Large-scale antibody immune response mapping of splenic B cells and bone marrow plasma cells in a transgenic mouse model

Abstract

Molecular characterization of antibody immunity and human antibody discovery is mainly carried out using peripheral memory B cells, and occasionally plasmablasts, that express B cell receptors (BCRs) on their cell surface. Despite the importance of plasma cells (PCs) as the dominant source of circulating antibodies in serum, PCs are rarely utilized because they do not express surface BCRs and cannot be analyzed using antigen-based fluorescence-activated cell sorting. Here, we studied the antibodies encoded by the entire mature B cell populations, including PCs, and compared the antibody repertoires of bone marrow and spleen compartments elicited by immunization in a human immunoglobulin transgenic mouse strain. To circumvent prior technical limitations for analysis of plasma cells, we applied single-cell antibody heavy and light chain gene capture from the entire mature B cell repertoires followed by yeast display functional analysis using a cytokine as a model immunogen. We performed affinity-based sorting of antibody yeast display libraries and large-scale next-generation sequencing analyses to follow antibody lineage performance, with experimental validation of 76 monoclonal antibodies against the cytokine antigen that identified three antibodies with exquisite double-digit picomolar binding affinity. We observed that spleen B cell populations generated higher affinity antibodies compared to bone marrow PCs and that antigen-specific splenic B cells had higher average levels of somatic hypermutation. A degree of clonal overlap was also observed between bone marrow and spleen antibody repertoires, indicating common origins of certain clones across lymphoid compartments. These data demonstrate a new capacity to functionally analyze antigen-specific B cell populations of different lymphoid organs, including PCs, for high-affinity antibody discovery and detailed fundamental studies of antibody immunity.

The rise of big data: deep sequencing-driven computational methods are transforming the landscape of synthetic antibody design

“…Because clonal abundances tend to correlate with affinity, the incorporation of clonal frequency rankings offers an indirect strategy for predicting Ab affinities. For instance, this was successfully demonstrated by Pan et al where the authors isolated bone marrow from immunized mice followed by Ab repertoire deep sequencing analysis [62]. Post computational screenings, they were able to effectively identify the distinct molecular features from antigen-specific clonal lineages.…”

“…Moreover, they also implemented calculations associated with enrichment rankings of the various Ab frequencies to effectively identify lead candidate Abs. Notably, when experimentally validated, all top-ranking identified clones proved to be selective for the target antigen and displayed high affinities [62]. Thus, the work presented by Pan et al successfully demonstrated that the application of deep sequencing-based clonal rankings can effectively formulate affinity predictions; this is accomplished by performing frequency cross-comparisons among the various clones.…”

“…Gallo Journal of Biomedical Science (2024) 31 :29 In this context, it can offer a statistical overview of the unique clonal repertoires. This capability is pertinent for conducting clonal frequency assessments to ascertain the efficacy of in vitro selections, including the presence of immuno-dominant high-affinity Abs [59][60][61][62]. Furthermore, this real-time feedback offers opportunity for instant responses regarding modifications in the Ab library design [67]; in turn, this can help avoid potentially cost and time-consuming molecular evaluations.…”

Antibodies, repertoires and microdevices in antibody discovery and characterization

“…Various organs, such as the spleen, lymph nodes, bone marrow, and blood, can be accessed, each displaying differences in antibody repertoire present in ASCs and MCs. [34][35][36] The quantity and quality of the induced repertoire in immunized animals highly depend on factors such as the chosen immunization protein (antigen), its quality, dose, administration, mouse strain, genetic model, harvest time, and immunization scheme, necessitating optimization for efficient and optimal screening 37,38 (unpublished data), but the frequency of antigen specificity in total cells is usually higher than in humans. Nevertheless, appropriately chosen conditions can lead to identifying high-affinity antibodies, as the affinity distribution tends to follow a normal distribution with outliers in the highaffinity range.…”