Profiling the Autoantibody Repertoire of Acquired Thrombotic Thrombocytopenic Purpura (aTTP) Patients By Single Cell Sorting and Deep Sequencing of Splenic B-Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 108-108
Author(s):  
Magdalena Skowronska ◽  
Monica M Schaller ◽  
Johanna A. Kremer Hovinga Strebel
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Memory B Cells ◽  
Light Chains ◽  
Cell Populations ◽  
Cell Repertoire ◽  
Specific Memory ◽  
B Cell Repertoire ◽  
Single Antigen

Abstract Introduction: Antibodies are the primary effector molecules in the humoral immune system. To create a diversity of receptors-antibodies B-cells undergo V(D)J gene recombination and somatic hypermutation. This allows the recognition of most of pathogens but also sometimes of self-antigen, which can lead to autoimmune diseases. Autoantibodies (Abs) neutralizing and/or accelerating the clearance of ADAMTS13 are present in nearly all acquired thrombotic thrombocytopenic purpura (aTTP) patients with severe ADAMTS13 activity (<5%). As increasing evidence points at the spleen as a major reservoir for antigen specific memory B-cells, we investigated the splenic B-cell repertoire. We sequenced heavy and corresponding light chains from single antigen-specific memory B-cells and deep sequenced global antibody repertoire among different B-cell populations. Combining both, the single antigen-specific repertoire and the general global repertoire from the same patients specimens allows an investigation of the V(D)J gene usage and abundance of specific V(D)J junction in the B-cell populations within one patient and also among different patients. Analysis of the repertoire enables better understanding of the humoral autoimmune response. Methods: Specimens were analyzed from 8 aTTP patients who were splenectomized due to a refractory course of the autoimmune disease. Splenic mononuclear cells were sorted by flow-cytometry into naïve and transitional (CD27-, IgD+), unswitched memory (CD27+, IgD+) and switched memory (CD27+, IgD-) as well as (CD27+, CD20-/CD19-, CD38+) plasma cells. The frequencies of highly positive anti-ADAMTS13 B-cells in each B-cell population were calculated. Library consisting of antibody cDNA from each B-cell population was deep sequenced on MiSeq Ilumina. Prime analysis were performed in CLC Genomics Workbench. High quality sequences were submitted to IMGT/high V-QUEST web-based analysis tool to determine for V(D)J genes alignments, and V(D)J junction (antigen binding region) and mutations analysis. The IMGT output was parsed into MySQL database for further analysis. In addition, from 4 patients anti-ADAMTS13 specific IgG bearing cells were individually sorted. Gene transcripts of single cells were reverse-transcribed followed by nested PCR of IgG heavy/light chains. V(D)J pairings were visualized with Circos software. In MySQL we compared single cell antigen specific sequence with global repertoire. Results: Anti-ADAMTS13 specific B-cells were detected in the spleen of all patients (average 0.01% of total B-cell population). Deep sequencing of the total B-cell repertoire revealed ~3 million productive sequences, from which ~2 million were unique. Splenic anti-ADAMTS13 specific B-cells of four aTTP patients revealed 80 antibodies with unique V(D)J junction. Among those most frequently used V-genes were IGHV1-69 and IGHV3-30 (15% and 12% respectively) in global repertoire 1.5% of antibodies were encoded with IGHV1-69 gene within respective population. The average identity to germline was lower in ADMTST13 specific B-cells (92% compare to 96%). Four V(D)J junctions were convergent in at least 2 patients (identical amino acid sequence). Conclusion: Anti-ADAMTS13 specific B-cells were found in all aTTP patients in different B-cell populations. We observed enrichment of some variable gene segments when comparing the specific anti-ADAMTS13 to the total splenic repertoire (mainly IGHV1-69). Finding convergent V(D)J junction is very promising and also confirms previous finding of our group where similar V(D)J junction were found among two patients. Currently we clone selected single sorted monoclonal Abs (Schaller et al, Blood 2014;124(23):3469-79). Functional testing will allow the selection of the inhibitory Abs to be used as tools to develop anti-idiotypic specific therapies for aTTP patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Shared HIV envelope-specific B cell clonotypes induced by a pox-protein vaccine regimen

2021 ◽  
Author(s):  
Kristen W. Cohen ◽  
Lamar Ballweber-Fleming ◽  
Michael Duff ◽  
Rachael E. Whaley ◽  
Aaron Seese ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Critical Role ◽  
Memory B Cells ◽  
Protein Vaccine ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Specific Igg

An effective HIV-1 vaccine will likely induce potent, broad neutralizing antibodies. No candidate vaccines have elicited these responses presumably because they fail to activate human B cell precursors that can affinity mature to generate broad neutralizing antibodies. To identify the B cell clonotypes that are elicited, we conducted in-depth analyses of the envelope-specific B cell repertoire in recipients of ALVAC-HIV vector (vCP2438) and bivalent subtype C gp120 protein (HVTN100). We observed high frequencies of envelope-specific IgG+ memory B cells with restricted immunogenetic diversity, relative to non-vaccine induced memory B cells, with preferential expansions of distinct variable genes but limited accumulation of mutations. Many envelope-specific clonotypes were shared across vaccinees, but did not overlap with the envelope-negative memory repertoire, within and across subjects. Single-cell sequencing of envelope-specific IgG+ memory B cells often revealed VH1-2*02 and VK3-20 sequence co-expression and in one case, contained a 5 amino acid CDRL3, the canonical signature of VRC01-class antibodies, confirming that these B cells are extremely rare but detectable. Our study provides evidence that immunogens play a critical role in selecting and restricting the responding B cell repertoire and supports the rational design of HIV vaccines targeting specific B cell lineages for induction of broadly-reactive neutralizing antibodies.

scholarly journals Long-term persistence of neutralizing memory B cells in SARS-CoV-2

2020 ◽  
Author(s):  
Rowena Bull ◽  
Arunasingam Abayasingam ◽  
Harikrishnan Balachandran ◽  
David Agapiou ◽  
Chaturaka Rodrigo ◽  
...  
Keyword(s):  
B Cells ◽  
Protective Immunity ◽  
Memory B Cells ◽  
Cell Repertoire ◽  
Neutralising Antibodies ◽  
Specific Memory ◽  
B Cell Repertoire ◽  
Antibody Titres ◽  
Selected Subset

Abstract Considerable concerns relating to the duration of protective immunity against SARS-CoV-2 have been raised, with evidence of antibody titres declining rapidly after infection and reports of reinfection. Here we monitored antibody responses against SARS-CoV-2 receptor binding domain (RBD) for up to six months after infection. While antibody titres were maintained, half of the cohort’s neutralising responses had returned to background. However, encouragingly in a selected subset of 13 participants, 12 had detectable RBD-specific memory B cells and these generally increased out to 6 months. Furthermore, we were able to generate monoclonal antibodies with SARS-CoV-2 neutralising capacity from these memory B cells. Overall our study suggests that the loss of neutralising antibodies in plasma may be countered by the maintenance of neutralising capacity in the memory B cell repertoire.

scholarly journals A public antibody class recognizes a novel S2 epitope exposed on open conformations of SARS-CoV-2 spike

2021 ◽  
Author(s):  
Mathieu Claireaux ◽  
Tom G Caniels ◽  
Marlon de Gast ◽  
Julianna Han ◽  
Denise Guerra ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Cell Repertoire ◽  
Fold Enrichment ◽  
B Cell Repertoire ◽  
High Fraction ◽  
Antibody Class

AbstractDelineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigated the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We found that ∼82% of SARS-CoV-2 S-reactive B cells show a naive phenotype, which represents an unusually high fraction of total human naive B cells (∼0.1%). Approximately 10% of these naive S-reactive B cells shared an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. A proportion of memory B cells, comprising switched (∼0.05%) and unswitched B cells (∼0.04%), was also reactive with S and some of these cells were reactive with ADAMTS13, which is associated with thrombotic thrombocytopenia. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

scholarly journals Antigen-Specific B Cell Memory

2000 ◽  
Vol 191 (7) ◽  
pp. 1149-1166 ◽  
Author(s):  
Louise J. McHeyzer-Williams ◽  
Melinda Cool ◽  
Michael G. McHeyzer-Williams
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Memory B Cells ◽  
Cell Memory ◽  
Specific Memory ◽  
B Cell Memory ◽  
Cellular Components

The mechanisms that regulate B cell memory and the rapid recall response to antigen remain poorly defined. This study focuses on the rapid expression of B cell memory upon antigen recall in vivo, and the replenishment of quiescent B cell memory that follows. Based on expression of CD138 and B220, we reveal a unique and major subtype of antigen-specific memory B cells (B220−CD138−) that are distinct from antibody-secreting B cells (B220+/−CD138+) and B220+CD138− memory B cells. These nonsecreting somatically mutated B220− memory responders rapidly dominate the splenic response and comprise &gt;95% of antigen-specific memory B cells that migrate to the bone marrow. By day 42 after recall, the predominant quiescent memory B cell population in the spleen (75–85%) and the bone marrow (&gt;95%) expresses the B220− phenotype. Upon adoptive transfer, B220− memory B cells proliferate to a lesser degree but produce greater amounts of antibody than their B220+ counterparts. The pattern of cellular differentiation after transfer indicates that B220− memory B cells act as stable self-replenishing intermediates that arise from B220+ memory B cells and produce antibody-secreting cells on rechallenge with antigen. Cell surface phenotype and Ig isotype expression divide the B220− compartment into two main subsets with distinct patterns of integrin and coreceptor expression. Thus, we identify new cellular components of B cell memory and propose a model for long-term protective immunity that is regulated by a complex balance of committed memory B cells with subspecialized immune function.

scholarly journals Broad Hemagglutinin-Specific Memory B Cell Expansion by Seasonal Influenza Virus Infection Reflects Early-Life Imprinting and Adaptation to the Infecting Virus

2019 ◽  
Vol 93 (8) ◽  
Author(s):  
Brenda L. Tesini ◽  
Preshetha Kanagaiah ◽  
Jiong Wang ◽  
Megan Hahn ◽  
Jessica L. Halliley ◽  
...  
Keyword(s):  
Influenza Virus ◽  
B Cells ◽  
Virus Infection ◽  
B Cell ◽  
Specific Antibody ◽  
Early Life ◽  
Influenza Virus Infection ◽  
Memory B Cells ◽  
Specific Memory ◽  
Original Antigenic Sin

ABSTRACTMemory B cells (MBCs) are key determinants of the B cell response to influenza virus infection and vaccination, but the effect of different forms of influenza antigen exposure on MBC populations has received little attention. We analyzed peripheral blood mononuclear cells and plasma collected following human H3N2 influenza infection to investigate the relationship between hemagglutinin-specific antibody production and changes in the size and character of hemagglutinin-reactive MBC populations. Infection produced increased concentrations of plasma IgG reactive to the H3 head of the infecting virus, to the conserved stalk, and to a broad chronological range of H3s consistent with original antigenic sin responses. H3-reactive IgG MBC expansion after infection included reactivity to head and stalk domains. Notably, expansion of H3 head-reactive MBC populations was particularly broad and reflected original antigenic sin patterns of IgG production. Findings also suggest that early-life H3N2 infection “imprints” for strong H3 stalk-specific MBC expansion. Despite the breadth of MBC expansion, the MBC response included an increase in affinity for the H3 head of the infecting virus. Overall, our findings indicate that H3-reactive MBC expansion following H3N2 infection is consistent with maintenance of response patterns established early in life, but nevertheless includes MBC adaptation to the infecting virus.IMPORTANCERapid and vigorous virus-specific antibody responses to influenza virus infection and vaccination result from activation of preexisting virus-specific memory B cells (MBCs). Understanding the effects of different forms of influenza virus exposure on MBC populations is therefore an important guide to the development of effective immunization strategies. We demonstrate that exposure to the influenza hemagglutinin via natural infection enhances broad protection through expansion of hemagglutinin-reactive MBC populations that recognize head and stalk regions of the molecule. Notably, we show that hemagglutinin-reactive MBC expansion reflects imprinting by early-life infection and that this might apply to stalk-reactive, as well as to head-reactive, MBCs. Our findings provide experimental support for the role of MBCs in maintaining imprinting effects and suggest a mechanism by which imprinting might confer heterosubtypic protection against avian influenza viruses. It will be important to compare our findings to the situation after influenza vaccination.

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 Memory B Cell Responses to Vibrio cholerae O1 Lipopolysaccharide Are Associated with Protection against Infection from Household Contacts of Patients with Cholera in Bangladesh

2012 ◽  
Vol 19 (6) ◽  
pp. 842-848 ◽  
Author(s):  
Sweta M. Patel ◽  
Mohammad Arif Rahman ◽  
M. Mohasin ◽  
M. Asrafuzzaman Riyadh ◽  
Daniel T. Leung ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Vibrio Cholerae ◽  
Memory B Cells ◽  
Content Type ◽  
Cell Responses ◽  
Household Contacts ◽  
Specific Memory ◽  
Specific Igg ◽  
Protection Against Infection

ABSTRACTVibrio choleraeO1 causes cholera, a dehydrating diarrheal disease. We have previously shown thatV. cholerae-specific memory B cell responses develop after cholera infection, and we hypothesize that these mediate long-term protective immunity against cholera. We prospectively followed household contacts of cholera patients to determine whether the presence of circulatingV. choleraeO1 antigen-specific memory B cells on enrollment was associated with protection againstV. choleraeinfection over a 30-day period. Two hundred thirty-six household contacts of 122 index patients with cholera were enrolled. The presence of lipopolysaccharide (LPS)-specific IgG memory B cells in peripheral blood on study entry was associated with a 68% decrease in the risk of infection in household contacts (P= 0.032). No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cells or IgA memory B cells specific to LPS. These results suggest that LPS-specific IgG memory B cells may be important in protection against infection withV. choleraeO1.

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.

Agonist for Toll-Like Receptor (TLR) 9 Amplifies as Well as Inhibits the Differentiation of FVIII-Specific Memory B Cells into Antibody-Producing Plasma Cells in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3382-3382
Author(s):  
Peter Allacher ◽  
Christina Hausl ◽  
Aniko Ginta Pordes ◽  
Rafi Uddin Ahmad ◽  
Hartmut J Ehrlich ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Memory B Cells ◽  
Memory B Cell ◽  
Cpg Dna ◽  
Cell Responses ◽  
Specific Memory ◽  
B Cell Responses

Abstract Memory B cells are essential for maintaining long-term antibody responses. They can persist for years even in the absence of antigen and are rapidly re-stimulated to differentiate into antibody-producing plasma cells when they encounter their specific antigen. Previously we demonstrated that ligands for TLR 7 and 9 amplify the differentiation of FVIII-specific memory B cells into anti-FVIII antibody-producing plasma cells at low concentrations of FVIII and prevent the inhibition of memory-B-cell differentiation at high concentrations of FVIII. The modulation of FVIII-specific memory-B-cell responses by agonists for TLR is highly relevant for the design of new immunotherapeutic approaches in patients with FVIII inhibitors because TLR are activated by a range of different viral and bacterial components. Specifically, TLR 7 is triggered by single-stranded RNA derived from viruses and TLR 9 is triggered by bacterial DNA containing unmethylated CpG motifs. We further explored the modulation of FVIII-specific memory-B-cell responses by agonists for TLRs by studying a broad range of concentrations of CpG DNA, a ligand for TLR 9, both in vitro and in vivo using the murine E17 model of hemophilia A. We used CpG-DNA in concentrations ranging from 0.1 to 10,000 ng/ml to study the modulation of FVIII-specific memory-B-cell responses in vitro and verified the specificity of the effects observed by including a blocking agent for TLR 9 and GpC-DNA, a non-stimulating negative control for CpG DNA. Furthermore, we used doses of CpG DNA ranging from 10 to 50,000 ng per dose to study the modulation of FVIII-specific memory-B-cell responses in vivo. E17 hemophilic mice were treated with a single intravenous dose of 200 ng FVIII to stimulate the generation of FVIII-specific memory B cells and were subsequently treated with another dose of FVIII that was given together with CpG DNA. We analyzed titers of anti-FVIII antibodies in the circulation of these mice one week after the second dose of FVIII. Previously we had shown that a single dose of 200 ng FVIII, given intravenously to E17 hemophilic mice, stimulates the formation of FVIII-specific memory B cells but is not sufficient to induce anti-FVIII antibodies that would be detectable in the circulation. Our results demonstrate a biphasic effect of CpG DNA on the re-stimulation of FVIII-specific memory B cells and their differentiation into antibody-producing plasma cells. Both in vitro and in vivo studies show that CpG DNA at high doses inhibits the re-stimulation and differentiation of FVIII-specific memory B cells. However, CpG DNA at low doses amplifies these processes. Amplification and inhibition of memory-B-cell responses are due to specific interactions of CpG DNA with TLR 9. Both effects are blocked by addition of a blocking agent for TLR 9 in vitro. We conclude that triggering of TLR 9 by bacterial DNA has a substantial influence on FVIII-specific memory-B-cell responses. The consequence of TLR 9 triggering can be inhibitory or stimulatory, depending on the actual concentration of the bacterial DNA. Our findings demonstrate the potential modulatory effects of bacterial infections on the regulation of FVIII inhibitor development.

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