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1.
MAbs ; 13(1): 1859049, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33487120

RESUMEN

Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, high-content imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins ß1 and ß6 or αV and ß1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Antígenos CD79/inmunología , Ensayos Analíticos de Alto Rendimiento/métodos , Fragmentos Fab de Inmunoglobulinas/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Anticuerpos Biespecíficos/aislamiento & purificación , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Citocinas/inmunología , Citocinas/metabolismo , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo
2.
Front Immunol ; 9: 637, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29636754

RESUMEN

Cellular activation in trans by interferons, cytokines, and chemokines is a commonly recognized mechanism to amplify immune effector function and limit pathogen spread. However, an optimal host response also requires that collateral damage associated with inflammation is limited. This may be particularly so in the case of granulomatous inflammation, where an excessive number and/or excessively florid granulomas can have significant pathological consequences. Here, we have combined transcriptomics, agent-based modeling, and in vivo experimental approaches to study constraints on hepatic granuloma formation in a murine model of experimental leishmaniasis. We demonstrate that chemokine production by non-infected Kupffer cells in the Leishmania donovani-infected liver promotes competition with infected KCs for available iNKT cells, ultimately inhibiting the extent of granulomatous inflammation. We propose trans-activation for chemokine production as a novel broadly applicable mechanism that may operate early in infection to limit excessive focal inflammation.


Asunto(s)
Granuloma/inmunología , Inflamación/inmunología , Macrófagos del Hígado/fisiología , Leishmania donovani/fisiología , Leishmaniasis Visceral/inmunología , Hígado/inmunología , Macrófagos/fisiología , Células T Asesinas Naturales/inmunología , Animales , Células Cultivadas , Quimiocinas/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Hígado/parasitología , Ratones , Ratones Endogámicos C57BL , Análisis de Sistemas , Activación Transcripcional
3.
J Hepatol ; 65(4): 758-768, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27262757

RESUMEN

BACKGROUND & AIMS: Kupffer cells (KCs), the resident tissue macrophages of the liver, play a crucial role in the clearance of pathogens and other particulate materials that reach the systemic circulation. Recent studies have identified KCs as a yolk sac-derived resident macrophage population that is replenished independently of monocytes in the steady state. Although it is now established that following local tissue injury, bone marrow derived monocytes may infiltrate the tissue and differentiate into macrophages, the extent to which newly differentiated macrophages functionally resemble the KCs they have replaced has not been extensively studied. METHODS: We studied the two populations of KCs using intravital microscopy, morphometric analysis and gene expression profiling. An ion homeostasis gene signature, including genes associated with scavenger receptor function and extracellular matrix deposition, allowed discrimination between these two KC sub-types. RESULTS: Bone marrow derived "KCs" accumulating as a result of genotoxic injury, resemble but are not identical to their yolk sac counterparts. Reflecting the differential expression of scavenger receptors, yolk sac-derived KCs were more effective at accumulating acetylated low density lipoprotein, whereas surprisingly, they were poorer than bone marrow-derived KCs when assessed for uptake of a range of bacterial pathogens. The two KC populations were almost indistinguishable in regard to i) response to lipopolysaccharide challenge, ii) phagocytosis of effete red blood cells and iii) their ability to contain infection and direct granuloma formation against Leishmania donovani, a KC-tropic intracellular parasite. CONCLUSIONS: Bone marrow-derived KCs differentiate locally to resemble yolk sac-derived KC in most but not all respects, with implications for models of infectious diseases, liver injury and bone marrow transplantation. In addition, the gene signature we describe adds to the tools available for distinguishing KC subpopulations based on their ontology. LAY SUMMARY: Liver macrophages play a major role in the control of infections in the liver and in the pathology associated with chronic liver diseases. It was recently shown that liver macrophages can have two different origins, however, the extent to which these populations are functionally distinct remains to be fully addressed. Our study demonstrates that whilst liver macrophages share many features in common, regardless of their origin, some subtle differences in function exist. DATA REPOSITORY: Gene expression data are available from the European Bioinformatics Institute ArrayExpress data repository (accession number E-MTAB-4954).


Asunto(s)
Médula Ósea , Humanos , Macrófagos del Hígado , Hígado , Macrófagos , Monocitos
4.
Adv Exp Med Biol ; 915: 329-46, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27193552

RESUMEN

Immune responses occur as a result of stochastic interactions between a plethora of different cell types and molecules that regulate the migration and function of innate and adaptive immune cells to drive protection from pathogen infection. The trafficking of immune cells into peripheral tissues during inflammation and then subsequent migration to draining lymphoid tissues has been quantitated using radiolabelled immune cells over 40 years ago. However, how these processes lead to efficient immune responses was unclear. Advances in physics (multi-photon), chemistry (probes) and biology (animal models) have provided immunologists with specialized tools to quantify the molecular and cellular mechanisms driving immune function in lymphoid tissues through directly visualising cellular behaviours in 3-dimensions over time. Through the temporal and spatial resolution of multi-photon confocal microscopy immunologists have developed new insights into normal immune homeostasis, host responses to pathogens, anti-tumour immune responses and processes driving development of autoimmune pathologies, by the quantification of the interactions and cellular migration involved in adaptive immune responses. Advances in deep tissue imaging, including new fluorescent proteins, increased resolution, speed of image acquisition, sensitivity, number of signals and improved data analysis techniques have provided unprecedented capacity to quantify immune responses at the single cell level. This quantitative information has facilitated development of high-fidelity mathematical and computational models of immune function. Together this approach is providing new mechanistic understanding of immune responses and new insights into how immune modulators work. Advances in biophysics have therefore revolutionised our understanding of immune function, directly impacting on the development of next generation immunotherapies and vaccines, and is providing the quantitative basis for emerging technology of simulation-guided experimentation and immunotherapeutic design.


Asunto(s)
Inmunidad Adaptativa , Ganglios Linfáticos/inmunología , Imagen Molecular , Animales , Biomarcadores/metabolismo , Comunicación Celular , Movimiento Celular , Difusión de Innovaciones , Predicción , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Imagenología Tridimensional , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Microscopía de Fluorescencia por Excitación Multifotónica , Imagen Molecular/historia , Imagen Molecular/métodos , Imagen Molecular/tendencias , Transducción de Señal , Factores de Tiempo
5.
Nat Commun ; 7: 11394, 2016 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-27099134

RESUMEN

Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ(+) ILC3s into wounded dermis; RORγ(+) ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ(+) ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair.


Asunto(s)
Epidermis/inmunología , Inmunidad Innata , Subgrupos Linfocitarios/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Receptor Notch1/inmunología , Heridas Penetrantes/inmunología , Animales , Movimiento Celular/inmunología , Quimiocina CCL20/genética , Quimiocina CCL20/inmunología , Quimiocina CXCL13/genética , Quimiocina CXCL13/inmunología , Epidermis/lesiones , Femenino , Regulación de la Expresión Génica , Interleucina-17/genética , Interleucina-17/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/deficiencia , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Receptor Notch1/genética , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Cicatrización de Heridas/genética , Cicatrización de Heridas/inmunología , Heridas Penetrantes/genética , Heridas Penetrantes/patología
6.
Sci Transl Med ; 6(225): 225ra29, 2014 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-24574340

RESUMEN

Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy.


Asunto(s)
Abietanos/farmacología , Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Inflamación/tratamiento farmacológico , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Pez Cebra , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Larva , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Investigación Biomédica Traslacional , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/inmunología , Pez Cebra/metabolismo
7.
PLoS One ; 7(4): e35466, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22496920

RESUMEN

Certain parasites have evolved to evade the immune response and establish chronic infections that may persist for many years. T cell responses in these conditions become muted despite ongoing infection. Upregulation of surface receptors with inhibitory properties provides an immune cell-intrinsic mechanism that, under conditions of chronic infection, regulates immune responses and limits cellular activation and associated pathology. The negative regulator, CD200 receptor, and its ligand, CD200, have been shown to regulate macrophage activation and reduce pathology following infection. We show that CD4 T cells also increase expression of inhibitory CD200 receptors (CD200R) in response to chronic infection. CD200R was upregulated on murine effector T cells in response to infection with bacterial, Salmonella enterica, or helminth, Schistosoma mansoni, pathogens that respectively drive predominant Th1- or Th2-responses. In vitro chronic and prolonged stimuli were required for the sustained upregulation of CD200R, and its expression coincided with loss of multifunctional potential in T effector cells during infection. Importantly, we show an association between IL-4 production and CD200R expression on T effector cells from humans infected with Schistosoma haematobium that correlated effectively with egg burden and, thus infection intensity. Our results indicate a role of CD200R:CD200 in T cell responses to helminths which has diagnostic and prognostic relevance as a marker of infection for chronic schistosomiasis in mouse and man.


Asunto(s)
Antígenos CD/inmunología , Antígenos de Superficie/biosíntesis , Linfocitos T CD4-Positivos/parasitología , Glicoproteínas de Membrana/biosíntesis , Receptores de Superficie Celular/biosíntesis , Esquistosomiasis/inmunología , Adolescente , Animales , Antihelmínticos/uso terapéutico , Antígenos de Superficie/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Niño , Enfermedad Crónica , Estudios de Cohortes , Femenino , Humanos , Interleucina-4/biosíntesis , Interleucina-4/inmunología , Masculino , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Orexina , Praziquantel/uso terapéutico , Receptores de Superficie Celular/inmunología , Schistosoma haematobium/inmunología , Schistosoma haematobium/aislamiento & purificación , Schistosoma mansoni/inmunología , Schistosoma mansoni/aislamiento & purificación , Esquistosomiasis/tratamiento farmacológico , Índice de Severidad de la Enfermedad
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