RESUMEN
The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.
Asunto(s)
Células Dendríticas/fisiología , Endosomas/metabolismo , Exonucleasas/metabolismo , Hepatitis/genética , Macrófagos/fisiología , Glicoproteínas de Membrana/metabolismo , Fosfolipasa D/metabolismo , Enfermedad de Alzheimer/genética , Animales , Artritis Reumatoide/genética , ADN de Cadena Simple/inmunología , Exonucleasas/genética , Estudio de Asociación del Genoma Completo , Humanos , Interferón gamma/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolipasa D/genética , Esclerodermia Sistémica/genética , Transducción de Señal , Receptor Toll-Like 9/metabolismoRESUMEN
Soluble thrombomodulin plasma concentrations are elevated in steroid-resistant graft-versus-host disease (GVHD), implying endothelial hypofunctioning for thrombomodulin-dependent generation of activated protein C's (APC) anticoagulant, anti-inflammatory, and antiapoptotic functions. Recombinant thrombomodulin or APC administration decreases acute GVHD, manifested by intense inflammation and tissue destruction. Here, we administered recombinant murine wild-type (WT) APC to mice with established chronic GVHD (cGVHD), a less-inflammatory autoimmune-like disease. WT APC normalized bronchiolitis obliterans-induced pulmonary dysfunction. Signaling-selective APC variants (3A-APC [APC with lysine 191-193 replaced with 3 alanines] or 5A-APC [APC with lysine 191-193 replaced with 3 alanines and arginine 229/230 replaced with 2 alanines]) with normal cytoprotective properties, but greatly reduced anticoagulant activity, provided similar results. Mechanistically, WT APC and signaling-selective variants reduced T follicular helper cells, germinal center formation, immunoglobulin, and collagen deposition. WT APC can potentially cleave protease-activated receptor 1 (PAR1) at Arg41 or Arg46, the latter causing anti-inflammatory signaling. cGVHD was reduced in recipients of T cells from WT PAR1 or mutated Gln41-PAR1 donors but not from mutated Gln46-PAR1 donors. These data implicate donor T-cell APC-induced noncanonical cleavage at Arg46-PAR1, which is known to confer cytoprotective and anti-inflammatory activities. Together, these data indicate that APC anticoagulant activity is dispensable, whereas anti-inflammatory signaling and cytoprotective cell signaling by APC are essential. Because a phase 2 ischemic stroke clinical trial did not raise any safety issues for 3A-APC treatment, our studies provide a foundational platform for testing in clinical cGVHD therapy.
Asunto(s)
Enfermedad Injerto contra Huésped/tratamiento farmacológico , Proteína C/uso terapéutico , Receptor PAR-1/metabolismo , Linfocitos T/efectos de los fármacos , Animales , Enfermedad Crónica , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/patología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Proteínas Recombinantes/uso terapéutico , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2-/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2-/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2-/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation.
Asunto(s)
Activación de Linfocitos , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Linfocitos T/inmunología , Linfocitos T/fisiología , Inmunidad Adaptativa , Animales , Muerte Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Genes p53/genética , Genes p53/inmunología , Enfermedad Injerto contra Huésped/inmunología , Inmunidad Innata , Isoantígenos/inmunología , Ratones , Proteína Adaptadora de Señalización NOD1/deficiencia , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/deficiencia , Proteína Adaptadora de Señalización NOD2/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/inmunología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de SeñalRESUMEN
Nucleotide-binding and oligomerization domain (NOD)-like receptors NOD1 and NOD2 are cytosolic innate immune receptors that recognize microbial peptidoglycans. Although studies have addressed the role of NOD proteins in innate immune responses, little attention has been given to their impact on the developing adaptive immune system. We have assessed the roles of NOD1 and NOD2 deficiency on T cell development in mice. Our results demonstrate that NOD1 and NOD2 promote the positive selection/maturation of CD8 single-positive thymocytes in a thymocyte-intrinsic manner. TCR-mediated ERK phosphorylation is significantly reduced in the absence of NOD proteins, but receptor-interacting protein 2 is not involved in CD8 single-positive thymocyte selection or ERK signaling. Commensal bacteria-free animals have thymocyte maturation defects, and exogenous NOD ligands can enhance thymocyte maturation in culture. These results raise the intriguing possibility that abnormal lymphocyte responses observed in NOD-dependent inflammatory diseases are not driven solely by microbial signals in the gut, but may also involve intrinsic lymphocyte defects resulting from impaired CD8 T cell thymic development.
Asunto(s)
Linfocitos T CD8-positivos/citología , Diferenciación Celular/inmunología , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Adaptadora de Señalización NOD2/inmunología , Timocitos/citología , Animales , Linfocitos T CD8-positivos/inmunología , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Timocitos/inmunología , Timo/citología , Timo/inmunologíaRESUMEN
B cell activation leads to proliferation and Ab production that can protect from pathogens or promote autoimmunity. Regulation of cell metabolism is essential to support the demands of lymphocyte growth and effector function and may regulate tolerance. In this study, we tested the regulation and role of glucose uptake and metabolism in the proliferation and Ab production of control, anergic, and autoimmune-prone B cells. Control B cells had a balanced increase in lactate production and oxygen consumption following activation, with proportionally increased glucose transporter Glut1 expression and mitochondrial mass upon either LPS or BCR stimulation. This contrasted with metabolic reprogramming of T cells, which had lower glycolytic flux when resting but disproportionately increased this pathway upon activation. Importantly, tolerance greatly affected B cell metabolic reprogramming. Anergic B cells remained metabolically quiescent, with only a modest increase in glycolysis and oxygen consumption with LPS stimulation. B cells chronically stimulated with elevated BAFF, however, rapidly increased glycolysis and Ab production upon stimulation. Induction of glycolysis was critical for Ab production, as glycolytic inhibition with the pyruvate dehydrogenase kinase inhibitor dichloroacetate sharply suppressed B cell proliferation and Ab secretion in vitro and in vivo. Furthermore, B cell-specific deletion of Glut1 led to reduced B cell numbers and impaired Ab production in vivo. Together, these data show that activated B cells require Glut1-dependent metabolic reprogramming to support proliferation and Ab production that is distinct from T cells and that this glycolytic reprogramming is regulated in tolerance.
Asunto(s)
Formación de Anticuerpos , Factor Activador de Células B/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Anergia Clonal/inmunología , Animales , Factor Activador de Células B/genética , Ácido Dicloroacético/farmacología , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Glucólisis/efectos de los fármacos , Homeostasis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Fcγ receptors (FcγRs) play key roles in the effector function of IgG, but their inappropriate activation plays a role in several disease etiologies. Therefore, it is critical to better understand how FcγRs are regulated. Numerous studies suggest that sialic acid-binding immunoglobulin-type lectins (Siglecs), a family of immunomodulatory receptors, modulate FcγR activity; however, it is unclear of the circumstances in which Siglecs can antagonize FcγRs and which Siglecs have this ability. Using liposomes displaying selective ligands to coengage FcγRs with a specific Siglec, we explore the ability of Siglec-3, Siglec-5, Siglec-7, and Siglec-9 to antagonize signaling downstream of FcγRs. We demonstrate that Siglec-3 and Siglec-9 can fully inhibit FcγR activation in U937 cells when coengaged with FcγRs. Cells expressing Siglec mutants reveal differential roles for the immunomodulatory tyrosine-based inhibitory motif (ITIM) and immunomodulatory tyrosine-based switch motif (ITSM) in this inhibition. Imaging flow cytometry enabled visualization of SHP-1 recruitment to Siglec-3 in an ITIM-dependent manner, while SHP-2 recruitment is more ITSM-dependent. Conversely, both cytosolic motifs of Siglec-9 contribute to SHP-1/2 recruitment. Siglec-7 poorly antagonizes FcγR activation for two reasons: masking by cis ligands and differences in its ITIM and ITSM. A chimera of the Siglec-3 extracellular domains and Siglec-5 cytosolic tail strongly inhibits FcγR when coengaged, providing evidence that Siglec-5 is more like Siglec-3 and Siglec-9 in its ability to antagonize FcγRs. Additionally, Siglec-3 and Siglec-9 inhibited FcγRs when coengaged by cells displaying ligands for both the Siglec and FcγRs. These results suggest a role for Siglecs in mediating FcγR inhibition in the context of an immunological synapse, which has important relevance to the effectiveness of immunotherapies.
RESUMEN
Phospholipase D4 (PLD4) is an endolysosomal exonuclease of ssRNA and ssDNA, rather than a phospholipase as its name suggests. Human polymorphisms in the PLD4 gene have been linked by genome-wide association studies to systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. However, B6.129 Pld4-/- mice develop features of a distinct disease, macrophage activation syndrome, which is reversed in mice mutated in TLR9. In this article, we compare a Pld4 null mutant identified on the BALB/c background, Pld4thss/thss, which has distinct phenotypes: short stature, thin hair, and features of systemic lupus erythematosus. All phenotypes analyzed were largely normalized in Pld4thss/thssTlr9-/- mice. Thus, Pld4thss/thss represents a rare model in which mouse lupus etiology is TLR9 dependent. Compared with PLD4-deficient B6 mice, Pld4thss/thss mice had elevated levels of serum IgG, IgG anti-dsDNA autoantibodies, BAFF, and IFN-γ and elevated B cell numbers. Overall, the data suggest that PLD4 deficiency can lead to a diverse array of rheumatological abnormalities depending upon background-modifying genes, and that these diseases of PLD4 deficiency are largely driven by TLR9 recognition of ssDNA.
Asunto(s)
Lupus Eritematoso Sistémico , Receptor Toll-Like 9 , Animales , Humanos , Ratones , Exonucleasas/genética , Estudio de Asociación del Genoma Completo , Inmunoglobulina G/genética , Lupus Eritematoso Sistémico/genética , Fosfolipasas , Receptor Toll-Like 9/genéticaRESUMEN
The TNF-family cytokine BAFF (BLyS) promotes B lymphocyte survival and is overexpressed in individuals with systemic lupus erythematosus and Sjögren's Syndrome. BAFF can rescue anergic autoreactive B cells from death, but only when competition from nonautoreactive B cells is lacking. Yet, high BAFF levels promote autoantibody formation in individuals possessing diverse B cells. To better understand how excess BAFF promotes autoimmunity in a polyclonal immune system, Ig L chain usage was analyzed in 3H9 site-directed IgH chain transgenic mice, whose B cells recognize DNA and chromatin when they express certain endogenous L chains. BAFF levels were manipulated in 3H9 mice by introducing transgenes expressing either BAFF or its natural inhibitor ΔBAFF. B cells in BAFF/3H9 mice were elevated in number, used a broad L chain repertoire, including L chains generating high-affinity autoreactivity, and produced abundant autoantibodies. Comparison of spleen and lymph node B cells suggested that highly autoreactive B cells were expanded. By contrast, ΔBAFF/3H9 mice had reduced B cell numbers with a repertoire similar to that of 3H9 mice, but lacking usage of a subset of Vκ genes. The results show that limiting BAFF signaling only slightly selects against higher affinity autoreactive B cells, whereas its overexpression leads to broad tolerance escape and positive selection of autoreactive cells. The results have positive implications for the clinical use of BAFF-depleting therapy.
Asunto(s)
Autoinmunidad/inmunología , Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Autotolerancia/inmunología , Animales , Autoanticuerpos/inmunología , Linfocitos B/citología , Linfocitos B/metabolismo , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
Transitional B cells turn over rapidly in vivo and are sensitive to apoptosis upon BCR ligation in vitro. However, little direct evidence addresses their tolerance sensitivity in vivo. A key marker used to distinguish these cells is IgD, which, through alternative RNA splicing of H chain transcripts, begins to be coexpressed with IgM at this stage. IgD is also expressed at high levels on naive follicular (B-2) and at lower levels on marginal zone and B-1 B cells. In this study, mice were generated to ubiquitously express a membrane-bound IgD-superantigen. These mice supported virtually no B-2 development, a greatly reduced marginal zone B cell population, but a relatively normal B-1 compartment. B cell development in the spleen abruptly halted at the transitional B cell population 1 to 2 stage, a block that could not be rescued by either Bcl-2 or BAFF overexpression. The developmentally arrested B cells appeared less mature and turned over more rapidly than nontransgenic T2 cells, exhibiting neither conventional features of anergy nor appreciable receptor editing. Paradoxically, type-2 T-independent responses were more robust in the transgenic mice, although T-dependent responses were reduced and had skewed IgL and IgH isotype usages. Nevertheless, an augmented memory response to secondary challenge was evident. The transgenic mice also had increased serum IgM, but diminished IgG, levels mirrored by the increased numbers of IgM(+) plasma cells. This model should facilitate studies of peripheral B cell tolerance, with the advantages of allowing analysis of polyclonal populations, and of B cells naturally lacking IgD.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Tolerancia Inmunológica/genética , Inmunoglobulina D/genética , Superantígenos/biosíntesis , Superantígenos/genética , Animales , Autoantígenos/biosíntesis , Autoantígenos/genética , Autoantígenos/metabolismo , Subgrupos de Linfocitos B/citología , Trasplante de Médula Ósea/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Humanos , Regiones Constantes de Inmunoglobulina/metabolismo , Inmunoglobulina D/biosíntesis , Inmunoglobulina D/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/metabolismo , Bazo/citología , Bazo/inmunología , Bazo/trasplante , Superantígenos/metabolismo , Transgenes/inmunologíaRESUMEN
During a T cell-dependent Ab response, B cells undergo Ab class switching and V region hypermutation, with the latter process potentially rendering previously innocuous B cells autoreactive. Class switching and hypermutation are temporally and anatomically linked with both processes dependent on the enzyme, activation-induced deaminase, and occurring principally, but not exclusively, in germinal centers. To understand tolerance regulation at this stage, we generated a new transgenic mouse model expressing a membrane-tethered gamma2a-reactive superantigen (gamma2a-macroself Ag) and assessed the fate of emerging IgG2a-expressing B cells that have, following class switch, acquired self-reactivity of the Ag receptor to the macroself-Ag. In normal mice, self-reactive IgG2a-switched B cells were deleted, leading to the selective absence of IgG2a memory responses. These findings identify a novel negative selection mechanism for deleting mature B cells that acquire reactivity to self-Ag. This process was only partly dependent on the Bcl-2 pathway, but markedly inefficient in MRL-Fas(lpr) lupus mice, suggesting that defective apoptosis of isotype-switched autoreactive B cells is central to Fas mutation-associated systemic autoimmunity.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina G/inmunología , Receptor fas/inmunología , Traslado Adoptivo , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Línea Celular , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Endogámicos , Ratones Transgénicos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Superantígenos/genética , Superantígenos/inmunología , Superantígenos/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
Phospholipase D3 (PLD3) and PLD4 polymorphisms have been associated with several important inflammatory diseases. Here, we show that PLD3 and PLD4 digest ssRNA in addition to ssDNA as reported previously. Moreover, Pld3-/-Pld4-/- mice accumulate small ssRNAs and develop spontaneous fatal hemophagocytic lymphohistiocytosis (HLH) characterized by inflammatory liver damage and overproduction of Interferon (IFN)-γ. Pathology is rescued in Unc93b13d/3dPld3-/-Pld4-/- mice, which lack all endosomal TLR signaling; genetic codeficiency or antibody blockade of TLR9 or TLR7 ameliorates disease less effectively, suggesting that both RNA and DNA sensing by TLRs contributes to inflammation. IFN-γ made a minor contribution to pathology. Elevated type I IFN and some other remaining perturbations in Unc93b13d/3dPld3-/-Pld4-/- mice requires STING (Tmem173). Our results show that PLD3 and PLD4 regulate both endosomal TLR and cytoplasmic/STING nucleic acid sensing pathways and have implications for the treatment of nucleic acid-driven inflammatory disease.
Asunto(s)
ADN/metabolismo , Exonucleasas/genética , Exonucleasas/metabolismo , Inflamación/metabolismo , Fosfolipasa D/genética , Fosfolipasa D/metabolismo , ARN/metabolismo , Animales , Células Dendríticas , Endosomas/metabolismo , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Receptores Toll-Like , TranscriptomaRESUMEN
It is not fully understood how the expression level of autoantigens in beta cells impacts autoimmune diabetes (T1D) development. Earlier studies using ovalbumin and also insulin had shown that secreted antigens could enhance diabetes development through facilitated presentation by antigen presenting cells. Here we sought to determine how the expression level of a membrane bound, non-secreted or cross-presented neo-antigen, the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), would influence T1D. We found that an RIP-LCMV transgenic mouse line exhibiting higher levels of beta cell GP expression developed more severe diabetes after LCMV infection or transfer of high numbers of activated autoreactive T cells. Importantly, all beta cells were lost and a significant increase in morbidity and mortality from T1D was noted. Insulitis and accumulation of autoaggressive CD8 cells was more profound in the RIP-LCMV-GP high-expressor line. Interestingly, the additional introduction of neo-antigen-specific CD4(+) helper or regulatory T cells was able to influence diabetogenesis positively or negatively. We conclude that a higher degree of autoantigen expression results in increased diabetes susceptibility. Therefore, autoantigens such as insulin that are expressed at higher levels in beta cells might have a more profound impact on diabetes pathogenesis.
Asunto(s)
Infecciones por Arenaviridae/inmunología , Linfocitos T CD8-positivos/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/metabolismo , Virus de la Coriomeningitis Linfocítica/fisiología , Traslado Adoptivo , Animales , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Infecciones por Arenaviridae/genética , Infecciones por Arenaviridae/fisiopatología , Autoantígenos/biosíntesis , Autoantígenos/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/trasplante , Células Cultivadas , Reactividad Cruzada/genética , Citotoxicidad Inmunológica/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/fisiopatología , Progresión de la Enfermedad , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Células Secretoras de Insulina/patología , Activación de Linfocitos/genética , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Little is know about the nature of peripheral B cell tolerance or how it may vary in distinct lineages. Although autoantibody transgenic studies indicate that anergy and apoptosis are involved, some studies claim that receptor editing occurs. To model peripheral B cell tolerance in a normal, polyclonal immune system, we generated transgenic mice expressing an Igκ-light chain-reactive superantigen targeted to the plasma membrane of hepatocytes (pAlb mice). In contrast to mice expressing κ superantigen ubiquitously, in which κ cells edit efficiently to λ, in pAlb mice, κ B cells underwent clonal deletion. Their κ cells failed to populate lymph nodes, and the remaining splenic κ cells were anergic, arrested at a semi-mature stage without undergoing receptor editing. In the liver, κ cells recognized superantigen, down-regulated surface Ig, and expressed active caspase 3, suggesting ongoing apoptosis at the site of B cell receptor ligand expression. Some, apparently mature, κ B1 and follicular B cells persisted in the peritoneum. BAFF (B cell-activating factor belonging to the tumor necrosis factor family) overexpression rescued splenic κ B cell maturation and allowed κ cells to populate lymph nodes. Our model facilitates analysis of tissue-specific autoimmunity, tolerance, and apoptosis in a polyclonal B cell population. The results suggest that deletion, not editing, is the major irreversible pathway of tolerance induction among peripheral B cells.
Asunto(s)
Linfocitos B/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Supresión Clonal/inmunología , Hígado/inmunología , Porinas/inmunología , Receptores Opioides kappa/inmunología , Receptores Virales/inmunología , Superantígenos/inmunología , Animales , Apoptosis/inmunología , Autoinmunidad/inmunología , Hepatocitos/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos/inmunologíaRESUMEN
Protein kinase C η (PKCη) is abundant in T cells and is recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell; however, its function in T cells is unknown. We showed that PKCη was required for the activation of mature CD8+ T cells through the T cell receptor. Compared with wild-type T cells, PKCη-/- T cells showed poor proliferation in response to antigen stimulation, a trait shared with T cells deficient in PKCθ, which is the most abundant PKC isoform in T cells and was thought to be the only PKC isoform with a specific role in T cell activation. In contrast, only PKCη-deficient T cells showed defective homeostatic proliferation, which requires self-antigen recognition. PKCη was dispensable for thymocyte development; however, thymocytes from mice doubly deficient in PKCη and PKCθ exhibited poor development, indicating some redundancy between the PKC isoforms. Deficiency in PKCη or PKCθ had opposing effects on the relative numbers of CD4+ and CD8+ T cells. PKCη-/- mice had a higher ratio of CD4+ to CD8+ T cells compared to that of wild-type mice, whereas PKCθ-/- mice had a lower ratio. Mice deficient in both isoforms exhibited normal cell ratios. Together, these data suggest that PKCη shares some redundant roles with PKCθ in T cell biology and also performs nonredundant functions that are required for T cell homeostasis and activation.
Asunto(s)
Proteína Quinasa C/inmunología , Linfocitos T/enzimología , Linfocitos T/inmunología , Animales , Secuencia de Bases , Relación CD4-CD8 , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Señalización del Calcio , Proliferación Celular , Homeostasis , Memoria Inmunológica/fisiología , Sinapsis Inmunológicas/enzimología , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Fenotipo , Proteína Quinasa C/deficiencia , Proteína Quinasa C/genética , Proteína Quinasa C-theta , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/inmunología , Linfocitos T/citologíaRESUMEN
Members of the Fgd (faciogenital dysplasia) gene family encode a group of critical guanine nucleotide exchange factors (GEFs), which, by specifically activating Cdc42, control cytoskeleton-dependent membrane rearrangements. In its first characterization, we find that FGD2 is expressed in antigen-presenting cells, including B lymphocytes, macrophages, and dendritic cells. In the B lymphocyte lineage, FGD2 levels change with developmental stage. In both mature splenic B cells and immature bone marrow B cells, FGD2 expression is suppressed upon activation through the B cell antigen receptor. FGD2 has a complex intracellular localization, with concentrations found in membrane ruffles and early endosomes. Although endosomal localization of FGD2 is dependent on a conserved FYVE domain, its C-terminal pleckstrin homology domain mediates recruitment to membrane ruffles. FGD2 overexpression promotes the activation of Cdc42 and leads to elevated JNK1 activity in a Cdc42- but not Rac1-dependent fashion. These findings are consistent with a role of FGD2 in leukocyte signaling and vesicle trafficking in cells specialized to present antigen in the immune system.
Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Membrana Celular/metabolismo , Endosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Animales , Presentación de Antígeno , Proteínas Sanguíneas/química , Células COS , Chlorocebus aethiops , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Macrófagos/metabolismo , Ratones , Fosfoproteínas/química , Transducción de SeñalRESUMEN
Marginal zone (MZ) B cells resemble fetally derived B1 B cells in their innate-like rapid responses to bacterial pathogens, but the basis for this is unknown. We report that the MZ is enriched in "fetal-type" B cell receptors lacking N regions (N(-)). Mixed bone marrow (BM) chimeras, made with adult terminal deoxynucleotidyl transferase (TdT)(+/+) and TdT(-/-) donor cells, demonstrate preferential repertoire-based selection of N(-) B cells into the MZ. Reconstitution of irradiated mice with adult TdT(+/+) BM reveals that the MZ can replenish N(-) B cells in adult life via repertoire-based selection and suggest the possibility of a TdT-deficient precursor population in the adult BM. The mixed chimera data also suggest repertoire-based bifurcations into distinct BM and splenic maturation pathways, with mature "recirculating" BM B cells showing a very strong preference for N(+) complementarity-determining region (CDR) 3 compared with follicular B cells. Because the T1 and MZ compartments are both the most enriched for N(-) H-CDR3, we propose a novel direct T1-->MZ pathway and identify a potential T1-MZ precursor intermediate. We demonstrate progressive but discontinuous repertoire-based selection throughout B cell development supporting multiple branchpoints and pathways in B cell development. Multiple differentiation routes leading to MZ development may contribute to the reported functional heterogeneity of the MZ compartment.
Asunto(s)
Linfocitos B/metabolismo , Animales , Linfocitos B/citología , Células de la Médula Ósea/citología , Diferenciación Celular , Separación Celular , Quimera , Citometría de Flujo , Inmunoglobulinas/metabolismo , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Fenotipo , Bazo/metabolismoRESUMEN
In mice carrying a synthetic Igkappa-reactive superantigen ("kappa macroself antigen"), low level expression induced split peripheral B cell tolerance in the sIgkappa+ compartment, with striking reductions in follicular and marginal zone (MZ) B cells and the retention of significant numbers of sIgkappa+ B-1a but not B-1b cells in the peritoneum. Here, we characterize the transgenic line pKkappa with this split tolerance phenotype and assess the effects of B cell competition and the survival cytokine BAFF (B cell activating factor belonging to the TNF family) on peripheral tolerance. In pKkappa mice the surviving peritoneal and splenic kappa+ B cells were largely lost in mice carrying one copy of the human Ckappa exon in place of the mouse version, a maneuver that generates additional antigen non-reactive competitor B cells in this model. Furthermore, overexpression of BAFF suppressed kappa-macroself antigen-induced deletion and promoted production of both IgM,kappa and IgA,kappa antibodies in mice with normal Igkappa alleles but not in mice carrying one copy of the human Ckappa allele. These findings suggest that BAFF overexpression has minimal effects on the survival of autoreactive B cells in a polyclonal immune system and that B cell:B cell competition plays a potent role in suppressing the survival of B-1 and splenic B cells with excessive autoreactivity.
Asunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Comunicación Celular/inmunología , Tolerancia Inmunológica/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Proteínas de la Membrana/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Factor Activador de Células B , Supervivencia Celular , Citometría de Flujo , Humanos , Proteínas de la Membrana/inmunología , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Peripheral B cell tolerance differs from central tolerance in anatomic location, in the stage of B cell development, and in the diversity of Ag-responsive cells. B cells in secondary lymphoid organs are heterogeneous, including numerous subtypes such as B-1, marginal zone, transitional, and follicular B cells, which likely respond differently from one another to ligand encounter. We showed recently that central B cell tolerance mediated by receptor editing was induced in mice carrying high levels of a ubiquitously expressed kappa-macroself Ag, a synthetic superantigen reactive to Igkappa. In this study, we characterize a new transgenic line that has a distinctly lower expression pattern from those described previously; the B cell tolerance phenotype of these mice is characterized by the presence of significant numbers of immature kappa+ B cells in the spleen, the loss of mature follicular and marginal zone B cells, the persistence of kappa+ B-1 cells in the peritoneal cavity, and significant levels of serum IgM,kappa. These findings suggest distinct signaling thresholds for tolerance among peripheral B cell subsets reactive with an identical ligand.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Tolerancia Inmunológica , Cadenas kappa de Inmunoglobulina/metabolismo , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciación Celular , Supresión Clonal , Genes de Inmunoglobulinas , Ligandos , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Quimera por Radiación/genética , Quimera por Radiación/inmunología , Transducción de Señal , Bazo/citología , Bazo/inmunologíaRESUMEN
Innate immune signals mediated by Toll-like receptors (TLRs) have been thought to contribute considerably to the antibody-enhancing effects of vaccine adjuvants. However, we report here that mice deficient in the critical signaling components for TLR mount robust antibody responses to T cell-dependent antigen given in four typical adjuvants: alum, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant. We conclude that TLR signaling does not account for the action of classical adjuvants and does not fully explain the action of a strong adjuvant containing a TLR ligand. This may have important implications in the use and development of vaccine adjuvants.
Asunto(s)
Adyuvantes Inmunológicos , Formación de Anticuerpos , Linfocitos B/inmunología , Transducción de Señal , Receptores Toll-Like/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Antígenos T-Independientes/inmunología , Esqueleto de la Pared Celular/inmunología , Factores Cordón/inmunología , Adyuvante de Freund/inmunología , Haptenos , Hemocianinas/inmunología , Inmunidad Innata , Inmunización , Inmunización Secundaria , Inmunoglobulinas/sangre , Ligandos , Lípido A/análogos & derivados , Lípido A/inmunología , Lipopolisacáridos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Picratos/inmunología , Linfocitos T/inmunología , Receptores Toll-Like/metabolismo , Vacunas/inmunologíaRESUMEN
Considerable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and DeltaBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and DeltaBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and DeltaBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and DeltaBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.