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.

scholarly journals Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus.

1990 ◽  
Vol 171 (1) ◽  
pp. 19-34 ◽  
Author(s):  
Y Ueki ◽  
I S Goldfarb ◽  
N Harindranath ◽  
M Gore ◽  
H Koprowski ◽  
...  
Keyword(s):  
B Cells ◽  
Antibody Response ◽  
B Cell ◽  
Rabies Virus ◽  
Gene Segment ◽  
Human Antibody ◽  
Cell Repertoire ◽  
High Affinity ◽  
Clonal Level ◽  
Vh Gene

We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0-2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human antibody response to foreign Ags and for generating human mAbs of predetermined specificity and high affinity.

scholarly journals Self-reactive VH4-34–expressing IgG B cells recognize commensal bacteria

2017 ◽  
Vol 214 (7) ◽  
pp. 1991-2003 ◽  
Author(s):  
Jean-Nicolas Schickel ◽  
Salomé Glauzy ◽  
Yen-Shing Ng ◽  
Nicolas Chamberlain ◽  
Christopher Massad ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Healthy Donor ◽  
Somatic Hypermutation ◽  
Gene Segment ◽  
Commensal Bacteria ◽  
Healthy Individuals ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Variable Heavy

The germline immunoglobulin (Ig) variable heavy chain 4–34 (VH4-34) gene segment encodes in humans intrinsically self-reactive antibodies that recognize I/i carbohydrates expressed by erythrocytes with a specific motif in their framework region 1 (FWR1). VH4-34–expressing clones are common in the naive B cell repertoire but are rarely found in IgG memory B cells from healthy individuals. In contrast, CD27+IgG+ B cells from patients genetically deficient for IRAK4 or MYD88, which mediate the function of Toll-like receptors (TLRs) except TLR3, contained VH4-34–expressing clones and showed decreased somatic hypermutation frequencies. In addition, VH4-34–encoded IgGs from IRAK4- and MYD88-deficient patients often displayed an unmutated FWR1 motif, revealing that these antibodies still recognize I/i antigens, whereas their healthy donor counterparts harbored FWR1 mutations abolishing self-reactivity. However, this paradoxical self-reactivity correlated with these VH4-34–encoded IgG clones binding commensal bacteria antigens. Hence, B cells expressing germline-encoded self-reactive VH4-34 antibodies may represent an innate-like B cell population specialized in the containment of commensal bacteria when gut barriers are breached.

scholarly journals Comparison of the fetal and adult functional B cell repertoires by analysis of VH gene family expression.

1988 ◽  
Vol 168 (2) ◽  
pp. 589-603 ◽  
Author(s):  
H D Jeong ◽  
J M Teale
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
B Cells ◽  
Gene Family ◽  
B Cell ◽  
Cell Lineage ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Vh Gene

The functional B cell repertoire in BALB/c mice was assessed at various stages in ontogeny. This was done by analyzing VH gene family expression using the sensitive technique of in situ hybridization. The B cell repertoire was probed with the mitogen, LPS, and the antigen DNP. DNP was chosen because B cells responsive to this hapten appear very early in ontogeny. The APCs that developed after stimulation with LPS or DNP were analyzed for VH gene expression by in situ hybridization of individual cells using radiolabeled VH gene family probes. The results indicated that VH gene expression in fetal B cells after stimulation was distinct from adult B cells in that there was a biased expression of D proximal families. The results indicated that this bias was associated with developmental age and not a given differentiation stage in the B cell lineage. In addition, stimulation of fetal B cells with DNP resulted in a large increase in expression of member(s) of VH 36-60, suggesting that the early appearance of DNP-responsive B cells is not strictly correlated with preferential rearrangement of D proximal families, VH 7183 and VH Q52. However, the results suggested that a large proportion of pre-B cells that preferentially rearrange D proximal families early in ontogeny become part of the functional developing repertoire.

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

1993 ◽  
Vol 177 (4) ◽  
pp. 1227-1227 ◽  
Author(s):  
A K Stewart ◽  
C Huang ◽  
B D Stollar ◽  
R S Schwartz
Keyword(s):  
B Cell ◽  
High Frequency ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Frequency Representation ◽  
Vh Gene ◽  
Human B Cell

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.

Analysis of VH gene expression in CD5+ and CD5- B-cell chronic lymphocytic leukemia

Blood ◽  
1995 ◽  
Vol 86 (10) ◽  
pp. 3883-3890 ◽  
Author(s):  
K Maloum ◽  
F Davi ◽  
C Magnac ◽  
O Pritsch ◽  
E McIntyre ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Random Distribution ◽  
Lymphocytic Leukemia ◽  
Mutation Pattern ◽  
Vh Gene ◽  
Vh Genes ◽  
Multiple Myelomas ◽  
Differentiation Stage ◽  
Follicular Lymphomas

In contrast to highly mutated follicular lymphomas and multiple myelomas, chronic lymphocytic leukemias (CLLs) frequently express VH genes in germline configuration. It is currently unclear whether this difference is related to the expression of CD5 or to the differentiation stage of the B cell when malignant transformation occurs. We have studied the VH sequence of 11 cases of CD5- B-CLL to address the question whether CD5- B-CLL are derived from naive pregerminal B cells (low mutation pattern) or from germinal center- derived memory B cells (high mutation pattern). Among the 12 detected rearrangements (2 distinct rearrangements in 1 case) VH1 family was found in 2, VH2 in 2, VH3 in 4, and VH4 in 4. Nine different VH genes were detected among the 12 rearrangements, including 2 cases expressing V1–69 (51p1) and 1 case expressing V4–39 (VH4.18), previously reported to be overexpressed in CD5+ B-CLL. A higher mutation pattern, following a random distribution, was observed when compared with classical CD5+ B- CLL. However, as reported in normal B cells, these results appeared to be related to membrane Ig phenotype (less mutations in membrane mu delta-expressing forms in leukemias expressing exclusively membrane mu). Overall, the differences found when comparing the mutational profile with classical CD5+ B-CLL were not clearcut and might be explained more by the membrane isotype (mu v mu delta) than by CD5 expression.

IgG-Secreting B-Cell Lymphoplasmocytoid Leukaemia : Molecular Characterization of Igh Rearrangements.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1778-1778
Author(s):  
Laurence Lode ◽  
Surinder S. Sahota ◽  
Soraya Wuilleme ◽  
Steven Richebourg ◽  
Marion Eveillard ◽  
...  
Keyword(s):  
B Cell ◽  
Elevated Serum ◽  
Chromosomal Translocation ◽  
Memory Cell ◽  
Fish Analysis ◽  
Cell Repertoire ◽  
Igh Locus ◽  
B Cell Repertoire ◽  
Vh Genes

Abstract Purpose : Elevated serum M component of IgG isotype is typically associated with multiple myeloma (MM). However, our group has previously reported cases with an elevated serum monoclonal IgG and a leukaemic B-cell lymphoplasmocytoid lymphoma (LPL) similar to Waldenstrom’s macroglobulinaemia (WM). The aim of this study was to extend analysis of IgH locus events in a larger series of IgG-secreting LPL. Patients and Methods : We investigated 20 patients with an elevated serum monoclonal IgG (>20g/l) and LPL (IgG-LPL). Morphological classification and immunophenotyping analysis were performed at diagnosis (serum IgG>4 g/l, CD19+ cells >30%, presence of lymphoplasmocytoid cells in blood and/or bone marrow). Histological classification and FISH analysis were performed when possible to further characterize those cases. Analysis of VH genes was carried out from RNA with VHLeader and CH primers. 14 patients were examined for both IgG and IgM transcripts; VH-Cμ and VH-Cg transcripts could be compared in 9 patients. Results : Of 25 Ig VH rearrangement sequences, 23 were functional and expressed in each case. VH3 family members appeared to be over-represented (19/21 patients (90.5%) as compared to 40/71 (56.3%) in normal B-cell repertoire (1). VH3-23 was the most frequently used segment (10/21 patients) and is frequently utilized in normal B-cells. IgG-LPL JH family use resembled the normal B-cell repertoire (predominance of JH4 and JH6 segments). The median CDR3 length was 10 amino acids [5–19]. However, and in contrast with features seen in other leukaemia, there was no evidence of homologous CDR3 motifs. All VH genes revealed highly somatically mutated sequences, with a median mutation rate 8.8% [0.7 – 11.1%] (IMGT database(2)). We compared pre (VH-Cμ) and post-switch (VH-Cg) transcripts, and 4/9 patients had identical clonally-derived sequences, and two 2/9 had divergent sequences. Interpretation and conclusion : This intended study of IgG-LPL reveals consistent features that argue for common origins of IgG-LPL with Waldenstrom’s macroglobulinemia. One feature is extensive somatic mutations in VH genes, suggesting origins from a cell that may have undergone successive rounds of mutation. Patterns of mutations in pre-and post-switched clonally derived sequences suggest that the final neoplastic event has occurred in a IgM+ memory cell undergoing isotype switch. However, no aberrant chromosomal translocation accrue at the IgH locus, as apparent from FISH data. This indicates that, unlike in typical MM, switch activity does not generate 14q32 abnormalities nor that such lesions play a role in the pathogenesis of LPL. Extensive mutations are also seen in VH genes in WM, and there is evidence that WM cells can undergo class switch events in vivo. In this tumor, switching occurs at a low subclonal level in some cells, which in rare cases can lead to the emergence of a dual population of clonally identical IgM and IgG expressing WM tumor cells at a later stage of disease (7). In typical WM, 14q32 abnormalities are also generally not seen. These data suggest that IgG-LPL and WM could be two variants of the same entity, with IgG-LPL exposed to persistent switch stimuli following transformation.

scholarly journals Automated analysis of high-throughput B-cell sequencing data reveals a high frequency of novel immunoglobulin V gene segment alleles

2015 ◽  
Vol 112 (8) ◽  
pp. E862-E870 ◽  
Author(s):  
Daniel Gadala-Maria ◽  
Gur Yaari ◽  
Mohamed Uduman ◽  
Steven H. Kleinstein
Keyword(s):  
B Cell ◽  
Large Scale ◽  
Gene Segment ◽  
Automated Analysis ◽  
Sequencing Data ◽  
Cell Repertoire ◽  
Accurate Analysis ◽  
V Genes ◽  
Repertoire Sequencing ◽  
Novel Alleles

Individual variation in germline and expressed B-cell immunoglobulin (Ig) repertoires has been associated with aging, disease susceptibility, and differential response to infection and vaccination. Repertoire properties can now be studied at large-scale through next-generation sequencing of rearranged Ig genes. Accurate analysis of these repertoire-sequencing (Rep-Seq) data requires identifying the germline variable (V), diversity (D), and joining (J) gene segments used by each Ig sequence. Current V(D)J assignment methods work by aligning sequences to a database of known germline V(D)J segment alleles. However, existing databases are likely to be incomplete and novel polymorphisms are hard to differentiate from the frequent occurrence of somatic hypermutations in Ig sequences. Here we develop a Tool for Ig Genotype Elucidation via Rep-Seq (TIgGER). TIgGER analyzes mutation patterns in Rep-Seq data to identify novel V segment alleles, and also constructs a personalized germline database containing the specific set of alleles carried by a subject. This information is then used to improve the initial V segment assignments from existing tools, like IMGT/HighV-QUEST. The application of TIgGER to Rep-Seq data from seven subjects identified 11 novel V segment alleles, including at least one in every subject examined. These novel alleles constituted 13% of the total number of unique alleles in these subjects, and impacted 3% of V(D)J segment assignments. These results reinforce the highly polymorphic nature of human Ig V genes, and suggest that many novel alleles remain to be discovered. The integration of TIgGER into Rep-Seq processing pipelines will increase the accuracy of V segment assignments, thus improving B-cell repertoire analyses.

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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