Vaccination elicits high affinity antibodies to pathogens and toxins to protect the individual. While T cells recognize antigen fragments with a fixed, low affinity, B cells initially generate low affinity antibodies and then undergo a process of focused hypermutation and selection to generate high affinity antibodies. The vital process of mutation and selection takes place in germinal centers (GCs), highly organized foci of antigen specific B cells within B follicles of the secondary lymphoid tissues. Experiments over the past 40 years have shown that GC contain two zones- the light zone in which B cells that have mutated their antibody genes by expressing activation-induced cytidine deaminase (AID) are selected by antigen to produce high affinity antibodies and a dark zone in which selected B cells proliferate. The selected and expanded B cells then differentiate into antibody producing plasma cells or memory B cells. The precise mechanism by which high affinity B cells are selected from a pool of B cells with diverse affinities is still unclear. Follicular dendritic cells (FDCs) found primarily in the light zone present intact antigens to B cells in the form of immune complexes. It has been proposed that B cells compete for antigen provided by FDCs and then present these antigens to follicular T cells to obtain survival and differentiation signals. The goal of the proposed work is to elucidate these mechanisms by investigating the cellular interactions of antigen specific B cells with T cells, FDCs and DCs during the GC reaction using a combination of molecular genetics and two-photon laser scanning intravital microscopy (TPLSIM), a method for submicron fluorescence imaging relatively deep in tissues (up to 400 mm). This will be a collaborative effort between the Nussenzweig and Dustin laboratories located at The Rockefeller University and NYU Medical School. Transgenic B- and T- cells expressing defined antigen receptor specificities, bone marrow-derived DCs and FDCs will be labeled with different fluorescent protein markers. In addition, antigen and immune complexes will be tagged with fluorescent dyes or semiconductor nanocrystals (Quantum dots(tm)). In the first Aim we will study the interactions of antigen specific B cells with antigen, Th cells and FDCs, and examine competition between low and high affinity B cells.
In Aim 2 we will use genetic tagging methods to study endogenous antibody responses and define the molecular mechanisms that produce light and dark zones and govern the entry and exit of follicular B cells into GCs. These studies will be directly applicable to optimizing vaccination strategies for high affinity antibody production. ? ? ?
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