RESUMEN
Protein conformational changes with fluctuations are fundamental aspects of protein-protein interactions (PPIs); understanding these motions is required for the rational design of PPI-regulating compounds. Src homology 2 (SH2) domains are commonly found in adapter proteins involved in signal transduction and specifically bind to consensus motifs of proteins containing phosphorylated tyrosine (pY). Here, we analysed the interaction between the N-terminal SH2 domain (nSH2) of the regulatory subunit in phosphoinositide 3-kinase (PI3K) and the cytoplasmic region of the T-cell co-receptor, CD28, using NMR and molecular dynamics (MD) simulations. First, we assigned the backbone signals of nSH2 on 1 H-15 N heteronuclear single quantum coherence spectra in the absence or presence of the CD28 phosphopeptide, SDpYMNMTPRRPG. Chemical shift perturbation experiments revealed allosteric changes at the BC loop and the C-terminal region of nSH2 upon CD28 binding. NMR relaxation experiments showed a conformational exchange associated with CD28 binding in these regions. The conformational stabilisation of the C-terminal region correlated with the regulation of PI3K catalytic function. Further, using 19 F- and 31 P-labelled CD28 phosphopeptide, we analysed the structural dynamics of CD28 and demonstrated that the aromatic ring of the pY residue fluctuated between multiple conformations upon nSH2 binding. Our MD simulations largely explained the NMR results and the structural dynamics of nSH2 and CD28 in both bound and unbound states. Notably, in addition to its major conformation, we detected a minor conformation of nSH2 in the CD28 bound state that may explain the allosteric conformational change in the BC loop.
Asunto(s)
Fosfatidilinositol 3-Quinasas , Dominios Homologos src , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Antígenos CD28/genética , Antígenos CD28/química , Antígenos CD28/metabolismo , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
Identification of the tyrosine phosphorylation (pY)-dependent interactome of immune co-receptors is crucial for understanding signal pathways involved in immunotherapy. However, identifying the motif-specific interactome for each pY commonly found on these multi-phosphorylated membrane proteins remains challenging. Here, we describe a photoaffinity-based chemical proteomic approach to dissect the motif-specific cytoplasmic interactomes of the critical immune co-receptor CD28. Various full-length CD28 cytoplasmic tails (CD28cyto) with defined pY and selectively replaced photo-methionine were synthesized and applied to explore three pY-motif-dependent CD28cyto interactomes. We identified a stand-alone interaction of phospholipase PLCG1 with the Y191 motif with enhanced affinity for the sequence neighboring the transmembrane domain. Importantly, taking advantage of native top-down mass spectrometry with a 193-nm laser, we discovered the direct association of a previously undefined pY218 motif with the kinase PKCθ through its C2 domain. This synthetic CD28cyto-based photoaffinity proteomic approach is generically applicable to the study of other immune co-receptors with multiple pY sites on their linear cytoplasmic tail.
Asunto(s)
Antígenos CD28 , Proteómica , Antígenos CD28/química , Antígenos CD28/metabolismo , Espectrometría de Masas , Fosforilación , Transducción de SeñalRESUMEN
B7H6, a stress-induced ligand which binds to the NK cell receptor NKp30, has recently emerged as a promising candidate for immunotherapy due to its tumor-specific expression on a broad array of human tumors. NKp30 can function as a chimeric antigen receptor (CAR) extracellular domain but exhibits weak binding with a fast on and off rate to B7H6 compared to the TZ47 anti-B7H6 single-chain variable fragment (scFv). Here, directed evolution using yeast display was employed to isolate novel NKp30 variants that bind to B7H6 with higher affinity compared to the native receptor but retain its fast association and dissociation profile. Two variants, CC3 and CC5, were selected for further characterization and were expressed as soluble Fc-fusion proteins and CARs containing CD28 and CD3ς intracellular domains. We observed that Fc-fusion protein forms of NKp30 and its variants were better able to bind tumor cells expressing low levels of B7H6 than TZ47, and that the novel variants generally exhibited improved in vitro tumor cell killing relative to NKp30. Interestingly, CAR T cells expressing the engineered variants produced unique cytokine signatures in response to multiple tumor types expressing B7H6 compared to both NKp30 and TZ47. These findings suggest that natural CAR receptors can be fine-tuned to produce more desirable signaling outputs while maintaining evolutionary advantages in ligand recognition relative to scFvs.
Asunto(s)
Antígenos B7/química , Receptor 3 Gatillante de la Citotoxidad Natural/química , Receptores Quiméricos de Antígenos/química , Animales , Antígenos CD28/química , Complejo CD3/química , Línea Celular Tumoral , Separación Celular , Citocinas/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Biblioteca de Genes , Variación Genética , Células HEK293 , Humanos , Inmunoterapia , Cinética , Ligandos , Ratones , Mutación , Conformación Proteica , Anticuerpos de Cadena Única/químicaRESUMEN
T-cell co-stimulation through CD28/CTLA4:B7-1/B7-2 axis is one of the extensively studied pathways that resulted in the discovery of several FDA-approved drugs for autoimmunity and cancer. However, many aspects of the signaling mechanism remain elusive, including oligomeric association and clustering of B7-2 on the cell surface. Here, we describe the structure of the IgV domain of B7-2 and its cryptic association into 1D arrays that appear to represent the pre-signaling state of B7-2 on the cell membrane. Super-resolution microscopy experiments on heterologous cells expressing B7-2 and B7-1 suggest, B7-2 form relatively elongated and larger clusters compared to B7-1. The sequence and structural comparison of other B7 family members, B7-1:CTLA4 and B7-2:CTLA-4 complex structures, support our view that the observed B7-2 1D zipper array is physiologically important. This observed 1D zipper-like array also provides an explanation for its clustering, and upright orientation on the cell surface, and avoidance of spurious signaling.
Asunto(s)
Antígeno B7-1/química , Antígeno B7-2/química , Antígenos CD28/química , Antígeno CTLA-4/química , Secuencia de Aminoácidos , Animales , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Sitios de Unión , Antígenos CD28/genética , Antígenos CD28/metabolismo , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Línea Celular Tumoral , Expresión Génica , Humanos , Ratones , Modelos Moleculares , Neuronas/citología , Neuronas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: The cell adhesion molecule IGPR-1 regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy and plays important roles in angiogenesis and tumor growth; however, the molecular mechanism governing the cell surface levels of IGPR-1 remains unknown. RESULTS: In the present study, we used an in vitro ubiquitination assay and identified ubiquitin E3 ligase NEDD4 and the ubiquitin conjugating enzyme UbcH6 involved in the ubiquitination of IGPR-1. In vitro GST-pulldown and in vivo co-immunoprecipitation assays demonstrated that NEDD4 binds to IGPR-1. Over-expression of wild-type NEDD4 downregulated IGPR-1 and deletion of WW domains (1-4) of NEDD4 revoked its effects on IGPR-1. Knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Deletion of 57 amino acids encompassing the polyproline rich (PPR) motifs on the C-terminus of IGPR-1 nullified its binding with NEDD4. Furthermore, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulates lysosomal-dependent degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine or ammonium chloride increased IGPR-1 levels ectopically expressed in HEK-293 cells and in multiple endogenously IGPR-1 expressing human skin melanoma cell lines. CONCLUSIONS: NEDD4 ubiquitin E3 ligase binds to and mediates polyubiquitination of IGPR-1 leading to its lysosomal-dependent degradation. NEDD4 is a key regulator of IGPR-1 expression with implication in the therapeutic targeting of IGPR-1 in human cancers.
Asunto(s)
Antígenos CD28/química , Membrana Celular/metabolismo , Lisosomas/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/genética , Células HEK293 , Humanos , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Estabilidad Proteica , UbiquitinaciónRESUMEN
Beta glucans are known to have immunomodulatory effects that mediated by a variety of mechanisms. In this article, we describe experiments and simulations suggesting that beta-1,3 glucans may promote activation of T cells by a previously unknown mechanism. First, we find that treatment of a T lymphoblast cell line with beta-1,3 oligoglucan significantly increases mRNA levels of T cell activation-associated cytokines, especially in the presence of the agonistic anti-CD3 antibody. This immunostimulatory activity was observed in the absence of dectin-1, a known receptor for beta-1,3 glucans. To clarify the molecular mechanism underlying this activity, we performed a series of molecular dynamics simulations and free-energy calculations to explore the interaction of beta-1,3 oligoglucans with potential immune receptors. While the simulations reveal little association between beta-1,3 oligoglucan and the immune receptor CD3, we find that beta-1,3 oligoglucans bind to CD28 near the region identified as the binding site for its natural ligands CD80 and CD86. Using a rigorous absolute binding free-energy technique, we calculate a dissociation constant in the low millimolar range for binding of 8-mer beta-1,3 oligoglucan to this site on CD28. The simulations show this binding to be specific, as no such association is computed for alpha-1,4 oligoglucan. This study suggests that beta-1,3 glucans bind to CD28 and may stimulate T cell activation collaboratively with T cell receptor activation, thereby stimulating immune function.
Asunto(s)
Antígenos CD28/metabolismo , Activación de Linfocitos/inmunología , Receptores Inmunológicos/metabolismo , Linfocitos T/inmunología , beta-Glucanos/metabolismo , Antígenos CD28/química , Citocinas/metabolismo , Humanos , Células Jurkat , Modelos Moleculares , Unión Proteica , Receptores Inmunológicos/química , Termodinámica , beta-Glucanos/químicaRESUMEN
Immune system plays essential role in functioning of higher organisms. Its hyperactivity can lead to autoimmune diseases or even anaphylactic shock while hypoactivity leads to proneness to infections or even cancer. T-cells play crucial role in immunity mechanisms and their activation and inhibition is strictly controlled by the regulatory proteins, such as CD28 and CTLA-4. Activity of these proteins is controlled by a pair of ligands, named CD80 and CD86, which can non-covalently bound to their receptors. While structure of human CTLA-4-CD86 complex in known, there is still no available structure for the CD28-CD86 system. To obtain the reliable structure of CD28-CD86 complex we first validated our methodology on the CTLA-4-CD86 system. Then coarse-grained UNRES-dock molecular docking simulation was performed followed by all-atom molecular dynamics simulations. As a result, we obtained a complete CD28-CD86 complex structure on atomistic level, in which interaction interface is consistent with available data. We also determined the kinetic properties for CTLA4-CD86 and CD28-CD86 complexes with use of coarse-grained model and determined the key residues for complex formation with use of Robetta, PPCheck and HawkDock servers. Our results not only verify high accuracy of the UNRES-dock method, but also provide a highly reliable model of the CD28-CD86 complex, which can be used in further studies and drug design.
Asunto(s)
Antígeno B7-2/química , Antígenos CD28 , Inmunoconjugados , Abatacept , Antígenos CD , Antígenos CD28/química , Humanos , Glicoproteínas de Membrana , Simulación del Acoplamiento Molecular , Conformación ProteicaRESUMEN
For the activation of T cells, it is necessary the specific recognition of the peptide by the T cell receptors (TCR) in the surface of antigen-presenting cells (APCs) and additional signals delivered by costimulatory receptors. In fish, knowledge about the presence of these costimulatory signals is limited and functional evidence almost absent. Thus, in this study, we have identified the stimulatory CD28 and the inhibitory cytotoxic T-lymphocyte-associated protein 4 (CTLA4) coreceptors in the European sea bass (Dicentrarchus labrax), and evaluated their transcription. In parallel, the transcription encoding for the T cell markers CD8α and CD4 was also evaluated. Both coreceptors showed the canonical architecture including a signal peptide, an immunoglobulin domain, a transmembrane region and a cytosolic tail. Protein predictions and phylogenetic tree identify them as true mammalian orthologues of CD28 and CTLA4. We found these genes constitutively expressed in all studied organs of European sea bass with high expression in lymphoid organs (thymus, spleen and head-kidney) and liver. The molecular expression pattern of these genes was up-regulated in head-kidney leucocytes stimulated with T mitogens as concanavalin A and phytohemagglutinin (PHA), but not with the B cell mitogen lipopolysaccharide (LPS). Fish challenged with nodavirus (NNV) evidenced a differential and opposing regulation of the cd28 and ctla4 transcription levels in the brain, the target organ for viral replication, and head-kidney. While cd28 transcription tends to decrease over the infection time in both organs the expression of the ctla4 gene tends to increase. Interestingly, the coreceptor expression is highly and significantly correlated to the transcription of the T cell markers. Our results highlight the important role of CD28 and CTLA4 as costimulatory receptors of T cells in European sea bass but further studies are deserved.
Asunto(s)
Inmunidad Adaptativa/genética , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Regulación de la Expresión Génica/inmunología , Perciformes/genética , Perciformes/inmunología , Secuencia de Aminoácidos , Animales , Lubina , Antígenos CD28/química , Antígenos CD28/genética , Antígenos CD28/inmunología , Antígeno CTLA-4/química , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Enfermedades de los Peces/virología , Proteínas de Peces/química , Perfilación de la Expresión Génica/veterinaria , Tejido Linfoide/inmunología , Nodaviridae/fisiología , Filogenia , Infecciones por Virus ARN/inmunología , Infecciones por Virus ARN/veterinaria , Infecciones por Virus ARN/virología , Alineación de Secuencia/veterinaria , Linfocitos T/inmunologíaRESUMEN
Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase ß activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase ß is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser220 The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy proteins ULK1 and Beclin-1 (BECN1), increased LC3-II levels, and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer and suggest that IGPR-1 may serve as a promising therapeutic target.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Antígenos CD28/metabolismo , Adhesión Celular , Secuencias de Aminoácidos , Animales , Autofagia/efectos de los fármacos , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Beclina-1/metabolismo , Antígenos CD28/química , Antígenos CD28/genética , Adhesión Celular/efectos de los fármacos , Células HEK293 , Humanos , Quinasa I-kappa B/deficiencia , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/farmacología , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación/efectos de los fármacos , Primates , ARN Guía de Kinetoplastida/metabolismo , Sirolimus/farmacología , Especificidad por SustratoRESUMEN
CD28 plays a critical role in regulating immune responses both by enhancing effector T cell activation and differentiation and controlling the development and function of regulatory T cells. CD28 is expressed at the cell surface as a disulfide linked homodimer that is thought to bind ligand monovalently. How ligand binding triggers CD28 to induce intracellular signaling as well as the proximal signaling pathways that are induced are not well-understood. In addition, recent data suggest inside-out signaling initiated by the T cell antigen receptor can enhance CD28 ligand binding, possibly by inducing a rearrangement of the CD28 dimer interface to allow for bivalent binding. To understand how possible conformational changes during ligand-induced receptor triggering and inside-out signaling are mediated, we examined the CD28 transmembrane domain. We identified an evolutionarily conserved YxxxxT motif that is shared with CTLA-4 and resembles the transmembrane dimerization motif within CD3ζ. We show that the CD28 transmembrane domain can drive protein dimerization in a bacterial expression system at levels equivalent to the well-known glycophorin A transmembrane dimerization motif. In addition, ectopic expression of the CD28 transmembrane domain into monomeric human CD25 can drive dimerization in murine T cells as detected by an increase in FRET by flow cytometry. Mutation of the polar YxxxxT motif to hydrophobic leucine residues (Y145L/T150L) attenuated CD28 transmembrane mediated dimerization in both the bacterial and mammalian assays. Introduction of the Y145L/T150L mutation of the CD28 transmembrane dimerization motif into the endogenous CD28 locus by CRISPR resulted in a dramatic loss in CD28 cell surface expression. These data suggest that under physiological conditions the YxxxxT dimerization motif within the CD28 transmembrane domain plays a critical role in the assembly and/or expression of stable CD28 dimers at the cell surface.
Asunto(s)
Secuencias de Aminoácidos , Antígenos CD28/química , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Secuencia de Aminoácidos , Animales , Antígenos CD28/genética , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Complejo CD3/química , Complejo CD3/inmunología , Complejo CD3/metabolismo , Membrana Celular/metabolismo , Secuencia Conservada , Expresión Génica Ectópica , Humanos , Ratones , Ratones Transgénicos , Posición Específica de Matrices de Puntuación , Transducción de SeñalRESUMEN
Chimeric antigen receptor modified T cells (CAR-T) have yielded impressive clinical outcomes in treating hematopoietic malignancies. However, relapses have occurred in a substantial number of patients and limited the development of CAR-T therapy. Most underlying reasons for these relapses can be attributed to poor persistence and rapid exhaustion of CAR-T cells in vivo. Despite multiple strategies having been developed, how to improve CAR-T persistence or resist exhaustion while maintaining sufficient cytotoxic functions is still a great challenge. Here we discuss engineering cytoplasmic signaling as an important strategy for CAR optimization. This review summarizes recent advances showing that the anti-tumor function of CAR-T cells can be improved by optimizing the CD3ζ domain or downstream signaling of CD28ζ CAR.
Asunto(s)
Antígenos CD28/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores Quiméricos de Antígenos/inmunología , Antígenos CD28/química , Complejo CD3/química , Complejo CD3/inmunología , Ingeniería Celular/métodos , Humanos , Activación de Linfocitos , Modelos Inmunológicos , Neoplasias/inmunología , Dominios Proteicos , Receptores Quiméricos de Antígenos/química , Transducción de Señal/inmunología , Linfocitos T/inmunologíaRESUMEN
Chimeric antigen receptor T (CAR-T) cell therapy is regarded as an effective solution for relapsed or refractory tumors, particularly for hematological malignancies. Although the initially approved anti-CD19 CAR-T therapy has produced impressive outcomes, setbacks such as high relapse rates and resistance were experienced, driving the need to discover engineered CAR-T cells that are more effective for therapeutic use. Innovations in the structure and manufacturing of CAR-T cells have resulted in significant improvements in efficacy and persistence, particularly with the development of fourth-generation CAR-T cells. Paired with an immune modifier, the use of fourth-generation and next-generation CAR-T cells will not be limited because of cytotoxic effects and will be an efficient tool for overcoming the tumor microenvironment. In this review, we summarize the recent transformations in the ectodomain, transmembrane domain, and endodomain of the CAR structure, which, together with innovative manufacturing technology and improved cell sources, improve the prospects for the future development of CAR-T cell therapy.
Asunto(s)
Ingeniería Celular/tendencias , Inmunoterapia Adoptiva/tendencias , Receptores Quiméricos de Antígenos/genética , Antígenos CD19/genética , Antígenos CD19/inmunología , Antígenos de Neoplasias/inmunología , Antígenos CD28/química , Antígenos CD28/inmunología , Quimiotaxis de Leucocito , Ensayos Clínicos como Asunto , Citocinas/metabolismo , Vectores Genéticos/genética , Humanos , Inmunoterapia Adoptiva/métodos , Lentivirus/genética , Linfoma de Células B Grandes Difuso/terapia , Neoplasias/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Unión Proteica , Dominios Proteicos , Ingeniería de Proteínas , Receptores de Quimiocina/inmunología , Receptores Quiméricos de Antígenos/agonistas , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T , Linfocitos T/inmunología , Linfocitos T/trasplante , Transducción Genética , Microambiente TumoralRESUMEN
HIV infection preferentially depletes HIV-specific CD4+ T cells, thereby impairing antiviral immunity. In this study, we explored the therapeutic utility of adoptively transferred CD4+ T cells expressing an HIV-specific chimeric antigen receptor (CAR4) to restore CD4+ T cell function to the global HIV-specific immune response. We demonstrated that CAR4 T cells directly suppressed in vitro HIV replication and eliminated virus-infected cells. Notably, CAR4 T cells containing intracellular domains (ICDs) derived from the CD28 receptor family (ICOS and CD28) exhibited superior effector functions compared to the tumor necrosis factor receptor (TNFR) family ICDs (CD27, OX40, and 4-1BB). However, despite demonstrating limited in vitro efficacy, only HIV-resistant CAR4 T cells expressing the 4-1BBζ ICD exhibited profound expansion, concomitant with reduced rebound viremia after antiretroviral therapy (ART) cessation and protection of CD4+ T cells (CAR-) from HIV-induced depletion in humanized mice. Moreover, CAR4 T cells enhanced the in vivo persistence and efficacy of HIV-specific CAR-modified CD8+ T cells expressing the CD28ζ ICD, which alone exhibited poor survival. Collectively, these studies demonstrate that HIV-resistant CAR4 T cells can directly control HIV replication and augment the virus-specific CD8+ T cell response, highlighting the therapeutic potential of engineered CD4+ T cells to engender a functional HIV cure.
Asunto(s)
Antígenos CD28/química , Linfocitos T CD4-Positivos/trasplante , Infecciones por VIH/terapia , VIH/fisiología , Proteína Coestimuladora de Linfocitos T Inducibles/química , Receptores Quiméricos de Antígenos/metabolismo , Animales , Antígenos CD28/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Farmacorresistencia Viral , VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Inmunoterapia Adoptiva , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Ratones , Dominios Proteicos , Receptores Quiméricos de Antígenos/genética , Resultado del Tratamiento , Replicación ViralRESUMEN
The hallmarks of the adaptive immune response are specificity and memory. The cellular response is mediated by T cells which express cell surface T cell receptors (TCRs) that recognize peptide antigens in complex with major histocompatibility complex (MHC) molecules on antigen presenting cells (APCs). However, binding of cognate TCRs with MHC-peptide complexes alone (signal 1) does not trigger optimal T cell activation. In addition to signal 1, the binding of positive and negative costimulatory receptors to their ligands modulates T cell activation. This complex signaling network prevents aberrant activation of T cells. CD28 is the main positive costimulatory receptor on nai¨ve T cells; upon activation, CTLA4 is induced but reduces T cell activation. Further studies led to the identification of additional negative costimulatory receptors known as checkpoints, e.g. PD1. This review chronicles the basic studies in T cell costimulation that led to the discovery of checkpoint inhibitors, i.e. antibodies to negative costimulatory receptors (e.g. CTLA4 and PD1) which reduce tumor growth. This discovery has been recognized with the award of the 2018 Nobel prize in Physiology/Medicine. This review highlights the structural and functional roles of costimulatory receptors, the mechanisms by which checkpoint inhibitors work, the challenges encountered and future prospects.
Asunto(s)
Receptores Coestimuladores e Inhibidores de Linfocitos T/fisiología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias/tratamiento farmacológico , Linfocitos T/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos CD28/química , Antígenos CD28/metabolismo , Antígeno CTLA-4/química , Antígeno CTLA-4/metabolismo , Receptores Coestimuladores e Inhibidores de Linfocitos T/química , Receptores Coestimuladores e Inhibidores de Linfocitos T/genética , Humanos , Activación de Linfocitos , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T/fisiologíaRESUMEN
BACKGROUND: CD28, a cell surface glycoprotein receptor, predominantly expressed on activated T cells, belongs to the Ig superfamily and provides a critical co-stimulatory signal. CTLA-4 has sequence homology to CD28, and is expressed on T cells after activation. It provides an inhibition signal coordinated with CD28 to regulate T cell activation. Both of them regulate T cell proliferation and differentiation and play an important role in the immune response pathway in vivo. OBJECTIVE: We studied the special role of different structural sites of CD28 in producing costimulatory signals. METHODS: We reviewed the relevant literature, mainly regarding the structure of CD28 to clarify its biological function, and its role in the immune response. RESULTS: In recent years, increasingly attention has been paid to CD28, which is considered as a key therapeutic target for many modern diseases, especially some immune diseases. CONCLUSION: In this paper, we mainly introduce the structure of CD28 and its related biological functions, as well as the application of costimulatory pathways targeting CD28 in disease treatment.
Asunto(s)
Anticuerpos/uso terapéutico , Antígenos CD28/química , Antígenos CD28/inmunología , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos B7/inmunología , Humanos , Activación de Linfocitos/efectos de los fármacosRESUMEN
Adaptive lymphocytes express a panel of immunoreceptors on the cell surface. Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids forming microdomains in the plasma membrane can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. We have shown that negatively charged acidic phospholipids can interact with positively charged juxtamembrane polybasic regions of immunoreceptors, such as TCR-CD3, CD28 and IgG-BCR, to regulate protein structure and function. Furthermore, we pay our attention to protein transmembrane domains. We show that a membrane-snorkeling Lys residue in integrin αLß2 regulates transmembrane heterodimer formation and integrin adhesion through ionic interplay with acidic phospholipids and calcium ions (Ca2+) in T cells, thus providing a new mechanism of integrin activation. Here, we review our recent progress showcasing the importance of both juxtamembrane and intramembrane ionic protein-lipid interactions.
Asunto(s)
Antígenos CD28/inmunología , Complejo CD3/inmunología , Membrana Celular/inmunología , Fosfolípidos/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Antígenos CD28/química , Antígenos CD28/metabolismo , Complejo CD3/química , Complejo CD3/metabolismo , Señalización del Calcio/inmunología , Membrana Celular/metabolismo , Humanos , Integrinas/inmunología , Integrinas/metabolismo , Iones/inmunología , Iones/metabolismo , Activación de Linfocitos , Ratones , Fosfolípidos/química , Fosfolípidos/metabolismo , Dominios Proteicos/genética , Dominios Proteicos/inmunología , Receptores de Antígenos de Linfocitos B/químicaRESUMEN
The co-stimulation and co-inhibition signal pathways, immune checkpoints, are among the central mechanisms to regulate the T-cell immunity. Optimal signals involve intricate interactions of numerous ligands and receptors. Manipulation of these signals offers great clinical opportunities and has revolutionized the cancer treatment therapies. The 2018 Nobel Prize in Physiology or Medicine was awarded to James P. Allison and Tasuku Honjo in recognition of their discovery of cancer immunotherapy by inhibition of immune checkpoint molecules. Despite the landmark discovery in cancer immunotherapy, the efforts to harness immunity against cancer are also restricted by the limited knowledge on the co-stimulation and co-inhibition signaling networks. Understanding the structures of these molecules, in particular, tackling the interaction paradigms from the structural perspective, help to provide more accurate insights into the signaling mechanisms, which may further facilitate the development of novel biologics and improve the efficacy of the existing biologics against these targets. Here we review our current understanding on the structures of these co-stimulatory and co-inhibitory molecules. Specifically, we focus on the structural basis of several checkpoint molecules among the CD28-B7 family and discuss the therapeutic drugs against these targets for the treatment of human cancers, autoimmune disorders, and transplantation.
Asunto(s)
Antígenos CD28 , Linfocitos T , Enfermedades Autoinmunes , Antígenos CD28/química , Antígenos CD28/inmunología , Humanos , Inmunoterapia , Neoplasias/terapia , Trasplante de Órganos , Transducción de Señal/inmunología , Linfocitos T/inmunologíaRESUMEN
Chimeric antigen receptors (CARs) have recently been approved for the treatment of hematological malignancies, but our lack of understanding of the basic mechanisms that activate these proteins has made it difficult to optimize and control CAR-based therapies. In this study, we use phosphoproteomic mass spectrometry and mechanistic computational modeling to quantify the in vitro kinetics of individual tyrosine phosphorylation on a variety of CARs. We show that each of the 10 tyrosine sites on the CD28-CD3ζ CAR is phosphorylated by lymphocyte-specific protein-tyrosine kinase (LCK) with distinct kinetics. The addition of CD28 at the N-terminal of CD3ζ increases the overall rate of CD3ζ phosphorylation. Our computational model identifies that LCK phosphorylates CD3ζ through a mechanism of competitive inhibition. This model agrees with previously published data in the literature and predicts that phosphatases in this system interact with CD3ζ through a similar mechanism of competitive inhibition. This quantitative modeling framework can be used to better understand CAR signaling and T cell activation.
Asunto(s)
Simulación por Computador , Receptores de Antígenos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Antígenos CD28/química , Antígenos CD28/metabolismo , Cinética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Mutación , Fosforilación , Proteómica , Receptores de Antígenos/química , Receptores de Antígenos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Especificidad por Sustrato , Tirosina/metabolismoRESUMEN
BACKGROUND: CD28 and CTLA-4 are homologous T-cell receptors that bind with B7-1 and produce two opposing immunological signals required for T-cell activation and inactivation, respectively. It has been clinically proven that specific blockade of these key protein-protein interactions at the synapse can offer immunotherapeutic benefits for cancers and autoimmune treatments. Hence, there is a growing interest towards developing anti-CD28 and anti-CTLA-4 small molecule inhibitors. To achieve this goal, it is important to understand unique molecular level fingerprint interactions that stabilize CTLA-4/B7-1 and CD28/B7-1 complexes. However, until recently, the structure of the human CD28/B7-1 complex has not been resolved experimentally, which remains a significant setback in achieving specific inhibitors against CTLA-4 or CD28. METHODS: Here, we employed a combination of advanced molecular modelling and extensive molecular dynamics (MD) simulations to model the CD28/B7-1 complex and characterize the key interactions that stabilize the complex. RESULTS: Ensemble protein-protein docking and MD-based binding-free energy calculations were used to obtain a comprehensive structural model of the CD28/B7-1 complex, which was validated with various mutation-based experimental data from literature. Our CD28/B7-1 model has much weaker binding affinity than the CTLA-4/B7-1 complex, which is in agreement with the results from our binding assay experiments and previous studies. CONCLUSIONS: Per-residue energy decomposition of the binding affinities of the two complexes revealed the unique fingerprint hot-spot sites in CTLA-4/B7-1 and CD28/B7-1 complexes. GENERAL SIGNIFICANCE: The results presented in this work will, on a long-run, be useful to develop new generation of specific CD28 and CTLA-4 inhibitors for targeted immunotherapy.
Asunto(s)
Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Antígeno CTLA-4/metabolismo , Simulación de Dinámica Molecular , Antígeno B7-1/química , Antígenos CD28/química , Análisis por Conglomerados , Humanos , Inmunoterapia , Ligandos , Activación de Linfocitos , Unión Proteica , Reproducibilidad de los ResultadosRESUMEN
Interaction of CD28 with CD80 or CD86 molecules provides a costimulatory signals required in T cell activation. In this study, we cloned and analyzed a CD28 gene (On-CD28) and a CD80/86 gene (On-CD80/86) from Nile tilapia (Oreochromis niloticus). Sequence analysis revealed the typical characteristics of On-CD28 protein; for instance, the proline-based motif (117TYPPPL122) is essential in binding of CD28 to CD80/86 ligands. Moreover, an extracellular Ig domain was found in On-CD80/86; this domain is responsible in binding of CD28 to CD80/86 receptors. Subcellular localization analysis showed that both On-CD28 and On-CD80/86 were distributed predominantly in the cytomembrane. Yeast two-hybrid assay showed that On-CD28 directly interacted with On-CD80/86. On-CD28 and On-CD80/86 transcripts were detected in all the examined tissues of healthy Nile tilapia, and the highest expression levels of On-CD28 and On-CD80/86 were detected in the brain and heart, respectively. Following a bacterial challenge using Streptococcus agalactiae in vivo, On-CD28 and On-CD80/86 were upregulated in head kidney, spleen, intestines, and brain. However, they showed different expression profiles in response to stimulation with inactivated S. agalactiae in vitro. These findings indicated that the interaction of On-CD28 with On-CD80/86 provides a costimulatory signals that possibly play an important role in T cell activation during S. agalactiae infection.