RESUMO
Though binding sites for the complement factor C1q and the canonical fragment crystallizable (Fc) gamma receptors (Fc[Formula: see text]Rs) on immunoglobulin G (IgG) molecules overlap, how C1q decoration of immune complexes (ICs) influences their ability to engage Fc[Formula: see text]Rs remains unknown. In this report, we use recombinant human Fc multimers as stable IC mimics to show that C1q engagement of ICs directly and transiently inhibits their interactions with Fc[Formula: see text]RIII (CD16) on human natural killer (NK) cells. This inhibition occurs by C1q engagement alone as well as in concert with other serum factors. Furthermore, the inhibition of Fc[Formula: see text]RIII engagement mediated by avid binding of C1q to ICs is directly associated with IC size and dependent on the concentrations of both C1q and Fc multimers present. Functionally, C1q-mediated Fc blockade limits the ability of NK cells to induce the upregulation of the cosignaling molecule, 4-1BB (CD137), and to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Although C1q is traditionally viewed as a soluble effector molecule, we demonstrate that C1q may also take on the role of an "immunologic rheostat," buffering Fc[Formula: see text]R-mediated activation of immune cells by circulating ICs. These data define a novel role for C1q as a regulator of immune homeostasis and add to our growing understanding that complement factors mediate pleiotropic effects.
Assuntos
Complemento C1q , Receptores de IgG , Humanos , Complemento C1q/metabolismo , Imunoglobulina G , Citotoxicidade Celular Dependente de Anticorpos , Células Matadoras NaturaisRESUMO
Non-secretor status due to homozygosity for the common FUT2 variant c.461G>A (p.Trp154∗) is associated with either risk for autoimmune diseases or protection against viral diarrhea and HIV. We determined the role of FUT2 in otitis media susceptibility by obtaining DNA samples from 609 multi-ethnic families and simplex case subjects with otitis media. Exome and Sanger sequencing, linkage analysis, and Fisher exact and transmission disequilibrium tests (TDT) were performed. The common FUT2 c.604C>T (p.Arg202∗) variant co-segregates with otitis media in a Filipino pedigree (LOD = 4.0). Additionally, a rare variant, c.412C>T (p.Arg138Cys), is associated with recurrent/chronic otitis media in European-American children (p = 1.2 × 10-5) and US trios (TDT p = 0.01). The c.461G>A (p.Trp154∗) variant was also over-transmitted in US trios (TDT p = 0.01) and was associated with shifts in middle ear microbiota composition (PERMANOVA p < 10-7) and increased biodiversity. When all missense and nonsense variants identified in multi-ethnic US trios with CADD > 20 were combined, FUT2 variants were over-transmitted in trios (TDT p = 0.001). Fut2 is transiently upregulated in mouse middle ear after inoculation with non-typeable Haemophilus influenzae. Four FUT2 variants-namely p.Ala104Val, p.Arg138Cys, p.Trp154∗, and p.Arg202∗-reduced A antigen in mutant-transfected COS-7 cells, while the nonsense variants also reduced FUT2 protein levels. Common and rare FUT2 variants confer susceptibility to otitis media, likely by modifying the middle ear microbiome through regulation of A antigen levels in epithelial cells. Our families demonstrate marked intra-familial genetic heterogeneity, suggesting that multiple combinations of common and rare variants plus environmental factors influence the individual otitis media phenotype as a complex trait.
Assuntos
Fucosiltransferases/genética , Variação Genética/genética , Otite Média/genética , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Orelha Média/microbiologia , Exoma/genética , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/fisiologia , Otite Média/microbiologia , Linhagem , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
We developed a fully recombinant anti-CD20 protein derived from cDNA encoding one Fab domain, two IgG1 Fc regions, the IgG2 hinge, and an isoleucine zipper. This protein, called GB4542, contained both the homodimer and higher-order multimers. Binding studies revealed that GB4542 preferentially bound CD20(+) cells yet also recognized CD20(-)FcγR(+) PBMC. In contrast, a control mAb containing the identical Fab region, GB4500, failed to bind CD20(-)FcγR(+) PBMC. Consistent with these findings, interactions between GB4542 and the canonical FcγRs had substantially lower KD values than correlate interfaces between GB4500 and these receptors. At low concentrations, GB4542 showed enhanced Ab-dependent cellular cytotoxicity, Ab-dependent cellular phagocytosis, and complement-dependent cytotoxicity compared with GB4500. However, at higher concentrations, an Fc analog of GB4542 inhibited anti-CD20 mAb-mediated B cell clearance through direct blocking of both Fc-FcγR interactions and C1q deposition on target cells. Furthermore, the higher-order multimer fraction of GB4542 demonstrated greater binding avidity with the canonical FcγRs and was associated with inhibitory effects observed in Ab-dependent cellular phagocytosis and complement-dependent cytotoxicity assays. These data suggest that GB4542 might have utility in the treatment of autoimmune diseases by combining both mAb-mediated B cell depletion and multimerized Fc-mediated tolerogenic effects.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Linfócitos B/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Doenças Autoimunes/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/imunologia , Receptores de IgG/imunologia , Proteínas Recombinantes/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
There is a lack of effective targeted therapies for the treatment of complement dependent diseases. We developed two recombinant Fc multimers, G207 and G211, with limited ability to interact with low/moderate affinity FcγRs, but with high avidity for C1q. These drugs effectively inhibited complement dependent cytotoxicity (CDC) in vitro, and prevented the deposition of C1q, C3b and MAC, on the surface of Ab-opsonized cells. Importantly, these inhibitory effects were both C1q dependent and independent. In order to determine the biologic relevance of our findings, we evaluated the clinical efficacy of these drugs in three different animal models, acute RBC hemolysis, anti-Thy-1 nephritis and passive Heymann's nephropathy (PHN), in which disease pathophysiology relies preferentially on complement activation. While G207 was protective in the anti-Thy-1 nephritis and PHN models, G211 was protective in all of the models tested and could effectively treat PHN. In the anti-Thy-1 nephritis model, G211 prevented the characteristic histologic changes associated with the disease and limited glomerular deposition of C3. Collectively, these data suggest that "complement preferential" Fc multimers offer a novel approach to the treatment of complement mediated diseases.
Assuntos
Complemento C1q/imunologia , Proteínas do Sistema Complemento/metabolismo , Eritrócitos/fisiologia , Doenças do Sistema Imunitário/terapia , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Animais , Células Cultivadas , Complemento C3/metabolismo , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Glomerulonefrite Membranosa , Hemólise , Humanos , Doenças do Sistema Imunitário/imunologia , Terapia de Alvo Molecular , Ligação Proteica , Multimerização Proteica , Receptores Fc/metabolismo , Antígenos Thy-1/imunologia , Transgenes/genéticaRESUMO
BACKGROUND: Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3-αGal antigen (GalTKO) and express human membrane cofactor protein (hCD46). Natural killer cells rapidly disappear from the blood during perfusion of GalTKO.hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA-E and ß2 microglobulin inhibits GalTKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models. METHODS: Lungs from pigs modified to express GalTKO.hCD46 (n=37) and GalTKO.hCD46.HLA-E (n=5) were harvested and perfused with human blood until failure or elective termination at 4 hours. Airway pressures and pulmonary artery hemodynamics were recorded in real time. Blood samples were also collected throughout the experiment for analysis. Porcine aortic endothelial cells (PAECs) from each genotype were cultured in monolayers in microfluidic channels and used in fluorescent cytotoxicity assays using human NK cells. RESULTS: HLA-E expression on GalTKO.hCD46 PAECs was associated with significantly decreased antibody-dependent and antibody-independent NK-mediated cytotoxicity under in vitro conditions simulating physiologic shear stress. Relative to GalTKO.hCD46 pig lungs perfused with human blood on an ex vivo platform, additional expression of HLA-E increased median lung survival (>4 hours, vs 162 minutes, P=.012), and was associated with attenuated rise in pulmonary vascular resistance, and decreased platelet activation and histamine elaboration. As expected, HLA-E expression was not associated with a significant difference in NK cell adhesion to endothelial cells in vitro, or NK cell and neutrophil sequestration during organ perfusion. CONCLUSIONS: We conclude human NK cell activation contributes significantly to GalTKO.hCD46 pig endothelial injury and lung inflammation and show that expression of HLA-E is associated with physiologically meaningful protection of GalTKO.hCD46 cells and organs exposed to human blood.
Assuntos
Células Endoteliais/imunologia , Sobrevivência de Enxerto/imunologia , Antígenos HLA/imunologia , Xenoenxertos/imunologia , Leucócitos/imunologia , Lesão Pulmonar/terapia , Proteína Cofatora de Membrana/imunologia , Animais , Animais Geneticamente Modificados , Citotoxicidade Imunológica/imunologia , Galactosiltransferases/genética , Sobrevivência de Enxerto/genética , Antígenos HLA/genética , Humanos , Células Matadoras Naturais/imunologia , Lesão Pulmonar/imunologia , Proteína Cofatora de Membrana/genética , Suínos , Transplante Heterólogo/métodosRESUMO
Altered B cell function is important in the pathogenesis of rheumatoid arthritis (RA). In this report, we show that patients with active RA have an increased frequency of CD32B low/neg cells in the CD27(+)IgD(-) memory B cell subset and that these changes are associated with phenotypic and functional B cell activation. Studies using PBMCs from healthy donors revealed that downregulation of CD32B on B cells is mediated by CD40-CD40L interactions and is potentiated by IL-4 and inhibited by both IL-10 and IL-21. These findings appear physiologically relevant because CD4 T cell expression of CD40L correlated with the frequency of CD32B low/neg cells in the CD27(+)IgD(-) memory B subset in patients with RA. Our data support a model in which high levels of CD40L, present on circulating T cells in patients with RA, causes B cell activation and CD32B downregulation, resulting in secondary protection of memory B cells from CD32B-mediated cell death.
Assuntos
Artrite Reumatoide/imunologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Antígenos CD40/imunologia , Receptores de IgG/imunologia , Adulto , Idoso , Artrite Reumatoide/metabolismo , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Regulação para Baixo , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de IgG/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Agonistic monoclonal antibodies (mAbs) directed against the co-signaling molecule CD137 (4-1BB) elicit potent anti-tumor immunity in mice. This anti-tumor immunity has traditionally been thought to result from the ability of the Fab portion of anti-CD137 to function as an analog for CD137L. Although binding of CD137 by anti-CD137 mAbs has the potential to cross-link the Fc fragments, enabling Fc engagement of low to moderate affinity Fc gamma receptors (FcγR), the relative import of such Fc-FcγR interactions in mediating anti-CD137 associated anti-tumor immunity is unknown. We studied the ability of a rat anti-mouse CD137 mAb (2A) to mediate the anti-tumor response against the EL4E7 lymphoma in WT and FcγR(-/-) strains. 2A-treated FcRγ(-/-) mice had improved anti-tumor immunity against EL4E7, which could be completely recapitulated in FcγRIII(-/-) animals. These improved anti-tumor responses were associated with increased splenic CD8ß T cell and dendritic cell (DC) populations. Furthermore, there was an increase in the number of DCs expressing high levels of the CD40, CD80, and CD86 molecules that are associated with more effective antigen presentation. Our results demonstrate an unexpected inhibitory role for FcγRIII in the anti-tumor function of anti-CD137 and underscore the need to consider antibody isotype when engineering therapeutic mAbs.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Linfoma/terapia , Receptores de IgG/deficiência , Receptores de IgG/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Imobilizados/imunologia , Anticorpos Imobilizados/metabolismo , Anticorpos Monoclonais/metabolismo , Feminino , Células HEK293 , Humanos , Linfoma/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Ratos , Receptores de IgG/metabolismo , Transfecção , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genéticaRESUMO
Given the heightened interest in manipulation of co-signaling cascades for cancer immunotherapy, we sought to determine how/whether tumors decorated with therapeutic monoclonal antibodies (mAbs) impact the expression of co-signaling molecules on human NK cells. Stimulation of NK cells with aggregated IgG1 resulted in the upregulation of HAVCR2 - the gene encoding T-cell immunoglobulin and mucin-containing domain (Tim)-3 - known to be involved in the induction of peripheral T cell tolerance. This upregulation of HAVCR2 was recapitulated at the protein level, following NK cell stimulation by either mAb opsonized tumors, recombinant human IgG1 Fc multimer, and/or non-Fc stimuli e.g. IL-12/IL-18. The patterns of Tim-3 expression were temporally distinct from the FcR mediated induction of the co-signaling molecule, 4-1BB (CD137), with Tim-3 increases observed twenty minutes following exposure to Fc multimers and remaining at high levels for at least six hours, while increases in CD137 expression were first observed at the four-hour time point. Importantly, these Tim-3+ NK cells were functionally diverse, as evidenced by the fact that their ability to produce IFN-γ in response to an NK cell responsive tumor was strictly dependent upon the stimuli employed for Tim-3 induction. These data suggest that Tim-3 upregulation is the common end-result of NK cell activation by a variety of unique and overlapping stimuli and is not an independent marker of NK cell exhaustion. Furthermore, our observations potentially explain the diverse functionality attributed to Tim-3+ NK cells and should be considered prior to use of anti-Tim-3 inhibitory mAbs for cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Imunoglobulina G/metabolismo , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Células Cultivadas , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Células K562 , Ativação Linfocitária , Neoplasias/imunologia , Multimerização Proteica , Agregação de Receptores , Receptores Fc/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Regulação para CimaRESUMO
The antiinflammatory effects of i.v. Ig (IVIG) in the treatment of autoimmune disease are due, in part, to the Fc fragments of Ig aggregates. In order to capitalize on the known antiinflammatory and tolerogenic properties of Ig Fc aggregates, we created a recombinant human IgG1 Fc multimer, GL-2045. In vitro, GL-2045 demonstrated high-avidity binding to Fc receptors, blocked the binding of circulating immune complexes from patients with rheumatoid arthritis to human Fcγ receptors (FcγRs), and inhibited antibody-mediated phagocytosis at log order-lower concentrations than IVIG. In vivo, administration of GL-2045 conferred partial protection against antibody-mediated platelet loss in a murine immune thrombocytopenic purpura (ITP) model. GL-2045 also suppressed disease activity in a therapeutic model of murine collagen-induced arthritis (CIA), which was associated with reduced circulating levels of IL-6. Furthermore, GL-2045 administration to nonhuman primates (NHPs) transiently increased systemic levels of the antiinflammatory cytokines IL-10 and IL-1RA, reduced the proinflammatory cytokine IL-8, and decreased surface expression of CD14 and HLA-DR on monocytes. These findings demonstrate the immunomodulatory properties of GL-2045 and suggest that it has potential as a treatment for autoimmune and inflammatory diseases, as a recombinant alternative to IVIG.
RESUMO
GL-2045 is a recombinant human immunoglobulin G1 (IgG1)-based Fc multimer designed to recapitulate the anti-inflammatory activities of intravenous immunoglobulin (IVIG) on the innate and adaptive immune responses. We used functional in vitro studies to determine if GL-2045 could mimic the modulatory activity of IVIG on complement activation. GL-2045, at log-order lower concentrations than heat-aggregated IgG (HAGG) and IVIG, protected antibody-opsonized cells from complement-dependent cytotoxicity. These protective effects were completely mediated by the higher order multimer fractions of GL-2045 and were partially dependent upon sequestration of C1q. Exposure of serum to GL-2045 and, to a lesser extent, IVIG, resulted in high levels of C4a, limited levels of C3a, and no C5a. In contrast, HAGG induced high levels of C4a, C3a, and C5a. The means by which GL-2045 governed complement activation was dependent on its ability to augment the function of factor H, alone and in combination with factor I, to indirectly limit the alternative form of C3 convertase, with resultant increases in the anti-inflammatory molecule, the "inactive" form of C3b, called iC3b. Although IVIG, like GL-2045, potentiated factor H function, it also directly inhibited the alternative form of C3 convertase. Our findings help elucidate how IVIG, GL-2045, and HAGG regulate complement function. Furthermore, the capacity of GL-2045 to sequester C1q and augment factor H activity, in combination with its ability to generate activation-induced immunomodulatory complement split products, such as iC3b, make it a viable drug candidate for the treatment of diverse complement-mediated diseases.
RESUMO
Mice with one thyroid hormone receptor (TR) alpha-1 allele encoding a dominant negative mutant receptor (TR alpha1(PV/+)) have persistently elevated serum T3 levels (1.9-fold above normal). They also have markedly increased hepatic type 1 iodothyronine deiodinase (D1) mRNA and enzyme activity (4- to 5-fold), whereas other hepatic T3-responsive genes, such as Spot14 and mitochondrial alpha-glycerol phosphate dehydrogenase (alpha-GPD), are only 0.7-fold and 1.7-fold that of wild-type littermates (TR alpha1+/+). To determine the cause of the disproportionate elevation of D1, TR alpha1+/+ and TR alpha1(PV/+) mice were rendered hypothyroid and then treated with T3. Hypothyroidism decreased hepatic D1, Spot14, and alpha-GPD mRNA to similar levels in TR alpha1+/+ and TR alpha1(PV/+) mice, whereas T3 administration caused an approximately 175-fold elevation of D1 mRNA but only a 3- to 6-fold increases in Spot14 and alpha-GPD mRNAs. Interestingly, the hypothyroidism-induced increase in cerebrocortical type 2 iodothyronine deiodinase activity was 3 times greater in the TR alpha1(PV/+) mice, and these mice had no T3-dependent induction of type 3 iodothyronine deiodinase. Thus, the marked responsiveness of hepatic D1 to T3 relative to other genes, such as Spot14 and alpha-GPD, explains the relatively large effect of the modest increase in serum T3 in the TR alpha1(PV/+) mice, and TR alpha plays a key role in T3-dependent positive and negative regulation of the deiodinases in the cerebral cortex.
Assuntos
Biomarcadores , Iodeto Peroxidase/biossíntese , Iodeto Peroxidase/química , Glândula Tireoide/metabolismo , Animais , Córtex Cerebral/metabolismo , Heterozigoto , Hipotireoidismo , Rim/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Mutação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiroxina/metabolismo , Fatores de Tempo , Tri-Iodotironina/metabolismoRESUMO
We have identified a cell type-specific, negative thyroid hormone-responsive element in the human type 1 iodothyronine deiodinase (hdio1) gene. This fragment, termed a JEG response element, bound tightly to a JEG-cell nuclear protein [JEG cell-specific transcription factor (JTF)] also present in placenta but not in COS-7, HeLa, or human embryonic kidney-293 cells. In JEG-3 cells, three copies of the JEG response element conferred a more than 40-fold transcriptional stimulation to the heterologous rat GH promoter which was further increased 2-fold by apo-thyroid hormone receptor (TR) and reduced 3-fold by T(3). Dimethyl sulfide footprinting showed overlapping contact sites for the high-affinity interaction of JTF and low-affinity binding of TR-retinoid X receptor. Expression of the same construct was unaffected by TR or T(3) in COS cells, indicating JTF was required for negative regulation by T(3)-TR. Mutations of the critical thyroid hormone responsive element binding P box amino acids EG to GS in TRalpha1 or TRbeta2 eliminated the apo-TR and T(3)-TR effects. These studies identify a novel mechanism for cell type-specific, promoter-independent negative regulation by T(3).
Assuntos
Regulação para Baixo , Iodeto Peroxidase/genética , Elementos de Resposta/genética , Fatores de Transcrição/fisiologia , Tri-Iodotironina/fisiologia , Animais , Linhagem Celular , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores dos Hormônios Tireóideos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tri-Iodotironina/farmacologiaRESUMO
The methods of cell detachment influence phenotype and function of human macrophages cultured in vitro. However, comparative studies defining the influence of cell detachment techniques on secondary characterization of M1 or M2 polarized macrophages are largely absent from the literature. In this study we evaluated the impact of trypsin, accutase, EDTA, PBS, and cell scraping on: A. cell recovery, B. phenotype and C. function of in vitro polarized macrophages. Our data demonstrate that while exposure to trypsin or accutase yields highly efficient recovery of viable cells, such chemical cleavage results in loss of select M2 cell surface markers with correlative changes in cell function. In contrast, phenotype and function are maintained following detachment by EDTA on ice. Our data suggest that seemingly "trivial" changes in methodologies for macrophage detachment induce both variable and profound changes on cell phenotype and function which can dramatically impact the results of polarization experiments.
Assuntos
Separação Celular/métodos , Colagenases/metabolismo , Ácido Edético/metabolismo , Macrófagos/imunologia , Peptídeo Hidrolases/metabolismo , Tripsina/metabolismo , Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Humanos , Lectinas Tipo C/biossíntese , Leucócitos Mononucleares/imunologia , Receptores de Lipopolissacarídeos/biossíntese , Receptor de Manose , Lectinas de Ligação a Manose/biossíntese , Fenótipo , Receptores de Superfície Celular/biossínteseRESUMO
In vitro assessment of lymphocyte and natural killer (NK) cell cytotoxicity typically employs density gradient centrifugation and magnetic cell separation to isolate effector cells, and chromium release to assess cytotoxicity. In order to improve the rapidity and scalability of in vitro cytotoxicity assessment, we evaluated the efficacy of a protocol utilizing tetrameric antibody complexes and SepMate™ isolation tubes to negatively select NK cells (TACs/Sep), and calcein-AM release to measure cytotoxicity. We compared the efficiency and accuracy of this protocol to a conventional approach employing density gradient centrifugation and magnetically labeled antibodies (DG/MACS) to isolate NK cells and chromium release to measure cytotoxicity. The TACs/Sep method significantly decreased the time required for NK cell isolation (1h vs. 4h), but resulted in higher red blood cell contamination. NK cell activation marker expression (including CD94, NKG2D, NKp30, NKp46, DNAM-1, 2B4, KIR2DL1/S1, KIR2DL2/L3, intracellular granzyme B, and perforin) was similar when comparing NK cells isolated by the TACs/Sep or DG/MACS methods, but the TACs/Sep method induced higher expression of CD16. In vitro cytotoxicity against HT29 colon cancer and K562 leukemia cells was not affected by the isolation method. Lastly, by combining the TACs/Sep NK cell isolation method with calcein-acetoxymethyl diacetylester (calcein-AM) release, the time required to assess in vitro cytotoxicity was reduced by 33% (4h) compared to protocols employing DG/MACS and chromium release. Altogether, these results provide the foundation for the development of a rapid, high throughput functional assay, and make it practical for the multiplexing of downstream applications, such as flow cytometric analysis and enzyme-linked immunosorbent assays (ELISAs).
Assuntos
Citotoxicidade Imunológica , Ensaios de Triagem em Larga Escala/métodos , Células Matadoras Naturais/imunologia , Linfócitos/imunologia , Centrifugação com Gradiente de Concentração , Células HT29 , Humanos , Separação Imunomagnética , Células K562RESUMO
Tumour antigen targeted antibodies (mAbs) can induce natural killer (NK) cells to kill tumours through antibody dependent cellular cytotoxicity (ADCC) upon engagement of NK cell expressed FcγRIIIa. FcγRIIIa polymorphisms partially dictate the potency of the ADCC response. The high affinity FcγRIIIa-158-valine (V) polymorphism is associated with more potent ADCC response than the low affinity FcγRIIIa-158-phenylalanine (F) polymorphism. Because approximately 45% of patients are homozygous for the FcγRIIIa-158-F polymorphism (FF genotype), their ability to mount ADCC is impaired. We investigated whether a novel mAb capable of binding multiple antigen specific targets and engaging multiple low affinity FcγRIIIa receptors could further enhance ADCC against colon cancer in vitro. Specifically, we generated a novel anti-epidermal growth factor receptor (EGFR) antibody (termed a stradobody) consisting of an unmodified Fab sequence and two Immunoglobulin G, subclass 1 (IgG1) Fc domains separated by an isoleucine zipper domain and the 12 amino-acid IgG2 hinge. The stradobody framework induced multimerisation and was associated with increased binding to the EGFR and FcγRIIIa. From a functional perspective, when compared to an unmodified anti-EGFR mAb with a sequence identical to cetuximab (a commercially available anti-EGFR mAb), stradobodies significantly enhanced ADCC. These effects were observed using both KRAS wild type HT29 and KRAS mutant SW480 colon cancer cells as targets, and by NK cells obtained from healthy donors and a cohort of patients with colon cancer. These data suggest that high avidity cross-linking of multiple tumour surface antigens and multiple NK cell associated FcγRIIIa molecules can enhance ADCC and partially overcome impaired ADCC by FF genotype individuals in vitro.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/terapia , Receptores ErbB/imunologia , Células Matadoras Naturais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Receptores ErbB/genética , Genótipo , Células HT29 , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Polimorfismo de Nucleotídeo Único , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
CD44 is an adhesion molecule in the extracellular matrix that shows various functions, including tumor genesis and metastasis. A recent study showed that CD44 expression level was strongly correlated with the generation of papillary thyroid carcinomas, the most prevalent malignancy of the thyroid gland. We report here that CD44 is negatively regulated by thyroid hormone (T(3)) through a novel mechanism. We demonstrate that nuclear receptor corepressor (NCoR) enhances thyroid hormone receptor (TR)-mediated basal transactivation by a weak TR.DNA interaction in the absence of T(3), which is repressed by T(3) through a transient TR .DNA interaction. Initially, we identified that CD44 was negatively directly transcriptionally T(3) -responsive. Deletion and mutation analysis indicated that both a weak TR and a GAGA-binding factor (GAF) binding sites on the CD44 promoter were required for negative regulation by T(3). The weak TR.DNA interaction was further confirmed by electrophoretic gel mobility shift assay, chromatin immunoprecipitation, and transfection assays using a non-DNA-binding TRalpha1 mutant. More interestingly, NCoR acted as a co-activator to enhance TR-mediated basal transactivation in the absence of T(3). This effect was eliminated by removal of TR or NCoR binding. Most strikingly, T(3) induced a remarkable increase in TR.DNA binding at 40-60 min after T(3) exposure that rapidly returned to basal levels, suggesting a T(3)-induced remodeling of chromatin structure at the early stage of T(3) stimulation resulting in repression. Therefore, we propose a mechanism by which NCoR, GAF, and TR interact with the CD44 negative T(3)-responsive element to enhance basal transactivation, whereas T(3) induces the remodeling of chromatin structure for repression.