RESUMEN
Dysregulation of autophagy has been implicated in the development of various disease indications including autoimmune diseases. Here we identified hitherto unsuspected molecular alterations of autophagy occurring at an early stage of the macroautophagy pathway in the salivary glands and spleen of NOD.H-2h4 mice that develop a primary Sjögren's-like syndrome. In this study we investigated the capacity of phosphopeptide P140 to correct immune alteration in NOD.H-2h4 mice and the effect on neogenesis of tertiary lymphoid structures in salivary glands, which is hallmark characteristic of SS. Phosphopeptide P140 known to lower excessive autophagy processes, rescued sick NOD.H-2h4 mice from some autophagy defects and significantly reduced formation of tertiary lymphoid structures in salivary glands. Mechanistically, the frequency of activated CD44high/CD62Llow CD4+ T cell populations was significantly decreased and this reduction was correlated with an increased number of CD44low/CD62Lhigh resting T cells. The CD8 T cell compartment was not affected. P140 down-regulated the maturation and differentiation of B cells into plasma cells, and decreased IgG and autoantibody secretion. It had no effect on germinal centers B cells (B220+ FAS+GL-7+) that are an important compound of the B cell humoral immune response. Together with previous data generated in MRL/lpr mice that develop some features of Sjögren's syndrome associated to other inflammatory and autoimmune defects, our present findings strongly reinforce the potential of autophagy modulators, such as P140, for treating patients with Sjögren's syndrome.
Asunto(s)
Autofagia , Susceptibilidad a Enfermedades , Síndrome de Sjögren/etiología , Animales , Autoanticuerpos/inmunología , Autoinmunidad , Autofagia/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Modelos Animales de Enfermedad , Centro Germinal/inmunología , Centro Germinal/metabolismo , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos MRL lpr , Ratones Endogámicos NOD , Glándulas Salivales/inmunología , Glándulas Salivales/metabolismo , Glándulas Salivales/patología , Síndrome de Sjögren/metabolismo , Síndrome de Sjögren/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
A growing body of evidence suggests that when B cells are chronically stimulated, a phenotypically unique subset expands. Data suggest that this atypical population contains B cell receptor (BCR) specificities capable of binding the antigen, or sets of antigens that initiated the expansion of these cells. These B cells have been given various names, including double negative B cells, atypical memory B cells, tissue-like memory B cells, or age associated B cells (ABCs). However, on close inspection these reports described B cell subsets that closely resemble B cells we refer to as CD11c+ B cells that often express T-bet. Here we will review the human studies that describe atypical memory B cells and compare and contrast their phenotype and suggested function in health and disease.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Antígeno CD11c/inmunología , Proteínas de Dominio T Box/inmunología , Animales , Enfermedades Autoinmunes/metabolismo , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Antígeno CD11c/metabolismo , Humanos , Memoria Inmunológica/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas de Dominio T Box/metabolismoRESUMEN
Chronic inflammatory conditions, such as in autoimmune disease, can disturb immune cell homeostasis and induce the expansion of normally rare cell populations. In our analysis of various murine models of lupus, we detect increased frequency of an uncommon subset identified as NK1.1(+)CD11c(+)CD122(+)MHC class II(+). These cells share characteristics with the NK cell lineage and with cells previously described as IFN-producing killer dendritic cells: 1) they depend on IL-15 and express E4BP4; 2) they are cytotoxic and produce type I and type II IFN upon activation; and 3) they are efficient APCs both through MHC class II expression and in cross-presentation to CD8s. These atypical NK cells are responsive to TLR stimulation and thus are most abundant in mice with high copy number of the Tlr7 gene. They are highly proliferative as assessed by in vivo BrdU incorporation. In adoptive transfer experiments they persist in high numbers for months and maintain their surface marker profile, indicating that this population is developmentally stable. Gene expression analyses on both mRNA and microRNAs show a modified cell cycle program in which various miR-15/16 family members are upregulated, presumably as a consequence of the proliferative signal mediated by the increased level of growth factors, Ras and E2F activity. Alternatively, low expression of miR-150, miR-181, and miR-744 in these cells implies a reduction in their differentiation capacity. These results suggest that cells of the NK lineage that undergo TLR stimulation might turn on a proliferative program in detriment of their full differentiation into mature NK cells.
Asunto(s)
Células Asesinas Naturales/inmunología , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos/inmunología , Subgrupos Linfocitarios/inmunología , Traslado Adoptivo , Animales , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Modelos Animales de Enfermedad , Femenino , Inmunohistoquímica , Células Asesinas Naturales/citología , Subgrupos Linfocitarios/citología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Quimera por TrasplanteRESUMEN
Several mouse models of systemic lupus erythematosus, including FcγRIIB-KO and TLR7tg mice, develop an expansion of an atypical NK cell subset with functional similarity to cells referred as IFN-producing killer DCs or pre-mature NKs in other systems. In this study, we show that atypical NKs purified from spleens of systemic lupus erythematosus-prone mice, and identified as NK1.1(+)CD11c(+)CD122(+)MHC-II(+), induce persistent autoimmune disease in an IFN-I- and CD40L-dependent manner when transferred to wild-type mice. A single transfer of 4 × 10(6) NK1.1(+) cells from TLR7tg into wild-type induces a 2-wk-long wave of inflammatory cytokines in the serum; a sustained increase in T cell activation and follicular helper cells for the following months; and a progressive expansion of dendritic cells, monocytes, and granulocytes. Furthermore, IL-15 deficiency, which impedes development of NK cells, ameliorates the autoimmune pathology of TLR7tg mice. These results suggest that cells of the NK lineage can develop into cytokine-producing/APCs that affect the priming and progression of systemic autoimmune disease.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Células Dendríticas/citología , Interferón Tipo I/inmunología , Células Asesinas Naturales/inmunología , Traslado Adoptivo , Animales , Ligando de CD40/inmunología , Citocinas/sangre , Células Dendríticas/inmunología , Granulocitos/inmunología , Inflamación/inmunología , Interferón Tipo I/biosíntesis , Interleucina-15/genética , Células Asesinas Naturales/trasplante , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Monocitos/inmunología , Receptores de IgG/genética , Linfocitos T Colaboradores-Inductores/inmunología , Receptor Toll-Like 7/genéticaRESUMEN
Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as "genomic storm" in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous "danger" signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.
RESUMEN
Toll-like receptor 7 (Tlr7) has been linked to systemic lupus disease incidence in humans and mice, but how TLR7 potentiates autoimmunity is unclear. We used a Tlr7 transgenic (tg) mouse model to investigate the cellular and molecular events required to induce spontaneous autoimmunity through increased TLR7 activity. We determined that Tlr7 exerts B-cell-intrinsic effects in promoting spontaneous germinal center (GC) and plasmablast B-cell development, and that these B-cell subsets are dependent on T-cell-derived signals through CD40L and SLAM-associated protein (SAP), but not IL-17. Antigen specificity also factored into TLR7-induced disease, as both a restricted T cell receptor (TCR) specificity and MHC haplotype H2(k/k) protected Tlr7tg mice from spontaneous lymphocyte activation and autoantibody production. Inflammatory myeloid cell expansion and autoimmunity did not develop in Tlr7tgIgH(-/-) mice, suggesting either that spontaneous TLR7 activation does not occur in dendritic cells, or, if it does occur, cannot drive these events in the absence of B-cell aid. These data indicate that autoimmune disease in Tlr7tg mice is contingent upon B cells receiving stimulation both through innate pathways and T-cell-derived signals and suggest a codependent relationship between B cells and T cells in the development of autoimmunity.
Asunto(s)
Inmunidad Adaptativa/inmunología , Autoinmunidad/inmunología , Linfocitos B/inmunología , Inmunidad Innata/inmunología , Lupus Eritematoso Sistémico/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 7/inmunología , Análisis de Varianza , Animales , Ligando de CD40/metabolismo , Citometría de Flujo , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína Asociada a la Molécula de Señalización de la Activación LinfocitariaRESUMEN
NK effector cells expressing a CAR construct may be used to target T-lineage markers. In this work, we compared the activity of a NK-specific CAR-NK and a CAR-T framework when expressed on NK effector cells to target CD3 and CD5 in T-cell malignancies. Our results show that CD3-CAR-T is more active than CD5-CAR-T to eliminate malignant T cells in vitro, however, CD3-CAR-T were less efficient to eliminate tumor cells in vivo, while CD5-CAR-T had antitumor activity in a diffuse xenograft model. Lack of in vivo efficacy correlated with downregulation of CD3 levels in target T cells after coculture with CD3-CAR effector cells. The CAR-NK framework greatly improved the efficacy of CARs leading to increased degranulation, cytokine secretion and elimination of the tumor xenograft by CD5-CAR-NK effector cells. Finally, all CAR constructs were similarly effective to eliminate malignant T cells in vitro. Our results show that the NK-CAR framework improves the activity of CARs in NK cells and that CD5 would be a better target than CD3 for T-cell malignancies, as dynamic downregulation of target expression may affect in vivo efficacy.
RESUMEN
Chimeric antigen receptor (CAR) T-cell therapy was envisioned as a mechanism to re-direct effector T-cells to eliminate tumor cells. CARs are composed of the variable region of an antibody that binds a native cancer antigen coupled to the signaling domain of a TCR and co-stimulatory molecules. Its success and approval by the U.S. Food and Drug Administration for the treatment of B-cell malignancies revolutionized the immunotherapy field, leading to extensive research on its possible application for other cancer types. In this review, we will focus on the evolution of CAR-T cell therapy outlining current technologies as well as major obstacles for its wide application. We will highlight achievements, the efforts to increase efficacy and to evolve into an off-the-shelf treatment, and as a possible future treatment for non-cancer related diseases.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Receptores Quiméricos de Antígenos/uso terapéutico , Animales , Ensayos Clínicos como Asunto , Humanos , Inmunoterapia Adoptiva , Ingeniería de Proteínas , Receptores Quiméricos de Antígenos/química , Estados Unidos , United States Food and Drug AdministrationRESUMEN
The only current curative treatment for chronic lymphocytic leukemia (CLL) is allogenic hematopoietic stem cell transplantation. Chimeric antigen receptor treatment targeting CD19 for CLL achieved some complete responses, suggesting the need for alternative or combinational therapies to achieve a more robust response. In this work, we evaluated CAR-T cells specific for Siglec-6, an antigen expressed in CLL, as a novel CAR-T cell treatment for CLL. We found that detection of SIGLEC6 mRNA and Siglec-6 protein is highly restricted to placenta and immune cells in other tissues and it is not expressed in hematopoietic stem cells. We generated CAR-T cells specific for Siglec-6 based on the sequence of the fully human anti-Siglec-6 antibody (JML1), which was identified in a CLL patient that was cured after allo-hematopoietic stem cell transplantation (alloHSCT), and observed that it specifically targeted CLL cells in vitro and in a xenograft mouse model. Interestingly, a short hinge region increased the activity of CAR-T cells to target cells expressing higher Siglec-6 levels but similarly targeted CLL cells expressing lower Siglec-6 levels in vitro and in vivo. Our results identify a novel CAR-T cell therapy for CLL and establish Siglec-6 as a possible target for immunotherapy.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Inmunoterapia Adoptiva/métodos , Lectinas/antagonistas & inhibidores , Leucemia Linfocítica Crónica de Células B/terapia , Receptores Quiméricos de Antígenos/inmunología , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Proliferación Celular , Terapia Combinada , Humanos , Lectinas/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The presently used approaches to silence autoreactive disease-associated B cells act indiscriminately and more specific therapies are obviously needed. In the present study, we analyze the ability of a chimeric antibody to suppress selectively pathological autoreactive B-lymphocytes in lupus-prone mice by cross-linking their surface Ig receptors with the inhibitory IgG Fc gamma RIIb receptors. The chimera was constructed by coupling an immunodominant mouse Histone 1 peptide to a rat monoclonal anti-mouse CD32 (Fc gamma RIIb) antibody. The administration of these chimeric molecules to MRL/lpr mice with initial and with full-blown disease resulted in the reduction of the levels of IgG anti-Histone 1 antibodies, of the albuminuria levels, of the size of lymphoid organs and in prevention of the development of skin lesions. The observed effect was limited to lupus-associated B cells only, as the treatment did not decrease the IgG antibody response to an administered foreign antigen. This study demonstrates the possibility to silence selectively autoreactive B cells and to delay the progression of an autoimmune disease using chimeric antibody molecules.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Autoanticuerpos/sangre , Linfocitos B/efectos de los fármacos , Histonas/uso terapéutico , Lupus Eritematoso Sistémico/terapia , Péptidos/uso terapéutico , Receptores de IgG/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos CD/metabolismo , Femenino , Histonas/química , Histonas/genética , Histonas/inmunología , Histonas/metabolismo , Lupus Eritematoso Sistémico/inmunología , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Ratas , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusión/inmunología , Resultado del TratamientoRESUMEN
There is an urgent and unmet need for therapeutic agents targeting selectively disease-associated B-lymphocytes in autoantibody-mediated diseases. We have constructed a chimeric molecule able to cross-link cell surface immunoglobulin with the inhibitory complement receptor type 1 (CD35) on DNA-specific B cells from SLE (systemic lupus erythematosus) patients with the aim of selectively silencing them. This engineered molecule is made of copies of the DNA-mimotope peptide DWEYSVWLSN coupled to a monoclonal anti-CD35 antibody. We found that the DNA-like peptide chimera induced a dose-dependent decrease in the number of IgG anti-dsDNA antibody producing cells when PBMCs of lupus patients were cultured in its presence. Our data present evidence that clustering BCR and the inhibitory CR1 on disease-associated autoreactive B-lymphocytes selectively suppresses autoantibody production.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , ADN/química , Lupus Eritematoso Sistémico/inmunología , Péptidos/química , Receptores de Complemento 3b/inmunología , Adulto , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antinucleares/efectos de los fármacos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Autoinmunidad/efectos de los fármacos , Estudios de Casos y Controles , Células Cultivadas , Epítopos , Femenino , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/efectos de los fármacos , Persona de Mediana Edad , Imitación Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
CD40/CD40L interactions play a critical role in immunity and autoimmunity. In this study, we sought to understand the requirement for CD40 signaling in the programmed cell death-1 (PD-1) checkpoint and CD28 costimulatory pathways important for maintenance of peripheral tolerance. Blocking either pathway can result in loss of self-tolerance and development of autoimmunity. We found that primary Sjögren's syndrome (pSS) and autoimmune thyroid diseases (ATDs) that develop spontaneously in CD28-deficient IFN-γ-/- NOD.H-2h4 (CD28-/-) mice required CD40 signaling. Specifically, blockade of CD40L with the anti-CD40L mAb, MR1, inhibited autoantibody production and inflammation in thyroid and salivary gland target tissues. Unexpectedly, however, ATD and pSS in PD-1-deficient IFN-γ-/- NOD.H-2h4 (PD-1-/-) mice developed independently of CD40/CD40L interactions. Treatment with MR1 had no effect and even exacerbated disease development in pSS and ATD, respectively. Most interesting, anti-thyroglobulin and pSS-associated autoantibodies were increased following anti-CD40L treatment, even though MR1 effectively inhibited the spontaneous splenic germinal centers that form in PD-1-deficient mice. Importantly, blockade of the PD-1 pathway by administration of anti-PD-1 mAb in CD28-/- mice recapitulated the PD-1-/- phenotype, significantly impacting the ability of MR1 to suppress ATD and pSS in these mice. These results indicate that there can be different pathways and requirements to autoimmune pathogenesis depending on the availability of specific checkpoint and costimulatory receptors, and an intact PD-1 pathway is apparently required for inhibition of autoimmunity by anti-CD40L.
RESUMEN
Uncontrolled secretion of type I IFN, as the result of endosomal TLR (i.e., TLR7 and TLR9) signaling in plasmacytoid DCs (pDCs), and abnormal production of autoantibodies by B cells are critical for systemic lupus erythematosus (SLE) pathogenesis. The importance of galectin-9 (Gal-9) in regulating various autoimmune diseases, including lupus, has been demonstrated. However, the precise mechanism by which Gal-9 mediates this effect remains unclear. Here, using spontaneous murine models of lupus (i.e., BXSB/MpJ and NZB/W F1 mice), we demonstrate that administration of Gal-9 results in reduced TLR7-mediated autoimmune manifestations. While investigating the mechanism underlying this phenomenon, we observed that Gal-9 inhibits the phenotypic maturation of pDCs and B cells and abrogates their ability to mount cytokine responses to TLR7/TLR9 ligands. Importantly, immunocomplex-mediated (IC-mediated) and neutrophil extracellular trap-mediated (NET-mediated) pDC activation was inhibited by Gal-9. Additionally, the mTOR/p70S6K pathway, which is recruited by both pDCs and B cells for TLR-mediated IFN secretion and autoantibody generation, respectively, was attenuated. Gal-9 was found to exert its inhibitory effect on both the cells by interacting with CD44.
Asunto(s)
Autoanticuerpos/inmunología , Linfocitos B/inmunología , Células Dendríticas/inmunología , Galectinas/inmunología , Lupus Eritematoso Sistémico/inmunología , Glicoproteínas de Membrana/inmunología , Receptor Toll-Like 7/inmunología , Animales , Linfocitos B/patología , Células Dendríticas/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Lupus Eritematoso Sistémico/patología , Masculino , Ratones , Proteínas Quinasas S6 Ribosómicas 70-kDa/inmunología , Transducción de Señal/inmunología , Serina-Treonina Quinasas TOR/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
B cells represent an important link between the adaptive and innate immune systems as they express both antigen-specific B-cell receptors (BCRs) as well as various Toll-like receptors (TLRs). Several checkpoints in B-cell development ensure that self-specific cells are eliminated from the mature B-cell repertoire to avoid harmful autoreactive responses. These checkpoints are controlled by BCR-mediated events but are also influenced by TLR-dependent signals from the innate immune system. Additionally, B-cell-intrinsic and extrinsic TLR signaling are critical for inflammatory events required for the clearance of microbial infections. Factors secreted by TLR-activated macrophages or dendritic cells directly influence the fate of protective and autoreactive B cells. Additionally, naive and memory B cells respond differentially to TLR ligands, as do different B-cell subsets. We review here recent literature describing intrinsic and extrinsic effects of TLR stimulation on the fate of B cells, with particular attention to autoimmune diseases.
Asunto(s)
Linfocitos B/inmunología , Tolerancia Inmunológica/fisiología , Inmunidad Innata/inmunología , Activación de Linfocitos/fisiología , Animales , Autoanticuerpos/metabolismo , Linfocitos B/metabolismo , Humanos , Tolerancia Inmunológica/inmunología , Ratones , Modelos Animales , Modelos Biológicos , Transducción de Señal/inmunologíaRESUMEN
A novel approach for the selective silencing of targeted autoreactive B lymphocytes is reviewed that mimics the physiological mechanisms for suppressing B cell activity. It is based on the use of bi- or tri-specific chimeric antibodies that cross-link BCRs with a pre-selected antigen-binding specificity with one or more inhibitory types of receptors on the surface of the same disease-associated B lymphocyte. The effect of these engineered antibodies was proved to be specific as they only suppressed the production of the targeted pathological antibodies while sparing those with other specificities. The administration of the chimeric molecules to lupus-prone MRL/lpr mice resulted in decreased levels of disease-associated IgG autoantibodies and of proteinuria, in the prevention of cutaneous lesions, in decreased sizes of the lymphoid organs and in prolonged survival. These results prove that it is indeed possible to selectively silence unwanted B lymphocytes as well as to significantly delay the natural course of a spontaneous antibody-mediated autoimmune disease.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Enfermedades Autoinmunes/terapia , Autoinmunidad , Linfocitos B/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Autoanticuerpos , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Linfocitos B/patología , Inmunoglobulina G/inmunología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/terapia , Ratones , Ratones Endogámicos MRL lpr , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de IgG/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunologíaRESUMEN
IgG molecules are exposed on a regular basis to acidic conditions during immunoaffinity purification procedures, as well as during the production of some therapeutic immunoglobulin preparations. This exposure is known to induce in them an antigen-binding polyreactivity. The molecular mechanisms and the possible biological significance of this phenomenon remain, however, poorly understood. In addition to the previously reported ability of these modified IgG antibodies to interact with a large panel of self-antigens, enhanced binding to non-self-antigens (bacterial), an increased ability to engage in F(ab')(2)/F(ab')(2) (idiotype/anti-idiotype) interactions and an increased functional antigen-binding affinity are reported here. The newly acquired 'induced polyreactivity' of low-pH buffer-exposed IgG is related to structural changes in the immunoglobulin molecules, and is at least partly attributable to the enhanced role of the hydrophobic effect in their interactions with antigen. Our results suggest that data from many previous studies on monoclonal and polyclonal IgG antibodies purified by low-pH buffer elution from protein A or protein G immunoaffinity columns should be reconsidered, as the procedure itself may have dramatically affected their antigen-binding behavior and biological activity. Low-pH buffer-treated pooled therapeutic immunoglobulins acquire novel beneficial properties, as passive immunotherapy with the pH 4.0 buffer-exposed, but not with the native therapeutic intravenous immunoglobulin preparation, improves the survival of mice with bacterial lipopolysaccharide-induced septic shock.
Asunto(s)
Inmunoglobulinas Intravenosas/química , Inmunoglobulinas Intravenosas/uso terapéutico , Sepsis/terapia , Naftalenosulfonatos de Anilina/química , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Antígenos Bacterianos/inmunología , Autoantígenos/inmunología , Western Blotting , Tampones (Química) , Proteína C-Reactiva/inmunología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/inmunología , Colorantes Fluorescentes/química , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulinas Intravenosas/inmunología , Interferón gamma/inmunología , Cinética , Ratones , Ratones Endogámicos ICR , Microscopía Fluorescente , Conformación Proteica , Proteínas Recombinantes , Análisis de SupervivenciaRESUMEN
DNA-specific B cells in SLE represent a logical target for therapeutic intervention. We hypothesize that it is possible to re-establish tolerance to native DNA in SCID mice with cells transferred from SLE patients or from lupus-prone MRL/lpr mice by administering chimeric molecules, containing a monoclonal antibody against inhibitory B cell receptors coupled to a peptide that antigenically mimics DNA. These protein-engineered molecules are able to co-crosslink selectively the antigen receptors of B cells possessing anti-native DNA specificity with the inhibitory surface receptors, thus delivering a strong suppressive signal.
Asunto(s)
Anticuerpos Antinucleares/genética , Anticuerpos Biespecíficos/uso terapéutico , Inmunoterapia , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/terapia , Animales , Anticuerpos Antinucleares/inmunología , Anticuerpos Biespecíficos/inmunología , Linfocitos B/inmunología , Biomimética , ADN/inmunología , Modelos Animales de Enfermedad , Retroalimentación Fisiológica , Humanos , Tolerancia Inmunológica , Inmunoterapia/tendencias , Ratones , Ingeniería de ProteínasRESUMEN
All B cell targeting therapeutic approaches used at present are unspecific and there is an urgent need for agents that silence selectively pathological autoreactive B lymphocytes only. We hypothesized that this aim could be achieved by chimeric antibodies that cross-link B cell immunoglobulin receptors with inhibitory receptors on the surface of the same targeted disease-associated cell. A hybrid molecule was constructed by coupling copies of the DNA-mimicking DWEYSVWLSN peptide and of the CD22-binding STN epitope with a free terminal sialic acid to a mouse monoclonal IgG antibody backbone. The DNA mimotope peptide binds to the immunoglobulin B cell receptor of pathological DNA-specific B cells of lupus mice, the STN epitope - to CD22 and the IgG by its Fc fragment - to FcgammaIIb on the surface of the same cell. Mass-spectra analysis showed that 4 STN epitopes plus 5 DNA mimotope peptides were coupled to a single light immunoglobulin chain and 4 STN - and 2 DNA mimotopes - to a heavy chain. Both FcgammaIIb and CD22 receptors on spleen cells from lupus MRL/lpr mice were phosphorylated after exposure to the chimeric antibody, indicating the involvement of both inhibitory pathways. The constructed chimera suppressed specifically in vitro as well as in vivo anti-DNA IgM and IgG antibody production and delayed the development of glomerulonephritis in the lupus-prone animals. The use of chimeric antibodies targeting two independent inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of pathological autoreactive B cells in autoimmune diseases.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Enfermedades Autoinmunes/tratamiento farmacológico , Linfocitos B/inmunología , Inmunidad Humoral/efectos de los fármacos , Receptores de IgG/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Anticuerpos Biespecíficos/uso terapéutico , Enfermedades Autoinmunes/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , ADN/inmunología , Epítopos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Inmunoterapia/métodos , Ratones , Ingeniería de Proteínas/métodos , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusión , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismoRESUMEN
The pathological DNA-specific B lymphocytes in lupus are logical targets for a selected therapeutic intervention. We have hypothesized that it should be possible to suppress selectively the activity of these B cells in lupus mice by administering to them an artificial molecule that cross-links their surface immunoglobulins with the inhibitory FcgammaIIb surface receptors. A hybrid molecule was constructed by coupling the DNA-mimicking DWEYSVWLSN peptide to a monoclonal anti-mouse FcgammaRIIb antibody. This chimeric antibody was added to cultured spleen cells from sick MRL/lpr mice, immunized with diphtheria toxoid, resulting in reduction of the numbers of anti-DNA but not of anti-diphtheria IgG antibody-producing cells. Intravenous infusions with the DNA-peptide antibody chimera to 7-wk-old animals prevented the appearance of IgG anti-DNA antibodies and of albuminuria in the next 2 months. The administration of the DNA-peptide chimeric antibody to 18 wk-old mice with full-blown disease resulted in the maintenance of a flat level of IgG anti-DNA antibodies and in delay of the aggravation of the lupus glomerulonephritis. The use of chimeric antibodies targeting inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of autoreactive disease-associated B cells in autoimmune diseases.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Subgrupos de Linfocitos B/efectos de los fármacos , Reactivos de Enlaces Cruzados/uso terapéutico , Inmunoconjugados/uso terapéutico , Inmunoglobulina G/biosíntesis , Inmunosupresores/uso terapéutico , Lupus Eritematoso Sistémico/terapia , Oligopéptidos/uso terapéutico , Receptores de Antígenos de Linfocitos B/efectos de los fármacos , Receptores de IgG/efectos de los fármacos , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antinucleares/inmunología , Especificidad de Anticuerpos , Apoptosis/efectos de los fármacos , Subgrupos de Linfocitos B/inmunología , Células Cultivadas/inmunología , Reactivos de Enlaces Cruzados/farmacología , ADN/inmunología , Toxoide Diftérico/inmunología , Modelos Animales de Enfermedad , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/prevención & control , Ratones , Ratones Endogámicos MRL lpr , Imitación Molecular , Oligopéptidos/administración & dosificación , Oligopéptidos/síntesis química , Oligopéptidos/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de IgG/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Killed viral vaccines are known to induce primarily antibody responses. By contrast DNA vaccination using naked DNA encoding viral antigens induces both humoral and cellular immune responses. Various approaches have been used to construct DNA vaccines with build-in adjuvanticity. We hypothesized that sequences encoding a common epitope of influenza A virus hemagglutinin jointed to sequences encoding a single-chain variable fragment (scFv) antibody fragment to a costimulatory B cell surface receptor would result in the in vivo expression of a chimeric viral peptide with increased immunogenicity. Such a hybrid DNA molecule was constructed by us, encoding a T and B cell epitope-containing influenza hemagglutinin peptide and a scFv antibody fragment binding to mouse complement receptors I and II (CR1 and CR2). A single immunization with a plasmid containing the described construct induced a strong anti-influenza cytotoxic response lasting for more than six months and a weak antibody response.