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T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells 1 – 3 . Here we developed a clinical-grade approach based on CRISPR–Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen–HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated. A first-in-human phase I clinical trial demonstrates the feasibility and safety of non-viral precision genome-engineering of a personalized adoptive cell transfer anticancer therapeutic.

B cell receptor (BCR) signalling is crucial for normal B cell development and adaptive immunity. BCR signalling also supports the survival and growth of malignant B cells in patients with B cell leukaemias or lymphomas. The mechanism of BCR pathway activation in these diseases includes continuous BCR stimulation by microbial antigens or autoantigens present in the tissue microenvironment, activating mutations within the BCR complex or downstream signalling components and ligand-independent tonic BCR signalling. The most established agents targeting BCR signalling are Bruton tyrosine kinase (BTK) inhibitors and PI3K isoform-specific inhibitors, and their introduction into the clinic is rapidly changing how B cell malignancies are treated. B cells and BCR-related kinases, such as BTK, also play a role in the microenvironment of solid tumours, such as squamous cell carcinoma and pancreatic cancer, and therefore targeting B cells or BCR-related kinases may have anticancer activity beyond B cell malignancies.

An adeno-associated virus (AAV) receptor protein essential for AAV2 entry into cells is identified; AAV receptor binds directly to the virus, and its ablation renders a diverse range of mammalian cell types and mice resistant to infection by AAV of multiple serotypes. A receptor for adeno-associated virus infection The recent revival of interest in gene therapy has been fueled by the availability of safer and more effective viral gene delivery methods, most notably adeno-associated virus (AAV) vectors. Jan Carette and colleagues now identify a protein that is essential for AAV entry into cells, subsequent to cell attachment. This protein, which they call AAVR, rapidly traffics from the plasma membrane to the trans -Golgi network. The authors show that the virus directly binds to AAVR and that genetic ablation of AAVR renders a diverse range of mammalian cell types and mice resistant to AAV infection. Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity 1 . They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2 ), and have been approved for treatment of lipoprotein lipase deficiency in Europe 3 . Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration 1 , 4 , yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans -Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr −/− (also known as Au040320 −/− and Kiaa0319l −/− ) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

Introducing chimeric antigen receptors into the endogenous T-cell receptor locus reduces tonic signalling, averts accelerated T-cell differentiation and delays T-cell exhaustion, leading to enhanced function and anti-tumour efficacy compared to random integrations. Making a CAR drive tumour rejection Using T cells transduced with synthetic chimeric antigen receptors (CARs) is a promising strategy for treating certain types of cancer. Here Michel Sadelain and colleagues provide evidence in a mouse tumour model that knocking the CAR into the endogenous T-cell receptor α constant locus reduces tonic signalling, avoids accelerated T-cell differentiation, and delays T-cell exhaustion. This results in enhanced function and anti-tumour efficacy compared with random integrations. Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumour rejection 1 . The most successful CARs used to date are those targeting CD19 (ref. 2 ), which offer the prospect of complete remission in patients with chemorefractory or relapsed B-cell malignancies 3 . CARs are typically transduced into the T cells of a patient using γ-retroviral 4 vectors or other randomly integrating vectors 5 , which may result in clonal expansion, oncogenic transformation, variegated transgene expression and transcriptional silencing 6 , 7 , 8 . Recent advances in genome editing enable efficient sequence-specific interventions in human cells 9 , 10 , including targeted gene delivery to the CCR5 and AAVS1 loci 11 , 12 . Here we show that directing a CD19-specific CAR to the T-cell receptor α constant ( TRAC ) locus not only results in uniform CAR expression in human peripheral blood T cells, but also enhances T-cell potency, with edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukaemia. We further demonstrate that targeting the CAR to the TRAC locus averts tonic CAR signalling and establishes effective internalization and re-expression of the CAR following single or repeated exposure to antigen, delaying effector T-cell differentiation and exhaustion. These findings uncover facets of CAR immunobiology and underscore the potential of CRISPR/Cas9 genome editing to advance immunotherapies.

Kawasaki disease (KD) is the most common cause of acquired heart disease in children in developed countries. Although functional and phenotypic changes of immune cells have been reported, a global understanding of immune responses underlying acute KD is unclear. Here, using single-cell RNA sequencing, we profile peripheral blood mononuclear cells from seven patients with acute KD before and after intravenous immunoglobulin therapy and from three age-matched healthy controls. The most differentially expressed genes are identified in monocytes, with high expression of pro-inflammatory mediators, immunoglobulin receptors and low expression of MHC class II genes in acute KD. Single-cell RNA sequencing and flow cytometry analyses, of cells from an additional 16 KD patients, show that although the percentage of total B cells is substantially decreased after therapy, the percentage of plasma cells among the B cells is significantly increased. The percentage of CD8 + T cells is decreased in acute KD, notably effector memory CD8 + T cells compared with healthy controls. Oligoclonal expansions of both B cell receptors and T cell receptors are observed after therapy. We identify biological processes potentially underlying the changes of each cell type. The single-cell landscape of both innate and adaptive immune responses provides insights into pathogenesis and therapy of KD. Immune cell changes are associated with Kawasaki disease (KD) pathogenesis. Here, using single cell RNA sequencing of PBMC, the authors show monocyte inflammatory genes are over-expressed in KD and TCR and BCR clonotype sequences show oligoclonal expansions after intravenous immunoglobulin therapy.

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer. Single cell RNA sequencing generates short reads from one end of a template, providing incomplete transcript coverage and limiting identification of diverse sequences such as antigen receptors. Here the authors combine long read nanopore sequencing with short read profiling of barcoded libraries to generate full-length antigen receptor sequences.

The authors characterize epitope-specific T cell repertoires, identify shared and recognizable features of TCRs, and develop tools to classify antigen specificity on the basis of sequence analysis. Defining T cell receptor repertoires In this study, Paul Thomas and colleagues use molecular genetic tools to analyse the diversity of epitope-specific T cell repertoires to characterize features that enable the prediction of T cell epitope specificity immunity based on sequence analysis. This manuscript is of broad interest to various fields ranging from basic immunology to applied immunotherapeutics and translational medicine. Elsewhere in this issue, Mark Davis and colleagues address the question of how T cell receptor sequences relate to antigen specificity and create an algorithm that predicts the human leukocyte antigen (HLA) restriction of the T cell receptor targets and helps to identify specific peptide major histocompatibility complex ligands. T cells are defined by a heterodimeric surface receptor, the T cell receptor (TCR), that mediates recognition of pathogen-associated epitopes through interactions with peptide and major histocompatibility complexes (pMHCs). TCRs are generated by genomic rearrangement of the germline TCR locus, a process termed V(D)J recombination, that has the potential to generate marked diversity of TCRs (estimated to range from 10 15 (ref. 1 ) to as high as 10 61 (ref. 2 ) possible receptors). Despite this potential diversity, TCRs from T cells that recognize the same pMHC epitope often share conserved sequence features, suggesting that it may be possible to predictively model epitope specificity. Here we report the in-depth characterization of ten epitope-specific TCR repertoires of CD8 + T cells from mice and humans, representing over 4,600 in-frame single-cell-derived TCRαβ sequence pairs from 110 subjects. We developed analytical tools to characterize these epitope-specific repertoires: a distance measure on the space of TCRs that permits clustering and visualization, a robust repertoire diversity metric that accommodates the low number of paired public receptors observed when compared to single-chain analyses, and a distance-based classifier that can assign previously unobserved TCRs to characterized repertoires with robust sensitivity and specificity. Our analyses demonstrate that each epitope-specific repertoire contains a clustered group of receptors that share core sequence similarities, together with a dispersed set of diverse ‘outlier’ sequences. By identifying shared motifs in core sequences, we were able to highlight key conserved residues driving essential elements of TCR recognition. These analyses provide insights into the generalizable, underlying features of epitope-specific repertoires and adaptive immune recognition.

Unlike conventional αβ T cells, γδ T cells typically recognize non-peptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This ‘alternate pathway’ of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties. B-1a B cells are innate-like cells with biased reactivity to bacteria and self-antigens. Here the authors show that reduced interleukin-7 in developing fetal liver-derived pro-B cells induces premature immunoglobulin κ rearrangement, alleviating the requirement for a pre-BCR selection stage and allowing the generation of autoreactive B1-a B cells.

The complete germline repertoires of the channel catfish, Ictalurus punctatus , T cell receptor (TR) loci, TRAD, TRB, and TRG were obtained by analyzing genomic data from PacBio sequencing. The catfish TRB locus spans 214 kb, and contains 112 TRBV genes, a single TRBD gene, 31 TRBJ genes and two TRBC genes. In contrast, the TRAD locus is very large, at 1,285 kb. It consists of four TRDD genes, one TRDJ gene followed by the exons for TRDC, 125 TRAJ genes and the exons encoding the TRAC. Downstream of the TRAC, are 140 TRADV genes, and all of them are in the opposite transcriptional orientation. The catfish TRGC locus spans 151 kb and consists of four diverse V-J-C cassettes. Altogether, this locus contains 15 TRGV genes and 10 TRGJ genes. To place our data into context, we also analyzed the zebrafish TR germline gene repertoires. Overall, our findings demonstrated that catfish possesses a more restricted repertoire compared to the zebrafish. For example, the 140 TRADV genes in catfish form eight subgroups based on members sharing 75% nucleotide identity. However, the 149 TRAD genes in zebrafish form 53 subgroups. This difference in subgroup numbers between catfish and zebrafish is best explained by expansions of catfish TRADV subgroups, which likely occurred through multiple, relatively recent gene duplications. Similarly, 112 catfish TRBV genes form 30 subgroups, while the 51 zebrafish TRBV genes are placed into 36 subgroups. Notably, several catfish and zebrafish TRB subgroups share ancestor nodes. In addition, the complete catfish TR gene annotation was used to compile a TR gene segment database, which was applied in clonotype analysis of an available gynogenetic channel catfish transcriptome. Combined, the TR annotation and clonotype analysis suggested that the expressed TRA, TRB, and TRD repertoires were generated by different mechanisms. The diversity of the TRB repertoire depends on the number of TRBV subgroups and TRBJ genes, while TRA diversity relies on the many different TRAJ genes, which appear to be only minimally trimmed. In contrast, TRD diversity relies on nucleotide additions and the utilization of up to four TRDD segments.