scholarly journals Trypanosoma cruzi Induces Regulatory B Cell Alterations in Patients With Chronic Chagas Disease

2021 ◽  
Vol 11 ◽  
Author(s):  
Magalí C. Girard ◽  
Micaela S. Ossowski ◽  
Arturo Muñoz-Calderón ◽  
Marisa Fernández ◽  
Yolanda Hernández-Vásquez ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Chagas Disease ◽  
Ex Vivo ◽  
Production Capacity ◽  
Cell Phenotype ◽  
Regulatory B Cell ◽  
B10 Cells ◽  
Chronic Chagas Disease

The clinical evolution of patients with chronic Chagas disease (CCD) is mainly associated with an excessive inflammation and a defective immunomodulatory profile caused by the interaction between T. cruzi and the host. Regulatory B (Breg) cells exert immune suppression mostly through IL-10 production (B10 cells), but also through IL-10-independent mechanisms. Previously, we demonstrated that CCD patients with cardiomyopathy show changes in the ex vivo Breg cell phenotypic distribution although maintain IL-10 production capacity. Here, we sought to identify potential alterations on Breg cells upon in vitro stimulation. Isolated B cells from CCD patients with or without cardiomyopathy and non-infected (NI) donors were stimulated with T. cruzi lysate or CpG + CD40L, and characterized by flow cytometry based on the expression of CD24, CD27, CD38, and the regulatory molecules IL-10 and PD-L1. IL-10 and IL-17 secretion in the supernatant of B cells was evaluated by ELISA. Data showed that T. cruzi stimulation diminished the expression of CD24 and CD38 on CD27− B cells while reducing the percentage of CD24high inside CD27+ B cells. Furthermore, T. cruzi induced a regulatory B cell phenotype by increasing B10 cells and IL-10 secretion in all the groups. The innate-like B10 cells expansion observed in patients with cardiomyopathy would be associated with CD27− B10 cell subsets, while no predominant phenotype was found in the other groups. Patients with cardiomyopathy also displayed higher IL-17 secretion levels in T. cruzi–activated B cells. CpG + CD40L stimulation revealed that B cells from CCD patients and NI donors had the same ability to differentiate into B10 cells and secrete IL-10 in vitro. Additionally, CCD patients showed an increased frequency of CD24−CD27− B cells and a reduction in the percentage of CD24highCD27+ Breg cells, which appeared to be inversely correlated with the presence of T. cruzi DNA in blood. Finally, CCD patients exhibited a higher frequency of PD-L1+ B cells in T. cruzi–stimulated samples, suggesting that IL-10-independent mechanisms could also be tangled in the control of inflammation. Altogether, our results provide evidence about the potential role of Breg cells in the immune response developed against T. cruzi and its contribution to chronic Chagas cardiomyopathy.

scholarly journals Ex vivo characterization of Breg cells in patients with chronic Chagas disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Magalí C. Girard ◽  
Gonzalo R. Acevedo ◽  
Micaela S. Ossowski ◽  
Marisa Fernández ◽  
Yolanda Hernández ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Chagas Disease ◽  
Peripheral Blood ◽  
Cardiac Involvement ◽  
Ex Vivo ◽  
Regulatory Function ◽  
B Cell Subsets ◽  
B10 Cells ◽  
Chronic Chagas Disease

AbstractDespite the growing importance of the regulatory function of B cells in many infectious diseases, their immunosuppressive role remains elusive in chronic Chagas disease (CCD). Here, we studied the proportion of different B cell subsets and their capacity to secrete IL-10 ex vivo in peripheral blood from patients with or without CCD cardiomyopathy. First, we immunophenotyped peripheral blood mononuclear cells from patients according to the expression of markers CD19, CD24, CD38 and CD27 and we showed an expansion of total B cell and transitional CD24highCD38high B cell subsets in CCD patients with cardiac involvement compared to non-infected donors. Although no differences were observed in the frequency of total IL-10 producing B cells (B10) among the groups, CCD patients with cardiac involvement showed an increased proportion of naïve B10 cells and a tendency to a higher frequency of transitional B10 cells compared to non-infected donors. Our research demonstrates that transitional B cells are greatly expanded in patients with the cardiac form of CCD and these cells retain the ability to secrete IL-10. These findings provide insight into the phenotypic distribution of regulatory B cells in CCD, an important step towards new strategies to prevent cardiomyopathy associated with T. cruzi infection.

scholarly journals Altered frequency of CD24highCD38high transitional B cells in patients with cardiac involvement of chronic Chagas disease

2019 ◽  
Author(s):  
Magalí C. Girard ◽  
Gonzalo R. Acevedo ◽  
Micaela S. Ossowski ◽  
Paula B. Alcaráz ◽  
Marisa Fernández ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Chagas Disease ◽  
Peripheral Blood ◽  
Cardiac Involvement ◽  
Mononuclear Cells ◽  
Regulatory Function ◽  
Chronic Patients ◽  
B10 Cells ◽  
Chronic Chagas Disease

ABSTRACTThe cardiomyopathy developed by patients with chronic Chagas disease (CCD), one of the most severe consequences of T. cruzi infection, is mainly associated with an imbalance between an excessive inflammatory reaction and a defective immunomodulatory profile cause by host-parasite interaction. Despite the growing importance of the regulatory function of B-cells in many malignancies, few studies have addressed their immunosuppressive role in chronic Chagas disease. In this work, we tackled this issue by studying the proportion of different B cell subpopulations and their capacity to secrete IL-10 in individuals with distinct clinical forms of CCD. Seven-colour flow cytometry was performed to examine the peripheral blood B cell compartment in chronic Chagas disease (CCD) patients with and without cardiac manifestations (n=10 for each group) and non-infected donors (n=9). Peripheral blood mononuclear cells (PBMC) were incubated for 5h with PMA, ionomicyn and brefeldin A. According to the expression of markers CD19, CD24 and CD38, we showed an expansion of total B cell and transitional CD24highCD38high B cell subsets in CCD patients with cardiac involvement compared to non-infected donors. Furthermore, although no differences were observed in the frequency of total IL-10 producing B cells (B10) among the groups, CCD patients with cardiac involvement showed a statistically significant increased proportion of naïve B10 cells and a tendency to an increased frequency of transitional B10 cells compared to non-infected donors. These findings suggest that immature transitional CD24highCD38high B cells are greatly expanded in patients with the cardiac form of chronic Chagas disease and these cells retain their ability to secrete IL-10 compared to non-infected donors. Furthermore, the distribution of naïve, transitional and memory B cells inside the B10 cells followed the same pattern in chronic patients without cardiac involvement and non-infected individuals. Our work provides insight into the phenotypic distribution of regulatory B cell in CCD, an important step towards new strategies to prevent cardiomiopathy associated with T. cruzi infection.

scholarly journals Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

2016 ◽  
Vol 213 (11) ◽  
pp. 2413-2435 ◽  
Author(s):  
Yi Wang ◽  
Cindy S. Ma ◽  
Yun Ling ◽  
Aziz Bousfiha ◽  
Yildiz Camcioglu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Loss Of Function ◽  
Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

scholarly journals Characterization of a rare IL-10–competent B-cell subset in humans that parallels mouse regulatory B10 cells

Blood ◽  
2011 ◽  
Vol 117 (2) ◽  
pp. 530-541 ◽  
Author(s):  
Yohei Iwata ◽  
Takashi Matsushita ◽  
Mayuka Horikawa ◽  
David J. DiLillo ◽  
Koichi Yanaba ◽  
...  
Keyword(s):  
B Cells ◽  
Autoimmune Disease ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Ex Vivo ◽  
Cell Subset ◽  
Cell Subpopulation ◽  
B10 Cells

Abstract Regulatory B cells control inflammation and autoimmunity in mice, including the recently identified IL-10–competent B10 cell subset that represents 1% to 3% of spleen B cells. In this study, a comparable IL-10–competent B10 cell subset was characterized in human blood. B10 cells were functionally identified by their ability to express cytoplasmic IL-10 after 5 hours of ex vivo stimulation, whereas progenitor B10 (B10pro) cells required 48 hours of in vitro stimulation before they acquired the ability to express IL-10. B10 and B10pro cells represented 0.6% and approximately 5% of blood B cells, respectively. Ex vivo B10 and B10pro cells were predominantly found within the CD24hiCD27+ B-cell subpopulation that was able to negatively regulate monocyte cytokine production through IL-10–dependent pathways during in vitro functional assays. Blood B10 cells were present in 91 patients with rheumatoid arthritis, systemic lupus erythematosus, primary Sjögren syndrome, autoimmune vesiculobullous skin disease, or multiple sclerosis, and were expanded in some cases as occurs in mice with autoimmune disease. Mean B10 + B10pro-cell frequencies were also significantly higher in patients with autoimmune disease compared with healthy controls. The characterization of human B10 cells will facilitate their identification and the study of their regulatory activities during human disease.

Glucocorticoid-induced leucine zipper (GILZ) inhibits B cell activation in systemic lupus erythematosus

2015 ◽  
Vol 75 (4) ◽  
pp. 739-747 ◽  
Author(s):  
Sarah A Jones ◽  
Andrew E J Toh ◽  
Dragana Odobasic ◽  
Marie-Anne Virginie Oudin ◽  
Qiang Cheng ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
B Cells ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Leucine Zipper ◽  
Germinal Centre ◽  
Cell Phenotype ◽  
Systemic Lupus ◽  
Human B Cells

ObjectivesSystemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of anti-inflammatory effects of glucocorticoids.MethodsWe conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice.ResultsReduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naïve B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naïve and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naïve B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli.ConclusionsOur findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.

In Vitro Co-Culture of CLL-B Cells Reveals Long-Term Survival, Proliferation, and Maintenance of Telomere Length

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 350-350
Author(s):  
Ceri H Jones ◽  
Thet Thet Lin ◽  
Elisabeth Jane Walsby ◽  
Guy E Pratt ◽  
Christopher Fegan ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Telomere Length ◽  
Erosion Rate ◽  
Ex Vivo ◽  
Effector Memory ◽  
Long Term Culture

Abstract Telomere length is a prognostic factor in Chronic Lymphocytic Leukemia (CLL) with short telomere length a powerful predictor of early time to first treatment and reduced overall survival. However, little is known about telomere dynamics through the course of an individual patient's disease. Our recent longitudinal analysis of CLL B-cell telomere length revealed very little dynamic change within individual patients with a mean erosion rate of -52bp/year (p=0.05). In marked contrast, T-cells derived from the same patients showed a significantly higher mean erosion rate of -119bp/year (p=0.02) with a median follow up time of 69 months. Here we present data derived from long-term in-vitro co-culture of peripheral blood from CLL patients coupled with temporal analysis of their telomere length dynamics. We utilized a multi-cellular co-culture system, comprised of autologous T-cells and CD40L-expressing mouse fibroblasts, to maintain CLL cells in long-term culture. Patient-derived peripheral blood mononuclear cells (n=16) were maintained for a median of 70 days (range 54-154); samples were analyzed every two weeks for tumor cell telomere length and evidence of proliferation. We used fluorescence-activated cell sorting (FACS) to sort populations of CD19+CD5+ CLL B-cells and CD3+ T-cells from each of the cultures. We then performed high-resolution single telomere length analysis (STELA) on these sorted subsets of cells and analyzed their telomere dynamics over this extended time course. Analysis of CLL B-cells from these cultures revealed significantly increased Ki-67+ at day 14 when compared to day 0 (p<0.001) and this was evident for the duration of the cultures. Despite sustained tumor cell proliferation, we observed no significant difference in the CLL B-cell telomere length with a mean TL at the start of 4.5kb vs 4.3kb at the end (p=0.14). The presence of T-cells was shown to be critical for the maintenance of the long-term cultures in two ways. Firstly, cultures that were treated with 4μM fludarabine showed a catastrophic reduction in T-cells (p=0.01), which was associated with a significantly shorter duration of survival of CLL B-cells when compared to untreated controls (median 17.5 days (range 7-70); p<0.001). Secondly, it proved impossible to maintain T-cell depleted, purified CLL B-cells, in long-term culture. T-cells isolated from the long-term cultures showed evidence of proliferation with Ki-67+ again being increased at day 14 in comparison to baseline (p=0.003). Furthermore, T-cells derived from these cultures showed a significant alteration in subset composition over time with a decrease in the numbers of naive CD4+ (p=0.05) and CD8+ (p=0.02) T-cells and a corresponding increase in effector memory (p=0.2) and terminally differentiated effector memory (EMRA) subsets (p=0.07). In conclusion, this study demonstrates that we have developed a robust, long-term culture method for the maintenance of CLL cells. Despite evidence of sustained CLL proliferation, CLL B-cells showed little telomere length erosion during long-term co-culture and this is compatible with our recent ex-vivo analysis, which showed that the telomere length of CLL B-cells are remarkably stable with a mean erosion rate of only -52bp/year. In both ex-vivo and in-vitro analysis, telomere erosion correlated with starting telomere length (r2=0.14, p=0.04 and r2=0.3 p=0.03 respectively). Taken together, our in-vitro and ex-vivo data imply that the radically short telomeres observed in some CLL patients are not the result of increased proliferation of the malignant B-cell, but rather the mutagenic event occurs in a B-cell which already has short telomeres. Furthermore, our novel long-term culture model has reinforced the vital role of T-cells in sustaining CLL B-cells viability and proliferation in-vitro. Given the consistent skewing of the T-cell pool towards a memory phenotype it seems unlikely that this is driven in-vitro by cognate TCR antigen recognition but rather a cytokine-mediated response. Disclosures Fegan: Gilead Sciences: Honoraria; Roche: Honoraria; AbbVie: Honoraria.

scholarly journals MHC class II cell-autonomously regulates self-renewal and differentiation of normal and malignant B cells

Blood ◽  
2019 ◽  
Vol 133 (10) ◽  
pp. 1108-1118 ◽  
Author(s):  
Julia Merkenschlager ◽  
Urszula Eksmond ◽  
Luca Danelli ◽  
Jan Attig ◽  
George R. Young ◽  
...  
Keyword(s):  
Stem Cell ◽  
B Cells ◽  
B Cell ◽  
Stem Cell Differentiation ◽  
Class Ii ◽  
Cell Phenotype ◽  
Self Renewal ◽  
Mhc Ii

Abstract Best known for presenting antigenic peptides to CD4+ T cells, major histocompatibility complex class II (MHC II) also transmits or may modify intracellular signals. Here, we show that MHC II cell-autonomously regulates the balance between self-renewal and differentiation in B-cell precursors, as well as in malignant B cells. Initiation of MHC II expression early during bone marrow B-cell development limited the occupancy of cycling compartments by promoting differentiation, thus regulating the numerical output of B cells. MHC II deficiency preserved stem cell characteristics in developing pro-B cells in vivo, and ectopic MHC II expression accelerated hematopoietic stem cell differentiation in vitro. Moreover, MHC II expression restrained growth of murine B-cell leukemia cell lines in vitro and in vivo, independently of CD4+ T-cell surveillance. Our results highlight an important cell-intrinsic contribution of MHC II expression to establishing the differentiated B-cell phenotype.

scholarly journals Maintenance of Human Germinal Center B Cells In Vitro

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 919-928 ◽  
Author(s):  
John D. Pound ◽  
John Gordon
Keyword(s):  
B Cells ◽  
Dna Synthesis ◽  
B Cell ◽  
Germinal Center ◽  
Culture System ◽  
Cell Phenotype ◽  
Common Component ◽  
Cell Numbers ◽  
L Cells

Abstract The ability to maintain germinal center (GC) B cells in culture should facilitate studies on the molecular and cellular events which accompany affinity maturation and the generation of memory in T-dependent responses. We have investigated the ability of cytokines to maintain human tonsillar GC B cells (IgD−/CD39−/CD38+/CD77+) in the “CD40 culture system.” In the absence of added cytokines, CD40 monoclonal antibody held on CD32-transfected L cells effectively sustained DNA synthesis in GC B cells for a maximum 3 to 4 days. Of the following cytokines (interleukin-1β [IL-1β], IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, and stem cell factor), only IL-2 and IL-4 provided a significant enhancement to DNA synthesis in the CD40 culture system; this was modest and shortterm. Following a study on the cooperative activity between pairs of cytokines, triple combinations were identified that could maintain high levels of GC B-cell stimulation for at least 10 days. IL-10 was a common component of these synergistic cytokine cocktails, which were IL-10 + IL-4 + IL-7; IL-10 + IL-3 + IL-7; IL-10 + IL-1β + IL-2; IL-10 + IL-1β + IL-3, and IL-10 + IL-3 + IL-6. Culture of GC B cells with these cytokine combinations resulted in a net increase in viable cell numbers of 50% to 100% whereas total cell numbers increased up to fourfold. Cells recovered from these cultures retained a GC B-cell phenotype with a significant proportion being CD38+/CD44−, features characteristic of centroblasts. Studies with metabolically inactive CD32-L cells supported a role for stromal cell-derived soluble factors in maintaining GC B cells in vitro.

Bone Marrow Derived Mesenchymal Stem Cells from Splenic Marginal Zone Lymphoma Patients Exhibit Altered Proliferative Potential and B Lymphocyte Immunomodulatory Properties

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2396-2396
Author(s):  
Athanasia Kalyva ◽  
Charalampos Pontikoglou ◽  
Christina Kalpadakis ◽  
Athina Trakaki ◽  
Nikitas Zorzos ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Ex Vivo ◽  
Proliferative Potential ◽  
Splenic Marginal Zone Lymphoma ◽  
Healthy Individuals ◽  
Healthy Donors ◽  
Normal Controls

Abstract Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. However, there is a concurrent high prevalence of bone marrow (BM) infiltration, suggesting that BM microenvironment dynamics could have a potential involvement in disease pathology. In this regard, we aim to characterise BM derived mesenchymal stem cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if MSCs show altered properties in SMZL patients compared to healthy controls. BM MSCs were isolated from 8 SMZL patients and 10 age- and sex-matched healthy controls. MSCs were in vitro expanded and re-seeded for a total of 5 passages (P). The colony forming unit-fibroblast (CFU-F) assay was used for the estimation of MSC frequency within the BM mononuclear cell (BMMC) fraction. Ex-vivo expanded MSCs were phenotypically characterized by flow cytometry (FC) using appropriate markers. In vitro differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. The proliferative potential of ex vivo expanded MSCs was evaluated by Methyl Triazolyl Tetrazolium (MTT)-based assay and survival characteristics were studied using FC and 7-Aminoactinomycin D (7-AAD) staining. To assess the effect of patient MSCs on B cell growth, B cells were immunomagnetically isolated (Miltenyi Biotec GmbH, Germany) from peripheral blood (PB) of normal individuals, labeled with carboxy fluorescein succinimidyl ester (CFSE; Gibco Invitrogen, Paisley, Scotland) and subsequently cultured in the absence or presence of confluent layers of allogeneic BM-MSCs from SMZL patients or normal controls in the presence of CpG oligonucleotide 2006 (Invivogen, France) and IL-2 (R&D Systems, Minneapolis, MN). In a separate set of experiments, B cell survival was evaluated via FC and 7-AAD staining, after co-culturing with BM-MSCs from patients or healthy donors. Finally, to study BM-MSC capacity to chemotactically attract B-cells, transwell migration assays were set. In the bottom chambers MSCs from patients or healthy individuals were grown until confluency and then isolated B cells from PB of either patients or controls were added into the upper chamber. Twelve hours later migrated cells were enumerated. Grouped data are expressed as means± 1 standard error of the mean (SEM). MSCs were successfully expanded from all participants in the study. Adherent cells from both study groups displayed the typical spindle-shape morphology and immunophenotypic analysis at the end of P2-P3-P4 demonstrated that cultures constituted of a homogeneous cell population, typically expressing CD29, CD44, CD73, CD90 and CD105 while being negative for CD14, CD34 and CD45. SMZL-derived MSCs were similar to their normal counterparts in the capacity to differentiate towards adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.5±0.68/105 ΒΜΜCs and 7.23±0.6/105 ΒΜΜCs, respectively; P=0.0032) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs displayed defective proliferative potential as compared to their normal counterparts at P2, as evidenced by the MTT assay (P<0.0001). To explore the influence of SMZL BM-MSCs in B cells survival we compared the viability of B cells isolated from the PB of healthy individuals cultured in medium alone to that of such cells co-cultured with either BM-MSCs derived from patients or normal controls. 43.85±1.46% of B cells cultured alone were apoptotic, while only 20.8±2.63% and 12±0.77% of B cells co-cultured with either normal MSCs or SMZL MSCs were apoptotic (P<0.0001 and P<0.0001, respectively). Notably patient MSCs confer a survival advantage in B cell viability over their normal counterparts (P=0.0374). Finally SMZL MSCs had a more potent chemotactic activity on B cells from healthy donors, as compared to MSCs from normal controls ( P<0.05). In conclusion we have shown for the first time that SMZL lymphoma MSCs are intrinsically defective in terms of proliferative potential and exert an altered modulation of B cell apoptosis and B cell chemotaxis. These preliminary results concerning the properties of SMZL MSCs merit further investigation and provide the theoretical background for exploring their potential implication in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ex vivo characterization and isolation of rare memory B cells with antigen tetramers

Blood ◽  
2011 ◽  
Vol 118 (2) ◽  
pp. 348-357 ◽  
Author(s):  
Bettina Franz ◽  
Kenneth F. May ◽  
Glenn Dranoff ◽  
Kai Wucherpfennig
Keyword(s):  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Memory B Cells ◽  
Cell Repertoire ◽  
Low Frequencies

Abstract Studying human antigen-specific memory B cells has been challenging because of low frequencies in peripheral blood, slow proliferation, and lack of antibody secretion. Therefore, most studies have relied on conversion of memory B cells into antibody-secreting cells by in vitro culture. To facilitate direct ex vivo isolation, we generated fluorescent antigen tetramers for characterization of memory B cells by using tetanus toxoid as a model antigen. Brightly labeled memory B cells were identified even 4 years after last immunization, despite low frequencies ranging from 0.01% to 0.11% of class-switched memory B cells. A direct comparison of monomeric to tetrameric antigen labeling demonstrated that a substantial fraction of the B-cell repertoire can be missed when monomeric antigens are used. The specificity of the method was confirmed by antibody reconstruction from single-cell sorted tetramer+ B cells with single-cell RT-PCR of the B-cell receptor. All antibodies bound to tetanus antigen with high affinity, ranging from 0.23 to 2.2 nM. Furthermore, sequence analysis identified related memory B cell and plasmablast clones isolated more than a year apart. Therefore, antigen tetramers enable specific and sensitive ex vivo characterization of rare memory B cells as well as the production of fully human antibodies.

Sign in / Sign up

Export Citation Format

Share Document