RESUMEN
There is a great need for pharmacological approaches to enhance neural progenitor cell (NPC) function particularly in neuroinflammatory diseases with failed neuroregeneration. In diseases such as multiple sclerosis and stroke, T-cell infiltration occurs in periventricular zones where NPCs are located and is associated with irreversible neuronal loss. We studied the effect of T-cell activation on NPC functions. NPC proliferation and neuronal differentiation were impaired by granzyme B (GrB) released by the T-cells. GrB mediated its effects by the activation of a Gi-protein-coupled receptor leading to decreased intracellular levels of cAMP and subsequent expression of the voltage-dependent potassium channel, Kv1.3. Importantly, blocking channel activity with margatoxin or blocking its expression reversed the inhibitory effects of GrB on NPCs. We have thus identified a novel pathway in neurogenesis. The increased expression of Kv1.3 in pathological conditions makes it a novel target for promoting neurorestoration.
Asunto(s)
Granzimas/metabolismo , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/fisiología , Activación de Linfocitos/fisiología , Inhibición Neural/fisiología , Neurogénesis/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Linfocitos T/metabolismo , Adulto , Animales , Células Cultivadas , Femenino , Feto , Humanos , Canal de Potasio Kv1.3/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Células Madre/efectos de los fármacos , Células Madre/enzimología , Células Madre/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimologíaRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.
RESUMEN
Through a combination of fluorescence microscopy and patch-clamp analysis we have identified a striking alteration in K(+) channel expression in terminally differentiated human CCR7(-)CD45RA(-) effector memory T lymphocytes (T(EM)). Following activation, T(EM) cells expressed significantly higher levels of the voltage-gated K(+) channel Kv1.3 and lower levels of the calcium-activated K(+) channel IKCa1 than naive and central memory T cells (T(CM)). Upon repeated in vitro antigenic stimulation, naive cells differentiated into Kv1.3(high)IKCa1(low) T(EM) cells, and the potent Kv1.3-blocking sea anemone Stichodactyla helianthus peptide (ShK) suppressed proliferation of T(EM) cells without affecting naive or T(CM) lymphocytes. Thus, the Kv1.3(high)IKCa1(low) phenotype is a functional marker of activated T(EM) lymphocytes. Activated myelin-reactive T cells from patients with MS exhibited the Kv1.3(high)IKCa1(low) T(EM) phenotype, suggesting that they have undergone repeated stimulation during the course of disease; these cells may contribute to disease pathogenesis due to their ability to home to inflamed tissues and exhibit immediate effector function. The Kv1.3(high)IKCa1(low) phenotype was not seen in glutamic acid decarboxylase, insulin-peptide or ovalbumin-specific and mitogen-activated T cells from MS patients, or in myelin-specific T cells from healthy controls. Selective targeting of Kv1.3 in T(EM) cells may therefore hold therapeutic promise for MS and other T cell-mediated autoimmune diseases.
Asunto(s)
Memoria Inmunológica , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Linfocitos T/metabolismo , Adulto , Animales , Complejo CD3/biosíntesis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Calcio/metabolismo , División Celular , Separación Celular , Cnidarios/metabolismo , Relación Dosis-Respuesta a Droga , Electrofisiología , Femenino , Citometría de Flujo , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio , Canal de Potasio Kv1.3 , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Péptidos/química , FenotipoRESUMEN
Neuroinflammatory diseases such as multiple sclerosis (MS) are characterized by focal regions of demyelination and axonal loss associated with infiltrating T cells. However, the role of activated T cells in causing neuronal injury remains unclear. CD4 and CD8 T cells were isolated from normal donors and polyclonally activated using plate-bound anti-CD3 and soluble anti-CD28. The conditioned T cell supernatants caused toxicity to cultured human fetal neurons, which could be blocked by immunodepleting the supernatants of granzyme B (GrB). Recombinant GrB also caused toxicity in neurons by caspase-dependent pathways but no toxicity was seen in astrocytes. The neurotoxicity was independent of perforin and could not be blocked by mannose-6-phosphate. However, GrB-induced neurotoxicity was sensitive to pertussis toxin, implicating the stimulation of Gialpha protein-coupled receptors. GrB caused a decrease in cAMP levels but only modest increases in intracellular calcium. The effect on intracellular calcium could be markedly potentiated by stromal-derived factor 1alpha. GrB-induced neurotoxicity could also be blocked by vitamin E and a neuroimmunophilin ligand. In conclusion, GrB may be an important mediator of neuronal injury in T cell-mediated neuroinflammatory disorders.
Asunto(s)
Neuronas/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Serina Endopeptidasas/toxicidad , Linfocitos T/enzimología , Antioxidantes/farmacología , Astrocitos/efectos de los fármacos , Calcio/metabolismo , Caspasas/metabolismo , Cromanos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Granzimas , Humanos , Activación de Linfocitos , Neuronas/metabolismo , Pirrolidinas/farmacología , Serina Endopeptidasas/metabolismoRESUMEN
BACKGROUND: Glatiramer acetate therapy is thought to be effective for multiple sclerosis (MS) by promoting T(H)2 cytokine deviation, possibly in the brain, but the exact mechanism and site of action are incompletely understood. Determining the site of action and effect of glatiramer on cell trafficking is of major importance in designing rational combination therapy clinical trials. OBJECTIVE: To determine whether glatiramer therapy will also act in the peripheral blood through bystander modulation of chemokine receptor (CKR) expression and cytokine production on T lymphocytes. DESIGN: Before-and-after trial. SETTING: A university MS specialty center. PATIENTS: Ten patients with relapsing-remitting MS. INTERVENTIONS: Treatment with glatiramer for 12 months and serial phlebotomy. MAIN OUTCOME MEASURES: Cytokine production, CKR expression, and cell migration. RESULTS: The glatiramer-reactive T cells were T(H)2 cytokine biased, consistent with previous studies. We found a significant reduction in the expression of the T(H)1 inflammation associated with the CKRs CXCR3, CXCR6, and CCR5 on glatiramer- and myelin-reactive T cells generated from patients with MS receiving glatiramer therapy vs baseline. Conversely, expression of the lymph node-homing CKR, CCR7, was markedly enhanced on the glatiramer-reactive T cells derived from patients with MS undergoing glatiramer therapy. There was a reduction in the percentage of CD4+ glatiramer-reactive T cells and an increase in the number of CD8+ glatiramer-reactive T cells. CONCLUSIONS: Glatiramer may suppress autoreactive CD4+ effector memory T cells and enhance CD8+ regulatory responses, and bystander modulation of CKRs may occur in the periphery.
Asunto(s)
Efecto Espectador/efectos de los fármacos , Efecto Espectador/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Péptidos/farmacología , Péptidos/uso terapéutico , Receptores de Quimiocina/biosíntesis , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Acetato de Glatiramer , Humanos , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/inmunología , Linfocitos T/metabolismoRESUMEN
Cytokine-polarized T cells have distinct chemokine receptor (CKR) expression patterns associated with their cytokine secretion profiles. In order to investigate this paradigm in autoreactive human T cells, we have determined the CKR expression pattern of myelin basic protein (MBP)-reactive T cell lines (TCL) and compared these profiles to those of TCL-generated in response to tetanus toxoid (TT). Expression of CXCR6 and CXCR3 on TCL was significantly positively correlated with IFNgamma, and inversely correlated with IL-5 production. TT TCL had significantly higher expression of CCR7(-)/CD45RA(-) T effector memory (Tem) cells than MBP TCL. However, in MBP-specific TCL, CXCR6 was found to be the best marker of conversion to the Tem phenotype. CXCR6 and CXCR3 are likely to be important in the migration of effector memory T cells in Th1-mediated inflammatory diseases such as multiple sclerosis (MS).
Asunto(s)
Interferón gamma/biosíntesis , Proteína Básica de Mielina/inmunología , Receptores de Citocinas/análisis , Receptores Acoplados a Proteínas G , Receptores Virales , Linfocitos T/química , Biomarcadores , División Celular/inmunología , Células Cultivadas , Epítopos , Citometría de Flujo , Humanos , Memoria Inmunológica/inmunología , Interleucina-5/análisis , Esclerosis Múltiple/inmunología , Receptores CXCR3 , Receptores CXCR6 , Receptores de Quimiocina/análisis , Receptores de Citocinas/inmunología , Linfocitos T/citología , Linfocitos T/metabolismo , Células TH1/química , Células TH1/inmunología , Células Th2/química , Células Th2/inmunologíaRESUMEN
We explored the kinetics of CCR7 expression on T(H)1 and T(H)2 polarized cells as well as on antigen-specific T cell lines at various stages of differentiation. A striking pattern of early (days 7-14) inducible CCR7 expression was seen preferentially on primary T(H)1 cell lines, as compared to T(H)2 cells, and was dependent on the strength and duration of the T cell receptor signal. Upon repeated restimulation (days 21-28) and differentiation, a switch occurred in which T(H)2 cells had high CCR7 expression, whereas T(H)1 cells lost CCR7 expression. Chronic (8 weeks and later) effector memory cell lines were 95% CCR7 negative. These data demonstrate an ordered pattern of CCR7 expression that suggest more rapid priming of T(H)1 cells in the lymph node, and delayed priming with prolonged CCR7 expression during T(H)2 responses, and may have implications for tracking T(H)1 effector T cells ex vivo in autoimmune diseases.
Asunto(s)
Presentación de Antígeno/inmunología , Inmunidad Celular/inmunología , Activación de Linfocitos/inmunología , Receptores de Quimiocina/inmunología , Células TH1/inmunología , Células Th2/inmunología , Antígenos de Superficie/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/fisiopatología , Linfocitos T CD8-positivos/inmunología , Polaridad Celular/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Cinética , Receptores CCR7 , Receptores de Quimiocina/metabolismo , Células TH1/metabolismo , Células Th2/metabolismoRESUMEN
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
A link between autoimmunity and improved antitumor immunity has long been recognized, although the exact mechanistic relationship between these two phenomena remains unclear. In the present study we have found that vitiligo, the autoimmune destruction of melanocytes, generates self antigen required for mounting persistent and protective memory CD8+ T cell responses to melanoma. Vitiligo developed in approximately 60% of mice that were depleted of regulatory CD4+ T cells and then subjected to surgical excision of large established B16 melanomas. Mice with vitiligo generated 10-fold larger populations of CD8+ memory T cells specific for shared melanoma/melanocyte antigens. CD8+ T cells in mice with vitiligo acquired phenotypic and functional characteristics of effector memory, suggesting that they were supported by ongoing antigen stimulation. Such responses were not generated in melanocyte-deficient mice, indicating a requirement for melanocyte destruction in maintaining CD8+ T cell immunity to melanoma. Vitiligo-associated memory CD8+ T cells provided durable tumor protection, were capable of mounting a rapid recall response to melanoma, and did not demonstrate phenotypic or functional signs of exhaustion even after many months of exposure to antigen. This work establishes melanocyte destruction as a key determinant of lasting melanoma-reactive immune responses, thus illustrating that immune-mediated destruction of normal tissues can perpetuate adaptive immune responses to cancer.
Asunto(s)
Linfocitos T CD8-positivos/citología , Melanocitos/citología , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Animales , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Homeostasis , Memoria Inmunológica , Interferón gamma/metabolismo , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vitíligo/metabolismoRESUMEN
Multiple Sclerosis (MS) is characterized by central nervous system perivenular and parenchymal mononuclear cell infiltrates consisting of activated T cells and macrophages. We recently demonstrated that elevated expression of the voltage-gated potassium channel, Kv1.3, is a functional marker of activated effector memory T (T(EM)) cells in experimental allergic encephalomyelitis and in myelin-specific T cells derived from the peripheral blood of patients with MS. Herein, we show that Kv1.3 is highly expressed in postmortem MS brain inflammatory infiltrates. The expression pattern revealed not only Kv1.3(+) T cells in the perivenular infiltrate but also high expression in the parenchyma of demyelinated MS lesions and both normal appearing gray and white matter. These cells were uniformly chemokine receptor 7 negative (CCR7(-)), consistent with an effector memory phenotype. Using double-labeling immunohistochemistry and confocal microscopy, we demonstrated colocalization of Kv1.3 with CD3, CD4, CD8, and some CD68 cells. The expression patterns mirrored in vitro experiments showing polarization of Kv1.3 to the immunological synapse. Kv1.3 was expressed in low to moderate levels on CCR7(+) central memory T cells from cerebrospinal fluid, but, when these cells were stimulated in vitro, they rapidly became Kv1.3(high)/CCR7(-) T(EM), suggesting that a subset of cerebrospinal fluid cells existed in a primed state ready to become T(EM). These studies provide further rationale for the use of specific Kv1.3 antagonists in MS.