RESUMEN
mAbs have revolutionized the treatment of autoimmune disorders. Even though mAbs have shown impressive efficacy in blocking T cell or B cell activation and/or recruitment to sites of inflammation, this group of biologicals are not devoid of adverse effects. The most serious adverse effects include infusion reactions, including the activation of the complement pathway. In this study, we present a detailed structure-function study of an anti-CCL20 humanized IgG1 mAb that neutralizes CCL20 chemokine and prevents the recruitment of Th17 cells to sites of inflammation. We demonstrate that the anti-CCL20 Ab changes significantly following administration to humans and monkeys and exposure to human serum. Analysis of the drug product revealed that the anti-CCL20 Ab has unexpectedly high C1q binding. This high binding was linked to immune complex formation in vivo but not during in vitro serum incubation. The immune complex contained multiple complement components. Anti-CCL20 Ab-mediated, complement-dependent cytotoxicity occurred when the Ab bound to CCL20 tethered to the cell membrane of target cells. Taken together, these results provide a likely cause for the animal toxicity observed. In addition, anti-CCL20 revealed progressive acidification because of N100 (located in CDR) deamidation over time, which did not directly impact Ag binding. Our study demonstrates that the safety profiling of mAbs should include the evaluation of effector functions in addition to typical stressed conditions.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Quimiocina CCL20/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Membrana Celular/inmunología , Proteínas del Sistema Complemento/inmunología , Humanos , Inmunoglobulina G/inmunología , Inflamación/inmunología , Macaca fascicularis , Células Th17/inmunologíaRESUMEN
OBJECTIVE: To investigate whether epigenetic cell counting represents a novel method to quantify immune cells in salivary glands of patients with different forms of Sjögren's and sicca syndrome and to capture immunopathology and potentially aid in diagnosis. METHODS: DNA from frozen salivary gland tissue sections of sicca patients was used for bisulphite conversion of demethylated DNA cytosine residues, followed by cell-specific quantitative PCR to calculate cell percentages in relation to total tissue cell numbers as quantified by housekeeping gene demethylation. The percentages of epigenetically quantified cells were correlated to RNA expression of matched salivary gland tissue and histological and clinical parameters. RESULTS: The percentages of epigenetically quantified CD3, CD4, CD8, T follicular helper (Tfh) cells, FoxP3+ regulatory T cells and B cells were significantly increased in the salivary glands of patients with SS. Unsupervised clustering using these percentages identified patient subsets with an increased lymphocytic focus score and local B cell hyperactivity and classifies patients different from conventional classification criteria. In particular, Tfh cells were shown to strongly correlate with the expression of CXCL13, lymphocytic focus scores, local B cell hyperactivity and anti-SSA positivity. CONCLUSION: Epigenetic cell counting is a promising novel tool to objectively and easily quantify immune cells in the labial salivary gland of sicca patients, with a relatively small amount of tissue needed. In view of the potential of this technique to include a huge number of (cell-specific) biomarkers, this opens up new standardized ways of salivary gland analysis with high relevance for patient classification, understanding of immunopathology and monitoring of drug responses in clinical trials.
Asunto(s)
Glándulas Salivales/inmunología , Síndrome de Sjögren/diagnóstico , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Epigénesis Genética , Femenino , Humanos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Glándulas Salivales/patología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/patología , Linfocitos T/patología , Linfocitos T Colaboradores-Inductores/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Megakaryoblastic leukemia 1 (MKL1), also known as MAL or myocardin-related transcription factor A (MRTF-A), is a coactivator of serum response factor, which regulates transcription of actin and actin cytoskeleton-related genes. MKL1 is known to be important for megakaryocyte differentiation and function in mice, but its role in immune cells is unexplored. Here we report a patient with a homozygous nonsense mutation in the MKL1 gene resulting in immunodeficiency characterized predominantly by susceptibility to severe bacterial infection. We show that loss of MKL1 protein expression causes a dramatic loss of filamentous actin (F-actin) content in lymphoid and myeloid lineage immune cells and widespread cytoskeletal dysfunction. MKL1-deficient neutrophils displayed reduced phagocytosis and almost complete abrogation of migration in vitro. Similarly, primary dendritic cells were unable to spread normally or to form podosomes. Silencing of MKL1 in myeloid cell lines revealed that F-actin assembly was abrogated through reduction of globular actin (G-actin) levels and disturbed expression of multiple actin-regulating genes. Impaired migration of these cells was associated with failure of uropod retraction likely due to altered contractility and adhesion, evidenced by reduced expression of the myosin light chain 9 (MYL9) component of myosin II complex and overexpression of CD11b integrin. Together, our results show that MKL1 is a nonredundant regulator of cytoskeleton-associated functions in immune cells and fibroblasts and that its depletion underlies a novel human primary immunodeficiency.
Asunto(s)
Codón sin Sentido , Síndromes de Inmunodeficiencia/genética , Infecciones por Pseudomonas/genética , Transactivadores/genética , Actinas/metabolismo , Actinas/ultraestructura , Línea Celular , Movimiento Celular , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Homocigoto , Humanos , Síndromes de Inmunodeficiencia/complicaciones , Síndromes de Inmunodeficiencia/diagnóstico , Síndromes de Inmunodeficiencia/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Pseudomonas/aislamiento & purificación , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/diagnóstico , Infecciones por Pseudomonas/metabolismoRESUMEN
AIMS: GSK3050002, a humanized IgG1κ antibody with high binding affinity to human CCL20, was administered in a first-in-human study to evaluate safety, pharmacokinetics (PK) and pharmacodynamics (PD). An experimental skin suction blister model was employed to assess target engagement and the ability of the compound to inhibit recruitment of inflammatory CCR6 expressing cells. METHODS: This study was a randomized, double-blind (sponsor open), placebo-controlled, single-centre, single ascending intravenous dose escalation trial in 48 healthy male volunteers. RESULTS: GSK3050002 (0.1-20 mg kg-1 ) was well tolerated and no safety concerns were identified. The PK was linear over the dose range, with a half-life of approximately 2 weeks. Complex of GSK3050002/CCL20 increased in serum and blister fluid with increasing doses of GSK3050002. There were dose-dependent decreases in CCR6+ cell recruitment to skin blisters with maximal effects at doses of 5 mg kg-1 and higher, doses at which GSK3050002/CCL20 complex in serum and blister fluid also appeared to reach maximum levels. CONCLUSIONS: These results indicate a relationship between PK, target engagement and PD, suggesting a selective inhibition of recruitment of CCR6+ cells by GSK3050002 and support further development of GSK3050002 in autoimmune and inflammatory diseases.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales/inmunología , Vesícula/inmunología , Quimiocina CCL20/inmunología , Receptores CCR6/inmunología , Adolescente , Adulto , Anciano , Vesícula/metabolismo , Recuento de Células , Quimiocina CCL20/sangre , Quimiocina CCL20/metabolismo , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Succión/efectos adversos , Adulto JovenRESUMEN
Patients deficient in the cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASp) are predisposed to varied autoimmunity, suggesting it has an important controlling role in participating cells. IL-10-producing regulatory B (Breg) cells are emerging as important mediators of immunosuppressive activity. In experimental, antigen-induced arthritis WASp-deficient (WASp knockout [WAS KO]) mice developed exacerbated disease associated with decreased Breg cells and regulatory T (Treg) cells, but increased Th17 cells in knee-draining LNs. Arthritic WAS KO mice showed increased serum levels of B-cell-activating factor, while their B cells were unresponsive in terms of B-cell-activating factor induced survival and IL-10 production. Adoptive transfer of WT Breg cells ameliorated arthritis in WAS KO recipients and restored a normal balance of Treg and Th17 cells. Mice with B-cell-restricted WASp deficiency, however, did not develop exacerbated arthritis, despite exhibiting reduced Breg- and Treg-cell numbers during active disease, and Th17 cells were not increased over equivalent WT levels. These findings support a contributory role for defective Breg cells in the development of WAS-related autoimmunity, but demonstrate that functional competence in other regulatory populations can be compensatory. A properly regulated cytoskeleton is therefore important for normal Breg-cell activity and complementation of defects in this lineage is likely to have important therapeutic benefits.
Asunto(s)
Artritis Experimental/inmunología , Subgrupos de Linfocitos B/inmunología , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Animales , Artritis Experimental/genética , Artritis Experimental/patología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Subgrupos de Linfocitos B/patología , Femenino , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Masculino , Ratones , Ratones Noqueados , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Células Th17/inmunología , Células Th17/patología , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Wiskott Aldrich syndrome (WAS), an X-linked immunodeficiency, results from loss-of-function mutations in the human hematopoietic cytoskeletal regulator gene WAS. Many missense mutations in the Ena Vasp homology1 (EVH1) domain preserve low-level WAS protein (WASp) expression and confer a milder clinical phenotype. Although disrupted binding to WASp-interacting protein (WIP) leads to enhanced WASp degradation in vivo, the intrinsic function of EVH1-mutated WASp is poorly understood. In the present study, we show that, despite mediating enhanced actin polymerization compared with wild-type WASp in vitro, EVH1 missense mutated proteins did not support full biologic function in cells, even when levels were restored by forced overexpression. Podosome assembly was aberrant and associated with dysregulated lamellipodia formation and impaired persistence of migration. At sites of residual podosome-associated actin polymerization, localization of EVH1-mutated proteins was preserved even after deletion of the entire domain, implying that WIP-WASp complex formation is not absolutely required for WASp localization. However, retention of mutant proteins in podosomes was significantly impaired and associated with reduced levels of WASp tyrosine phosphorylation. Our results indicate that the EVH1 domain is important not only for WASp stability, but also for intrinsic biologic activity in vivo.
Asunto(s)
Células Dendríticas/patología , Mutación Missense , Proteína del Síndrome de Wiskott-Aldrich/genética , Actinas/metabolismo , Animales , Biopolímeros , Proteínas Portadoras/metabolismo , Movimiento Celular , Células Cultivadas , Proteínas del Citoesqueleto , Células Dendríticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Polimerizacion , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Estructura Terciaria de Proteína , Seudópodos/patología , Proteínas Recombinantes de Fusión/fisiología , Eliminación de Secuencia , Organismos Libres de Patógenos Específicos , Proteína del Síndrome de Wiskott-Aldrich/química , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/fisiologíaRESUMEN
Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell-intrinsic mechanisms critically contribute to WAS-associated autoimmunity.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Animales , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Recuento de Células , Modelos Animales de Enfermedad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by absence of Wiskott-Aldrich syndrome protein (WASP) expression, resulting in defective function of many immune cell lineages and susceptibility to severe bacterial, viral, and fungal infections. Despite a significant proportion of patients with WAS having recurrent viral infections, surprisingly little is known about the effects of WASP deficiency on antiviral immunity. OBJECTIVE: We sought to evaluate the antiviral immune response in patients with WASP deficiency in vivo. METHODS: Viral clearance and associated immunopathology were measured after infection of WASP-deficient (WAS KO) mice with lymphocytic choriomeningitis virus (LCMV). Induction of antiviral CD8(+) T-cell immunity and cytotoxicity was documented in WAS KO mice by means of temporal enumeration of total and antigen-specific T-cell numbers. Type I interferon (IFN-I) production was measured in serum in response to LCMV challenge and characterized in vivo by using IFN-I reporter mice crossed with WAS KO mice. RESULTS: WAS KO mice showed reduced viral clearance and enhanced immunopathology during LCMV infection. This was attributed to both an intrinsic CD8(+) T-cell defect and defective priming of CD8(+) T cells by dendritic cells (DCs). IFN-I production by WAS KO DCs was reduced both in vivo and in vitro. CONCLUSIONS: These studies use a well-characterized model of persistence-prone viral infection to reveal a critical deficiency of CD8(+) T-cell responses in murine WASP deficiency, in which abrogated production of IFN-I by DCs might play an important contributory role. These findings might help us to understand the immunodeficiency of WAS.
Asunto(s)
Infecciones por Arenaviridae/inmunología , Células Dendríticas/inmunología , Interferón Tipo I/inmunología , Infecciones por Rhabdoviridae/inmunología , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Humanos , Virus de la Coriomeningitis Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Virus de la Estomatitis Vesicular Indiana , Síndrome de Wiskott-Aldrich/inmunología , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.
Asunto(s)
Citoesqueleto/ultraestructura , Células Dendríticas/ultraestructura , Sinapsis Inmunológicas/ultraestructura , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Proteína del Síndrome de Wiskott-Aldrich/fisiología , Animales , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Señalización del Calcio/inmunología , Movimiento Celular , Cruzamientos Genéticos , Genes Reporteros , Prueba de Complementación Genética , Humanos , Sinapsis Inmunológicas/inmunología , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Linfocitos T/ultraestructura , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patología , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/inmunologíaRESUMEN
Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca(2+) responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell-T cell signaling machinery that regulates T cell activation.
Asunto(s)
Conexina 43/inmunología , Uniones Comunicantes/inmunología , Sinapsis Inmunológicas/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Animales , Comunicación Celular/inmunología , Separación Celular , Conexina 43/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Uniones Comunicantes/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Sinapsis Inmunológicas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Receptor Cross-Talk/inmunología , Transducción de Señal/inmunología , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Selective depletion of T cells expressing LAG-3, an immune checkpoint receptor that is upregulated on activated T cells, has been investigated in pre-clinical models as a potential therapeutic approach in inflammatory and autoimmune diseases where activated T cells are implicated. AIMS: GSK2831781, a depleting monoclonal antibody that specifically binds LAG-3 proteins, may deplete activated LAG-3+ cells in ulcerative colitis (UC). METHODS: Patients with moderate to severe UC were randomised to GSK2831781 or placebo. Safety, tolerability, efficacy, pharmacokinetics and pharmacodynamics of GSK2831781 were evaluated. RESULTS: One hundred four participants across all dose levels were randomised prior to an interim analysis indicating efficacy futility criteria had been met. Efficacy results focus on the double-blind induction phase of the study (GSK2831781 450 mg intravenously [IV], N = 48; placebo, N = 27). Median change from baseline (95% credible interval [CrI]) in complete Mayo score was similar between groups (GSK2831781 450 mg IV: -1.4 [-2.2, -0.7]; placebo: -1.4 [-2.4, -0.5]). Response rates for endoscopic improvement favoured placebo. Clinical remission rates were similar between groups. In the 450-mg IV group, 14 (29%) participants had an adverse event of UC versus 1 (4%) with placebo. LAG-3+ cells were depleted to 51% of baseline in blood; however, there was no reduction in LAG-3+ cells in the colonic mucosa. Transcriptomic analysis of colon biopsies showed no difference between groups. CONCLUSION: Despite evidence of target cell depletion in blood, GSK2831781 failed to reduce inflammation in the colonic mucosa suggesting no pharmacological effect. The study was terminated early (NCT03893565).
Asunto(s)
Colitis Ulcerosa , Humanos , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/patología , Anticuerpos Monoclonales/efectos adversos , Método Doble Ciego , Linfocitos T , Inducción de Remisión , Resultado del TratamientoRESUMEN
The Wiskott-Aldrich syndrome protein (WASp) is a key cytoskeletal regulator in hematopoietic cells. Covalent modification of a conserved tyrosine by phosphorylation has emerged as an important potential determinant of activity, although the physiological significance remains uncertain. In a murine knockin model, mutation resulting in inability to phosphorylate Y293 (Y293F) mimicked many features of complete WASp-deficiency. Although a phosphomimicking mutant Y293E conferred enhanced actin-polymerization, the cellular phenotype was similar due to functional dysregulation. Furthermore, steady-state levels of Y293E-WASp were markedly reduced compared to wild-type WASp and Y293F-WASp, although partially recoverable by treatment of cells with proteasome inhibitors. Consequently, tyrosine phosphorylation plays a critical role in normal activation of WASp in vivo, and is indispensible for multiple tasks including proliferation, phagocytosis, chemotaxis, and assembly of adhesion structures. Furthermore, it may target WASp for proteasome-mediated degradation, thereby providing a default mechanism for self-limiting stimulation of the Arp2/3 complex.
Asunto(s)
Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Células COS , Línea Celular , Movimiento Celular , Chlorocebus aethiops , Hematopoyesis , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Fagocitosis , Fosforilación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tirosina/química , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/patología , Proteína del Síndrome de Wiskott-Aldrich/química , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
This study investigated ethnic differences in the safety, tolerability, pharmacokinetics, and pharmacodynamics of GSK2831781, an anti-lymphocyte activation gene 3 (LAG3) monoclonal antibody, in healthy participants, and determined local tolerability and bioavailability following subcutaneous (SC) administration. A double-blind, randomized study of (A) single intravenous (IV) doses of GSK2831781 450 mg or placebo in Japanese and White participants; and (B) single SC doses of GSK2831781 150 or 450 mg, or placebo in White participants, was conducted. Blood samples for analyses were collected before dosing and over 112 days after dosing. GSK2831781 was well tolerated in Japanese and White participants after both IV and SC doses, with the adverse event profile in Japanese being consistent with other populations. There were no injection site adverse events. There was no evidence of differences in systemic exposure among Japanese and White participants. Systemic exposure did not vary with body weight. SC bioavailability was 76.5%, as estimated using population pharmacokinetic modeling. Full and sustained target engagement and evidence of LAG3+ cell depletion (≈53%-66%) were observed in both populations and after both administration routes. No evidence of reduced circulating regulatory T cells (CD4+ CD25+ CD127low FoxP3+ ) was observed. Following IV and SC administration, GSK2831781 depleted circulating LAG3+ T cells with no interethnic difference observed. There were no major impacts on circulating regulatory T cells.
Asunto(s)
Relación Dosis-Respuesta a Droga , Humanos , Japón , Método Doble Ciego , Área Bajo la Curva , Voluntarios SanosRESUMEN
Immune mediated inflammatory diseases (IMIDs) are a heterogeneous group of debilitating, multifactorial and unrelated conditions featured by a dysregulated immune response leading to destructive chronic inflammation. The immune dysregulation can affect various organ systems: gut (e.g., inflammatory bowel disease), joints (e.g., rheumatoid arthritis), skin (e.g., psoriasis, atopic dermatitis), resulting in significant morbidity, reduced quality of life, increased risk for comorbidities, and premature death. As there are no reliable disease progression and therapy response biomarkers currently available, it is very hard to predict how the disease will develop and which treatments will be effective in a given patient. In addition, a considerable proportion of patients do not respond sufficiently to the treatment. ImmUniverse is a large collaborative consortium of 27 partners funded by the Innovative Medicine Initiative (IMI), which is sponsored by the European Union (Horizon 2020) and in-kind contributions of participating pharmaceutical companies within the European Federation of Pharmaceutical Industries and Associations (EFPIA). ImmUniverse aims to advance our understanding of the molecular mechanisms underlying two immune-mediated diseases, ulcerative colitis (UC) and atopic dermatitis (AD), by pursuing an integrative multi-omics approach. As a consequence of the heterogeneity among IMIDs patients, a comprehensive, evidence-based identification of novel biomarkers is necessary to enable appropriate patient stratification that would account for the inter-individual differences in disease severity, drug efficacy, side effects or prognosis. This would guide clinicians in the management of patients and represent a major step towards personalized medicine. ImmUniverse will combine the existing and novel advanced technologies, including multi-omics, to characterize both the tissue microenvironment and blood. This comprehensive, systems biology-oriented approach will allow for identification and validation of tissue and circulating biomarker signatures as well as mechanistic principles, which will provide information about disease severity and future disease progression. This truly makes the ImmUniverse Consortium an unparalleled approach.
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Dermatitis Atópica , Medicina de Precisión , Humanos , Calidad de Vida , Biomarcadores , Progresión de la EnfermedadRESUMEN
Culturing of human peripheral blood CD14 positive monocytes is a method for generation of dendritic cells (DCs) for experimental purposes or for use in clinical grade vaccines. When culturing human DCs in this manner for clinical vaccine production, we noticed that 5-10% of cells within the bulk culture were binuclear or multiple nuclear, but had typical dendritic cell morphology and immunophenotype. We refer to the cells as binuclear cells in dendritic cell cultures (BNiDCs). By using single cell PCR analysis of mitochondrial DNA polymorphisms we demonstrated that approximately 20-25% of cells in DC culture undergo a fusion event. Flow sorted BNiDC express low HLA-DR and IL-12p70, but high levels of IL-10. In mixed lymphocyte reactions, purified BNiDC suppressed lymphocyte proliferation. Blockade of dendritic cell-specific transmembrane protein (DC-STAMP) decreased the number of binuclear cells in DC cultures. BNiDC represent a potentially tolerogenic population within DC preparations for clinical use.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Dendríticas/inmunología , Inmunidad , Terapia de Inmunosupresión/métodos , Proteínas de la Membrana/antagonistas & inhibidores , Monocitos/inmunología , Proteínas Adaptadoras Transductoras de Señales , Anticuerpos/farmacología , Diferenciación Celular/inmunología , Fusión Celular , Núcleo Celular , Células Dendríticas/citología , Células Dendríticas/metabolismo , Citometría de Flujo , Antígenos HLA-DR/biosíntesis , Antígenos HLA-DR/inmunología , Humanos , Tolerancia Inmunológica , Inmunohistoquímica , Inmunofenotipificación , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Interleucina-12/biosíntesis , Interleucina-12/inmunología , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Lipopolisacáridos/inmunología , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Monocitos/citología , Monocitos/metabolismo , Análisis de la Célula IndividualRESUMEN
Neutrophils are amongst the first immune cells to arrive at sites of infection and play an important role as the host's first line of defence against invading pathogens. Defects of neutrophil number or function are usually recognized clinically by recurrent infections that often are life-threatening. Over the last few years, a number of genetic mutations have been discovered to be the basis for congenital neutropenia, adding to our understanding of the molecular basis of these diseases. While many genetic mutations that cause severe congenital neutropenia result in a differentiation block at the promyelocyte stage, defects of neutrophil function are more heterogeneous on clinical, genetic and mechanistic levels. In this review we discuss recent advances in our understanding of the genetic and molecular basis of human neutrophil disorders.
Asunto(s)
Neutropenia/genética , Neutrófilos/fisiología , Diferenciación Celular/genética , Movimiento Celular , Citoesqueleto/fisiología , Humanos , Síndromes de Inmunodeficiencia/genética , Neutropenia/congénito , Neutrófilos/patología , Estallido Respiratorio/genética , Transducción de Señal/genética , Receptores Toll-Like/fisiologíaRESUMEN
Lentiviral vectors (lentivectors) are effective for stimulation of cell-mediated and humoral immunity following subcutaneous and intramuscular immunization. However, lentivector genome integration carries a risk of perturbation of host gene expression. Here, we demonstrate that lentivectors with multiple mutations that prevent integration are also effective immunogens. First, systemic CD8(+) T-cell responses to the model antigen ovalbumin were detected following subcutaneous injection of nonintegrating lentivectors. Transfer of transgenic OT1 T cells demonstrated that antigen presentation persisted for at least 30 days. Furthermore, an enhanced CD8(+) T-cell response, peaking at 7 days, was stimulated by coexpression of p38 MAP kinase or an NF-kappaB activator from the same vector. Second, we demonstrated systemic CD8(+) T-cell and antibody responses to the secreted hepatitis B virus (HBV) surface antigen expressed from a nonintegrating lentivector injected intramuscularly. The induction, specificity, and kinetics of antibody production closely mimicked those of natural HBV infection. In this case, both the vector genome and the immune response were maintained for at least 2 months. Together, our data indicate that nonintegrating lentivectors can be employed to generate effective vaccines.
Asunto(s)
Anticuerpos Antineoplásicos/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Cáncer/inmunología , Vectores Genéticos , Vacunas contra Hepatitis B/inmunología , Lentivirus/genética , Linfocitos T/inmunología , Animales , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Neoplasias/patología , Neoplasias/prevención & control , Integración ViralRESUMEN
To more precisely identify the B-cell phenotype in Wiskott-Aldrich syndrome (WAS), we used 3 distinct murine in vivo models to define the cell intrinsic requirements for WAS protein (WASp) in central versus peripheral B-cell development. Whereas WASp is dispensable for early bone marrow B-cell development, WASp deficiency results in a marked reduction in each of the major mature peripheral B-cell subsets, exerting the greatest impact on marginal zone and B1a B cells. Using in vivo bromodeoxyuridine labeling and in vitro functional assays, we show that these deficits reflect altered peripheral homeostasis, partially resulting from an impairment in integrin function, rather than a developmental defect. Consistent with these observations, we also show that: (1) WASp expression levels increase with cell maturity, peaking in those subsets exhibiting the greatest sensitivity to WASp deficiency; (2) WASp(+) murine B cells exhibit a marked selective advantage beginning at the late transitional B-cell stage; and (3) a similar in vivo selective advantage is manifest by mature WASp(+) human B cells. Together, our data provide a better understanding of the clinical phenotype of WAS and suggest that gene therapy might be a useful approach to rescue altered B-cell homeostasis in this disease.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Homeostasis/inmunología , Proteína del Síndrome de Wiskott-Aldrich/inmunología , Animales , Homeostasis/genética , Ratones , Ratones Noqueados , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Quimiocina CCL20/inmunología , Proteínas del Sistema Complemento/inmunología , Enfermedades del Complejo Inmune/tratamiento farmacológico , Enfermedades del Complejo Inmune/inmunología , Inmunoconjugados/uso terapéutico , Animales , Anticuerpos Monoclonales/toxicidad , Enfermedad Crónica , Cristalización , Determinación de Punto Final , Femenino , Humanos , Inflamación/inmunología , Inflamación/patología , Macaca fascicularisRESUMEN
Wiskott-Aldrich syndrome (WAS) is an X-linked hematological disease characterized by immunodeficiency, eczema, and thrombocytopaenia, and shows promise for treatment with hematopoietic stem cell gene therapy. The immunopathology of WAS is attributable at least in part to defects of cell migration and localization as a result of chemotactic, adhesive, and chemokinetic defects. Whereas previous studies using either gammaretroviral or lentiviral vectors have demonstrated variable correction of T-cell proliferation and dendritic cell (DC) cytoarchitecture, we have used a lentiviral vector expressing an eGFP-WASp fusion protein to test the potential for restoration of cell migratory defects. Multilineage expression of the fusion transgene was present for up to 10 months after primary engraftment, and also in secondary recipients analyzed after a further 9 months. Transduced bone marrow-derived dendritic cells (BMDCs) demonstrated recovery of podosome numbers and turnover, while B cells, BMDCs, and Langerhans cells (LCs) exhibited enhanced chemotactic responses to specific stimuli. As an indication of functionality in vivo, splenic marginal zone B cells and a cutaneous contact hypersensitivity (CHS) response to dinitrofluorobenzene (DNFB) were both partially restored. These proof of principle experiments demonstrate that WAS protein (WASp) transgene expression can be successfully maintained long term in primary and secondary recipients, and that it is associated with a significant repair of migratory defects.