Exploring T Cell Receptor Repertoires in Myocardial Diseases.

Mounting experimental and clinical evidence has revealed that adaptive immune mechanisms targeting myocardial antigens are triggered by different forms of cardiac injury and impact disease progression. B and T lymphocytes recognize specific antigens via unique adaptive immune receptors generated through a somatic rearrangement process that generates a potential repertoire of 1019 unique receptors. While the adaptive immune receptor repertoire diversity provides the basis for immunologic specificity, making sense of it can be a challenging task. In the present review, we discuss key aspects underlying the generation of TCRs (T cell receptors) and emerging tools for their study in the context of myocardial diseases. Moreover, we outline how exploring TCR repertoires could lead to a deeper understanding of myocardial pathophysiological principles and potentially serve as diagnostic tools.

Cellular architecture shapes the naïve T cell response.

After antigen stimulation, naïve T cells display reproducible population-level responses, which arise from individual T cells pursuing specific differentiation trajectories. However, cell-intrinsic predeterminants controlling these single-cell decisions remain enigmatic. We found that the subcellular architectures of naïve CD8 T cells, defined by the presence (TØ) or absence (TO) of nuclear envelope invaginations, changed with maturation, activation, and differentiation. Upon T cell receptor (TCR) stimulation, naïve TØ cells displayed increased expression of the early-response gene Nr4a1, dependent upon heightened calcium entry. Subsequently, in vitro differentiation revealed that TØ cells generated effector-like cells more so compared with TO cells, which proliferated less and preferentially adopted a memory-precursor phenotype. These data suggest that cellular architecture may be a predeterminant of naïve CD8 T cell fate.

Bulk T-cell receptor sequencing confirms clonality in obstetric antiphospholipid syndrome and may as a potential biomarker.

The heterogeneity of the T cell receptor (TCR) repertoire critically influences the autoimmune response in obstetric antiphospholipid syndrome (OAPS) and is intimately associated with the prophylaxis of autoimmune disorders. Investigating the TCR diversity patterns in patients with OAPS is thus of paramount clinical importance. This investigation procured peripheral blood specimens from 31 individuals with OAPS, 21 patients diagnosed with systemic lupus erythematosus (SLE), and 22 healthy controls (HC), proceeding with TCR repertoire sequencing. Concurrently, adverse pregnancy outcomes in the OAPS cohort were monitored and documented over an 18-month timeframe. We paid particular attention to disparities in V/J gene utilisation and the prevalence of shared clonotypes amongst OAPS patients and the comparative groups. When juxtaposed with observations from healthy controls and SLE patients, immune repertoire sequencing disclosed irregular T- and B-cell profiles and a contraction of diversity within the OAPS group. Marked variances were found in the genomic rearrangements of the V gene, J gene, and V/J combinations. Utilising a specialised TCRß repertoire, we crafted a predictive model for OAPS classification with robust discriminative capability (AUC = 0.852). Our research unveils alterations in the TCR repertoire among OAPS patients for the first time, positing potential covert autoimmune underpinnings. These findings nominate the TCR repertoire as a prospective peripheral blood biomarker for the clinical diagnosis of OAPS and may offer valuable insights for advancing the understanding of OAPS immunologic mechanisms and prognostic outcomes.

TULIP: A transformer-based unsupervised language model for interacting peptides and T cell receptors that generalizes to unseen epitopes.

The accurate prediction of binding between T cell receptors (TCR) and their cognate epitopes is key to understanding the adaptive immune response and developing immunotherapies. Current methods face two significant limitations: the shortage of comprehensive high-quality data and the bias introduced by the selection of the negative training data commonly used in the supervised learning approaches. We propose a method, Transformer-based Unsupervised Language model for Interacting Peptides and T cell receptors (TULIP), that addresses both limitations by leveraging incomplete data and unsupervised learning and using the transformer architecture of language models. Our model is flexible and integrates all possible data sources, regardless of their quality or completeness. We demonstrate the existence of a bias introduced by the sampling procedure used in previous supervised approaches, emphasizing the need for an unsupervised approach. TULIP recognizes the specific TCRs binding an epitope, performing well on unseen epitopes. Our model outperforms state-of-the-art models and offers a promising direction for the development of more accurate TCR epitope recognition models.

Single-cell analysis of bronchoalveolar cells in inflammatory and fibrotic post-COVID lung disease.

Background: Persistent radiological lung abnormalities are evident in many survivors of acute coronavirus disease 2019 (COVID-19). Consolidation and ground glass opacities are interpreted to indicate subacute inflammation whereas reticulation is thought to reflect fibrosis. We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis. Methods: We compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern, targeting lung segments affected by the predominant abnormality. Results: CD4 central memory T cells and CD8 effector memory T cells were significantly more abundant in those with inflammatory radiology. Clustering of similar TCRs from multiple donors was a striking feature of both phenotypes, consistent with tissue localised antigen-specific immune responses. There was no enrichment for known SARS-CoV-2-reactive TCRs, raising the possibility of T cell-mediated immunopathology driven by failure in immune self-tolerance. Conclusions: Post-COVID radiological inflammation and fibrosis show evidence of shared antigen-specific T cell responses, suggesting a role for therapies targeting T cells in limiting post-COVID lung damage.

Chimeric antigen receptor T-cell therapy in childhood acute myeloid leukemia: how far are we from a clinical application?

Recurrent and/or refractory (R/R) pediatric acute myeloid leukemia (AML) remains a recalcitrant disease with poor outcomes. Cell therapy with genetically modified immune effector cells holds the promise to improve outcomes for R/R AML since it relies on cytotoxic mechanisms that are distinct from chemotherapeutic agents. While T cells expressing chimeric antigen receptors (CAR T cells) showed significant anti-AML activity in preclinical models, early phase clinical studies have demonstrated limited activity, irrespective of the targeted AML antigen. Lack of efficacy is most likely multifactorial, including: (i) a limited array of AML-specific targets and target antigen heterogeneity; (ii) the aggressive nature of R/R AML and heavy pretreatment of patients; (iii) T-cell product manufacturing, and (iv) limited expansion and persistence of the CAR T cells, which is in part driven by the immunosuppressive AML microenvironment. Here we review the results of early phase clinical studies with AML-specific CAR T cells, and avenues investigators are exploring to improve their effector function.

Chimeric antigen receptor T-cell therapy for T-cell acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell therapy is a new and effective treatment for patients with hematologic malignancies. Clinical responses to CAR T cells in leukemia, lymphoma, and multiple myeloma have provided strong evidence of the antitumor activity of these cells. In patients with refractory or relapsed B-cell acute lymphoblastic leukemia (ALL), the infusion of autologous anti-CD19 CAR T cells is rapidly gaining standard-of-care status and might eventually be incorporated into frontline treatment. In T-ALL, however, leukemic cells generally lack surface molecules recognized by established CAR, such as CD19 and CD22. Such deficiency is particularly important, as outcome is dismal for patients with T-ALL that is refractory to standard chemotherapy and/or hematopoietic stem cell transplant. Recently, CAR T-cell technologies directed against T-cell malignancies have been developed and are beginning to be tested clinically. The main technical obstacles stem from the fact that malignant and normal T cells share most surface antigens. Therefore, CAR T cells directed against T-ALL targets might be susceptible to self-elimination during manufacturing and/or have suboptimal activity after infusion. Moreover, removing leukemic cells that might be present in the cell source used for CAR T-cell manufacturing might be problematic. Finally, reconstitution of T cells and natural killer cells after CAR T-cell infusion might be impaired. In this article, we discuss potential targets for CAR T-cell therapy of T-ALL with an emphasis on CD7, and review CAR configurations as well as early clinical results.

Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations.

Hotspot driver mutations presented by human leukocyte antigens might be recognized by anti-tumor T cells. Based on their advantages of tumor-specificity and immunogenicity, neoantigens derived from hotspot mutations, such as PIK3CAH1047L, may serve as emerging targets for cancer immunotherapies. NetMHCpan V4.1 was utilized for predicting neoepitopes of PIK3CA hotspot mutation. Using in vitro stimulation, antigen-specific T cells targeting the HLA-A*11:01-restricted PIK3CA mutation were isolated from healthy donor-derived peripheral blood mononuclear cells. T cell receptors (TCRs) were cloned using single-cell PCR and sequencing. Their functionality was assessed through T cell activation markers, cytokine production and cytotoxic response to cancer cell lines pulsed with peptides or transduced genes of mutant PIK3CA. Immunogenic mutant antigens from PIK3CA and their corresponding CD8+ T cells were identified. These PIK3CA mutation-specific CD8+ T cells were subsequently enriched, and their TCRs were isolated. The TCR clones exhibited mutation-specific and HLA-restricted reactivity, demonstrating varying degrees of functional avidity. Identified TCR genes were transferred into CD8+ Jurkat cells and primary T cells deficient of endogenous TCRs. TCR-expressing cells demonstrated specific recognition and reactivity against the PIK3CAH1047L peptide presented by HLA-A*11:01-expressing K562 cells. Furthermore, mutation-specific TCR-T cells demonstrated an elevation in cytokine production and profound cytotoxic effects against HLA-A*11:01+ malignant cell lines harboring PIK3CAH1047L. Our data demonstrate the immunogenicity of an HLA-A*11:01-restricted PIK3CA hotspot mutation and its targeting therapeutic potential, together with promising candidates of TCR-T cell therapy.

HeteroTCR: A heterogeneous graph neural network-based method for predicting peptide-TCR interaction.

Identifying interactions between T-cell receptors (TCRs) and immunogenic peptides holds profound implications across diverse research domains and clinical scenarios. Unsupervised clustering models (UCMs) cannot predict peptide-TCR binding directly, while supervised predictive models (SPMs) often face challenges in identifying antigens previously unencountered by the immune system or possessing limited TCR binding repertoires. Therefore, we propose HeteroTCR, an SPM based on Heterogeneous Graph Neural Network (GNN), to accurately predict peptide-TCR binding probabilities. HeteroTCR captures within-type (TCR-TCR or peptide-peptide) similarity information and between-type (peptide-TCR) interaction insights for predictions on unseen peptides and TCRs, surpassing limitations of existing SPMs. Our evaluation shows HeteroTCR outperforms state-of-the-art models on independent datasets. Ablation studies and visual interpretation underscore the Heterogeneous GNN module's critical role in enhancing HeteroTCR's performance by capturing pivotal binding process features. We further demonstrate the robustness and reliability of HeteroTCR through validation using single-cell datasets, aligning with the expectation that pMHC-TCR complexes with higher predicted binding probabilities correspond to increased binding fractions.

Combining SiRPα decoy-coengineered T cells and antibodies augments macrophage-mediated phagocytosis of tumor cells.

The adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells (ACT) targeting the HLA-A2-restricted cancer-testis epitope NY-ESO-1157-165 (A2/NY) has yielded favorable clinical responses against several cancers. Two approaches to improve ACT are TCR affinity optimization and T cell coengineering to express immunomodulatory molecules that can exploit endogenous immunity. By computational design we previously developed a panel of binding-enhanced A2/NY-TCRs including A97L, which augmented the in vitro function of gene-modified T cells as compared with WT. Here, we demonstrated higher persistence and improved tumor control by A97L-T cells. In order to harness macrophages in tumors, we further coengineered A97L-T cells to secrete a high-affinity signal regulatory protein α (SiRPα) decoy (CV1) that blocks CD47. While CV1-Fc-coengineered A97L-T cells mediated significantly better control of tumor outgrowth and survival in Winn assays, in subcutaneous xenograft models the T cells, coated by CV1-Fc, were depleted. Importantly, there was no phagocytosis of CV1 monomer-coengineered T cells by human macrophages. Moreover, avelumab and cetuximab enhanced macrophage-mediated phagocytosis of tumor cells in vitro in the presence of CV1 and improved tumor control upon coadministration with A97L-T cells. Taken together, our study indicates important clinical promise for harnessing macrophages by combining CV1-coengineered TCR-T cells with targeted antibodies to direct phagocytosis against tumor cells.

qPCR assay for detection of Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Elements from CAR-T and TCR-T cells in fresh and formalin-fixed tissue.

As adoptive cellular therapies become more commonplace in cancer care, there is a growing need to monitor site-specific localization of engineered cells-such as chimeric antigen receptor T (CAR-T) cells and T-cell receptor T (TCR-T) cells-in patients' tissues to understand treatment effectiveness as well as associated adverse events. Manufacturing CAR-T and TCR-T cells involves transduction with viral vectors commonly containing the WPRE gene sequence to enhance gene expression, providing a viable assay target unique to these engineered cells. Quantitative PCR (qPCR) is currently used clinically in fresh patient tissue samples and blood with target sequences specific to each immunotherapy product. Herein, we developed a WPRE-targeted qPCR assay that is broadly applicable for detection of engineered cell products in both fresh and archival formalin-fixed paraffin embedded (FFPE) tissues. Using both traditional PCR and SYBR Green PCR protocols, we demonstrate the use of this WPRE-targeted assay to successfully detect two CAR-T cell and two TCR-T cell products in FFPE tissue. Standard curve analysis reported a reproducible limit of detection at 100 WPRE copies per 20µL PCR reaction. This novel and inexpensive technique could provide better understanding of tissue abundance of engineered therapeutic T cells in both tumor and second-site toxicity tissues and provide quantitative assessment of immune effector cell trafficking in archival tissue.

Immunoglobulin and T cell receptor repertoire changes induced by a prototype vaccine against Chagas disease in naïve rhesus macaques.

BACKGROUND: A vaccine against Trypanosoma cruzi, the agent of Chagas disease, would be an excellent additional tool for disease control. A recombinant vaccine based on Tc24 and TSA1 parasite antigens was found to be safe and immunogenic in naïve macaques. METHODS: We used RNA-sequencing and performed a transcriptomic analysis of PBMC responses to vaccination of naïve macaques after each vaccine dose, to shed light on the immunogenicity of this vaccine and guide the optimization of doses and formulation. We identified differentially expressed genes and pathways and characterized immunoglobulin and T cell receptor repertoires. RESULTS: RNA-sequencing analysis indicated a clear transcriptomic response of PBMCs after three vaccine doses, with the up-regulation of several immune cell activation pathways and a broad non-polarized immune profile. Analysis of the IgG repertoire showed that it had a rapid turnover with novel IgGs produced following each vaccine dose, while the TCR repertoire presented several persisting clones that were expanded after each vaccine dose. CONCLUSIONS: These data suggest that three vaccine doses may be needed for optimum immunogenicity and support the further evaluation of the protective efficacy of this vaccine.

T Cell Activators Exhibit Distinct Downstream Effects on Chimeric Antigen Receptor T Cell Phenotype and Function.

T cell activation is an essential step in chimeric Ag receptor (CAR) T (CAR T) cell manufacturing and is accomplished by the addition of activator reagents that trigger the TCR and provide costimulation. We explore several T cell activation reagents and examine their effects on key attributes of CAR T cell cultures, such as activation/exhaustion markers, cell expansion, gene expression, and transduction efficiency. Four distinct activators were examined, all using anti-CD3 and anti-CD28, but incorporating different mechanisms of delivery: Dynabeads (magnetic microspheres), TransAct (polymeric nanomatrix), Cloudz (alginate hydrogel), and Microbubbles (lipid membrane containing perfluorocarbon gas). Clinical-grade lentiviral vector was used to transduce cells with a bivalent CD19/CD22 CAR, and cell counts and flow cytometry were used to monitor the cells throughout the culture. We observed differences in CD4/CD8 ratio when stimulating with the Cloudz activator, where there was a significant skewing toward CD8 T cells. The naive T cell subset expressing CD62L+CCR7+CD45RA+ was the highest in all donors when stimulating with Dynabeads, whereas effector/effector memory cells were highest when using the Cloudz. Functional assays demonstrated differences in killing of target cells and proinflammatory cytokine secretion, with the highest killing from the Cloudz-stimulated cells among all donors. This study demonstrates that the means by which these stimulatory Abs are presented to T cells contribute to the activation, resulting in differing effects on CAR T cell function. These studies highlight important differences in the final product that should be considered when manufacturing CAR T cells for patients in the clinic.

Advances in research on factors affecting chimeric antigen receptor T-cell efficacy.

Chimeric antigen receptor T-cell (CAR-T) therapy is becoming an effective technique for the treatment of patients with relapsed/refractory hematologic malignancies. After analyzing patients with tumor progression and sustained remission after CAR-T cell therapy, many factors were found to be associated with the efficacy of CAR-T therapy. This paper reviews the factors affecting the effect of CAR-T such as tumor characteristics, tumor microenvironment and immune function of patients, CAR-T cell structure, construction method and in vivo expansion values, lymphodepletion chemotherapy, and previous treatment, and provides a preliminary outlook on the corresponding therapeutic strategies.

DIRMC: a database of immunotherapy-related molecular characteristics.

Cancer immunotherapy has brought about a revolutionary breakthrough in the field of cancer treatment. Immunotherapy has changed the treatment landscape for a variety of solid and hematologic malignancies. To assist researchers in efficiently uncovering valuable information related to cancer immunotherapy, we have presented a manually curated comprehensive database called DIRMC, which focuses on molecular features involved in cancer immunotherapy. All the content was collected manually from published literature, authoritative clinical trial data submitted by clinicians, some databases for drug target prediction such as DrugBank, and some experimentally confirmed high-throughput data sets for the characterization of immune-related molecular interactions in cancer, such as a curated database of T-cell receptor sequences with known antigen specificity (VDJdb), a pathology-associated TCR database (McPAS-TCR) et al. By constructing a fully connected functional network, ranging from cancer-related gene mutations to target genes to translated target proteins to protein regions or sites that may specifically affect protein function, we aim to comprehensively characterize molecular features related to cancer immunotherapy. We have developed the scoring criteria to assess the reliability of each MHC-peptide-T-cell receptor (TCR) interaction item to provide a reference for users. The database provides a user-friendly interface to browse and retrieve data by genes, target proteins, diseases and more. DIRMC also provides a download and submission page for researchers to access data of interest for further investigation or submit new interactions related to cancer immunotherapy targets. Furthermore, DIRMC provides a graphical interface to help users predict the binding affinity between their own peptide of interest and MHC or TCR. This database will provide researchers with a one-stop resource to understand cancer immunotherapy-related targets as well as data on MHC-peptide-TCR interactions. It aims to offer reliable molecular characteristics support for both the analysis of the current status of cancer immunotherapy and the development of new immunotherapy. DIRMC is available at http://www.dirmc.tech/. Database URL: http://www.dirmc.tech/.

Self-sufficient primary natural killer cells engineered to express T cell receptors and interleukin-15 exhibit improved effector function and persistence.

Background: NK cells can be genetically engineered to express a transgenic T-cell receptor (TCR). This approach offers an alternative strategy to target heterogenous tumors, as NK:TCR cells can eradicate both tumor cells with high expression of HLA class I and antigen of interest or HLA class I negative tumors. Expansion and survival of NK cells relies on the presence of IL-15. Therefore, autonomous production of IL-15 by NK:TCR cells might improve functional persistence of NK cells. Here we present an optimized NK:TCR product harnessed with a construct encoding for soluble IL-15 (NK:TCR/IL-15), to support their proliferation, persistence and cytotoxic capabilities. Methods: Expression of tumor-specific TCRs in peripheral blood derived NK-cells was achieved following retroviral transduction. NK:TCR/IL-15 cells were compared with NK:TCR cells for autonomous cytokine production, proliferation and survival. NK:BOB1-TCR/IL-15 cells, expressing a HLA-B*07:02-restricted TCR against BOB1, a B-cell lineage specific transcription factor highly expressed in all B-cell malignancies, were compared with control NK:BOB1-TCR and NK:CMV-TCR/IL-15 cells for effector function against TCR antigen positive malignant B-cell lines in vitro and in vivo. Results: Viral incorporation of the interleukin-15 gene into engineered NK:TCR cells was feasible and high expression of the TCR was maintained, resulting in pure NK:TCR/IL-15 cell products generated from peripheral blood of multiple donors. Self-sufficient secretion of IL-15 by NK:TCR cells enables engineered NK cells to proliferate in vitro without addition of extra cytokines. NK:TCR/IL-15 demonstrated a marked enhancement of TCR-mediated cytotoxicity as well as enhanced NK-mediated cytotoxicity resulting in improved persistence and performance of NK:BOB1-TCR/IL-15 cells in an orthotopic multiple myeloma mouse model. However, in contrast to prolonged anti-tumor reactivity by NK:BOB1-TCR/IL-15, we observed in one of the experiments an accumulation of NK:BOB1-TCR/IL-15 cells in several organs of treated mice, leading to unexpected death 30 days post-NK infusion. Conclusion: This study showed that NK:TCR/IL-15 cells secrete low levels of IL-15 and can proliferate in an environment lacking cytokines. Repeated in vitro and in vivo experiments confirmed the effectiveness and target specificity of our product, in which addition of IL-15 supports TCR- and NK-mediated cytotoxicity.

Controlling the Potency of T Cell Activation Using an Optically Tunable Chimeric Antigen Receptor.

The ability of biological systems to convert inputs from their environment into information to guide future decisions is central to life and a matter of great importance. While we know the components of many of the signaling networks that make these decisions, our understanding of the dynamic flow of information between these parts remains far more limited. T cells are an essential white blood cell type of an adaptive immune response and can discriminate between healthy and infected cells with remarkable sensitivity. This chapter describes the use of a synthetic T-cell receptor (OptoCAR) that is optically tunable within cell conjugates, providing control over the duration, and intensity of intracellular T-cell signaling dynamics. Optical control can also provide control over signaling with high spatial precision, and the OptoCAR is likely to find application more generally when modulating T-cell function with imaging approaches.

Measurement of Forces Acting on Single T-Cell Receptors.

Molecular forces are increasingly recognized as an important parameter to understand cellular signaling processes. In the recent years, evidence accumulated that also T-cells exert tensile forces via their T-cell receptor during the antigen recognition process. To measure such intercellular pulling forces, one can make use of the elastic properties of spider silk peptides, which act similar to Hookean springs: increased strain corresponds to increased stress applied to the peptide. Combined with Förster resonance energy transfer (FRET) to read out the strain, such peptides represent powerful and versatile nanoscopic force sensing tools. In this paper, we provide a detailed protocol how to synthesize a molecular force sensor for application in T-cell antigen recognition and hands-on guidelines on experiments and analysis of obtained single molecule FRET data.