# VH-replacement shapes the antibody repertoire by removing the genes of non-functional heavy-chains

## TL;DR

This paper explains how the immune system removes non-functional antibody genes during early B-cell development, shaping the diversity of antibodies.

## Contribution

The study identifies a physiological role for VH-replacement in removing non-functional heavy-chain genes during B-cell development.

## Key findings

- VH-replacement recombination products follow the pattern of selection for functional and non-functional VDJ rearrangements.
- Certain VH/JH gene combinations are heavily counter-selected during antibody heavy-chain selection.
- VH-replacement helps release stalled B-cells by removing non-functional heavy-chain genes.

## Abstract

The diversity of antibodies underpins robust immune responses. During the formation of the antibody repertoire in early bone marrow B-cells, random antibody heavy-chain proteins are generated from recombined VH, DH, and JH gene segments. Many are non-functional and are negatively selected. To understand this process in normal mice, we have undertaken an in-depth analysis of heavy-chain selection at this pre-B cell transition. We find independent selection acting on three regions of the complementarity-determining region 3 (CDR3) antigen-binding site, with particularly heavy counter-selection against certain productive VH/JH combinations. This led us to hypothesise that VH-replacement, where the VH gene segment in an existing VDJ combination is replaced, targets productive VDJ rearrangements that code for non-functional heavy chains. We detect VH-replacement recombination products that closely follow the pattern of selection of functional and non-functional VDJ rearrangements. This reveals a physiological role for VH-replacement in the developmental release of B-cells that are stalled by non-functional heavy-chains. This leads to re-modelling of the restricted early VDJ repertoire toward the use of other VH gene segments throughout the IgH locus.

Antibody heavy-chains are selected for fitness during the transition from pro-B to pre-B cells in the bone marrow. Detailed analysis of VDJ recombination during this process characterizes how selection occurs both before and after pre-B cell proliferation.

Particular variable (VH)/joining (JH) gene combinations are strongly counter-selected.The common A and R residues at the 5’ end of complementarity-determining region 3 (CDR3) are further selected demonstrating their importance for CDR3 functionality.VH-replacement, where the VH gene segment in an existing VDJ gene rearrangement is replaced, is associated with removal of VDJ gene rearrangements coding for non-functional heavy-chains.

Particular variable (VH)/joining (JH) gene combinations are strongly counter-selected.

The common A and R residues at the 5’ end of complementarity-determining region 3 (CDR3) are further selected demonstrating their importance for CDR3 functionality.

VH-replacement, where the VH gene segment in an existing VDJ gene rearrangement is replaced, is associated with removal of VDJ gene rearrangements coding for non-functional heavy-chains.

During early B-cell development, VH-replacement is involved in the counter-selection of antibody genes coding for heavy-chains that cannot form a pre-B cell receptor.