scholarly journals Clonal analysis of primary B cells responsive to the pathogenic bacterium Salmonella typhimurium.

1987 ◽  
Vol 165 (2) ◽  
pp. 340-358 ◽  
Author(s):  
L W Duran ◽  
E S Metcalf
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lipid A ◽  
Predominant Role ◽  
Cell Repertoire ◽  
Cell Responses ◽  
B Cell Repertoire ◽  
Cell Clones ◽  
B Cell Subsets ◽  
Synthetic Antigens

In the present study, a modification of the splenic focus system is used to analyze the S. typhimurium strain TML (TML)-specific B cell repertoire. The results show that the frequency of primary TML-specific splenic B cells in CBA/Ca mice is approximately 1 per 10(5) B cells and less than 30% of these B cells are specific for LPS. In contrast, the frequency of memory TML-specific cells is approximately 1 per 5-8 X 10(3) splenic B cells and greater than 95% of these B cells are specific for LPS. These results suggest that the frequency of primary TML-specific B cells is extremely low and that it expands 15-20-fold after antigen exposure. It is interesting that less than 30% of the primary B cells are specific for the LPS molecule since it is considered to be the major antigenic determinant on Salmonella organisms. Furthermore, the majority of the LPS-specific anti-TML antibody-producing clones are directed against the LPS O antigen region. Conversely, more than half to two-thirds of the memory LPS-specific anti-TML B cell clones are directed against the KDO or lipid A region of the LPS molecule. These results indicate that the preferential expansion of LPS-specific B cell clones observed after immunization resides primarily in the B cell subsets responsive to the KDO/lipid A moieties on the LPS molecule. Finally, unlike B cell responses to chemically defined antigens, TML stimulates very little IgG1 antibody. IgG2 and IgA isotypes appear to play a predominant role in anti-TML antibody responses, although all H chain classes are produced to some extent. Collectively, these findings are consistent with the responses reported for two other natural antigens, HA and PC. Hence, the pattern of stimulation by infectious agents, such as S. typhimurium, appears to be distinct from that of synthetic antigens. Thus, the studies presented herein have begun to provide insights into those subsets of B cells responsive to S. typhimurium and other infectious disease organisms.

scholarly journals Low Numbers of CD27- IgD- 'Double Negative' Senescent B-Cells Early after Reduced Intensity T Cell Depleted Haematopoietic Stem Cell Transplantation Are Predictive of Subsequent Chronic GvHD

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4529-4529
Author(s):  
Francesca Kinsella ◽  
Harriet Protheroe ◽  
Hayden Pearce ◽  
Charlotte F Inman ◽  
Suzy A Eldershaw ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Chronic Gvhd ◽  
Subsequent Development ◽  
Haematopoietic Stem Cell ◽  
Cell Repertoire ◽  
Graft Versus Host ◽  
Post Transplant ◽  
B Cell Repertoire ◽  
B Cell Subsets

Chronic graft versus host disease (cGvHD) is an important complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). It is now believed that B cells play a major role in the development of cGvHD as evidenced by the efficacy of B-cell directed therapies, and the disturbances in B cell subsets found in patients. This is characterised by a relative lack of naive B cells and increase in CD27+ (antigen experienced) mature B cells that can mediate host directed responses. Despite this the cellular mechanisms underlying the pathogenesis of cGvHD remain unclear. We investigated the phenotype and function of CD19+ B-cell subsets in a cohort of 46 patients undergoing reduced intensity-conditioned allo-HSCT with alemtuzumab. 76% of patients had myeloid disease and 24% lymphoid disease. All patients received PBMC grafts, fludarabine and melphalan conditioning and 10mg/day alemtuzumab from day -5 for 5 days. Overall survival at 3 years was 65% and relapse free survival was 62%. 16% patients experienced acute GvHD (grade 2+) and 29% developed chronic GvHD. B cell subsets were examined at 6 weeks post-transplant and CD27-IgD- 'double negative' (DN) B-cells were seen to dominate the B cell repertoire (mean 55% of B-cells). DN B-cells are an ill-defined memory B cell subset associated with immune senescence, auto-immune inflammatory conditions and cancer but their role in allo-HSCT has not been described. The frequency of DN B-cells decreased over time, as the naive B cell compartment regenerated, but still represented 31% of B cells at 1 year. However the mean number of DN B cells remained remarkably stable (0.033x109/L at 6 weeks v 0.037x109/L at 1 year). 14% of DN B-cells early post-transplant were transitional (CD24high CD38high) whilst 10% were plasma cells (CD24- CD38high). The remaining 76% had increased CD86 expression compared to memory B-cells (16% v 9.6%; *p=0.039) and lower levels of surface immunoglobulin. DN B-cells were less functional than other B cell subsets with decreased proportions of cells producing IL-2 (20%), IL-6 (17%), IFN-γ (6.2%), and IL-10 (6.2%) compared to naive or memory B cells. They also demonstrated reduced proliferation to in vitro stimulation (Ki-67 5.3%). Importantly, the proportion of DN B cells early post transplant was then studied in relation to transplant outcome and a lower frequency of cells was found to be correlated with subsequent development of cGvHD (38% v 62%; *p=0.04). These data show that senescent CD27-IgD- DN B-cells dominate the B cell repertoire in the early period after lymphocyte-depleted allo-HSCT. Furthermore, they are significantly reduced in patients who subsequently develop cGvHD. These data suggest that early B cell senescence can directly regulate the subsequent development of chronic graft versus host disease and indicate that the composition of the B cell repertoire at an early time-point post-transplant may be used to predict subsequent clinical outcome. Disclosures No relevant conflicts of interest to declare.

scholarly journals Thymus-derived B cell clones persist in the circulation after thymectomy in myasthenia gravis

2020 ◽  
Vol 117 (48) ◽  
pp. 30649-30660 ◽  
Author(s):  
Ruoyi Jiang ◽  
Kenneth B. Hoehn ◽  
Casey S. Lee ◽  
Minh C. Pham ◽  
Robert J. Homer ◽  
...  
Keyword(s):  
B Cells ◽  
Myasthenia Gravis ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Cell Clones ◽  
Autoantibody Titer ◽  
Repertoire Sequencing ◽  
Symptom Measures

Myasthenia gravis (MG) is a neuromuscular, autoimmune disease caused by autoantibodies that target postsynaptic proteins, primarily the acetylcholine receptor (AChR) and inhibit signaling at the neuromuscular junction. The majority of patients under 50 y with AChR autoantibody MG have thymic lymphofollicular hyperplasia. The MG thymus is a reservoir of plasma cells that secrete disease-causing AChR autoantibodies and although thymectomy improves clinical scores, many patients fail to achieve complete stable remission without additional immunosuppressive treatments. We speculate that thymus-associated B cells and plasma cells persist in the circulation after thymectomy and that their persistence could explain incomplete responses to resection. We studied patients enrolled in a randomized clinical trial and used complementary modalities of B cell repertoire sequencing to characterize the thymus B cell repertoire and identify B cell clones that resided in the thymus and circulation before and 12 mo after thymectomy. Thymus-associated B cell clones were detected in the circulation by both mRNA-based and genomic DNA-based sequencing. These antigen-experienced B cells persisted in the circulation after thymectomy. Many circulating thymus-associated B cell clones were inferred to have originated and initially matured in the thymus before emigration from the thymus to the circulation. The persistence of thymus-associated B cells correlated with less favorable changes in clinical symptom measures, steroid dose required to manage symptoms, and marginal changes in AChR autoantibody titer. This investigation indicates that the diminished clinical response to thymectomy is related to persistent circulating thymus-associated B cell clones.

scholarly journals AB0146 DRUG DEPENDENT ALTERATIONS IN B-CELL REPERTOIRE IN SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS WITH LOW DISEASE ACTIVITY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1373.2-1373
Author(s):  
S. Zenz ◽  
B. Dreo ◽  
B. Prietl ◽  
S. Kofler ◽  
H. Sourij ◽  
...  
Keyword(s):  
B Cells ◽  
Disease Activity ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Cell Repertoire ◽  
Low Disease Activity ◽  
B Cell Repertoire ◽  
Drug Dependent ◽  
B Cell Subsets

Background:B-cells play a major role in the pathogenesis and perpetuation of the immune response in systemic lupus erythematosus (SLE). So far, B-cell subtypes have been studied well, but the precise mechanisms of the B-cell alterations during disease activity and during remission, depending on different medication, are still unclear.Objectives:The aim of our study was to investigate the drug dependent alterations in the B-cell repertoire of SLE patients with low disease activity (SLEDAI – 2K ≤4).Methods:Peripheral blood samples from 39 patients suffering from SLE (mean±SD; age 43±13 years, 87.2% females, disease duration 11.1±7 years) were drawn over 2 years. All SLE patients were in remission or low disease activity (median±SE, SLEDAI of 2.0±1.5). B-cells were characterized using CD19, CD20, CD5, CD27 antibodies and were grouped in naïve (IgD+27-), non-switched memory (IgD+, CD27+), memory (IgD-,CD27+), B1 (CD5+27-) and MBL-like (CD5++) B-cells. A quantitative flow cytometric bead-based assay (QuantiBRITE PE kit from Becton Dickinson) was used for the estimation of CD19 antibodies bound per cell. Further, CD38 and CD86 antibodies were used to characterize the B-cell subsets. All cytometric measurements were performed using a standardized BD LSR Fortessa platform. After 3 years of follow-up, patients’ data about disease activity and current medication were obtained.Results:22 SLE patients were treated with hydroxychloroquine (85.8%) and 19 patients received mycophenolate mofetil (MMF; n=14; 54.6%) or azathioprine (AZA; n= 5; 19.5 %). 5 patients were treated with other DMARDs. Independently of hydroxychloroquine and/or MMF, no significant differences were seen in naïve, non-switched memory, post-switched memory, plasma blasts, B1- or MBL-like B-cells. Patients treated with AZA had significantly lower naïve B-cells (mean±SD, 39.3±6.7vs. 73.1±19.3 %; p = 0.028), but had significantly higher IgD-post switched B-cells (31.2±9.1 vs.12.5 ±9.2 %; p = 0.028, respectively) compared with no AZA-treatment. Interestingly, activated B-cells (5.5±1.5 vs. 1.8±1.1%; p = 0.009) were significantly higher in AZA-treated. After 3 years of follow-up, almost all patients were in remission (median±SE, SLEDAI of 2.0±2.0), except of 3 patients with a SLEDAI of ≥ 6. Interestingly, those patients had at baseline, statistically higher naïve B-cells (p = 0.041) and lower B1-like B-cells (p =0.020) compared with patients with low disease activity.Conclusion:Our results suggest that independently of hydroxychloroquine and/or MMF treatment, all patients with low disease activity had similar normal B-cell subsets. Interestingly, in the small group of patients who were treated with AZA, a reduced regeneration of B-cells was shown. Patients with higher disease and high naïve B-cells showed an increased disease activity after three years.Acknowledgments:The research was performed in “CBmed” and funded by the Austrian Federal Government within the COMET K1 Centre Program, Land Steiermark and Land Wien.Disclosure of Interests:None declared

scholarly journals SARS-CoV-2 mRNA vaccines induce a robust germinal centre reaction in humans

2021 ◽  
Author(s):  
Ali Ellebedy ◽  
Jackson Turner ◽  
Jane O'Halloran ◽  
Elizaveta Kalaidina ◽  
Wooseob Kim ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Messenger Rna ◽  
Neutralizing Antibodies ◽  
Germinal Centre ◽  
S Protein ◽  
Fine Needle Aspirates ◽  
Cell Responses ◽  
Cell Clones ◽  
Shared Epitopes

Abstract Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA)-based vaccines are ~95% effective in preventing coronavirus disease 2019. However, the dynamics of antibody secreting plasmablasts (PBs) and germinal centre (GC) B cells induced by these vaccines in SARS-CoV-2 naïve and antigen-experienced humans remains unclear. Here we examined peripheral blood and/or lymph node (LN) antigen-specific B cell responses in 32 individuals who received two doses of BNT162b2, an mRNA-based vaccine encoding the full-length SARS-CoV-2 spike (S) gene. Circulating IgG- and IgA-secreting PBs targeting the S protein peaked one week after the second immunization then declined and were undetectable three weeks later. PB responses coincided with maximal levels of serum anti-S binding and neutralizing antibodies to a historical strain as well as emerging variants, especially in individuals previously infected with SARS-CoV-2, who produced the most robust serological responses. Fine needle aspirates of draining axillary LNs identified GC B cells that bind S protein in all participants sampled after primary immunization. GC responses increased after boosting and were detectable in two distinct LNs in several participants. Remarkably, high frequencies of S-binding GC B cells and PBs were maintained in draining LNs for up to seven weeks after first immunization, with a substantial fraction of the PB pool class-switched to IgA. GC B cell-derived monoclonal antibodies predominantly targeted the RBD, with fewer clones binding to the N-terminal domain or shared epitopes within the S proteins of human betacoronaviruses OC43 and HKU1. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a robust and persistent GC B cell response that engages pre-existing as well as new B cell clones, which enables generation of high-affinity, broad, and durable humoral immunity.

scholarly journals Efficient Peripheral Clonal Elimination of B Lymphocytes in MRL/lpr Mice Bearing Autoantibody Transgenes

1998 ◽  
Vol 188 (5) ◽  
pp. 909-917 ◽  
Author(s):  
Jennifer A. Kench ◽  
David M. Russell ◽  
David Nemazee
Keyword(s):  
B Cells ◽  
B Cell ◽  
Genetic Background ◽  
Cell Tolerance ◽  
Cell Repertoire ◽  
B Cell Tolerance ◽  
B Cell Repertoire ◽  
Nuclear Antigens ◽  
Large Numbers ◽  
Liver And Kidney

Peripheral B cell tolerance was studied in mice of the autoimmune-prone, Fas-deficient MRL/ lpr.H-2d genetic background by introducing a transgene that directs expression of membrane-bound H-2Kb antigen to liver and kidney (MT-Kb) and a second transgene encoding antibody reactive with this antigen (3-83μδ, anti-Kk,b). Control immunoglobulin transgenic (Ig-Tg) MRL/lpr.H-2d mice lacking the Kb antigen had large numbers of splenic and lymph node B cells bearing the transgene-encoded specificity, whereas B cells of the double transgenic (Dbl-Tg) MRL/lpr.H-2d mice were deleted as efficiently as in Dbl-Tg mice of a nonautoimmune B10.D2 genetic background. In spite of the severely restricted peripheral B cell repertoire of the Ig-Tg MRL/lpr.H-2d mice, and notwithstanding deletion of the autospecific B cell population in the Dbl-Tg MRL/lpr.H-2d mice, both types of mice developed lymphoproliferation and exhibited elevated levels of IgG anti-chromatin autoantibodies. Interestingly, Dbl-Tg MRL/lpr.H-2d mice had a shorter lifespan than Ig-Tg MRL/lpr.H-2d mice, apparently as an indirect result of their relative B cell lymphopenia. These data suggest that in MRL/lpr mice peripheral B cell tolerance is not globally defective, but that certain B cells with receptors specific for nuclear antigens are regulated differently than are cells reactive to membrane autoantigens.

scholarly journals Complexity of the human memory B-cell compartment is determined by the versatility of clonal diversification in germinal centers

2015 ◽  
Vol 112 (38) ◽  
pp. E5281-E5289 ◽  
Author(s):  
Bettina Budeus ◽  
Stefanie Schweigle de Reynoso ◽  
Martina Przekopowitz ◽  
Daniel Hoffmann ◽  
Marc Seifert ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Clone ◽  
Human Memory ◽  
Memory B Cells ◽  
Sequencing Analysis ◽  
Cell Compartment ◽  
Memory B Cell ◽  
Cell Clones ◽  
B Cell Subsets

Our knowledge about the clonal composition and intraclonal diversity of the human memory B-cell compartment and the relationship between memory B-cell subsets is still limited, although these are central issues for our understanding of adaptive immunity. We performed a deep sequencing analysis of rearranged immunoglobulin (Ig) heavy chain genes from biological replicates, covering more than 100,000 memory B lymphocytes from two healthy adults. We reveal a highly similar B-cell receptor repertoire among the four main human IgM+ and IgG+ memory B-cell subsets. Strikingly, in both donors, 45% of sequences could be assigned to expanded clones, demonstrating that the human memory B-cell compartment is characterized by many, often very large, B-cell clones. Twenty percent of the clones consisted of class switched and IgM+(IgD+) members, a feature that correlated significantly with clone size. Hence, we provide strong evidence that the vast majority of Ig mutated B cells—including IgM+IgD+CD27+ B cells—are post-germinal center (GC) memory B cells. Clone members showed high intraclonal sequence diversity and high intraclonal versatility in Ig class and IgG subclass composition, with particular patterns of memory B-cell clone generation in GC reactions. In conclusion, GC produce amazingly large, complex, and diverse memory B-cell clones, equipping the human immune system with a versatile and highly diverse compartment of IgM+(IgD+) and class-switched memory B cells.

scholarly journals The allogeneic bisection of carrier-specific enhancement of monoclonal B-cell responses.

1975 ◽  
Vol 142 (5) ◽  
pp. 1165-1179 ◽  
Author(s):  
S K Pierce ◽  
N R Klinman
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Immunoglobulin Class ◽  
Cell Responses ◽  
Cell Clones ◽  
Ia Antigens ◽  
Collaborative Interactions ◽  
B Cell Responses

The ability of T cells to enhance the response of syngeneic and allogeneic B cells to thymus-dependent hapten-carrier conjugates was analyzed. This analysis was carried out on individual primary B cells in splenic fragment cultures derived from irradiated reconstituted mice. This system has several advantages: (a) the response of the B cells is entirely dependent on carrier priming of the irradiated recipient; (b) this B-cell response can be quantitated in terms of the number of responding cells; and (c) very small B-cell responses can be readily detected and analyzed. The results indicate that the majority of hapten-specific B cells were stimulated in allogeneic and syngeneic recipients only if these recipients were previously carrier primed. The number of B cells responding in carrier-primed allogeneic recipients was 60-70% of that in syngeneic carrier-primed recipients. The antibody-forming cell clones resulting from B cells stimulated in the allogeneic environment produced small amounts of antibody and antibody solely of the IgM immunoglobulin class, while the larger responses in syngeneic recipients were predominantly IgG1 or IgM plus IgG1. The capacity of collaborative interactions between carrier-primed T cells and primary B cells to yield IgG1 antibody-producing clones was shown to be dependent on syngeny between these cells in the H-2 gene complex. It is concluded that: (a) B cells can be triggered by T-dependent antigens to clone formation through collaboration with T cells which differ at the H-2 complex as long as these T cells recognize the antigen; (b) the immunoglobulin class produced by the progeny of stimulated B cells generally depends on the nature of the stimulatory event rather than the nature of the B cell itself; and (c) stimulation to IgG1 production is dependent on syngeny between the collaborating T and B cells probably within the Ir-1A region. The role of the Ia antigens in the formation of IgG1-producing clones is not yet clear; Ia identity could permit IgG1 production or, conversely, nonidentity of Ia could induce all allogeneic interactions which prohibit IgG1 production.

scholarly journals High-frequency representation of a single VH gene in the expressed human B cell repertoire.

1993 ◽  
Vol 177 (2) ◽  
pp. 409-418 ◽  
Author(s):  
A K Stewart ◽  
C Huang ◽  
B D Stollar ◽  
R S Schwartz
Keyword(s):  
B Cells ◽  
B Cell ◽  
High Frequency ◽  
Gene Segment ◽  
Normal Subjects ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
V Genes ◽  
Vh Gene ◽  
Vh Genes

Idiotype (Id) 16/6 marks a variable (V) region structure that occurs frequently in the human immunoglobulin repertoire. The basis of the Id has been traced to a germline heavy chain gene segment, VH18/2 (VH26). To pursue the molecular basis for the frequency of Id 16/6, we have analyzed polymerase chain reaction-generated C mu, C gamma, and VH3 family V gene libraries derived from the circulating and tonsillar B cells of four normal individuals and from the B cells of two patients with active systemic lupus erythematosus (SLE). The frequency of VH18/2 in these libraries was compared with three control VH genes, VH56P1, VH21/28, and VHA57. Plaque lifts from C mu and C gamma VH cDNA libraries were screened with gene-specific oligonucleotide probes. The frequency of VH18/2 ranged from 4 to 10% of JH+ plaques (two of five times that of control VH genes). In four VH3 family-specific libraries derived from rearranged DNA, VH18/2 represented 19-33% of VH3+ plaques. Hybridizing VH18/2 plaques were 98-100% homologous to the germline VH gene; mutations when present were often in framework 3. Extensive variation was seen in the complementarity determining region 3 sequences of these rearranged V genes. The high frequency of VH18/2 expression in the B cell repertoire was confirmed by sequencing randomly picked JH+ plaques. In two patients with active SLE the frequency of use of VH18/2 was not greater than that observed in normal subjects. These results show that VH18/2 is overrepresented in the B cell repertoire of normal subjects and suggest that the immune repertoire may be dominated by relatively few V genes.

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