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Anti-viral T-cell responses are usually directed against a limited set of antigens, but often contain many T cells expressing different T-cell receptors (TCRs). Identical TCRs found within virus-specific T-cell populations in different individuals are known as public TCRs, but also TCRs highly-similar to these public TCRs, with only minor variations in amino acids on specific positions in the Complementary Determining Regions (CDRs), are frequently found. However, the degree of freedom at these positions was not clear. In this study, we used the HLA-A*02:01-restricted EBV-LMP2(FLY)-specific public TCR as model and modified the highly-variable position 5 of the CDR3 beta sequence with all 20 amino acids. Our results demonstrate that amino acids at this particular position in the CDR3 beta region of this TCR are completely inter-changeable, without loss of TCR function. We show that the inability to find certain variants in individuals is explained by their lower recombination probability rather than by steric hindrance.

A common genetic variant within the T cell receptor alpha (TCRA)-T cell receptor delta (TCRD) locus (rs2204985) has been recently found to associate with thymic function. Aim of this study was to investigate the potential impact of donor rs2204985 genotype on patient’s outcome after unrelated hematopoietic stem cell transplantation (uHSCT). 2016 adult patients were retrospectively analyzed. rs2204985 genotyping was performed by next generation sequencing, p < 0.05 was considered significant and donor rs2204985 GG/AG genotypes were set as reference vs. the AA genotype. Multivariate analysis of the combined cohort regarding the impact of donor’s rs2204985 genotype indicated different risk estimates in 10/10 and 9/10 HLA matched transplantations. A subanalysis on account of HLA incompatibility revealed that donor AA genotype in single HLA mismatched cases (n = 624) associated with significantly inferior overall- (HR: 1.48, p = 0.003) and disease-free survival (HR: 1.50, p = 0.001). This effect was driven by a combined higher risk of relapse incidence (HR: 1.40, p = 0.026) and non-relapse mortality (HR: 1.38, p = 0.042). This is the first study to explore the role of rs2204985 in a clinical uHSCT setting. Our data suggest that donor rs2204985 AA genotype in combination with single HLA mismatches may adversely impact post-HSCT outcome and should thus be avoided., publishedVersion

The ability of individual T and B cells to display Ag receptors of unique uniform specificity is the molecular basis of adaptive immunity. Most αβ T cells achieve uniform specificity by assembling in-frame genes on only one allelic copy of TCRβ and TCRα loci, while others prevent incorporation of TCRα protein from both alleles into TCRs. Analysis of mice expressing TCR proteins from a restricted combination of transgenes showed that TCR protein pairing restrictions achieve uniform specificity of cells expressing two types of TCRβ protein. However, whether this mechanism operates in the physiological context where each dual-TCRβ cell expresses one set of a vast number of different TCRβ proteins remains an open question, largely because there is a low, but significant, portion of cells carrying two in-frame TCRβ genes. To resolve this issue, we inactivated one allelic copy of the TCRα locus in a new mouse strain that assembles two in-frame TCRβ genes in an elevated fraction of cells. This genetic manipulation has no effect on the frequency of cells that display multiple types of αβ TCR, yet increases the representation of cells displaying TCRβ proteins that generate more highly expressed TCRs. Our data demonstrate that some TCRβ proteins exhibit differential functional pairing with TCRα proteins, but these restrictions have negligible contribution for ensuring uniform specificity of cells that express two types of TCRβ protein. Therefore, we conclude that mechanisms governing monogenic assembly and expression of TCRβ genes in individual cells are paramount for uniform specificity of αβ T lymphocytes.

In mammals, the T lymphocyte receptor (TCR) is a multiprotein complex formed by the proteins TCRα, TCRβ, CD3ε, CD3γ, CD3δ, and CD3ζ. It is responsible for recognizing antigens processed and presented by antigen-presenting cells (APC). The TCR is located at the cytoplasmic membrane of the T lymphocyte but is functional assembled in the rough endoplasmic reticulum (RER). Most of the available information on TCR constituents in salmonids comes from numerous nucleotide sequences available in different databases. In this work, by in silico homology modeling, we generated the TCRαβ/CD3 complex of rainbow trout (Oncorhynchus mykiss) and characterized the structure of the different proteins and their potential interactions. The results show that the main structural features described in mammalian TCR/CD3 are present in the model predicted for trout. Furthermore, we highlighted several aminoacidic interactions between TCRα, TCRβ, CD3γδ, and CD3ε. In silico structural analyses suggest that trout TCRαβ complex would fit similarly to that described for mammals. Herein, we explore the implications of the modeled trout complex and the leukocyte phenotypes, mainly associated with different regulation mechanisms of trout TCRαβ/CD3 subunits gene expression or may be due to differences in the assembly process of the complex in the RER. However, further studies will be needed to study deeper the mechanisms involved.

TCRαβ+ CD8α+ CD8β− intraepithelial lymphocytes (CD8αα IEL) differentiate from thymic IEL precursors (IELp) and contribute to gut homeostasis. However, their development remains poorly understood. Here we show that the zinc finger transcription factor LRF, encoded by Zbtb7a, is highly expressed in both CD8αα+ IELs and in IELp and serves in a cell-intrinsic manner to promote IEL differentiation. Mice genetically deficient for LRF (Cd4-Cre Zbtb7afl/fl) lack CD8αα+ IELs, contrasting with largely conserved numbers of conventional CD8αβ+ T cells in the thymus and the secondary lymphoid organs. Unlike their wild-type counterparts, LRF-deficient IELp failed to prevent colitis caused by adoptive transfer of CD4+ T cells in T cell-deficient mice. LRF disruption affects neither the development of thymic IELp, nor their in vitro expansion or differentiation in response to IL-15. However, Cd4-Cre Zbtb7afl/fl mice accumulate CD8αα+ T cells in the spleen, unlike wild-type controls, suggesting impaired migration of IELp to the gut. Single-cell RNA sequencing found LRF necessary for the expression of genes characteristic of the most mature IELp, including Itgb7, encoding the β7 subunit of α4β7. Chromatin immunoprecipitation and gene regulatory network analyses both defined Itgb7 as an LRF target. Our study identifies LRF as an essential transcriptional regulator of IELp maturation in the thymus and subsequent migration to the intestinal epithelium.

Enhancers activate transcription through long-distance interactions with their cognate promoters within a particular subtopologically associated domain (sub-TAD). The TCRα enhancer (Eα) is located at the sub-TAD boundary between the TCRα and DAD1 genes and regulates transcription toward both sides in an ∼1-Mb region. Analysis of Eα activity in transcribing the unrearranged TCRα gene at the 5′-sub-TAD has defined Eα as inactive in CD4−CD8− thymocytes, active in CD4+CD8+ thymocytes, and strongly downregulated in CD4+ and CD8+ thymocytes and αβ T lymphocytes. Despite its strongly reduced activity, Eα is still required for high TCRα transcription and expression of TCRαβ in mouse and human T lymphocytes, requiring collaboration with distant sequences for such functions. Because VαJα rearrangements in T lymphocytes do not induce novel long-range interactions between Eα and other genomic regions that remain in cis after recombination, strong Eα connectivity with the 3′-sub-TAD might prevent reduced transcription of the rearranged TCRα gene. Our analyses of transcriptional enhancer dependence during T cell development and non-T lineage tissues at the 3′-sub-TAD revealed that Eα can activate the transcription of specific genes, even when it is inactive to transcribe the TCRα gene at the 5′-sub-TAD. Hence distinct requirements for Eα function are necessary at specific genes at both sub-TADs, implying that enhancers do not merely function as chromatin loop anchors that nucleate the formation of factor condensates to increase gene transcription initiated at their cognate promoters. The observed different regulated Eα activity for activating specific genes at its flanking sub-TADs may be a general feature for enhancers located at sub-TAD boundaries.

Unrelated donor (URD) hematopoietic stem cell transplant (HSCT) is associated with an increased risk of severe graft-versus-host disease (GVHD). TCRαβ/CD19 depletion may reduce this risk, whereas maintaining graft-versus-leukemia. Outcome data with TCRαβ/CD19 depletion generally describe haploidentical donors, with relatively few URDs. We hypothesized that TCRαβ/CD19-depletion would attenuate the risks of GVHD and relapse for URD HSCT. Sixty pediatric and young adult (YA) patients with hematologic malignancies who lacked a matched-related donor were enrolled at 2 large pediatric transplantation centers between October 2014 and September 2019. All patients with acute leukemia had minimal residual disease testing, and DP typing was available for 77%. All patients received myeloablative total body irradiation– or busulfan-based conditioning with no posttransplant immune suppression. Engraftment occurred in 98%. Four-year overall survival was 69% (95% confidence interval [CI], 52%-81%), and leukemia-free survival was 64% (95% CI, 48%-76%), with no difference between lymphoid and myeloid malignancies (P = .6297 and P = .5441, respectively). One patient (1.7%) experienced primary graft failure. Relapse occurred in 11 patients (3-year cumulative incidence, 21%; 95% CI, 11-34), and 8 patients (cumulative incidence, 15%; 95% CI, 6.7-26) experienced nonrelapse mortality. Grade III to IV acute GVHD was seen in 8 patients (13%), and 14 patients (26%) developed chronic GVHD, of which 6 (11%) had extensive disease. Nonpermissive DP mismatch was associated with higher likelihood of acute GVHD (odds ratio, 16.50; 95% CI, 1.67-163.42; P = .0166) but not with the development of chronic GVHD. URD TCRαβ/CD19-depleted peripheral HSCT is a safe and effective approach to transplantation for children/YAs with leukemia. This trial was registered at www.clinicaltrials.gov as #NCT02323867.

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired H and L chains of Igs and VDJ and VJ chains of TCRs from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single-cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. We used custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single-cell immune repertoire profiling. Using these rhesus-specific assays, we sequenced Ig and TCR repertoires in >60,000 cells from cryopreserved rhesus PBMCs, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single-cell level.

Peripheral T-cell lymphoma (PTCL) is a type of non-Hodgkin lymphoma that progresses aggressively with poor survival rate. CAR T cell targeting T-cell receptor β-chain constant domains 1 (TRBC1) of malignant T cells has been developed recently by using JOVI.1 monoclonal antibody as a template. However, the mode of JOVI.1 binding is still unknown. This study aimed to investigate the molecular interaction between JOVI.1 antibody and TRBC1 by using computational methods and molecular docking. Therefore, the TRBC protein crystal structures (TRBC1 and TRBC2) as well as the sequences of JOVI.1 CDR were chosen as the starting materials. TRBC1 and TRBC2 epitopes were predicted, and molecular dynamic (MD) simulation was used to visualize the protein dynamic behavior. The structure of JOVI.1 antibody was also generated before the binding mode was predicted using molecular docking with an antibody mode. Epitope prediction suggested that the N3K4 region of TRBC1 may be a key to distinguish TRBC1 from TCBC2. MD simulation showed the major different surface conformation in this area between two TRBCs. The JOVI.1-TRBC1 structures with three binding modes demonstrated JOVI.1 interacted TRBC1 at N3K4 residues, with the predicted dissociation constant (Kd) ranging from 1.5 × 108 to 1.1 × 1010 M. The analysis demonstrated JOVI.1 needed D1 residues of TRBC1 for the interaction formation to N3K4 in all binding modes. In conclusion, we proposed the three binding modes of the JOVI.1 antibody to TRBC1 with the new key residue (D1) necessary for N3K4 interaction. This data was useful for JOVI.1 redesign to improve the PTCL-targeting CAR T cell.

MHC-related protein 1 (MR1) presents microbial riboflavin metabolites to mucosal-associated invariant T (MAIT) cells for surveillance of microbial presence. MAIT cells express a semi-invariant T-cell receptor (TCR), which recognizes MR1-antigen complexes in a pattern-recognition-like manner. Recently, diverse populations of MR1-restricted T cells have been described that exhibit broad recognition of tumor cells and appear to recognize MR1 in association with tumor-derived self-antigens, though the identity of these antigens remains unclear. Here, we have used TCR gene transfer and engineered MR1-expressing antigen-presenting cells to probe the MR1 restriction and antigen reactivity of a range of MR1-restricted TCRs, including model tumor-reactive TCRs. We confirm MR1 reactivity by these TCRs, show differential dependence on lysine at position 43 of MR1 (K43) and demonstrate competitive inhibition by the MR1 ligand 6-formylpterin. TCR-expressing reporter lines, however, failed to recapitulate the robust tumor specificity previously reported, suggesting an importance of accessory molecules for MR1-dependent tumor reactivity. Finally, MR1-mutant cell lines showed that distinct residues on the α1/α2 helices were required for TCR binding by different MR1-restricted T cells and suggested central but distinct docking modes by the broad family of MR1-restricted αβ TCRs. Collectively, these data are consistent with recognition of distinct antigens by diverse MR1-restricted T cells.

Double-negative (DN) T cells are present at relatively low frequencies in human peripheral blood, and are characterized as expressing the alpha-beta or gamma-delta T-cell receptor (TCR), but not the CD4 nor the CD8 co-receptors. Despite their low frequencies, these cells are potent producers of cytokines and, thus, are key orchestrators of immune responses. DN T cells were initially associated with induction of peripheral immunological tolerance and immunomodulatory activities related to disease prevention. However, other studies demonstrated that these cells can also display effector functions associated with pathology development. This apparent contradiction highlighted the heterogeneity of the DN T-cell population. Here, we review phenotypic and functional characteristics of DN T cells, emphasizing their role in human diseases. The need for developing biomarkers to facilitate the translation of studies from animal models to humans will also be discussed. Finally, we will examine DN T cells as promising therapeutic targets to prevent or inhibit human disease development.

The potential protective or pathogenic role of the adaptive immune response to SARS‐CoV‐2 infection has been vigorously debated. While COVID‐19 patients consistently generate a T lymphocyte response to SARS‐CoV‐2 antigens, evidence of significant immune dysregulation in these patients continues to accumulate. In this study, next generation sequencing of the T cell receptor beta chain (TRB) repertoire was conducted in hospitalized COVID‐19 patients to determine if immunogenetic differences of the TRB repertoire contribute to disease course severity. Clustering of highly similar TRB CDR3 amino acid sequences across COVID‐19 patients yielded 781 shared TRB sequences. The TRB sequences were then filtered for known associations with common diseases such as EBV and CMV. The remaining sequences were cross‐referenced to a publicly accessible dataset that mapped COVID‐19 specific TCRs to the SARS‐CoV‐2 genome. We identified 158 SARS‐CoV‐2 specific TRB sequences belonging to 134 clusters in our COVID‐19 patients. Next, we investigated 113 SARS‐CoV‐2 specific clusters binding only one peptide target in relation to disease course. Distinct skewing of SARS‐CoV‐2 specific TRB sequences toward the nonstructural proteins (NSPs) encoded within ORF1a/b of the SARS‐CoV‐2 genome was observed in clusters associated with critical disease course when compared to COVID‐19 clusters associated with a severe disease course. These data imply that T‐lymphocyte reactivity towards peptides from NSPs of SARS‐CoV‐2 may not constitute an effective adaptive immune response and thus may negatively affect disease severity., Graphical Abstract The T cell receptor beta chain (TRB) repertoire of hospitalized COVID‐19 patients revealed that TRB sequences targeting nonstructural proteins of SARS‐CoV‐2 may negatively affect disease severity.

Contains fulltext : 248576.pdf (Publisher’s version ) (Open Access) Single-cell antigen-receptor gene amplification and sequencing platforms have been used to characterize T cell receptor (TCR) repertoires but typically fail to generate paired full-length gene products for direct expression cloning and do not enable linking this data to cell phenotype information. To overcome these limitations, we established a high-throughput platform for the quantitative and qualitative analysis of human TCR repertoires that provides insights into the clonal and functional composition of human CD4(+) and CD8(+) αβ T cells at the molecular and cellular level. The strategy is a powerful tool to qualitatively assess differences between antigen receptors of phenotypically defined αβ T cell subsets, e.g. in immune responses to cancer, vaccination, or infection, and in autoimmune diseases.

Key Points TCRαβ/CD19-depleted HLA-haploidentical HSCT is an effective strategy for children with several nonmalignant disorders.Patients given this type of transplant benefit from a low incidence of GVHD and TRM, with graft failure being the main obstacle., Visual Abstract, Several nonmalignant disorders (NMDs), either inherited or acquired, can be cured by allogeneic hematopoietic stem cell transplantation (HSCT). Between January 2012 and April 2020, 70 consecutive children affected by primary immunodeficiencies, inherited/acquired bone marrow failure syndromes, red blood cell disorders, or metabolic diseases, lacking a fully matched donor or requiring urgent transplantation underwent TCRαβ/CD19-depleted haploidentical HSCT from an HLA-partially matched relative as part of a prospective study. The median age at transplant was 3.5 years (range 0.3-16.1); the median time from diagnosis to transplant was 10.5 months (2.7 for SCID patients). Primary engraftment was obtained in 51 patients, while 19 and 2 patients experienced either primary or secondary graft failure (GF), the overall incidence of this complication being 30.4%. Most GFs were observed in children with disease at risk for this complication (eg, aplastic anemia, thalassemia). All but 5 patients experiencing GF were successfully retransplanted. Six patients died of infectious complications (4 had active/recent infections at the time of HSCT), the cumulative incidence of transplant-related mortality (TRM) being 8.5%. Cumulative incidence of grade 1-2 acute GVHD was 14.4% (no patient developed grade 3-4 acute GVHD). Only one patient at risk developed mild chronic GVHD. With a median follow-up of 3.5 years, the 5-year probability of overall and disease-free survival was 91.4% and 86.8%, respectively. In conclusion, TCRαβ/CD19-depleted haploidentical HSCT from an HLA-partially matched relative is confirmed to be an effective treatment of children with NMDs. Prompt donor availability, low incidence of GVHD, and TRM make this strategy an attractive option in NMDs patients. The study is registered at ClinicalTrial.gov as NCT01810120.

Early T-cell development from CD4

SummaryDiversity of the T-cell receptor (TCR) repertoire is central to adaptive immunity. The TCR is composed of α and β chains, encoded by the TRA and TRB genes, of which the variable regions determine antigen specificity. To generate novel biological insights into the complex functioning of immune cells, combined capture of variable regions and single-cell transcriptomes provides a compelling approach. Recent developments enable the enrichment of TRA and TRB variable regions from widely used technologies for 3’-biased single-cell RNA-sequencing (scRNA-seq). However, a comprehensive computational pipeline to process TCR-enriched data from 3’ scRNA-seq is not available. Here we present an analysis pipeline to process TCR variable regions enriched from 3’ scRNA-seq cDNA. The tool reports TRA and TRB nucleotide and amino acid sequences linked to cell barcodes, enabling the reconstruction of T-cell clonotypes with associated transcriptomes. We demonstrate the software using peripheral blood mononuclear cells (PBMCs) from a healthy donor and detect TCR sequences in a high proportion of single T-cells. Detection of TCR sequences is negligible in non-T-cell populations, demonstrating specificity. Finally, we show that TCR clones are larger in CD8 Memory T-cells than other T-cell types, indicating an association between T-cell clonotypes and differentiation states.Availability and implementationThe Workflow for Association of T-cell receptors from 3’ single-cell RNA-seq (WAT3R), including test data, is available on GitHub (https://github.com/mainciburu/WAT3R), Docker Hub (https://hub.docker.com/r/mainciburu/wat3r), and a workflow on the Terra platform (https://app.terra.bio). The test dataset is available on GEO (accession number pending).

CMV infection is a significant complication after solid organ transplantation. We used single cell TCR αβ sequencing to determine how memory inflation impacts clonality and diversity of the CMV-responsive CD8 and CD4 T cell repertoire in the first year after transplantation in human subjects. We observed CD8 T cell inflation but no changes in clonal diversity, indicating homeostatic stability in clones. In contrast, the CD4 repertoire was diverse and stable over time, with no evidence of CMV-responsive CD4 T cell expansion. We identified shared CDR3 TCR motifs among patients but no public CMV-specific TCRs. Temporal changes in clonality in response to transplantation and in the absence of detectable viral reactivation suggest changes in the repertoire immediately after transplantation followed by an expansion with stable clonal competition that may mediate protection.

The B cell and T cell antigen receptors (BCR and TCR) share a common architecture in which variable dimeric antigen-binding modules assemble with invariant dimeric signaling modules to form functional receptor complexes. In the TCR, a highly conserved T cell receptor αβ (TCRαβ) transmembrane (TM) interface forms a rigid structure around which its three dimeric signaling modules assemble through well-characterized polar interactions. Noting that the key features stabilizing this TCRαβ TM interface also appear with high evolutionary conservation in the TM sequences of the membrane immunoglobulin (mIg) heavy chains that form the BCR’s homodimeric antigen-binding module, we asked whether the BCR contained an analogous TM structure. Using an unbiased biochemical and computational modeling approach, we found that the mouse IgM BCR forms a core TM structure that is remarkably similar to that of the TCR. This structure is reinforced by a network of interhelical hydrogen bonds, and our model is nearly identical to the arrangement observed in the just-released cryo-electron microscopy (cryo-EM) structures of intact human BCRs. Our biochemical analysis shows that the integrity of this TM structure is vital for stable assembly with the BCR signaling module CD79AB in the B cell endoplasmic reticulum, and molecular dynamics simulations indicate that BCRs of all five isotypes can form comparable structures. These results demonstrate that, despite their many differences in composition, complexity, and ligand type, TCRs and BCRs rely on a common core TM structure that has been shaped by evolution for optimal receptor assembly and stability in the cell membrane.

While the functions of αβ T cells in host resistance to pathogen infection are understood in far more detail than those of γδ lineage T cells, γδ T cells perform critical, essential functions during immune responses that cannot be compensated for by αβ T cells. Accordingly, it is critical to understand how the development of γδ T cells is controlled so that their generation and function might be manipulated in future for therapeutic benefit. This introductory chapter will focus primarily on the basic processes that underlie γδ T cell development in the thymus, as well as the current understanding of how they are controlled.

Quantitative real-time PCR and next-generation sequencing (NGS) are invaluable techniques to analyze T cell receptor (Tcr) gene rearrangements in mouse lymphocyte populations. Although these approaches are powerful, they also have limitations that must be accounted for in experimental design and data interpretation. Here, we provide relevant background required for understanding these limitations and then outline established quantitative real-time PCR and NGS methods that can be used for analysis of mouse Tcra and Tcrb gene rearrangements in mice.

T lymphocytes (T cells) are essential components of the adaptive immune system; they serve multiple functions in responses to pathogens and to ensure immune homeostasis. Written for readers first entering this field of study, this chapter is a brief overview of the development of T cells in the thymus, from the entry of thymus-settling bone marrow-derived precursors to the egress of mature T cells. Surveyed topics include the differentiation and expansion of early precursors, the generation of the T cell antigen receptor repertoire, the selection of αβ T cell precursors, and their acquisition of functional competency.

Antibodies and T-cell receptors have been a subject of much interest due to their central role in the immune system and their potential applications in several biotechnological and medical applications from cancer therapy to vaccine development. A unique feature of these two lymphocyte receptors is their ability to bind a huge variety of different (pathogen) targets. This ability stems from six short loops in the binding domain that have hypervariable sequence due to genetic recombination mechanism. Particularly one of these loops, the third complementarity determining region (CDR3), has the highest sequence variability and a dominant role in binding the target. However, it has also been proven the most difficult to be modeled structurally, which is vitally important for downstream tasks such as binding prediction. This difficulty stems from its variability in sequence that both reduces the possibility of finding homologues and introduces unique structural features in the loop. We present here a general protocol for modeling such loops in antibodies and T-cell receptors. We also discuss the difficulties in loop modeling and the advantages and limitations of different modeling methods.

Inborn errors of IFN-γ immunity can underlie tuberculosis (TB). We report three patients from two kindreds without EBV viremia or disease but with severe TB and inherited complete ITK deficiency, a condition associated with severe EBV disease that renders immunological studies challenging. They have CD4+ αβ T lymphocytopenia with a concomitant expansion of CD4−CD8− double-negative (DN) αβ and Vδ2− γδ T lymphocytes, both displaying a unique CD38+CD45RA+T-bet+EOMES− phenotype. Itk-deficient mice recapitulated an expansion of the γδ T and DN αβ T lymphocyte populations in the thymus and spleen, respectively. Moreover, the patients’ T lymphocytes secrete small amounts of IFN-γ in response to TCR crosslinking, mitogens, or forced synapse formation with autologous B lymphocytes. Finally, the patients’ total lymphocytes secrete small amounts of IFN-γ, and CD4+, CD8+, DN αβ T, Vδ2+ γδ T, and MAIT cells display impaired IFN-γ production in response to BCG. Inherited ITK deficiency undermines the development and function of various IFN-γ–producing T cell subsets, thereby underlying TB.

Genome editing of allogeneic T cells can provide “off-the-shelf” alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor α chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator–like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3’ long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 10 6 to 2.0 × 10 6 CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy.

Funding Information: We are deeply grateful to all patients who participated in the study and generously contributed samples. We acknowledge the computational resources provided by the Aalto Science-IT project and all the personnel in the Hematology Research Unit Helsinki for insightful conversations. We especially thank Dr. Nathalie Labarriere for providing information of MAA-specific TCR sequences of their study; Dr. Michael Durante and Dr. J. William Harbor for providing information related to their study; Dr. Olli Dufva for his help with interpreting the data. We additionally acknowledge Ms. Lotta Länsman for providing administrative advice. This study was supported by the European Research Council (Project: M-IMM 647355)(SM), Academy of Finland (314442, S.M., H.L. and 335527 S.M.), Sigrid Juselius Foundation (S.M.), Signe and Ane Gyllenberg Foundation (S.M.), Helsinki Institute for Life Science (S.M.), Cancer Foundation Finland (S.M.) and State funding for the University-level Health Research in Finland(S.M.). J.H. was supported by Finnish Hematology Association, Blood Disease Research Foundation, Helsinki Institute for Life Science, Biomedicum Helsinki Foundation, Finnish Medical Foundation, K. Albin Johansson Foundation, Kaute Foundation, Suomalais-Norjalainen Lääketieteen Säätiö, and Emil Aaltonen Foundation. T.L. was supported by Academy of Finland (Decision 311081). Funding Information: M.H. has received honoraria from Pierre Fabre, Novartis, Bristol-Myers Squibb (B.M.S.), Merck Sharp & Dohme (M.S.D.), Roche, Amgen, Sanofi, and Incyte. S.M. has received honoraria and research funding from B.M.S. and research funding from Novartis and Pfizer. M.M.D. has received honoraria and/or equity from Amgen, Chugai, Vir, IGMS, Janux, A2, 3T, Mozart, Red Tree, and research funding from Sanofi. The remaining authors declare no competing interests. Publisher Copyright: © 2022, The Author(s). Analyzing antigen-specific T cell responses at scale has been challenging. Here, we analyze three types of T cell receptor (TCR) repertoire data (antigen-specific TCRs, TCR-repertoire, and single-cell RNA + TCRαβ-sequencing data) from 515 patients with primary or metastatic melanoma and compare it to 783 healthy controls. Although melanoma-associated antigen (MAA) -specific TCRs are restricted to individuals, they share sequence similarities that allow us to build classifiers for predicting anti-MAA T cells. The frequency of anti-MAA T cells distinguishes melanoma patients from healthy and predicts metastatic recurrence from primary melanoma. Anti-MAA T cells have stem-like properties and frequent interactions with regulatory T cells and tumor cells via Galectin9-TIM3 and PVR-TIGIT -axes, respectively. In the responding patients, the number of expanded anti-MAA clones are higher after the anti-PD1(+anti-CTLA4) therapy and the exhaustion phenotype is rescued. Our systems immunology approach paves the way for understanding antigen-specific responses in human disorders.

T cell receptor (TCR) sequences are exceptionally diverse and can now be comprehensively measured with next-generation sequencing technologies. However, a thorough investigation of longitudinal TCR repertoires throughout childhood in health and during development of a common childhood disease, type 1 diabetes (T1D), has not been undertaken. Here, we deep sequenced the TCR-β chain repertoires from longitudinal peripheral blood DNA samples at 4 time points beginning early in life (median age of 1.4 years) from children who progressed to T1D (n = 29) and age/sex-matched islet autoantibody-negative controls (n = 25). From 53 million TCR-β sequences, we show that the repertoire is extraordinarily diverse early in life and narrows with age independently of disease. We demonstrate the ability to identify specific TCR sequences, including those known to recognize influenza A and, separately, those specific for insulin and its precursor, preproinsulin. Insulin-reactive TCR-β sequences were more common and frequent in number as the disease progressed in those who developed T1D compared with genetically at risk nondiabetic children, and this was not the case for influenza-reactive sequences. As an independent validation, we sequenced and analyzed TCR-β repertoires from a cohort of new-onset T1D patients (n = 143), identifying the same preproinsulin-reactive TCRs. These results demonstrate an enrichment of preproinsulin-reactive TCR sequences during the progression to T1D, highlighting the importance of using disease-relevant TCR sequences as powerful biomarkers in autoimmune disorders.

Previous studies have demonstrated the regulatory functions of Ly49(+)CD8(+) T cells towards self-reactive CD4(+) T cells in mice. Recently, we found KIR(+)CD8(+) T cells are the equivalent of mouse Ly49(+)CD8(+) T cells in humans. They are increased in patients with autoimmune or infectious diseases as a negative feedback mechanism to suppress the arising pathogenic cells and maintain peripheral tolerance. Here, we describe the methods how we characterize the KIR(+)CD8(+) T cells from different diseases using single-cell RNA and TCR sequencing.

The quantitative tracking of the dynamics of T cells is challenging in human immunology. Although bulk sequencing of T cell receptor (TCR) α- and β-chains has been widely used for determining the clonality of T cells, such methods are limited in unveiling the phenotypic differences of T cells with the same clonotypes. Here, we describe a bioinformatics framework, STARTRAC, that integrates the single-cell transcriptome and TCR sequences as lineage-specific markers to quantitatively assess the dynamics of T cells, including their clonal expansion, tissue migration, and developmental transition properties.

T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical trap combined with fluorescence permits direct monitoring of T-cell triggering in response to force application at various concentrations of peptide-bound major histocompatibility complex molecules (pMHC). The technique mimics physiological shear forces applied as cells crawl across antigen-presenting surfaces during immune surveillance. True single molecule studies performed on single cells profile force-bond lifetime, typically seen as a catch bond, and conformational change at the TCR-pMHC bond on the surface of the cell upon force loading. Together, activation and single molecule single cell studies provide chemical and physical triggering thresholds as well as insight into catch bond formation and quaternary structural changes of single TCRs. The present methods detail assay design, preparation, and execution, as well as data analysis. These methods may be applied to a wide range of pMHC-TCR interactions and have potential for adaptation to other receptor-ligand systems.

T cell differentiation in the thymus generates CD4+helper and cytotoxic CD8+cells as the two principal T cell lineages. Curiously, at the end of this complex selection process, CD4+cells invariably outnumber CD8+cells. Here, we examine the dynamics of repertoire formation and the emergence of the skewed CD4/CD8 ratio using high-resolution endogenous CRISPR/Cas9 barcoding that indelibly marks immature T cells at the DN2/DN3 pre-TCR stage. In wild-type mice, greater clone size of CD4+cells and an intrinsically greater probability of Tcr β clonotypes for pMHCII interactions are major contributors to the skewed CD4/CD8 ratio. Clonal perturbations of thymocyte differentiation following the precocious expression of a rearranged iNKT invariant TCR α chain are due to loss of Tcr β clonotypes from the CD4 lineage-committed pre-selection repertoire. The present barcoding scheme offers a novel means to examine the clonal dynamics of lymphocyte differentiation orthogonal to that using TCR clonotypes.