RESUMEN
Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.
Asunto(s)
Interleucina-15/metabolismo , Neoplasias Experimentales/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/fisiología , Animales , Antígenos CD19/metabolismo , Humanos , Inmunoterapia Adoptiva , Activación de Linfocitos , Ratones , Células Precursoras de Linfocitos T/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR(+)γδ T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in >10(9) CAR(+)γδ T cells from <10(6) total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR(+)γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)γδ T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.
Asunto(s)
Antígenos CD19/inmunología , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos CD19/metabolismo , Línea Celular Tumoral , Electroporación , Humanos , Leucemia/terapia , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Transposasas/genética , Transposasas/metabolismoRESUMEN
Although the phenomenon that omega-3 polyunsaturated fatty acids (n-3 PUFAs) shows to have a beneficial effect in patients suffering from multiple sclerosis (MS) and other autoimmune diseases has been empirically well-documented, the molecular mechanisms that underline the anti-inflammatory effects of n-3 PUFAs are yet to be understood. In experimental autoimmune encephalomyelitis (EAE), a model for MS, we show that one of the underlying mechanisms by which dietary docosahexaenoic acid (DHA) exerts its anti-inflammatory effect is regulating the functional activities of dendritic cells (DCs). In DHA-treated EAE mice, DCs acquire a regulatory phenotype characterized by low expression of co-stimulatory molecules, decreased production of pro-inflammatory cytokines, and enhanced capability of regulatory T-cell induction. The effect of DHA on DCs is mediated by the lipid-sensing receptor, G protein-coupled receptor 120 (GPR120). A GPR120-specific small-molecule agonist could ameliorate the autoimmune inflammation by regulating DCs, while silencing GPR120 in DCs strongly increased the immunogenicity of DCs. Stimulation of GPR120 induces suppressor of cytokine signaling 3 (SOCS3) expression and down-regulates signal transducer and activator of transcription 3 (STAT3) phosphorylation, explaining the molecular mechanism for regulatory DC induction.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Receptores Acoplados a Proteínas G/agonistas , Animales , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ácidos Docosahexaenoicos/uso terapéutico , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Quinasas Janus/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Receptores Acoplados a Proteínas G/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Proteína 3 Supresora de la Señalización de Citocinas/metabolismoRESUMEN
The CD56 antigen (NCAM-1) is highly expressed on several malignancies with neuronal or neuroendocrine differentiation, including small-cell lung cancer and neuroblastoma, tumor types for which new therapeutic options are needed. We hypothesized that CD56-specific chimeric antigen receptor (CAR) T cells could target and eliminate CD56-positive malignancies. Sleeping Beauty transposon-generated CD56R-CAR T cells exhibited αßT-cell receptors, released antitumor cytokines upon co-culture with CD56+ tumor targets, demonstrated a lack of fratricide, and expression of cytolytic function in the presence of CD56+ stimulation. The CD56R-CAR+ T cells are capable of killing CD56+ neuroblastoma, glioma, and SCLC tumor cells in in vitro co-cultures and when tested against CD56+ human xenograft neuroblastoma models and SCLC models, CD56R-CAR+ T cells were able to inhibit tumor growth in vivo. These results indicate that CD56-CARs merit further investigation as a potential treatment for CD56+ malignancies.
Asunto(s)
Antígeno CD56/metabolismo , Glioma/terapia , Neoplasias Pulmonares/terapia , Neuroblastoma/terapia , Receptores Quiméricos de Antígenos/metabolismo , Carcinoma Pulmonar de Células Pequeñas/terapia , Linfocitos T/inmunología , Linfocitos T/trasplante , Animales , Antígeno CD56/genética , Línea Celular Tumoral , Glioma/patología , Humanos , Neoplasias Pulmonares/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Neuroblastoma/patología , Carcinoma Pulmonar de Células Pequeñas/patología , Transposasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have demonstrated that downregulation of proliferation by CD4(+) T-cells in mice fed n-3 PUFA diets is dependent on the involvement of CD28. Therefore, we hypothesized that the balance of co-stimulatory and downregulatory properties of CD28 and CTLA-4, respectively, would be altered by diet. Mice were fed a control corn oil (CO)-enriched diet devoid of n-3 PUFA, or diets enriched with either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 14d. The proliferation of splenic CD4(+) T-cells was suppressed by DHA and EPA following stimulation with anti-CD3 and anti-CD28. Surprisingly, the number of surface CD28 molecules was not reduced in activated CD4(+) T-cells from either group of n-3 PUFA-fed mice. However, in mice fed EPA, CTLA-4 protein levels were enhanced significantly 72 h post-activation (P<0.01). Therefore, we conclude that dietary EPA may suppress CD4(+) T-cell activation by enhancing the downregulatory co-receptor CTLA-4, while not altering the levels of CD28.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Grasas Insaturadas en la Dieta/farmacología , Ácido Eicosapentaenoico/farmacología , Animales , Antígenos CD , Antígenos de Diferenciación/genética , Antígenos CD28/genética , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígeno CTLA-4 , Proliferación Celular/efectos de los fármacos , Aceite de Maíz/farmacología , Grasas Insaturadas en la Dieta/administración & dosificación , Ácidos Docosahexaenoicos/farmacología , Ácidos Grasos/análisis , Ácidos Grasos Omega-3/administración & dosificación , Ácidos Grasos Omega-3/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Mismatch of human leukocyte antigens (HLA) adversely impacts the outcome of patients after allogeneic hematopoietic stem-cell transplantation (alloHSCT). This translates into the clinical requirement to timely identify suitable HLA-matched donors which in turn curtails the chances of recipients, especially those from a racial minority, to successfully undergo alloHSCT. We thus sought to broaden the existing pool of registered unrelated donors based on analysis that eliminating the expression of the HLA-A increases the chance for finding a donor matched at HLA-B, -C, and -DRB1 regardless of a patient's race. Elimination of HLA-A expression in HSC was achieved using artificial zinc finger nucleases designed to target HLA-A alleles. Significantly, these engineered HSCs maintain their ability to engraft and reconstitute hematopoiesis in immunocompromised mice. This introduced loss of HLA-A expression decreases the need to recruit large number of donors to match with potential recipients and has particular importance for patients whose HLA repertoire is under-represented in the current donor pool. Furthermore, the genetic engineering of stem cells provides a translational approach to HLA-match a limited number of third-party donors with a wide number of recipients.
Asunto(s)
Desoxirribonucleasas/genética , Eliminación de Gen , Antígenos HLA-A/genética , Trasplante de Células Madre Hematopoyéticas/etnología , Células Madre Hematopoyéticas/inmunología , Alelos , Animales , Desoxirribonucleasas/metabolismo , Selección de Donante/ética , Expresión Génica , Ingeniería Genética/métodos , Antígenos HLA-A/inmunología , Antígenos HLA-B/genética , Antígenos HLA-B/inmunología , Antígenos HLA-C/genética , Antígenos HLA-C/inmunología , Cadenas HLA-DRB1/genética , Cadenas HLA-DRB1/inmunología , Accesibilidad a los Servicios de Salud/ética , Trasplante de Células Madre Hematopoyéticas/ética , Células Madre Hematopoyéticas/citología , Prueba de Histocompatibilidad , Humanos , Ratones , Grupos Raciales , Trasplante Heterólogo , Trasplante Homólogo , Donante no Emparentado , Dedos de ZincRESUMEN
PURPOSE: The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. EXPERIMENTAL DESIGN: Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR(+) T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR(+) T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. RESULTS: We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K(+) AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env(+) tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env-specific CAR(+) T cells, we observed a significant antitumor effect. CONCLUSIONS: Adoptive cellular immunotherapy with HERV-K env-specific CAR(+) T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors.
Asunto(s)
Terapia Genética/métodos , Inmunoterapia Adoptiva/métodos , Melanoma/virología , Linfocitos T/trasplante , Proteínas Virales/inmunología , Animales , Ingeniería Genética/métodos , Humanos , Inmunohistoquímica , Melanoma/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Many tumors overexpress tumor-associated antigens relative to normal tissue, such as EGFR. This limits targeting by human T cells modified to express chimeric antigen receptors (CAR) due to potential for deleterious recognition of normal cells. We sought to generate CAR(+) T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies that differ in affinity. T cells with low-affinity nimotuzumab-CAR selectively targeted cells overexpressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high-affinity cetuximab-CAR was not affected by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from nonmalignant cells.
Asunto(s)
Antígenos de Neoplasias/inmunología , Receptores ErbB/inmunología , Neoplasias/inmunología , Receptores de Antígenos/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Línea Celular Tumoral , Cetuximab/administración & dosificación , Epítopos/inmunología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/biosíntesis , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoterapia Adoptiva , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Receptores de Antígenos/uso terapéutico , Transducción de Señal , Linfocitos T/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In recent years, our understanding of the plasma membrane has changed considerably as our knowledge of lipid microdomains has expanded. Lipid microdomains include structures known as lipid rafts and caveolae, which are readily identified by their unique lipid constituents. Cholesterol, sphingolipids and phospholipids with saturated fatty acyl chain moieties are highly enriched in these lipid microdomains. Lipid rafts and caveolae have been shown to play an important role in the compartmentalization, modulation and integration of cell signaling. Therefore, these microdomains may have an influential role in human disease. Dietary n-3 polyunsaturated fatty acids (PUFA) ameliorate a number of human diseases including coronary heart disease, autoimmune and inflammatory disorders, diabetes, obesity and cancer, which has been generally linked to its membrane remodeling properties. Recent in vitro evidence suggests that perturbations in membrane composition alter the function of resident proteins and, consequently, cellular responses. This review examines the role of n-3 PUFA in modulating the lipid composition and functionality of lipid microdomains and its potential significance to human health.
Asunto(s)
Ácidos Grasos Volátiles/química , Microdominios de Membrana/química , Animales , Caveolas/fisiología , Grasas Insaturadas en la Dieta/farmacología , Humanos , Modelos BiológicosRESUMEN
Dietary n-3 PUFA have been shown to attenuate T-cell-mediated inflammation, in part, by suppressing T-cell activation and proliferation. n-3 PUFA have also been shown to promote apoptosis, another important mechanism for the prevention of chronic inflammation by maintaining T-cell homeostasis through the contraction of populations of activated T cells. Recent studies have specifically examined Fas death receptor-mediated activation-induced cell death (AICD), since it is the form of apoptosis associated with peripheral T-cell deletion involved in immunological tolerance and T-cell homeostasis. Data from our laboratory indicate that n-3 PUFA promote AICD in T helper 1 polarized cells, which are the mediators of chronic inflammation. Since Fas and components of the death-inducing signaling complex are recruited to plasma membrane microdomains (rafts), the effect of dietary n-3 PUFA on raft composition and resident protein localization has been the focus of recent investigations. Indeed, there is now compelling evidence that dietary n-3 PUFA are capable of modifying the composition of T-cell membrane microdomains (rafts). Because the lipids found in membrane microdomains actively participate in signal transduction pathways, these results support the hypothesis that dietary n-3 PUFA influence signaling complexes and modulate T-cell cytokinetics in vivo by altering T-cell raft composition.
Asunto(s)
Membrana Celular/química , Ácidos Grasos Omega-3/farmacología , Linfocitos T/química , Animales , Apoptosis , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Proliferación Celular , Grasas de la Dieta/farmacología , Ácidos Grasos Omega-3/fisiología , Ácidos Grasos Insaturados/fisiología , Humanos , Linfocitos T/citología , Linfocitos T/fisiologíaRESUMEN
PURPOSE: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. EXPERIMENTAL DESIGN: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. RESULTS: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1(neg)Vδ2(neg) subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1(neg)Vδ2(neg)>Vδ2) of survival of mice with ovarian cancer xenografts. CONCLUSIONS: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans.
Asunto(s)
Activación de Linfocitos/inmunología , Neoplasias/inmunología , Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Traslado Adoptivo , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Citocinas/biosíntesis , Citocinas/farmacología , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Interferón gamma/biosíntesis , Ratones , Ratones Transgénicos , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Neoplasias/mortalidad , Neoplasias/terapia , ARN Mensajero , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Subgrupos de Linfocitos T/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Investigational therapy can be successfully undertaken using viral- and nonviral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently, the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR(+) T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease.
Asunto(s)
Elementos Transponibles de ADN/genética , Terapia Genética/efectos adversos , Mutagénesis Insercional/genética , Animales , Ensayos Clínicos como Asunto , Humanos , Neoplasias/etiología , Neoplasias/patología , Factores de RiesgoRESUMEN
Improving the therapeutic efficacy of T cells expressing a chimeric antigen receptor (CAR) represents an important goal in efforts to control B-cell malignancies. Recently an intrinsic strategy has been developed to modify the CAR itself to improve T-cell signaling. Here we report a second extrinsic approach based on altering the culture milieu to numerically expand CAR(+) T cells with a desired phenotype, for the addition of interleukin (IL)-21 to tissue culture improves CAR-dependent T-cell effector functions. We used electrotransfer of Sleeping Beauty system to introduce a CAR transposon and selectively propagate CAR(+) T cells on CD19(+) artificial antigen-presenting cells (aAPC). When IL-21 was present, there was preferential numeric expansion of CD19-specific T cells which lysed and produced IFN-γ in response to CD19. Populations of these numerically expanded CAR(+) T cells displayed an early memory surface phenotype characterized as CD62L(+)CD28(+) and a transcriptional profile of naïve T cells. In contrast, T cells propagated with only exogenous IL-2 tended to result in an overgrowth of CD19-specific CD4(+) T cells. Furthermore, adoptive transfer of CAR(+) T cells cultured with IL-21 exhibited improved control of CD19(+) B-cell malignancy in mice. To provide coordinated signaling to propagate CAR(+) T cells, we developed a novel mutein of IL-21 bound to the cell surface of aAPC that replaced the need for soluble IL-21. Our findings show that IL-21 can provide an extrinsic reprogramming signal to generate desired CAR(+) T cells for effective immunotherapy.
Asunto(s)
Antígenos CD19/biosíntesis , Linfocitos B/citología , Neoplasias Hematológicas/metabolismo , Inmunoterapia Adoptiva/métodos , Interleucina-12/metabolismo , Linfocitos T/inmunología , Animales , Antígenos CD19/metabolismo , Antígenos CD28/metabolismo , Linaje de la Célula , Técnicas de Cocultivo , Humanos , Interferón gamma/metabolismo , Interleucinas/metabolismo , Células K562 , Selectina L/metabolismo , Ratones , Ratones Endogámicos NOD , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Linfocitos T/metabolismoRESUMEN
Copolymer-I (COP-I) is an unique immune regulatory polymer that has been shown to suppress experimental autoimmune encephalomyelitis (EAE) and is a treatment option for multiple sclerosis (MS). To investigate whether its immune suppressive effects can be extended to other autoimmune diseases, we treated mice with COP-I during the induction of collagen-induced arthritis (CIA). Our results show that COP-I treatment exacerbated CIA, leading to faster onset, more severe and longer-lasting disease. The mechanisms underlying the exacerbation of CIA by COP-I treatment include enhanced activation and inflammatory cytokine production by autoreactive T cells and elevated production of autoreactive antibodies. In addition, germinal center response was significantly enhanced by COP-I treatment. Thus, great caution should be taken when COP-I is to be used in MS patients with other autoimmune complications or its potential therapeutic effects are to be extended beyond autoimmune demyelinating diseases.
Asunto(s)
Artritis Experimental/inducido químicamente , Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/tratamiento farmacológico , Inmunosupresores/efectos adversos , Péptidos/efectos adversos , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Colágeno/inmunología , Centro Germinal/efectos de los fármacos , Acetato de Glatiramer , Inmunidad Celular , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-4/biosíntesis , Masculino , Ratones , Ratones Endogámicos DBA , Linfocitos T Reguladores/efectos de los fármacos , Células TH1/efectos de los fármacosRESUMEN
In aging, both primary and secondary antibody responses are impaired. One of the most notable changes in age-associated immune deficiency is the diminished germinal center (GC) reaction. This impaired GC response reduces antibody affinity maturation, decreases memory B cell development, and prevents the establishment of long-term antibody-forming cells in the bone marrow. It is of great importance to explore novel strategy in improving GC response in the elderly. In this study, the efficacy of immunization with immune complexes in overcoming age-associated deficiency in GC response was investigated. We show that the depressed GC response in aged mice can be significantly elevated by immunization with immune complexes. Importantly, there is a significant improvement of B cell memory response and long-lived plasma cells. Our results demonstrate that immune complex immunization may represent a novel strategy to elicit functional GC response in aging, and possibly, to overcome age-related immune deficiency in general.
Asunto(s)
Envejecimiento/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Centro Germinal/inmunología , Animales , Formación de Anticuerpos/inmunología , Complejo Antígeno-Anticuerpo/farmacología , Médula Ósea/inmunología , Femenino , Inmunización , Ratones , Ratones Endogámicos C57BL , Nitrofenoles/inmunología , Fenilacetatos , Células Plasmáticas/inmunología , Linfocitos T/inmunologíaRESUMEN
Decline in cellular immunity in aging compromises protection against infectious diseases and leads to the increased susceptibility of the elderly to infection. In particular, Ag-specific cytotoxic T lymphocyte (CTL) response against virus is markedly reduced in an aged immune system. It is of great importance to explore novel strategy in eliciting effective antiviral CTL activity in the elderly. In this study, the efficacy and mechanisms of immunization with immune complexes in overcoming age-associated deficiency in cellular immunity were investigated. In this study, we show that the severely depressed CTL response to influenza A in aged mice can be significantly restored by immunization with immune complexes consisting of influenza A virus and mAb to influenza A nucleoprotein. The main mechanisms underlying this recovery of CTL response induced by immune complex immunization in aged mice are enhanced dendritic cell function and elevated production of IFN-gamma in both CD4(+) Th1 and CD8(+) CTLs. Thus, these results demonstrate that immune complex immunization may represent a novel strategy to elicit effective virus-specific cytotoxic response in an aged immune system, and possibly, to overcome age-related immune deficiency in general.
Asunto(s)
Envejecimiento/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Hemaglutininas/inmunología , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Nucleoproteínas/inmunologíaRESUMEN
B cells play a pathogenic or regulatory role in many autoimmune diseases through production of autoantibodies, cytokine production, and Ag presentation. However, the mechanisms that regulate these B cell functions under different autoimmune settings remain unclear. In the current study, we found that when B cells overexpress an antiapoptotic gene, Bcl(XL), they significantly increased production of IFN-gamma and enhanced Th1 response. Consistently, Bcl-x(L) transgenic mice developed more severe and sustained collagen-induced arthritis due to the enhanced Th1 response. The production of autoantibodies in Bcl(XL) transgenic mice was comparable to that in wild-type mice. Thus, our results indicate a novel role of Bcl(XL) in regulating B cell functions and immune responses. In patients with rheumatoid arthritis, arthritogenic B cells often up-regulate Bcl(XL) expression, which may not only render B cells resistant to apoptosis but also alter the ability of the autoreactive B cells to produce cytokines and modulate the inflammatory response. This may have therapeutic implications if Bcl(XL) expression can be down-regulated in autoreactive B cells.
Asunto(s)
Artritis Experimental/inmunología , Linfocitos B/inmunología , Interferón gamma/inmunología , Células TH1/inmunología , Proteína bcl-X/inmunología , Animales , Formación de Anticuerpos , Artritis Experimental/genética , Artritis Experimental/terapia , Autoanticuerpos/inmunología , Linfocitos B/patología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Inflamación/terapia , Ratones , Ratones Transgénicos , Células TH1/patología , Proteína bcl-X/genéticaRESUMEN
Dietary n-3 PUFAs have been shown to attenuate T-cell-mediated inflammation. To investigate whether dietary n-3 PUFAs promote activation-induced cell death (AICD) in CD4+ T-cells induced in vitro to a polarized T-helper1 (Th1) phenotype, C57BL/6 mice were fed diets containing either 5% corn oil (CO; n-6 PUFA control) or 4% fish oil (FO) plus 1% CO (n-3 PUFA) for 2 weeks. Splenic CD4+ T-cells were cultured with alpha-interleukin-4 (alphaIL-4), IL-12, and IL-2 for 2 days and then with recombinant (r) IL-12 and rIL-2 for 3 days in the presence of diet-matched homologous mouse serum (HMS) to prevent loss of cell membrane fatty acids, or with fetal bovine serum. After polarization, Th1 cells were reactivated and analyzed for interferon-gamma and IL-4 by intracellular cytokine staining and for apoptosis by Annexin V/propidium iodide. Dietary FO enhanced Th1 polarization by 49% (P = 0.0001) and AICD by 24% (P = 0.0001) only in cells cultured in the presence of HMS. FO enhancement of Th1 polarization and AICD after culture was associated with the maintenance of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) in plasma membrane lipid rafts. In conclusion, n-3 PUFAs enhance the polarization and deletion of proinflammatory Th1 cells, possibly as a result of alterations in membrane microdomain fatty acid composition.
Asunto(s)
Grasas Insaturadas en la Dieta/metabolismo , Ácidos Grasos Insaturados/metabolismo , Células TH1/metabolismo , Triglicéridos/metabolismo , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , Ácidos Grasos Omega-3 , Microdominios de Membrana/metabolismo , Ratones , Células TH1/citologíaRESUMEN
Previous studies showing dietary (n-3) polyunsaturated fatty acids (PUFA) attenuate T cell immune-mediated inflammatory diseases led us to hypothesize that (n-3) PUFA promote activation-induced cell death (AICD) in T cells. Because T cell subsets display a differential resistance to AICD, we compared the effects of (n-3) PUFA feeding on T cells stimulated in vitro to express different cytokine profiles. Mice were fed either diets lacking (n-3) PUFA (control) or (n-3) PUFA-containing diets for 14 d. Splenic T cells were stimulated with alphaCD3/alphaCD28, phorbol myristate acetate (PMA)/Ionomycin or alphaCD3/PMA for 48 h, followed by reactivation with the same stimuli for 5 h. Apoptosis was measured using Annexin V/propidium iodide. (n-3) PUFA were selectively incorporated into membrane phospholipid pools. Cytokine analyses revealed that (n-3) PUFA enhanced AICD only in T cells expressing a T helper cell (Th)1-like cytokine profile after stimulation with PMA/Ionomycin compared to mice fed the (n-6) PUFA control diet (P = 0.0008). In contrast, no increase in apoptosis was seen in T cells stimulated with alphaCD3/PMA, which exhibited a Th2 cytokine profile. These data demonstrate that the ability of (n-3) PUFA to promote AICD is dependent on the activation stimulus. In conclusion, we have identified a novel mechanism by which (n-3) PUFA modulate T cell-mediated immunity by selective deletion of Th1-like cells while maintaining or enhancing the Th2-mediated humoral immune response.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Grasos Omega-3/farmacología , Linfocitos T/efectos de los fármacos , Animales , Dieta , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
We have previously demonstrated that immunoglobulin A (IgA)(-/-) knockout (KO) mice exhibit levels of susceptibility to influenza virus infection that are similar to those of their normal IgA(+/+) littermates. To understand the mechanism of this apparent mucosal immunity without IgA, immunoglobulin isotype and T helper 1 (Th1)-type [interferon-gamma (IFN-gamma)] and Th2-type [interleukin (IL)-4, IL-5)] cytokine responses to influenza vaccine were evaluated. Intranasal immunization with influenza virus subunit vaccine plus cholera toxin/cholera toxin B subunit (CT/CTB) induced significant influenza virus-specific immunoglobulin G (IgG) antibody in the serum and nasal passages of both IgA(-/-) and IgA(+/+) mice, while IgA antibodies were induced only in IgA(+/+) mice. IgA KO mice exhibited an IgG1 subclass haemagglutinin (HA)-specific response but no detectable IgG2a and IgG2b responses. In contrast, IgA(+/+) mice exhibited significant IgG1 as well as IgG2a responses. This indicates a predominant Th2-type response in IgA KO mice compared to normal mice. Following stimulation with influenza virus in vitro, splenic lymphocytes from immunized IgA(-/-) mice produced significantly lower levels of IFN-gamma than IgA(+/+) mice (P < 0.001), but elaborated similar levels of IL-4 and IL-5. This was true at both protein and mRNA levels. Immunized mice were challenged intranasally with a small inoculum of influenza virus to allow deposition of virus in the nasal mucosal passages. Compared to non-immunized mice, immunized IgA(-/-) and IgA(+/+) mice exhibited significant, but similar levels of reduction in virus titres in the nose and lung. These results demonstrate that in addition to IgA deficiency, IgA gene deletion also resulted in down-regulated Th1-type immune responses and confirm our previous data that IgA antibody is not indispensable for the prevention of influenza virus infection.