Humans who have recovered from infectious disease possess a memory B cell pool that contains highly-potent antibodies against the cleared pathogenic agent. This pool is a valuable source of therapeutic antibodies but identifying these requires a method that can screen a large number of cells rapidly to identify promising candidates. We used Barcode Enabled Antigen Mapping (BEAM) to screen 100 million peripheral blood mononuclear cells (PBMCs) from a donor who had recovered from COVID-19. This method labels antigens of interest with unique reporter oligonucleotides and uses these labeled antigens to stain lymphocytes according to the binding specificity of their antigen receptors. Individual antigen-bound cells are then captured and analysed using the 10x Genomics Single Cell Immune Profiling Solution. This allows us to identify the antigen-specific cells while also generating transcriptomic profiles and natively paired, heavy and light chain full-length B-cell receptor sequences from each cell. We used BEAM to identify B cells that bound to the spike protein of SARS-CoV-2 and discovered 222 antibodies with high binding affinity confirmed by SPR. The majority of these antibodies bound to multiple variants of concern with some also recognising endemic coronaviruses. We then investigated the potential for these antibodies to neutralize live SARS-CoV-2 and confirmed that 55 exhibited potent neutralization activity. Finally, we performed epitope binning and discovered that the collection of antibodies bound to multiple different epitopes on the spike protein. Compared with hybridoma or other approaches, single cell methods have the potential to discover therapeutic antibodies more rapidly and with a higher diversity of leads.

Barcoding strategies are fundamental to droplet-based single-cell sequencing, and understanding the biases and caveats between approaches is essential. Here, we comprehensively evaluated both short and long reads of the cDNA obtained through the two marketed approaches from 10x Genomics, the “3’ assay” and the “5’ assay”, which attach barcodes at different ends of the mRNA molecule. Although the barcode detection, cell-type identification, and gene expression profile are similar in both assays, the 5’ assay captured more exonic molecules and fewer intronic molecules compared to the 3’ assay. We found that 13.7% of genes sequenced have longer average read lengths and are more complete (spanning both polyA-site and TSS) in the long reads from the 5’ assay compared to the 3’ assay. These genes are characterized by long average transcript length, high intron number, and low expression overall. Despite these differences, cell-type-specific isoform profiles observed from the two assays remain highly correlated. This study provides a benchmark for choosing the single-cell assay for the intended research question, and insights regarding platform-specific biases to be mindful of when analyzing data, particularly across samples and technologies.

A more complete understanding of immune responses to disease requires consideration of multiple different cell types across both the innate and adaptive branches of the immune system along with the detection of many different analytes. We used Barcode Enabled Antigen Mapping (BEAM) and Immune Profiling technology to perform simultaneous multimodal profiling at single cell resolution in hundreds of thousands of peripheral blood mononuclear cells (PBMCs) from a human donor following recovery from COVID-19. In addition to measuring gene and protein expression, we generated full-length, paired sequences of the rearranged T- and B-cell receptors while also screening their specificity for a wide range of antigens from SARS-CoV-2 and other viral pathogens. These data provide insights into the entirety of the immune landscape after recovery from acute viral disease. The scale and throughput of our experiments gave us high-resolution data from all cell types from the innate and adaptive immune systems. We identified antigen-specific clones of both B and T lymphocytes, with the high cellular throughput enabling detection of rare clones. Analysis of all PBMC cell types allowed us to place these antigen-specific clones within the overall transcriptional landscape of the post-viral immune system. Experiments such as these will underpin new systems immunology approaches and will continue to reveal the complex interplay between the components of the immune system. We envisage that these methods will be valuable in the analysis of the immune response to vaccination, infectious disease, cancer, allergy, autoimmune conditions, and ageing that can potentially lead to the development of novel diagnostic and therapeutic approaches.

Half a billion years of evolutionary battle forged the vertebrate adaptive immune system, an astonishingly versatile factory for molecules that can adapt to arbitrary attacks. The history of an individual encounter is chronicled within a clonotype: the descendants of a single fully rearranged adaptive immune cell. For B cells, reading this immune history for an individual remains a fundamental challenge of modern immunology. Identification of such clonotypes is a magnificently challenging problem for three reasons: It is thus impossible to determine clonotypes exactly. All solutions to this problem make a trade-off between sensitivity and specificity; useful solutions must address actual artifacts found in real data. We present enclone [1][1] , a system for computing approximate clonotypes from single cell data, and demonstrate its use and value with the 10x Genomics Immune Profiling Solution. To test it, we generate data for 1.6 million individual B cells, from four humans, including deliberately enriched memory cells, to tax the algorithm and provide a resource for the community. We analytically determine the specificity of enclone ’s clonotyping algorithm, showing that on this dataset the probability of co-clonotyping two unrelated B cells is around 10−9. We prove that using only heavy chains increases the error rate by two orders of magnitude. enclone comprises a comprehensive toolkit for the analysis and display of immune receptor data. It is ultra-fast, easy to install, has public source code, comes with public data, and is documented at [bit.ly/enclone][2]. It has three “flavors” of use: (1) as a command-line tool run from a terminal window, that yields visual output; (2) as a command-line tool that yields parseable output that can be fed to other programs; and (3) as a graphical version (GUI). ### Competing Interest Statement The authors were employees and shareholders of 10x Genomics, Inc at the time of publication. D.B.J. and W.J.M. are inventors on multiple patent applications assigned to 10x Genomics, Inc. related to algorithms and visualization schemas described and displayed in this manuscript. W.J.M., P.S., B.A.A., and D.B.J. are inventors on multiple patent applications assigned to 10x Genomics, Inc. related to technologies for the study of the adaptive immune repertoire. [1]: #fn-3 [2]: http://bit.ly/enclone

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens 1 . In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1–CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens 2 – 22 . Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes 23 , 24 . We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Continued advances in single cell gene detection sensitivity and the ability to analyze data quickly with biological context are critical for discoveries in therapeutic research. The second version of the Chromium Single Cell Immune Profiling Solution by 10x Genomics enables highly sensitive detection of gene expression, full-length paired T-cell alpha- and beta-chain and immunoglobulin sequences, T-cell antigen specificity, and cell surface protein expression from the same single cells, allowing a comprehensive view of the immune response at the cellular level. The new workflow provides a 45% increase in the number of genes detected per cell and up to a 25% increase in the cells detected with paired full-length V(D)J receptor sequences in melanoma tumor derived cells. Using a new version of Cell Ranger (v5.0), clonotypes were grouped from >30,000 cells, increasing the power to detect small clonotype expansions with fewer total cells. In addition, the new software enabled comparison of pre- and post-influenza vaccination B-cell receptor sequences from a single donor, identifying post-vaccination specific clonotypes. These technological and informatics advancements enhance a researcher’s ability to perform a broad characterization of immune cell populations at unprecedented throughput and resolution.