ABSTRACT
BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.
Subject(s)
Cancer Vaccines , Glioma , Animals , Glioma/drug therapy , Glioma/genetics , Glioma/pathology , Glutarates , HLA-A2 Antigen/genetics , HLA-DR1 Antigen/genetics , Humans , Immune Checkpoint Inhibitors , Isocitrate Dehydrogenase/genetics , Mice , Mice, Transgenic , Tumor Microenvironment , Up-Regulation , Vaccines, SubunitABSTRACT
Ag-specific immunotherapy is a long-term goal for the treatment of autoimmune diseases; however developing a means of therapeutically targeting autoimmune T cells in an Ag-specific manner has been difficult. Through the engineering of an HLA-DR1 chimeric Ag receptor (CAR), we have produced CD8+ CAR T cells that target CD4+ T cells in an Ag-specific manner and tested their ability to inhibit the development of autoimmune arthritis in a mouse model. The DR1 CAR molecule was engineered to contain CD3ζ activation and CD28 signaling domains and a covalently linked autoantigenic peptide from type II collagen (CII; DR1-CII) to provide specificity for targeting the autoimmune T cells. Stimulation of the DR1-CII CAR T cells by an anti-DR Ab induced cytokine production, indicating that the DR1-CAR functions as a chimeric molecule. In vitro CTL assays using cloned CD4+ T cells as target cells demonstrated that the DR1-CII CAR T cells efficiently recognize and kill CD4+ T cells that are specific for the CII autoantigen. The CTL function was highly specific, as no killing was observed using DR1-restricted CD4+ T cells that recognize other Ags. When B6.DR1 mice, in which autoimmune arthritis had been induced, were treated with the DR1-CII CAR T cells, the CII-specific autoimmune CD4+ T cell response was significantly decreased, autoantibody production was suppressed, and the incidence and severity of the autoimmune arthritis was diminished. These data demonstrate that HLA-DR CAR T cells have the potential to provide a highly specific therapeutic approach for the treatment of autoimmune disease.
Subject(s)
Arthritis, Experimental/therapy , Arthritis, Rheumatoid/therapy , Autoimmune Diseases/therapy , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immunotherapy, Adoptive/methods , Receptors, Chimeric Antigen/genetics , Animals , Arthritis, Experimental/immunology , Arthritis, Rheumatoid/immunology , Autoimmune Diseases/immunology , Cells, Cultured , Cytotoxicity, Immunologic , Disease Models, Animal , Genetic Engineering , HLA-DR1 Antigen/genetics , HLA-DR1 Antigen/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Chimeric Antigen/metabolism , T-Cell Antigen Receptor SpecificityABSTRACT
OBJECTIVES: To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. RESULTS: When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR12xHis) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested. CONCLUSIONS: Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.
Subject(s)
Cell Culture Techniques/methods , HLA-DR1 Antigen/biosynthesis , Recombinant Proteins/biosynthesis , Animals , Bioreactors , Biotechnology/methods , Cell Line , Cell Proliferation , Drosophila , HLA-DR1 Antigen/genetics , Recombinant Proteins/geneticsABSTRACT
The immune system focuses on and responds to very few representative immunodominant epitopes from pathogenic insults. However, due to the complexity of the antigen processing, understanding the parameters that lead to immunodominance has proved difficult. In an attempt to uncover the determinants of immunodominance among several dominant epitopes, we utilized a cell free antigen processing system and allowed the system to identify the hierarchies among potential determinants. We then tested the results in vivo; in mice and in human. We report here, that immunodominance of known sequences in a given protein can change if two or more proteins are being processed and presented simultaneously. Surprisingly, we find that new spacer/tag sequences commonly added to proteins for purification purposes can distort the capture of the physiological immunodominant epitopes. We warn against adding tags and spacers to candidate vaccines, or recommend cleaving it off before using for vaccination.
Subject(s)
Antigen Presentation , Immunodominant Epitopes , Amino Acid Sequence , Animals , Antigen Presentation/genetics , Antigen Presentation/immunology , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , HLA-DR1 Antigen/genetics , HLA-DR1 Antigen/immunology , Humans , Immunodominant Epitopes/genetics , Immunodominant Epitopes/immunology , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/immunology , Mice , Mice, Transgenic , Recombinant Proteins/genetics , Recombinant Proteins/immunology , VaccinationABSTRACT
PURPOSE OF REVIEW: The review provides updates on novel risk markers for the development of pediatric inflammatory uveitis and a severe disease course, on treatment of refractory disease, and on the measurement of visual outcomes. RECENT FINDINGS: There are several new genetic markers, biomarkers, and clinical factors that may influence a child's uveitis disease course. It is important to identify children at risk for poor visual outcomes and who are refractory to traditional therapy. Racial disparities have recently been reported. We describe agents of potential benefit. In addition, we discuss the importance of patient reported outcomes in this population. SUMMARY: Uveitis can lead to vision-threatening complications. Timely and aggressive treatment of children identified to be at risk for a severe uveitis course may lead to improved outcomes.
Subject(s)
Adrenal Cortex Hormones/therapeutic use , Antirheumatic Agents/therapeutic use , Autoimmune Diseases/drug therapy , Uveitis/drug therapy , Adolescent , Black or African American , Aqueous Humor/chemistry , Arthritis, Juvenile/epidemiology , Autoimmune Diseases/epidemiology , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Blood Sedimentation , Child , HLA-B27 Antigen/genetics , HLA-DR1 Antigen/genetics , HLA-DRB1 Chains/genetics , Humans , Methotrexate/therapeutic use , Recurrence , Risk Factors , S100 Proteins/immunology , Treatment Outcome , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Uveitis/epidemiology , Uveitis/genetics , Uveitis/immunology , Visual AcuityABSTRACT
The rapid occurrence of emerging infectious diseases demonstrates an urgent need for a new preclinical experimental model that reliably replicates human immune responses. Here, a new homozygous humanized human leukocyte antigen (HLA)-A11/DR1 transgenic mouse (HLA-A11(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-)) was generated by crossing HLA-A11 transgenic (Tg) mice with HLA-A2(+/+)/DR01(+/+)/H-2-ß2m(-/-)/IAß(-/-) mice. The HLA-A11-restricted immune response of this mouse model was then examined. HLA-A11 Tg mice expressing a chimeric major histocompatibility complex (MHC) molecule comprising the α1, α2, and ß2m domains of human HLA-A11 and the α3 transmembrane and cytoplasmic domains of murine H-2D(b) were generated. The correct integration of HLA-A11 and HLA-DR1 into the genome of the HLA-A11/DR1 Tg mice (which lacked the expression of endogenous H-2-I/II molecules) was then confirmed. Immunizing mice with a recombinant HBV vaccine or a recombinant HIV-1 protein resulted in the generation of IFN-γ-producing cytotoxic T lymphocyte (CTL) and antigen-specific antibodies. The HLA-A11-restricted CTL response was directed at HLA immunodominant epitopes. These mice represent a versatile animal model for studying the immunogenicity of HLA CTL epitopes in the absence of a murine MHC response. The established animal model will also be useful for evaluating and optimizing T cell-based vaccines and for studying differences in antigen processing between mice and humans.
Subject(s)
AIDS Vaccines/immunology , Drug Evaluation, Preclinical/methods , HLA-A11 Antigen/genetics , HLA-DR1 Antigen/genetics , Hepatitis B Vaccines/immunology , Mice, Transgenic , AIDS Vaccines/administration & dosage , Animals , Crosses, Genetic , HIV Antibodies/blood , Hepatitis B Antibodies/blood , Hepatitis B Vaccines/administration & dosage , Homozygote , Humans , Interferon-gamma/metabolism , Models, Animal , T-Lymphocytes/immunologyABSTRACT
Ag presentation by MHC class II (MHC II) molecules to CD4(+) T cells plays a key role in the regulation of the adaptive immune response. Loading of antigenic peptides onto MHC II is catalyzed by HLA-DM (DM), a nonclassical MHC II molecule. The mechanism of DM-facilitated peptide loading is an outstanding problem in the field of Ag presentation. In this study, we systemically explored possible kinetic mechanisms for DM-catalyzed peptide association by measuring real-time peptide association kinetics using fluorescence polarization assays and comparing the experimental data with numerically modeled peptide association reactions. We found that DM does not facilitate peptide association by stabilizing peptide-free MHC II against aggregation. Moreover, DM does not promote transition of an inactive peptide-averse conformation of MHC II to an active peptide-receptive conformation. Instead, DM forms an intermediate with MHC II that binds peptide with faster kinetics than MHC II in the absence of DM. In the absence of peptides, interaction of MHC II with DM leads to inactivation and formation of a peptide-averse form. This study provides novel insights into how DM efficiently catalyzes peptide loading during Ag presentation.
Subject(s)
Antigen Presentation , HLA-D Antigens/chemistry , HLA-DR1 Antigen/chemistry , Models, Chemical , Peptides/chemistry , Amino Acid Sequence , Binding Sites , Fluorescence Polarization Immunoassay , Gene Expression Regulation , HLA-D Antigens/genetics , HLA-DR1 Antigen/genetics , Humans , Kinetics , Molecular Sequence Data , Peptides/genetics , Protein Binding , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Signal Transduction , SolutionsABSTRACT
Peptides bind MHC class II molecules through a thermodynamically nonadditive process consequent to the flexibility of the reactants. Currently, how the specific outcome of this binding process affects the ensuing epitope selection needs resolution. Calorimetric assessment of binding thermodynamics for hemagglutinin 306-319 peptide variants to the human MHC class II HLA-DR1 (DR1) and a mutant DR1 reveals that peptide/DR1 complexes can be formed with different enthalpic and entropic contributions. Complexes formed with a smaller entropic penalty feature circular dichroism spectra consistent with a non-compact form, and molecular dynamics simulation shows a more flexible structure. The opposite binding mode, compact and less flexible, is associated with greater entropic penalty. These structural variations are associated with rearrangements of residues known to be involved in HLA-DR (DM) binding, affinity of DM for the complex, and complex susceptibility to DM-mediated peptide exchange. Thus, the thermodynamic mechanism of peptide binding to DR1 correlates with the structural rigidity of the complex, and DM mediates peptide exchange by "sensing" flexible complexes in which the aforementioned residues are rearranged at a higher frequency than in more rigid ones.
Subject(s)
HLA-D Antigens/metabolism , HLA-DR1 Antigen/metabolism , Hemagglutinins/metabolism , Circular Dichroism , HLA-D Antigens/immunology , HLA-DR1 Antigen/genetics , HLA-DR1 Antigen/immunology , Hemagglutinins/genetics , Hemagglutinins/immunology , Humans , Molecular Dynamics Simulation , Protein Binding/immunology , Protein Conformation , Surface Plasmon Resonance , Temperature , ThermodynamicsABSTRACT
BACKGROUND: Active immunization against Aß was reported to have a therapeutic effect in murine models of Alzheimer's disease. Clinical Aß vaccination trial AN1792 was interrupted due to the development in 6 % of the patients of meningoencephalitis likely involving pro-inflammatory CD4(+) T cells. However, the potential implication of auto-aggressive anti-Aß CD8(+) T cells has been poorly investigated. METHODS: Potential MHC-I-restricted Aß-derived epitopes were first analyzed for their capacity to recruit functional CD8(+) T cell responses in mouse models. Their impact on migration of CD8(+) T cells into the brain parenchyma and potential induction of meningoencephalitis and/or neuronal damage was investigated upon vaccination in the APPPS1 mouse model of AD. RESULTS: We identified one nonamer peptide, Aß33-41, which was naturally processed and presented in association with H-2-D(b) molecule on neurons and CD11b(+) microglia. Upon optimization of anchor residues for enhanced binding to H-2-D(b), immunization with the modified Aß33-41NP peptide elicited Aß-specific IFNγ-secreting CD8(+) T cells, which are cytotoxic towards Aß-expressing targets. Whereas T cell infiltration in the brain of APPPS1 mice is dominated by CD3(+)CD8(-) T cells and increases with disease evolution between 4 and 7 months of age, a predominance of CD3(+)CD8(+) over CD3(+)CD8(-) cells was observed in 6- to 7-month-old APPPS1 but not in WT animals, only after vaccination with Aß33-41NP. The number of CD11b(+) mononuclear phagocytes, which significantly increases with age in the brain of APPPS1 mice, was reduced following immunization with Aß33-41NP. Despite peripheral activation of Aß-specific CD8(+) cytotoxic effectors and enhanced infiltration of CD8(+) T cells in the brain of Aß33-41NP-immunized APPPS1 mice, no clinical signs of severe autoimmune neuroinflammation were observed. CONCLUSIONS: Altogether, these results suggest that Aß-specific CD8(+) T cells are not major contributors to meningoencephalitis in response to Aß vaccination.
Subject(s)
Alzheimer Disease/complications , Alzheimer Disease/therapy , Amyloid beta-Peptides/metabolism , Encephalitis/etiology , Encephalitis/pathology , Immunotherapy, Active/adverse effects , Alzheimer Disease/genetics , Alzheimer Disease/immunology , Amyloid beta-Peptides/immunology , Amyloid beta-Protein Precursor/genetics , Animals , Antibodies/analysis , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Epitopes, T-Lymphocyte/immunology , HLA-A2 Antigen/genetics , HLA-A2 Antigen/metabolism , HLA-DR1 Antigen/genetics , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/immunology , Microglia/metabolism , Mutation/genetics , Peptide Fragments/immunology , Presenilin-1/geneticsABSTRACT
BACKGROUND: Rheumatic heart disease (RHD), the only long term consequence of acute rheumatic fever, remains a leading cause of morbidity and mortality among young adults in Uganda. An inherited susceptibility to acute rheumatic fever centers around the major histocompatibility class II human leucocyte antigens. However, there is paucity of data from sub-Saharan Africa. This study compares the frequency of HLA class II DR alleles between RHD cases and normal controls in Uganda. METHODS: One hundred ninety-nine participants including 96 established RHD cases aged 5-60 years and 103 age and sex matched normal controls were recruited for participation. DNA was manually extracted from buffy coat samples and HLA analysis was performed. HLA-DR allelic frequency comparison between cases and controls were estimated using conditional logistic regression with 95% confidence intervals. P -values were corrected for multiple hypothesis testing. RESULTS: 199 participants (103 female, 51.8%) completed the study. The mean (SD) age in years for cases and controls were 29.6 (10.2) and 29(18), respectively. After conditional logistic regression and multiple hypothesis testing, HLA-DR1was associated with a decreased risk of RHD (OR = 0.42, CI 0.21-085, P = 0.01, Corrected P value (PC) = 0.09,) while HLA-DR11 was associated with increased risk of RHD (OR = 3.31, CI 1.57-6.97, P = <0.001, Pc < 0.001). No other significant associations were found. CONCLUSION: In this first study of HLA genetic susceptibility to RHD in Uganda, HLA- DR1 was more common in normal controls while HLA- DR11 was more common among RHD cases suggesting a disease susceptibility association. In future studies, high resolution HLA analysis and genome wide studies should be carried out to confirm this pattern.
Subject(s)
HLA-DR Serological Subtypes/genetics , HLA-DR1 Antigen/genetics , Rheumatic Heart Disease/genetics , Adolescent , Adult , Case-Control Studies , Child , Child, Preschool , Female , Gene Frequency , Genetic Predisposition to Disease , Humans , Logistic Models , Male , Middle Aged , Odds Ratio , Rheumatic Heart Disease/immunology , Risk Factors , Uganda , Young AdultABSTRACT
INTRODUCTION: T cells orchestrate joint inflammation in rheumatoid arthritis (RA), yet they are difficult to study due to the small numbers of antigen-specific cells. The goal of this study was to characterize a new humanized model of autoimmune arthritis and to describe the phenotypic and functional changes that occur in autoimmune T cells following the induction of pathological events. METHODS: We developed a double transgenic mouse containing both the HLA-DR1 transgene and an HLA-DR1-restricted collagen-specific TCR in order to obtain large numbers of antigen-specific T cells that can be used for immunologic studies. RESULTS: In vitro, CII-specific T cells from this mouse proliferated vigorously in response to the CII immunodominant peptide A2 and the cells altered their phenotype to become predominately CD62Llow and CD44high "activated" T cells. The response was accompanied by the production of Th1, Th2, and Th17-type cytokines. Following immunization with bovine CII/CFA, these mice develop an accelerated arthritis compared to single transgenic HLA-DR1 mice. On the other hand, when the mice were treated orally with the analog peptide A12, (a suppressive analog of collagen we have previously described), arthritis was significantly suppressed, despite the fact that >90% of the CD4+ T cells express the TCR Tg. In GALT tissues taken from the A12-treated mice, IL-2, IFN-γ, and IL-17 production to the autoimmune collagen determinant dropped while high levels of IL-10 and IL-4 were produced. CONCLUSIONS: We have developed a humanized model of autoimmune arthritis that will be useful for the study of T cell directed therapies as well as T cell mediated mechanisms of autoimmune diseases.
Subject(s)
Arthritis, Experimental/immunology , Arthritis, Rheumatoid/immunology , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Cytokines/immunology , Flow Cytometry , HLA-DR1 Antigen/genetics , Humans , Immunophenotyping , Mice , Mice, Transgenic , Phenotype , Receptors, Antigen, T-Cell/geneticsABSTRACT
Genetic, molecular and biological studies indicate that rheumatoid arthritis (RA), a severe arthritic disorder affecting approximately 1% of the population in developed countries, is caused by an upper urinary tract infection by the microbe, Proteus mirabilis. Elevated levels of specific antibodies against Proteus bacteria have been reported from 16 different countries. The pathogenetic mechanism involves six stages triggered by cross-reactive autoantibodies evoked by Proteus infection. The causative amino acid sequences of Proteus namely, ESRRAL and IRRET, contain arginine doublets which can be acted upon by peptidyl arginine deiminase thereby explaining the early appearance of anti-citrullinated protein antibodies in patients with RA. Consequently, RA patients should be treated early with anti-Proteus antibiotics as well as biological agents to avoid irreversible joint damages.
Subject(s)
Arthritis, Rheumatoid/microbiology , Proteus Infections/microbiology , Urinary Tract Infections/microbiology , Antibodies, Bacterial/blood , Arthritis, Rheumatoid/etiology , Arthritis, Rheumatoid/genetics , Autoantibodies/blood , Chondrocytes/immunology , Cross Reactions , HLA-DR1 Antigen/genetics , HLA-DR1 Antigen/metabolism , Humans , Lymph Nodes/immunology , Proteus Infections/complications , Proteus mirabilis/pathogenicity , Urinary Tract Infections/complicationsABSTRACT
Regulatory T cells (Tregs) are critical homeostatic components in preventing the development of autoimmunity, and are a major focus for their therapeutic potential for autoimmune diseases. To enhance the efficacy of Tregs in adoptive therapy, we developed a strategy for generating engineered Tregs that have the capacity to target autoimmune T cells in an Ag-specific manner. Using a retroviral expression system encoding Foxp3 and HLA-DR1 covalently linked to the immunodominant peptide of the autoantigen type II collagen (DR1-CII), naive T cells were engineered to become Tregs that express DR1-CII complexes on their surface. When these cells were tested for their ability to prevent the development of collagen induced arthritis, both the engineered DR1-CII-Foxp3 and Foxp3 only Tregs significantly reduced the severity and incidence of disease. However, the mechanism by which these two populations of Tregs inhibited disease differed significantly. Disease inhibition by the DR1-CII-Foxp3 Tregs was accompanied by significantly lower numbers of autoimmune CII-specific T cells in vivo and lower levels of autoantibodies in comparison with engineered Tregs expressing Foxp3 alone. In addition, the numbers of IFN-γ- and IL-17-expressing T cells in mice treated with DR1-CII-Foxp3 Tregs were also significantly reduced in comparison with mice treated with Foxp3 engineered Tregs or vector control cells. These data indicate that the coexpression of class II autoantigen-peptide complexes on Tregs provides these cells with a distinct capacity to regulate autoimmune T cell responses that differs from that used by conventional Tregs.
Subject(s)
Gene Expression , HLA-DR1 Antigen/genetics , HLA-DR1 Antigen/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Animals , Arthritis/genetics , Arthritis/immunology , Arthritis/prevention & control , Arthritis, Experimental , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Autoimmune Diseases/prevention & control , Autoimmunity , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Line , Cell Survival , Collagen Type II/genetics , Collagen Type II/immunology , Collagen Type II/metabolism , Cytokines/immunology , Cytokines/metabolism , Gene Order , Genetic Vectors/genetics , Humans , Mice , Mice, Transgenic , Peptides/immunology , Phenotype , Retroviridae/genetics , Transduction, GeneticABSTRACT
Hepatitis B virus (HBV) persistence may be due to impaired HBV-specific immune responses being unable to eliminate efficiently or cure infected hepatocytes. The immune mechanisms that lead to HBV persistence have not been completely identified, and no appropriate animal model is available for such studies. Therefore, we established a chronic HBV infection model in a mouse strain with human leukocyte antigen A2/DR1 (HLA-A2/DR1) transgenes and an H-2 class I/class II knockout. The liver of these mice was transduced with adeno-associated virus serotype 2/8 (AAV2/8) carrying a replication-competent HBV DNA genome. In all AAV2/8-transduced mice, hepatitis B virus surface antigen, hepatitis B virus e antigen, and HBV DNA persisted in serum for at least 1 year. Viral replication intermediates and transcripts were detected in the livers of the AAV-injected mice. The hepatitis B core antigen was expressed in 60% of hepatocytes. No significant inflammation was observed in the liver. This was linked to a higher number of regulatory T cells in liver than in controls and a defect in HBV-specific functional T-cell responses. Despite the substantial tolerance resulting from expression of HBV antigens in hepatocytes, we succeeded in priming functional HBV-specific T-cell responses in peripheral tissues, which subsequently reached the liver. This AAV2/8-HBV-transduced HLA-A2/DR1 murine model recapitulates virological and immunological characteristics of chronic HBV infection, and it could be useful for the development of new treatments and immune-based therapies or therapeutic vaccines for chronic HBV infections.
Subject(s)
Disease Models, Animal , HLA-A2 Antigen/metabolism , HLA-DR1 Antigen/metabolism , Hepatitis B virus/pathogenicity , Virus Replication , Animals , DNA, Viral/blood , Dependovirus/genetics , Female , Gene Deletion , Genetic Vectors , H-2 Antigens/genetics , HLA-A2 Antigen/genetics , HLA-DR1 Antigen/genetics , Hepatitis B Surface Antigens/blood , Hepatitis B e Antigens/blood , Hepatitis B virus/physiology , Humans , Liver/virology , Male , Mice , Mice, Knockout , Mice, Transgenic , TransgenesABSTRACT
The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. However, while it has long been the primary molecular target of metastatic prostate cancer therapies, it has not been explored as an immunotherapeutic target. In particular, the AR ligand-binding domain (LBD) is a potentially attractive target, as it has an identical sequence among humans as well as among multiple species, providing a logical candidate for preclinical evaluation. In this report, we evaluated the immune and anti-tumor efficacy of a DNA vaccine targeting the AR LBD (pTVG-AR) in relevant rodent preclinical models. We found immunization of HHDII-DR1 mice, which express human HLA-A2 and HLA-DR1, with pTVG-AR augmented AR LBD HLA-A2-restricted peptide-specific, cytotoxic immune responses in vivo that could lyse human prostate cancer cells. Using an HLA-A2-expressing autochthonous model of prostate cancer, immunization with pTVG-AR augmented HLA-A2-restricted immune responses that could lyse syngeneic prostate tumor cells and led to a decrease in tumor burden and an increase in overall survival of tumor-bearing animals. Finally, immunization decreased prostate tumor growth in Copenhagen rats that was associated with a Th1-type immune response. These data show that the AR is as a prostate cancer immunological target antigen and that a DNA vaccine targeting the AR LBD is an attractive candidate for clinical evaluation.
Subject(s)
Prostatic Neoplasms/therapy , Receptors, Androgen/immunology , Adenocarcinoma/prevention & control , Animals , Cancer Vaccines/therapeutic use , Cell Line, Tumor , Cytotoxicity, Immunologic , HLA-A2 Antigen/genetics , HLA-DR1 Antigen/genetics , Humans , Immunization , Male , Mice , Mice, Transgenic , Prostatic Neoplasms/immunology , Prostatic Neoplasms/mortality , Prostatic Neoplasms/prevention & control , Protein Structure, Tertiary , Rats , Vaccines, DNA/therapeutic useABSTRACT
BACKGROUND: Renal transplant patients have a higher incidence of non-melanoma skin cancer (NMSC). Previous studies hypothesized that human leukocyte antigen (HLA), especially types DR1, DR4, and DR7, may influence the incidence of these tumors. This study investigates the association between NMSC and the presence of HLA DR1, DR4, and DR7 in renal transplant patients in southern Brazil. METHODS: In a historical cohort study, 1032 patients who underwent renal transplantation during the period from January 1993 to December 2006 were examined to identify occurrences of NMSC and HLA status prior to transplant. RESULTS: Of the 1032 patients examined, 59 (5.71%) developed NMSC (squamous cell carcinoma [SCC]: 2.42%; basal cell carcinoma [BCC]: 1.74%; both: 1.55%). The presence of HLA DR1 was associated with a higher probability of developing any NMSC and particularly with developing BCC (P < 0.05). There was no statistically significant association between the presence of HLA DR4 or DR7 and the occurrence of NMSC in this sample. CONCLUSIONS: HLA DR1 appears to be associated with the development of BCC, as well as with the higher number of NMSC lesions in renal transplant patients. This study supports the trend to associate the DR1 allele with BCC and not with SCC.
Subject(s)
Carcinoma, Basal Cell/genetics , HLA-DR1 Antigen/genetics , Kidney Transplantation , Skin Neoplasms/genetics , Adult , Brazil/epidemiology , Carcinoma, Basal Cell/epidemiology , Carcinoma, Squamous Cell/epidemiology , Carcinoma, Squamous Cell/genetics , Cohort Studies , Female , Genetic Predisposition to Disease/epidemiology , Genetic Predisposition to Disease/genetics , Humans , Incidence , Kidney Transplantation/statistics & numerical data , Male , Prevalence , Retrospective Studies , Risk Factors , Skin Neoplasms/epidemiologyABSTRACT
BACKGROUND AND OBJECTIVES: The HLA system plays a central role in the distinction between self antigens and non-self antigens. This study aimed to investigate the association between HLA genes and the outcomes of patients with ANCA-associated vasculitis (AAV). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study recruited 152 consecutive Chinese patients with AAV. The predictive value of the HLA alleles for renal outcome, response to treatment, and all-cause mortality were analyzed. RESULTS: The proportion of patients with treatment failure was significantly higher in DRB1*0405-positive patients than in DRB1*0405-negative patients (41.7% versus 12.9%; P=0.008; corrected P=0.02). After adjusting for the other potential predictors, DRB1*0405 was still an independent predictor for the poor response to treatment (hazard ratio [HR], 5.91; 95% confidence interval [95% CI], 1.23-28.52; P=0.03). Renal survival was significantly worse in patients with DRB1*0405 than those without DRB1*0405 (P<0.001; corrected P<0.001). After adjusting for the other potential predictors, DRB1*0405 was still an independent predictor for ESRD (HR, 5.50; 95% CI, 2.18-13.88; P<0.001). The probability of all-cause mortality in patients with DPB1*0402 was significantly higher than those without DPB1*0402 (P=0.02; corrected P=0.04). After adjusting for the other potential predictors, DPB1*0402 was still an independent predictor for all-cause mortality (HR, 2.52; 95% CI, 1.21-5.28; P=0.01). CONCLUSIONS: In AAV patients, DRB1*0405 might be an independent risk factor for the poor response to treatment and the deterioration of renal function, whereas DPB1*0402 might be an independent risk factor for all-cause mortality.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/genetics , HLA-DR1 Antigen/genetics , Kidney Failure, Chronic/genetics , Adolescent , Adrenal Cortex Hormones/therapeutic use , Adult , Aged , Aged, 80 and over , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/mortality , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/physiopathology , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/therapy , Chi-Square Distribution , China , Disease Progression , Drug Therapy, Combination , Female , Gene Frequency , Genetic Predisposition to Disease , HLA-DP beta-Chains/genetics , HLA-DRB1 Chains/genetics , Humans , Immunosuppressive Agents/therapeutic use , Kaplan-Meier Estimate , Kidney/physiopathology , Kidney Failure, Chronic/immunology , Kidney Failure, Chronic/mortality , Kidney Failure, Chronic/physiopathology , Kidney Failure, Chronic/therapy , Logistic Models , Male , Middle Aged , Multivariate Analysis , Phenotype , Plasma Exchange , Proportional Hazards Models , Risk Assessment , Risk Factors , Time Factors , Treatment Failure , Young AdultABSTRACT
INTRODUCTION: We used DR1 transgenic mice and covalently linked DR1 multimers to characterize analog-specific inhibitory T cells in collagen-induced arthritis (CIA). Because of the low numbers of antigen-specific T cells in wild-type mice, functional T-cell studies in autoimmune arthritis have been challenging. The use of T-cell receptor (TCR) transgenic mice has provided useful information, but such T cells may not represent the heterogeneous T-cell response that occurs in natural settings. Our focus was to develop tools to identify and characterize the population of immunoregulatory T cells induced in wild-type mice by an analog peptide of CII259-273, which contains amino acid substitutions at positions 263 (N) and 266 (D) (analog peptide A12). METHODS: DR1 multimers, developed by loading empty class II molecules with exogenous peptide, provide a method for visualizing antigen-specific T cells with flow cytometry. However, the low binding avidity of A12 for the major histocompatibility complex (MHC) made this strategy untenable. To overcome this problem, we generated DR1 multimers in which the analog peptide A12 was covalently linked, hoping that the low-avidity analog would occupy enough binding clefts to allow detection of the responsive T cells. RESULTS: Staining with the tetramer revealed that A12-specific T cells were readily detectable at 10 days after immunization. These CD4(+) T cells are a highly selective subset of the TCR repertoire and have a limited clonality. Analysis of cytokine expression showed that cells detected by tetramer (A12) expressed primarily suppressive cytokines (interleukin-4 (IL-4) and IL-10) in response to collagen, compared with control cells. Although they did not express Fox-p3, they were extremely effective in preventing and suppressing inflammatory arthritis. CONCLUSIONS: In summary, our studies showed that the use of covalently linked multimers allows characterization of analog-specific T cells that are otherwise difficult to detect. The suppressive character of the analog-specific T-cell response suggests that these cells attenuate autoimmunity and differ significantly in phenotype from the inflammatory T cells predominantly found in arthritic joints. Such reagents will become powerful tools to study T-cell responses in RA patients in upcoming clinical trials.
Subject(s)
Arthritis, Experimental/immunology , Collagen Type II/immunology , HLA-DR1 Antigen , T-Lymphocytes/immunology , Animals , Arthritis, Experimental/genetics , Arthritis, Rheumatoid/immunology , Flow Cytometry , HLA-DR1 Antigen/genetics , HLA-DR1 Antigen/immunology , Humans , Mice , Mice, Knockout , Mice, TransgenicABSTRACT
Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT+ (neuT+) triple transgenic mice represent an improvement over neuT+ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85-94) (p85) CTL and HER-2(776-790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT+ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8+ CTLs and CD4+ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4+ and CD8+ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4+ and CD8+ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.
Subject(s)
Diphtheria Toxin/pharmacology , Interleukin-2/pharmacology , Mammary Neoplasms, Experimental/therapy , Receptor, ErbB-2/immunology , Vaccination/methods , Animals , Antineoplastic Agents/immunology , Antineoplastic Agents/pharmacology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/administration & dosage , Cancer Vaccines/immunology , Cell Line, Tumor , Diphtheria Toxin/immunology , Female , HLA-A2 Antigen/genetics , HLA-A2 Antigen/immunology , HLA-DR1 Antigen/genetics , HLA-DR1 Antigen/immunology , Humans , Immune Tolerance/drug effects , Immune Tolerance/immunology , Immunosuppressive Agents/immunology , Immunosuppressive Agents/pharmacology , Interleukin-2/immunology , Male , Mammary Neoplasms, Experimental/immunology , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Rats , Receptor, ErbB-2/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , Tumor Burden/drug effects , Tumor Burden/immunology , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
Helper T-cell activation generally requires the coreceptor CD4, which binds MHC class II molecules. A remarkable feature of the CD4-MHC class II interaction is its exceptionally low affinity, which ranges from K(D) = â¼200 µM to >2 mM. Investigating the biological role of the much lower affinity of this interaction than those of other cell-cell recognition molecules will require CD4 mutants with enhanced binding to MHC class II for testing in models of T-cell development. To this end, we used in vitro-directed evolution to increase the affinity of human CD4 for HLA-DR1. A mutant CD4 library was displayed on the surface of yeast and selected using HLA-DR1 tetramers or monomers, resulting in isolation of a CD4 clone containing 11 mutations. Reversion mutagenesis showed that most of the affinity increase derived from just two substitutions, Gln40Tyr and Thr45Trp. A CD4 variant bearing these mutations bound HLA-DR1 with K(D) = 8.8 µM, compared with >400 µM for wild-type CD4. To understand the basis for improved affinity, we determined the structure of this CD4 variant in complex with HLA-DR1 to 2.4 Å resolution. The structure provides an atomic-level description of the CD4-binding site on MHC class II and reveals how CD4 recognizes highly polymorphic HLA-DR, -DP, and -DQ molecules by targeting invariant residues in their α2 and ß2 domains. In addition, the CD4 mutants reported here constitute unique tools for probing the influence of CD4 affinity on T-cell activation and development.