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1.
J Biol Chem ; 300(6): 107325, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38685532

RESUMEN

Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.


Asunto(s)
Antígeno B7-H1 , Inhibidores de Puntos de Control Inmunológico , Humanos , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Polímeros/química , Línea Celular Tumoral
2.
EMBO J ; 33(17): 1928-40, 2014 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-24963146

RESUMEN

Natural killer (NK) cells are involved in immune responses against tumors and microbes. NK-cell activation is regulated by intrinsic and extrinsic mechanisms that ensure NK tolerance and efficacy. Here, we show that the cytoplasmic signaling molecules Dok1 and Dok2 are tyrosine phosphorylated upon NK-cell activation. Overexpression of Dok proteins in human NK cells reduces cell activation induced by NK-cell-activating receptors. Dok1 and Dok2 gene ablation in mice induces an NK-cell maturation defect and leads to increased IFN-γ production induced by activating receptors. Taken together, these results reveal that Dok1 and Dok2 proteins are involved in an intrinsic negative feedback loop downstream of NK-cell-activating receptors in mouse and human.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Humanos , Interferón gamma/metabolismo , Ratones
3.
Front Immunol ; 14: 1139123, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37006259

RESUMEN

The propagation and diversification of signals downstream of the T cell receptor (TCR) involve several adaptor proteins that control the assembly of multimolecular signaling complexes (signalosomes). The global characterization of changes in protein-protein interactions (PPI) following genetic perturbations is critical to understand the resulting phenotypes. Here, by combining genome editing techniques in T cells and interactomics studies based on affinity purification coupled to mass spectrometry (AP-MS) analysis, we determined and quantified the molecular reorganization of the SLP76 interactome resulting from the ablation of each of the three GRB2-family adaptors. Our data showed that the absence of GADS or GRB2 induces a major remodeling of the PPI network associated with SLP76 following TCR engagement. Unexpectedly, this PPI network rewiring minimally affects proximal molecular events of the TCR signaling pathway. Nevertheless, during prolonged TCR stimulation, GRB2- and GADS-deficient cells displayed a reduced level of activation and cytokine secretion capacity. Using the canonical SLP76 signalosome, this analysis highlights the plasticity of PPI networks and their reorganization following specific genetic perturbations.


Asunto(s)
Transducción de Señal , Linfocitos T , Linfocitos T/metabolismo , Transducción de Señal/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Mapas de Interacción de Proteínas
4.
Eur J Immunol ; 41(12): 3443-54, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21918970

RESUMEN

The human butyrophilin (BTN) 3 or CD277 molecules belong to the B7 family members and are expressed in various immune cells such as T and NK cells. Here, we show that CD277 triggering considerably enhances TCR-induced cytokine production and cell proliferation, even when another co-stimulatory molecule, CD28, is engaged. These CD277-induced additive functional effects are in accordance with the detection of early T-cell activation events such as TCR-induced cell signaling being increased upon CD277 engagement. However, we found that CD277 triggering is not involved in CD16- or NKp46-induced NK cell activation. BTN3/CD277 comprises three structurally related members, BTN3A1, BTN3A2 and BTN3A3. CD277 antibodies recognize all isoforms and we describe a differential expression of BTN3 isoforms between T and NK cells that could explain differential CD277 functions between T and NK cells. Our results show that, while T cells express all BTN3/CD277 transcripts, NK cells express mostly BTN3A2, which lacks the B30.2 intracellular domain. Furthermore, NKp30-induced cytokine production is decreased by the specific engagement of BTN3A2, but not by BTN3A1 triggering. Thus, we provide new insights into the CD277 co-stimulatory pathway that may differentially participate in the regulation of various cell-mediated immune responses.


Asunto(s)
Antígenos CD/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Asesinas Naturales/inmunología , Animales , Butirofilinas , Antígenos CD28/inmunología , Células COS , Línea Celular Transformada , Proliferación Celular , Chlorocebus aethiops , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Interferón gamma/inmunología , Activación de Linfocitos/inmunología , Receptor 3 Gatillante de la Citotoxidad Natural/inmunología , Isoformas de Proteínas , Receptores de Antígenos de Linfocitos T/inmunología , Regulación hacia Arriba
5.
J Exp Med ; 219(2)2022 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-35061003

RESUMEN

We exploited traceable gene tagging in primary human T cells to establish the composition and dynamics of seven canonical TCR-induced protein signaling complexes (signalosomes) using affinity purification coupled with mass spectrometry (AP-MS). It unveiled how the LAT adaptor assembles higher-order molecular condensates and revealed that the proximal TCR-signaling network has a high degree of qualitative and quantitative conservation between human CD4+ and CD8+ T cells. Such systems-level conservation also extended across human and mouse T cells and unexpectedly encompassed protein-protein interaction stoichiometry. Independently of evolutionary considerations, our study suggests that a drug targeting the proximal TCR signaling network should behave similarly when applied to human and mouse T cells. However, considering that signaling differences likely exist between the distal TCR-signaling pathway of human and mouse, our fast-track AP-MS approach should be favored to determine the mechanism of action of drugs targeting human T cell activation. An opportunity is illustrated here using an inhibitor of the LCK protein tyrosine kinase as a proof-of-concept.


Asunto(s)
Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Biomarcadores , Comunicación Celular/inmunología , Edición Génica , Humanos , Inmunofenotipificación , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Modelos Biológicos , Fosforilación , Mapeo de Interacción de Proteínas , Especificidad de la Especie , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo
6.
J Exp Med ; 218(2)2021 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-33125054

RESUMEN

To determine the respective contribution of the LAT transmembrane adaptor and CD5 and CD6 transmembrane receptors to early TCR signal propagation, diversification, and termination, we describe a CRISPR/Cas9-based platform that uses primary mouse T cells and permits establishment of the composition of their LAT, CD5, and CD6 signalosomes in only 4 mo using quantitative mass spectrometry. We confirmed that positive and negative functions can be solely assigned to the LAT and CD5 signalosomes, respectively. In contrast, the TCR-inducible CD6 signalosome comprised both positive (SLP-76, ZAP70, VAV1) and negative (UBASH3A/STS-2) regulators of T cell activation. Moreover, CD6 associated independently of TCR engagement to proteins that support its implication in inflammatory pathologies necessitating T cell transendothelial migration. The multifaceted role of CD6 unveiled here accounts for past difficulties in classifying it as a coinhibitor or costimulator. Congruent with our identification of UBASH3A within the CD6 signalosome and the view that CD6 constitutes a promising target for autoimmune disease treatment, single-nucleotide polymorphisms associated with human autoimmune diseases have been found in the Cd6 and Ubash3a genes.


Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Secuencia de Bases , Femenino , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Polimorfismo de Nucleótido Simple/inmunología , Transducción de Señal/inmunología
7.
Cell Rep ; 27(11): 3315-3330.e7, 2019 06 11.
Artículo en Inglés | MEDLINE | ID: mdl-31189114

RESUMEN

Deciphering how TCR signals are modulated by coinhibitory receptors is of fundamental and clinical interest. Using quantitative interactomics, we define the composition and dynamics of the PD-1 and BTLA coinhibitory signalosomes in primary effector T cells and at the T cell-antigen-presenting cell interface. We also solve the existing controversy regarding the role of the SHP-1 and SHP-2 protein-tyrosine phosphatases in mediating PD-1 coinhibition. PD-1 predominantly recruits SHP-2, but when absent, it recruits SHP-1 and remains functional. In contrast, BTLA predominantly recruits SHP-1 and to a lesser extent SHP-2. By separately analyzing the PD-1-SHP-1 and PD-1-SHP-2 complexes, we show that both dampen the TCR and CD28 signaling pathways equally. Therefore, our study illustrates how comparison of coinhibitory receptor signaling via quantitative interactomics in primary T cells unveils their extent of redundancy and provides a rationale for designing combinations of blocking antibodies in cancer immunotherapy on the basis of undisputed modes of action.


Asunto(s)
Receptor de Muerte Celular Programada 1/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Animales , Femenino , Humanos , Inmunoterapia , Células Jurkat , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/genética , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo
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