scholarly journals Potential anti-EBV effects associated with elevated interleukin-21 levels: a case report

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Kristian Assing ◽  
Christian Nielsen ◽  
Marianne Jakobsen ◽  
Charlotte B. Andersen ◽  
Kristin Skogstrand ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Formation ◽  
Memory B Cells ◽  
Memory B Cell

Abstract Background Germinal center derived memory B cells and plasma cells constitute, in health and during EBV reactivation, the largest functional EBV reservoir. Hence, by reducing germinal center derived formation of memory B cells and plasma cells, EBV loads may be reduced. Animal and in-vitro models have shown that IL-21 can support memory B and plasma cell formation and thereby potentially contribute to EBV persistence. However, IL-21 also displays anti-viral effects, as mice models have shown that CD4+ T cell produced IL-21 is critical for the differentiation, function and survival of anti-viral CD8+ T cells able to contain chronic virus infections. Case presentation We present immunological work-up (flow-cytometry, ELISA and genetics) related to a patient suffering from a condition resembling B cell chronic active EBV infection, albeit with moderately elevated EBV copy numbers. No mutations in genes associated with EBV disease, common variable immunodeficiency or pertaining to the IL-21 signaling pathway (including hypermorphic IL-21 mutations) were found. Increased (> 5-fold increase 7 days post-vaccination) CD4+ T cell produced (p < 0.01) and extracellular IL-21 levels characterized our patient and coexisted with: CD8+ lymphopenia, B lymphopenia, hypogammaglobulinemia, compromised memory B cell differentiation, absent induction of B-cell lymphoma 6 protein (Bcl-6) dependent peripheral follicular helper T cells (pTFH, p = 0.01), reduced frequencies of peripheral CD4+ Bcl-6+ T cells (p = 0.05), compromised plasmablast differentiation (reduced protein vaccine responses (p < 0.001) as well as reduced Treg frequencies. Supporting IL-21 mediated suppression of pTFH formation, pTFH and CD4+ IL-21+ frequencies were strongly inversely correlated, prior to and after vaccination, in the patient and in controls, Spearman’s rho: − 0.86, p < 0.001. Conclusions To the best of our knowledge, this is the first report of elevated CD4+ IL-21+ T cell frequencies in human EBV disease. IL-21 overproduction may, apart from driving T cell mediated anti-EBV responses, disrupt germinal center derived memory B cell and plasma cell formation, and thereby contribute to EBV disease control.

scholarly journals Multiscale Modeling of Germinal Center Recapitulates the Temporal Transition From Memory B Cells to Plasma Cells Differentiation as Regulated by Antigen Affinity-Based Tfh Cell Help

2021 ◽  
Vol 11 ◽  
Author(s):  
Elena Merino Tejero ◽  
Danial Lashgari ◽  
Rodrigo García-Valiente ◽  
Xuefeng Gao ◽  
Fabien Crauste ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Asymmetric Division ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Germinal Center Reaction

Germinal centers play a key role in the adaptive immune system since they are able to produce memory B cells and plasma cells that produce high affinity antibodies for an effective immune protection. The mechanisms underlying cell-fate decisions are not well understood but asymmetric division of antigen, B-cell receptor affinity, interactions between B-cells and T follicular helper cells (triggering CD40 signaling), and regulatory interactions of transcription factors have all been proposed to play a role. In addition, a temporal switch from memory B-cell to plasma cell differentiation during the germinal center reaction has been shown. To investigate if antigen affinity-based Tfh cell help recapitulates the temporal switch we implemented a multiscale model that integrates cellular interactions with a core gene regulatory network comprising BCL6, IRF4, and BLIMP1. Using this model we show that affinity-based CD40 signaling in combination with asymmetric division of B-cells result in switch from memory B-cell to plasma cell generation during the course of the germinal center reaction. We also show that cell fate division is unlikely to be (solely) based on asymmetric division of Ag but that BLIMP1 is a more important factor. Altogether, our model enables to test the influence of molecular modulations of the CD40 signaling pathway on the production of germinal center output cells.

scholarly journals Coupled Antigen and BLIMP1 Asymmetric Division With a Large Segregation Between Daughter Cells Recapitulates the Temporal Transition From Memory B Cells to Plasma Cells and a DZ-to-LZ Ratio in the Germinal Center

2021 ◽  
Vol 12 ◽  
Author(s):  
Elena Merino Tejero ◽  
Danial Lashgari ◽  
Rodrigo García-Valiente ◽  
Jiaojiao He ◽  
Philippe A. Robert ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Germinal Centers ◽  
Memory B Cells ◽  
Plasma Cell Differentiation ◽  
Daughter Cells

Memory B cells and antibody-secreting plasma cells are generated within germinal centers during affinity maturation in which B-cell proliferation, selection, differentiation, and self-renewal play important roles. The mechanisms behind memory B cell and plasma cell differentiation in germinal centers are not well understood. However, it has been suggested that cell fate is (partially) determined by asymmetric cell division, which involves the unequal distribution of cellular components to both daughter cells. To investigate what level and/or probability of asymmetric segregation of several fate determinant molecules, such as the antigen and transcription factors (BCL6, IRF4, and BLIMP1) recapitulates the temporal switch and DZ-to-LZ ratio in the germinal center, we implemented a multiscale model that combines a core gene regulatory network for plasma cell differentiation with a model describing the cellular interactions and dynamics in the germinal center. Our simulations show that BLIMP1 driven plasma cell differentiation together with coupled asymmetric division of antigen and BLIMP1 with a large segregation between the daughter cells results in a germinal center DZ-to-LZ ratio and a temporal switch from memory B cells to plasma cells that have been observed in experiments.

Effects Of Bendamustine Plus Rituximab On The Distribution Of Normal Peripheral Blood Leucocyte Populations In Advanced-Stage Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5289-5289 ◽  
Author(s):  
Georgiana Grigore ◽  
Martin Perez-Andres ◽  
Susana Barrena ◽  
Rosa Ana Rivas ◽  
Marcos González ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Lymphocytic Leukemia ◽  
Cd4 T Cell ◽  
Advanced Stage ◽  
Time Point

Abstract Introduction Management of B-cell chronic lymphocytic leukemia (CLL) is currently undergoing profound changes. Accordingly, new treatment options with an expected less toxicity than standard regimens are been explored. Recent results show that chemoimmunotherapy may improve the life expectancy of CLLpatients and has proven to be more efficient than chemotherapy alone in depleting malignant cells. Despite its efficacy, little is known about its precise immunomodulatory effects. Aim To evaluate the effects of chemoimmunotherapy with bendamustine plusrituximab (BR) on the distribution of normal residual leucocyte populations in peripheral blood (PB) from advanced-stage CLL patients, with special emphasis on maturation-associated B-cell subsets (immature, naïve, memory IgM/IgG/IgA and plasma cells). Material and Methods Distribution of PB neoplastic cells and residual normal immune cell subpopulations were analyzed in 72 CLL patients with advanced disease (Binet B/C), before therapy (M0) and after 1 course of BR (M1). The same analysis was repeated 3 months after completing treatment (M3) in 31/72 patients. PB leucocyte cell subsets were identified at each time-point by 8-color flow cytometry with monoclonal antibody reagents against CD3, CD4, CD5, CD8, TCRgd, CD19, CD20, CD27, CD38, CD45, CD56, sIgM, sIgA, sIgG, sIgLambda and sIgKappa. Results After the first BR course, absolute counts of all PB myeloid subsets were significantly decreased as compared to time M0, including neutrophils (2,744±1,830 vs 4,764±2,906 cells/uL, p<0.001), eosinophils (132±185 vs 215±245 cells/uL; p<0.001), basophils (37±28 vs 59±47 cells/uL, p<0.001), monocytes (334±280 vs 504±424 cells/uL, p=0.001) and dendritic cells (DCs, 41±40 vs 89±168 cells/uL, p=0.02), as well as NK cells (120±147 vs 550±599 cells/uL, p<0.001). At M3, all these populations remained decreased when compared to M0, but at similar levels to M1 (except for the absolute number of DCs, found to be increased vs. M1 -74±46 vs 41±40 cells/uL, p=0.008- and closer to M0). In turn, total T cells were reduced in M1 as compared to M0 values (818±655 vs 3,905±2,375 cells/uL, p<0.001), due to decreased numbers of CD4+ (424±376 vs 1,573±1,204 cells/uL, p<0.001), CD8+ (342±330 vs 1,334±1,218 cells/uL, p<0.001) and TCRgd (21±28 vs 141±289 cells/uL, p=0.001) T cells, leading to an increased CD4/CD8 ratio (1.8±1.3 vs 1.4±0.8, p=0.004). Also, decreased levels of CD4 (222±156 cells/uL), CD8 (501±544 cells/uL) and TCRgd (21±40 cells/uL) T cells were observed at time M3 vs. baseline values. No changes (p>0.05) were observed for CD8 and TCRgd for M3 vs. M1, while CD4+ T-cell numbers were significantly reduced (p=0.006), resulting in an inverted CD4/CD8 ratio (0.9±1.0 vs. 1.8±1.3, p=0.005) at the M3 time-point. As regards B cells, the absolute count of both neoplastic and normal B lymphocytes were significantly decreased at time M1 vs. M0 (3,363±9,353 vs 53,521±56,602 CLL cells/uL and 2±6 vs 58±107 normal B-cells/uL, p=0.006 and p<0.001, respectively). Within the normal residual B-cell compartment, we found significantly decreased numbers of immature (0.07±0.22 vs 6.55±21.64 cells/uL, p=0.01) and memory (1.3±14.7 vs 35.1±43.6 cells/uL, p<0.001) B cells -including sIgM (0.5±2.3 vs 14.5±24.8 cells/uL, p<0.001), sIgG (0.2±1.0 vs 11.5±17.2 cells/uL; p<0.001) and sIgA (0.6±3.1 vs 9.5±12.5 cells/uL, p<0.001) memory B cells-. At time M3, decreased (p<0.01) naïve (0.46±2.58 cells/uL) and memory B-cells (1.34±6.75 cells/uL), including IgM (0.46±2.58 cells/uL), IgG (0.34±1.69 cells/uL) and IgA (0.09±0.31 cells/uL), but not immature cells (2.28±8.84 cells/uL, p=0.9), were observed as compared to time M0. Differences did not reach statistical significance when comparing M3 vs. M1. The number of circulating plasma cells did not significantly vary during treatment. Conclusions All PB leucocyte subsets are affected by BR treatment in advanced-stage CLL. Interestingly, at time M3 the CD4+ T-cell subset continues to be decreased, while the other T-cell compartments seem to remain stable. Also, normal B cells are affected by BR treatment, and the depletion induced after one course therapy is maintained even three months after finishing BR therapy, except for immature B cells, that seem to be the first to recover in PB. Further studies will offer a more accurate insight into the biology of cell recovery during and after BR therapy in CLL patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activation of Virus-specific Memory B Cells in the Absence of T Cell Help

2004 ◽  
Vol 199 (4) ◽  
pp. 593-602 ◽  
Author(s):  
Barbara J. Hebeis ◽  
Karin Klenovsek ◽  
Peter Rohwer ◽  
Uwe Ritter ◽  
Andrea Schneider ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Human Herpesvirus ◽  
Memory B Cells ◽  
Specific Memory ◽  
T Cell Help

Humoral immunity is maintained by long-lived plasma cells, constitutively secreting antibodies, and nonsecreting resting memory B cells that are rapidly reactivated upon antigen encounter. The activation requirements for resting memory B cells, particularly the role of T helper cells, are unclear. To analyze the activation of memory B cells, mice were immunized with human cytomegalovirus, a complex human herpesvirus, and tick-born encephalitis virus, and a simple flavivirus. B cell populations devoid of Ig-secreting plasma cells were adoptively transferred into T and B cell–deficient RAG-1−/− mice. Antigenic stimulation 4–6 d after transfer of B cells resulted in rapid IgG production. The response was long lasting and strictly antigen specific, excluding polyclonal B cell activation. CD4+ T cells were not involved since (a) further depletion of CD4+ T cells in the recipient mice did not alter the antibody response and (b) recipient mice contained no detectable CD4+ T cells 90 d posttransfer. Memory B cells could not be activated by a soluble viral protein without T cell help. Transfer of memory B cells into immunocompetent animals indicated that presence of helper T cells did not enhance the memory B cell response. Therefore, our results indicate that activation of virus-specific memory B cells to secrete IgG is independent of cognate or bystander T cell help.

Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells

Blood ◽  
2010 ◽  
Vol 115 (9) ◽  
pp. 1718-1726 ◽  
Author(s):  
Ghyath Maarof ◽  
Laurence Bouchet-Delbos ◽  
Hélène Gary-Gouy ◽  
Ingrid Durand-Gasselin ◽  
Roman Krzysiek ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Plasma Cell ◽  
Germinal Center ◽  
Molecular Mechanisms ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Plasma Cell Differentiation

Abstract Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27+ memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)–driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

Requirements for Co-Stimulation in T-Cell Dependent and T-Cell Independent Re-Stimulation of FVIII-Specific Memory B Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 238-238 ◽  
Author(s):  
Aniko Ginta Pordes ◽  
Christina Hausl ◽  
Peter Allacher ◽  
Rafi Uddin Ahmad ◽  
Eva M Muchitsch ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Memory B Cells ◽  
Specific Memory ◽  
Stimulation Of

Abstract Memory B cells specific for factor VIII (FVIII) are critical for maintaining FVIII inhibitors in patients with hemophilia A. They are precursors of anti-FVIII antibody-producing plasma cells and are highly efficient antigen-presenting cells for the activation of T cells. The eradication of FVIII-specific memory B cells will be a prerequisite for any successful new approach to induce immune tolerance in patients with FVIII inhibitors. Little is known about the regulation of these cells. Previously we showed that ligands for toll-like receptors (TLR) 7 and 9 are able to re-stimulate FVIII-specific memory B cells in the absence of T-cell help. However, alternative “helper cells” such as dendritic cells are essential for providing help to memory B cells under such conditions. Based on these findings, we asked which co-stimulatory interactions are required for the restimulation of memory B cells in the presence of dendritic cells and ligands for TLR and whether these co-stimulatory interactions are the same as those required for the restimulation of memory B cells in the presence of activated T cells. We used spleen cells from hemophilic mice treated with human FVIII to generate highly purified populations of memory B cells, CD4+ T cells and dendritic cells. The required purity was achieved by a combination of magnetic bead separation and fluorescence-activated cell sorting. The memory B cell compartment was specified by the expression of CD19 together with IgG and the absence of surface IgM and IgD. Memory B cells were cultured in the presence of FVIII to stimulate their differentiation into anti-FVIII antibody-producing plasma cells. Different combinations of CD4+ T cells, ligands for TLR 7 and 9 and dendritic cells were added to the memory-B-cell cultures. Blocking antibodies and competitor proteins were used to specify the co-stimulatory interactions required for the re-stimulation of memory B cells in the presence of either CD4+ T cells or dendritic cells and ligands for TLR 7 and 9. Our results demonstrate that the blockade of B7-1 and B7-2 as well as the blockade of CD40L inhibit the re-stimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing plasma cells in the presence of T-cell help. Similar requirements apply for the re-stimulation of memory B cells in the presence of dendritic cells and ligands for TLR 7 or 9. Dendritic cells in the absence of ligands for TLR are not able to provide help for the re-stimulation of memory B cells, which indicates that dendritic cells need to be activated. Furthermore, ligands for TLR 7 or 9 were not able to re-stimulate memory B cells in the complete absence of dendritic cells. Based on these results we conclude that dendritic cells activated by ligands for TLR 7 or 9 can substitute for activated CD4+ T cells in providing co-stimulatory help for memory-B-cell re-stimulation. CD40-CD40L interactions seem to be the most important co-stimulatory interactions for the re-stimulation of memory B cells, not only in the presence of activated CD4+ T cells but also in the presence of ligands for TLR and dendritic cells.

The Impact of VWF on FVIII Immune Responses in Hemophilia a Mice with Pre-Existing Anti-FVIII Immunity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 84-84
Author(s):  
Juan Chen ◽  
Jocelyn A. Schroeder ◽  
Xiaofeng Luo ◽  
Robert R. Montgomery ◽  
Qizhen Shi
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Specific Memory ◽  
The Impact

Abstract Development of inhibitory antibodies (inhibitors) against FVIII is the significant complication in protein replacement therapy for hemophilia A (HA). Currently, immune tolerance induction (ITI) with aggressive infusion of high-dose FVIII represents the only effective therapeutic approach for eradication of FVIII inhibitors and results in restoration of normal FVIII pharmacokinetics in inhibitor patients. Whether the use of FVIII products containing VWF will affect the efficacy of the ITI is still a debated issue in the treatment of inhibitor patients. In this study, we explored the impact of VWF on FVIII immune responses in HA with pre-existing anti-F8 immunity using both in vitro and in vivomodels. Since the FVIII immune response is CD4+ T cell dependent, we first investigated how VWF affects FVIII-primed CD4+ T cells in response to FVIII restimulation. To address this question, we used a T cell proliferation assay. FVIIInull (F8null) mice were immunized with recombinant human FVIII (rhF8) to induce inhibitor development. Splenocytes from primed mice were labeled with CellTrace™ Violet and cultured with rhF8 with or without rhVWF. Four days later, cells were analyzed by FACS to assess the daughter (proliferated) cell population. The percentage of daughter CD4+ T cells (14.0±7.5%) in the condition cultured with 1 U/ml of rhF8 was significantly higher than without rhF8 (3.7±1.7%, n=6). The daughter cells further increased to 21.5±10.3% when cells were incubated with 10 U/ml of rhF8. However, when rhVWF was added to the culture media in addition to rhF8, percentages of daughter CD4+ T cells were significantly decreased in both the 1 U/ml and 10 U/ml rhF8 treatment groups (10.4±7.1% and 15. 8±8.4%, respectively). To further explore how VWF affects the FVIII immune response, we analyzed cytokine profiles in T cell culture supernants using a multiplex ELISA assay. The levels of IFNg and IL10 in the groups cultured with rhF8 in the presence of rhVWF were significantly lower than in the groups cultured with rhF8 only. The levels of TNFa, IL4, IL5, and IL12 in the groups cultured with rhF8 together with rhVWF were not significantly different than those in rhF8 groups without VWF. These results demonstrated that VWF significantly suppresses rhF8-primed CD4+ T cell proliferation in response to rhF8 restimulation and the inhibition is via the Th1 pathway. In a setting of pre-existing anti-F8 immunity, how FVIII-specific memory B cells respond to FVIII-restimulation and mature to antibody secreting cells (ASCs) is the critical pathway in terms of the clinical efficacy of FVIII infusion. To investigate how VWF affects memory B cell maturation upon FVIII restimulation, we used ELISPOT-based assay. Splenocytes from rhF8-primed HA mice were used as the source to prepare F8-specific memory B cell pools. CD138+ cells were depleted and the remaining cells were used as a pool of memory B cells. To stimulate the maturation of F8-specific memory B cells into ASCs, memory B cell pools from primed F8null mice were cultured with rhF8 with or without rhVWF for 6 days. After culture, newly formed ASCs were assessed by the ELISPOT assay. There were 54.4±19.5 ASCs/106 cells when cells from memory B cell pool were cultured with 0.05 U/ml rhF8. In contrast, there was only 15.6±1.6 ASCs/106cells after the cells were cultured with rhF8 together with rhVWF, indicating that memory B cell maturation is suppressed in the presence of rhVWF. We then used an in vivo model to further evaluate the impact of VWF on the immunogenicity of FVIII in HA with pre-existing immunity. Since we are unable to mimic the human ITI in F8null mice, we transferred memory B cells from rhF8-primed F8null splenocytes into immunocompromised F8null mice followed by rhF8 immunization in the presence or absence of rhVWF. Blood samples were collected one week after immunization for analysis. The inhibitor titer in animals that received rhF8-primed memory B cell pool followed by rhF8 immunization was 45.9±63.0 BU/ml (n=11), which was significantly higher than the titer in animals immunized with rhF8 together with rhVWF (23.9±38.4, P<.01). These results demonstrate that VWF suppressed the anti-F8 memory response in vivo. In summary, our ex vivo and in vivo data demonstrated that VWF attenuates F8-primed CD4+T cells and memory B cells in response to rhF8 restimulation, suggesting that infusion of FVIII together with VWF might reduce anti-F8 memory responses in HA with inhibitors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Molecular footprints of a germinal center derivation of human IgM+(IgD+)CD27+ B cells and the dynamics of memory B cell generation

2009 ◽  
Vol 206 (12) ◽  
pp. 2659-2669 ◽  
Author(s):  
Marc Seifert ◽  
Ralf Küppers
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Clone ◽  
Human Peripheral Blood ◽  
Large Population ◽  
Memory B Cells ◽  
Cell Repertoire ◽  
Memory B Cell

The origin of IgM+CD27+ B lymphocytes with mutated IgV genes, which account for ∼20% of human peripheral blood (PB) B cells, is controversially discussed. A generation in a primary diversification pathway, in T cell–independent immune responses, or in T cell–dependent germinal center (GC) reactions has been proposed. We show here that IgM+IgD+CD27+ and IgM+IgD−/lowCD27+ B cell subsets carry, like class-switched memory B cells, mutations in the Bcl6 gene as a genetic trait of a GC experience. Moreover, the identification of PB IgM+IgD+CD27+ B cells clonally related to GC-derived IgG+ memory B cells with shared and distinct IgV gene mutations demonstrates the GC origin also of the former subset. These findings provide genetic evidence for a GC derivation of somatically mutated IgM+ B cells and indicate that adult humans harbor a large population of IgM+IgD+ post-GC memory B cells. Furthermore, the analysis revealed that a highly diverse and often very large population of memory B cells is generated from a given GC B cell clone, and that (preferentially IgM) memory B cells are generated already early in the GC reaction. This provides novel insights into the dynamics of GC reactions and the generation of a memory B cell repertoire.

scholarly journals Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

2021 ◽  
Author(s):  
Casper Marsman ◽  
Dorit Verhoeven
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Formation ◽  
B Cell Differentiation ◽  
Differentiation Potential ◽  
Human B Cell

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

Sign in / Sign up

Export Citation Format

Share Document