Abstract P812: Characterizing NMDA Receptor Subunits on B Cells

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Mary K Malone ◽  
Vanessa Torres ◽  
Jadwiga Turchan-Cholewo ◽  
Ann M Stowe
Keyword(s):  
B Cells ◽  
B Cell ◽  
Critical Role ◽  
Extracellular Glutamate ◽  
Future Studies ◽  
B Subunit ◽  
Baseline Levels ◽  
B Subunits ◽  
Post Hoc ◽  
2D Images

Background: N-methyl-D-aspartate (NMDARs) play a critical role in neuronal excitotoxicity after stroke. The actions of NMDARs have been shown mostly in obligatory GluN1 subunits on neurons and not GluN2A/B subunits. In B cells, these subunits have not been highly characterized though the presence of NMDARs has been shown. The function of the GluN2A/B subunits can be neuroprotective or pro-death in neurons, respectively. We hypothesized that GluN2A and GluN2B subunit presence on B cells would be affected by exposure to extracellular glutamate. Methods: Splenic B cells were isolated from 3-4mo-old C57BL/6 male mice via magnetic separation and treated with physiologic levels of L-glutamate (glu; 1uM) in the presence or absence of 5ug/mL LPS. B cell cytospins were stained for B220, GluN2A, and GluN2B, imaged using confocal microscopy, and quantified in FIJI. An average of 10.7 B cells were quantified per image at 80-157x magnification. RGB channels of the z-stacks were quantified to identify positive B220 expression. The z-stacks were split into 2D images and quantified plane-by-plane to identify GluN2A/B subunit clusters. Each cluster of subunits was recorded per cell in view across all planes of the original z-stack to yield total subunit count. Groups included 14-43 B cells quantified, and the number of subunits per cell were analyzed via ordinary two-way ANOVA, Sidak post-hoc test (Graphpad Prism). Significance was p<0.05. Results: There was an average of 19.3±7.2 GluN2A subunits and 19.0±5.0 GluN2B subunits per cell for unstimulated, untreated B cells. Neither glu treatment (p=0.23) nor LPS stimulation (p= 0.10) impacted the number of GluN2A subunits per B cell. LPS decreased GluN2B subunits when compared to unstimulated B cells (11.1±5.1 subunits; p=0.02). Glu treatment normalized GluN2B subunits per B cell near untreated baseline levels (18.2±11 subunits per cell; p=0.01), resulting in an interaction between LPS stimulation and glu treatment in B cells (F (1, 86) =6.180, P=0.015). Conclusions: Our data suggests activated B cells downregulate GluN2B-containing NMDARs following LPS stimulation. This downregulation mimics that of NMDAR activity on neurons upon excitoxicity (PMID: 24361499) but future studies should confirm GluN2B internalization.

scholarly journals Hyaluronate-CD44 Interactions Can Induce Murine B-Cell Activation

Blood ◽  
1997 ◽  
Vol 89 (8) ◽  
pp. 2901-2908 ◽  
Author(s):  
Asimah Rafi ◽  
Mitzi Nagarkatti ◽  
Prakash S. Nagarkatti
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Critical Role ◽  
Surface Glycoprotein ◽  
B Cell Differentiation ◽  
B Cell Activation ◽  
Cell Surface Glycoprotein ◽  
Immunodeficient Mice

Abstract CD44 is a widely distributed cell surface glycoprotein whose principal ligand has been identified as hyaluronic acid (HA), a major component of the extracellular matrix (ECM). Recent studies have demonstrated that activation through CD44 leads to induction of effector function in T cells and macrophages. In the current study, we investigated whether HA or monoclonal antibodies (MoAbs) against CD44 would induce a proliferative response in mouse lymphocytes. Spleen cells from normal and nude, but not severe combined immunodeficient mice, exhibited strong proliferative responsiveness to stimulation with soluble HA or anti-CD44 MoAbs. Furthermore, purified B cells, but not T cells, were found to respond to HA. HA was unable to stimulate T cells even in the presence of antigen presenting cells (APC) and was unable to act as a costimulus in the presence of mitogenic or submitogenic concentrations of anti-CD3 MoAbs. In contrast, stimulation of B cells with HA in vitro, led to B-cell differentiation as measured by production of IgM antibodies in addition to increased expression of CD44 and decreased levels of CD45R. The fact that the B cells were responding directly to HA through its binding to CD44 and not to any contaminants or endotoxins was demonstrated by the fact that F(ab)2 fragments of anti-CD44 MoAbs or soluble CD44 fusion proteins could significantly inhibit the HA-induced proliferation of B cells. Also, HA-induced proliferation of B cells was not affected by the addition of polymixin B, and B cells from lipopolysaccharide (LPS)-unresponsive C3H/HeJ strain responded strongly to stimulation with HA. Furthermore, HA, but not chondroitin-sulfate, another major component of the ECM, induced B-cell activation. It was also noted that injection of HA intraperitoneally, triggered splenic B cell proliferation in vivo. Together, the current study demonstrates that interaction between HA and CD44 can regulate murine B-cell effector functions and that such interactions may play a critical role during normal or autoimmune responsiveness of B cells.

scholarly journals Splenic T Zone Development Is B Cell Dependent

2001 ◽  
Vol 194 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Vu N. Ngo ◽  
Richard J. Cornall ◽  
Jason G. Cyster
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Critical Role ◽  
Early Postnatal Development ◽  
Cell Accumulation ◽  
Zone Development ◽  
Cell Expression ◽  
Immune Defects ◽  
B Cell Deficiency

The factors regulating growth and patterning of the spleen are poorly defined. We demonstrate here that spleens from B cell–deficient mice have 10-fold reduced expression of the T zone chemokine, CCL21, a threefold reduction in T cell and dendritic cell (DC) numbers, and reduced expression of the T zone stromal marker, gp38. Using cell transfer and receptor blocking approaches, we provide evidence that B cells play a critical role in the early postnatal development of the splenic T zone. This process involves B cell expression of lymphotoxin (LT)α1β2, a cytokine that is required for expression of CCL21 and gp38. Introduction of a B cell specific LTα transgene on to the LTα-deficient background restored splenic CCL21 and gp38 expression, DC numbers, and T zone size. This work also demonstrates that the role of B cells in T zone development is distinct from the effect of B cells on splenic T cell numbers, which does not require LTα1β2. Therefore, B cells influence spleen T zone development by providing: (a) signals that promote T cell accumulation, and: (b) signals, including LTα1β2, that promote stromal cell development and DC accumulation. Defects in these parameters may contribute to the immune defects associated with B cell deficiency in mice and humans.

A New Vaccine Paradigm To Break Tolerance with Potential Applications for the Immunotherapy of B Cell Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1411-1411
Author(s):  
Ronald P. Taylor ◽  
Emily C. Whipple ◽  
Margaret A. Lindorfer ◽  
Andrew H. Ditto ◽  
Ryan S. Shanahan
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
B Cells ◽  
B Cell ◽  
Humoral Immune Response ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Breakdown Product ◽  
Humoral Immune ◽  
Mouse Igg2a

Abstract Complement (C) plays a critical role in the immune response by opsonizing immune complexes (IC) and thymus-independent type 2 antigens with C3 breakdown product C3dg. We investigated the in vivo fate and handling in mice of anti-CR1/CR2 mAb 7G6. We used this rat IgG mAb as a surrogate for C3dg-opsonized IC; mAb 7G6 binds to CR1/CR2 with high affinity, blocks C3dg binding and saturates mouse B cell CR2 at inputs of only 2 ug. RIA, flow cytometry, and fluorescence immunohistochemistry were used to examine the disposition of 0.5–2 ug quantities of mAb 7G6 infused i.v. in mice. The mAb binds to circulating B cells and in the spleen binds preferentially to marginal zone (MZ) B cells. However, within 24 h MZ B cells relocate and transfer the mAb to regions rich in follicular dendritic cells (FDC). Localization of intact antigen to FDC should induce a substantial immune response, and therefore we immunized mice and monkeys i.v. with low doses (1–20 ug/kg) of prototype antigens constructed with anti-CR1/2 mAb 7G6 or anti-CR2 mAb HB135, respectively. We observed a strong immune response characterized by early development of IgG antibodies and long-lasting immunity extending out to at least one year. We applied our immunization paradigm to mouse IgG idiotypes, based on i.v. infusion of mouse IgG2a mAbs which were cross-linked with mAb 7G6. The purpose of these experiments was to determine if tolerance can be broken in order to develop a more powerful vaccine strategy to induce a cytotoxic humoral immune response to malignant B cells based on targeting the idiotype of immunoglobulin molecules expressed on their surfaces. I.V. immunization with the constructs indeed generated a mouse IgG1 immune response to two different mouse IgG2a mAbs, as demonstrated by ELISA. The immune response was idiotype specific, but some anti-isotype antibodies were also detected. Moreover, sera from immunized mice immunoprecipitated the specific radiolabeled mouse mAbs in the presence of 7.5% polyethylene glycol. This humoral immune response was also demonstrable in flow cytometry assays in which IgG1 in sera of immunized mice bound to erythrocytes opsonized with bispecific mAb constructs consisting of the IgG2a mAb crosslinked with an anti-CR1 mAb. The present approach, based on coupling the targeted immunoglobulin to an anti-CR2 mAb for delivery to FDC, may lead to a more effective immunotherapeutic vaccine compared to methods currently in clinical trials which require use of glutaraldehyde to effect crosslinking of the targeted immunoglobulin to KLH.

ZAP-70 Expression in Human B cells: Analysis of Normal Cells and Acute Lymphoblastic Leukemia (ALL) Cells with Pro/Pre B Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4443-4443
Author(s):  
Marta Crespo ◽  
Neus Villamor ◽  
Eva Gine ◽  
Dolors Colomer ◽  
Teresa Marafioti ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Protein Tyrosine Kinase ◽  
Lymphoblastic Leukemia ◽  
Human Lymphocytes ◽  
Critical Role ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Qrt Pcr ◽  
Human B Cells

Abstract ZAP-70 is a protein tyrosine kinase of the Syk/ZAP-70 family that plays a critical role in the signal transduction from the T-cell receptor. In human lymphocytes, ZAP-70 gene has been reported to be expressed in T and NK derived cells, and in IgVH unmutated B-chronic lymphocytic leukemia cells. More recently, ZAP-70 expression has been shown to be required for the development of pro-B cells to pre-B cells in mice. To ascertain the expression of ZAP-70 gene in human immature B-cell stages, we analyzed ZAP-70 protein and/or mRNA in normal human B cells at different stages of B cell maturation, including pro/pre-B cells and tumoral cells from 20 B-ALL. ZAP-70 expression was assessed by flow cytometry (FC), immunofluorescence (IF), and/or by quantitative real time RT-PCR (QRT-PCR). In normal bone marrow, ZAP-70 expression was found only in T and in immature B cells (CD19+/CD10+/CD20 −). Moreover, T cells -but no mature B cells- from normal tonsil expressed ZAP-70, as assessed by QRT-PCR and IF. In B-ALLs, a high ZAP-70 expression by FC was observed in 9/13 cases (mean, 82.6%, range 60–99%), whereas in 4 cases ZAP-70 was barely detectable (mean, 13%). By QRT-PCR, 10/16 B-ALLs showed levels of expression similar to ZAP-70 non-expressing cell lines and normal B-cells, whereas in the remaining cases ZAP-70 expression was 3–4 times higher than in normal mature B-cells. Taken together, a high expression of ZAP-70 was found in 11/21 (52%) B-ALLs. No relationship was observed between the level of ZAP-70 expression and the B-ALL maturation status. In conclusion, among normal B cell subsets ZAP-70 expression is restricted to B-cells with pro/pre phenotype. In addition, ZAP-70 is expressed in 52% of B-ALLs, probably as a reflection of their B-cell origin.

Critical Role for CD81 in B Cell Activation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1342-1342
Author(s):  
Mrinmoy Sanyal ◽  
Rosemary Fernandez ◽  
Shoshana Levy
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Cell Function ◽  
Calcium Influx ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Free Calcium ◽  
B Cell Activation ◽  
Deficient Mice

Abstract CD81 is a component of the CD19/CD21 signaling complex in B cells. CD81 was originally discovered as target of an anti-proliferative antibody in a human B cell lymphoma. However, the exact role of CD81 in B cell function is not known. Here we studied B cells from CD81 knockout mice. We demonstrate that upon BCR induction these B cells flux higher intracellular free calcium ion; increase the phosphorylation of BCR-related proximal and distal substrates and increase their proliferation. Similarly, polyclonal activation of CD81-deficient B cells with LPS induced increased proliferation and antibody secretion. Consistent with these intrinsic B cell capabilities, CD81-deficient mice mounted significantly higher immune response upon antigenic stimulation. In addition, bone marrow perisinusoidal B cells (IgM+IgD+) capable of mounting T-independent immune responses against blood-borne pathogens were over represented in CD81-deficient mice. These cells also displayed increased calcium influx kinetics as splenic B cells and produced higher amounts of antibody after polyclonal stimulation. Taken together, these results suggest that CD81 is involved in suppressing B cell activation.

DNA Methyltransferase 1 Contributes to Epigenetic Signatures and Biological Phenotype during Normal B-Cell Differentiation and Lymphomagenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 685-685 ◽  
Author(s):  
Rita Shaknovich ◽  
Leandro Cerchietti ◽  
Maria E. Figueroa ◽  
Ari Melnick
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Dna Methyltransferase ◽  
Critical Role ◽  
Epigenetic Programming ◽  
Differential Digestion ◽  
Epigenetic Signatures

Abstract Normal hematopoiesis requires incremental changes in gene expression in order to establish cellular phenotypes with specialized functions. We are particularly interested in the transcriptional and epigenetic programming of germinal center (GC) B-cells, which acquire unusual biological features normally associated with cancer. Specifically, GC B-cells (i.e. centroblasts - CB) undergo rapid DNA replication while at the same time undergoing genetic recombination, and give rise to a majority of B-cell lymphomas. We hypothesized that epigenetic programming would play a critical role in the CB stage of development, and that gene-specific and genome-wide DNA methyltransferase activity is critical for these cells. We first examined the CpG methylation levels of 24,000 gene promoters in five sets of primary human B-cells just prior to (i.e. naïve B-cells - NBC) and upon entering the GC reaction (i.e. CBs). This was achieved using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, which relies on differential digestion of genomic DNA by the isoschizomer enzymes HpaII and Msp. HELP is a robust and reproducible method that provides accurate and quantitative measurement of DNA methylation levels throughout the genome. Remarkably, we found that the DNA methylation profile of B-cells undergoes a significant shift as readily appreciated by hierarchical clustering. The epigenetic signatures of NBC and CB are differentiation-stage dependent and do not vary significantly between individuals. The coefficient of correlation between individuals was 0.98, as compared to the NBC vs. CB fractions 0.92–0.95. Supervised analysis demonstrated that 266 genes (P<0.001) were differentially methylated upon entry of NB-cells into the GC reaction. We further correlated the methylation status of these genes with their gene expression level. The most heavily affected pathways by differential methylation and concordant expression in naïve B-cells were the Jak/STAT and MAP3K signaling pathways, while in CBs the p38 MAPK pathway and Ikaros family of genes were most affected. Given the epigenetic reprogramming observed in CBs vs. NBCs, along with the need for maintenance of methylation during rapid replication, we predicted that DNA methyltransferase (DNMT) enzymes play a critical role in centroblasts. By performing QPCR and Western blots on isolated fractions of human tonsilar lymphocytes and anatomical localization by immunohistochemistry, we found that DNMTs have a complex temporal and combinatorial expression pattern whereby DNMT1 was the main methyltransferase detectable in centroblasts. Additionally we studied 10 DLBCL cell lines and a panel of primary DLBCL (n=176 for mRNA and 70 for protein) for DNMTs expression. Spearman Rank correlation analysis revealed that DNMT1 was preferentially highly expressed in GCB vs. ABC primary DLBCLs, as well as in BCR vs. OxPhos DLBCLs. Taken together, our data suggest that i) dynamic changes in epigenetic programming contribute to formation of GCs, ii) that DNMT1 may play both a de novo and maintenance methylation role in GC cells, iii) that DNMT1 is markedly upregulated in normal centroblasts and in DLBCLs with the BCR or GCB gene expression profiles and iv) specific therapeutic targeting of DNMT1 rather than non-specific global inhibition of DNA methylation could be a useful anti-lymphoma strategy for germinal center-derived DLBCLs.

scholarly journals Antibody to a Lytic Cycle Viral Protein Decreases Gammaherpesvirus Latency in B-Cell-Deficient Mice

2002 ◽  
Vol 76 (22) ◽  
pp. 11460-11468 ◽  
Author(s):  
Shivaprakash Gangappa ◽  
Sharookh B. Kapadia ◽  
Samuel H. Speck ◽  
Herbert W. Virgin
Keyword(s):  
B Cells ◽  
B Cell ◽  
Viral Genome ◽  
Ex Vivo ◽  
Critical Role ◽  
Passive Transfer ◽  
Lytic Cycle ◽  
Peritoneal Cells ◽  
Latently Infected ◽  
Immunocompromised Hosts

ABSTRACT While antiviral antibody plays a key role in resistance to acute viral infection, the contribution of antibody to the control of latent virus infection is less well understood. Gammaherpesvirus 68 (γHV68) infection of mice provides a model well suited to defining contributions of specific immune system components to the control of viral latency. B cells play a critical role in regulating γHV68 latency, but the mechanism(s) by which B cells regulate latency is not known. In the experiments reported here, we determined the effect of passively transferred antibody on established γHV68 latency in B-cell-deficient (B-cell−/−) mice. Immune antibody decreased the frequency of cells reactivating ex vivo from latency in splenocytes (>10-fold) and peritoneal cells (>100-fold) and the frequency of cells carrying latent viral genome in splenocytes (>5-fold) and peritoneal cells (>50-fold). This effect required virus-specific antibody and was observed when total and virus-specific serum antibody concentrations in recipient B-cell−/− mice were <8% of those in normal mice during latent infection. Passive transfer of antibody specific for the lytic cycle γHV68 RCA protein, but not passive transfer of antibody specific for the v-cyclin protein or the latent protein M2, decreased both the frequency of cells reactivating ex vivo from latency and the frequency of cells carrying the latent viral genome. Therefore, antibody specific for lytic cycle viral antigens can play an important role in the control of gammaherpesvirus latency in immunocompromised hosts. Based on these findings, we propose a model in which ongoing productive replication is essential for maintaining high levels of latently infected cells in immunocompromised hosts. We confirmed this model by the treatment of latently infected B-cell−/− mice with the antiviral drug cidofovir.

scholarly journals Formalin-Inactivated Coxiella burnetii Phase I Vaccine-Induced Protection Depends on B Cells To Produce Protective IgM and IgG

2013 ◽  
Vol 81 (6) ◽  
pp. 2112-2122 ◽  
Author(s):  
Guoquan Zhang ◽  
Ying Peng ◽  
Laura Schoenlaub ◽  
Alexandra Elliott ◽  
William Mitchell ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Phase I ◽  
B Cell ◽  
Coxiella Burnetii ◽  
Critical Role ◽  
Partial Protection ◽  
Content Type ◽  
Deficient Mice

ABSTRACTTo further understand the mechanisms of formalin-inactivatedCoxiella burnetiiphase I (PI) vaccine (PIV)-induced protection, we examined if B cell, T cell, CD4+T cell, or CD8+T cell deficiency in mice significantly affects the ability of PIV to confer protection against aC. burnetiiinfection. Interestingly, compared to wild-type (WT) mice, PIV conferred comparable levels of protection in CD4+T cell- or CD8+T cell-deficient mice and partial protection in T cell-deficient mice but did not provide measurable protection in B cell-deficient mice. These results suggest that PIV-induced protection depends on B cells. In addition, anti-PI-specific IgM was the major detectable antibody (Ab) in immune sera from PIV-vaccinated CD4+T cell-deficient mice, and passive transfer of immune sera from PIV-vaccinated CD4+T cell-deficient mice conferred significant protection. These results suggest that T cell-independent anti-PI-specific IgM may contribute to PIV-induced protection. Our results also suggested that PIV-induced protection may not depend on complement activation and Fc receptor-mediated effector functions. Furthermore, our results demonstrated that both IgM and IgG from PIV-vaccinated WT mouse sera were able to inhibitC. burnetiiinfectionin vivo, but only IgM from PIV-vaccinated CD4+T cell-deficient mouse sera inhibitedC. burnetiiinfection. Collectively, these findings suggest that PIV-induced protection depends on B cells to produce protective IgM and IgG and that T cell-independent anti-PI-specific IgM may play a critical role in PIV-induced protection againstC. burnetiiinfection.

scholarly journals Circadian Clock Protein CRY Controls B-Cell Intrinsic Tolerance

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1029-1029 ◽  
Author(s):  
Qi Cao ◽  
Xuan Zhao ◽  
Sigal Gery ◽  
Zhengshan Chen ◽  
Ruishu Deng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Signaling Pathway ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Adaptive Immune System ◽  
Cell Tolerance ◽  
Double Knockout ◽  
B Cell Tolerance ◽  
Physiological Processes

Abstract The circadian system regulates numerous physiological processes including adaptive immune system. Here we show that mice deficient for the circadian genes Cry1 and Cry2, (Cry double knockout [DKO]) display an autoimmune phenotype including higher serum IgG concentration than wild type (WT) mice, presence of serum anti-nuclear antibodies, precipitation of IgG, IgM and complement 3 (C3) in glomeruli, and massive infiltrations of leukocytes into the lung and kidney. A large panel of autoantigens demonstrated that the sera of the Cry DKO mice but not the WT mice, had autoantibodies covering most of the specificities reported to be present in patients with SLE, rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome and other autoimmune disorders. Taken together, lost of the CRY circadian protein leds to severe autoimmunity. Furthermore, flow cytometry analysis of lymphoid organs showed lower pre-B cell numbers and higher mature recirculating B cells in the bone marrow as well as increased number of B2 B cells in the peritoneal cavity of Cry DKO mice. The BCR-proximal signaling pathway plays a critical role in peripheral B cell tolerance and activation. Activation of splenic B cells from the Cry DKO mice elicited markedly enhanced and prolonged tyrosine phosphorylation of cellular proteins compared to WT mice, suggesting that a very active BCR signaling pathway may contribute to impaired B cell tolerance in the Cry DKO mice. In summary, our results suggest that B cell development, as well as the intrinsic checkpoints of immune tolerance, are under direct circadian control. 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.

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