TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.

Background: In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. Methods: We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. Results: We show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. Conclusion: These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

Phospho-mimetic CD3ε variants prevent TCR and CAR signaling.

Introduction: Antigen binding to the T cell antigen receptor (TCR) leads to the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex, and thereby to T cell activation. The CD3ε subunit plays a unique role in TCR activation by recruiting the kinase LCK and the adaptor protein NCK prior to ITAM phosphorylation. Here, we aimed to investigate how phosphorylation of the individual CD3ε ITAM tyrosines impacts the CD3ε signalosome. Methods: We mimicked irreversible tyrosine phosphorylation by substituting glutamic acid for the tyrosine residues in the CD3ε ITAM. Results: Integrating CD3ε phospho-mimetic variants into the complete TCR-CD3 complex resulted in reduced TCR signal transduction, which was partially compensated by the involvement of the other TCR-CD3 ITAMs. By using novel CD3ε phospho-mimetic Chimeric Antigen Receptor (CAR) variants, we avoided any compensatory effects of other ITAMs in the TCR-CD3 complex. We demonstrated that irreversible CD3ε phosphorylation prevented signal transduction upon CAR engagement. Mechanistically, we demonstrated that glutamic acid substitution at the N-terminal tyrosine residue of the CD3ε ITAM (Y39E) significantly reduces NCK binding to the TCR. In contrast, mutation at the C-terminal tyrosine of the CD3ε ITAM (Y50E) abolished LCK recruitment to the TCR, while increasing NCK binding. Double mutation at the C- and N-terminal tyrosines (Y39/50E) allowed ZAP70 to bind, but reduced the interaction with LCK and NCK. Conclusions: The data demonstrate that the dynamic phosphorylation of the CD3ε ITAM tyrosines is essential for CD3ε to orchestrate optimal TCR and CAR signaling and highlights the key role of CD3ε signalosome to tune signal transduction.

The self-reactive FVIII T cell repertoire in healthy individuals relies on a short set of epitopes and public clonotypes.

Non-mutated FVIII-specific CD4 T cell epitopes have been recently found to contribute to the development of inhibitors in patients with hemophilia A (HA), while auto-reactive CD4 T cells specific to FVIII circulate in the blood of healthy individuals at a frequency close to the foreign protein ovalbumin. Thus, although FVIII is a self-protein, the central tolerance raised against FVIII appears to be low. In this study, we conducted a comprehensive analysis of the FVIII CD4 T cell repertoire in 29 healthy donors. Sequencing of the CDR3ß TCR region from isolated FVIII-specific CD4 T cells revealed a limited usage and pairing of TRBV and TRBJ genes as well as a mostly hydrophobic composition of the CDR3ß region according to their auto-reactivity. The FVIII repertoire is dominated by a few clonotypes, with only 13 clonotypes accounting for half of the FVIII response. Through a large-scale epitope mapping of the full-length FVIII sequence, we identified 18 immunodominant epitopes located in the A1, A3, C1, and C2 domains and covering half of the T cell response. These epitopes exhibited a broad specificity for HLA-DR or DP molecules or both. T cell priming with this reduced set of peptides revealed that highly expanded clonotypes specific to these epitopes were responsible individually for up to 32% of the total FVIII repertoire. These FVIII T cell epitopes and clonotypes were shared among HLA-unrelated donors tested and previously reported HA patients. Our study highlights the role of the auto-reactive T cell response against FVIII in HA and its similarity to the response observed in healthy individuals. Thus, it provides valuable insights for the development of new tolerance induction and deimmunization strategies.

Tracking of activated cTfh cells following sequential influenza vaccinations reveals transcriptional profile of clonotypes driving a vaccine-induced immune response.

Introduction: A vaccine against influenza is available seasonally but is not 100% effective. A predictor of successful seroconversion in adults is an increase in activated circulating T follicular helper (cTfh) cells after vaccination. However, the impact of repeated annual vaccinations on long-term protection and seasonal vaccine efficacy remains unclear. Methods: In this study, we examined the T cell receptor (TCR) repertoire and transcriptional profile of vaccine-induced expanded cTfh cells in individuals who received sequential seasonal influenza vaccines. We measured the magnitude of cTfh and plasmablast cell activation from day 0 (d0) to d7 post-vaccination as an indicator of a vaccine response. To assess TCR diversity and T cell expansion we sorted activated and resting cTfh cells at d0 and d7 post-vaccination and performed TCR sequencing. We also single cell sorted activated and resting cTfh cells for TCR analysis and transcriptome sequencing. Results and discussion: The percent of activated cTfh cells significantly increased from d0 to d7 in each of the 2016-17 (p < 0.0001) and 2017-18 (p = 0.015) vaccine seasons with the magnitude of cTfh activation increase positively correlated with the frequency of circulating plasmablast cells in the 2016-17 (p = 0.0001) and 2017-18 (p = 0.003) seasons. At d7 post-vaccination, higher magnitudes of cTfh activation were associated with increased clonality of cTfh TCR repertoire. The TCRs from vaccine-expanded clonotypes were identified and tracked longitudinally with several TCRs found to be present in both years. The transcriptomic profile of these expanded cTfh cells at the single cell level demonstrated overrepresentation of transcripts of genes involved in the type-I interferon pathway, pathways involved in gene expression, and antigen presentation and recognition. These results identify the expansion and transcriptomic profile of vaccine-induced cTfh cells important for B cell help.

Molecular techniques drive cutting edge advancements in management of cutaneous T cell lymphoma.

Cutaneous 5T cell lymphoma (CTCL), characterized by malignant T cells infiltrating the skin with potential for dissemination, remains a challenging disease to diagnose and treat due to disease heterogeneity, treatment resistance, and lack of effective and standardized diagnostic and prognostic clinical tools. Currently, diagnosis of CTCL practically relies on clinical presentation, histopathology, and immunohistochemistry. These methods are collectively fraught with limitations in sensitivity and specificity. Fortunately, recent advances in flow cytometry, polymerase chain reaction, high throughput sequencing, and other molecular techniques have shown promise in improving diagnosis and treatment of CTCL. Examples of these advances include T cell receptor clonotyping via sequencing to detect CTCL earlier in the disease course and single-cell RNA sequencing to identify gene expression patterns that commonly drive CTCL pathogenesis. Experience with these techniques has afforded novel insights which may translate into enhanced diagnostic and therapeutic approaches for CTCL.

On the feasibility of using TCR sequencing to follow a vaccination response - lessons learned.

T cells recognize pathogens by their highly specific T-cell receptor (TCR), which can bind small fragments of an antigen presented on the Major Histocompatibility Complex (MHC). Antigens that are provided through vaccination cause specific T cells to respond by expanding and forming specific memory to combat a future infection. Quantification of this T-cell response could improve vaccine monitoring or identify individuals with a reduced ability to respond to a vaccination. In this proof-of-concept study we use longitudinal sequencing of the TCRß repertoire to quantify the response in the CD4+ memory T-cell pool upon pneumococcal conjugate vaccination. This comes with several challenges owing to the enormous size and diversity of the T-cell pool, the limited frequency of vaccine-specific TCRs in the total repertoire, and the variation in sample size and quality. We defined quantitative requirements to classify T-cell expansions and identified critical parameters that aid in reliable analysis of the data. In the context of pneumococcal conjugate vaccination, we were able to detect robust T-cell expansions in a minority of the donors, which suggests that the T-cell response against the conjugate in the pneumococcal vaccine is small and/or very broad. These results indicate that there is still a long way to go before TCR sequencing can be reliably used as a personal biomarker for vaccine-induced protection. Nevertheless, this study highlights the importance of having multiple samples containing sufficient T-cell numbers, which will support future studies that characterize T-cell responses using longitudinal TCR sequencing.

Case Report: An unclassified T cell lymphoma subtype with co-expression of TCR αß and γ chains revealed by single cell sequencing.

Background: T cell lymphomas (TCL) are a group of heterogeneous diseases with over 40 subtypes. In this study, we identified a novel TCL subtype which was featured by a unique T cell receptor (TCR) presentation, α, ß and γ chains were co-existing in a single malignant T cell. Case presentation: A 45-year-old male patient was diagnosed T cell lymphoma after 2-month of abdominal distension and liver enlargement. Combining histology review, PET-CT scanning and immunophenotyes, the patient was not classified to any existing TCL subtypes. To better understand this unclassified TCL case, we performed single cell RNA sequencing paired with TCR sequencing on the patient's PBMC and bone marrow samples. To our surprise, we identified that the malignant T cells had a very rare TCR combination, by expressing two α chains, one ß chain and one γ chain simultaneously. We further studied the molecular pathogenesis and tumor cell heterogeneity of this rare TCL subtype. A set of potential therapeutic targets were identified from the transcriptome data, such as CCL5, KLRG1 and CD38. Conclusions: We identified the first TCL case co-expressing α, ß and γ chains and dissected its molecular pathogenesis, providing valuable information for precision medicine options for this novel TCL subtype.

From bench to bedside: the history and progress of CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy represents a major breakthrough in cancer care since the approval of tisagenlecleucel by the Food and Drug Administration in 2017 for the treatment of pediatric and young adult patients with relapsed or refractory acute lymphocytic leukemia. As of April 2023, six CAR T cell therapies have been approved, demonstrating unprecedented efficacy in patients with B-cell malignancies and multiple myeloma. However, adverse events such as cytokine release syndrome and immune effector cell-associated neurotoxicity pose significant challenges to CAR T cell therapy. The severity of these adverse events correlates with the pretreatment tumor burden, where a higher tumor burden results in more severe consequences. This observation is supported by the application of CD19-targeted CAR T cell therapy in autoimmune diseases including systemic lupus erythematosus and antisynthetase syndrome. These results indicate that initiating CAR T cell therapy early at low tumor burden or using debulking strategy prior to CAR T cell infusion may reduce the severity of adverse events. In addition, CAR T cell therapy is expensive and has limited effectiveness against solid tumors. In this article, we review the critical steps that led to this groundbreaking therapy and explore ongoing efforts to overcome these challenges. With the promise of more effective and safer CAR T cell therapies in development, we are optimistic that a broader range of cancer patients will benefit from this revolutionary therapy in the foreseeable future.

Deep characterization of human γδ T cell subsets defines shared and lineage-specific traits.

Under non-pathological conditions, human γδ T cells represent a small fraction of CD3+ T cells in peripheral blood (1-10%). They constitute a unique subset of T lymphocytes that recognize stress ligands or non-peptide antigens through MHC-independent presentation. Major human γδ T cell subsets, Vδ1 and Vδ2, expand in response to microbial infection or malignancy, but possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that differences at the gene, phenotypic, and functional level would provide evidence that γδ T cell subpopulations belong to distinct lineages. Comparisons between each subset and the identification of the molecular determinants that underpin their differences has been hampered by experimental challenges in obtaining sufficient numbers of purified cells. By utilizing a stringent FACS-based isolation method, we compared highly purified human Vδ1 and Vδ2 cells in terms of phenotype, gene expression profile, and functional responses. We found distinct genetic and phenotypic signatures that define functional differences in γδ T cell populations. Differences in TCR components, repertoire, and responses to calcium-dependent pathways suggest that Vδ1 and Vδ2 T cells are different lineages. These findings will facilitate further investigation into the ligand specificity and unique role of Vδ1 and Vδ2 cells in early immune responses.

Challenges and opportunities in analyzing and modeling peptide presentation by HLA-II proteins.

The human leukocyte antigen (HLA) proteins are an indispensable component of adaptive immunity because of their role in presenting self and foreign peptides to T cells. Further, many complex diseases are associated with genetic variation in the HLA region, implying an important role for specific HLA-presented peptides in the etiology of these diseases. Identifying the specific set of peptides presented by an individual's HLA proteins in vivo, as a whole being referred to as the immunopeptidome, has therefore gathered increasing attention for different reasons. For example, identifying neoepitopes for cancer immunotherapy, vaccine development against infectious pathogens, or elucidating the role of HLA in autoimmunity. Despite the tremendous progress made during the last decade in these areas, several questions remain unanswered. In this perspective, we highlight five remaining key challenges in the analysis of peptide presentation and T cell immunogenicity and discuss potential solutions to these problems. We believe that addressing these questions would not only improve our understanding of disease etiology but will also have a direct translational impact in terms of engineering better vaccines and in developing more potent immunotherapies.

Viral infection reveals hidden sharing of TCR CDR3 sequences between individuals.

The T cell receptor is generated by a process of random and imprecise somatic recombination. The number of possible T cell receptors which this process can produce is enormous, greatly exceeding the number of T cells in an individual. Thus, the likelihood of identical TCRs being observed in multiple individuals (public TCRs) might be expected to be very low. Nevertheless such public TCRs have often been reported. In this study we explore the extent of TCR publicity in the context of acute resolving Lymphocytic choriomeningitis virus (LCMV) infection in mice. We show that the repertoire of effector T cells following LCMV infection contains a population of highly shared TCR sequences. This subset of TCRs has a distribution of naive precursor frequencies, generation probabilities, and physico-chemical CDR3 properties which lie between those of classic public TCRs, which are observed in uninfected repertoires, and the dominant private TCR repertoire. We have named this set of sequences "hidden public" TCRs, since they are only revealed following infection. A similar repertoire of hidden public TCRs can be observed in humans after a first exposure to SARS-COV-2. The presence of hidden public TCRs which rapidly expand following viral infection may therefore be a general feature of adaptive immunity, identifying an additional level of inter-individual sharing in the TCR repertoire which may form an important component of the effector and memory response.

The recombinase activating genes: architects of immune diversity during lymphocyte development.

The mature lymphocyte population of a healthy individual has the remarkable ability to recognise an immense variety of antigens. Instead of encoding a unique gene for each potential antigen receptor, evolution has used gene rearrangements, also known as variable, diversity, and joining gene segment (V(D)J) recombination. This process is critical for lymphocyte development and relies on recombination-activating genes-1 (RAG1) and RAG2, here collectively referred to as RAG. RAG serves as powerful genome editing tools for lymphocytes and is strictly regulated to prevent dysregulation. However, in the case of dysregulation, RAG has been implicated in cases of cancer, autoimmunity and severe combined immunodeficiency (SCID). This review examines functional protein domains and motifs of RAG, describes advances in our understanding of the function and (dys)regulation of RAG, discuss new therapeutic options, such as gene therapy, for RAG deficiencies, and explore in vitro and in vivo methods for determining RAG activity and target specificity.

Haematopoietic Stem Cell Transplantation Results in Extensive Remodelling of the Clonal T Cell Repertoire in Multiple Sclerosis.

Autologous haematopoietic stem cell transplantation (AHSCT) is a vital therapeutic option for patients with highly active multiple sclerosis (MS). Rates of remission suggest AHSCT is the most effective form of immunotherapy in controlling the disease. Despite an evolving understanding of the biology of immune reconstitution following AHSCT, the mechanism by which AHSCT enables sustained disease remission beyond the period of lymphopenia remains to be elucidated. Auto-reactive T cells are considered central to MS pathogenesis. Here, we analyse T cell reconstitution for 36 months following AHSCT in a cohort of highly active MS patients. Through longitudinal analysis of sorted naïve and memory T cell clones, we establish that AHSCT induces profound changes in the dominant T cell landscape of both CD4+ and CD8+ memory T cell clones. Lymphopenia induced homeostatic proliferation is followed by clonal attrition; with only 19% of dominant CD4 (p <0.025) and 13% of dominant CD8 (p <0.005) clones from the pre-transplant repertoire detected at 36 months. Recovery of a thymically-derived CD4 naïve T cell repertoire occurs at 12 months and is ongoing at 36 months, however diversity of the naïve populations is not increased from baseline suggesting the principal mechanism of durable remission from MS after AHSCT relates to depletion of putative auto-reactive clones. In a cohort of MS patients expressing the MS risk allele HLA DRB1*15:01, public clones are probed as potential biomarkers of disease. AHSCT appears to induce sustained periods of disease remission with dynamic changes in the clonal T cell repertoire out to 36 months post-transplant.

Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data.

Advances in next-generation sequencing (NGS) have improved the resolution of T-cell receptor (TCR) repertoire analysis, and recent single-cell sequencing has made it possible to obtain information about TCR pairs. In our previous study, cytomegalovirus (CMV) pp65-specific T-cell response restricted by a single human leukocyte antigen (HLA) class I allotype was observed in an individual. Therefore, to effectively clone an antigen-specific TCR from these T cells, we developed a TCR cloning system that does not require a single cell level. First, we established the improved Jurkat reporter cell line, which was TCRαß double knock-out and expressed CD8αß molecules. Furthermore, functional TCRs were directly obtained by reverse TCR cloning using unique CDR3-specific PCR primers after bulk TCR sequencing of activation marker-positive CD8 T cells by NGS. A total of 15 TCRα and 14 TCRß strands were successfully amplified by PCR from cDNA of 4-1BB-positive CD8 T cells restricted by HLA-A*02:01, HLA-A*02:06, HLA-B*07:02, and HLA-B*40:06. The panels with combinations of TCRα and TCRß genes were investigated using Jurkat reporter cell line and artificial antigen-presenting cells (APCs). In two TCR pairs restricted by HLA-A*02:01, one TCR pair by HLA-A*02:06, four TCR pairs by HLA-B*07:02, and one TCR pair by HLA-B*40:06, their specificity and affinity were confirmed. The TCR pair of A*02:01/1-1 showed alloreactivity to HLA-A*02:06. The one TCR pair showed a higher response to the naturally processed antigen than that of the peptide pool. This reverse TCR cloning system will not only provide functional information to TCR repertoire analysis by NGS but also help in the development of TCR-T therapy.

On TCR binding predictors failing to generalize to unseen peptides.

Several recent studies investigate TCR-peptide/-pMHC binding prediction using machine learning or deep learning approaches. Many of these methods achieve impressive results on test sets, which include peptide sequences that are also included in the training set. In this work, we investigate how state-of-the-art deep learning models for TCR-peptide/-pMHC binding prediction generalize to unseen peptides. We create a dataset including positive samples from IEDB, VDJdb, McPAS-TCR, and the MIRA set, as well as negative samples from both randomization and 10X Genomics assays. We name this collection of samples TChard. We propose the hard split, a simple heuristic for training/test split, which ensures that test samples exclusively present peptides that do not belong to the training set. We investigate the effect of different training/test splitting techniques on the models' test performance, as well as the effect of training and testing the models using mismatched negative samples generated randomly, in addition to the negative samples derived from assays. Our results show that modern deep learning methods fail to generalize to unseen peptides. We provide an explanation why this happens and verify our hypothesis on the TChard dataset. We then conclude that robust prediction of TCR recognition is still far for being solved.

pyTCR: A comprehensive and scalable solution for TCR-Seq data analysis to facilitate reproducibility and rigor of immunogenomics research.

T cell receptor (TCR) studies have grown substantially with the advancement in the sequencing techniques of T cell receptor repertoire sequencing (TCR-Seq). The analysis of the TCR-Seq data requires computational skills to run the computational analysis of TCR repertoire tools. However biomedical researchers with limited computational backgrounds face numerous obstacles to properly and efficiently utilizing bioinformatics tools for analyzing TCR-Seq data. Here we report pyTCR, a computational notebook-based solution for comprehensive and scalable TCR-Seq data analysis. Computational notebooks, which combine code, calculations, and visualization, are able to provide users with a high level of flexibility and transparency for the analysis. Additionally, computational notebooks are demonstrated to be user-friendly and suitable for researchers with limited computational skills. Our tool has a rich set of functionalities including various TCR metrics, statistical analysis, and customizable visualizations. The application of pyTCR on large and diverse TCR-Seq datasets will enable the effective analysis of large-scale TCR-Seq data with flexibility, and eventually facilitate new discoveries.

Divide and Conquer: Phenotypic and Temporal Heterogeneity Within CD8+ T Cell Responses.

The advent of technologies that can characterize the phenotypes, functions and fates of individual cells has revealed extensive and often unexpected levels of diversity between cells that are nominally of the same subset. CD8+ T cells, also known as cytotoxic T lymphocytes (CTLs), are no exception. Investigations of individual CD8+ T cells both in vitro and in vivo have highlighted the heterogeneity of cellular responses at the levels of activation, differentiation and function. This review takes a broad perspective on the topic of heterogeneity, outlining different forms of variation that arise during a CD8+ T cell response. Specific attention is paid to the impact of T cell receptor (TCR) stimulation strength on heterogeneity. In particular, this review endeavors to highlight connections between variation at different cellular stages, presenting known mechanisms and key open questions about how variation between cells can arise and propagate.

Public T-Cell Receptors (TCRs) Revisited by Analysis of the Magnitude of Identical and Highly-Similar TCRs in Virus-Specific T-Cell Repertoires of Healthy Individuals.

Since multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of T cells in a human body, the likelihood that many public identical (PUB-I) TCR-sequences frequently contribute to immune responses has been estimated to be low. Here, we quantitatively analyzed the TCR-repertoires of 190 purified virus-specific memory T-cell populations, directed against 21 epitopes of Cytomegalovirus, Epstein-Barr virus and Adenovirus isolated from 29 healthy individuals, and determined the magnitude, defined as prevalence within the population and frequencies within individuals, of PUB-I TCR and of TCR-sequences that are highly-similar (PUB-HS) to these PUB-I TCR-sequences. We found that almost one third of all TCR nucleotide-sequences represented PUB-I TCR amino-acid (AA) sequences and found an additional 12% of PUB-HS TCRs differing by maximally 3 AAs. We illustrate that these PUB-I and PUB-HS TCRs were structurally related and contained shared core-sequences in their TCR-sequences. We found a prevalence of PUB-I and PUB-HS TCRs of up to 50% among individuals and showed frequencies of virus-specific PUB-I and PUB-HS TCRs making up more than 10% of each virus-specific T-cell population. These findings were confirmed by using an independent TCR-database of virus-specific TCRs. We therefore conclude that the magnitude of the contribution of PUB-I and PUB-HS TCRs to these virus-specific T-cell responses is high. Because the T cells from these virus-specific memory TCR-repertoires were the result of successful control of the virus in these healthy individuals, these PUB-HS TCRs and PUB-I TCRs may be attractive candidates for immunotherapy in immunocompromised patients that lack virus-specific T cells to control viral reactivation.

Single-Cell TCR and Transcriptome Analysis: An Indispensable Tool for Studying T-Cell Biology and Cancer Immunotherapy.

T cells have been known to be the driving force for immune response and cancer immunotherapy. Recent advances on single-cell sequencing techniques have empowered scientists to discover new biology at the single-cell level. Here, we review the single-cell techniques used for T-cell studies, including T-cell receptor (TCR) and transcriptome analysis. In addition, we summarize the approaches used for the identification of T-cell neoantigens, an important aspect for T-cell mediated cancer immunotherapy. More importantly, we discuss the applications of single-cell techniques for T-cell studies, including T-cell development and differentiation, as well as the role of T cells in autoimmunity, infectious disease and cancer immunotherapy. Taken together, this powerful tool not only can validate previous observation by conventional approaches, but also can pave the way for new discovery, such as previous unidentified T-cell subpopulations that potentially responsible for clinical outcomes in patients with autoimmunity or cancer.

Binding of Staphylococcal Enterotoxin B (SEB) to B7 Receptors Triggers TCR- and CD28-Mediated Inflammatory Signals in the Absence of MHC Class II Molecules.

The inflammatory activity of staphylococcal enterotoxin B (SEB) relies on its capacity to trigger polyclonal T-cell activation by binding both T-cell receptor (TCR) and costimulatory receptor CD28 on T cells and MHC class II and B7 molecules on antigen presenting cells (APC). Previous studies highlighted that SEB may bind TCR and CD28 molecules independently of MHC class II, yet the relative contribution of these interactions to the pro-inflammatory function of SEB remained unclear. Here, we show that binding to MHC class II is dispensable for the inflammatory activity of SEB, whereas binding to TCR, CD28 and B7 molecules is pivotal, in both human primary T cells and Jurkat T cell lines. In particular, our finding is that binding of SEB to B7 molecules suffices to trigger both TCR- and CD28-mediated inflammatory signalling. We also provide evidence that, by strengthening the interaction between CD28 and B7, SEB favours the recruitment of the TCR into the immunological synapse, thus inducing lethal inflammatory signalling.