RESUMEN
T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to self-peptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward.
Asunto(s)
Sinapsis Inmunológicas/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Comunicación Celular , Humanos , Sinapsis Inmunológicas/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca(2+)]i) elevation. TCR activation triggers increased [Ca(2+)]i and can arrest T-cell motility in vitro. However, the requirement for [Ca(2+)]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca(2+) release-activated Ca(2+) (CRAC) channel pathway required for [Ca(2+)]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant-negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4(+) T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca(2+)]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen-specific ORAI1-DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Canales de Calcio/inmunología , Calcio/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Canales de Calcio/genética , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Proteína ORAI1 , Péptidos/inmunología , Péptidos/farmacología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Molécula de Interacción Estromal 1RESUMEN
T Cell Receptor (TCR) antigen binding underlies a key mechanism of the adaptive immune response yet the vast diversity of TCRs and the complexity of protein interactions limits our ability to build useful low dimensional representations of TCRs. To address the current limitations in TCR analysis we develop a capacity-controlled disentangling variational autoencoder trained using a dataset of approximately 100 million TCR sequences, that we name TCR-VALID. We design TCR-VALID such that the model representations are low-dimensional, continuous, disentangled, and sufficiently informative to provide high-quality TCR sequence de novo generation. We thoroughly quantify these properties of the representations, providing a framework for future protein representation learning in low dimensions. The continuity of TCR-VALID representations allows fast and accurate TCR clustering and is benchmarked against other state-of-the-art TCR clustering tools and pre-trained language models.
Asunto(s)
Receptores de Antígenos de Linfocitos T , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Humanos , Aprendizaje Profundo , Algoritmos , Análisis por Conglomerados , Biología Computacional/métodos , Secuencia de AminoácidosRESUMEN
Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30-1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ T cells in effector sites.
Asunto(s)
Inmunidad Adaptativa , Inmunidad Innata , Listeria monocytogenes/patogenicidad , Listeriosis/patología , Linfocitos T Citotóxicos/patología , Animales , Citotoxicidad Inmunológica , Células Dendríticas/inmunología , Células Dendríticas/patología , Femenino , Técnicas de Sustitución del Gen , Interacciones Huésped-Patógeno , Listeriosis/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/patología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The essential role of the Delta-like ligand 4 (Dll4)-Notch signaling pathway in T-lymphocyte development is well established. It has been shown that specific inactivation of Dll4 on thymic stromal cells during early post-natal development leads to a deregulation in T-cell differentiation. However, whether ongoing Dll4-Notch signaling is required for T-cell development in the adult thymus is unknown. The use of anti-Dll4 Abs allowed us to confirm and expand previous studies by examining the kinetics and the reversibility of Dll4-Notch signaling blockade in T-cell development in adult mice. We found that anti-Dll4 treatment reduced thymic cellularity after 7 days, as a consequence of a developmental delay in T-cell maturation at the pro-T-cell double negative 1 (CD4(-) CD8(-) c-kit(+) CD44(+) CD25(-) ) stage, leading to decreased numbers of immature double-positive (CD4(+) CD8(+) ) T cells without affecting the frequency of mature single positive CD4(+) and CD8(+) thymocytes, while promoting alternative thymic B-cell expansion. This cellular phenotype was similarly observed in both young adult and aged mice (>1.5 years), extending our understanding of the ongoing role for Dll4-Notch signaling during T-cell development in the adult thymus. Finally, after cessation of Dll4 Ab treatment, thymic cellularity and thymocyte subset ratios returned to normal levels, indicating reversibility of this phenotype in both adult and aged mice, which has important implications for potential clinical use of Dll4-Notch inhibitors.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas de la Membrana/inmunología , Receptores Notch/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Timo/inmunología , Proteínas Adaptadoras Transductoras de Señales , Factores de Edad , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Atrofia/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas de Unión al Calcio , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Citometría de Flujo , Perfilación de la Expresión Génica , Homeostasis/inmunología , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Notch/genética , Receptores Notch/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Linfocitos T/metabolismo , Timo/metabolismo , Timo/patología , Factores de TiempoRESUMEN
Monocytes are highly plastic immune cells that modulate antitumor immunity. Therefore, identifying factors that regulate tumor monocyte functions is critical for developing effective immunotherapies. Here, we determine that endogenous cancer cell-derived type I interferons (IFNs) control monocyte functional polarization. Guided by single-cell transcriptomic profiling of human and mouse tumors, we devise a strategy to distinguish and separate immunostimulatory from immunosuppressive tumor monocytes by surface CD88 and Sca-1 expression. Leveraging this approach, we show that cGAS-STING-regulated cancer cell-derived IFNs polarize immunostimulatory monocytes associated with anti-PD-1 immunotherapy response in mice. We also demonstrate that immunosuppressive monocytes convert into immunostimulatory monocytes upon cancer cell-intrinsic cGAS-STING activation. Consistently, we find that human cancer cells can produce type I IFNs that polarize monocytes, and our immunostimulatory monocyte gene signature is enriched in patient tumors that respond to anti-PD-1 immunotherapy. Our work exposes a role for cancer cell-derived IFNs in licensing monocyte functions that influence immunotherapy outcomes.
Asunto(s)
Interferón Tipo I , Neoplasias , Humanos , Ratones , Animales , MonocitosRESUMEN
Specificity of neurotrophin factor signaling is dictated through the action of Trk receptor tyrosine kinases. Once activated, Trk receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin receptors are differentially regulated by ubiquitination to modulate the survival of neurons.
Asunto(s)
Supervivencia Celular/fisiología , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Receptor trkA/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Western Blotting , Células Cultivadas , Complejos de Clasificación Endosomal Requeridos para el Transporte , Técnica del Anticuerpo Fluorescente , Humanos , Datos de Secuencia Molecular , Ubiquitina-Proteína Ligasas Nedd4 , ARN Interferente Pequeño , Ratas , Receptor trkA/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Ubiquitina/metabolismoRESUMEN
The chemokine receptor CXCR4 is expressed in B cells at multiple stages of their development. CXCR4 function in humoral immunity has not been fully investigated. We have generated gene-targeted mice in which CXCR4 can be selectively inactivated in B cells and have shown that it is required for retention of B cell precursors in the bone marrow. CXCR4-deficient B cell precursors that migrated prematurely became localized in splenic follicles despite their unresponsiveness to CXCL13. Concomitantly, mature B cell populations were reduced in the splenic marginal zone and primary follicles, and in the peritoneal cavity in the mutant animals, as were T-independent antibody responses. In addition, aberrant B cell follicles formed ectopically in intestinal lamina propria around Peyer's patches. These findings establish an important role for CXCR4 in regulating homeostasis of B cell compartmentalization and humoral immunity.
Asunto(s)
Formación de Anticuerpos/inmunología , Linfocitos B/citología , Homeostasis/inmunología , Receptores CXCR4/inmunología , Animales , Anexina A5 , Linfocitos B/metabolismo , Southern Blotting , Quimiocina CXCL13 , Quimiocinas CXC , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Mutantes , Ganglios Linfáticos Agregados/inmunología , Reacción en Cadena de la Polimerasa , Bazo/inmunologíaRESUMEN
Short-lived TCR microclusters and a longer-lived protein kinase Ctheta-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCtheta, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca(2+) signaling and the number and size of the CD80 clusters, a characteristic of TCR microclusters. Thus, the T cell-DC interface contains dynamic costimulatory foci that share characteristics of microclusters and cSMACs.
Asunto(s)
Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Células Dendríticas/inmunología , Sinapsis Inmunológicas/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Subgrupos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígeno B7-1/genética , Antígeno B7-1/fisiología , Antígenos CD28/genética , Antígenos CD28/fisiología , Células CHO , Cromosomas Artificiales Bacterianos/genética , Cricetinae , Cricetulus , Células Dendríticas/enzimología , Células Dendríticas/metabolismo , Proteínas Fluorescentes Verdes/genética , Sinapsis Inmunológicas/enzimología , Sinapsis Inmunológicas/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Proteína Quinasa C-theta , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Receptores de Antígenos de Linfocitos T/genética , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Clinical observations suggest that responses to cancer immunotherapy are correlated with intra-tumoral T cell receptor (TCR) clonality, tumor mutation burden (TMB) and host HLA genotype, highlighting the importance of host T cell recognition of tumor antigens. However, the dynamic interplay between T cell activation state and changes in TCR repertoire in driving the identification of potential immunodominant antigen(s) remains largely unexplored. Here, we performed single-cell RNA-sequencing on CD8+ tumor-infiltrating T cells (TILs) using the murine colorectal tumor model MC38 to identify unique TCR sequences and validate their tumor reactivity. We found that the majority of clonally expanded TILs are tumor-reactive and their TCR repertoire is unique amongst individual MC38 tumor-bearing mice. Our query identified that multiple expanded TCR clones recognized the retroviral epitope p15E as an immunodominant antigen. In addition, we found that the endogenous retroviral genome encoding for p15E is highly expressed in MC38 tumors, but not in normal tissues, due to epigenetic derepression. Further, we demonstrated that the p15E-specific TILs exhibit an activated phenotype and an increase in frequency upon treatment with anti-41BB and anti-PD-1 combination immunotherapy. Importantly, we showed that although p15E-specific TILs are not required to mount a primary anti-tumor response, they contributed to the development of strong immune memory. Overall our results revealed that endogenous retroviral antigens expressed by tumor cells may represent an important and underappreciated category of tumor antigens that could be readily targeted in the clinic.
Asunto(s)
Retrovirus Endógenos , Neoplasias , Animales , Inmunoterapia , Activación de Linfocitos , Ratones , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Monoclonal antibodies that block the programmed cell death 1 (PD-1) checkpoint have revolutionized cancer immunotherapy. However, many major tumor types remain unresponsive to anti-PD-1 therapy, and even among responsive tumor types, most of the patients do not develop durable antitumor immunity. It has been shown that bispecific antibodies activate T cells by cross-linking the TCR/CD3 complex with a tumor-specific antigen (TSA). The class of TSAxCD3 bispecific antibodies have generated exciting results in early clinical trials. We have recently described another class of "costimulatory bispecifics" that cross-link a TSA to CD28 (TSAxCD28) and cooperate with TSAxCD3 bispecifics. Here, we demonstrate that these TSAxCD28 bispecifics (one specific for prostate cancer and the other for epithelial tumors) can also synergize with the broader anti-PD-1 approach and endow responsiveness-as well as long-term immune memory-against tumors that otherwise do not respond to anti-PD-1 alone. Unlike CD28 superagonists, which broadly activate T cells and induce cytokine storm, TSAxCD28 bispecifics display little or no toxicity when used alone or in combination with a PD-1 blocker in genetically humanized immunocompetent mouse models or in primates and thus may provide a well-tolerated and "off the shelf" combination approach with PD-1 immunotherapy that can markedly enhance antitumor efficacy.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Animales , Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD28 , Humanos , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1RESUMEN
T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide-major histocompatibility complexes ("signal 1"); activation is enhanced by engagement of a second "costimulatory" receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell ("signal 2"). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Neoplasias/inmunología , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Proliferación Celular , Citocinas/metabolismo , Citotoxicidad Inmunológica , Femenino , Células HEK293 , Humanos , Sinapsis Inmunológicas/metabolismo , Activación de Linfocitos/inmunología , Macaca fascicularis , Ratones , Neoplasias/patología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Profiling T cell receptor (TCR) repertoire via short read transcriptome sequencing (RNA-Seq) has a unique advantage of probing simultaneously TCRs and the genome-wide RNA expression of other genes. However, compared to targeted amplicon approaches, the shorter read length is more prone to mapping error. In addition, only a small percentage of the genome-wide reads may cover the TCR loci and thus the repertoire could be significantly under-sampled. Although this approach has been applied in a few studies, the utility of transcriptome sequencing in probing TCR repertoires has not been evaluated extensively. Here we present a systematic assessment of RNA-Seq in TCR profiling. We evaluate the power of both Fluidigm C1 full-length single cell RNA-Seq and bulk RNA-Seq in characterizing the repertoires of different diversities under either naïve conditions or after immunogenic challenges. Standard read length and sequencing coverage were employed so that the evaluation was conducted in accord with the current RNA-Seq practices. Despite high sequencing depth in bulk RNA-Seq, we encountered difficulty quantifying TCRs with low transcript abundance (<1%). Nevertheless, top enriched TCRs with an abundance of 1-3% or higher can be faithfully detected and quantified. When top TCR sequences are of interest and transcriptome sequencing is available, it is worthwhile to conduct a TCR profiling using the RNA-Seq data.
Asunto(s)
ARN/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Sitios Genéticos , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones , Ratones Endogámicos C57BL , ARN/química , ARN/aislamiento & purificación , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Bazo/citología , Bazo/inmunología , Bazo/virología , TranscriptomaRESUMEN
Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8+ T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8+ T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8+ T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1-Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Memoria Inmunológica/inmunología , Inmunoterapia , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/patología , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/inmunología , FenotipoRESUMEN
The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti-PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1-dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861-70. ©2017 AACR.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Proliferación Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/genética , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Línea Celular Tumoral , Técnicas de Sustitución del Gen , Humanos , Inmunoterapia , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
A central mechanism of tumour progression and metastasis involves the generation of an immunosuppressive 'macroenvironment' mediated in part through tumour-secreted factors. Here we demonstrate that upregulation of the Inhibitor of Differentiation 1 (Id1), in response to tumour-derived factors, such as TGFß, is responsible for the switch from dendritic cell (DC) differentiation to myeloid-derived suppressor cell expansion during tumour progression. Genetic inactivation of Id1 largely corrects the myeloid imbalance, whereas Id1 overexpression in the absence of tumour-derived factors re-creates it. Id1 overexpression leads to systemic immunosuppression by downregulation of key molecules involved in DC differentiation and suppression of CD8 T-cell proliferation, thus promoting primary tumour growth and metastatic progression. Furthermore, advanced melanoma patients have increased plasma TGFß levels and express higher levels of ID1 in myeloid peripheral blood cells. This study reveals a critical role for Id1 in suppressing the anti-tumour immune response during tumour progression and metastasis.
Asunto(s)
Proteína 1 Inhibidora de la Diferenciación/fisiología , Melanoma Experimental/inmunología , Células Mieloides/fisiología , Animales , Diferenciación Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Factores Reguladores del Interferón/metabolismo , Leucocitos Mononucleares/metabolismo , Melanoma Experimental/metabolismo , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The proinflammatory activity of T helper 17 (Th17) cells can be beneficial to the host during infection. However, uncontrolled or inappropriate Th17 activation has been linked to several autoimmune and autoinflammatory pathologies. Indeed, preclinical and clinical data show that Th17 cells are associated with several autoimmune diseases such as arthritis, multiple sclerosis, psoriasis, and lupus. Furthermore, targeting the interleukin-17 (IL-17) pathway has attenuated disease severity in preclinical models of autoimmune diseases. Interestingly, a recent report brings to light a potential role for Th17 cells in the autoinflammatory disorder adult-onset Still's disease (AOSD). Whether Th17 cells are the cause or are directly involved in AOSD remains to be shown. In this paper, we discuss the biology of Th17 cells, their role in autoimmune disease development, and in AOSD in particular, as well as the growing interest of the pharmaceutical industry in their use as therapeutic targets.
RESUMEN
Delta-like ligand 4 (Dll4)-Notch signaling is essential for T cell development and alternative thymic lineage decisions. How Dll4-Notch signaling affects pro-T cell fate and thymic dendritic cell (tDC) development is unknown. We found that Dll4 pharmacological blockade induces accumulation of tDCs and CD4(+)CD25(+)FoxP3(+) regulatory T cells (T(reg) cells) in the thymic cortex. Both genetic inactivation models and anti-Dll4 antibody (Ab) treatment promote de novo natural T(reg) cell expansion by a DC-dependent mechanism that requires major histocompatibility complex II expression on DCs. Anti-Dll4 treatment converts CD4(-)CD8(-)c-kit(+)CD44(+)CD25(-) (DN1) T cell progenitors to immature DCs that induce ex vivo differentiation of naive CD4(+) T cells into T(reg) cells. Induction of these tolerogenic DN1-derived tDCs and the ensuing expansion of T(reg) cells are Fms-like tyrosine kinase 3 (Flt3) independent, occur in the context of transcriptional up-regulation of PU.1, Irf-4, Irf-8, and CSF-1, genes critical for DC differentiation, and are abrogated in thymectomized mice. Anti-Dll4 treatment fully prevents type 1 diabetes (T1D) via a T(reg) cell-mediated mechanism and inhibits CD8(+) T cell pancreatic islet infiltration. Furthermore, a single injection of anti-Dll4 Ab reverses established T1D. Disease remission and recurrence are correlated with increased T(reg) cell numbers in the pancreas-draining lymph nodes. These results identify Dll4-Notch as a novel Flt3-alternative pathway important for regulating tDC-mediated T(reg) cell homeostasis and autoimmunity.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/fisiología , Diabetes Mellitus Tipo 1/prevención & control , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Anticuerpos/farmacología , Western Blotting , Proteínas de Unión al Calcio , Cartilla de ADN/genética , Células Dendríticas/inmunología , Diabetes Mellitus Tipo 1/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Genes MHC Clase II/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Páncreas/patología , Reacción en Cadena de la Polimerasa , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Timo/citología , Timo/inmunología , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
CXCR4 regulates cell proliferation, enhances cell survival and induces chemotaxis, yet molecular mechanisms underlying its signaling remain elusive. Like all other G-protein coupled receptors (GPCRs), CXCR4 delivers signals through G-protein-dependent and -independent pathways, the latter involving its serine-rich cytoplasmic tail. To evaluate the signaling and biological contribution of this G-protein-independent pathway, we generated mutant mice that express cytoplasmic tail-truncated CXCR4 (ΔT) by a gene knock-in approach. We found that ΔT mice exhibited multiple developmental defects, with not only G-protein-independent but also G-protein-dependent signaling events completely abolished, despite ΔT's ability to still associate with G-proteins. These results reveal an essential positive regulatory role of the cytoplasmic tail in CXCR4 signaling and suggest the tail is crucial for mediating G-protein activation and initiating crosstalk between G-protein-dependent and G-protein-independent pathways for correct GPCR signaling.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Organogénesis , Receptores CXCR4/fisiología , Transducción de Señal , Animales , Adhesión Celular/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacología , Quimiotaxis/efectos de los fármacos , Femenino , Citometría de Flujo , Proteínas de Unión al GTP/genética , Mucosa Gástrica/metabolismo , Células HEK293 , Humanos , Immunoblotting , Inmunoprecipitación , Masculino , Ratones , Ratones Noqueados , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/fisiología , Unión Proteica , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Estómago/embriología , Factores de TiempoRESUMEN
T cells survey antigen-presenting dendritic cells (DCs) by migrating through DC networks, arresting and maintaining contact with DCs for several hours after encountering high-potency complexes of peptide and major histocompatibility complex (pMHC), leading to T cell activation. The effects of low-potency pMHC complexes on T cells in vivo, however, are unknown, as is the mechanism controlling T cell arrest. Here we evaluated T cell responses in vivo to high-, medium- and low-potency pMHC complexes and found that regardless of potency, pMHC complexes induced upregulation of CD69, anergy and retention of T cells in lymph nodes. However, only high-potency pMHC complexes expressed by DCs induced calcium-dependent T cell deceleration and calcineurin-dependent anergy. The pMHC complexes of lower potency instead induced T cell anergy by a biochemically distinct process that did not affect T cell dynamics.