generation-discriminative-human-monoclonal-antibodies-from-rare

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Université de Nantes

LabEx IGO, "Immunotherapy, Graft, Oncology"

The impact of MHC phenotype on the shaping of the peripheral naive T cell repertoire in humans remains unknown. To address this, we compared the frequency and antigenic avidity of naive T cells specific for immunodominant self-, viral, and tumor Ags presented by a human MHC class I allele (HLA-A*02, referred to as A2) in individuals expressing or not this allele. Naive T cell frequencies varied from one Ag specificity to another but were restrained for a given specificity. Although A2-restricted T cells showed similar repertoire features and antigenic avidities in A2+ and A2- donors, A2 expression had either a positive, neutral, or negative impact on the frequency of A2-restricted naive CD8 T cells, depending on their fine specificity. We also identified in all donors CD4 T cells specific for A2/peptide complexes, whose frequencies were not affected by MHC class I expression, but nevertheless correlated with those of their naive CD8 T cell counterparts. Therefore, both selection by self-MHC and inherent TCR reactivity regulate the frequency of human naive T cell precursors. Moreover this study also suggests that T cell repertoire shaping by a given self-MHC allele is dispensable for generation of immunodominant T cell responses restricted by this particular allele.

Impact of TCR reactivity and HLA phenotype on naive CD8 T cell frequency in humans.

In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27(-)IgD(+) (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells.

Characterization of antigen-specific B cells using nominal antigen-coated flow-beads.

There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by reconstruction of human mAbs by RT-PCR and expression cloning.

Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies.

Xavier Saulquin

CRCINA,
Inserm,
CNRS,
Université d'Angers,
CRCINA, Inserm, CNRS, Université d'Angers

Université de Nantes

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Xavier Saulquin

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Université de Nantes

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

CRCINA,
Inserm,
CNRS,
Université d'Angers,
CRCINA, Inserm, CNRS, Université d'Angers