High-throughput profiling of antigen-specific antibody secreting cells using an antibody secretion trap (TRAPnSeq)

Following activation by cognate antigen, B cells undergo fine-tuning of their antigen receptors and may ultimately differentiate into antibody-secreting cells (ASCs). While antigen-specific antibodies from B cell receptor (BCR) expressing B cells can be readily cloned and sequenced following flow sorting, antigen-specific plasma cells that lack surface BCR cannot be easily profiled in a high-throughput way. Here, we report an approach, TRAPnSeq (antigen specificity mapping through Ig secretionTRAPandSequencing), that allows capture of secreted antibodies on the surface of ASCs, which in turn enables high-throughput screening of single ASCs against large antigen panels and recovery of paired VH:VL antibody sequences. This approach incorporates flow cytometry, standard microfluidic platforms and DNA barcoding technologies to isolate and characterize antigen-specific ASCs through single cell V(D)J, RNA and antigen barcode sequencing. We show the utility of TRAPnSeq by profiling antigen-specific IgG and IgE plasma cells from mouse and humans and validate antigen binding by ELISA. TRAPnSeq can easily be combined with existing B cell platforms to accelerate antibody discovery from ASCs and can further be expanded to any protein secreting-cells. The TRAPnSeq protocol was performed on bone marrow from hIL-4Rα immunized mice to identify and sort IgG+/hIL-4Rα binding plasma cells. The spleen and draining lymph nodes were also processed from these mice, stained using conventional B cell surface markers, and IgG+/hIL-4Rα-binding B cells were sorted. All sorted cells were processed using the 10x genomics platform. >>> Submitter statement concerning raw data: The VELOCIMMUNE V3 mouse strain is a patented and proprietary genome. We have been advised by our legal counsel not to release raw sequencing (fastq) files. However, we are depositing the processed single cell count tables to enable reproducibility of our analysis. <<<<