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A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells.

ABSTRACT: Pathologic rheumatoid factor (RF) levels are hallmarks of several human diseases. Production of monoclonal RF in vitro is essential for studies of the antigenic specificities of RF, as well as for a dissection of the mechanisms of aberrant RF+ B cell activation. We have expanded upon previous methods to develop a flow cytometry-based method to efficiently clone monoclonal antibodies (mAbs) from humans with expansions of RF-like, immunoglobulin heavy chain variable region (IgVH) 1-69 gene segment-containing B cells. The cloned variable regions are expressed as IgM and produced during culture at concentrations between 5 and 20 ?g/ml. Using this system, we show that clonal Igs from patients with HCV-related mixed cryoglobulinemia, when expressed as IgM, have RF activity. We anticipate that this system will be useful for the cloning and expression of mAbs partially encoded by VH1-69 and for determination of the reactivity patterns of polyspecific, low-affinity IgMs of human pathogenic importance.

SUBMITTER: Charles ED

PROVIDER: S-EPMC3003765 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells.

Charles Edgar D ED Orloff Michael I M MI Dustin Lynn B LB

Journal of immunological methods 20100924 2

Pathologic rheumatoid factor (RF) levels are hallmarks of several human diseases. Production of monoclonal RF in vitro is essential for studies of the antigenic specificities of RF, as well as for a dissection of the mechanisms of aberrant RF+ B cell activation. We have expanded upon previous methods to develop a flow cytometry-based method to efficiently clone monoclonal antibodies (mAbs) from humans with expansions of RF-like, immunoglobulin heavy chain variable region (IgVH) 1-69 gene segment ...[more]

PMID: 20875420

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Project description:Multiple antibody effector functions arise in HIV-1 infection that could be harnessed to protect against infection or clear the persistent reservoir. Here, we have investigated the genetic and functional memory B cell and antibody landscape present during early infection in six individuals infected with either subtype A, C, or an A/C recombinant HIV-1. These individuals demonstrated varying levels of plasma autologous neutralization (nAb) against the transmitted/founder envelope (T/F Env) pseudovirus and non-neutralizing Fc-mediated effector function (nnFc) antibody-dependent cell-mediated cytotoxicity (ADCC) against the T/F Env gp120 protein at ~7 months after infection. Genetic analysis of the immunoglobulin heavy (VH) and light (VL) chain variable domain gene segments from 352 autologous T/F Env gp120-specific single B cells recovered at this same 7-month time-point revealed an over-representation of the VH1-69 germline in five of six individuals. A defining feature of the VH1-69 utilizing gp120-specific antibodies was their significantly more hydrophobic complementarity-determining region-2 (CDRH2) regions compared to other VH CDRH2 sequences from each individual. While none of the VH1-69 antibodies possessed strong neutralizing activity against virions pseudotyped with the autologous T/F Env, almost a third were capable of mediating high ADCC activity, as assayed by intracellular granzyme B activity in CEM.NKr.CCR5 target cells coated with autologous T/F Env gp120. High ADCC mediating VH1-69 antibodies exhibited shorter complementarity-determining region-3 (CDRH3) lengths and a more neutral isoelectric point than antibodies lacking this function. In the individual that developed the highest autologous ADCC responses, the high granzyme B producing antibodies bound to surface expressed envelope in the absence of CD4 and were not enhanced by the addition of soluble CD4. Overall, VH1-69 utilizing antibodies are commonly induced against gp120 in diverse HIV-1 infections and a subset of these antibodies can mediate ADCC functions, serving as a bridge between the innate and adaptive immune response to HIV-1.

Dataset Information

A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells.

Publications

A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells.

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