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1.
Nat Immunol ; 24(4): 676-689, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36914891

RESUMEN

Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LATG135D) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LATG135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LATG135D T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LATG135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Linfocitos T , Ratones , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Receptores de Antígenos de Linfocitos T/metabolismo , Activación de Linfocitos , Fosforilación , Fosfoproteínas/genética
2.
Nat Immunol ; 20(11): 1481-1493, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31611699

RESUMEN

Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Femenino , Ligandos , Masculino , Proteínas de la Membrana/inmunología , Ratones , Fosfolipasa C gamma/metabolismo , Fosforilación/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/metabolismo , Tirosina/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo
3.
Nat Immunol ; 19(7): 733-741, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29915297

RESUMEN

T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Secuencias de Aminoácidos , Animales , Células HEK293 , Humanos , Células Jurkat , Proteínas de la Membrana/química , Ratones , Ratones Endogámicos C57BL , Fosforilación , Prolina/análisis , Receptores de Antígenos de Linfocitos T/metabolismo , Timo/inmunología
4.
Nature ; 631(8022): 867-875, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38987588

RESUMEN

Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.


Asunto(s)
Linfocitos T CD8-positivos , Hepatitis B Crónica , Hígado , Animales , Humanos , Masculino , Ratones , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Hepatocitos/inmunología , Hepatocitos/virología , Hígado/inmunología , Hígado/virología , Fosforilación , Transducción de Señal , Activación de Linfocitos
6.
Proc Natl Acad Sci U S A ; 121(30): e2407159121, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39012820

RESUMEN

Mutations in the tyrosine phosphatase Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting autoinhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8 to 10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine-binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.


Asunto(s)
Simulación de Dinámica Molecular , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Dominios Homologos src , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Humanos , Dominios Homologos src/genética , Unión Proteica , Mutación , Fosforilación , Sitios de Unión/genética , Fosfotirosina/metabolismo , Ligandos
7.
Nat Immunol ; 15(3): 266-74, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24487322

RESUMEN

Interactions of T cell antigen receptors (TCRs) with complexes of self peptide and major histocompatibility complex (MHC) are crucial to T cell development, but their role in peripheral T cell responses remains unclear. Specific and nonspecific stimulation of LLO56 and LLO118 T cells, which transgenically express a TCR specific for the same Listeria monocytogenes epitope, elicited distinct interleukin 2 (IL-2) and phosphorylated kinase Erk responses, the strength of which was set in the thymus and maintained in the periphery in proportion to the avidity of the binding of the TCR to the self peptide-MHC complex. Deprivation of self peptide-MHC substantially compromised the population expansion of LLO56 T cells in response to L. monocytogenes in vivo. Despite their very different self-reactivity, LLO56 T cells and LLO118 T cells bound cognate peptide-MHC with an identical affinity, which challenges associations made between these parameters. Our findings highlight a crucial role for selecting ligands encountered during thymic 'education' in determining the intrinsic functionality of CD4+ T cells.


Asunto(s)
Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Separación Celular , Citometría de Flujo , Humanos , Immunoblotting , Listeriosis/inmunología , Ratones , Ratones Noqueados , Resonancia por Plasmón de Superficie , Timo/citología , Timo/inmunología , Transfección
8.
Immunol Rev ; 307(1): 145-160, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-34923645

RESUMEN

Establishing both central and peripheral tolerance requires the appropriate TCR signaling strength to discriminate self- from agonist-peptide bound to self MHC molecules. ZAP70, a cytoplasmic tyrosine kinase, directly interacts with the TCR complex and plays a central and requisite role in TCR signaling in both thymocytes and peripheral T cells. By studying ZAP70 hypomorphic mutations in mice and humans with a spectrum of hypoactive or hyperactive activities, we have gained insights into mechanisms of central and peripheral tolerance. Interestingly, both hypoactive and hyperactive ZAP70 can lead to the development of autoimmune diseases, albeit through distinct mechanisms. Immature thymocytes and mature T cells rely on normal ZAP70 function to complete their development in the thymus and to modulate T cell responses in the periphery. Hypoactive ZAP70 function compromises key developmental checkpoints required to establish central tolerance, allowing thymocytes with potentially self-reactive TCRs a greater chance to escape negative selection. Such 'forbidden clones' may escape into the periphery and may pose a greater risk for autoimmune disease development since they may not engage negative regulatory mechanisms as effectively. Hyperactive ZAP70 enhances thymic negative selection but some thymocytes will, nonetheless, escape negative selection and have greater sensitivity to weak and self-ligands. Such cells must be controlled by mechanisms involved in anergy, expansion of Tregs, and upregulation of inhibitory receptors or signaling molecules. However, such potentially autoreactive cells may still be able to escape control by peripheral negative regulatory constraints. Consistent with findings in Zap70 mutants, the signaling defects in at least one ZAP70 substrate, LAT, can also lead to autoimmune disease. By dissecting the similarities and differences among mouse models of patient disease or mutations in ZAP70 that affect TCR signaling strength, we have gained insights into how perturbed ZAP70 function can lead to autoimmunity. Because of our work and that of others on ZAP70, it is likely that perturbations in other molecules affecting TCR signaling strength will be identified that also overcome tolerance mechanisms and cause autoimmunity. Delineating these molecular pathways could lead to the development of much needed new therapeutic targets in these complex diseases.


Asunto(s)
Enfermedades Autoinmunes , Autoinmunidad , Proteínas Tirosina Quinasas/metabolismo , Animales , Humanos , Tolerancia Inmunológica , Ratones , Receptores de Antígenos de Linfocitos T/metabolismo , Timocitos , Timo
9.
Nat Immunol ; 13(9): 880-7, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22842345

RESUMEN

The sustained entry of Ca(2+) into CD4(+)CD8(+) double-positive thymocytes is required for positive selection. Here we identified a voltage-gated Na(+) channel (VGSC) that was essential for positive selection of CD4(+) T cells. Pharmacological inhibition of VGSC activity inhibited the sustained Ca(2+) influx induced by positively selecting ligands and the in vitro positive selection of CD4(+) but not CD8(+) T cells. In vivo short hairpin RNA (shRNA)-mediated knockdown of the gene encoding a regulatory ß-subunit of a VGSC specifically inhibited the positive selection of CD4(+) T cells. Ectopic expression of VGSC in peripheral AND CD4(+) T cells bestowed the ability to respond to a positively selecting ligand, which directly demonstrated that VGSC expression was responsible for the enhanced sensitivity. Thus, active VGSCs in thymocytes provide a mechanism by which a weak positive selection signal can induce the sustained Ca(2+) signals required for CD4(+) T cell development.


Asunto(s)
Linfocitos T CD4-Positivos/citología , Diferenciación Celular/inmunología , Canales de Sodio/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Citometría de Flujo , Humanos , Activación del Canal Iónico , Ratones , Ratones Transgénicos , Canal de Sodio Activado por Voltaje NAV1.5 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subunidad beta-4 de Canal de Sodio Activado por Voltaje
10.
J Immunol ; 206(10): 2322-2337, 2021 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-33931484

RESUMEN

The costimulatory receptor CD28 synergizes with the TCR to promote IL-2 production, cell survival, and proliferation; yet the obligatory interdependence of TCR and CD28 signaling is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 Y173 and PLC-γ1 Y783. In this study, we identified TCR-inducible, Itk-mediated phosphorylation of Gads Y45 in a human T cell line and in mouse primary T cells. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the dimerization-dependent binding of Gads to phospho-LAT. We provide evidence that Itk acts through SLP-76 and Gads to promote the TCR/CD28-induced activation of the RE/AP transcriptional element from the IL-2 promoter. Two Itk-related features of SLP-76, Y173 and a proline-rich Itk SH3 binding motif on SLP-76, were dispensable for activation of NFAT but selectively required for the TCR/CD28-induced increase in cytoplasmic and nuclear c-Rel and consequent RE/AP activation. We provide evidence that unphosphorylated, monomeric Gads mediates an RE/AP-directed inhibitory activity that is mitigated upon Gads dimerization and Y45 phosphorylation. This study illuminates a new, to our knowledge, regulatory module, in which TCR-induced, Itk-mediated phosphorylation sites on SLP-76 and Gads control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD28/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Dimerización , Vectores Genéticos , Humanos , Interleucina-2/metabolismo , Células Jurkat , Ratones , Ratones Endogámicos BALB C , Fosfoproteínas/genética , Fosforilación/genética , Unión Proteica , Transfección
11.
Proc Natl Acad Sci U S A ; 117(42): 26020-26030, 2020 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-33020303

RESUMEN

T cells exhibit remarkable sensitivity and selectivity in detecting and responding to agonist peptides (p) bound to MHC molecules in a sea of self pMHC molecules. Despite much work, understanding of the underlying mechanisms of distinguishing such ligands remains incomplete. Here, we quantify T cell discriminatory capacity using channel capacity, a direct measure of the signaling network's ability to discriminate between antigen-presenting cells (APCs) displaying either self ligands or a mixture of self and agonist ligands. This metric shows how differences in information content between these two types of peptidomes are decoded by the topology and rates of kinetic proofreading signaling steps inside T cells. Using channel capacity, we constructed numerically substantiated hypotheses to explain the discriminatory role of a recently identified slow LAT Y132 phosphorylation step. Our results revealed that in addition to the number and kinetics of sequential signaling steps, a key determinant of discriminatory capability is spatial localization of a minimum number of these steps to the engaged TCR. Biochemical and imaging experiments support these findings. Our results also reveal the discriminatory role of early negative feedback and necessary amplification conferred by late positive feedback.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Presentadoras de Antígenos/inmunología , Activación de Linfocitos/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Proteínas de la Membrana/metabolismo , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Humanos , Células Jurkat , Cinética , Ligandos , Modelos Teóricos , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal
12.
Trends Biochem Sci ; 43(2): 108-123, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29269020

RESUMEN

The mechanisms by which a T cell detects antigen using its T cell antigen receptor (TCR) are crucial to our understanding of immunity and the harnessing of T cells therapeutically. A hallmark of the T cell response is the ability of T cells to quantitatively respond to antigenic ligands derived from pathogens while remaining inert to similar ligands derived from host tissues. Recent studies have revealed exciting properties of the TCR and the behaviors of its signaling effectors that are used to detect and discriminate between antigens. Here we highlight these recent findings, focusing on the proximal TCR signaling molecules Zap70, Lck, and LAT, to provide mechanistic models and insights into the exquisite sensitivity and specificity of the TCR.


Asunto(s)
Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Animales , Humanos
13.
Nat Immunol ; 10(11): 1155-61, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19801984

RESUMEN

Although CD4(+) and CD8(+) T cells differ in the strength of their positively selecting signal, endogenous positively selecting ligands have been identified only for major histocompatibility complex (MHC) class I-restricted T cell antigen receptors (TCRs). Here we screened for ligands able to positively select MHC class II-restricted TCRs using thymocytes from four I-E(k)-restricted TCR-transgenic mice and a large panel of self peptides. One peptide, gp250, induced positive selection of AND CD4(+) T cells, had no homology with the AND TCR agonist ligand and was recognized with a high degree of specificity. The gp250 peptide acted as a coagonist to initiate the activation and enhance the survival of peripheral AND CD4(+) T cells. Thus, positively selecting ligands are critical in thymocyte development and in the activation and maintenance of peripheral T cells.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Activación de Linfocitos , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Sustitución de Aminoácidos , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD4-Positivos/citología , Línea Celular , Proliferación Celular , Lectinas Tipo C , Ligandos , Ratones , Ratones Transgénicos , Unión Proteica , Timo/citología , Timo/inmunología
14.
Curr Top Microbiol Immunol ; 373: 49-67, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-23612987

RESUMEN

The vertebrate antigen receptors are anticipatory in their antigen recognition and display a vast diversity. Antigen receptors are assembled through V(D)J recombination, in which one of each Variable, (Diverse), and Joining gene segment are randomly utilized and recombined. Both gene rearrangement and mutational insertion are generated through randomness; therefore, the process of antigen receptors generation requires a rigorous testing system to select every receptor which is useful to recognize foreign antigens, but which would cause no harm to self cells. In the case of T cell receptors (TCR), such a quality control responsibility rests in thymic positive and negative selection. In this review, we focus on the critical involvement of self-peptides in the generation of a T cell repertoire, discuss the role of T cell thymic development in shaping the specificity of TCR repertoire, and directing function fitness of mature T cells in periphery. Here, we consider thymic positive selection to be not merely a one-time maturing experience for an individual T cell, but a life-long imprinting which influences the function of each individual T cell in periphery.


Asunto(s)
Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Timo/inmunología , Animales , Epítopos , Humanos
15.
J Exp Med ; 221(10)2024 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-39167074

RESUMEN

αß T cells are critical components of the adaptive immune system; they maintain tissue and immune homeostasis during health, provide sterilizing immunity after pathogen infection, and are capable of eliminating transformed tumor cells. Fundamental to these distinct functions is the ligand specificity of the unique antigen receptor expressed on each mature T cell (TCR), which endows lymphocytes with the ability to behave in a cell-autonomous, disease context-specific manner. Clone-specific behavioral properties are initially established during T cell development when thymocytes use TCR recognition of major histocompatibility complex (MHC) and MHC-like ligands to instruct survival versus death and to differentiate into a plethora of inflammatory and regulatory T cell lineages. Here, we review the ligand specificity of the preselection thymocyte repertoire and argue that developmental stage-specific alterations in TCR signaling control cross-reactivity and foreign versus self-specificity of T cell sublineages.


Asunto(s)
Timo , Humanos , Animales , Timo/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Timocitos/inmunología , Timocitos/metabolismo , Linfocitos T/inmunología , Diferenciación Celular/inmunología , Complejo Mayor de Histocompatibilidad/inmunología
16.
bioRxiv ; 2024 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-37502916

RESUMEN

Mutations in the tyrosine phosphatase SHP2 are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting auto-inhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that, while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8-10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.

17.
J Immunol ; 184(7): 3628-38, 2010 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-20194718

RESUMEN

Chemokines promote lymphocyte motility by triggering F-actin rearrangements and inducing cellular polarization. Chemokines can also enhance cell-cell adhesion and costimulate T cells. In this study, we establish a requirement for the actin-bundling protein L-plastin (LPL) in CCR7- and sphingosine-1-phosphate-mediated T cell chemotaxis using LPL(-/-) mice. Disrupted motility of mature LPL(-/-) thymocytes manifested in vivo as diminished thymic egress. Two-photon microscopy of LPL(-/-) lymphocytes revealed reduced velocity and motility in lymph nodes. Defective migration resulted from defective cellular polarization following CCR7 ligation, as CCR7 did not polarize to the leading edge in chemokine-stimulated LPL(-/-) T cells. However, CCR7 signaling to F-actin polymerization and CCR7-mediated costimulation was intact in LPL(-/-) lymphocytes. The differential requirement for LPL in CCR7-induced cellular adhesion and CCR7-induced motility allowed assessment of the contribution of CCR7-mediated motility to positive selection of thymocytes and lineage commitment. Results suggest that normal motility is not required for CCR7 to function in positive selection and lineage commitment. We thus identify LPL as a molecule critical for CCR7-mediated motility but dispensable for early CCR7 signaling. The requirement for actin bundling by LPL for polarization reveals a novel mechanism of regulating actin dynamics during T cell motility.


Asunto(s)
Quimiotaxis de Leucocito/inmunología , Fosfoproteínas/inmunología , Receptores CCR7/inmunología , Transducción de Señal/inmunología , Animales , Adhesión Celular/inmunología , Polaridad Celular/inmunología , Separación Celular , Proteínas del Citoesqueleto , Citometría de Flujo , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos , Microscopía Confocal , Fosfoproteínas/metabolismo , Receptores CCR7/metabolismo
18.
Front Immunol ; 12: 673196, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33936119

RESUMEN

Self- and non-self ligand discrimination is a core principle underlying T cell-mediated immunity. Mature αß T cells can respond to a foreign peptide ligand presented by major histocompatibility complex molecules (pMHCs) on antigen presenting cells, on a background of continuously sensed self-pMHCs. How αß T cells can properly balance high sensitivity and high specificity to foreign pMHCs, while surrounded by a sea of self-peptide ligands is not well understood. Such discrimination cannot be explained solely by the affinity parameters of T cell antigen receptor (TCR) and pMHC interaction. In this review, we will discuss how T cell ligand discrimination may be molecularly defined by events downstream of the TCR-pMHC interaction. We will discuss new evidence in support of the kinetic proofreading model of TCR ligand discrimination, and in particular how the kinetics of specific phosphorylation sites within the adaptor protein linker for activation of T cells (LAT) determine the outcome of TCR signaling. In addition, we will discuss emerging data regarding how some kinases, including ZAP-70 and LCK, may possess scaffolding functions to more efficiently direct their kinase activities.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Activación de Linfocitos/inmunología , Proteínas de la Membrana/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Autotolerancia/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/inmunología , Proteína Tirosina Quinasa ZAP-70/metabolismo
19.
Oncogene ; 40(34): 5253-5261, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34290401

RESUMEN

The exploitation of T cell-based immunotherapies and immune checkpoint blockade for cancer treatment has dramatically shifted oncological treatment paradigms and broadened the horizons of cancer immunology. Dendritic cells have emerged as the critical tailors of T cell immune responses, which initiate and coordinate anti-tumor immunity. Importantly, genetic alterations in cancer cells, cytokines and chemokines produced by cancer and stromal cells, and the process of tumor microenvironmental regulation can compromise dendritic cell-T cell cross-talk, thereby disrupting anti-tumor T cell responses. This review summarizes how T cell activation is controlled by dendritic cells and how the tumor microenvironment alters dendritic cell properties in the context of the anti-tumor immune cycle. Furthermore, we will highlight therapeutic options for tailoring dendritic cell-mediated decision-making in T cells for cancer treatment.


Asunto(s)
Anticuerpos , Microambiente Tumoral , Humanos , Inmunoterapia , Activación de Linfocitos
20.
Mol Biol Evol ; 25(10): 2189-98, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18667440

RESUMEN

Human c-Myb proto-oncogene is highly expressed in hematopoietic progenitors as well as leukemia and certain solid tumor. However, the regulatory mechanisms of its expression and biological functions remain largely unclear. Recently, c-Myb has been shown to be targeted by microRNA-150 (miR-150) which thereby controls B cell differentiation in mice. In this study, we demonstrated that c-Myb is an evolutionary conserved target of miR-150 in human and zebrafish, using reporter assays. Ectopic expression of miR-150 in breast cancer and leukemic cells repressed endogenous c-Myb at both messenger RNA (mRNA) and protein levels. Among several leukemia cell lines, primary leukemia cells, and normal lymphocytes, expression levels of miR-150 inversely correlated with c-Myb. The miR-150 overexpression or c-Myb silencing in zebrafish zygotes led to similar and serious phenotypic defects in zebrafish, and the phenotypic aberrations induced by miR-150 could be reversed by coinjection of c-Myb mRNA. Our findings suggest that c-Myb is an evolutionally conserved target of miR-150 and miR-150/c-Myb interaction is important for embryonic development and possibly oncogenesis.


Asunto(s)
Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Línea Celular Tumoral , Aberraciones Cromosómicas , Humanos , Linfocitos/metabolismo , Ratones , Datos de Secuencia Molecular , Fenotipo , Proto-Oncogenes Mas , Pez Cebra
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