RESUMEN
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS that has been linked with defects in regulatory T cell function. Therefore, strategies to selectively target pathogenic cells via enhanced regulatory T cell activity may provide therapeutic benefit. Kv1.3 is a voltage-gated potassium channel expressed on myelin-reactive T cells from MS patients. Kv1.3-knockout (KO) mice are protected from experimental autoimmune encephalomyelitis, an animal model of MS, and Kv1.3-KO Th cells display suppressive capacity associated with increased IL-10. In this article, we demonstrate that myelin oligodendrocyte glycoprotein-specific Kv1.3-KO Th cells exhibit a unique regulatory phenotype characterized by high CD25, CTLA4, pSTAT5, FoxO1, and GATA1 expression without a corresponding increase in Foxp3. These phenotypic changes result from increased signaling through IL-2R. Moreover, myelin oligodendrocyte glycoprotein-specific Kv1.3-KO Th cells can ameliorate experimental autoimmune encephalomyelitis following transfer to wild-type recipients in a manner that is partially dependent on IL-2R and STAT5 signaling. The present study identifies a population of Foxp3(-) T cells with suppressive properties that arises in the absence of Kv1.3 and enhances the understanding of the molecular mechanism by which these cells are generated. This increased understanding could contribute to the development of novel therapies for MS patients that promote heightened immune regulation.
Asunto(s)
Antígenos/inmunología , Factores de Transcripción Forkhead/metabolismo , Canal de Potasio Kv1.3/deficiencia , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Calcio/metabolismo , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Expresión Génica , Inmunomodulación , Inmunofenotipificación , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones Noqueados , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Glicoproteína Mielina-Oligodendrócito/inmunología , Factores de Transcripción NFATC/metabolismo , Fenotipo , Fosforilación , Factor de Transcripción STAT5/metabolismo , Transducción de SeñalRESUMEN
Multiple sclerosis (MS) is a demyelinating disease of the CNS characterized by inflammation and neurodegeneration. Animal models that enable the study of remyelination in the context of ongoing inflammation are greatly needed for the development of novel therapies that target the pathological inhibitory cues inherent to the MS plaque microenvironment. We report the development of an innovative animal model combining cuprizone-mediated demyelination with transfer of myelin-reactive CD4(+) T cells. Characterization of this model reveals both Th1 and Th17 CD4(+) T cells infiltrate the CNS of cuprizone-fed mice, with infiltration of Th17 cells being more efficient. Infiltration correlates with impaired spontaneous remyelination as evidenced by myelin protein expression, immunostaining, and ultrastructural analysis. Electron microscopic analysis further reveals that demyelinated axons are preserved but reduced in caliber. Examination of the immune response contributing to impaired remyelination highlights a role for peripheral monocytes with an M1 phenotype. This study demonstrates the development of a novel animal model that recapitulates elements of the microenvironment of the MS plaque and reveals an important role for T cells and peripheral monocytes in impairing endogenous remyelination in vivo. This model could be useful for testing putative MS therapies designed to enhance remyelination in the setting of active inflammation, and may also facilitate modeling the pathophysiology of denuded axons, which has been a challenge in rodents because they typically remyelinate very quickly.
Asunto(s)
Sistema Nervioso Central/patología , Cuprizona/toxicidad , Enfermedades Desmielinizantes/terapia , Inhibidores de la Monoaminooxidasa/toxicidad , Vaina de Mielina/metabolismo , Células Th17/fisiología , Traslado Adoptivo , Animales , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Sistema Nervioso Central/ultraestructura , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Adyuvante de Freund/toxicidad , Interleucina-17/metabolismo , Antígenos Comunes de Leucocito/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocitos/patología , Monocitos/ultraestructura , Proteínas de la Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidad , Infiltración Neutrófila , Fragmentos de Péptidos/toxicidad , Regeneración/efectos de los fármacos , Células Th17/ultraestructura , Factores de TiempoRESUMEN
Pharmacologic targeting of T helper (TH) cell trafficking poses an attractive opportunity for amelioration of autoimmune diseases such as multiple sclerosis (MS). MS risk is associated with vitamin D deficiency, and its bioactive form, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to prevent experimental autoimmune encephalomyelitis, a mouse model of MS, via an incompletely understood mechanism. Herein, we systematically examined 1,25(OH)2D3 effects on TH cells during their migration from the lymph nodes to the CNS. Our data demonstrate that myelin-reactive TH cells are successfully generated in the presence of 1,25(OH)2D3, secrete proinflammatory cytokines, and do not preferentially differentiate into suppressor T cells. These cells are able to leave the lymph node, enter the peripheral circulation, and migrate to the s.c. immunization sites. However, TH cells from 1,25(OH)2D3-treated mice are unable to enter the CNS parenchyma but are instead maintained in the periphery. Upon treatment cessation, mice rapidly develop experimental autoimmune encephalomyelitis, demonstrating that 1,25(OH)2D3 prevents the disease only temporarily likely by halting TH cell migration into the CNS.
Asunto(s)
Calcitriol/farmacología , Movimiento Celular/efectos de los fármacos , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Animales , Movimiento Celular/inmunología , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/prevención & control , Ensayo de Inmunoadsorción Enzimática , Mediciones Luminiscentes , Ratones , Ratones Endogámicos C57BL , Estadísticas no Paramétricas , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Half of all patients with multiple sclerosis (MS) experience cognitive impairment, for which there is no pharmacological treatment. Using magnetic resonance spectroscopy (MRS), we examined metabolic changes in the hippocampi of MS patients, compared the findings to performance on a neurocognitive test battery, and found that N-acetylaspartylglutamate (NAAG) concentration correlated with cognitive functioning. Specifically, MS patients with cognitive impairment had low hippocampal NAAG levels, whereas those with normal cognition demonstrated higher levels. We then evaluated glutamate carboxypeptidase II (GCPII) inhibitors, known to increase brain NAAG levels, on cognition in the experimental autoimmune encephalomyelitis (EAE) model of MS. Whereas GCPII inhibitor administration did not affect physical disabilities, it increased brain NAAG levels and dramatically improved learning and memory test performance compared with vehicle-treated EAE mice. These data suggest that NAAG is a unique biomarker for cognitive function in MS and that inhibition of GCPII might be a unique therapeutic strategy for recovery of cognitive function.
Asunto(s)
Disfunción Cognitiva/enzimología , Encefalomielitis Autoinmune Experimental/complicaciones , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Hipocampo/metabolismo , Esclerosis Múltiple/complicaciones , Adulto , Análisis de Varianza , Animales , Disfunción Cognitiva/etiología , Dipéptidos/metabolismo , Femenino , Citometría de Flujo , Humanos , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Pruebas Neuropsicológicas , Compuestos OrganofosforadosRESUMEN
Increasing evidence suggests ion channels have critical functions in the differentiation and plasticity of T cells. Kv1.3, a voltage-gated K(+) channel, is a functional marker and a pharmacological target for activated effector memory T cells. Selective Kv1.3 blockers have been shown to inhibit proliferation and cytokine production by human and rat effector memory T cells. We used Kv1.3 knockout (KO) mice to investigate the mechanism by which Kv1.3 blockade affects CD4(+) T cell differentiation during an inflammatory immune-mediated disease. Kv1.3 KO animals displayed significantly lower incidence and severity of myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis. Kv1.3 was the only K(V) channel expressed in MOG 35-55-specific CD4(+) T cell blasts, and no K(V) current was present in MOG-specific CD4(+) T cell-blasts from Kv1.3 KO mice. Fewer CD4(+) T cells migrated to the CNS in Kv1.3 KO mice following disease induction, and Ag-specific proliferation of CD4(+) T cells from these mice was impaired with a corresponding cell-cycle delay. Kv1.3 was required for optimal expression of IFN-γ and IL-17, whereas its absence led to increased IL-10 production. Dendritic cells from Kv1.3 KO mice fully activated wild-type CD4(+) T cells, indicating a T cell-intrinsic defect in Kv1.3 KO mice. The loss of Kv1.3 led to a suppressive phenotype, which may contribute to the mechanism by which deletion of Kv1.3 produces an immunotherapeutic effect. Skewing of CD4(+) T cell differentiation toward Ag-specific regulatory T cells by pharmacological blockade or genetic suppression of Kv1.3 might be beneficial for therapy of immune-mediated diseases such as multiple sclerosis.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Canal de Potasio Kv1.3/metabolismo , Activación de Linfocitos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Western Blotting , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Electrofisiología , Encefalomielitis Autoinmune Experimental/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/citologíaRESUMEN
The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (T(EM)) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (T(CM)) into T(EM). Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of T(EM) into T(CM), via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21(cip1) with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in T(EM)-mediated autoimmune diseases.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/inmunología , Memoria Inmunológica , Canal de Potasio Kv1.3/inmunología , Transducción de Señal/inmunología , Proteína smad3/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/inmunología , Proteína Quinasa CDC2/metabolismo , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/inmunología , División Celular/genética , División Celular/inmunología , Células Cultivadas , Ciclina B1/genética , Ciclina B1/inmunología , Ciclina B1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Fase G2/genética , Fase G2/inmunología , Humanos , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Fosforilación/genética , Fosforilación/inmunología , Transducción de Señal/genética , Proteína smad3/genética , Proteína smad3/metabolismoRESUMEN
Double-negative T (DNT) cells are αßTCR(+)CD3(+)CD4(-)CD8(-)NK1.1(-) cells that constitute a small but significant proportion of the αßTCR(+) T cells. Their developmental pathway and pathological significance remain unclear. In the present study, we utilized chronic in vitro stimulation of CD4(+) T cells to mimic immune hyper-activation of autoimmune lymphoproliferative syndrome and systemic lupus erythematosus, conditions characterized by DNT cells accumulation. After approximately 4-5 rounds of stimulation, the CD3(+)CD4(-) population became apparent. These cells did not express CD8, NK1.1, γδTCR, or B220, exhibited a highly proliferative effector phenotype, and were dependent on T cell receptor (TCR) stimulation for survival. Moreover, CD3(+)CD4(-) cells expressed MHC class II-restricted αßTCR, indicative of their origin from a CD4(+) T cell population. The results presented herein illustrate a novel method of DNT cell generation in vitro and suggest that immune hyper-activation could also be implicated in the genesis of the disease-associated DNT cells in vivo.
Asunto(s)
Síndrome Linfoproliferativo Autoinmune/inmunología , Linfocitos T CD4-Positivos/inmunología , Lupus Eritematoso Sistémico/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Proliferación Celular , Regulación hacia Abajo , Femenino , Citometría de Flujo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FenotipoRESUMEN
Fingolimod (FTY720) is a multiple sclerosis (MS) therapeutic that upon phosphorylation causes the internalization of sphingosine-1-phosphate receptors (S1PR) and traps CCR7+ T-cells in lymph nodes but relatively spares CCR7-effector T-cells. Nonetheless, FTY720-treated patients are more susceptible to viral infections, indicating a CD8 T-cell defect. Thus, the effects of FTY720 on CD8 T-cells were investigated. To this end, we utilized experimental autoimmune encephalomyelitis (EAE) and a murine influenza model. CD8 T-cell trafficking, IFNγ and Granzyme B (GrB) production were assessed by flow cytometry. CD8 T-cell cytotoxic function was assessed in vitro by an LDH release assay. FTY720 not only ameliorated EAE by sequestering T-cells, but also reduced IFNγ and Granzyme B (GrB) in splenic CD8 T-cells. Murine influenza infection was exacerbated and mortality was increased, as FTY720 inhibited CD8 T-cell GrB production and lung infiltration. Remarkably, only the unphosphorylated compound was able to reduce IFNγ and GrB levels in CD8 T-cells and inhibits their cytotoxic function in vitro. The phosphorylated moiety had no effect in vitro, indicating that CD8 T-cell suppression by FTY720 is independent of S1PR modulation. The addition of arachidonic acid rescued CD8 T-cell function, suggesting that this effect may be mediated via inhibition of cytosolic phospholipase A2. Herein, we demonstrate that FTY720 suppresses CD8 T-cells independently of its trafficking effects and S1PR modulation. This provides a novel explanation not only for the increased rate of viral infections in FTY720-treated patients, but also for its efficacy in MS, as CD8 T-cells have emerged as crucial mediators of MS pathogenesis.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/inmunología , Inmunosupresores/farmacología , Infecciones por Orthomyxoviridae/inmunología , Glicoles de Propileno/farmacología , Esfingosina/análogos & derivados , Animales , Linfocitos T CD8-positivos/inmunología , Quimiotaxis de Leucocito/efectos de los fármacos , Femenino , Clorhidrato de Fingolimod , Citometría de Flujo , Granzimas/biosíntesis , Subtipo H1N1 del Virus de la Influenza A , Interferón gamma/biosíntesis , Lisofosfolípidos/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Esfingosina/metabolismo , Esfingosina/farmacologíaRESUMEN
We investigated CD45RA and CCR7 expression in CD4+ and CD8+ subsets of cerebrospinal fluid (CSF) lymphocytes, both immediately ex vivo and after stimulation, from 134 patients with a variety of inflammatory and non-inflammatory neurological diseases. Most inflammatory diseases had a higher CD4+:CD8+ ratio and higher percentage of effector memory T cells (T(EM)) than non-inflammatory controls, excluding active infection. Moreover, we found that patients with highly elevated cell counts in the CSF tended to have a lower percentage of central memory T cells (T(CM)) than patients with low or absent pleocytosis, with a concomitant increase in T(EM). We also found that samples with elevated IgG index or presence of oligoclonal bands had a significantly higher CD4+:CD8+ ratio than normal samples, consistent with increased CD4+ help for intrathecal IgG synthesis by B cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Enfermedades del Sistema Nervioso Central/líquido cefalorraquídeo , Subgrupos de Linfocitos T/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Enfermedades del Sistema Nervioso Central/inmunología , Enfermedades del Sistema Nervioso Central/metabolismo , Niño , Preescolar , Femenino , Humanos , Inmunoglobulina G/líquido cefalorraquídeo , Lactante , Inflamación/líquido cefalorraquídeo , Inflamación/inmunología , Inflamación/metabolismo , Antígenos Comunes de Leucocito/biosíntesis , Antígenos Comunes de Leucocito/líquido cefalorraquídeo , Antígenos Comunes de Leucocito/inmunología , Masculino , Persona de Mediana Edad , Receptores CCR7/análisis , Receptores CCR7/biosíntesis , Receptores CCR7/inmunología , Subgrupos de Linfocitos T/metabolismo , Adulto JovenRESUMEN
INTRODUCTION: Solid pseudopapillary neoplasms are rare pancreatic neoplasms with low malignant potential and favorable prognosis that are typically seen in young women. PRESENTATION OF CASE: We report a case of two large solid pseudopapillary neoplasms in a 23-year old woman who was treated successfully with a total pancreatectomy. CONCLUSION: To the best of our knowledge, this is the first report of two discrete solid pseudopapillary neoplasms in the same patient.