RESUMEN
Food allergy is a prevalent, potentially deadly disease caused by inadvertent sensitization to benign food antigens. Pathogenic Th2 cells are a major driver for disease, and allergen-specific immunotherapies (AIT) aim to increase the allergen threshold required to elicit severe allergic symptoms. However, the majority of AIT approaches require lengthy treatments and convey transient disease suppression, likely due to insufficient targeting of pathogenic Th2 responses. Here, the ability of allergen-encapsulating nanoparticles to directly suppress pathogenic Th2 responses and reactivity is investigated in a mouse model of food allergy. NPs associate with pro-tolerogenic antigen presenting cells, provoking accumulation of antigen-specific, functionally suppressive regulatory T cells in the small intestine lamina propria. Two intravenous doses of allergen encapsulated in poly(lactide-co-glycolide) nanoparticles (NPs) significantly reduces oral food challenge (OFC)-induced anaphylaxis. Importantly, NP treatment alters the fates of pathogenic allergen-specific Th2 cells, reprogramming these cells toward CD25+FoxP3+ regulatory and CD73+FR4+ anergic phenotypes. NP-mediated reductions in the frequency of effector cells in the gut and mast cell degranulation following OFC are also demonstrated. These studies reveal mechanisms by which an allergen-encapsulating NP therapy and, more broadly, allergen-specific immunotherapies, can rapidly attenuate allergic responses by targeting pathogenic Th2 cells.
RESUMEN
Cancer treatment utilizing infusion therapies to enhance the patient's own immune response against the tumor have shown significant functionality in a small subpopulation of patients. Additionally, advances have been made in the utilization of nanotechnology for the treatment of disease. We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic-co-glycolic acid) (PLGA) nanoparticles (IMPs; named ONP-302) for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.
Asunto(s)
Melanoma Experimental , Nanopartículas , Animales , Humanos , Inmunoterapia/métodos , Interleucina-15 , Melanoma Experimental/terapia , Proteínas de la Membrana/metabolismo , Células Mieloides/metabolismo , Nucleotidiltransferasas/metabolismo , Microambiente TumoralRESUMEN
Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by inflammation, demyelination, and neurodegeneration. The ideal MS therapy would both specifically inhibit the underlying autoimmune response and promote repair/regeneration of myelin as well as maintenance of axonal integrity. Currently approved MS therapies consist of non-specific immunosuppressive molecules/antibodies which block activation or CNS homing of autoreactive T cells, but there are no approved therapies for stimulation of remyelination nor maintenance of axonal integrity. In an effort to repurpose an FDA-approved medication for myelin repair, we chose to examine the effectiveness of digoxin, a cardiac glycoside (Na+ /K+ ATPase inhibitor), originally identified as pro-myelinating in an in vitro screen. We found that digoxin regulated multiple genes in oligodendrocyte progenitor cells (OPCs) essential for oligodendrocyte (OL) differentiation in vitro, promoted OL differentiation both in vitro and in vivo in female naïve C57BL/6J (B6) mice, and stimulated recovery of myelinated axons in B6 mice following demyelination in the corpus callosum induced by cuprizone and spinal cord demyelination induced by lysophosphatidylcholine (LPC), respectively. More relevant to treatment of MS, we show that digoxin treatment of mice with established MOG35-55 -induced Th1/Th17-mediated chronic EAE combined with tolerance induced by the i.v. infusion of biodegradable poly(lactide-co-glycolide) nanoparticles coupled with MOG35-55 (PLG-MOG35-55 ) completely ameliorated clinical disease symptoms and stimulated recovery of OL lineage cell numbers. These findings provide critical pre-clinical evidence supporting future clinical trials of myelin-specific tolerance with myelin repair/regeneration drugs, such as digoxin, in MS patients.
Asunto(s)
Glicósidos Cardíacos , Enfermedades Desmielinizantes , Esclerosis Múltiple , Animales , Glicósidos Cardíacos/efectos adversos , Diferenciación Celular , Cuprizona , Enfermedades Desmielinizantes/inducido químicamente , Digoxina/efectos adversos , Modelos Animales de Enfermedad , Reposicionamiento de Medicamentos , Femenino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/tratamiento farmacológico , Vaina de Mielina/fisiología , Oligodendroglía/fisiologíaRESUMEN
Type 1 diabetes (T1D) is an autoimmune disease characterized by T and B cell responses to proteins expressed by insulin-producing pancreatic ß cells, inflammatory lesions within islets (insulitis), and ß cell loss. We previously showed that Ag-specific tolerance targeting single ß cell protein epitopes is effective in preventing T1D induced by transfer of monospecific diabetogenic CD4 and CD8 transgenic T cells to NOD.scid mice. However, tolerance induction to individual diabetogenic proteins, for example, GAD65 (glutamic acid decarboxylase 65) or insulin, has failed to ameliorate T1D both in wild-type NOD mice and in the clinic. Initiation and progression of T1D is likely due to activation of T cells specific for multiple diabetogenic epitopes. To test this hypothesis, recombinant insulin, GAD65, and chromogranin A proteins were encapsulated within poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (COUR CNPs) to assess regulatory T cell induction, inhibition of Ag-specific T cell responses, and blockade of T1D induction/progression in NOD mice. Whereas treatment of NOD mice with CNPs containing a single protein inhibited the corresponding Ag-specific T cell response, inhibition of overt T1D development only occurred when all three diabetogenic proteins were included within the CNPs (CNP-T1D). Blockade of T1D following CNP-T1D tolerization was characterized by regulatory T cell induction and a significant decrease in both peri-insulitis and immune cell infiltration into pancreatic islets. As we have recently published that CNP treatment is both safe and induced Ag-specific tolerance in a phase 1/2a celiac disease clinical trial, Ag-specific tolerance induced by nanoparticles encapsulating multiple diabetogenic proteins is a promising approach to T1D treatment.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Nanopartículas , Animales , Diabetes Mellitus Experimental/patología , Epítopos , Insulina , Ratones , Ratones Endogámicos NOD , Ratones SCID , ProteínasRESUMEN
Methods to test both the functionality and mechanism of action for human recombinant proteins and antibodies in vitro have been limited by multiple factors. To test the functionality of a recombinant protein or antibody, the receptor, the receptor-associated ligand, or both must be expressed by the cells present within the in vitro culture. While the use of transfected cell lines can circumvent this gap, the use of transfected cell lines does not allow for studying the native signaling pathway(s) modulated by the specific recombinant protein or antibody in primary cells. The present protocol utilizes sort purified CD14+ monocytes and T cells, both CD4+ T cells and CD8+ T cells, from healthy donors in a co-culture system. This methodology is particularly relevant for testing recombinant proteins or antibodies that are putative therapeutics for the treatment of autoimmune disease and cancer. While the current protocol focuses on co-cultures containing B7-H4 expressing monocytes plus either autologous CD4+ T cells or CD8+ T cells, the protocol can be modified for the user's specific needs.
RESUMEN
The potent immune regulatory function of an agonistic B7-H4-Ig fusion protein (B7-H4Ig) has been demonstrated in multiple experimental autoimmune models; however, the identity of a functional B7-H4 receptor remained unknown. The biological activity of B7-H4 is associated with decreased inflammatory CD4+ T cell responses as supported by a correlation between B7-H4-expressing tumor-associated macrophages and Foxp3+ T cells within the tumor microenvironment. Recent data indicate that members of the semaphorin (Sema)/plexin/neuropilin (Nrp) family of proteins both positively and negatively modulate immune cell function. In this study, we show that B7-H4 binds the soluble Sema family member Sema3a. Additionally, B7-H4Ig-induced inhibition of inflammatory CD4+ T cell responses is lost in both Sema3a functional mutant mice and mice lacking Nrp-1 expression in Foxp3+ T cells. These findings indicate that B7-H4Ig binds to Sema3a, which acts as a functional bridge to stimulate an Nrp-1/Plexin A4 heterodimer to form a functional immunoregulatory receptor complex resulting in increased levels of phosphorylated PTEN and enhanced regulatory CD4+ T cell number and function.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Neuropilina-1/metabolismo , Receptores de Superficie Celular/metabolismo , Semaforina-3A/metabolismo , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfohidrolasa PTEN/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
ILDR2 is a member of the Ig superfamily, which is implicated in tricellular tight junctions, and has a putative role in pancreatic islet health and survival. We recently found a novel role for ILDR2 in delivering inhibitory signals to T cells. In this article, we show that short-term treatment with ILDR2-Fc results in long-term durable beneficial effects in the relapsing-remitting experimental autoimmune encephalomyelitis and NOD type 1 diabetes models. ILDR2-Fc also promotes transplant engraftment in a minor mismatch bone marrow transplantation model. ILDR2-Fc displays a unique mode of action, combining immunomodulation, regulation of immune homeostasis, and re-establishment of Ag-specific immune tolerance via regulatory T cell induction. These findings support the potential of ILDR-Fc to provide a promising therapeutic approach for the treatment of autoimmune diseases.
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Antígenos/inmunología , Homeostasis/inmunología , Tolerancia Inmunológica/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Proteínas de la Membrana/inmunología , Animales , Trasplante de Médula Ósea/métodos , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NODRESUMEN
Germline mutations in ATM (encoding the DNA-damage signaling kinase, ataxia-telangiectasia-mutated) increase Familial Pancreatic Cancer (FPC) susceptibility, and ATM somatic mutations have been identified in resected human pancreatic tumors. Here we investigated how Atm contributes to pancreatic cancer by deleting this gene in a murine model of the disease expressing oncogenic Kras (KrasG12D). We show that partial or total ATM deficiency cooperates with KrasG12D to promote highly metastatic pancreatic cancer. We also reveal that ATM is activated in pancreatic precancerous lesions in the context of DNA damage and cell proliferation, and demonstrate that ATM deficiency leads to persistent DNA damage in both precancerous lesions and primary tumors. Using low passage cultures from primary tumors and liver metastases we show that ATM loss accelerates Kras-induced carcinogenesis without conferring a specific phenotype to pancreatic tumors or changing the status of the tumor suppressors p53, p16Ink4a and p19Arf. However, ATM deficiency markedly increases the proportion of chromosomal alterations in pancreatic primary tumors and liver metastases. More importantly, ATM deficiency also renders murine pancreatic tumors highly sensitive to radiation. These and other findings in our study conclusively establish that ATM activity poses a major barrier to oncogenic transformation in the pancreas via maintaining genomic stability.
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Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Animales , Biomarcadores de Tumor , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Daño del ADN , Modelos Animales de Enfermedad , Inestabilidad Genómica , Humanos , Hibridación Fluorescente in Situ , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Neoplasias Pancreáticas/mortalidad , Tolerancia a Radiación/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Leukocyte trafficking into the CNS is a prominent feature driving the immunopathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Blocking the recruitment of inflammatory leukocytes into the CNS represents an exploitable therapeutic target; however, the adhesion molecules that specifically regulate the step of leukocyte diapedesis into the CNS remain poorly understood. We report that CD99 is critical for lymphocyte transmigration without affecting adhesion in a human blood-brain barrier model. CD99 blockade in vivo ameliorated experimental autoimmune encephalomyelitis and decreased the accumulation of CNS inflammatory infiltrates, including dendritic cells, B cells, and CD4(+) and CD8(+) T cells. Anti-CD99 therapy was effective when administered after the onset of disease symptoms and blocked relapse when administered therapeutically after disease symptoms had recurred. These findings underscore an important role for CD99 in the pathogenesis of CNS autoimmunity and suggest that it may serve as a novel therapeutic target for controlling neuroinflammation.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/inmunología , Linfocitos T CD8-positivos/inmunología , Moléculas de Adhesión Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Antígeno 12E7 , Animales , Antígenos CD/fisiología , Linfocitos B , Barrera Hematoencefálica/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/fisiología , Adhesión Celular , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/inmunología , Células Dendríticas , Modelos Animales de Enfermedad , Humanos , Inflamación/inmunología , Inflamación/terapia , RatonesRESUMEN
We evaluated the therapeutic efficacy and mechanisms of action of both mouse and human B7-H4 Immunoglobulin fusion proteins (mB7-H4Ig; hB7-H4Ig) in treating EAE. The present data show that mB7-H4Ig both directly and indirectly (via increasing Treg function) inhibited CD4⺠T-cell proliferation and differentiation in both Th1- and Th17-cell promoting conditions while inducing production of IL-10. B7-H4Ig treatment effectively ameliorated progression of both relapsing (R-EAE) and chronic EAE correlating with decreased numbers of activated CD4⺠T-cells within the CNS and spleen, and a concurrent increase in number and function of Tregs. The functional requirement for Treg activation in treating EAE was demonstrated by a loss of therapeutic efficacy of hB7-H4Ig in R-EAE following inactivation of Treg function either by anti-CD25 treatment or blockade of IL-10. Significant to the eventual translation of this treatment into clinical practice, hB7-H4Ig similarly inhibited the in vitro differentiation of naïve human CD4⺠T-cells in both Th1- and Th17-promoting conditions, while promoting the production of IL-10. B7-H4Ig thus regulates pro-inflammatory T-cell responses by a unique dual mechanism of action and demonstrates significant promise as a therapeutic for autoimmune diseases, including MS.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inmunoglobulinas/farmacología , Interleucina-10/inmunología , Linfocitos T/efectos de los fármacos , Inhibidor 1 de la Activación de Células T con Dominio V-Set/farmacología , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Inmunoglobulinas/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Linfocitos T/inmunología , Inhibidor 1 de la Activación de Células T con Dominio V-Set/inmunologíaRESUMEN
In search of autoantigen-presenting cells that prime the pathogenic autoantibody-inducing Th cells of lupus, we found that CD41(+)CD151(+) cells among Lineage(-) (Lin(-)) CD117(+) (c-Kit(+)) CX3CR1(-) splenocytes depleted of known APCs were most proficient in presenting nuclear autoantigens from apoptotic cells to induce selectively an autoimmune Th17 response in different lupus-prone mouse strains. The new APCs have properties resembling megakaryocyte and/or bipotent megakaryocyte/erythroid progenitors of bone marrow, hence they are referred to as MM cells in this study. The MM cells produce requisite cytokines, but they require contact for optimal Th17 induction upon nucleosome feeding, and can induce Th17 only before undergoing differentiation to become c-Kit(-)CD41(+) cells. The MM cells expand up to 10-fold in peripheral blood of lupus patients and 49-fold in spleens of lupus mice preceding disease activity; they accelerate lupus in vivo and break tolerance in normal mice, inducing autoimmune Th17 cells. MM cells also cause Th17 skewing to foreign Ag in normal mice without Th17-polarizing culture conditions. Several molecules in MM cells are targets for blocking of autoimmunization. This study advances our understanding of lupus pathogenesis and Th17 differentiation biology by characterizing a novel category of APC.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos/inmunología , Células Progenitoras de Megacariocitos/inmunología , Células Th17/inmunología , Adulto , Animales , Presentación de Antígeno/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Diferenciación Celular/inmunología , Separación Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Mutantes , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Th17/citologíaRESUMEN
Tolerance therapy with nucleosomal histone peptides H4(71-94), H4(16-39), or H1'(22-42) controls disease in lupus-prone SNF1 mice. It would be clinically important to determine whether a cocktail of the above epitopes would be superior. Herein, we found that compared with cocktail peptides, H4(71-94) monotherapy more effectively delayed nephritis onset, prolonged lifespan, diminished immunoglobulin G autoantibody levels, reduced autoantigen-specific Th1 and Th17 responses and frequency of T(FH) cells in spleen and the helper ability of autoimmune T cells to B cells, by inducing potent CD8 Treg cells. H4(71-94) therapy was superior in "tolerance spreading," suppressing responses to other autoepitopes, nucleosomes, and ribonucleoprotein. We also developed an in vitro assay for therapeutic peptides (potentially in humans), which showed that H4(71-94), without exogenous transforming growth factor (TGF)-ß, was efficient in inducing stable CD4(+)CD25(+)Foxp3(+) T cells by decreasing interleukin 6 and increasing TGF-ß production by dendritic cells that induced ALK5-dependent Smad-3 phosphorylation (TGF-ß signal) in target autoimmune CD4(+) T cells.
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Autoinmunidad/efectos de los fármacos , Histonas/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Inmunoensayo , Factores Inmunológicos/farmacología , Nefritis Lúpica/tratamiento farmacológico , Péptidos/farmacología , Animales , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Citocinas/inmunología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Combinación de Medicamentos , Epítopos/inmunología , Femenino , Citometría de Flujo , Histonas/síntesis química , Histonas/inmunología , Tolerancia Inmunológica/inmunología , Factores Inmunológicos/síntesis química , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Nucleosomas/inmunología , Nucleosomas/metabolismo , Péptidos/síntesis química , Péptidos/inmunologíaRESUMEN
A recessive mutation named Justy was found that abolishes B lymphopoiesis but does not impair other major aspects of hematopoiesis. Transplantation experiments showed that homozygosity for Justy prevented hematopoietic progenitors from generating B cells but did not affect the ability of bone marrow stroma to support B lymphopoiesis. In bone marrow from mutant mice, common lymphoid progenitors and pre-pro-B cells appeared normal, but cells at subsequent stages of B lymphopoiesis were dramatically reduced in number. Under culture conditions that promoted B lymphopoiesis, mutant pre-pro-B cells remained alive and began expressing the B cell marker CD19 but failed to proliferate. In contrast, these cells were able to generate myeloid or T/NK precursors. Genetic and molecular analysis demonstrated that Justy is a point mutation within the Gon4-like (Gon4l) gene, which encodes a protein with homology to transcriptional regulators. This mutation was found to disrupt Gon4l pre-mRNA splicing and dramatically reduce expression of wild-type Gon4l RNA and protein. Consistent with a role for Gon4l in transcriptional regulation, the levels of RNA encoding C/EBPalpha and PU.1 were abnormally high in mutant B cell progenitors. Our findings indicate that the Gon4l protein is required for B lymphopoiesis and may function to regulate gene expression during this process.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Linfopoyesis/genética , Mutación/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Biosíntesis de Proteínas , Empalme del ARN/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
In recent years, restriction-less recombination cloning systems based on site-specific recombinase with high efficiency have been proven to be very successful. Thus, it is desirable to convert existing conventional vectors to recombination vectors. In this report, we describe the conversion of a set of widely used conventional vectors to Gateway recombination expression vectors. An attB cassette flanked by several restriction enzyme sites was inserted in a cloning vector, and then subcloned into existing vectors to be converted to construct intermediate vectors containing the attB cassette, which were then converted to recombination expression vectors by in vitro recombination. The intermediate vectors generated in this study can be used for releasing the attB cassette to convert other vectors using the same protocol described here. With the increasing number of recombination vectors constructed with this protocol, the likeliness of releasing the attB cassette from an existing vector, rather than synthesizing it with PCR, will increase. The final expression vectors can also be used for releasing the attR cassette for constructing new vectors.