Somatic hypermutation generates antibody specificities beyond the primary repertoire

B cell somatic hypermutation (SHM) and selection in germinal centers (GCs) enhance antibody affinity for antigen. Here, we investigated whether SHM-based antibody evolution is restricted to specificities established through V(D)J recombination in the primary repertoire. Tracking pre-defined non-specific B cells across multiple immunization models revealed that non-cognate B cells within GCs undergo SHM. Under conditions of limited B cell competition, these B cells generated de novo antigen recognition to multiple epitopes across diverse model antigens. Phylogenetic analyses identified diverse mutational pathways leading to new antigen affinities, and enhanced T cell co-stimulation further promoted new antigen recognition. Our data support a model in which B cell competition-rather than an intrinsic requirement for specific affinity-limits the emergence of new affinities through SHM, highlighting the mammalian adaptive immune system's ability to explore antibody-antigen interactions beyond those encoded by the V(D)J-dependent primary repertoire, demonstrating the flexibility of SHM in not only ripening but also reshaping specificity. Overall design: Different tissues (spleen, mesenteric lymph node and peyer's patches) of unimmunized mice were harvested, processed and cells were counted. 1-2 x 106 cells, whenever possible, were aliquoted and stained with 0.5 ug of cell hashing antibodies (BioLegend Total-Seq C antibodies, Hashtag 1-9, different hashtag antibodies per tissue sample) after blocking with TruStain FcX™ PLUS (anti-mouse CD16/32, BioLegend, Cat# 156604) according to manufacturer's protocol. After incubating with hashing antibodies, cells were washed and stained with FACS staining panel as mentioned above - DAPI, Dump (CD4, CD8, F4/80 and Gr1), B220. Total B cells were sorted (upto 50,000 cells per tissue and cells from all three tissues of one mouse were pooled) and proceeded for scRNA-seq library preparation using Chromium GEM-X Single Cell 5' Reagent Kits v3 (10X Genomics) according to the manufacturer's protocol. Briefly, sorted B cells were pooled, counted, and 33,000 to 50,000 cells were loaded on the GEM-X Chip to run on Chromium X for gel-beads in emulsion (GEMs) generation. Thereafter RT-PCR, cDNA amplification, fragmentation, end repair, A-tailing and sample indexing were performed. Libraries were quantitated and analysed at Tape station (Agilent Bioanalyzer) during intermediate steps of library preparation. Final libraries were quantified by qPCR (KAPA Library Quantification Kit Illumina® Platforms, Kapa Biosystems # KR0405), diluted to 2 nM and pooled. 650 pM of pooled libraries were mixed with 15% of PhiX control library and loaded on NextSeq™ 2000 P4 XLEAP-SBS™ Reagent Kit (100 Cycles, Illumina # 20100994. Libraries were sequenced on Illumina NextSeq™ 2000 sequencer with paired-end reads for 28 cycles of read 1, 10 cycles of i7 index, 10 cycles of i5 index, and 90 cycles of read 2, targeting a median depth of 10,000 reads per cell (5,000 reads per cell for BCR libraries and 5,000 reads per cell for hashtag libraries).