte development, but their role in regulating differentiation and maintenance of B-cell subsets during an immune response is unclear. Using stage-specific deletion of monocytic leukemia zinc finger protein (MOZ), a histone acetyltransferase, we demonstrate that mutation of this chromatin modifier alters fate decisions in both primary and secondary responses. In the absence of MOZ, germinal center B cells were significantly impaired in their ability to generate dark zone centroblasts, with a concomitant decrease in both cell-cycle progression and BCL-6 expression. In contrast, there was increased differentiation to IgM and low-affinity IgG1(+) memory B cells. The lack of MOZ affected the functional outcome of humoral immune responses, with an increase in secondary germinal centers and a corresponding decrease in secondary high-affinity antibody-secreting cell formation. Therefore, these data provide strong evidence that manipulating epigenetic modifiers can regulate fate decisions during humoral responses, and thus could be targeted for therapeutic intervention.

Phenotypes and distribution of mucosal memory B-cell populations in the SIV/SHIV rhesus macaque model.

As vaccine-elicited antibodies have now been associated with HIV protective efficacy, a thorough understanding of mucosal and systemic B-cell development and maturation is needed. We phenotyped mucosal memory B-cells, investigated isotype expression and homing patterns, and defined plasmablasts and plasma cells at three mucosal sites (duodenum, jejunum and rectum) in rhesus macaques, the commonly used animal model for pre-clinical vaccine studies. Unlike humans, macaque mucosal memory B-cells lacked CD27 expression; only two sub-populations were present: naïve (CD21(+)CD27(-)) and tissue-like (CD21(-)CD27(-)) memory. Similar to humans, IgA was the dominant isotype expressed. The homing markers CXCR4, CCR6, CCR9 and α4β7 were differentially expressed between naïve and tissue-like memory B-cells. Mucosal plasmablasts were identified as CD19(+)CD20(+/-)HLA-DR(+)Ki-67(+)IRF4(+)CD138(+/-) and mucosal plasma cells as CD19(+)CD20(-)HLA-DR(-)Ki-67(-)IRF4(+)CD138(+). Both populations were CD39(+/-)CD27(-). Plasma cell phenotype was confirmed by spontaneous IgA secretion by ELISpot of positively-selected cells and J-chain expression by real-time PCR. Duodenal, jejunal and rectal samples were similar in B-cell memory phenotype, isotype expression, homing receptors and plasmablast/plasma cell distribution among the three tissues. Thus rectal biopsies adequately monitor B-cell dynamics in the gut mucosa, and provide a critical view of mucosal B-cell events associated with development of vaccine-elicited protective immune responses and SIV/SHIV pathogenesis and disease control.

Intrahepatic B‐cell follicles of chronically hepatitis C virus‐infected individuals lack signs of an ectopic germinal center reaction

Chronic infection with hepatitis C virus (HCV) often affects the B-cell compartment, leading to the occurrence of autoimmunity and B-cell lymphoproliferation, in particular mixed cryoglobulinemia and B-cell lymphomas. HCV presumably causes these lymphoproliferations by chronic antigenic stimulation and/or direct mutagenic effects on B cells. It has been speculated that the interaction of HCV with B cells and the expansion of antigen-triggered B cells happens in germinal center-like structures in the livers of HCV carriers. We studied rearranged immunoglobulin V(H) genes from seven B-cell follicles microdissected from the livers of three unselected chronic HCV patients. The follicles consisted of polyclonal naive and memory B-cell populations with only rare indication of minor clonal expansions and no evidence for active somatic hypermutation. Frequent detection of V(H) rearrangements using the VH1-69 gene segment nevertheless indicated that at least a fraction of the B cells is HCV-specific and/or autoreactive. Thus, the typical intrahepatic B-cell follicles in chronic HCV carriers do not function as ectopic germinal centers for clonal expansion and affinity maturation of B cells. Hence, autoreactive and HCV-specific B-cell clones might either develop in secondary lymphoid organs or in intrahepatic follicles only under particular, yet undefined, circumstances.

Evidence For Immunoglobulin D In Patients With Chronic Rhinosinusitis

Immunoglobulin D (IgD) is an enigmatic antibody isotype typically coexpressed with IgM in naïve B cells. However, monomeric IgD comprises approximately 8% of human nasal secreted immunoglobulin and an IgM-IgD+CD27+ memory B-cell population has been recently described in nasal mucosa and tonsils. A role for IgD in chronic rhinosinusitis (CRS) remains unexplored and we tested the hypothesis that IgD is elevated in patients with CRS and characterized the IgD+ cells.

An Inactivated Cell Culture Japanese Encephalitis Vaccine (JE-ADVAX) Formulated with Delta Inulin Adjuvant Provides Robust Heterologous Protection against West Nile Encephalitis via Cross-Protective Memory B Cells and Neutralizing Antibody

West Nile virus (WNV), currently the cause of a serious U.S. epidemic, is a mosquito-borne flavivirus and member of the Japanese encephalitis (JE) serocomplex. There is currently no approved human WNV vaccine, and treatment options remain limited, resulting in significant mortality and morbidity from human infection. Given the availability of approved human JE vaccines, this study asked whether the JE-ADVAX vaccine, which contains an inactivated cell culture JE virus antigen formulated with Advax delta inulin adjuvant, could provide heterologous protection against WNV infection in wild-type and β2-microglobulin-deficient (β2m(-/-)) murine models. Mice immunized twice or even once with JE-ADVAX were protected against lethal WNV challenge even when mice had low or absent serum cross-neutralizing WNV titers prior to challenge. Similarly, β2m(-/-) mice immunized with JE-ADVAX were protected against lethal WNV challenge in the absence of CD8(+) T cells and prechallenge WNV antibody titers. Protection against WNV could be adoptively transferred to naive mice by memory B cells from JE-ADVAX-immunized animals. Hence, in addition to increasing serum cross-neutralizing antibody titers, JE-ADVAX induced a memory B-cell population able to provide heterologous protection against WNV challenge. Heterologous protection was reduced when JE vaccine antigen was administered alone without Advax, confirming the importance of the adjuvant to induction of cross-protective immunity. In the absence of an approved human WNV vaccine, JE-ADVAX could provide an alternative approach for control of a major human WNV epidemic.

Dynamics of memory B-cell populations in blood, lymph nodes, and bone marrow during antiretroviral therapy and envelope boosting in simian immunodeficiency virus SIVmac251-infected rhesus macaques.

Human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection causes B-cell dysregulation and the loss of memory B cells in peripheral blood mononuclear cells (PBMC). These effects are not completely reversed by antiretroviral treatment (ART). To further elucidate B-cell changes during chronic SIV infection and treatment, we investigated memory B-cell subpopulations and plasma cells/plasmablasts (PC/PB) in blood, bone marrow, and lymph nodes of rhesus macaques during ART and upon release from ART. Macaques previously immunized with SIV recombinants and the gp120 protein were included to assess the effects of prior vaccination. ART was administered for 11 weeks, with or without gp120 boosting at week 9. Naïve and resting, activated, and tissue-like memory B cells and PC/PB were evaluated by flow cytometry. Antibody-secreting cells (ASC) and serum antibody titers were assessed. No lasting changes in B-cell memory subpopulations occurred in bone marrow and lymph nodes, but significant decreases in numbers of activated memory B cells and increases in numbers of tissue-like memory B cells persisted in PBMC. Macaque PC/PB were found to be either CD27(+) or CD27(-) and therefore were defined as CD19(+) CD38(hi) CD138(+). The numbers of these PC/PB were transiently increased in both PBMC and bone marrow following gp120 boosting of the unvaccinated and vaccinated macaque groups. Similarly, ASC numbers in PBMC and bone marrow of the two macaque groups also transiently increased following envelope boosting. Nevertheless, serum binding titers against SIVgp120 remained unchanged. Thus, even during chronic SIV infection, B cells respond to antigen, but long-term memory does not develop, perhaps due to germinal center destruction. Earlier and/or prolonged treatment to allow the generation of virus-specific long-term memory B cells should benefit ART/therapeutic vaccination regimens.

Pneumococcal Conjugate and Plain Polysaccharide Vaccines Have Divergent Effects on Antigen-Specific B Cells

Background. A 23-valent unconjugated pneumococcal polysaccharide vaccine (23vP), routinely administered at the age of 65, has limited effectiveness, and revaccination induces attenuated antibody responses. It is not known whether pneumococcal polysaccharide-protein conjugated vaccines (PCV), although highly effective in infants, offer any immunological advantages over 23vP in adults.Methods. We immunized adults with schedules combining both PCV and 23vP and investigated B-cell responses to establish whether PCV7 (a 7-valent PCV) induced T-dependent responses in adults, to assess the role of memory B cells in 23vP-induced antibody hyporesponsiveness, and to identify the B-cell subtypes involved.Results. A single dose of PCV7 induced significant increases in serotype-specific memory B-cell populations in peripheral blood indicating a T-dependent response. Conversely, immunization with 23vP resulted in a decrease in memory B-cell frequency. Furthermore, memory B-cell responses to subsequent immunization with PCV7, when given after 23vP, were attenuated. Notably, B1b cells, a subset important in protecting mice against pneumococci, were also depleted following immunization with 23vP in humans.Conclusions. This study indicates that PCV7 may have an immunological advantage over 23vP in adults and that 23vP-induced depletion of memory and B1b-cell subsets may provide a basis for antibody hyporesponsiveness and the limited effectiveness of 23vP.Clinical Trials Registration. ISRCTN: 78768849.

Multiple routes to B-cell memory

B-cell memory describes the populations of cells that provide long-term humoral immunity: long-lived antibody-secreting plasma cells that reside mainly in the bone marrow and memory B cells. Interestingly, the memory B-cell population is heterogenous, although the importance of this heterogeneity has been unclear. Recent studies have investigated the formation and function of memory in different settings. In particular, T-independent memory-like cells and T-dependent (TD) IgM memory B cells qualitatively differ from canonical TD class-switched memory B cells; however, these studies suggest that IgM memory cells preserve the memory population over long periods of time. These subsets are evocative of the evolution of the humoral immune response, with memory-like cells appearing before acquisition of germinal centers, suggesting that there are multiple pathways to producing B-cell memory.

Human antibody and memory B and T-cell responses after primary and booster immunisation against Neisseria meningitidis B

Vaccination against disease aims at the induction of long-lasting cellular and humoral immune responses. Few studies have addressed the mechanisms by which meningococcal vaccines generate and sustain immunological memory. The goal of this study was to investigate the development of long-term humoral and cellular memory to Neisseria meningitidis serogroup B (MenB) in health subjects after immunisation with the Cuban outer membrane protein (OMP) vaccine (VA-MENGOC-BC®). The results showed that three doses of vaccine were necessary to induce a detectable memory B-cell response (mean of 0.46%) which became undetectable 6 months later. After boosting, only 2 of 5 individuals responded with an increase in memory B-cell frequencies (values of 0.15% and 0.34%). Bactericidal and opsonic antibody levels were higher after primary immunisation (log2 mean and median of 4.7 and 1212, respectively) when compared with post-booster response (log2 mean of 2.6 and median of 285, respectively). Together, these data suggest a failure of vaccine to induce long-term memory B-cell and serological memory in adults. However, we observed a significant and functional memory T-cell response specially after boosting, with a predominance of activated (CD69+) central memory T-cell (CD4+CD45−CCR7+) response. Therefore, this study suggests that vaccination with the MenB vaccine induced the generation and activation of memory T-cells but failed to maintain the memory B-cell population at a stable size and/or function.

Epigenetic and Transcriptional Changes Which Follow Epstein-Barr Virus Infection of Germinal Center B Cells and Their Relevance to the Pathogenesis of Hodgkin's Lymphoma

Although Epstein-Barr virus (EBV) usually establishes an asymptomatic lifelong infection, it is also implicated in the development of germinal center (GC) B-cell-derived malignancies, including Hodgkin's lymphoma (HL). Following primary infection, EBV remains latent in the memory B-cell population, where host-driven methylation of viral DNA contributes to the repression of viral gene expression. However, it is still unclear how EBV harnesses the cell's methylation machinery in B cells, how this contributes to viral persistence, and what impact this has on the methylation of cellular genes. We show that EBV infection of GC B cells is followed by upregulation of the DNA methyltransferase DNMT3A and downregulation of DNMT3B and DNMT1. We show that the EBV latent membrane protein 1 (LMP1) oncogene downregulates DNMT1 and that DNMT3A binds to the viral promoter Wp. Genome-wide promoter arrays performed with these cells showed that EBV-associated methylation changes in cellular genes were not randomly distributed across the genome but clustered at chromosomal locations, consistent with an instructive pattern of methylation, and were in part determined by promoter CpG content. Both DNMT3B and DNMT1 were downregulated and DNMT3A was upregulated in HL cell lines, recapitulating the pattern of expression observed following EBV infection of GC B cells. We also found, by using gene expression profiling, that genes differentially expressed following EBV infection of GC B cells were significantly enriched for those reported to be differentially expressed in HL. These observations suggest that EBV-infected GC B cells are a useful model for studying virus-associated changes contributing to the pathogenesis of HL.

Phenotype and Function of CD25-Expressing B Lymphocytes Isolated from Human Umbilical Cord Blood

Background. We have shown that approximately 30% of human peripheral blood B-cells express CD25. B cells expressing CD25 display a mature phenotype belonging to the memory B-cell population and have a better proliferative and antigen-presenting capacity. The aim of the present study was to characterize the CD25-expressing subset of B cells in human cord blood. Material and Methods. Mononuclear cell fraction from human cord blood (n = 34) and peripheral adult blood (n = 22) was sorted into CD20+CD25+ and CD20+CD25− B-cell populations. Phenotype and function of these B-cell populations were compared using flow cytometry, proliferation, cytokine production, and immunoglobulin secretion. Results. CD25-expressing B cells are a limited population of cord blood mononuclear cells representing 5% of the CD20+ B cells. They are characterised by high expression of CD5 in cord blood and CD27 in adult blood. CD25-expressing B cells express a functional IL-2 receptor and high levels of CC-chemokine receptors and spontaneously produce antibodies of IgG and IgM subclass. Conclusions. CD25 expression is a common denominator of a specific immunomodulatory B-cell subset ready to proliferate upon IL-2 stimulation, possibly ready to migrate and home into the peripheral tissue for further differentiation/action.

High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations

AbstractB-cell receptor (BCR) diversity is achieved centrally by rearrangement of Variable, Diversity, and Joining genes, and peripherally by somatic hypermutation and class-switching of the rearranged genes. Peripheral B-cell populations are subject to both negative and positive selection events in the course of their development that have the potential to shape the BCR repertoire. The origin of IgM+IgD+CD27+ (IgM memory) cells is controversial. It has been suggested that they may be a prediversified, antigen-independent, population of cells or that they are a population of cells that develop in response to T-independent antigens. Most recently, it was suggested that the majority of IgM memory cells are directly related to switched memory cells and are early emigrants from the germinal center reaction. Advances in sequencing technology have enabled us to undertake large scale IGH repertoire analysis of transitional, naive, IgM memory and switched memory B-cell populations. We find that the memory B-cell repertoires differ from the transitional and naive repertoires, and that the IgM memory repertoire is distinct from that of class-switched memory. Thus we conclude that a large proportion of IgM memory cells develop in response to different stimuli than for class-switched memory cell development.

Functional Characterization of Murine CD25 Expressing B Cells

B cells are an important part of both innate and adaptive immune system. Their ability to produce antibodies, cytokines and to present antigen makes them a crucial part in defence against pathogens. In this study, we have in naïve Naval Medical Research Institute mice functionally characterized a subpopulation of splenic B cells expressing CD25, which comprise about 1% of the total B cell compartment. Murine spleen cells were sorted into two highly purified B cell populations either CD19(+) CD25(+) or CD19(+) CD25(-). We found that CD25(+) B cells secreted higher levels of IL-6, IL-10 and INFgamma in response to different TLR-agonists, and were better at presenting alloantigen to CD4(+) T cells. CD25 expressing B cells spontaneously secreted immunoglobulins of IgA, IgG and IgM subclass and had better migratory ability when compared with CD25(-) B cells. In conclusion, our results demonstrate that CD25(+) B cells are highly activated and functionally mature. Therefore, we suggest that this population plays a major role in the immune system and may belong to the memory B-cell population.

Patients Treated with Rituximab-Containing Immunochemotherapy Have a Significant and Prolonged Lack of Humoral Response to Influenza Vaccine Associated with a Persistent Depletion of B Memory Cells.

Abstract 936▪▪This icon denotes an abstract that is clinically relevant. 1 Background.Rituximab, anti-CD20 mAb, has become an essential drug for the treatment of Non Hodgkin Lymphoma (NHL). Although transient B cells depletion frequently occurs after Rituximab treatment, it usually resolves after 6-9 months. Nevertheless, high frequency of non-neutropenic infections and persistent hypogammaglobulinaemia during follow-up period have been recently reported. However, impaired humoral response to the recall and primary antigens was found in NHL patients during (or few months after), Rituximab treatment.Influenza vaccination is generally recommended in Lymphoma patients, but no data are available about the activity of this vaccine after Rituximab-based chemotherapy (RIT). 2 Objective.To assess humoral response to influenza vaccine after RIT in complete remission (CR) NHL pts, as compared to healthy subjects. 3 Patients and Methods.Considered that disease status might affect the immune response, only NHL pts without evidence of disease and that had completed RIT no less than 6 months before the accrual were eligible. Healthy volunteers served as an age-matched control group. All the subjects were vaccinated with the same commercially available influenza vaccine (contained 2 A and 1 B viral strains). Hemagglutinin inhibition assays were performed before and 4 weeks after vaccination. The EMEA parametres for assessment of vaccines were determined. Seroconversion rate (SC), seroprotection rate (SP), and mean fold increase after Beyer correction/logarithmic transformation (BMFI) were evaluated to compare the two groups. Circulating lymphocytic subpopulations, NK and Dendritic cells, were assessed by immuno-cytofluorometry to evaluate the presence of potentially relevant phenotypic perturbations after vaccination and between the two groups. 4 Results.During 2008/09 epidemic season, 31 patients (PTS) and 34 healthy controls (CTR) were enrolled and analyzed. The median period after RIT administration was 29 months (range 7- 54).SC, SP and BMFI were lower in PTS group compared with CTR group (p <0.05 or less in 7/9 evaluated parameters). However, according with the EMEA criteria after age stratification, only the PTS weren't sufficiently protected. PTS that received Fludarabine-Rituximab regime had a high probability not to respond to any of the 3 vaccine strains and a lower SC as compared to PTS treated with the others regimes (p<0.05).Interestingly, while peripheral CD27- naïve B-cells were present, CD27+ memory B-cell populations were significantly depleted in the patients (p<0.0001) [Fig]. 5 Conclusions.Patients treated with RIT, especially those treated with Rituximab-Fludarabine regimens, have a significant lack of humoral response to influenza vaccine compared with healthy controls, even long time after treatment administration. In these patients the vaccination does not appear to confer adequate protection. The profound depletion in CD27+ B memory cells observed in these patients may explain, in part, this humoral failure. These results raise the concern that PTS treated with RIT, even if in complete remission or on follow since a long time, may be at particular risk of infection, therefore needing careful surveillance, during this period of H1N1 swine-flu spreading.TabAntigensParametresPTSCTRP valueAH1N1SC %29.041.2nsSP %74.294.1<0.05BMFI1.84±0.924.69±1.54<0.0001AH3N2SC %22.652.9<0.05SP %64.594.1<0.01BMFI0.6±0.91.9±1.25<0.0001BSC %3.229.4<0.01SP %22.644.1NsBMFI0.59±0.71.84±0.91<0.0001 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Defining origins of malignant B cells: a new circulating normal human IgM+D+ B-cell subset lacking CD27 expression and displaying somatically mutated IGHV genes as a relevant memory population

In probing the cell of origin in malignant B cells, an imprint of somatic hypermutation (SHM) in immunoglobulin (Ig) variable (V) region genes delineates antigen encounter, and identifying the precise pathway generating SHM in the normal B-cell counterpart becomes relevant. SHM remains the definitive memory imprint in normal human B cells, but CD27 expression also delineates memory. Recently, dye extrusion adenosine triphosphate-binding transporter assays identified circulating isotype-switched memory B cells that lacked CD27, yet exhibited low levels of SHM. To extend findings, we report a pre-switched CD27(-ve) circulating memory B-cell population in normal blood using comparable assays, and isolated CD19(+)IgM(+)D(+)CD27(-ve) cells (>99% purity) for the analysis of IGHV5/IGHV3-IGHM transcripts. Of these (n=334), approximately 78% were germ line and naive B cell derived. Strikingly, 21.9% of the transcripts were mutated. They showed 3-5 mutations (13.5% of sequences) and >5 mutations (8.4% of sequences) per transcript. Accrual of mutations in a subset of CD19(+)IgM(+)D(+)CD27(-ve) cells define a new circulating pre-switched memory B-cell pool, present in substantial numbers in the population harboring naive B cells. These CD19(+)IgM(+)D(+)CD27(-ve) memory B cells may have a distinct lineage and function, and seem relevant to understanding origins of malignant B cells, in particular those of hairy cell leukemia cells, which display mutated V genes yet lack CD27 expression.

Refined characterization and reference values of the pediatric T- and B-cell compartments

Work in the past years has led to a refined phenotypical description of functionally distinct T- and B-cell subsets. Since both lymphocyte compartments are established and undergo dramatic changes during childhood, redefined pediatric reference values of both compartments are needed. In a cohort of 145 healthy children, aged 0-18 years, the relative and absolute numbers of the various T- and B-cell subsets were determined. In addition, we found that besides thymic output, naive (CD27(+)CD45RO(-)) T-cell proliferation contributed significantly to the establishment of the naive T-cell compartment. At birth, regulatory (CD25(+)CD127(-)CD4(+)) T cells (Tregs) mainly had a naive (CD27(+)CD45RO(-)) phenotype whereas 'memory or effector-like' (CD45RO(+)) Tregs accumulated slowly during childhood. Besides the CD27(+)IgM(+)IgD(+) memory B-cell population, the recently identified CD27(-)IgG(+) and CD27(-)IgA(+) memory B-cell populations were already present at birth. These data provide reference values of the T- and B-cell compartments during childhood for studies of immunological disorders or immune reconstitution in children.

Physiological up-regulation of inhibitory receptors Fc RII and CR1 on memory B cells is lacking in SLE patients

Under physiological conditions immune complexes (IC) are efficiently cleared from the circulation and meanwhile provide important feedback signals for the immune system via Fc gamma Rs and complement receptors. Dysregulation of these mechanisms have been implicated in conditions where IC concentrations reach pathological levels and inflict diseases, like systemic lupus erythematosus (SLE). Our aim was to compare distinct sub-populations of CD19(+) B cells of healthy individuals and SLE patients with regard to their expression of Fc gamma R type II (Fc gamma RII, CD32), complement receptor type 1 (CR1, CD35) and complement receptor type 2 (CR2, CD21) and sIgG/IgM. The following four groups of peripheral CD19(+) B cells were investigated: IgM(+)/CD27(-) naive, IgM(+)/CD27(+) and IgM(-)/CD27(+) memory cells and CD27(high) plasmablasts. We demonstrate that the expression of the inhibitory receptors Fc gamma RII and CR1 is up-regulated on peripheral memory B cells of healthy controls, whereas this up-regulation is considerably impaired on the memory B cells of SLE patients. This reduction affects both the IgM(+) and switched memory B cells. We found a striking difference between the expression of complement receptors CD21 and CD35; namely, no up-regulation of CD21 occurred on the memory B cells of healthy donors, and its decreased expression in SLE patients was characteristic for both the CD27(-) naive and the CD27(+) memory B-cell populations. Our results clearly demonstrate that the previously reported reduced expression of IC-binding receptors is mainly due to the disturbed memory compartment; however, the higher frequency of CD19(+)/CD27(high)/sIg(low) plasmablasts expressing minimal levels of these receptors also contributes to this diminution.

Clonogenic Multiple Myeloma Progenitors, Stem Cell Properties, and Drug Resistance

Many agents are active in multiple myeloma, but the majority of patients relapse. This clinical pattern suggests most cancer cells are eliminated, but cells with the clonogenic potential to mediate tumor regrowth are relatively chemoresistant. Our previous data suggested that CD138(+) multiple myeloma plasma cells cannot undergo long-term proliferation but rather arise from clonogenic CD138(neg) B cells. We compared the relative sensitivity of these distinct cell types to clinical antimyeloma agents and found that dexamethasone, lenadilomide, bortezomib, and 4-hydroxycyclophosphamide inhibited CD138(+) multiple myeloma plasma cells but had little effect on CD138(neg) precursors in vitro. We further characterized clonogenic multiple myeloma cells and stained cell lines using the Hoechst side population and Aldefluor assays. Each assay identified CD138(neg) cells suggesting that they possess high drug efflux capacity and intracellular drug detoxification activity. We also found that multiple myeloma cells expressing the memory B-cell markers CD20 and CD27 could give rise to clonogenic multiple myeloma growth in vitro and engraft immunodeficient nonobese diabetes/severe combined immunodeficient mice during both primary and secondary transplantation. Furthermore, both the side population and Aldefluor assays were capable of identifying circulating clonotypic memory B-cell populations within the peripheral blood of multiple myeloma patients. Our results suggest that circulating clonotypic B-cell populations represent multiple myeloma stem cells, and the relative drug resistance of these cells is mediated by processes that protect normal stem cells from toxic injury.