High-affinity antibodies that are protective against infection evolve from lower-affinity precursors in the germinal center (GC). This evolution is thought to occur by selective expansion of higher-affinity B cell mutants while lower-affinity ones are eliminated by apoptosis. However, the degree to which affinity-based selection restricts clonal diversity in the antibody response is unknown, as are the precise kinetics and strength of clonal evolution in GCs. Thus, we do not know how (or indeed whether) the GC generates the ideal balance between antibody affinity and clonal diversity necessary for a protective response. This parameter is important to consider when devising vaccination strategies, especially those aimed at fostering the development of rare B cell clones with ?broadly-neutralizing? potential against HIV and influenza. This gap in knowledge is largely due to our technical inability to precisely measure clonal diversity and the strength of selection over time in individual GCs. We have recently developed two microscopy-based techniques that greatly improve our ability to measure the evolution of clonal diversity during the GC response in mice: (i) multicolor fate-mapping, which relies on stochastic recombination of a ?Brainbow? allele to quantify the extent of clonal selection in individual GCs by imaging; and (ii) in situ photoactivation, which allows us to isolate hundreds of B and T cells from individual GCs by flow cytometry. We propose to use these methods to further our understanding of the role of T cell help in controlling GC clonal diversity, to understand the difference in clonal dynamics between primary and recall GCs, and to investigate the how clonal dynamics change over time in chronic GCs induced by retroviral infection. Upon fulfillment of our specific aims, we expect to have increased our understanding of how clonal competition in GCs affects the diversity and immunodominance of the antibody response. We expect that our findings will inform future vaccine development, especially against highly diverse pathogens such as HIV and influenza.
The proposed research is relevant to public health because it addresses the question of why vaccination, which is effective against many infections, fails to induce broadly protective antibodies against HIV and influenza. We propose that understanding the competition between different antibody-producing cells may lead to answers to this question. Thus, the proposed research is relevant to the NIH's mission of developing basic knowledge that will help reduce the burden of human disease.
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