De novo antibody identification in human blood from full-length single B cell transcriptomics and matching haplotype-resolved germline assemblies

Abstract

Immunoglobulin (IGH, IGK, IGL) loci in the human genome are highly polymorphic regions that encode the building blocks of the light and heavy chain IG proteins that dimerize to form antibodies. The processes of V(D)J recombination and somatic hypermutation in B cells are responsible for creating an enormous reservoir of highly specific antibodies capable of binding a vast array of possible antigens. However, the antibody repertoire is fundamentally limited by the set of variable (V), diversity (D), and joining (J) alleles present in the germline IG loci. To better understand how the germline IG haplotypes contribute to the expressed antibody repertoire, we combined genome sequencing of the germline IG loci with single-cell transcriptome sequencing of B cells from the same donor. Sequencing and assembly of the germline IG loci captured the IGH locus in a single fully phased contig where the maternal and paternal contributions to the germline V, D, and J repertoire can be fully resolved. The B cells were collected following a measles, mumps, and rubella (MMR) vaccination, resulting in a population of cells that were activated in response to this specific immune challenge. Single-cell, full-length transcriptome sequencing of these B cells results in whole transcriptome characterization of each cell, as well as highly accurate consensus sequences for the somatically rearranged and hypermutated light and heavy chain IG transcripts. A subset of antibodies synthesized based on their consensus heavy and light chain transcript sequences demonstrate binding to measles antigens and neutralization of authentic measles virus.

Figure 1.

Experimental design schematic. Peripheral blood mononuclear cells (PBMCs) collected from a donor at 6 days post-MMR vaccination were separated into B cells for single-cell sequencing and monocytes for germline sequencing and assembly.

Figure 2.

A donor-specific, haploid assembly was created using Flye and HapDup (Methods). Colored bars indicate the position on the contig of functional V, D, J, and C gene segments. (A) The fully phased IGH locus, with the dashed lines depicting alternative structures in sections where the assembly differs structurally between the two haplotypes, such as the IGHV3-9 and IGHV1-8 structural variant. The D gene cluster is expanded to show that a block of D gene segments is missing from each haplotype. (B) The IGL locus, where the IGL C V-CLUSTER was assembled into a separate contig from the remaining V-CLUSTERs and the J-C-CLUSTERs. All gene segments were shared between both haplotypes.

Figure 3.

Single-cell expression data from the MBC gate. (A) Uniform Manifold Approximation and Projection (UMAP) visualization of cells clustered by gene expression quantified by short-read sequencing. (B) UMAP visualization of cells clustered by isoform expression quantified by nanopore sequencing. (C) Volcano plot showing isoforms that are differentially expressed in one cluster relative to the entire population of cells, with Bonferroni-adjusted P-values. One differentially expressed isoform of the apoptosis regulating gene BCL2 is highlighted. (D) Cluster-specific expression of the BCL2 gene and the differential transcript usage of three BCL2 isoforms. BCL2–202 isoforms make up only 4% of BCL2 transcripts in cluster 3 but represent 32% of BCL2 transcripts in cluster 8.

Figure 4.

Single-cell expression data from ASC gate. (A) UMAP visualization of cells clustered by gene expression quantified by nanopore sequencing. (B) Expression of marker genes differentiating in each cluster. (C) Proportion of reads assigned to IGHG1 and IGHG3 within each cell from the ASC gate in both the short-read and nanopore sequencing data sets. (D) Schematic illustrating the genes involved in glycosylation of the conserved asparagine residue in the CH2 domain of the heavy chain. (E) Differential expression of two genes in the glycosylation pathway, B4GALT1 and FUT8, in certain antibody isotypes of plasmablasts and plasma cells from the ASC gate. Each point represents a single cell.

Figure 5.

Characterizing single-cell IG sequences and antibody synthesis. (A) The percentage of IGH reads in each cell possessing the M exons indicative of a membrane-bound antibody, stratified by cell type. (B) Proportions of each isotype identified from consensus IGH sequences, stratified by cell type. (C) CDR3 amino acid length distributions for all productive IGH consensus sequences, stratified by cell type. CDR3 lengths are enumerated between the junction anchor residues: Cys 104 and Try 118 (IMGT numbering) for each sequence. (D) Mutation frequency compared to the germline sequences in the FWR1–4 and CDR1–3 regions for each cell type and isotype. (E) Dandelion plot of sequences clustered within each nonsingleton clone by VDJ amino acid sequence similarity. (F) Putative positive recombinantly expressed antibodies sequenced from the ASC gate were screened by flow cytometry for binding to MeV surface expressed proteins (F or RBP/H) on virus infected Raji-DC-SIGN B cells (multiplicity of infection [MOI] 0.01). MMR-vaccinated, hyper-immune sera from the donor at the time of cell collection was used as a positive control. (G) Neutralization assay in Vero cells for MMR-82 (MeV+) and hyper-immune sera against a GFP-expressing MeV-Edmonston recombinant virus (1000 IU/96-well). Data are presented as percent infection (mean ± SD) compared to infection in media alone (set as 100%). Experiments were performed in triplicate. Anti-HNV monoclonal antibody (mAb) is an irrelevant IgG1 mAb against an unrelated virus and serves a negative control.

Figure 6.

Donor-specific V gene usage and mutation rates. (A) Overall IGHV gene usage frequency for all complete and productive heavy chain consensus sequences with an in-frame CDR3. Cells with a low V gene identity, high mutation rates (≥15%) are stratified (light blue), as they are sometimes filtered out by programs such as Hi-V-QUEST (IMGT). (B) Statistics for ambiguous IGH and IGL V gene calls when using IgBLAST with various databases. (C) V gene usage for cells expressing one of the heterozygous V alleles (left), or that expressed a homozygous V allele but were haplotype-discriminable by a heterozygous J allele (right). (D) Ratio of replacement to silent mutations (R/S) in framework regions (FWRs) versus CDRs. The dotted line represents theoretical R/S ratios for unselected CDRs at R/S = 2.9. The red lines are the mean R/S values. The elevated replacement mutations in the CDRs suggest cells that have undergone antigen selection.