Identifocation the B Cell Subsets Responsible for Anti-pneumococcal Response

Pneumococcal Diseases

Immunology, human, B-lymphocytes subsets, Antibody-Producing Cells, 23 valent pneumococcal capsular polysaccharide vaccine

The purpose of this study is to determine which B lymphocytes subsets are responsible for the production of IgM, IgG2 and IgA anti-pneumococcal capsular polysaccharides after vaccination with a 23-valent pneumococcal polysaccharide vaccine.

The aim of this study is to identify which specific B cell subset(s) is/are responsible for the production of protective IgM, IgG2 and IgA anti-pneumococcal capsular polysaccharides (capPS) in response to immunization of healthy individuals with the Pneumovax, a prototypical T-independent type 2 vaccine. In other words, from which B cells are IgM, G2 and A-secreting plasma cells derived? To address this question, it will be taken advantage of the unique Ig heavy chain VDJ signature expressed by each B cell clone and the strategy will rely on the search of clonal filiations between plasmablasts (PB)/plasma cells (PC) and different B cell subpopulations. Therefore, healthy individuals will be vaccinated with Pneumovax, and blood samples will be collected at day 0, 7, 14, 28 and 56. Starting from these blood samples, different B cells subsets (in particular IgM+IgD+CD27+ and switched memory IgM-IgD-CD27+ B cells) and the PB/PC peaking at day 7 after vaccination will be isolated by cell sorting. CapPS-secreting PB cannot be specifically isolated, but the investigators assume that they will represent the majority of the isolated PB/PC at the peak of the response. Thus, day 7-PB/PC will be sorted both in bulk or as single cells in 96-well PCR plates. RNA will be extracted from bulk sorted PB/PC, and VDJ-µ, -alpha and -gamma Ig transcripts will be amplified by RT-PCR and analyzed by the H-CDR spectratyping method in order to identify the sequence of dominant VDJ signatures that most probably correspond to anti-capPS-secreting cells. In parallel, for each PB/PC single cell, Ig heavy and corresponding Ig light chain gene transcripts will be amplified by nested RT-PCR, sequenced, and provided that the heavy chain contains a dominant VDJ signature, they will be cloned into eukaryotic expression vectors to produce monoclonal human Abs. The recombinant antibodies will then be tested for reactivity against capPS from the vaccine by ELISA. When VDJ signatures of capPS will be validated, with this powerful molecular tool, the investigators will look for the presence of these signatures through VDJ-specific PCR on cDNA prepared from the different B cell subsets.

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