RESUMEN
The B-cell surface protein CD19 is present throughout the cell life cycle and is uniformly expressed in leukemias, making it a target for chimeric antigen receptor engineered immune cell therapy. Identifying the sequence dependence of the binding of CD19 to antibodies empowers fundamental study and more tailored development of CD19-targeted therapeutics. To identify the antibody-binding epitopes on CD19, we screened a comprehensive single-site saturation mutation library of the human CD19 extracellular domain to identify mutations detrimental to binding FMC63-the dominant CD19 antibody used in chimeric antigen receptor development-as well as 4G7-2E3 and 3B10, which have been used in various types of CD19 research and development. All three antibodies had partially overlapping, yet distinct, epitopes near the published epitope of antibody B43. The FMC63 conformational epitope spans spatially adjacent, but genetically distant, loops in exons 3 and 4. The 3B10 epitope is a linear peptide sequence that binds CD19 with 440 pM affinity. Along with their primary goal of epitope mapping, the mutational tolerance data also empowered additional CD19 variant design and analysis. A designed CD19 variant with all N-linked glycosylation sites removed successfully bound antibody in the yeast display context, which provides a lead for aglycosylated applications. Screening for thermally stable variants identified mutations to guide further CD19 stabilization for fusion protein applications and revealed evolutionary affinity-stability trade-offs. These fundamental insights into CD19 sequence-function relationships enhance our understanding of antibody-mediated CD19-targeted therapeutics.
Asunto(s)
Antígenos CD19/química , Antígenos CD19/inmunología , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Antígenos CD19/genética , Mapeo Epitopo , Exones , Humanos , Mutación , Dominios ProteicosRESUMEN
CD19-targeted chimeric antigen receptor (CAR) T-cells (CAR19s) show remarkable efficacy in the treatment of relapsed/refractory acute lymphocytic leukemia and Non-Hodgkin's lymphoma. However, the use of CAR T-cell therapy against CD19-negative hematological cancers and solid tumors has been challenging. We propose CD19-fusion proteins (CD19-FPs) to leverage the benefits of CAR19s while retargeting this validated cellular therapy to alternative tumor antigens. We demonstrate the ability of a fusion of CD19 extracellular domain (ECD) and a human epidermal growth factor receptor 2 (HER2) single-chain antibody fragment to retarget CAR19s to kill HER2+ CD19- tumor cells. To enhance the modularity of this technology, we engineered a more robust CD19 ECD via deep mutational scanning with yeast display and flow cytometric selections for improved protease resistance and anti-CD19 antibody binding. These enhanced CD19 ECDs significantly increase, and in some cases recover, fusion protein expression while maintaining target antigen affinity. Importantly, CD19-FPs retarget CAR19s to kill tumor cells expressing multiple distinct antigens, including HER2, CD20, EGFR, BCMA, and Clec12A as N- or C-terminal fusions and linked to both antibody fragments and fibronectin ligands. This study provides fundamental insights into CD19 sequence-function relationships and defines a flexible and modular platform to retarget CAR19s to any tumor antigen.
Asunto(s)
Antígenos CD19/metabolismo , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/metabolismo , Linfocitos T/inmunología , Antígenos CD19/genética , Antígenos CD19/inmunología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Células HEK293 , Humanos , Mutagénesis , Neoplasias/inmunología , Neoplasias/patología , Dominios Proteicos/genética , Ingeniería de Proteínas , Receptor ErbB-2/antagonistas & inhibidores , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplanteRESUMEN
Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Virales/metabolismo , Linfocitos T CD4-Positivos/virología , Membrana Celular/metabolismo , Membrana Celular/virología , Técnicas de Silenciamiento del Gen , Células HEK293 , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , Células HeLa , Receptor Celular 1 del Virus de la Hepatitis A , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Células Jurkat , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Fosfatidilserinas/metabolismo , ARN Interferente Pequeño/genética , Receptores Virales/genética , Virión/crecimiento & desarrollo , Virión/metabolismo , Replicación Viral/fisiologíaRESUMEN
UNLABELLED: T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent internalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indicating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. IMPORTANCE: T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members are recently identified phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance virus entry by binding the phospholipid, PtdSer, present on the viral membrane. While it is known that the PtdSer binding is essential for the PVEER function of TIM-1, TIM-3 shares this binding activity but does not enhance virus entry. No comprehensive studies have been done to characterize the other domains of TIM-1. In this study, using a variety of chimeric proteins and deletion mutants, we define the features necessary for a functional PVEER. With these features in mind, we generated a TIM-1 mimic using functionally similar domains from other proteins. This mimic, like TIM-1, effectively enhanced transduction. These studies provide insight into the key features necessary for PVEERs and will allow for more effective identification of unknown PVEERs.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Receptores Virales/metabolismo , Virosis/metabolismo , Internalización del Virus , Fenómenos Fisiológicos de los Virus , Línea Celular , Ebolavirus/genética , Ebolavirus/fisiología , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Fosfatidilserinas/metabolismo , Estructura Terciaria de Proteína , Receptores Virales/química , Receptores Virales/genética , Acoplamiento Viral , Virosis/genética , Virosis/virología , Virus/genéticaRESUMEN
The cell surface receptor T cell immunoglobulin mucin domain 1 (TIM-1) dramatically enhances filovirus infection of epithelial cells. Here, we showed that key phosphatidylserine (PtdSer) binding residues of the TIM-1 IgV domain are critical for Ebola virus (EBOV) entry through direct interaction with PtdSer on the viral envelope. PtdSer liposomes but not phosphatidylcholine liposomes competed with TIM-1 for EBOV pseudovirion binding and transduction. Further, annexin V (AnxV) substituted for the TIM-1 IgV domain, supporting a PtdSer-dependent mechanism. Our findings suggest that TIM-1-dependent uptake of EBOV occurs by apoptotic mimicry. Additionally, TIM-1 enhanced infection of a wide range of enveloped viruses, including alphaviruses and a baculovirus. As further evidence of the critical role of enveloped-virion-associated PtdSer in TIM-1-mediated uptake, TIM-1 enhanced internalization of pseudovirions and virus-like proteins (VLPs) lacking a glycoprotein, providing evidence that TIM-1 and PtdSer-binding receptors can mediate virus uptake independent of a glycoprotein. These results provide evidence for a broad role of TIM-1 as a PtdSer-binding receptor that mediates enveloped-virus uptake. Utilization of PtdSer-binding receptors may explain the wide tropism of many of these viruses and provide new avenues for controlling their virulence.
Asunto(s)
Ebolavirus/fisiología , Glicoproteínas de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Virales/metabolismo , Internalización del Virus , Alphavirus/química , Alphavirus/fisiología , Animales , Anexina A5/metabolismo , Baculoviridae/química , Baculoviridae/fisiología , Línea Celular , Ebolavirus/química , Receptor Celular 1 del Virus de la Hepatitis A , Interacciones Huésped-Patógeno , Humanos , Transducción GenéticaRESUMEN
Semaphorins are axon guidance factors that assist growing axons in finding appropriate targets and forming synapses. Emerging evidence suggests that semaphorins are involved not only in embryonic development but also in immune responses. Semaphorin 7A (Sema7A; also known as CD108), which is a glycosylphosphatidylinositol-anchored semaphorin, promotes axon outgrowth through beta1-integrin receptors and contributes to the formation of the lateral olfactory tract. Although Sema7A has been shown to stimulate human monocytes, its function as a negative regulator of T-cell responses has also been reported. Thus, the precise function of Sema7A in the immune system remains unclear. Here we show that Sema7A, which is expressed on activated T cells, stimulates cytokine production in monocytes and macrophages through alpha1beta1 integrin (also known as very late antigen-1) as a component of the immunological synapse, and is critical for the effector phase of the inflammatory immune response. Sema7A-deficient (Sema7a-/-) mice are defective in cell-mediated immune responses such as contact hypersensitivity and experimental autoimmune encephalomyelitis. Although antigen-specific and cytokine-producing effector T cells can develop and migrate into antigen-challenged sites in Sema7a-/- mice, Sema7a-/- T cells fail to induce contact hypersensitivity even when directly injected into the antigen-challenged sites. Thus, the interaction between Sema7A and alpha1beta1 integrin is crucial at the site of inflammation. These findings not only identify a function of Sema7A as an effector molecule in T-cell-mediated inflammation, but also reveal a mechanism of integrin-mediated immune regulation.
Asunto(s)
Antígenos CD/metabolismo , Inflamación/inmunología , Integrina alfa1beta1/metabolismo , Semaforinas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos CD/genética , Citocinas/metabolismo , Inmunidad/inmunología , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/metabolismo , Semaforinas/deficiencia , Semaforinas/genética , Transducción de SeñalRESUMEN
B cell lymphoma therapy has been transformed by CD19-targeting cellular therapeutics that induce high clinical response rates and impressive remissions in relapsed and refractory patients. However, approximately half of all patients who respond to CD19-directed cell therapy relapse, the majority within 6 months. One characteristic of relapse is loss or reduction of CD19 expression on malignant B cells. We designed a unique therapeutic to prevent and reverse relapses due to lost or reduced CD19 expression. This novel biologic, a CAR T Engager, binds CD20 and displays the CD19 extracellular domain. This approach increases the apparent CD19 antigen density on CD19-positive/CD20-positive lymphoma cells, and prevents antigen-loss induced relapse, as CD19 bound to CD20 remains present on the cell surface. We demonstrate that this novel therapeutic prevents and reverses lymphoma relapse in vitro and prevents CD19-negative lymphoma growth and relapse in vivo.
Asunto(s)
Linfoma , Receptores Quiméricos de Antígenos , Antígenos CD19 , Antígenos CD20 , Humanos , Linfoma/terapia , Recurrencia Local de Neoplasia , Receptores de Antígenos de Linfocitos T , Linfocitos TRESUMEN
B cell activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) are closely related ligands within the TNF superfamily that play important roles in B lymphocyte biology. Both ligands share two receptors--transmembrane activator and calcium signal--modulating cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA)--that are predominantly expressed on B cells. In addition, BAFF specifically binds BAFF receptor, whereas the nature of a postulated APRIL-specific receptor remains elusive. We show that the TNF homology domain of APRIL binds BCMA and TACI, whereas a basic amino acid sequence (QKQKKQ) close to the NH2 terminus of the mature protein is required for binding to the APRIL-specific "receptor." This interactor was identified as negatively charged sulfated glycosaminoglycan side chains of proteoglycans. Although T cell lines bound little APRIL, the ectopic expression of glycosaminoglycan-rich syndecans or glypicans conferred on these cells a high binding capacity that was completely dependent on APRIL's basic sequence. Moreover, syndecan-1-positive plasma cells and proteoglycan-rich nonhematopoietic cells displayed high specific, heparin-sensitive binding to APRIL. Inhibition of BAFF and APRIL, but not BAFF alone, prevented the survival and/or the migration of newly formed plasma cells to the bone marrow. In addition, costimulation of B cell proliferation by APRIL was only effective upon APRIL oligomerization. Therefore, we propose a model whereby APRIL binding to the extracellular matrix or to proteoglycan-positive cells induces APRIL oligomerization, which is the prerequisite for the triggering of TACI- and/or BCMA-mediated activation, migration, or survival signals.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Proteoglicanos/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Factor Activador de Células B , Receptor del Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Línea Celular , Movimiento Celular/genética , Proliferación Celular , Citometría de Flujo , Heparina/metabolismo , Humanos , Inmunoprecipitación , Ratones , Células Plasmáticas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transfección , Proteína Activadora Transmembrana y Interactiva del CAML , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Refractory acute myeloid leukemia (AML) remains an incurable malignancy despite the clinical use of novel targeted therapies, new antibody-based therapies, and cellular therapeutics. Here, we describe the preclinical development of a novel cell therapy that targets the antigen CLEC12A with a biparatopic bridging protein. Bridging proteins are designed as "CAR-T cell engagers," with a CAR-targeted protein fused to antigen binding domains derived from antibodies. Here, we created a CD19-anti-CLEC12A bridging protein that binds to CAR19 T cells and to the antigen CLEC12A. Biparatopic targeting increases the potency of bridging protein-mediated cytotoxicity by CAR19 T cells. Using CAR19 T cells that secrete the bridging protein we demonstrate potent activity against aggressive leukemic cell lines in vivo This CAR-engager platform is facile and modular, as illustrated by activity of a dual-antigen bridging protein targeting CLEC12A and CD33, designed to counter tumor heterogeneity and antigen escape, and created without the need for extensive CAR T-cell genetic engineering. CAR19 T cells provide an optimal cell therapy platform with well-understood inherent persistence and fitness characteristics.
Asunto(s)
Antígenos CD19/inmunología , Inmunoterapia Adoptiva/métodos , Inmunoterapia/métodos , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptores Mitogénicos/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Linfocitos T/inmunología , Animales , Deriva y Cambio Antigénico , Apoptosis , Proliferación Celular , Citotoxicidad Inmunológica/inmunología , Humanos , Técnicas In Vitro , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo. Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.
Asunto(s)
Antígenos CD19/genética , Inmunoterapia Adoptiva/métodos , Linfoma de Células B/terapia , Receptor ErbB-2/genética , Receptores Quiméricos de Antígenos/genética , Linfocitos T/inmunología , Animales , Antígenos CD19/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Línea Celular Tumoral , Proliferación Celular , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Receptores ErbB/genética , Receptores ErbB/inmunología , Expresión Génica , Vectores Genéticos/inmunología , Vectores Genéticos/metabolismo , Humanos , Lentivirus/genética , Lentivirus/inmunología , Activación de Linfocitos , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones SCID , Unión Proteica , Receptor ErbB-2/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/citología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The transcription factor nuclear factor (NF)-kappaB has been suggested to be a key mediator of the development of lymph nodes and Peyer's patches. However, targeted deletion of NF-kappaB/ Rel family members has not yet corroborated such a function. Here we report that when mice lacking the RelA subunit of NF-kappaB are brought to term by breeding onto a tumor necrosis factor receptor (TNFR)1-deficient background, the mice that are born lack lymph nodes, Peyer's patches, and an organized splenic microarchitecture, and have a profound defect in T cell-dependent antigen responses. Analyses of TNFR1/RelA-deficient embryonic tissues and of radiation chimeras suggest that the dependence on RelA is manifest not in hematopoietic cells but rather in radioresistant stromal cells needed for the development of secondary lymphoid organs.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Tejido Linfoide , FN-kappa B/fisiología , Animales , Presentación de Antígeno/fisiología , Proteínas de Unión al ADN/genética , Desarrollo Embrionario y Fetal/fisiología , Citometría de Flujo , Inmunohistoquímica , Tejido Linfoide/embriología , Tejido Linfoide/fisiología , Ratones , Ratones Noqueados , FN-kappa B/genética , Linfocitos T/inmunología , Factor de Transcripción ReIARESUMEN
T cell Ig and mucin domain (TIM)-4 is preferentially expressed on antigen-presenting cells, and its counter-ligand, TIM-1, is thought to deliver co-stimulating signals to T cells. However, the physiological functions of TIM-4 remain unclear. Here, we demonstrate that TIM-4 inhibits naive T cell activation through a ligand other than TIM-1. The inhibitory effect of TIM-4 was specific to naive T cells which do not express TIM-1, and the effect disappeared in pre-activated T cells. Conversely, antibody-mediated blockade of TIM-4 in vivo substantially suppressed T cell-mediated inflammatory responses despite enhanced generation of antigen-specific T cells. Furthermore, treatment with anti-TIM-4 reduced the inflammatory responses developed in mice that were adoptively transferred with antigen-primed T cells. These results suggest that TIM-4 exerts bimodal functions depending on the activation status of T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Receptor Celular 1 del Virus de la Hepatitis A , Inflamación/inmunología , Inflamación/metabolismo , Ligandos , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratas , Ratas Endogámicas LewRESUMEN
Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory signals and enabling patients to produce an effective antitumour response. Inhibitors of CTLA4, PD1 or PDL1 administered as single agents have resulted in durable tumour regression in some patients, and combinations of PD1 and CTLA4 inhibitors may enhance antitumour benefit. Numerous additional immunomodulatory pathways as well as inhibitory factors expressed or secreted by myeloid and stromal cells in the tumour microenvironment are potential targets for synergizing with immune checkpoint blockade. Given the breadth of potential targets in the immune system, critical questions to address include which combinations should move forward in development and which patients will benefit from these treatments. This Review discusses the leading drug targets that are expressed on tumour cells and in the tumour microenvironment that allow enhancement of the antitumour immune response.
Asunto(s)
Antineoplásicos/farmacología , Inmunoterapia/métodos , Neoplasias/terapia , Animales , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/inmunología , Humanos , Terapia Molecular Dirigida , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
T cell, immunoglobulin domain and mucin domain-1 (TIM-1) is the nominant member of a small family of related proteins that regulate immune cell activities. TIM-1 was initially characterized in a mouse congenic analysis of Th2 T cell responses and related pathology. Data accumulated to date suggest that TIM-1 regulates effector T cell function, and may play distinct roles in the activities of B cells, invariant NKT cells and epithelial cells. In addition, a variety of ligands for TIM-1 have been proposed. In this review I discuss recent data that have accumulated on the function of TIM-1, propose a model to explain how TIM-1 regulates effector T cell activity through recognition of distinct ligands, and review others functions of this increasingly fascinating protein. Of considerable interest are the novel findings that TIM-1 mediates virus entry and virulence.
Asunto(s)
Asma/inmunología , Glicoproteínas de Membrana , Proteínas de la Membrana , Receptores Inmunológicos/metabolismo , Receptores Virales , Linfocitos T , Animales , Células Dendríticas/inmunología , Filoviridae/metabolismo , Receptor Celular 1 del Virus de la Hepatitis A , Virus de la Hepatitis A/metabolismo , Humanos , Hipótesis de la Higiene , Infecciones/inmunología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/inmunología , Isoformas de Proteínas , Receptores Virales/inmunología , Receptores Virales/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The gene encoding T cell immunoglobulin and mucin domain-1 (Tim-1) is linked to atopy and asthma susceptibility in mice and humans. Tim-1 is a transmembrane protein expressed on activated lymphocytes and appears to have a role as a co-stimulatory receptor in T cells. The protein has not been shown to have enzymatic activity but contains a site within its cytoplasmic tail predicted to be a target for tyrosine kinases. Here, we show that Tim-1 can associate with the kinase Fyn, a member of the Src family of tyrosine kinases. This association does not require Fyn's kinase activity and is independent of the phosphorylation of a conserved tyrosine present within the cytoplasmic tail of Tim-1. Fyn is necessary for phosphorylation of this tyrosine in Tim-1 and the phosphorylation of Tim-1 varies with the levels of Fyn present in cells. These data suggest a role for Fyn in the signaling downstream of Tim-1.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Receptores Virales/metabolismo , Linfocitos T/metabolismo , Animales , Linfocitos B/metabolismo , Línea Celular , Células Epiteliales , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Fosforilación , Proteínas Proto-Oncogénicas c-fyn/genética , ARN Interferente Pequeño , Receptores Virales/genética , Transducción de SeñalRESUMEN
Studies in mice and humans have revealed that the T cell, immunoglobulin, mucin (TIM) genes are associated with several atopic diseases. TIM-1 is a type I membrane protein that is expressed on T cells upon stimulation and has been shown to modulate their activation. In addition to a recently described interaction with dendritic cells, TIM-1 has also been identified as a phosphatidylserine recognition molecule, and several protein ligands have been proposed. Our understanding of its activity is complicated by the possibility that TIM-1 possesses multiple and diverse binding partners. In order to delineate the function of TIM-1, we generated monoclonal antibodies directed to a cleft formed within the IgV domain of TIM-1. We have shown here that antibodies that bind to this defined cleft antagonize TIM-1 binding to specific ligands and cells. Notably, these antibodies exhibited therapeutic activity in a humanized SCID model of experimental asthma, ameliorating inflammation, and airway hyperresponsiveness. Further experiments demonstrated that the effects of the TIM-1-specific antibodies were mediated via suppression of Th2 cell proliferation and cytokine production. These results demonstrate that modulation of the TIM-1 pathway can critically influence activated T cells in a humanized disease model, suggesting that TIM-1 antagonists may provide potent therapeutic benefit in asthma and other immune-mediated disorders.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Asma/prevención & control , Glicoproteínas de Membrana/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Receptores Virales/antagonistas & inhibidores , Animales , Asma/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Femenino , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Activación de Linfocitos , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Ratones , Ratones SCID , Fosfatidilserinas/metabolismo , Receptores Virales/fisiologíaRESUMEN
Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as alpha2beta1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express alpha2beta1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether alpha2beta1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the alpha2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the alpha2beta1-integrin. Newborn mice were pretreated with a monoclonal antibody against the alpha2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed alpha2beta1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-alpha2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the alpha2beta1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.
Asunto(s)
Conductos Biliares/citología , Conductos Biliares/metabolismo , Atresia Biliar/virología , Integrina alfa2beta1/metabolismo , Rotavirus/fisiología , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales , Anticuerpos Antivirales , Línea Celular , Modelos Animales de Enfermedad , Silenciador del Gen , Hepatocitos/metabolismo , Integrina alfa2beta1/genética , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Replicación ViralRESUMEN
Topical transcutaneous immunization (TCI) presents many clinical advantages, but its underlying mechanism remains unknown. TCI induced Ag-specific IgA Ab-secreting cells expressing CCR9 and CCR10 in the small intestine in a retinoic acid-dependent manner. These intestinal IgA Abs were maintained in Peyer's patch-null mice but abolished in the Peyer's patch- and lymph node-null mice. The mesenteric lymph node (MLN) was shown to be the site of IgA isotype class switching after TCI. Unexpectedly, langerin(+)CD8alpha(-) dendritic cells emerged in the MLN after TCI; they did not migrate from the skin but rather differentiated rapidly from bone marrow precursors. Depletion of langerin(+) cells impaired intestinal IgA Ab responses after TCI. Taken together, these findings suggest that MLN is indispensable for the induction of intestinal IgA Abs following skin immunization and that cross-talk between the skin and gut immune systems might be mediated by langerin(+) dendritic cells in the MLN.
Asunto(s)
Antígenos de Superficie/inmunología , Células Dendríticas/inmunología , Intestinos/inmunología , Lectinas Tipo C/inmunología , Ganglios Linfáticos/inmunología , Lectinas de Unión a Manosa/inmunología , Mesenterio/inmunología , Piel/inmunología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Cultivadas , Inmunización , Inmunoglobulina A/inmunología , Ratones , Ratones Endogámicos C57BL , Tretinoina/farmacologíaRESUMEN
The T cell, Ig domain, and mucin domain-1 (TIM-1) gene is associated with Th2 T cell responses and human atopic diseases. The mechanism by which TIM-1 influences T cell responses remains unknown. We demonstrate that TIM-1 is recruited to the TCR-signaling complex via association with CD3. TIM-1 up-regulates TCR-associated signaling events, including phosphorylation of Zap70 and IL-2-inducible T cell kinase. This activity requires TIM-1 tyrosine phosphorylation. TIM-1 expression induces formation of a novel complex that includes PI3K and ITK. Finally, the consequences of TIM-1 activation include increased expression of effector cytokines. These results demonstrate that TIM-1 is a critical component of the human T cell response and provide a mechanistic hypothesis for the role of TIM-1 in disease.
Asunto(s)
Complejo CD3/metabolismo , Activación de Linfocitos , Glicoproteínas de Membrana/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Virales/metabolismo , Linfocitos T/inmunología , Complejo CD3/análisis , Células Cultivadas , Citocinas/metabolismo , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Receptores Virales/análisis , Receptores Virales/genética , Linfocitos T/química , Tirosina/genética , Tirosina/metabolismo , Regulación hacia Arriba , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
The TAPR locus containing the TIM gene family is implicated in the development of atopic inflammation in mouse, and TIM-1 allelic variation has been associated with the incidence of atopy in human patient populations. In this study, we show that manipulation of the TIM-1 pathway influences airway inflammation and pathology. Anti-TIM-1 mAbs recognizing distinct epitopes differentially modulated OVA-induced lung inflammation in the mouse. The epitopes recognized by these Abs were mapped, revealing that mAbs to both the IgV and stalk domains of TIM-1 have therapeutic activity. Unexpectedly, mAbs recognizing unique epitopes spanning exon 4 of the mucin/stalk domains exacerbated immune responses. Using Ag recall response studies, we demonstrate that the TIM-1 pathway acts primarily by modulating the production of T(H)2 cytokines. Furthermore, ex vivo cellular experiments indicate that TIM-1 activity controls CD4(+) T cell activity. These studies validate the genetic hypothesis that the TIM-1 locus is linked to the development of atopic disease and suggest novel therapeutic strategies for targeting asthma and other atopic disorders.