RESUMEN
Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) share a developmental relationship with Foxp3(+) regulatory T cells (T(reg) cells). Here we show that a T(H)-17 population differentiates in the thymus in a manner influenced by recognition of self antigen and by the cytokines IL-6 and transforming growth factor-beta (TGF-beta). Like previously described T(H)-17 cells, the T(H)-17 cells that developed in the thymus expressed the transcription factor RORgamma t and the IL-23 receptor. These cells also expressed alpha(4)beta(1) integrins and the chemokine receptor CCR6 and were recruited to the lung, gut and liver. In the liver, these cells secreted IL-22 in response to self antigen and mediated host protection during inflammation. Thus, T(H)-17 cells, like T(reg) cells, can be selected by self antigens in the thymus.
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Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Interleucina-17/metabolismo , Subgrupos de Linfocitos T/inmunología , Timo/citología , Animales , Linfocitos T CD4-Positivos/citología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inflamación/inmunología , Integrina alfa4beta1/biosíntesis , Interleucina-23/biosíntesis , Interleucina-6/inmunología , Interleucina-6/metabolismo , Interleucinas/metabolismo , Ratones , Ratones Transgénicos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Reacción en Cadena de la Polimerasa , Receptores CCR6/biosíntesis , Receptores de Ácido Retinoico/biosíntesis , Receptores de Hormona Tiroidea/biosíntesis , Subgrupos de Linfocitos T/citología , Timo/inmunología , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Interleucina-22RESUMEN
AIMS/HYPOTHESIS: Validated biomarkers are needed to monitor the effects of immune intervention in individuals with type 1 diabetes. Despite their importance, few options exist for monitoring antigen-specific T cells. Previous reports described a combinatorial approach that enables the simultaneous detection and quantification of multiple islet-specific CD8+ T cell populations. Here, we set out to evaluate the performance of a combinatorial HLA-A2 multimer assay in a multi-centre setting. METHODS: The combinatorial HLA-A2 multimer assay was applied in five participating centres using centralised reagents and blinded replicate samples. In preliminary experiments, samples from healthy donors were analysed using recall antigen multimers. In subsequent experiments, samples from healthy donors and individuals with type 1 diabetes were analysed using beta cell antigen and recall antigen multimers. RESULTS: The combinatorial assay was successfully implemented in each participating centre, with CVs between replicate samples that indicated good reproducibility for viral epitopes (mean %CV = 33.8). For beta cell epitopes, the assay was very effective in a single-centre setting (mean %CV = 18.4), but showed sixfold greater variability across multi-centre replicates (mean %CV = 119). In general, beta cell antigen-specific CD8+ T cells were detected more commonly in individuals with type 1 diabetes than in healthy donors. Furthermore, CD8+ T cells recognising HLA-A2-restricted insulin and glutamate decarboxylase epitopes were found to occur at higher frequencies in individuals with type 1 diabetes than in healthy donors. CONCLUSIONS/INTERPRETATION: Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting.
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Linfocitos T CD8-positivos/metabolismo , Antígeno HLA-A2/metabolismo , Adulto , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Adulto JovenRESUMEN
Allergy and type 1 diabetes are immune mediated diseases that, despite being etiologically distinct, each have inappropriate activation and effector function of antigen-specific T cells in the pathogenic process. Understanding changes in the frequency and phenotype of these cells is critical to improve assessment of disease diagnosis and prognosis and effectively assess immunological response to therapy. In the setting of antigen-specific therapy in allergy and type 1 diabetes, assays to monitor the immunological mechanisms of disease have been improving in recent years, and we are getting closer to an accurate understanding of how the cellular immune response is modulated during treatment. In this review, we summarize the current state of cell-based immune monitoring of antigen therapy trials. We then discuss emerging advances in antigen-specific biomarkers that are transforming our knowledge about allergy and that have the potential to dramatically impact our understanding of T cell-mediated autoimmune diseases, such as type 1 diabetes.
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Antígenos/inmunología , Biomarcadores/análisis , Diabetes Mellitus Tipo 1/terapia , Hipersensibilidad/terapia , Inmunoterapia/métodos , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/inmunología , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/inmunología , Inmunidad Celular/inmunología , Modelos Inmunológicos , Linfocitos T/inmunologíaRESUMEN
As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.
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Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Lentivirus/genética , Virus Sindbis/genética , Proteínas del Envoltorio Viral/genética , Vectores Genéticos/administración & dosificación , Células HEK293 , Humanos , Inmunidad Celular/inmunología , Distribución TisularRESUMEN
BACKGROUND: Checkpoint inhibitors targeting the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) pathway are effective therapies in a range of immunogenic cancer types. Blocking this pathway with an oral therapy could benefit patients through greater convenience, particularly in combination regimens, and allow flexible management of immune-mediated toxicities. METHODS: PD-L1 binding activity was assessed in engineered dimerization and primary cell target occupancy assays. Preclinical antitumor activity was evaluated in ex vivo and in vivo human PD-L1-expressing tumor models. Human safety, tolerability, pharmacokinetics, and biomarker activity were evaluated in an open-label, multicenter, sequential dose-escalation study in patients with advanced solid tumors. Biomarkers evaluated included target occupancy, flow cytometric immunophenotyping, plasma cytokine measurements, and T-cell receptor sequencing. RESULTS: GS-4224 binding caused dimerization of PD-L1, blocking its interaction with PD-1 and leading to reversal of T-cell inhibition and increased tumor killing in vitro and in vivo. The potency of GS-4224 was dependent on the density of cell surface PD-L1, with binding being most potent on PD-L1-high cells. In a phase 1 dose-escalation study in patients with advanced solid tumors, treatment was well tolerated at doses of 400-1,500 mg once daily. Administration of GS-4224 was associated with a dose-dependent increase in plasma GS-4224 exposure and reduction in free PD-L1 on peripheral blood T cells, an increase in Ki67 among the PD-1-positive T-cell subsets, and elevated plasma cytokines and chemokines. CONCLUSIONS: GS-4224 is a novel, orally bioavailable small molecule inhibitor of PD-L1. GS-4224 showed evidence of expected on-target biomarker activity, including engagement of PD-L1 and induction of immune-related pharmacodynamic responses consistent with PD-L1 blockade. TRIAL REGISTRATION NUMBER: NCT04049617.
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Inhibidores de Puntos de Control Inmunológico , Neoplasias , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Antígeno B7-H1 , Receptor de Muerte Celular Programada 1 , Neoplasias/tratamiento farmacológico , Linfocitos T/metabolismoRESUMEN
Follicular helper T (T(FH)) cells, defined by expression of the surface markers CXCR5 and programmed death receptor-1 (PD-1) and synthesis of IL-21, require upregulation of the transcriptional repressor Bcl6 for their development and function in B cell maturation in germinal centers. We have explored the role of B cells and the cytokines IL-6 and IL-21 in the in vivo regulation of Bcl6 expression and T(FH) cell development. We found that T(FH) cells are characterized by a Bcl6-dependent downregulation of P-selectin glycoprotein ligand 1 (PSGL1, a CCL19- and CCL21-binding protein), indicating that, like CXCR5 and PD-1 upregulation, modulation of PSGL1 expression is part of the T(FH) cell program of differentiation. B cells were neither required for initial upregulation of Bcl6 nor PSGL1 downregulation, suggesting these events preceded T-B cell interactions, although they were required for full development of the T(FH) cell phenotype, including CXCR5 and PD-1 upregulation, and IL-21 synthesis. Bcl6 upregulation and T(FH) cell differentiation were independent of IL-6 and IL-21, revealing that either cytokine is not absolutely required for development of Bcl6(+) T(FH) cells in vivo. These data increase our understanding of Bcl6 regulation in T(FH) cells and their differentiation in vivo and identifies a new surface marker that may be functionally relevant in this subset.
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Diferenciación Celular/inmunología , Proteínas de Unión al ADN/fisiología , Bazo/inmunología , Bazo/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Comunicación Celular/inmunología , Cricetinae , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/inmunología , Femenino , Inmunofenotipificación , Cooperación Linfocítica/inmunología , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-bcl-6 , Bazo/citología , Subgrupos de Linfocitos T/citología , Linfocitos T Colaboradores-Inductores/citología , Regulación hacia Arriba/inmunologíaRESUMEN
Renal pathology in systemic lupus erythematosus involves both autoantibody deposition and a cellular inflammatory response, both of which are mediated by effector CD4 T cells. MRL(lpr) mice spontaneously develop massive perivascular infiltrates, but the pathways that regulate the development, trafficking, and effector functions of kidney-infiltrating T cells are poorly defined. To address these questions, we first surveyed inflammatory chemokine protein levels in nephritic kidneys from lupus-prone MRL(lpr) mice. After identifying highly elevated levels of the CXCR3 ligand CXCL9, we found that kidney-infiltrating effectors are enriched for expression of CXCR3, as well as P-selectin ligand and ICOS. Using genetic ablation, we demonstrate that ICOS plays an essential role in the establishment of renal perivascular infiltrates, although a small number of infiltrating cells remain around the blood vessels. Interestingly, though IgG autoantibody production is substantially reduced in Icos(-/-) MRL(lpr) mice, the progression of immune complex glomerulonephritis is only modestly diminished and the production of inflammatory chemokines, such as CXCL9, remains high in the kidney. We find that Icos(-/-) effector cell numbers are only slightly reduced and these have normal expression of CXCR3 and P-selectin ligand with intact migration to CXCL9. However, they have impaired production of inflammatory cytokines and fail to show evidence of efficient proliferation in the kidney. Thus, while dispensable for acquisition of renal trafficking receptor expression, ICOS is strictly required for local inflammatory functions of autoreactive CD4 T cells in murine lupus.
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Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD4-Positivos/inmunología , Quimiotaxis de Leucocito/inmunología , Riñón/inmunología , Lupus Eritematoso Sistémico/inmunología , Animales , Antígenos de Diferenciación de Linfocitos T/metabolismo , Autoanticuerpos/sangre , Autoantígenos/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/metabolismo , Quimiocina CXCL9/inmunología , Quimiocina CXCL9/metabolismo , Citocinas/biosíntesis , Citocinas/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteína Coestimuladora de Linfocitos T Inducibles , Riñón/patología , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos MRL lpr , Ratones Transgénicos , Selectina-P/inmunología , Receptores CCR5/inmunología , Receptores CCR5/metabolismo , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismoRESUMEN
At diagnosis, most people with type 1 diabetes (T1D) produce measurable levels of endogenous insulin, but the rate at which insulin secretion declines is heterogeneous. To explain this heterogeneity, we sought to identify a composite signature predictive of insulin secretion, using a collaborative assay evaluation and analysis pipeline that incorporated multiple cellular and serum measures reflecting ß cell health and immune system activity. The ability to predict decline in insulin secretion would be useful for patient stratification for clinical trial enrollment or therapeutic selection. Analytes from 12 qualified assays were measured in shared samples from subjects newly diagnosed with T1D. We developed a computational tool (DIFAcTO, Data Integration Flexible to Account for different Types of data and Outcomes) to identify a composite panel associated with decline in insulin secretion over 2 years following diagnosis. DIFAcTO uses multiple filtering steps to reduce data dimensionality, incorporates error estimation techniques including cross-validation and sensitivity analysis, and is flexible to assay type, clinical outcome, and disease setting. Using this novel analytical tool, we identified a panel of immune markers that, in combination, are highly associated with loss of insulin secretion. The methods used here represent a potentially novel process for identifying combined immune signatures that predict outcomes relevant for complex and heterogeneous diseases like T1D.
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Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/inmunología , Progresión de la Enfermedad , Secreción de Insulina/fisiología , Adolescente , Adulto , Niño , Biología Computacional , Femenino , Humanos , Hipoglucemiantes/farmacología , Inmunoterapia/métodos , Células Secretoras de Insulina/metabolismo , Masculino , Adulto JovenRESUMEN
BACKGROUND/OBJECTIVES: There is currently no licensed prophylactic or therapeutic vaccine for HSV-2 infection. METHODS: We developed a novel preclinical vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with the potent synthetic TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. The vaccine was tested for immunogenicity and efficacy in pre-clinical models for preventative and therapeutic vaccination. RESULTS: Vaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. Supporting the use of G103 therapeutically, the vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score. CONCLUSIONS: Taken together, the data show that G103 is a viable candidate for development of a novel prophylactic and therapeutic HSV-2 vaccine.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Glucósidos/administración & dosificación , Herpes Genital/prevención & control , Herpes Genital/terapia , Herpesvirus Humano 2/inmunología , Vacunas contra Herpesvirus/inmunología , Lípido A/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Modelos Animales de Enfermedad , Femenino , Cobayas , Herpes Genital/inmunología , Herpesvirus Humano 2/genética , Vacunas contra Herpesvirus/administración & dosificación , Vacunas contra Herpesvirus/aislamiento & purificación , Memoria Inmunológica , Ratones Endogámicos C57BL , Resultado del Tratamiento , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/aislamiento & purificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificaciónRESUMEN
Recognizing an increasing need for biomarkers that predict clinical outcomes in type 1 diabetes (T1D), JDRF, a major funding organization for T1D research, recently instituted the Core for Assay Validation (CAV) to accelerate the translation of promising assays from discovery to clinical implementation via a process of coordinated evaluation of biomarkers. In this model, the CAV facilitates the validation of candidate assay methods as well as qualification of proposed biomarkers for a specific clinical use in well-characterized patients. We describe here a CAV-driven pilot project aimed at identifying biomarkers that predict the rate of decline in beta cell function after diagnosis. In a formalized pipeline, candidate assays are first assessed for general rationale, technical precision, and biological associations in a cross-sectional cohort. Those with the most favorable characteristics are then applied to placebo arm subjects of T1D intervention trials to assess their predictive correlation with beta cell function. We outline a go/no-go process for advancing candidate assays in a defined qualification pipeline that also allows for the discovery of novel predictive biomarker combinations. This strategy could be a model for other collaborative biomarker development efforts in and beyond T1D.
RESUMEN
Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and therefore attractive targets for cancer immunotherapy. We have developed an integration-deficient lentiviral vector termed ID-VP02 that is designed to deliver antigen-encoding nucleic acids selectively to human DCs in vivo. ID-VP02 utilizes a genetically and glycobiologically engineered Sindbis virus glycoprotein to target human DCs through the C-type lectin DC-SIGN (CD209) and also binds to the homologue murine receptor SIGNR1. Specificity of ID-VP02 for antigen-presenting cells in the mouse was confirmed through biodistribution studies showing that following subcutaneous administration, transgene expression was only detectable at the injection site and the draining lymph node. A single immunization with ID-VP02 induced a high level of antigen-specific, polyfunctional effector and memory CD8 T-cell responses that fully protected against vaccinia virus challenge. Upon homologous readministration, ID-VP02 induced a level of high-quality secondary effector and memory cells characterized by stable polyfunctionality and expression of IL-7Rα. Importantly, a single injection of ID-VP02 also induced robust cytotoxic responses against an endogenous rejection antigen of CT26 colon carcinoma cells and conferred both prophylactic and therapeutic antitumor efficacy. ID-VP02 is the first lentiviral vector which combines integration deficiency with DC targeting and is currently being investigated in a phase I trial in cancer patients.
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Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer , Carcinoma/terapia , Neoplasias del Colon/terapia , Células Dendríticas/inmunología , Vectores Genéticos , Inmunoterapia Adoptiva , Lentivirus/genética , Virus Sindbis/genética , Virus Vaccinia/inmunología , Vaccinia/inmunología , Animales , Carcinoma/inmunología , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Ensayos Clínicos Fase I como Asunto , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica , Células Dendríticas/trasplante , Células Dendríticas/virología , Ingeniería Genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Memoria Inmunológica , Lectinas Tipo C/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Unión Proteica , Receptores de Superficie Celular/metabolismo , Receptores de Interleucina-7/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Integración Viral/genéticaRESUMEN
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of T cell tolerance to nuclear antigens. Studies in mice and humans have demonstrated that T cells from individuals with lupus are abnormal. Here, we review the known T cell defects in lupus and their possible biochemical nature, genetic causes, and significance for lupus pathogenesis.
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Lupus Eritematoso Sistémico/inmunología , Linfocitos T/inmunología , Animales , Humanos , Lupus Eritematoso Sistémico/genética , RatonesRESUMEN
Posttranslational modification (PTM) of self-proteins has been shown to elicit clinically relevant immune responses in rheumatoid arthritis and celiac disease. Accumulating evidence suggests that recognition of modified self-proteins may also be important in type 1 diabetes. Our objective was to identify posttranslationally modified GAD65 peptides, which are recognized by subjects with type 1 diabetes, and to assess their disease relevance. We show that citrullination and transglutamination of peptides can enhance their binding to DRB1*04:01, a diabetes-susceptible HLA allele. These and corresponding modifications to amino acids at T-cell contact positions modulated the recognition of multiple GAD65 peptides by self-reactive T cells. Using class II tetramers, we verified that memory T cells specific for these modified epitopes were detectable directly ex vivo in the peripheral blood of subjects with type 1 diabetes at significantly higher frequencies than healthy controls. Furthermore, T cells that recognize these modified epitopes were either less responsive or nonresponsive to their unmodified counterparts. Our findings suggest that PTM contributes to the progression of autoimmune diabetes by eliciting T-cell responses to new epitope specificities that are present primarily in the periphery, thereby circumventing tolerance mechanisms.
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Diabetes Mellitus Tipo 1/inmunología , Epítopos de Linfocito T/inmunología , Glutamato Descarboxilasa/inmunología , Adulto , Secuencia de Aminoácidos , Linfocitos T CD4-Positivos/inmunología , Glutamato Descarboxilasa/metabolismo , Cadenas HLA-DRB1/inmunología , Humanos , Células Secretoras de Insulina/inmunología , Procesamiento Proteico-PostraduccionalRESUMEN
Complement component C4 (C4) is a highly variable complement pathway gene situated â¼500 kb from DRB1 and DQB1, the genes most strongly associated with many autoimmune diseases. Variations in C4 copy number (CN), length, and isotype create a highly diverse gene cluster in which insertion of an endogenous retrovirus in the ninth intron of C4, termed HERV-K(C4), is a notable component. We investigated the relationship between C4 variation/CN and type 1 diabetes. We found that individuals with type 1 diabetes have significantly fewer copies of HERV-K(C4) and that this effect is not solely due to linkage with known major histocompatibility complex class II susceptibility alleles. We show that HERV-K(C4) is a novel marker of type 1 diabetes that accounts for the disease association previously attributed to some key HLA-DQB1 alleles, raising the possibility that this retroviral insertion element contributes to functional protection against type 1 diabetes.
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Complemento C4/genética , Diabetes Mellitus Tipo 1/genética , Retrovirus Endógenos/genética , Predisposición Genética a la Enfermedad , Adolescente , Adulto , Alelos , Niño , Variaciones en el Número de Copia de ADN , Femenino , Dosificación de Gen , Variación Genética , Humanos , Complejo Mayor de Histocompatibilidad/genética , Masculino , Polimorfismo GenéticoRESUMEN
Lentiviral vectors (LVs) are being developed for clinical use in humans for applications including gene therapy and immunotherapy. A safety concern for use of LVs in humans is the generation of replication-competent lentivirus (RCL), which may arise due to recombination between the split genomes of third-generation LVs. Although no RCL has been detected to date, design optimizations that minimize recombination events between split genome vectors would provide an added safety benefit that may further reduce the risk of RCL formation. Here we describe design elements introduced to the gag/pol plasmid with the intention of eliminating psi-gag recombination between the vector genome and gag/pol. These design changes, consisting of codon optimization of the gag/pol sequence and the deletion of the Rev-responsive element, abrogate the requirement for Rev in expression of Gag protein, thus the resulting gag/pol construct being Rev independent (RI gag/pol). We show that generating vector using the RI gag/pol construct has no effect on particle production or transduction titers. The RI and wild-type gag/pol vectors function equivalently as antigen-specific immunotherapy, potently inducing antigen-specific CD8 T cells that protect against challenge with vaccinia virus. Most importantly, the designed RI gag/pol eliminated detectable psi-gag recombination. Interestingly, we detected recombination between the vector genome and gag/pol from regions without sequence homology. Our findings imply that although unpredictable recombination events may still occur, the RI gag/pol design is sufficient to prevent psi-gag recombination.
RESUMEN
The role of specialized follicular helper T (T(FH)) cells in the germinal center has become well recognized, but it is less clear how effector T cells govern the extrafollicular response, the dominant pathway of high-affinity, isotype-switched autoantibody production in the MRL/MpJ-Fas(lpr) (MRL(lpr)) mouse model of lupus. MRL(lpr) mice lacking the Icos gene have impaired extrafollicular differentiation of immunoglobulin (Ig) G(+) plasma cells accompanied by defects in CXC chemokine receptor (CXCR) 4 expression, interleukin (IL) 21 secretion, and B cell helper function in CD4 T cells. These phenotypes reflect the selective loss of a population of T cells marked by down-regulation of P-selectin glycoprotein ligand 1 (PSGL-1; also known as CD162). PSGL-1(lo) T cells from MRL(lpr) mice express CXCR4, localize to extrafollicular sites, and uniquely mediate IgG production through IL-21 and CD40L. In other autoimmune strains, PSGL-1(lo) T cells are also abundant but may exhibit either a follicular or extrafollicular phenotype. Our findings define an anatomically distinct extrafollicular population of cells that regulates plasma cell differentiation in chronic autoimmunity, indicating that specialized humoral effector T cells akin to T(FH) cells can occur outside the follicle.
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Antígenos de Diferenciación de Linfocitos T/inmunología , Autoinmunidad/inmunología , Inmunoglobulina G/biosíntesis , Interleucinas/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/genética , Diferenciación Celular/fisiología , Quimiocinas/inmunología , Modelos Animales de Enfermedad , Centro Germinal/citología , Centro Germinal/inmunología , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles , Lupus Eritematoso Sistémico/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Noqueados , Células Plasmáticas/citología , Células Plasmáticas/fisiología , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: To test the therapeutic effect of DCB-3503, a synthetic compound derived from a natural product that inhibits NF-kappaB, on end-organ disease in the MRL-Fas(lpr) murine model of systemic lupus erythematosus (SLE). METHODS: Eight-week-old female MRL/Fas(lpr) mice were treated intraperitoneally with a low (2 mg/kg) or high (6 mg/kg) dose of DCB-3503 for 10 weeks. Control groups were administered vehicle treatment alone (negative control) or 25 mg/kg cyclophosphamide (positive control). Mice were bled before (8 weeks) and during (13 weeks) treatment, and when they were killed (20 weeks), and serum samples were analyzed for total IgM and IgG levels and autoantibody titers. When the mice were killed, spleen and lymph nodes (axillary, brachial, and cervical) were examined by flow cytometric analysis. The presence of skin and renal disease was determined by histopathologic analysis. RESULTS: DCB-3503 reduced anti-double-stranded DNA and antichromatin autoantibodies and nearly abrogated inflammatory skin disease in MRL/Fas(lpr) mice; however, it had little effect on histologic kidney disease. Treated mice did not have hematologic or hepatic toxicity. These data indicate that end-organ disease in MRL/Fas(lpr) mice responds differentially to NF-kappaB inhibitor. CONCLUSION: DCB-3503 causes significant abrogation of skin disease in MRL/Fas(lpr) mice and may potentially be beneficial in the treatment of inflammatory skin disease in SLE.