RESUMO
Aging is a natural process that compromises the immune system's functionality increasing the risk of infectious, tumors, and autoimmune diseases. The thymus involution is an age-dependent process characterized by decreased cellularity, peripheral lymphocyte infiltration into the perivascular space, and expansion of adipose tissue. All those modifications hamper the functionality of the organ and lead to a decline of naïve T-cell production with a shrinking of the T-cell repertoire. Thymus atrophy is described in several disorders including autoimmune diseases. The quantification of T-cell receptor excision circles (TRECs) in recent thymus emigrants is a standard procedure to investigate the thymic function. In this chapter, we discuss the methodology used to quantify this molecule in peripheral blood mononuclear cells and isolated CD4+ and CD8+ T cells.
Assuntos
Linfócitos T CD8-Positivos , Receptores de Antígenos de Linfócitos T , Timo , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Timo/imunologia , Timo/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/imunologiaRESUMO
The T-cell receptor (TCR) is the key molecule involved in the adaptive immune response. It is generated by the V(D)J recombination, responsible of the enormous diversity of the TCR repertoire, a crucial feature determining the individual capability to response to antigens and to build immunological memory. A pivotal role in the recognition of antigen is played by the hypervariable complementarity-determining region 3 (CDR3) of the V-beta chain of TCR. Investigating the CDR3 supports the understanding of the adaptive immune system dynamics in physiological processes, such as immune aging, and in disease, especially autoimmune disorders in which T cells are main actors. High-throughput sequencing (HTS) paved the way for a great progress in the investigation of TCR repertoire, enhancing the read depth in the process of library generation of sequencing and the number of samples that can be analyzed simultaneously. Therefore, the leverage of big datasets stressed the need to develop computational approach, by bioinformatics, to unravel the characteristics of the TCR repertoire.
Assuntos
Regiões Determinantes de Complementaridade , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Receptores de Antígenos de Linfócitos T , Linfócitos T , Fluxo de Trabalho , Biologia Computacional/métodos , Humanos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Regiões Determinantes de Complementaridade/genética , Separação Celular/métodos , Recombinação V(D)JRESUMO
BACKGROUND: Cytomegalovirus (CMV) reactivation is a serious problem in recipients of allogeneic hematopoietic stem cell transplantation. Long-term latency depends on specific T cell immune reconstitution, which identifies various pathogens by T cell receptors (TCRs). However, the mechanisms underlying the selection of CMV-specific TCRs in recipients after transplantation remain unclear. METHODS: Using high-throughput sequencing and bioinformatics analysis, the T cell immune repertoire of seven CMV reactivated recipients (CRRs) were analyzed and compared to those of seven CMV non-activated recipients (CNRs) at an early stage after transplant. RESULTS: The counts of unique complementarity-determining region 3 (CDR3) were significantly higher in CNRs than in CRRs. The CDR3 clones in the CNRs exhibit higher homogeneity compared to the CRRs. With regard to T cell receptor ß-chain variable region (TRBV) and joint region (TRBJ) genotypes, significant differences were observed in the frequencies of TRBV6, BV23, and BV7-8 between the two groups. In addition to TRBV29-1/BJ1-2, TRBV2/BJ2-2, and TRBV12-4/BJ1-5, 11 V-J combinations had significantly different expression levels between CRRs and CNRs. CONCLUSIONS: The differences in TCR diversity, TRBV segments, and TRBV-BJ combinations observed between CNRs and CRRs might be associated with post-transplant CMV reactivation and could serve as a foundation for further research.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Transplante de Células-Tronco Hematopoéticas , Receptores de Antígenos de Linfócitos T , Transplante Homólogo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Citomegalovirus/imunologia , Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/imunologia , Masculino , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Adulto , Feminino , Pessoa de Meia-Idade , Transplante Homólogo/efeitos adversos , Regiões Determinantes de Complementaridade/genética , Transplantados , Sequenciamento de Nucleotídeos em Larga Escala , Adulto Jovem , Ativação Viral , Genótipo , Linfócitos T/imunologia , Adolescente , Biologia Computacional/métodosRESUMO
Cancers eventually kill hosts even when infiltrated by cancer-specific T cells. We examined whether cancer-specific T cell receptors of CD4+ T cells (CD4TCRs) from tumor-bearing hosts can be exploited for adoptive TCR therapy. We focused on CD4TCRs targeting an autochthonous mutant neoantigen that is only presented by stroma surrounding the MHC class II-negative cancer cells. The 11 most common tetramer-sorted CD4TCRs were tested using TCR-engineered CD4+ T cells. Three TCRs were characterized by convergent recombination for which multiple T cell clonotypes differed in their nucleotide sequences but encoded identical TCR α and ß chains. These preferentially selected TCRs destroyed tumors equally well and halted progression through reprogramming of the tumor stroma. TCRs represented by single T cell clonotypes were similarly effective only if they shared CDR elements with preferentially selected TCRs in both α and ß chains. Selecting candidate TCRs on the basis of these characteristics can help identify TCRs that are potentially therapeutically effective.
Assuntos
Linfócitos T CD4-Positivos , Imunoterapia Adotiva , Linfócitos T CD4-Positivos/imunologia , Animais , Imunoterapia Adotiva/métodos , Camundongos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Camundongos Endogâmicos C57BL , Humanos , Camundongos Transgênicos , Feminino , Recombinação Genética/imunologiaRESUMO
The thymus plays a pivotal role in generating a highly-diverse repertoire of T lymphocytes while preventing autoimmunity. Thymus seeding progenitors (TSPs) are a heterogeneous group of multipotent progenitors that migrate to the thymus via CCR7 and CCR9 receptors. While NOTCH guides thymus progenitors toward T cell fate, the absence or disruption of NOTCH signaling renders the thymus microenvironment permissive to other cell fates. Following T cell commitment, developing T cells undergo multiple selection checkpoints by engaging with the extracellular matrix, and interacting with thymic epithelial cells (TECs) and other immune subsets across the different compartments of the thymus. The different selection checkpoints assess the T cell receptor (TCR) performance, with failure resulting in either repurposing (agonist selection), or cell death. Additionally, environmental cues such as inflammation and endocrine signaling induce acute thymus atrophy, contributing to the demise of most developing T cells during thymic selection. We discuss the occurrence of acute thymus atrophy in response to systemic inflammation. The thymus demonstrates high plasticity, shaping inflammation by abrogating T cell development and undergoing profound structural changes, and facilitating regeneration and restoration of T cell development once inflammation is resolved. Despite the challenges, thymic selection ensures a highly diverse T cell repertoire capable of discerning between self and non-self antigens, ultimately egressing to secondary lymphoid organs where they complete their maturation and exert their functions.
Assuntos
Atrofia , Linfócitos T , Timo , Timo/imunologia , Timo/patologia , Humanos , Animais , Linfócitos T/imunologia , Movimento Celular/imunologia , Transdução de Sinais , Diferenciação Celular/imunologia , Inflamação/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
BACKGROUND: T cells, the "superstar" of the immune system, play a crucial role in antitumor immunity. T-cell receptors (TCR) are crucial molecules that enable T cells to identify antigens and start immunological responses. The body has evolved a unique method for rearrangement, resulting in a vast diversity of TCR repertoires. A healthy TCR repertoire is essential for the particular identification of antigens by T cells. METHODS: In this article, we systematically summarized the TCR creation mechanisms and analysis methodologies, particularly focusing on the application of next-generation sequencing (NGS) technology. We explore the TCR repertoire in health and cancer, and discuss the implications of TCR repertoire analysis in understanding carcinogenesis, cancer progression, and treatment. RESULTS: The TCR repertoire analysis has enormous potential for monitoring the emergence and progression of malignancies, as well as assessing therapy response and prognosis. The application of NGS has dramatically accelerated our comprehension of TCR diversity and its role in cancer immunity. CONCLUSIONS: To substantiate the significance of TCR repertoires as biomarkers, more thorough and exhaustive research should be conducted. The TCR repertoire analysis, enabled by advanced sequencing technologies, is poised to become a crucial tool in the future of cancer diagnosis, monitoring, and therapy evaluation.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias , Receptores de Antígenos de Linfócitos T , Humanos , Neoplasias/imunologia , Neoplasias/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Biomarcadores Tumorais/genética , PrognósticoRESUMO
CD8+ T cells destroy insulin-producing pancreatic ß cells in type 1 diabetes through HLA class I-restricted presentation of self-antigens. Combinatorial peptide library screening was used to produce a preferred peptide recognition landscape for a patient-derived T cell receptor (TCR) that recognized the preproinsulin-derived (PPI-derived) peptide sequence LWMRLLPLL in the context of disease risk allele HLA A*24:02. Data were used to generate a strong superagonist peptide, enabling production of an autoimmune HLA A*24:02-peptide-TCR structure by crystal seeding. TCR binding to the PPI epitope was strongly focused on peptide residues Arg4 and Leu5, with more flexibility at other positions, allowing the TCR to strongly engage many peptides derived from pathogenic bacteria. We confirmed an epitope from Klebsiella that was recognized by PPI-reactive T cells from 3 of 3 HLA A*24:02+ patients. Remarkably, the same epitope selected T cells from 7 of 8 HLA A*24+ healthy donors that cross-reacted with PPI, leading to recognition and killing of HLA A*24:02+ cells expressing PPI. These data provide a mechanism by which molecular mimicry between pathogen and self-antigens could have resulted in the breaking of self-tolerance to initiate disease.
Assuntos
Diabetes Mellitus Tipo 1 , Antígeno HLA-A24 , Insulina , Precursores de Proteínas , Receptores de Antígenos de Linfócitos T , Humanos , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/genética , Precursores de Proteínas/imunologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Insulina/imunologia , Insulina/metabolismo , Antígeno HLA-A24/imunologia , Antígeno HLA-A24/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/genética , Linfócitos T CD8-Positivos/imunologia , Feminino , MasculinoRESUMO
Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic malignancies, offering remarkable remission rates in otherwise refractory conditions. However, its expansion into broader oncological applications faces significant hurdles, including limited efficacy in solid tumors, safety concerns related to toxicity, and logistical challenges in manufacturing and scalability. This review critically examines the latest advancements aimed at overcoming these obstacles, highlighting innovations in CAR T-cell engineering, novel antigen targeting strategies, and improvements in delivery and persistence within the tumor microenvironment. We also discuss the development of allogeneic CAR T cells as off-the-shelf therapies, strategies to mitigate adverse effects, and the integration of CAR T cells with other therapeutic modalities. This comprehensive analysis underscores the synergistic potential of these strategies to enhance the safety, efficacy, and accessibility of CAR T-cell therapies, providing a forward-looking perspective on their evolutionary trajectory in cancer treatment.
Assuntos
Imunoterapia Adotiva , Neoplasias , Receptores de Antígenos Quiméricos , Biologia Sintética , Microambiente Tumoral , Humanos , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/economia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Neoplasias/terapia , Neoplasias/imunologia , Biologia Sintética/métodos , Microambiente Tumoral/imunologia , Animais , Linfócitos T/imunologia , Linfócitos T/transplante , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
CD8+ T cells exert immunological pressure against immunodeficiency lentiviruses. In previous studies, we examined the TCR repertoire of CD8+ T cells specific for a single SIV immunodominant epitope, Gag-CM9, throughout SIV infection or after vaccination, and across multiple anatomic sites. We identified both tissue specific TCR sequences and TCRs shared by multiple anatomical sites. Here we use single cell RNA sequencing to evaluate if the tissue localization or TCR sequence of a CM9-specific CD8+ T cell corresponds with unique transcriptomics. CM9-specific CD8+ T cells were sorted from blood, lymph nodes, spleen, and liver from SIV infected rhesus macaques with progressive SIV infection and in animals who spontaneously control SIV replication after cessation of antiretroviral therapy. The cells were processed through a single cell sequencing protocol, creating a TCR amplified library and an RNA gene expression library corresponding to individual cells. Gene set enrichment analysis revealed no distinct transcriptional profiles for CM9 specific CD8+ T cells between different anatomical sites and between cells with shared or tissue specific TCRs. Similarly, no clear transcriptional profiles were associated with clonotypes which were shared across individual animals. However, CM9 specific CD8+ T cells from posttreatment controllers did exhibit enrichment of pathways associated with cellular activation compared to progressively infected animals, suggesting that altered transcription in distinct cellular pathways in antigen specific CD8+ T cells may associate with viral control. Together, these studies represent a thorough analysis of the relationship between anatomical and clonal origin, and the transcriptional profile of antigen specific CD8+ T cells and unravel pathways that may be important for CD8+ T cell mediated control of SIV replication.
Assuntos
Linfócitos T CD8-Positivos , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Linfócitos T CD8-Positivos/imunologia , Vírus da Imunodeficiência Símia/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Receptores de Antígenos de Linfócitos T/imunologia , MultiômicaRESUMO
Multiple synchronous lung cancers (MSLCs) constitute a unique subtype of lung cancer. To explore the genomic and immune heterogeneity across different pathological stages of MSLCs, we analyse 16 MSLCs from 8 patients using single-cell RNA-seq, single-cell TCR sequencing, and bulk whole-exome sequencing. Our investigation indicates clonally independent tumours with convergent evolution driven by shared driver mutations. However, tumours from the same individual exhibit few shared mutations, indicating independent origins. During the transition from pre-invasive to invasive adenocarcinoma, we observe a shift in T cell phenotypes characterized by increased Treg cells and exhausted CD8+ T cells, accompanied by diminished cytotoxicity. Additionally, invasive adenocarcinomas exhibit greater neoantigen abundance and a more diverse TCR repertoire, indicating heightened heterogeneity. In summary, despite having a common genetic background and environmental exposure, our study emphasizes the individuality of MSLCs at different stages, highlighting their unique genomic and immune characteristics.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Mutação , Análise de Célula Única , Humanos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Sequenciamento do Exoma , Feminino , Genômica , Masculino , Linfócitos T CD8-Positivos/imunologia , Pessoa de Meia-Idade , Heterogeneidade Genética , Idoso , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Neoplasias Primárias Múltiplas/genética , Neoplasias Primárias Múltiplas/imunologia , Neoplasias Primárias Múltiplas/patologiaRESUMO
Mechanical force has repeatedly been highlighted to be involved in T cell activation. However, the biological significance of mechanical force for T cell receptor signaling remains under active consideration. Here, guided by theoretical analysis, we provide a perspective on how mechanical forces between a T cell and an antigen-presenting cell can influence the bond of a single T cell receptor major histocompatibility complex during early T cell activation. We point out that the lifetime of T cell receptor bonds and thus the degree of their phosphorylation which is essential for T cell activation depends considerably on the T cell receptor rigidity and the average magnitude and frequency of an applied oscillatory force. Such forces could be, for example, produced by protrusions like microvilli during early T cell activation or invadosomes during full T cell activation. These features are suggestive of mechanical force being exploited by T cells to advance self-nonself discrimination in early T cell activation.
Assuntos
Ativação Linfocitária , Receptores de Antígenos de Linfócitos T , Linfócitos T , Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Humanos , Animais , Fosforilação , Transdução de Sinais/imunologia , Fenômenos Biomecânicos , Células Apresentadoras de Antígenos/imunologiaRESUMO
Programming cell signaling during T-cell activation represents a simple strategy for improving the potency of therapeutic T-cell products. Stim-R technology (Lyell Immunopharma) is a customizable, degradable synthetic cell biomimetic that emulates physiologic, cell-like presentation of signal molecules to control T-cell activation. A breadth of Stim-R formulations with different anti-CD3/anti-CD28 (αCD3/αCD28) antibody densities and stoichiometries were screened for their effects on multiple metrics of T-cell function. We identified an optimized formulation that produced receptor tyrosine kinase-like orphan receptor 1 (ROR1)-targeted chimeric antigen receptor (CAR) T cells with enhanced persistence and polyfunctionality in vitro, as assessed in repeat-stimulation assays, compared with a benchmark product generated using a conventional T-cell-activating reagent. In transcriptomic analyses, CAR T cells activated with Stim-R technology showed downregulation of exhaustion-associated gene sets and retained a unique subset of stem-like cells with effector-associated gene signatures following repeated exposure to tumor cells. Compared with the benchmark product, CAR T cells activated using the optimized Stim-R technology formulation exhibited higher peak expansion, prolonged persistence, and improved tumor control in a solid tumor xenograft model. Enhancing T-cell products with Stim-R technology during T-cell activation may help improve therapeutic efficacy against solid tumors.
Assuntos
Ativação Linfocitária , Receptores de Antígenos Quiméricos , Transdução de Sinais , Linfócitos T , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/genética , Humanos , Animais , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Camundongos , Imunoterapia Adotiva/métodos , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Antígenos CD28/imunologia , Antígenos CD28/metabolismoRESUMO
High-throughput immune repertoire (IR) sequencing provides direct insight into the diversity of B cell receptor (BCR) and T cell receptor (TCR), with great potential to revolutionize the diagnosis, monitoring, and prevention of immune system-related disorders. In this study, multiplex PCR was applied to amplify the complementarity-determining regions of BCR and TCR, followed by comprehensive analysis by high-throughput sequencing. We compare the TCR (BCR) of bone marrow fluid (BMF) and peripheral blood (PB) samples from 17 patients in the Epstein-Barr and immunodeficiency groups, respectively. Our study shows that the diversity of the IR of blood samples is very similar to that of bone marrow samples statistically. However, the distributions of the monoclonal genes are significantly different in these 2 samples of most patients. This suggests that the BMFs can be replaced by the PB samples in diversity detection of IR to monitor the immune status of the body, while the detection of the BMFs is unreplaceable when the monoclonal change occurs. We used high-throughput sequencing to assess the TCR and BCR of the patients and provide a basis for the clinical analysis of PB and bone marrow samples and selection of disease diagnosis and monitoring methods.
Assuntos
Medula Óssea , Infecções por Vírus Epstein-Barr , Sequenciamento de Nucleotídeos em Larga Escala , Receptores de Antígenos de Linfócitos T , Humanos , Estudos Retrospectivos , Infecções por Vírus Epstein-Barr/imunologia , Masculino , Feminino , Medula Óssea/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Criança , Adulto , Adolescente , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/genética , Pré-Escolar , Pessoa de Meia-Idade , Adulto JovemRESUMO
Long COVID occurs in a small but important minority of patients following COVID-19, reducing quality of life and contributing to healthcare burden. Although research into underlying mechanisms is evolving, immunity is understudied. SARS-CoV-2-specific T cell responses are of key importance for viral clearance and COVID-19 recovery. However, in long COVID, the establishment and persistence of SARS-CoV-2-specific T cells are far from clear, especially beyond 12 mo postinfection and postvaccination. We defined ex vivo antigen-specific B cell and T cell responses and their T cell receptors (TCR) repertoires across 2 y postinfection in people with long COVID. Using 13 SARS-CoV-2 peptide-HLA tetramers, spanning 11 HLA allotypes, as well as spike and nucleocapsid probes, we tracked SARS-CoV-2-specific CD8+ and CD4+ T cells and B-cells in individuals from their first SARS-CoV-2 infection through primary vaccination over 24 mo. The frequencies of ORF1a- and nucleocapsid-specific T cells and B cells remained stable over 24 mo. Spike-specific CD8+ and CD4+ T cells and B cells were boosted by SARS-CoV-2 vaccination, indicating immunization, in fully recovered and people with long COVID, altered the immunodominance hierarchy of SARS-CoV-2 T cell epitopes. Meanwhile, influenza-specific CD8+ T cells were stable across 24 mo, suggesting no bystander-activation. Compared to total T cell populations, SARS-CoV-2-specific T cells were enriched for central memory phenotype, although the proportion of central memory T cells decreased following acute illness. Importantly, TCR repertoire composition was maintained throughout long COVID, including postvaccination, to 2 y postinfection. Overall, we defined ex vivo SARS-CoV-2-specific B cells and T cells to understand primary and recall responses, providing key insights into antigen-specific responses in people with long COVID.
Assuntos
Linfócitos T CD8-Positivos , COVID-19 , Receptores de Antígenos de Linfócitos T , SARS-CoV-2 , Humanos , Linfócitos T CD8-Positivos/imunologia , SARS-CoV-2/imunologia , COVID-19/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Epitopos de Linfócito T/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Pessoa de Meia-Idade , Masculino , Feminino , Síndrome de COVID-19 Pós-Aguda , Fenótipo , Linfócitos B/imunologia , Memória Imunológica/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , IdosoRESUMO
Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRß H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.
Assuntos
Fragmentos Fab das Imunoglobulinas , Transdução de Sinais , Linfócitos T , Humanos , Cinética , Ligantes , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , DNA/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Ativação Linfocitária , Mutação PuntualRESUMO
INTRODUCTION: Dual-expressing lymphocytes (DEs) are unique immune cells that express both B cell receptors (BCRs, surface antibody) and T cell receptors (TCRs). In type 1 diabetes, DE antibodies are predominated by one antibody (x-mAb), an IgM monoclonal antibody with a germline-encoded CDR3 that recognizes self-reactive TCRs. We explored if x-mAb and its interacting TCRs have distinct structural features. METHODS: Using bioinformatics, we compared x-mAb and its most common interacting TCRαß to billions of antigen receptor sequences to determine if they were unique or randomly generated. RESULTS: X-mAb represents a unique class of human antibodies with a conserved CDR3 sequence (CARx1-4DTAMVYYFYDW), consisting of a fixed DJH motif (DTAMVYYFDYW) paired with various VH genes. A public TCRß clonotype (CASSPGTEAFF) associated with x-mAb on DEs features two invariant segments, VßD (CASSPGT) and DJß (PGTEAFF), key to two large families of public TCRß clonotypes-CASSPGT-Jßx and CASSPGT-Jßx-formed by recombining the VßD motif with Jß genes and the DJß motif with Vß genes. B cells also use CASSPGT as a VHD motif for public IGH clonotypes (CASSPGT-Jßx). DISCUSSION: DEs, unlike conventional T and B cells, use invariant motifs to create public antibodies and TCRs, a trait previously seen only in cartilaginous fish.
Assuntos
Anticorpos Monoclonais , Humanos , Anticorpos Monoclonais/imunologia , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/genética , Biologia Computacional/métodos , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Motivos de Aminoácidos , Imunoglobulina M/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de AminoácidosAssuntos
Antígeno de Maturação de Linfócitos B , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/métodos , Antígeno de Maturação de Linfócitos B/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
As an adoptive cellular therapy, Chimeric Antigen Receptor T cell (CAR T cell) therapy has shown remarkable success in hematological malignancies but only limited efficacy against solid tumors. Compared with blood cancers, solid tumors present a series of challenges that ultimately combine to neutralize the function of CAR T cells. These challenges include, but are not limited to, antigen heterogeneity - variability in the expression of the antigen on tumor cells, as well as trafficking and infiltration into the solid tumor tissue. A critical question for solving the heterogeneity problem is whether CAR T therapy induces bystander effects, such as antigen spreading. Antigen spreading occurs when CAR T cells activate other endogenous antitumor CD8 T cells against antigens that were not originally targeted. In this work, we develop a mathematical model of CAR T cell therapy for solid tumors that considers both antigen heterogeneity and bystander effects. Our model is based on in vivo treatment data that includes a mixture of target antigen-positive and target antigen-negative tumor cells. We use our model to simulate large cohorts of virtual patients to better understand the relationship involving bystander killing. We also investigate several strategies for enhancing bystander effects, thus increasing CAR T cell therapy's overall efficacy for solid tumors.
Assuntos
Efeito Espectador , Imunoterapia Adotiva , Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Efeito Espectador/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/imunologia , Modelos Teóricos , Linfócitos T CD8-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Immunotherapy represents a transformative shift in cancer treatment. Among myriad immune-based approaches, chimeric antigen receptor (CAR) T-cell therapy has shown promising results in treating hematological malignancies. Despite aggressive treatment options, the prognosis for patients with malignant brain tumors remains poor. Research leveraging CAR T-cell therapy for brain tumors has surged in recent years. Pre-clinical models are crucial in evaluating the safety and efficacy of these therapies before they advance to clinical trials. However, current models recapitulate the human tumor environment to varying degrees. Novel in vitro and in vivo techniques offer the opportunity to validate CAR T-cell therapies but also have limitations. By evaluating the strengths and weaknesses of various pre-clinical glioma models, this review aims to provide a roadmap for the development and pre-clinical testing of CAR T-cell therapies for brain tumors.