RESUMEN
Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell receptor signaling and as such is an attractive target for cancer immunotherapy. Although the role of the HPK1 kinase domain (KD) has been extensively characterized, the function of its citron homology domain (CHD) remains elusive. Through a combination of structural, biochemical, and mechanistic studies, we characterize the structure-function of CHD in relationship to KD. Crystallography and hydrogen-deuterium exchange mass spectrometry reveal that CHD adopts a seven-bladed ß-propellor fold that binds to KD. Mutagenesis associated with binding and functional studies show a direct correlation between domain-domain interaction and negative regulation of kinase activity. We further demonstrate that the CHD provides stability to HPK1 protein in cells as well as contributes to the docking of its substrate SLP76. Altogether, this study highlights the importance of the CHD in the direct and indirect regulation of HPK1 function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Serina-Treonina Quinasas , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/química , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/química , Fosfoproteínas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Dominios Proteicos , Cristalografía por Rayos X , Células HEK293RESUMEN
Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.
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Sistemas CRISPR-Cas , Reparación del ADN por Recombinación , Humanos , Edición Génica/métodos , Reparación del ADN , Reparación del ADN por Unión de ExtremidadesRESUMEN
Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.
Asunto(s)
Linfocitos T CD8-positivos , Neoplasias , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos CD28/metabolismo , Humanos , Neoplasias/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Inmunológicos/metabolismoRESUMEN
Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-small cell lung cancer (NSCLC) patients, based on their significant overall survival (OS) benefit. Using transcriptomic analysis of 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy from two large randomized clinical trials, we find a significant B cell association with extended OS with PD-L1 blockade, independent of CD8+ T cell signals. We then derive gene signatures corresponding to the dominant B cell subsets present in NSCLC from single-cell RNA sequencing (RNA-seq) data. Importantly, we find increased plasma cell signatures to be predictive of OS in patients treated with atezolizumab, but not chemotherapy. B and plasma cells are also associated with the presence of tertiary lymphoid structures and organized lymphoid aggregates. Our results suggest an important contribution of B and plasma cells to the efficacy of PD-L1 blockade in NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antígeno B7-H1/genética , Antígeno B7-H1/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Inhibidores de Puntos de Control Inmunológico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Células Plasmáticas/patologíaRESUMEN
BACKGROUND: CD8+ tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, are thought to actively suppress cancer progression, leading to the hypothesis that their presence in tumors may predict response to immunotherapy. METHODS: Here, we test this by combining high-dimensional single-cell modalities with bulk tumor transcriptomics from 1868 patients enrolled in lung and bladder cancer clinical trials of atezolizumab (anti-programmed cell death ligand 1 (PD-L1)). RESULTS: ITGAE was identified as the most significantly upregulated gene in inflamed tumors. Tumor CD103+ CD8+ TRM cells exhibited a complex phenotype defined by the expression of checkpoint regulators, cytotoxic proteins, and increased clonal expansion. CONCLUSIONS: Our analyses indeed demonstrate that the presence of CD103+ CD8+ TRM cells, quantified by tracking intratumoral CD103 expression, can predict treatment outcome, suggesting that patients who respond to PD-1/PD-L1 blockade are those who exhibit an ongoing antitumor T-cell response.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos CD/genética , Antígeno B7-H1/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Linfocitos T CD8-positivos/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Cadenas alfa de Integrinas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Anticuerpos Monoclonales Humanizados/efectos adversos , Antígeno B7-H1/inmunología , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Humanos , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Fenotipo , Ensayos Clínicos Controlados Aleatorios como Asunto , Factores de Tiempo , Resultado del Tratamiento , Microambiente Tumoral , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/inmunologíaRESUMEN
Although elevated plasma interleukin-8 (pIL-8) has been associated with poor outcome to immune checkpoint blockade 1, this has not been comprehensively evaluated in large randomized studies. Here we analyzed circulating pIL-8 and IL8 gene expression in peripheral blood mononuclear cells and tumors of patients treated with atezolizumab (anti-PD-L1 monoclonal antibody) from multiple randomized trials representing 1,445 patients with metastatic urothelial carcinoma (mUC) and metastatic renal cell carcinoma. High levels of IL-8 in plasma, peripheral blood mononuclear cells and tumors were associated with decreased efficacy of atezolizumab in patients with mUC and metastatic renal cell carcinoma, even in tumors that were classically CD8+ T cell inflamed. Low baseline pIL-8 in patients with mUC was associated with increased response to atezolizumab and chemotherapy. Patients with mUC who experienced on-treatment decreases in pIL-8 exhibited improved overall survival when treated with atezolizumab but not with chemotherapy. Single-cell RNA sequencing of the immune compartment showed that IL8 is primarily expressed in circulating and intratumoral myeloid cells and that high IL8 expression is associated with downregulation of the antigen-presentation machinery. Therapies that can reverse the impacts of IL-8-mediated myeloid inflammation will be essential for improving outcomes of patients treated with immune checkpoint inhibitors.
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Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Interleucina-8/metabolismo , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Adulto , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígeno B7-H1/inmunología , Biomarcadores Farmacológicos/sangre , Biomarcadores Farmacológicos/metabolismo , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/diagnóstico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Transicionales/diagnóstico , Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/metabolismo , Carcinoma de Células Transicionales/mortalidad , Resistencia a Antineoplásicos , Femenino , Humanos , Interleucina-8/sangre , Neoplasias Renales/diagnóstico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Neoplasias Renales/mortalidad , Masculino , Neoplasias/metabolismo , Neoplasias/mortalidad , Pronóstico , Análisis de Supervivencia , Insuficiencia del Tratamiento , Neoplasias Urológicas/diagnóstico , Neoplasias Urológicas/tratamiento farmacológico , Neoplasias Urológicas/metabolismo , Neoplasias Urológicas/mortalidadRESUMEN
CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ T cell activation and effector function.
Asunto(s)
Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Activación de Linfocitos , Sistema de Señalización de MAP Quinasas/inmunología , Modelos Inmunológicos , Animales , Antígenos CD/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones NoqueadosRESUMEN
Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.
Asunto(s)
Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias/patología , Variantes Farmacogenómicas , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/citología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Células Clonales , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Linfocitos T/metabolismo , TranscriptomaRESUMEN
Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part because of the challenges associated to the study of membrane-expressed proteins. Here, to enable sensitive detection of receptor-ligand interactions in high throughput, we implement a new platform, the Conditioned Media AlphaScreen, for interrogation of a library consisting of most single transmembrane human proteins. Using this method to study key immune receptors, we identify and further validate the interleukin receptor IL20RA as the first binding partner for the checkpoint inhibitor B7-H3. Further, KIR2DL5, a natural killer cell protein that had remained orphan, is uncovered as a functional binding partner for the poliovirus receptor (PVR). This interaction is characterized using orthogonal assays, which demonstrate that PVR specifically engages KIR2DL5 on natural killer cells leading to inhibition of cytotoxicity. Altogether, these results reveal unappreciated links between protein families that may importantly influence receptor-driven functions during disease. Applicable to any target of interest, this technology represents a versatile and powerful approach for elucidation of receptor-ligand interactomes, which is essential to understand basic aspects of the biology of the plasma membrane proteins and ultimately inform the development of novel therapeutic strategies.
Asunto(s)
Antígenos B7/metabolismo , Matriz Extracelular/metabolismo , Células Asesinas Naturales/metabolismo , Receptores de Interleucina/metabolismo , Receptores KIR2DL5/metabolismo , Receptores Virales/metabolismo , Comunicación Celular , Células HEK293 , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ligandos , Unión Proteica , Mapas de Interacción de ProteínasRESUMEN
Myeloid-derived suppressor cells (MDSCs) are found in most cancer malignancies and support tumorigenesis by suppressing immunity and promoting tumor growth. Here we identify the bromodomain (BRD) of CBP/EP300 as a critical regulator of H3K27 acetylation (H3K27ac) in MDSCs across promoters and enhancers of pro-tumorigenic target genes. In preclinical tumor models, in vivo administration of a CBP/EP300-BRD inhibitor (CBP/EP300-BRDi) alters intratumoral MDSCs and attenuates established tumor growth in immunocompetent tumor-bearing mice, as well as in MDSC-dependent xenograft models. Inhibition of CBP/EP300-BRD redirects tumor-associated MDSCs from a suppressive to an inflammatory phenotype through downregulation of STAT pathway-related genes and inhibition of Arg1 and iNOS. Similarly, CBP/EP300-BRDi decreases differentiation and suppressive function of human MDSCs in vitro. Our findings uncover a role of CBP/EP300-BRD in intratumoral MDSCs that may be targeted therapeutically to boost anti-tumor immunity.
Asunto(s)
Carcinogénesis/metabolismo , Histonas/metabolismo , Células Mieloides/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Arginasa/genética , Arginasa/metabolismo , Línea Celular Tumoral , Células Cultivadas , Elementos de Facilitación Genéticos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Regiones Promotoras Genéticas , Dominios Proteicos , Factores de Transcripción STAT/metabolismo , Factores de Transcripción p300-CBP/químicaRESUMEN
Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.
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Dominio Catalítico , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/química , Animales , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Células Sf9 , SpodopteraRESUMEN
Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226, with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells, with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore, inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors.
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Antígenos de Diferenciación de Linfocitos T/metabolismo , Proteína Forkhead Box O1/antagonistas & inhibidores , Proteína Forkhead Box O1/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Línea Celular Tumoral , Citotoxicidad Inmunológica , Regulación Neoplásica de la Expresión Génica , Humanos , Ligandos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones , Ratones Noqueados , Nectinas/metabolismo , Fosforilación , Receptores Virales/metabolismo , Transducción de Señal/inmunologíaRESUMEN
We examined hematopoietic protein kinase 1 (HPK1), whose reliance on scaffold versus kinase functions for negative immune cell regulation is poorly understood and critical to its assessment as a viable drug target. We identify kinase-dependent roles for HPK1 in CD8 T cells that restrict their anti-viral and anti-tumor responses by using HPK1 kinase-dead (HPK1.kd) knockin mice. Loss of HPK1 kinase function enhanced T cell receptor signaling and cytokine secretion in a T-cell-intrinsic manner. In response to chronic lymphocytic choriomeningitis virus (LCMV) infection or tumor challenge, viral clearance and tumor growth inhibition were enhanced in HPK1.kd mice, accompanied by an increase in effector CD8 T cell function. Co-blockade of PD-L1 further enhanced T effector cell function, resulting in superior anti-viral and anti-tumor immunity over single target blockade. These results identify the importance of HPK1 kinase activity in the negative regulation of CD8 effector functions, implicating its potential as a cancer immunotherapy target.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Neoplasias del Colon/inmunología , Neoplasias del Colon/terapia , Femenino , Glioma/inmunología , Glioma/terapia , Inmunoterapia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/inmunología , Distribución Aleatoria , Transducción de SeñalRESUMEN
The contribution of the kidney-draining lymph node (KLN) to the pathogenesis of ischemia-reperfusion injury (IRI) of the kidney and its subsequent recovery has not been explored in depth. In addition, the mechanism by which repetitive IRI contributes to renal fibrosis remains poorly understood. Herein, we have found that IRI of the kidney is associated with expansion of high endothelial venules (HEVs) and activation of fibroblastic reticular cells (FRCs) in the KLN, as demonstrated by significant expansion in the extracellular matrix. The lymphotoxin α signaling pathway mediates activation of FRCs, and chronic treatment with lymphotoxin ß receptor-immunoglobulin fusion protein (LTßr-Ig) resulted in marked alteration of the KLN as well as augmentation of renal fibrosis. Depletion of FRCs reduced T cell activation in the KLN and ameliorated renal injury in acute IRI. Repetitive renal IRI was associated with senescence of FRCs, fibrosis of the KLN, and renal scarring, which were ameliorated by FRC administration. Therefore, our study emphasizes the critical role of FRCs in both the initiation and repair phases of injury following IRI of the kidney.
Asunto(s)
Lesión Renal Aguda/complicaciones , Fibrosis/complicaciones , Riñón/patología , Ganglios Linfáticos/patología , Daño por Reperfusión/complicaciones , Lesión Renal Aguda/inmunología , Lesión Renal Aguda/patología , Animales , Modelos Animales de Enfermedad , Fibrosis/inmunología , Proteínas de Homeodominio/genética , Inmunoglobulinas , Receptor beta de Linfotoxina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión/inmunología , Transducción de SeñalRESUMEN
Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.
Asunto(s)
Antineoplásicos/farmacología , Proteína de Unión a CREB/antagonistas & inhibidores , Pirazoles/farmacología , Piridinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Proteína de Unión a CREB/química , Perros , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Ratones , Dominios Proteicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pirazoles/síntesis química , Pirazoles/química , Pirazoles/farmacocinética , Piridinas/síntesis química , Piridinas/química , Piridinas/farmacocinética , ARN/genética , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Potenciales de la Membrana/fisiología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Técnicas de Placa-Clamp/métodos , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Animales , Células CHO , Cricetulus , Evaluación Preclínica de Medicamentos , Expresión Génica , Ensayos Analíticos de Alto Rendimiento/instrumentación , Canal de Potasio Kv1.3/deficiencia , Canal de Potasio Kv1.3/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.3/genética , Canal de Sodio Activado por Voltaje NAV1.3/metabolismo , Canal de Sodio Activado por Voltaje NAV1.4/genética , Canal de Sodio Activado por Voltaje NAV1.4/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Técnicas de Placa-Clamp/instrumentación , Cultivo Primario de Células , Ratas , Canales de Sodio/genética , Canales de Sodio/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , TransgenesRESUMEN
This report is a summary of 'New Cancer Immunotherapy Agents in Development' program, which took place in association with the 31st Annual Meeting of the Society for Immunotherapy of Cancer (SITC), on November 9, 2016 in National Harbor, Maryland. Presenters gave brief overviews of emerging clinical and pre-clinical immune-based agents and combinations, before participating in an extended panel discussion with multidisciplinary leaders, including members of the FDA, leading academic institutions and industrial drug developers, to consider topics relevant to the future of cancer immunotherapy.
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Vacunas contra el Cáncer/uso terapéutico , Inmunoterapia , Neoplasias/tratamiento farmacológico , Microambiente Tumoral/inmunología , Vacunas contra el Cáncer/inmunología , Humanos , Neoplasias/inmunología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Voltage-gated Kv1.3 and Ca2+-dependent KCa3.1 are the most prevalent K+ channels expressed by human and rat T cells. Despite the preferential upregulation of Kv1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether Kv1.3 is required for their induction and function is unclear. Here we show, using Kv1.3-deficient rats, that Kv1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in Kv1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of Kv1.3 under these specific settings. However, experiments with Kv1.3 KO rats and Kv1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both Kv1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on Kv1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.
Asunto(s)
Epítopos/inmunología , Memoria Inmunológica , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canal de Potasio Kv1.3/metabolismo , Linfocitos T/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/deficiencia , Activación de Linfocitos/inmunología , Fenotipo , Bloqueadores de los Canales de Potasio/farmacología , ARN Interferente Pequeño/metabolismo , Ratas , Linfocitos T/efectos de los fármacosRESUMEN
Expression of PD-L1, the ligand for T-cell inhibitory receptor PD-1, is one key immunosuppressive mechanism by which cancer avoids eradication by the immune system. Therapeutic use of blocking antibodies to PD-L1 or its receptor PD-1 has produced unparalleled, durable clinical responses, with highest likelihood of response seen in patients whose tumour or immune cells express PD-L1 before therapy. The significance of PD-L1 expression in each cell type has emerged as a central and controversial unknown in the clinical development of immunotherapeutics. Using genetic deletion in preclinical mouse models, here we show that PD-L1 from disparate cellular sources, including tumour cells, myeloid or other immune cells can similarly modulate the degree of cytotoxic T-cell function and activity in the tumour microenvironment. PD-L1 expression in both the host and tumour compartment contribute to immune suppression in a non-redundant fashion, suggesting that both sources could be predictive of sensitivity to therapeutic agents targeting the PD-L1/PD-1 axis.