Antigen-binding B cells and polyreactive antibodies

We generated a novel human neutralizing human mAb RabD4 against rabies virus glycoprotein using in vitro stimulation human peripheral B cells produced from immunized donor. It was revealed that the human mAb RabD4 demonstrated high antigen-binding activity and virus-neutralizing activity in the FAVN test with the CVS-11 rabies virus.

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Relationship between Fc receptors, antigen-binding sites on T and B cells, and H-2 complex-associated determinants.

Journal of Experimental Medicine ◽

10.1084/jem.141.3.547 ◽

1975 ◽

Vol 141 (3) ◽

pp. 547-560 ◽

Cited By ~ 60

Author(s):

A Basten ◽

J F Miller ◽

R Abraham

Keyword(s):

T Cells ◽

B Cells ◽

Specific Antigen ◽

Fc Receptors ◽

Fc Receptor ◽

Antigen Binding ◽

T And B Cells ◽

Spleen Cells ◽

Fab Fragment ◽

Thymus Cells

The relationship between H-2 complex-associated determinants, Fc receptors, and specific antigen-recognition sites on T and B cells was examined by binding and functional assays. The Fc receptor was detected by radiolabeled immune complexes or aggregated human IgG. Both these reagents selectively bound to B cells, not to T cells. When spleen cells, from mice primed to several antigens, were exposed to highly substituted radioactive aggregates, their capacity to transfer both a direct and indirect plaque-forming cell response to these antigens was abrogated. Addition of B cells, but not of T cells, restored responsiveness. Complexed Ig binding to Fc receptors was prevented by pretreatment of mixed lymphoid cell populations with antisera directed against membrane components on the same cell (e.g., H-2) and on other cells (e.g., theta). The lack of specificity of inhibition was thought to be due to the formation on cell surfaces of antigen-antibody complexes which would then attach to the Fc receptor during the incubation precedure. Specific blockade of the Fc receptor during the incubation procedure. Specific blockade of the Fc receptor however occurred when B cells were pretreated with the Fab fragments of anti-H-2 antibody. This was demonstrated autoradiographically and by inhibition of aggregate-induced suicide. The blocking activity of ante-H-2 Fab was removed by absorption with spleen cells from thymectomized irradiated mice but not with thymus cells of appropriate specificity. This suggested that the antibodies involved had specificity for determinants on the B-cell membrane distinct from those coded by the K or D end of the H-2 complex, and either absent from, or poorly represented on, thymus cells. Specific antigen-induced suicide of B cells was achieved simply by incubating the cells with radioactive antigen in the cold. T-cell suicide on the other hand required that the 125I-labeled antigen be presented to the T cells at 37 degrees-C on the surface of spleen cells from antigen-primed mice. Pretreatment of T cells with the Fab fragment of anti-H-2 antibody protected them from the suicide effect. By contrast no such protection of B cells could be achieved by this procedure. In other words H-2 (? Ir)-associated determinants may not only be in close proximity to the antigen-binding site on T cells but, in addition, may be involved in the effective operation of the receptor.

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Molecular Analysis of Immunoglobulin Variable Genes in HIV-Related Non-Hodgkin Lymphoma: Implications for Disease Pathogenesis and Histogenesis.

Blood ◽

10.1182/blood.v106.11.330.330 ◽

2005 ◽

Vol 106 (11) ◽

pp. 330-330

Author(s):

Daniela Capello ◽

Eva Berra ◽

Michaela Cerri ◽

Annunziata Gloghini ◽

Davide Rossi ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Molecular Analysis ◽

Light Chain ◽

Mutation Frequency ◽

Aggressive Lymphoma ◽

Antigen Binding ◽

High Affinity ◽

Aggressive Lymphomas ◽

Selection For

Abstract Molecular analysis of immunoglobulin variable region (IGV) genes can provide insights into the histogenesis and clonal history of B-cell NHL. We investigated 67 HIV-related NHL (HIV-NHL), including 30 HIV-diffuse large B cell lymphomas (DLBCL), 21 HIV-Burkitt lymphoma (BL), 6 HIV-primary effusion lymphomas (PEL) and 10 HIV-plasmablastic lymphomas (PBL) for usage, mutation frequency and intratumoral heterogeneity of clonal IGV rearrangements as well as mutation profile and CDR3 structure of IGHV, IGKV and IGLV genes. Results were compared to 200 IGV rearrangements from aggressive lymphomas of immunocompetent hosts and to the normal B-cell repertoire. We identified a total of 65 IGHV and 56 IGV light chain rearrangements in HIV-NHL. A functional IGHV rearrangement was found in 60/67 (90%) cases, a functional IGKV chain rearrangement in 17/38 (44.7%) cases and a functional IGLV rearrangement in 21/38 cases (55.3%). Fifty-three of 60 HIV-NHL (88.3%) showed somatic hypermutation in IGHV and/or IGV light chain genes. The average mutation frequency was 9.42% (median 7.50%, range 2.04%–23.3%) for IGHV genes and 5.42% (median 4.20%, range 2.01%–12.5%) for IGV light chain genes. IGV germline rearrangements selectively associated with HIV-PBL (p<0.001). Among mutated cases, average mutation frequencies did not differ among HIV-NHL groups. HIV-NHL showed a significant overrepresentation of the IGHV4 family (28/60; 46.6%) and a significant underrepresentation of IGHV3 family (18/60, 30.0%) compared to aggressive lymphomas of immunocompetent hosts (p<0.05) and to normal B-cells (p<0.05). IGHV4–34 was the IGHV gene most frequently rearranged (17/60; 28.3%) and was overrepresented in HIV-NHL versus aggressive lymphoma of immunocompetent hosts (17%; p<0.03) and normal B-cells (4%; p<0.001). IGHV4-34 expressing cells preferentially associated with lambda chain rearrangements (70%). The IGKV4-1 gene was the IGKV segment most frequently rearranged (6/17; 35.3%) and its usage was biased in HIV-NHL compared to normal B-cells (5.30; p<0.001). The single IGLV gene most frequently encountered was IGLV1-44 (6/17; 35,3%). Distribution of replacement and silent mutations in IGHV sequences showed tendency to conserve FR sequences and maintain antigen binding in 34/52 (65.4%) cases. A higher than expected number of CDR replacement mutations, suggesting selection for high affinity antigen binding, occurred in 17/52 (32.7%) cases. Analysis of intraclonal heterogeneity showed the presence of ongoing mutations in only 1 HIV-BL and 2 HIV-DLBCL. Implications of these data are multifold. First, most HIV-NHL derive from B-cells persistently subjected to GC reaction, suggesting a potential role for antigen stimulation in the pathogenesis of these lymphomas. This hypothesis is supported by the finding of antigen binding preservation in the majority of HIV-NHL and selection for high affinity antigen binding in a fraction of cases. Second, the preferential usage of IGHV4-34 and IGKV4-1 genes in a fraction of HIV-NHL may suggest a role for stimulation of pre-neoplastic B-cells with polyreactive and/or autoreactive antigens. Finally, at variance with NHL of immucompetent hosts, the presence of intraclonal heterogeneity is a rare finding in HIV-NHL, suggesting a derivation from B-cells that have concluded the GC-reaction.

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Highly Restricted Usage of Immunoglobulin Light Chain IGKV3-20 with Stereotyped Sequence in Primary Cold-Agglutinin Disease (CAD)

Blood ◽

10.1182/blood.v126.23.4137.4137 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4137-4137 ◽

Cited By ~ 3

Author(s):

Agnieszka Malecka ◽

Gunhild Trøen ◽

Anne Tierens ◽

Ingunn Østlie ◽

Ulla Randen ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Heavy Chain ◽

Light Chain ◽

Cold Agglutinin ◽

Antigen Binding ◽

Gene Rearrangements ◽

Cold Agglutinin Disease ◽

Immunoglobulin Light Chain ◽

Thermal Amplitude

Abstract Primary cold agglutinin disease (CAD) is a type of hemolytic anemia mediated by anti-I autoantibodies. Patients suffer from anemia as well as circulatory problems. However, the severity of disease differs greatly between patients. We recently demonstrated that primary CAD is caused by an underlying low grade B cell lymphoproliferative disease of the bone marrow with a typical histology that is different from lymphoplasmacytic lymphoma and, accordingly, does not display the MYD88 L265P mutation (Randen et al., Haematologica, 2013). The majority of patients display circulating monoclonal antibodies encoded by the immunoglobulin heavy chain gene IGHV4-34. The disease severity does not correlate with antibody titers, but seems to be determined by the thermal amplitude, i.e., the highest temperature at which the cold agglutinin binds to the antigen. The framework region 1 of IGHV4-34 encodes for a sequence that binds to I antigen. However, this does not explain the molecular basis of disease heterogeneity. We studied 27 patients with well-characterized primary CAD and sequenced immunoglobulin heavy as well as immunoglobulin light chains to find additional consensus regions that may determine anti-I reactivity. Bone marrow aspirates, or frozen bone marrow trephine biopsies and blood from 27 patients with well-documented primary CAD were collected. Monoclonal B cells were isolated by flow sorting (FACS Aria Ilu High speed sorter, Becton Dickinson). Viable cells were detected using the forward scatter versus side scatter dot plot. Subsequently, CD45 bright events with low side scatter features representing lymphocytes, were selected. Then, CD5 positive and CD19 negative events, i.e. T cells, were gated out using a CD5 versus CD19 dot plot leaving only B cells. Finally, monoclonal B cells were selected using the immunoglobulin light chain gate, either k or l. Clonally rearranged IGH genes were detected using the Somatic Hypermutation Assay v2.0 (Invivoscribe) and were then sequenced. Immunoglobulin light chain genes (IGL) were amplified by an in-house diagnostic protocol based on Biomed-2 primers (van Dongen et al., Leukemia, 2003). All sequences were analyzed using the IMGT database (www.imgt.org). Productive IGHV4-34 gene rearrangements were identified in 22/27 patients. In 4 patients, no productive rearrangement was identified, while in one patient a productive IGHV3-23 was seen. No significant homology of complementarity determining region 3 (CDR3) regions was found between IGHV sequences. The N-glycosylation sequence within the CDR2 region, affecting antigen-binding, was mutated in 8 patients whereas no mutations were present in 7 patients and mutations in flanking residues were seen in 6 patients. The latter mutations may modulate glycosylation efficacy. Clonal rearrangement of the IGKV3-20 was detected in 16/27 patients, clonal IGKV3-15 gene rearrangements were identified in 4/27 patients whereas other IGL genes were rearranged in 4/27 patients. No clonal IGL gene rearrangement was found in 3/27 patients. Of interest, 7 of the patients with IGKV3-20 rearrangement displayed highly homologous CDR3 regions. The latter was highly associated with an un-mutated N-glycosylation sequence of the respective IGHV4-34 sequence. In conclusion, our data show that in addition to IGHV, also IGLV usage is highly restricted in CAD. Furthermore, stereotyped IGLV sequences are seen that are mutually exclusive with mutated N-glycosylation sequences in the IGHV CDR2 sequence. These data indicate that multiple regions within the immunoglobulin heavy chain as well as immunoglobulin light chain contribute to I-antigen binding. The data suggest that subtle differences in these multiple binding sequences may contribute to the differences in thermal amplitude of I antigen binding of the antibody. The highly restricted usage of IGKV3-20 provides a rationale for vaccination with IGKV3-20 proteins, known to be immunogenic and being considered for treatment in other lymphoproliferative diseases (Martorelli et al., Clin Cancer Res, 2012). Disclosures No relevant conflicts of interest to declare.

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Impaired early B cell tolerance in patients with rheumatoid arthritis

Journal of Experimental Medicine ◽

10.1084/jem.20042321 ◽

2005 ◽

Vol 201 (10) ◽

pp. 1659-1667 ◽

Cited By ~ 208

Author(s):

Jonathan Samuels ◽

Yen-Shing Ng ◽

Claire Coupillaud ◽

Daniel Paget ◽

Eric Meffre

Keyword(s):

Rheumatoid Arthritis ◽

B Cells ◽

B Cell ◽

Cell Tolerance ◽

B Cell Tolerance ◽

Autoantibody Production ◽

Autoreactive B Cells ◽

Healthy Donors ◽

Polyreactive Antibodies

Autoantibody production is a characteristic of most autoimmune diseases including rheumatoid arthritis (RA). The role of these autoantibodies in the pathogenesis of RA remains elusive, but they appear in the serum many years before the onset of clinical disease suggesting an early break in B cell tolerance. The stage of B cell development at which B cell tolerance is broken in RA remains unknown. We previously established in healthy donors that most polyreactive developing B cells are silenced in the bone marrow, and additional autoreactive B cells are removed in the periphery. B cell tolerance in untreated active RA patients was analyzed by testing the specificity of recombinant antibodies cloned from single B cells. We find that autoreactive B cells fail to be removed in all six RA patients and represent 35–52% of the mature naive B cell compartment compared with 20% in healthy donors. In some patients, RA B cells express an increased proportion of polyreactive antibodies that can recognize immunoglobulins and cyclic citrullinated peptides, suggesting early defects in central B cell tolerance. Thus, RA patients exhibit defective B cell tolerance checkpoints that may favor the development of autoimmunity.

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Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1517612113 ◽

2016 ◽

Vol 113 (5) ◽

pp. E558-E567 ◽

Cited By ~ 19

Author(s):

Jing Wang ◽

Shan Tang ◽

Zhengpeng Wan ◽

Yiren Gao ◽

Yiyun Cao ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Single Molecule ◽

Cell Activation ◽

Cell Receptor ◽

B Cell Receptor ◽

Antigen Binding ◽

B Cell Activation ◽

Class I Mhc ◽

Antigen System

Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.

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Light chain editing generates polyreactive antibodies in chronic graft-versus-host reaction

Journal of Experimental Medicine ◽

10.1084/jem.20060075 ◽

2006 ◽

Vol 203 (7) ◽

pp. 1761-1772 ◽

Cited By ~ 33

Author(s):

Esther J. Witsch ◽

Hong Cao ◽

Hidehiro Fukuyama ◽

Martin Weigert

Keyword(s):

T Cells ◽

B Cells ◽

Transgenic Mice ◽

Marginal Zone ◽

Basic Protein ◽

Autoimmune Response ◽

Graft Versus Host Reaction ◽

Host Reaction ◽

Graft Versus Host ◽

Polyreactive Antibodies

The chronic graft-versus-host (cGvH) reaction is a model of induced lupus caused by alloreactive CD4+ T cells from a Bm-12 mouse in a C57BL/6 recipient. We used this cGvH reaction in C57BL/6 anti-DNA H chain transgenic mice, 56R/B6, to understand the structure, specificity, and origin of the induced autoantibodies (auto-Abs). We found anti-DNA Abs that reacted to several different antigens, such as phosphatidylserine, myelin basic protein, thyroglobulin, histone, insulin, cytochrome C, and β-galactosidase. This polyreactivity was found for Abs from B cells that expressed the 56R H chain transgene with “editor” L chains that did not completely veto autoreactivity. We suggest that such incomplete editing results in polyreactivity and that incompletely edited polyreactive B cells influence the subsequent expression of pathogenic auto-Abs in disease. We also found B cells that coexpress κ and λ L chain. These B cells contributed to the autoimmune response and are possibly in the marginal zone of the spleen.

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Acquisition of Mannoses on the Surface Immunoglobulin Binding Site Reveals Functional Status and Cell of Origin in Diffuse Large B Cell Lymphomas

Blood ◽

10.1182/blood-2018-99-118318 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 677-677

Author(s):

Giorgia Chiodin ◽

Philip Rock ◽

Enrica Antonia Martino ◽

Beatriz Valle Argos ◽

Graham Packham ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Binding Site ◽

Tumor Cells ◽

Antigen Binding ◽

Cell Of Origin ◽

B Cell Lymphomas ◽

Antigen Binding Site ◽

Surface Immunoglobulin ◽

Large B Cell

Abstract Acquisition of mannosylated glycans in the surface immunoglobulin (sIg) variable region (sIgV) antigen-binding site is a unique tumor-specific structural change of certain lymphomas, including all follicular lymphomas (FL) and ~40% diffuse large B-cell lymphomas (DLBCL). Mannosylation of the sIgV allows binding to environmental lectins including DC-SIGN (Coelho V et al, PNAS 2010). SIgV engagement is generally required for survival of DLBCL cells (Young RM et al, PNAS 2015), but how sIgV mannosylation distributes and affects behavior in the two germinal center B-cell-like (GCB-like) or activated B-cell-like (ABC-like) DLBCL subsets is unknown. While the mannosylation of the sIgV is tumor specific and irreversible, there are other natural N-glycosylation sites in the sIg constant region (sIgC). In secreted IgM these are mainly fully glycosylated and that is seen in sIgM of normal B cells (Krysov S et al, Blood 2010). However, engagement of sIgM by anti-IgM leads to expression ofan immature (mannosylated) form in both tumor and normal B cells. This conversion is dynamic, and tumor B cells restore expression of sIgC with mature glycans following BCR disengagement in vitro(Krysov S et al, Blood 2010). In this study, the glycosylation patterns of sIgV and sIgC were analyzed in GCB-like (n=6) vs ABC-like DLBCL lines (n=2) and primary samples (n=8) by IGHV-D-J sequencing, DC-SIGN binding and immunoblot of the biotinylated sIg following digestion by EndoH (specific for the mannosylated sugars) or by PNGase (removes all sugars). We found acquisition of N-mannosylation sequence motifs in the IGHV-D-J transcripts of all GCB-DLBCL lines with t(14;18), indicating a likely relationship with FL. In contrast, neither of the ABC-DLBCL lines had acquired sites, confirming a separate origin. DC-SIGN binding, which is specific for mannosylated IgV structures on the tumor cells, was observed in all GCB-DLBCL and not in the ABC-DLBCL, confirming that the acquired sites were glycosylated. These results allowed us to discriminate DLBCL cases into "DC-SIGN binders" (DB-DLBCL) vs "DC-SIGN non-binders" DLBCL (NB-DLBCL). Analysis of the carbohydrate structures on the sIgC revealed that the immature form was confined to the NB-DLBCL lines (2/2), while the DB-DLBCL expressed a mature fully glycosylated form (6/6). Consistent with the nature of ABC-DLBCL, these results revealed an activated BCR status of the NB-DLBCL. This was confirmed in the 8 primary samples (5/8 DB, 3/8 NB), which expressed an immature (activated) sIgC in 3/3 NB-DLBCL and a mature sIgC in 5/5 DB-DLBCL. However, engagement of anti-IgM F(ab')2 polyclonal antibody converted the inactive sIg form of DB-DLBCL into an activated sIg with relative increase of the immature sugars. It was evident that the mannosylated sites on the sIgC were not available for DC-SIGN binding, which is confined to the sIgV sites. We verified BCR activation status by investigating constitutive phosphorylation of SYK, BTK and PLCγ2, which are recruited to the membrane upon BCR activation, prior to endosome formation (Phelan JD et al, Nature 2018), in 2 DB-DLBCL lines (NU-DHL1 and SU-DHL6) and 2 NB-DLBCL (HBL-1 and TMD8). Basal phosphorylation of SYK, BTK and PLCγ2 was higher in the NB-DLBCL, consistent with the activated status associated with an immature sIgC. Our results reveal a functional dichotomy in DLBCL, which indicates: first, the cell of origin dictates whether sIgV carries mannoses in the antigen-binding site; second, reversible sIgC mannosylation associates with activation via sIg. Interestingly, this feature of activation is in ABC-DLBCL, which lacks IgV mannosylation. It is consistent with the suggestion that occupation of the antigen-binding sites with mannoses blocks further engagement of the receptor by 'antigen'. However, acquisition of mannoses in the sIgV sites appears to confer an ability to interact with environmental lectins such as DC-SIGN, whereas the sIgC sites fail to do this, suggesting an alternative function. Clearly, the post-translational modification targets several sites in sIg. Sites in the sIgC have a similar, possibly maturational, function in normal B cells, but in tumor cells the irreversible addition of mannoses to the sIgV adds a tumor-specific function. Disclosures Packham: Aquinox: Research Funding. Forconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy.

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