RESUMO
T cell receptors (TCRs) are protein complexes formed by six different polypeptides. In most T cells, TCRs are composed of αß subunits displaying immunoglobulin-like variable domains that recognize peptide antigens associated with major histocompatibility complex molecules expressed on the surface of antigen-presenting cells. TCRαß subunits are associated with the CD3 complex formed by the γ, δ, ε, and ζ subunits, which are invariable and ensure signal transduction. Here, we review how the expression and function of TCR complexes are orchestrated by several fine-tuned cellular processes that encompass (a) synthesis of the subunits and their correct assembly and expression at the plasma membrane as a single functional complex, (b) TCR membrane localization and dynamics at the plasma membrane and in endosomal compartments, (c) TCR signal transduction leading to T cell activation, and (d) TCR degradation. These processes balance each other to ensure efficient T cell responses to a variety of antigenic stimuli while preventing autoimmunity.
Assuntos
Regulação da Expressão Gênica , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Complexo CD3/genética , Complexo CD3/metabolismo , Membrana Celular/metabolismo , Endocitose/genética , Endocitose/imunologia , Endossomos/metabolismo , Humanos , Imunomodulação , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteólise , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Relação Estrutura-AtividadeRESUMO
Cancer immunotherapy has transformed treatment possibilities, but its effectiveness differs significantly among patients, indicating the presence of alternative pathways for immune evasion. Here, we show that ITPRIPL1 functions as an inhibitory ligand of CD3ε, and its expression inhibits T cells in the tumor microenvironment. The binding of ITPRIPL1 extracellular domain to CD3ε on T cells significantly decreased calcium influx and ZAP70 phosphorylation, impeding initial T cell activation. Treatment with a neutralizing antibody against ITPRIPL1 restrained tumor growth and promoted T cell infiltration in mouse models across various solid tumor types. The antibody targeting canine ITPRIPL1 exhibited notable therapeutic efficacy against naturally occurring tumors in pet clinics. These findings highlight the role of ITPRIPL1 (or CD3L1, CD3ε ligand 1) in impeding T cell activation during the critical "signal one" phase. This discovery positions ITPRIPL1 as a promising therapeutic target against multiple tumor types.
Assuntos
Complexo CD3 , Ativação Linfocitária , Linfócitos T , Evasão Tumoral , Microambiente Tumoral , Animais , Complexo CD3/metabolismo , Complexo CD3/imunologia , Humanos , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral/imunologia , Cães , Neoplasias/imunologia , Linhagem Celular Tumoral , Feminino , Ligação Proteica , Proteína-Tirosina Quinase ZAP-70/metabolismo , Anticorpos Neutralizantes/imunologia , Camundongos Endogâmicos C57BLRESUMO
A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.
Assuntos
Complexo CD3/metabolismo , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Complexo CD3/química , Proteína Tirosina Quinase CSK/metabolismo , Linhagem Celular , Citocinas/metabolismo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Sobrevida , Vanadatos/farmacologiaRESUMO
Initiation of T cell antigen receptor (TCR) signaling involves phosphorylation of CD3 cytoplasmic tails by the tyrosine kinase Lck. How Lck is recruited to the TCR to initiate signaling is not well known. We report a previously unknown binding motif in the CD3ε cytoplasmic tail that interacts in a noncanonical mode with the Lck SH3 domain: the receptor kinase (RK) motif. The RK motif is accessible only upon TCR ligation, demonstrating how ligand binding leads to Lck recruitment. Binding of the Lck SH3 domain to the exposed RK motif resulted in local augmentation of Lck activity, CD3 phosphorylation, T cell activation and thymocyte development. Introducing the RK motif into a well-characterized 41BB-based chimeric antigen receptor enhanced its antitumor function in vitro and in vivo. Our findings underscore how a better understanding of the functioning of the TCR might promote rational improvement of chimeric antigen receptor design for the treatment of cancer.
Assuntos
Complexo CD3/metabolismo , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Motivos de Aminoácidos/imunologia , Animais , Complexo CD3/imunologia , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Gamma delta (γδ) T cells, a unique T cell subgroup, are crucial in various immune responses and immunopathology1-3. The γδ T cell receptor (TCR), which is generated by γδ T cells, recognizes a diverse range of antigens independently of the major histocompatibility complex2. The γδ TCR associates with CD3 subunits, initiating T cell activation and holding great potential in immunotherapy4. Here we report the structures of two prototypical human Vγ9Vδ2 and Vγ5Vδ1 TCR-CD3 complexes5,6, revealing two distinct assembly mechanisms that depend on Vγ usage. The Vγ9Vδ2 TCR-CD3 complex is monomeric, with considerable conformational flexibility in the TCRγ-TCRδ extracellular domain and connecting peptides. The length of the connecting peptides regulates the ligand association and T cell activation. A cholesterol-like molecule wedges into the transmembrane region, exerting an inhibitory role in TCR signalling. The Vγ5Vδ1 TCR-CD3 complex displays a dimeric architecture, whereby two protomers nestle back to back through the Vγ5 domains of the TCR extracellular domains. Our biochemical and biophysical assays further corroborate the dimeric structure. Importantly, the dimeric form of the Vγ5Vδ1 TCR is essential for T cell activation. These findings reveal organizing principles of the γδ TCR-CD3 complex, providing insights into the unique properties of γδ TCR and facilitating immunotherapeutic interventions.
Assuntos
Complexo CD3 , Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T , Humanos , Complexo CD3/química , Complexo CD3/imunologia , Complexo CD3/metabolismo , Complexo CD3/ultraestrutura , Colesterol/metabolismo , Colesterol/química , Microscopia Crioeletrônica , Ligantes , Ativação Linfocitária/imunologia , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/ultraestrutura , Linfócitos T/química , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução de Sinais , Membrana Celular/química , Membrana Celular/metabolismoRESUMO
Cholesterol molecules specifically bind to the resting αßTCR to inhibit cytoplasmic CD3ζ ITAM phosphorylation through sequestering the TCR-CD3 complex in an inactive conformation. The mechanisms of cholesterol-mediated inhibition of TCR-CD3 and its activation remain unclear. Here, we present cryoelectron microscopy structures of cholesterol- and cholesterol sulfate (CS)-inhibited TCR-CD3 complexes and an auto-active TCR-CD3 variant. The structures reveal that cholesterol molecules act like a latch to lock CD3ζ into an inactive conformation in the membrane. Mutations impairing binding of cholesterol molecules to the tunnel result in the movement of the proximal C terminus of the CD3ζ transmembrane helix, thereby activating the TCR-CD3 complex in human cells. Together, our data reveal the structural basis of TCR inhibition by cholesterol, illustrate how the cholesterol-binding tunnel is allosterically coupled to TCR triggering, and lay a foundation for the development of immunotherapies through directly targeting the TCR-CD3 complex.
Assuntos
Complexo Receptor-CD3 de Antígeno de Linfócitos T , Linfócitos T , Complexo CD3/genética , Complexo CD3/metabolismo , Colesterol/metabolismo , Microscopia Crioeletrônica , Humanos , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
Kinase recruitment to membrane receptors is essential for signal transduction. However, the underlying regulatory mechanisms are poorly understood. We investigated how conformational changes control T cell receptor (TCR) association and activity of the kinase Zap70. Structural analysis showed that TCR binding or phosphorylation of Zap70 triggers a transition from a closed, autoinhibited conformation to an open conformation. Using Zap70 mutants with defined conformations, we found that TCR dwell times controlled Zap70 activity. The closed conformation minimized TCR dwell times and thereby prevented activation by membrane-associated kinases. Parallel recruitment of coreceptor-associated Lck kinase to the TCR ensured Zap70 phosphorylation and stabilized Zap70 TCR binding. Our study suggests that the dynamics of cytosolic enzyme recruitment to the plasma membrane regulate the activity and function of receptors lacking intrinsic catalytic activity.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Complexo CD3/metabolismo , Membrana Celular/metabolismo , Medição da Troca de Deutério , Humanos , Espectrometria de Massas , Simulação de Dinâmica Molecular , Mutação , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/genética , Fatores de Tempo , Proteína-Tirosina Quinase ZAP-70/genéticaRESUMO
Initial molecular details of cellular activation following αßT cell antigen receptor (TCR) ligation by peptide-major histocompatibility complexes (pMHC) remain unexplored. We determined the nuclear magnetic resonance (NMR) structure of the TCRα subunit transmembrane (TM) domain revealing a bipartite helix whose segmentation fosters dynamic movement. Positively charged TM residues Arg251 and Lys256 project from opposite faces of the helix, with Lys256 controlling immersion depth. Their modification caused stepwise reduction in TCR associations with CD3ζζ homodimers and CD3εγ plus CD3εδ heterodimers, respectively, leading to an activated transcriptome. Optical tweezers revealed that Arg251 and Lys256 mutations altered αßTCR-pMHC bond lifetimes, while mutations within interacting TCRα connecting peptide and CD3δ CxxC motif juxtamembrane elements selectively attenuated signal transduction. Our findings suggest that mechanical forces applied during pMHC ligation initiate T cell activation via a dissociative mechanism, shifting disposition of those basic sidechains to rearrange TCR complex membrane topology and weaken TCRαß and CD3 associations.
Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Domínios Proteicos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Biomarcadores , Complexo CD3/química , Sequência Conservada , Perfilação da Expressão Gênica , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transdução de Sinais , TranscriptomaRESUMO
CD247, also known as CD3ζ, is a crucial signaling molecule that transduces signals delivered by TCR through its three ITAMs. CD3ζ is required for successful thymocyte development. Three additional alternatively spliced variants of murine CD247 have been described, that is, CD3ι, CD3θ, and CD3η, that differ from CD3ζ in the C terminus such that the third ITAM is lost. Previous studies demonstrated defects in T cell development in mice expressing CD3η, but the TCR signaling pathways affected by CD3η and the impacts of the CD3ι and CD3θ on T cell development were not explored. In this study, we used a retrovirus-mediated gene transfer technique to express these three isoforms individually and examined the roles of them on T cell development and activation. Rag1-/- mice reconstituted with CD3θ-expressing bone marrow failed to develop mature T cells. CD3ι-expressing T cells exhibited similar development and activation as cells expressing CD3ζ. In contrast, thymic development was severely impaired in CD3η-reconstituted mice. Single-positive but not double-positive CD3η-expressing thymocytes had reduced TCR expression, and CD5 expression was decreased at the double-positive stage, suggesting a defect in positive selection. Peripheral CD3η-expressing T cells had expanded CD44hi populations and upregulation of exhaustion markers seen by flow cytometry and RNA sequencing analysis. Analysis of early signaling events demonstrated significantly reduced activation of both the PLCγ1 and Akt/mTOR signaling pathways. There was also a reduction in the frequency of activation of CD3η-expressing T cells. These studies reveal the importance of the CD3ζ C-terminal region in T cell development and activation.
Assuntos
Receptores de Antígenos de Linfócitos T , Timócitos , Animais , Camundongos , Complexo CD3/genética , Complexo CD3/metabolismo , Diferenciação Celular/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Timócitos/metabolismoRESUMO
Chronic hepatitis B (CHB) virus infection, which can be divided into immune-tolerant (IT), immune-active (IA), inactive carrier (IC) phases, and HBeAg-negative hepatitis (ENEG), can induce liver cirrhosis and eventually hepatocellular carcinoma (HCC). CD3+CD56+ NKT-like cells play an important role in antiviral immune response. However, the mechanism of NKT-like cells to mediate immune tolerance remains largely elusive. In this study, we observed circulating NKT-like cells from IC and IT CHB patients were phenotypically and functionally impaired, manifested by increased expression of inhibitory receptor TIGIT and decreased capacity of secreting antiviral cytokines. Besides, TIGIT+ NKT-like cells of IC and IT CHB patients expressed lower levels of cytotoxic cytokines than the TIGIT- subset. Furthermore, increased expression of CD155, the ligand of TIGIT, on plasmacytoid dendritic cells (pDCs) was detected in IC and IT CHB patients. Importantly, the co-culture of NKT-like cells and pDCs showed that NKT-like cells restored their antiviral ability after TIGIT blockade upon HBV peptide stimulation in IC and IT CHB patients. In conclusion, our findings suggest that the TIGIT pathway may mediate immune tolerance in IT CHB patients and lead to functional impairment in IC patients, indicating that TIGIT may be a potential therapeutic checkpoint for immunotherapy of CHB patients.
Assuntos
Complexo CD3 , Antígeno CD56 , Células Dendríticas , Vírus da Hepatite B , Hepatite B Crônica , Tolerância Imunológica , Células T Matadoras Naturais , Receptores Imunológicos , Humanos , Receptores Imunológicos/metabolismo , Hepatite B Crônica/imunologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Antígeno CD56/metabolismo , Masculino , Vírus da Hepatite B/imunologia , Feminino , Células T Matadoras Naturais/imunologia , Adulto , Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Pessoa de Meia-Idade , Receptores Virais/metabolismo , Receptores Virais/imunologia , Citocinas/metabolismo , Citocinas/imunologiaRESUMO
The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.
Assuntos
Complexo CD3/imunologia , Citocinas/biossíntese , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Complexo CD3/metabolismo , Linhagem Celular , Proliferação de Células , Células HEK293 , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismoRESUMO
Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.
Assuntos
Anticorpos Biespecíficos , Antígenos CD19 , Plasmócitos , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Anticorpos Biespecíficos/farmacologia , Animais , Camundongos , Antígenos CD19/imunologia , Antígenos CD19/genética , Antígenos CD19/metabolismo , Plasmócitos/metabolismo , Plasmócitos/imunologia , Linhagem Celular Tumoral , Linfócitos T/imunologia , Linfócitos T/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Complexo CD3/genética , Ativação Linfocitária/imunologia , Citotoxicidade ImunológicaRESUMO
Anti-cluster of differentiation (CD) 3 × α programmed death-ligand 1 (PD-L1) bispecific T-cell engager (BsTE)-bound T-cells (BsTE:T) are a promising new cancer treatment agent. However, the mechanisms of action of bispecific antibody-armed activated T-cells are poorly understood. Therefore, this study aimed to investigate the anti-tumor mechanism and efficacy of BsTE:T. The BsTE:T migration was assessed in vivo and in vitro using syngeneic and xenogeneic tumor models, flow cytometry, immunofluorescence staining, transwell migration assays, microfluidic chips, Exo View R100, western blotting, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. In murine B16 melanoma, MC38 colon cancer, and human multiple myeloma cells, BsTE:T exhibited superior tumor elimination relative to that of T-cells or BsTE alone. Moreover, BsTE:T migration into tumors was significantly enhanced owing to the presence of PD-L1 in tumor cells and secretion of PD-L1-containing exosomes. Furthermore, increased infiltration of CD44highCD62Llow effector memory CD8+ T-cells into tumors was closely associated with the anti-tumor effect of BsTE:T. Therefore, BsTE:T is an innovative potential anti-tumor therapy, and exosomal PD-L1 plays a crucial role both in vitro and in vivo in the anti-tumor activity of BsTE:T.
Assuntos
Anticorpos Biespecíficos , Antígeno B7-H1 , Exossomos , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/imunologia , Exossomos/metabolismo , Exossomos/imunologia , Camundongos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Humanos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Camundongos Endogâmicos C57BL , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linhagem Celular Tumoral , Feminino , Movimento Celular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
T-cell therapy has emerged as an effective approach for treating viral infections and cancers. However, a significant challenge is the selection of T-cell receptors (TCRs) that exhibit the desired functionality. Conventionally in vitro techniques, such as peptide sensitivity measurements and cytotoxicity assays, provide valuable insights into TCR potency but are labor-intensive. In contrast, measuring ligand binding properties (z-Movi technology) could provide an accelerated processing while showing robust correlations with T-cell functions. In this study, we assessed whether cell avidity can predict functionality also in the context of TCR-engineered T cells. To this end, we developed a flexible system for TCR re-expression by generating a Jurkat-derived T cell clone lacking TCR and CD3 expression through CRISPR-Cas9-mediated TRBC knockout. The knockin of a transgenic TCR into the TRAC locus restored TCR/CD3 expression, allowing for CD3-based purification of TCR-engineered T cells. Subsequently, we characterized these engineered cell lines by functional readouts, and assessment of binding properties through the z-Movi technology. Our findings revealed a strong correlation between the cell avidities and functional sensitivities of Jurkat TCR-T cells. Altogether, by integrating cell avidity measurements with our versatile T cell engineering platform, we established an accelerated system for enhancing the in vitro selection of clinically relevant TCRs.
Assuntos
Receptores de Antígenos de Linfócitos T , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Células Jurkat , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/citologia , Sistemas CRISPR-Cas/genética , Complexo CD3/metabolismo , Complexo CD3/imunologiaRESUMO
CD8 T cells recognize infected and cancerous cells via their T-cell receptor (TCR), which binds peptide-MHC complexes on the target cell. The affinity of the interaction between the TCR and peptide-MHC contributes to the antigen sensitivity, or functional avidity, of the CD8 T cell. In response to peptide-MHC stimulation, the TCR-CD3 complex and CD8 co-receptor are downmodulated. We quantified CD3 and CD8 downmodulation following stimulation of human CD8 T cells with CMV, EBV, and HIV peptides spanning eight MHC restrictions, observing a strong correlation between the levels of CD3 and CD8 downmodulation and functional avidity, regardless of peptide viral origin. In TCR-transduced T cells targeting a tumor-associated antigen, changes in TCR-peptide affinity were sufficient to modify CD3 and CD8 downmodulation. Correlation analysis and generalized linear modeling indicated that CD3 downmodulation was the stronger correlate of avidity. CD3 downmodulation, simply measured using flow cytometry, can be used to identify high-avidity CD8 T cells in a clinical context.
Assuntos
Linfócitos T CD8-Positivos , Receptores de Antígenos de Linfócitos T , Humanos , Regulação para Baixo , Receptores de Antígenos de Linfócitos T/genética , Antígenos CD8/metabolismo , Peptídeos/metabolismo , Complexo CD3/metabolismoRESUMO
BACKGROUND: In oral squamous cell carcinoma (OSCC), the tumor-node-metastasis (TNM) staging system is a significant factor that influences prognosis and treatment decisions for OSCC patients. Unfortunately, TNM staging does not consistently predict patient prognosis and patients with identical clinicopathological characteristics may have vastly different survival outcomes. Host immunity plays an important role in tumor progression but is not included in the TNM staging system. Tumor-infiltrating lymphocytes (TILs) are part of the host immune response that recognizes tumor cells; and the presence of TILs has emerged as potential candidates for prognostic markers for many types of cancers. The present study aims to determine the association of T cell-specific markers (CD3, CD4, CD8, and FOXP3) with clinicopathological characteristics and survival outcomes in OSCC patients. The prognostic value of CD3, CD4, and CD8 will also be evaluated based on tumor stage. METHODS: Tissue microarrays were constructed containing 231 OSCC cases and analyzed by immunohistochemical staining for the expression of CD3, CD4, CD8, and FOXP3. The expression scores for each marker were correlated with clinicopathological parameters and survival outcomes. The prognostic impact of CD3, CD4 and CD8 were further analyzed based on tumor stage (early or advanced). RESULTS: CD3, CD4, and CD8 were found to be significantly associated with both overall survival and progression-free survival using univariate analysis. However, none of these markers were found to independently predict the survival outcomes of OSCC using multivariate analysis. Only conventional factors such as nodal status, tumor differentiation and perineural invasion (PNI) were independent predictors of survival outcomes, with nodal status being the strongest independent predictor. Additionally, low CD4 (but not CD3 or CD8) expression was found to identify early-stage OSCC patients with exceptionally poor prognosis which was similar to that of advanced staged OSCC patients. CONCLUSIONS: TIL markers such as CD3, CD4, CD8, and FOXP3 can predict the survival outcomes of OSCC patients, but do not serve as independent prognostic markers as found with conventional factors (i.e. nodal status, tumor differentiation and PNI). CD4 expression may assist with risk stratification in early-stage OSCC patients which may influence treatment planning and decision making for early-stage OSCC patients.
Assuntos
Carcinoma de Células Escamosas , Linfócitos do Interstício Tumoral , Neoplasias Bucais , Estadiamento de Neoplasias , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/imunologia , Neoplasias Bucais/mortalidade , Masculino , Feminino , Prognóstico , Pessoa de Meia-Idade , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/mortalidade , Idoso , Fatores de Transcrição Forkhead/metabolismo , Adulto , Biomarcadores Tumorais/metabolismo , Idoso de 80 Anos ou mais , Complexo CD3/metabolismoRESUMO
The eight-subunit T cell receptor (TCR)-CD3 complex is the primary determinant for T cell fate decisions. Yet how it relays ligand-specific information across the cell membrane for conversion to chemical signals remains unresolved. We hypothesized that TCR engagement triggers a change in the spatial relationship between the associated CD3ζζ subunits at the junction where they emerge from the membrane into the cytoplasm. Using three in situ proximity assays based on ID-PRIME, FRET, and EPOR activity, we determined that the cytosolic juxtamembrane regions of the CD3ζζ subunits are spread apart upon assembly into the TCR-CD3 complex. TCR engagement then triggered their apposition. This mechanical switch resides upstream of the CD3ζζ intracellular motifs that initiate chemical signaling, as well as the polybasic stretches that regulate signal potentiation. These findings provide a framework from which to examine triggering events for activating immune receptors and other complex molecular machines.
Assuntos
Complexo CD3/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Complexos Multiproteicos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Animais , Complexo CD3/genética , Humanos , Hibridomas , Mecanotransdução Celular , Camundongos , Complexos Multiproteicos/genética , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Estrutura Terciária de Proteína/genética , Receptor Cross-Talk , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Signaling by toll-like receptors (TLRs) initiates important immune responses against viral infection. The role of TLRs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not well elucidated. Thus, we investigated the interaction of TLRs agonists and SARS-COV-2 antigens with immune cells in vitro. MATERIAL & METHODS: 30 coronavirus disease 2019 (COVID-19) patients (15 severe and 15 moderate) and 10 age and sex-matched healthy control (HC) were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated and activated with TLR3, 7, 8, and 9 agonists, the spike protein (SP) of SARS-CoV-2, and the receptor binding domain (RBD) of SP. Frequencies of CD3+IFN-ß+ T cells, and CD3+IFN-γ+ T cells were evaluated by flow cytometry. Interferon (IFN)-ß gene expression was assessed by qRT-PCR. RESULTS: The frequency of CD3+IFN-ß+ T cells was higher in PBMCs from moderate (p < 0.0001) and severe (p = 0.009) patients at baseline in comparison with HCs. The highest increase in the frequency of CD3+IFN-ß+ T cells in cell from moderate patients was induced by TLR8 agonist and SP (p < 0.0001 for both) when compared to HC, while, the highest increase of the frequency of CD3+IFN-ß+ T cells in sample of severe patients was seen with TLR8 and TLR7 agonists (both p = 0.002). The frequency of CD3+IFN-γ+ T cells was significantly increased upon stimulation with TLR agonists in cell from patients with moderate and severe COVID-19, compared with HC (all p < 0.01), except with TLR7 and TLR8 agonists. The TLR8 agonist did not significantly increase the frequency of CD3+IFN-γ+ T cells in PBMCs of severe patients, but did so in cells from patients with moderate disease (p = 0.01). Moreover, IFN-ß gene expression was significantly upregulated in CD3+T cells from moderate (p < 0.0001) and severe (p = 0.002) COVID-19 patients, compared to HC after stimulation with the TLR8 agonist, while, stimulation of T cells with SP, significantly up-regulated IFN-ß mRNA expression in cells from patients with moderate (p = 0.0003), but not severe disease. CONCLUSION: Stimulation of PBMCs from COVID-19 patients, especially patients with moderate disease, with TLR8 agonist and SP increased the frequency of IFN-ß-producing T cells and IFN-ß gene expression.
Assuntos
Complexo CD3 , COVID-19 , SARS-CoV-2 , Linfócitos T , Receptores Toll-Like , Humanos , COVID-19/imunologia , COVID-19/virologia , SARS-CoV-2/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos , Adulto , Interferon gama/metabolismo , Interferon gama/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Interferon beta/genética , Interferon beta/imunologia , Idoso , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Agonistas do Receptor Semelhante a TollRESUMO
Cytomegalovirus (CMV) infection detrimentally influences graft survival in kidney transplant recipients, with the risk primarily determined by recipient and donor serostatus. However, recipient CD8+ T cells play a crucial role in CMV control. The optimal preventive strategy (prophylaxis vs. pre-emptive treatment), particularly for seropositive (intermediate risk) recipients, remains uncertain. We investigated CD8+ T cell subpopulation dynamics and CMV occurrence (DNAemia ≥ 100 IU/mL) in 65 kidney transplant recipients, collecting peripheral blood mononuclear cells before (T1) and 1 year after transplantation (T2). Comparing the two timepoints, we found an increase in granulocyte, monocyte and CD3+CD8+ T cells numbers, while FoxP3+CD25+, LAG-3+ and PD-1+ frequencies were reduced at T2. CMV DNAemia occurred in 33 recipients (55.8%) during the first year. Intermediate risk patients were disproportionally affected by posttransplant CMV (N = 29/45, 64.4%). Intermediate risk recipients developing CMV after transplantation exhibited lower leukocyte, monocyte, and granulocyte counts and higher FoxP3+CD25+ frequencies in CD3+CD8+ T cells pre-transplantation compared to patients staying CMV negative. Pre-transplant FoxP3+CD25+ in CD3+CD8+ T cells had the best discriminatory potential for CMV infection prediction within the first year after transplantation (AUC: 0.746). The FoxP3+CD25+ CD3+CD8+ T cell subset may aid in selecting intermediate risk kidney transplant recipients for CMV prophylaxis.
Assuntos
Linfócitos T CD8-Positivos , Infecções por Citomegalovirus , Fatores de Transcrição Forkhead , Subunidade alfa de Receptor de Interleucina-2 , Transplante de Rim , Humanos , Transplante de Rim/efeitos adversos , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Feminino , Masculino , Linfócitos T CD8-Positivos/imunologia , Pessoa de Meia-Idade , Fatores de Transcrição Forkhead/metabolismo , Adulto , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Idoso , Complexo CD3/metabolismo , Citomegalovirus/imunologia , Fatores de Risco , Transplantados , Sobrevivência de Enxerto/imunologiaRESUMO
BACKGROUND: The association between p16INK4a and p21, a marker of cellular senescence, and the Immunoscore, an immunological prognostic indicator, in rectal cancer patients undergoing curative surgery were investigated. METHODS: A total of 82 patients who underwent curative surgery for rectal cancer were evaluated. The resected specimens were analyzed for p16INK4a, p21, CD3 and CD8 expression by immunohistochemistry. Immunoscore was calculated on the basis of CD3 and CD8 expressions. The clinicopathological characteristics and long-term outcomes were evaluated. RESULTS: Among the 82 patients, 24 (29.3%) were p16INK4a-positive and 11 (13.4%) were p21-positive. The patients were classified into the following five Immunoscore groups (IS0-5). IS0, IS1 and IS2 were classified as the low Immunoscore group (45 patients, 54.9%) and IS3 and IS4 as the high Immunoscore group (37 patients, 45.1%). There was no significant difference in age, sex, body mass index, American Society of Anesthesiologists physical status, depth of invasion of the tumor, lymph node metastasis and histological classification of the tumor with p16INK4a or p21 expression or Immunoscore. p16INK4a-positive expression and low Immunoscore each showed a tendency to indicate poor prognosis of disease-free survival (DFS). Patients with the combination of p16INK4a and p21 positivity and with p16INK4a positivity and low Immunoscore showed significantly poor prognosis of DFS. Patients with p21 positive positivity and low Immunoscore tended to have worse DFS. CONCLUSIONS: p16INK4a, p21 and Immunoscore may be prognostic indicators of rectal cancer. The combination of them may provide more accurate prognostic prediction than either factor alone.