RESUMEN
OBJECTIVE: To discuss the advantages and technical limitations of various molecular genetic techniques in the diagnosis of two infants featuring all-round developmental retardation. METHODS: The two patients were initially screened by using chromosomal microarray analysis (CMA). For patient 1, his parents were also subjected to CMA analysis, and the data was analyzed by using ChAS and UPD-tool software. For patient 2, methylation-specific PCR (MS-PCR) was carried out. RESULTS: Patient 1 was diagnosed with maternal uniparental disomy (UPD) type Prader-Willi syndrome (PWS) by CMA and UPD-tool family analysis. His chromosomes 15 were of maternal UPD with homology/heterology. Patient 2 was diagnosed with deletion type PWS by combined CMA and MS-PCR. CONCLUSION: Correct selection of laboratory methods based on the advantages and limitations of various molecular techniques can help with diagnosis of genomic imprinting disorders and enable better treatment and prognosis through early intervention.
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Pruebas Genéticas/métodos , Síndrome de Prader-Willi , Cromosomas Humanos Par 15/genética , Impresión Genómica , Humanos , Lactante , Masculino , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , Disomía Uniparental/diagnóstico , Disomía Uniparental/genéticaRESUMEN
Herpes simplex virus 1 (HSV-1) is one of the most prevalent herpesviruses in humans and represents a constant health threat to aged and immunocompromised populations. How HSV-1 interacts with the host immune system to efficiently establish infection and latency is only partially known. CD1d-restricted NKT cells are a critical arm of the host innate immune system and play potent roles in anti-infection and antitumor immune responses. We discovered previously that upon infection, HSV-1 rapidly and efficiently downregulates CD1d expression on the cell surface and suppresses the function of NKT cells. Furthermore, we identified the viral serine/threonine protein kinase US3 as a major viral factor downregulating CD1d during infection. Interestingly, neither HSV-1 nor its US3 protein efficiently inhibits mouse CD1d expression, suggesting that HSV-1 has coevolved with the human immune system to specifically suppress human CD1d (hCD1d) and NKT cell function for its pathogenesis. This is consistent with the fact that wild-type mice are mostly resistant to HSV-1 infection. On the other hand, in vivo infection of CD1d-humanized mice (hCD1d knock-in mice) showed that HSV-1 can indeed evade hCD1d function and establish infection in these mice. We also report here that US3-deficient viruses cannot efficiently infect hCD1d knock-in mice but infect mice lacking all NKT cells at a higher efficiency. Together, these studies supported HSV-1 evasion of human CD1d and NKT cell function as an important pathogenic factor for the virus. Our results also validated the potent roles of NKT cells in antiherpesvirus immune responses and pointed to the potential of NKT cell ligands as adjuvants for future vaccine development.IMPORTANCE Herpes simplex virus 1 (HSV-1) is among the most common human pathogens. Little is known regarding the exact mechanism by which this virus evades the human immune system, particularly the innate immune system. We reported previously that HSV-1 employs its protein kinase US3 to modulate the expression of the key antigen-presenting molecule, CD1d, so as to evade the antiviral function of NKT cells. Here we demonstrated that the virus has coevolved with the human CD1d and NKT cell system and that NKT cells indeed play potent roles in anti-HSV immune responses. These studies point to the great potential of exploring NKT cell ligands as adjuvants for HSV vaccines.
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Presentación de Antígeno/inmunología , Antígenos CD1d/fisiología , Células Dendríticas/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Células T Asesinas Naturales/inmunología , Animales , Regulación hacia Abajo , Femenino , Herpes Simple/inmunología , Herpes Simple/patología , Interacciones Huésped-Patógeno , Humanos , Evasión Inmune , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , VirulenciaRESUMEN
Invariant NKT (iNKT) cells are unconventional innate-like T cells demonstrating potent antitumor function in conventional mouse models. However, the iNKT cell ligands have had limited efficacy in human antitumor clinical trials, mostly due to the profound differences in the properties and compositions of iNKT cells between the two species, including the presence of a CD8(+) subset of iNKT cells only in humans. To build reliable in vivo models for studying human iNKT cells, we recently developed the first humanized mouse model (hCD1d-KI) with human CD1d knocked in. To further humanize the mouse model, we now introduced the human invariant NKT TCRα-chain (Vα24Jα18) into the hCD1d-knockin mice. Similar to humans, this humanized mouse model developed a subset of CD8αß(+) iNKT cells among other human-like iNKT subsets. The presence of the CD8αß(+) iNKT cells in the thymus suggests that these cells developed in the thymus. In the periphery, these NKT cells showed a strong Th1-biased cytokine response and potent cytotoxicity for syngeneic tumor cells upon activation, as do human CD8αß(+) iNKT cells. The low binding avidity of iNKT TCRs to the human CD1d/lipid complex and high prevalence of Vß7 TCRß among the CD8(+) iNKT cells strongly point to a low avidity-based developmental program for these iNKT cells, which included the suppression of Th-POK and upregulation of eomesodermin transcriptional factors. Our establishment of this extensively humanized mouse model phenotypically and functionally reflecting the human CD1d/iNKT TCR system will greatly facilitate the future design and optimization of iNKT cell-based immunotherapies.
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Antígenos CD8/metabolismo , Células T Asesinas Naturales/metabolismo , Subgrupos de Linfocitos T/metabolismo , Animales , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Citotoxicidad Inmunológica , Humanos , Memoria Inmunológica , Inmunofenotipificación , Ratones , Ratones Noqueados , Modelos Animales , Células T Asesinas Naturales/inmunología , Fenotipo , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Subgrupos de Linfocitos T/inmunología , Transcripción GenéticaRESUMEN
In this article, we characterize a novel Ag for invariant NKT (iNKT) cells capable of producing an especially robust Th1 response. This glycosphingolipid, DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), with the only change being a single atom: the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared with αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by dendritic cells in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB06-1 compared with αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Therefore, our data are consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result, in part, from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10-producing iNKT cells, which could counteract the benefits of increased early IFN-γ production.
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Galactosilceramidas/inmunología , Glicoesfingolípidos/inmunología , Interferón gamma/biosíntesis , Activación de Linfocitos/inmunología , Células T Asesinas Naturales/inmunología , Células TH1/inmunología , Animales , Antígenos CD1d/inmunología , Sitios de Unión/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Galactosilceramidas/química , Glicoesfingolípidos/química , Humanos , Interleucina-10/biosíntesis , Interleucina-12/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/inmunologíaRESUMEN
UNLABELLED: Herpes simplex virus 1 (HSV-1) causes one of the most prevalent herpesviral infections in humans and is the leading etiological agent of viral encephalitis and eye infections. Our understanding of how HSV-1 interacts with the host at the cellular and organismal levels is still limited. We and others previously reported that, upon infection, HSV-1 rapidly and efficiently downregulates CD1d cell surface expression and suppresses the function of NKT cells, a group of innate T cells with critical immunoregulatory function. The viral protein kinase US3 plays a major role in this immune evasion mechanism, and its kinase activity is required for this function. In this study, we investigated the cellular substrate(s) phosphorylated by US3 and how it mediates US3 suppression of CD1d recycling. We identified the type II kinesin motor protein KIF3A as a critical kinesin factor in the cell surface expression of CD1d. Interestingly, KIF3A is phosphorylated by US3 both in vitro and in infected cells. Mass spectrometry analysis of purified KIF3A showed that it is phosphorylated predominantly at serine 687 by US3. Ablation of this phosphorylation abolished US3-mediated downregulation of CD1d expression, suggesting that phosphorylation of KIF3A is the primary mechanism of HSV-1 suppression of CD1d expression by US3 protein. Understanding of the precise mechanism of viral modulation of CD1d expression will help to develop more efficient vaccines in the future to boost host NKT cell-mediated immune responses against herpesviruses. IMPORTANCE: Herpes simplex virus 1 (HSV-1) is among the most common human pathogens. Little is known regarding the exact mechanism by which this virus evades the human immune system, particularly the innate immune system. We previously reported that HSV-1 employs its protein kinase US3 to modulate the expression of the key antigen-presenting molecule CD1d to evade the antiviral function of NKT cells. Here we identified the key cellular motor protein KIF3A as a cellular substrate phosphorylated by US3, and this phosphorylation event mediates US3-induced immune evasion.
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Antígenos CD1d/biosíntesis , Regulación hacia Abajo , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Células HeLa , Humanos , Evasión Inmune , Células Asesinas Naturales/inmunología , Espectrometría de Masas , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Despite a high degree of conservation, subtle but important differences exist between the CD1d antigen presentation pathways of humans and mice. These differences may account for the minimal success of natural killer T (NKT) cell-based antitumor therapies in human clinical trials, which contrast strongly with the powerful antitumor effects in conventional mouse models. To develop an accurate model for in vivo human CD1d (hCD1d) antigen presentation, we have generated a hCD1d knock-in (hCD1d-KI) mouse. In these mice, hCD1d is expressed in a native tissue distribution pattern and supports NKT cell development. Reduced numbers of invariant NKT (iNKT) cells were observed, but at an abundance comparable to that in most normal humans. These iNKT cells predominantly expressed mouse Vß8, the homolog of human Vß11, and phenotypically resembled human iNKT cells in their reduced expression of CD4. Importantly, iNKT cells in hCD1d knock-in mice exert a potent antitumor function in a melanoma challenge model. Our results show that replacement of mCD1d by hCD1d can select a population of functional iNKT cells closely resembling human iNKT cells. These hCD1d knock-in mice will allow more accurate in vivo modeling of human iNKT cell responses and will facilitate the preclinical assessment of iNKT cell-targeted antitumor therapies.
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Antígenos CD1d/inmunología , Células Asesinas Naturales/inmunología , Melanoma Experimental/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD1d/genética , Secuencia de Bases , Línea Celular , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Metástasis de la Neoplasia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Autoantibody production is a hallmark of autoimmune diseases, such as lupus and rheumatoid arthritis. Accumulating evidence suggests a role of invariant NKT (iNKT) cells in their pathogenesis. Mechanisms underlying the role of iNKT cells in these diseases, however, remain unclear. In this study, we show that iNKT cells suppress IgG anti-DNA Ab and rheumatoid factor production and reduce IL-10-secreting B cells in a contact-dependent manner, but increase total IgG production and enhance activation markers on B cells via soluble factors. In vivo reconstitution with iNKT cells also reduces autoantibody production in iNKT-deficient mice and in SCID mice implanted with B cells. Using an anti-DNA transgenic model, we found that autoreactive B cells spontaneously produce IL-10 and are activated in vivo. In the presence of activated iNKT cells, these autoreactive B cells are selectively reduced, whereas nonautoreactive B cells are markedly activated. Because iNKTs recognize CD1d, we reasoned that CD1d might play a role in the differential regulation of autoreactive versus nonautoreactive B cells by iNKT cells. Indeed, autoreactive B cells express more CD1d than nonautoreactive B cells, and CD1d deficiency in lupus mice exacerbates autoantibody production and enhances Ab response to a self-peptide but not to a foreign peptide. Importantly, iNKT cells fail to inhibit autoantibody production by CD1d-deficient B cells. Thus, iNKT cells inhibit autoreactive B cells in a contact- and CD1d-dependent manner but activate nonautoreactive B cells via cytokines. Such ability of iNKTs to suppress autoantibody production, without causing global suppression of B cells, has important implications for the development of iNKT-based therapy for autoimmune diseases.
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Antígenos CD1d/fisiología , Autoanticuerpos/metabolismo , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Adhesión Celular/inmunología , Células T Asesinas Naturales/inmunología , Animales , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Autoanticuerpos/biosíntesis , Adhesión Celular/genética , Galactosilceramidas/farmacología , Inmunoglobulina G/metabolismo , Inmunosupresores/farmacología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Depleción Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NZB , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Células T Asesinas Naturales/metabolismoRESUMEN
Galectin-1 is a galactoside-binding lectin expressed in multiple tissues that has pleiotropic immunomodulatory functions. We previously showed that galectin-1 activates human monocyte-derived dendritic cells (MDDCs) and triggers a specific genetic program that up-regulates DC migration through the extracellular matrix, an integral property of mucosal DCs. Here, we identify the galectin-1 receptors on MDDCs and immediate downstream effectors of galectin-1-induced MDDC activation and migration. Galectin-1 binding to surface CD43 and CD45 on MDDCs induced an unusual unipolar co-clustering of these receptors and activates a dose-dependent calcium flux that is abrogated by lactose. Using a kinome screen and a systems biology approach, we identified Syk and protein kinase C tyrosine kinases as mediators of the DC activation effects of galectin-1. Galectin-1, but not lipopolysaccharide, stimulated Syk phosphorylation and recruitment of phosphorylated Syk to the CD43 and CD45 co-cluster on MDDCs. Inhibitors of Syk and protein kinase C signaling abrogated galectin-1-induced DC activation as monitored by interleukin-6 production; and MMP-1, -10, and -12 gene up-regulation; and enhanced migration through the extracellular matrix. The latter two are specific features of galectin-1-activated DCs. Interestingly, we also found that galectin-1 can prime DCs to respond more quickly to low dose lipopolysaccharide stimulation. Finally, we underscore the biological relevance of galectin-1-enhanced DC migration by showing that intradermal injection of galectin-1 in MRL-fas mice, which have a defect in skin DC emigration, increased the in vivo migration of dermal DCs to draining lymph nodes.
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Células Dendríticas/metabolismo , Galectina 1/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Leucosialina/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Células Dendríticas/citología , Galectina 1/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/biosíntesis , Transporte Iónico/efectos de los fármacos , Lipopolisacáridos/farmacología , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 12 de la Matriz/genética , Ratones , Ratones Endogámicos MRL lpr , Fosforilación/efectos de los fármacos , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Quinasa Syk , Tirosina/metabolismoRESUMEN
Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α-galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly proinflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here, we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α-galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6â³ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings and suggested basic principles for capturing useful properties of sphinganine analogs of synthetic iNKT cell activators in the design of immunotherapeutic agents.
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Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacología , Galactosilceramidas/química , Galactosilceramidas/farmacología , Activación de Linfocitos/efectos de los fármacos , Células T Asesinas Naturales/efectos de los fármacos , Neoplasias/terapia , Adolescente , Adulto , Anciano , Animales , Antígenos CD1d/inmunología , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Inmunoterapia , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Células T Asesinas Naturales/inmunología , Neoplasias/inmunologíaRESUMEN
To examine whether cytokine production of CD4(+)immune T cells and CD8(+)immune T cells in Toxoplasma gondii-infected mice differ in their responses to infected cells and to soluble antigens of the parasite, we compared the production of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-10 by the immune T cell populations following in vitro stimulation with tachyzoite-infected macrophages and tachyzoite lysate antigens (TLA). Both CD4(+)and CD8(+)immune T cells produced large amounts of IFN-gamma in response to either infected macrophages or TLA, but the CD4(+)T cells produced greater amounts of the cytokine than did the CD8(+)T cells with both stimulations. Both T cell populations also produced IL-2 after stimulation with infected macrophages, whereas only CD4(+)T cells did when stimulated with TLA. CD4(+)immune T cells also produced large amounts of IL-4 and IL-10 after stimulation with infected macrophages, but CD8(+)T cells did not. These results indicate that CD4(+)immune T cells produce IFN-gamma, IL-2, IL-4, and IL-10 in response to infected macrophages, whereas CD8(+)immune T cells produce predominantly IFN-gamma and IL-2. Since IL-4 and IL-10 could suppress IFN-gamma-mediated protective mechanisms against the parasite, the production of these cytokines by CD4(+)immune T cells in response to infected cells could negatively affect their protective activity in vivo.
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Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Macrófagos Peritoneales/inmunología , Células TH1/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Linfocitos T CD8-positivos/parasitología , Células Cultivadas , Técnicas de Cocultivo , Femenino , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos BALB C , Células TH1/parasitologíaRESUMEN
CD1d-restricted invariant NKT (iNKT) cells are a group of innate-like regulatory T cells that recognize lipid antigens. Both mouse modeling experiments and human clinical studies have suggested a key role for iNKT cells in anti-HBV immunity and these potent T cells can be explored as a novel therapeutic target for anti-HBV treatment. We aim to humanize mice in the CD1d/iNKT cell lipid presentation system and provide new research tools for identifying novel anti-HBV agents.
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Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.
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Antígenos Virales/inmunología , Vacuna BCG/inmunología , Glucolípidos/inmunología , Mycobacterium bovis/inmunología , Células T Asesinas Naturales/inmunología , Animales , Vacuna BCG/administración & dosificación , Vacuna BCG/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Anergia Clonal/inmunología , Modelos Animales de Enfermedad , Femenino , Galactosilceramidas/inmunología , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Humanos , Memoria Inmunológica , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunologíaRESUMEN
The CD1d-dependent presentation of lipid antigens to natural killer T (NKT) cells is an integral part of the innate immune system. However, the development of anticancer therapies based on NKT-cell agonists has had limited success so far. Humanizing mice with respect to the CD1d/NKT antigen presentation system will provide a tool to identify novel lipids that exert antineoplastic functions by targeting NKT cells before the initiation of costly and lengthy clinical trials.
RESUMEN
Marginal zone B cells (MZB) mount a rapid antibody response, potently activate naïve T cells, and are enriched in autoreactive B cells. MZBs express high levels of CD1d, the restriction element for invariant natural killer T cells (iNKT). Here, we examined the effect of iNKT cells on MZB cell activation and numbers in vitro and in vivo in normal and autoimmune mice. Results show that iNKT cells activate MZBs, but restrict their numbers in vitro and in vivo in normal BALB/c and C57/BL6 mice. iNKT cells do so by increasing the activation-induced cell death and curtailing proliferation of MZB cells, whereas they promote the proliferation of follicular B cells. Sorted iNKT cells can directly execute this function, without help from other immune cells. Such MZB regulation by iNKTs is mediated, at least in part, via CD1d on B cells in a contact-dependent manner, whereas iNKT-induced proliferation of follicular B cells occurs in a contact- and CD1d-independent manner. Finally, we show that iNKT cells reduce 'autoreactive' MZB cells in an anti-DNA transgenic model, and limit MZB cell numbers in autoimmune-prone (NZB×NZW)F1 and non-obese diabetic mice, suggesting a potentially new mechanism whereby iNKT cells might regulate pathologic autoimmunity. Differential regulation of follicular B cells versus potentially autoreactive MZBs by iNKT cells has important implications for autoimmune diseases as well as for conditions that require a rapid innate B cell response.
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Subgrupos de Linfocitos B/citología , Homeostasis/inmunología , Células T Asesinas Naturales/fisiología , Animales , Enfermedades Autoinmunes/inmunología , Subgrupos de Linfocitos B/inmunología , Muerte Celular , Proliferación Celular , Inmunidad Innata , Activación de Linfocitos , RatonesRESUMEN
We examined the role of interferon-γ (IFN-γ) in expression of chemokine mRNA and proteins in the brain during chronic infection with Toxoplasma gondii using BALB/c and BALB/c-background IFN-γ knockout (IFN-γ(-/-)) mice. BALB/c mice are genetically resistant to development of toxoplasmic encephalitis and establish a latent, chronic infection in the brain through IFN-γ-mediated immune responses. Amounts of mRNA for CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CCL2/MCP-1, CCL3/MIP-1α, and CCL5/RANTES significantly increased in the brains of wild-type mice after infection. CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES mRNA were most abundant among these chemokines. An increase in amounts of mRNA for CXCL10/IP-10, CCL2/MCP-1, CCL3/MIP-1α, and CCL5/RANTES was also observed in the brains of IFN-γ(-/-) mice after infection, although CXCL10/I-10 and CCL5/RANTES mRNA levels in infected IFN-γ(-/-) mice were significantly lower than those of infected wild-type animals. Amounts of mRNA for CXCL9/MIG and CXCL11/I-TAC remained at the basal levels in infected IFN-γ(-/-) mice. When amounts of the chemokine proteins were examined in the brain homogenates of uninfected and infected mice of both strains, large amounts of CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES were detected only in infected wild-type animals. These results indicate that CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES are the chemokines predominantly induced in the brains of genetically resistant BALB/c mice during chronic infection with T. gondii, and their expression is dependent on IFN-γ.
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Encéfalo/metabolismo , Interferón gamma/metabolismo , ARN Mensajero/análisis , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Encéfalo/inmunología , Encéfalo/patología , Quimiocina CCL5/biosíntesis , Quimiocina CCL5/genética , Quimiocina CXCL10/biosíntesis , Quimiocina CXCL10/genética , Quimiocina CXCL9/biosíntesis , Quimiocina CXCL9/genética , Enfermedad Crónica , Encefalitis , Regulación de la Expresión Génica , Interferón gamma/genética , Interferón gamma/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Toxoplasma/patogenicidad , Toxoplasmosis/genética , Toxoplasmosis/fisiopatologíaRESUMEN
OBJECTIVE: CD1d-reactive invariant natural killer T (iNKT) cells secrete multiple cytokines upon T cell receptor (TCR) engagement and modulate many immune-mediated conditions. The purpose of this study was to examine the role of these cells in the development of autoimmune disease in genetically lupus-prone (NZBxNZW)F1 (BWF1) mice. METHODS: The CD1d1-null genotype was crossed onto the NZB and NZW backgrounds to establish CD1d1-knockout (CD1d0) BWF1 mice. CD1d0 mice and their wild-type littermates were monitored for the development of nephritis and assessed for cytokine responses to CD1d-restricted glycolipid alpha-galactosylceramide (alphaGalCer), anti-CD3 antibody, and concanavalin A (Con A). Thymus and spleen cells were stained with CD1d tetramers that had been loaded with alphaGalCer or its analog PBS-57 to detect iNKT cells, and the cells were compared between BWF1 mice and class II major histocompatibility complex-matched nonautoimmune strains, including BALB/c, (BALB/cxNZW)F1 (CWF1), and NZW. RESULTS: CD1d0 BWF1 mice had more severe nephritis than did their wild-type littermates. Although iNKT cells and iNKT cell responses were absent in CD1d0 BWF1 mice, the CD1d0 mice continued to have significant numbers of interferon-gamma-producing NKT-like (CD1d-independent TCRbeta+,NK1.1+ and/or DX5+) cells. CD1d deficiency also influenced cytokine responses by conventional T cells: upon in vitro stimulation of splenocytes with Con A or anti-CD3, type 2 cytokine levels were reduced, whereas type 1 cytokine levels were increased or unchanged in CD1d0 mice as compared with their wild-type littermates. Additionally, numbers of thymic iNKT cells were lower in young wild-type BWF1 mice than in nonautoimmune strains. CONCLUSION: Germline deletion of CD1d exacerbates lupus in BWF1 mice. This finding, together with reduced thymic iNKT cells in young BWF1 mice as compared with nonautoimmune strains, implies a regulatory role of CD1d and iNKT cells during the development of lupus.
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Antígenos CD1/inmunología , Predisposición Genética a la Enfermedad , Células Asesinas Naturales/inmunología , Lupus Eritematoso Sistémico/genética , Nefritis Lúpica/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD1/genética , Antígenos CD1d , Células Cultivadas , Modelos Animales de Enfermedad , Galactosilceramidas/farmacología , Silenciador del Gen , Interferón gamma/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/patología , Nefritis Lúpica/genética , Nefritis Lúpica/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NZB , Ratones Noqueados , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/patología , Linfocitos T/patología , Timo/metabolismo , Timo/patologíaRESUMEN
Cytokine responses of lymphocytes against Toxoplasma gondii are usually studied by stimulating the cells from infected animals with tachyzoite lysate antigens (TLA) in vitro. We examined whether strains (genotypes) of the parasite used for TLA affect the production of IL-2, IL-4, IL-10 and IFN-gamma in the culture of spleen cells obtained from mice infected with the ME49 (type II) strain. The amounts of IL-2 and IL-4 detected in the culture supernatants following stimulation with TLA of the RH (type I) strain were significantly greater than those detected following stimulation with TLA of the ME49 strain at 2 and 4 weeks after infection, respectively. These results suggest that antigen variations among the strains of T. gondii affect cytokine production of lymphocytes of infected mice. Therefore, to obtain accurate information on cytokine production by immune lymphocytes of infected hosts, it is important to use the identical strain of T. gondii for both infection of animals and preparation of TLA to stimulate the lymphocytes in vitro.
Asunto(s)
Antígenos de Protozoos/inmunología , Citocinas/metabolismo , Activación de Linfocitos/inmunología , Bazo/inmunología , Toxoplasma/clasificación , Toxoplasma/inmunología , Animales , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/parasitologíaRESUMEN
IL-5 preferentially activates B1 cells to produce natural antibodies cross-reactive to self antigens. To determine the role of IL-5 in antibody-mediated autoimmune disease, we generated systemic lupus erythematosus (SLE)-prone (NZB x NZW)F1 mice congenic for IL-5 transgene (TG-F1). The transgene unexpectedly reduced the incidence of lupus nephritis. Anti-DNA antibodies in sera and those produced by splenic B cells in vitro were markedly decreased in TG-F1 mice, while total polyclonal Ig levels were comparable to those in IL-5 transgene-negative (NZB x NZW)F1 (non-TG-F1) littermates. Flow cytometry-sorted splenic B1 cells showed a significant reduction of anti-DNA antibody synthesis in response to IL-5, while proliferative responses to IL-5 did not significantly differ between TG-F1 and non-TG-F1 mice. As TG-F1 mice aged, frequencies of peripheral B1 cells progressively increased, and the mice frequently developed B cell chronic lymphocytic leukemia (B-CLL). Our results suggest that dysregulated, continuous high expression of IL-5 in SLE-prone mice may directly or indirectly mediate a skewed signaling of proliferation/differentiation of self-antigen-activated B1 cells, leading to suppression of autoimmune disease, but instead to aberrant expansion of B1 cells, giving rise to B-CLL. Thus, this model may provide a clue to the pathogenesis of both SLE and B-CLL.