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Principal Investigator: Victoria Celine Rosado
Organization: HARVARD MEDICAL SCHOOL
Fiscal Year: 2024
Award: $43,456
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <19S Gamma Globulin><7S Gamma Globulin><Ab response><Affinity><Antibodies><Antibody Affinity><Antibody Formation><Antibody Production><Antibody Response><Antigens><Antiseptics><Avidity><B blood cells><B cell><B cell receptor><B cells><B-Cell Antigen Receptor><B-Cell Development><B-Cell Receptor Binding><B-Cell Subsets><B-Cells><B-Lymphocyte Subsets><B-Lymphocytes><B-cell><Binding><Binding Sites><Biotinylation><Blood><Blood Reticuloendothelial System><Bone Marrow><Bone Marrow Reticuloendothelial System><Buffers><Cell Body><Cell Surface Antigens><Cell membrane><Cell surface><Cells><Combining Site><Complex><Cytoplasmic Membrane><Data><Development><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Frequencies><Gene Transcription><Genetic Transcription><Germinal Center><Grippe><Homo sapiens><Human><Humoral Immunities><IgD><IgG><IgM><Immune><Immune Globulins><Immune response><Immunes><Immunoglobulin D><Immunoglobulin G><Immunoglobulin M><Immunoglobulins><Immunologic Surface Markers><Immunological Surface Markers><Immunological response><Individual><Infection><Influenza><Influenza HA><Influenza Hemagglutinin><Label><Ligands><Light><Link><Local Anti-Infective Agents><Lung><Lung Respiratory System><Lymph Node Reticuloendothelial System><Lymph node proper><Lymphatic nodes><Mature B-Cell><Mature B-Lymphocyte><Measures><Mediastinal Lymph Node><Mediastinal lymph node group><Membrane><Messenger RNA><Mice><Mice Mammals><Modeling><Modern Man><Molecular><Molecular Interaction><Murine><Mus><Output><Photoradiation><Plasma Membrane><RNA Expression><RNA Splicing><Reaction><Reactive Site><Reagent><Recombinants><Regulation><Site><Spleen><Spleen Reticuloendothelial System><Splicing><Strepavidin><Streptavidin><Structure><Structure of germinal center of lymph node><Surface><Surface Antigens><Survey Instrument><Surveys><T-Cells><T-Lymphocyte><Testing><Time><Topical Anti-Infective Agents><Transcription><Variant><Variation><Viral Antigens><Work><analyzing longitudinal><antibody biosynthesis><antibody-based immunity><antigen antibody affinity><antigen binding><antigen bound><developmental><flow cytophotometry><flu HA><flu hemagglutinin><flu infection><flu virus infection><host response><immune system response><immunogen><immunoglobulin biosynthesis><immunoresponse><infected with flu><infected with flu virus><infected with influenza><infected with influenza virus><influenza infection><influenza viral HA><influenza viral hemagglutinin><influenza virus HA><influenza virus hemagglutinin><influenza virus infection><innovate><innovation><innovative><longitudinal analysis><lymph gland><lymph nodes><lymph organ><lymphatic organ><lymphnodes><lymphoid organ><mRNA><membrane structure><molecular scale><monomer><mouse model><murine model><nanobodies><nanobody><pathogen><plasmalemma><pulmonary><receptor binding><receptor bound><recruit><sdAb><secondary lymph organ><secondary lymphatic organ><secondary lymphoid organ><single domain antibodies><single molecule><thymus derived lymphocyte><tool><virtual><virus antigen>