Regulation of B cell receptor avidity to organize antigen recognition

Harvard Medical School, Boston MA

Investigators

Abstract

Project Summary / Abstract This application proposes to define how B cell receptor binding site (BCRbs) copy number at the plasma membrane surface is an actively regulated parameter that is developmentally tuned to optimize antigen recognition and support affinity-driven antibody selection with B cell germinal centers. At the molecular scale, increasing receptor binding site number can markedly increase affinity for ligand through the avidity effect, formally defined as a reduction in binding off-rate. To define an analogous principle at the whole B cell level, I have constructed a recombinant BCRbs counter, which for the first time measures the absolute number of antigen binding sites on B lymphocytes. This counter consists of 1:1:1 stoichiometric complex of: one molecule of a nanobody VHH (specific to the BCR light chain constant domain) + one fluorescent molecule linked through one molecule of monomeric streptavidin. Using this tool, I will test the central hypothesis that BCR avidity is developmentally regulated to promote antigen reception, and that this principle provides substrate for B cell affinity selection following immune challenge. Aim 1 builds from my discovery that BCRbs number is progressively increased during B lymphocyte development: first as IgM BCR on immature B cells emerging from the bone marrow, through to transitional development in the periphery, and then to the mature IgM+/IgD+ antigen receptive state that populates the lymphoid organs (e.g. lymph node and spleen). I will define how this developmental enhancement of BCRbs avidity is achieved molecularly and whether it is conserved across both mice and humans. Additionally, I will evaluate how BCRbs avidity can be differentially applied in mature B cells, wherein I find that avidity is maximized in marginal zone B cells, which respond rapidly to bloodborne antigen in the absence of affinity maturation. In Aim 2, I will evaluate BCRbs avidity as a parameter that organizes affinity- driven selection within B cell germinal centers (GCs). Application of the BCRbs counter to GCs induced within a mouse model of influenza virus infection reveals that antigen-specific B cells with higher BCRbs number are selected for over time. Avidity (artificially increasing affinity through reduction of binding off rate) has the potential ‘buffer’ antibody binding strength by enabling selection of BCRs with weak monomeric affinity, and ultimately contribute to the affinity ceiling long noted in antibody output. I will define this relationship by comparing copy number of the BCR selected in the GC vs the actual in solution affinity value of its monomeric antigen binding site. Collectively this proposal seeks to define BCR avidity as new parameter organizing B cell antigen recognition.