RESUMO
IMPORTANCE: HIV-1-specific CD8+ T cells are anticipated to become effector cells for curative treatment using the "shock and kill" approach in people living with HIV-1 (PLWH) under combined antiretroviral therapy (cART). Previous studies demonstrated that the frequency of HIV-1-specific CD8+ T cells is reduced under cART and their functional ability remains impaired. These studies analyzed T-cell responses to a small number of HIV-1 epitopes or overlapping HIV-1 peptides. Therefore, the features of CD8+ T cells specific for HIV-1 epitopes under cART remain only partially clarified. Here, we analyzed CD8+ T cells specific for 63 well-characterized epitopes in 90 PLWH. We demonstrated that CD8+ T cells specific for large numbers of HIV-1 epitopes were maintained in an epitope-dependent fashion under long-term cART and that long-term cART enhanced or restored the ability of HIV-1-specific T cells to proliferate in vitro. This study implies that some HIV-1-specific T cells would be useful as effector cells for curative treatment.
Assuntos
Fármacos Anti-HIV , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD8-Positivos , Epitopos de Linfócito T , Infecções por HIV , HIV-1 , Humanos , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , HIV-1/imunologiaRESUMO
A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant-free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)-restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell-penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor-specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat12.5 nanofiber) increased antigen cross-presentation by bone marrow-derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat12.5 nanofibers exhibited increased presentation of the H2kb-epitope (reminiscent for cross-presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor-infiltrating IFN-γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber-based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.
Assuntos
Vacinas Anticâncer/imunologia , Peptídeos Penetradores de Células/farmacologia , Imunidade/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Adjuvantes Imunológicos/farmacologia , Animais , Células da Medula Óssea/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Vacinas Anticâncer/farmacologia , Peptídeos Penetradores de Células/química , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Humanos , Imunidade/imunologia , Interferon gama/genética , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Nanofibras/química , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
Tofacitinib is an oral small molecule targeting the intracellular Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways approved for the treatment of active rheumatoid arthritis (RA). We investigated the effects of tofacitinib on the response of RA lymphocytes to B and T cell collagen epitopes in their native and post-translationally modified forms. In particular, peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy subjects were cultured with type II collagen peptides (T261-273, B359-369, carT261-273, citB359-369) or with phorbol myristate acetate (PMA)/ionomycin/CD40L in the presence or absence of 100 nM tofacitinib for 20 h and analyzed by fluorescence activated cell sorter (FACS). Cultures without brefeldin A were used for cytokine supernatant enzyme-linked immunosorbent assay (ELISA) analysis. Tofacitinib down-regulated inflammatory cytokines by stimulated B [interleukin (IL)-6 and tumor necrosis factor (TNF)-α] and T [interferon (IFN)-γ, IL-17 or TNF-α] cells in the short term, while a significant reduction of IL-17 and IL-6 levels in peripheral blood mononuclear cell (PBMC) supernatant was also observed. IL-10 was significantly reduced in collagen-stimulated B cells from patients with RA and increased in controls, thus mirroring an altered response to collagen self-epitopes in RA. Tofacitinib partially prevented the IL-10 down-modulation in RA B cells stimulated with collagen epitopes. In conclusion, the use of tofacitinib exerts a rapid regulatory effect on B cells from patients with RA following stimulation with collagen epitopes while not reducing inflammatory cytokine production by lymphocytes.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Linfócitos/efeitos dos fármacos , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Idoso , Artrite Reumatoide/metabolismo , Colágeno Tipo II/metabolismo , Citocinas/metabolismo , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/metabolismo , Feminino , Humanos , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Janus Quinases/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. RESULTS: Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-ß, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28 < 2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells. CONCLUSIONS: Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterisation of antigen-specific T cells in ex vivo patient samples including RA 'at risk' subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Citrulina/uso terapêutico , Antígenos de Histocompatibilidade Classe II/metabolismo , Adulto , Idoso , Artrite Reumatoide/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrinogênio/metabolismo , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Pirofosfatases/metabolismo , Vimentina/uso terapêuticoRESUMO
Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells, but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation, the process by which pathogen Ags are internalized, degraded, and presented by MHC class I, is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article, we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs, dendritic cells and macrophages, and CD4 T cells, three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities, reducing NADPH oxidase 2 activation, and lowering phagolysosomal reactive oxygen species production and pH, which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell-mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification, to our knowledge, of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Catepsinas/metabolismo , Apresentação Cruzada/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Endossomos/efeitos dos fármacos , Endossomos/imunologia , Endossomos/fisiologia , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Hidrólise/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/virologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Immunoactivation depends upon the antigen potential to modulate T-cell repertoires. The present study has enumerated the effect of 61 kDa recombinant Leishmania donovani co-factor-independent phosphoglycerate mutase (rLd-iPGAM) on mononuclear cells of healthy and treated visceral leishmaniasis subjects as well as on THP-1 cell line. rLd-iPGAM stimulation induced higher expression of interleukin-1ß (IL-1ß) in the phagocytic cell, its receptor and CD69 on T-cell subsets. These cellular activations resulted in upregulation of host-protective cytokines IL-2, IL-12, IL-17, tumour necrosis factor-α and interferon-γ, and downregulation of IL-4, IL-10 and tumour growth factor-ß. This immune polarization was also evidenced by upregulation of nuclear factor-κ light-chain enhancer of activated B cells p50 and regulated expression of suppressor of mother against decapentaplegic protein-4. rLd-iPGAM stimulation also promoted lymphocyte proliferation and boosted the leishmaniacidal activity of macrophages by upregulating reactive oxygen species. It also induced 1·8-fold higher release of nitric oxide (NO) by promoting the transcription of inducible nitric oxide synthase gene. Besides, in silico analysis suggested the presence of major histocompatibility complex class I and II restricted epitopes, which can proficiently trigger CD8+ and CD4+ cells, respectively. This study reports rLd-iPGAM as an effective immunoprophylactic agent, which can be used in future vaccine design.
Assuntos
Epitopos de Linfócito T/imunologia , Leishmania donovani/enzimologia , Leishmania donovani/imunologia , Macrófagos/imunologia , Fosfoglicerato Mutase/imunologia , Proteínas Recombinantes/farmacologia , Linhagem Celular , Coenzimas/deficiência , Coenzimas/genética , Simulação por Computador , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Epitopos de Linfócito T/efeitos dos fármacos , Genes MHC Classe I/imunologia , Genes MHC da Classe II/imunologia , Humanos , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/imunologia , Leishmaniose Visceral/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/parasitologia , Subunidade p50 de NF-kappa B/efeitos dos fármacos , Subunidade p50 de NF-kappa B/genética , Óxido Nítrico , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Th1RESUMO
Japanese cedar pollinosis is a seasonal allergic disease caused by two major pollen allergens: Cry j 1 and Cry j 2 antigens. To develop an oral vaccine to treat pollinosis, we constructed recombinant Lactococcus lactis harboring the gene encoding fused T cell epitopes from the Cry j 1 and Cry j 2 antigens. The recombinant T cell epitope peptide was designed to contain the fused cholera toxin B subunit as an adjuvant and a FLAG tag at the C-terminus. An expression plasmid was constructed by inserting the T cell epitope peptide gene into the multiple cloning sites of plasmid pNZ8148, an Escherichia coli-L. lactis shuttle vector. The constructed plasmid was transformed into L. lactis NZ9000 for expression induced by nisin, an antibacterial peptide from L. lactis. The expression of the epitope peptide was induced with 10-40 ng/mL nisin, and the expressed T cell epitope peptide was detected by western blot analysis using an anti-FLAG antibody and an antibody against the T cell epitopes. The concentration of the epitope peptide was estimated to be ~ 22 mg/L of culture in the presence of 40 ng/mL nisin, although it varied depending on the nisin concentration, the culture time, and the bacterial concentration when nisin was added. The expression of the recombinant epitope peptide in L. lactis, an organism generally recognized as safe, as demonstrated in this study, may contribute to the development of an oral vaccine for the treatment of pollinosis.
Assuntos
Alérgenos/imunologia , Epitopos de Linfócito T/metabolismo , Lactococcus lactis/efeitos dos fármacos , Nisina/farmacologia , Rinite Alérgica Sazonal/terapia , Adjuvantes Imunológicos/administração & dosagem , Alérgenos/genética , Vacinas Bacterianas/imunologia , Toxina da Cólera/administração & dosagem , Toxina da Cólera/genética , Cryptomeria/imunologia , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Escherichia coli/genética , Humanos , Imunoglobulina E/imunologia , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Nisina/administração & dosagem , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Plasmídeos , Pólen/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rinite Alérgica Sazonal/imunologia , Rinite Alérgica Sazonal/prevenção & controleRESUMO
BACKGROUND: The identification of fragment sequences, or motifs, within a therapeutic protein that may elicit an immune response when processed by T-cells can be provided by computer-aided approaches. Immunogenicity is a significant problem associated with protein therapeutics and should be investigated in the early stage of protein-based drug development to avoid treatment resistance and potentially life-threatening immune responses. PURPOSE: To provide a combined computer-aided protocol for investigating the immunogenic profile of a recombinant Kunitz-type inhibitor, which has been reported as promising antitumor agent by our research group. METHODS: The combination of databases searching (IEDB and SYFPEITHI) and molecular docking simulations was exploited, herein. This combined protocol has allowed the identification of potential epitopes before in vitro/in vivo evaluation. Predictors of human proteasome cleavage transport and major histocompatibility complex (MHC) binding were considered as overall score assigning the corresponding intrinsic potential of being a T cell epitope to each fragment sequence. The peptides or motifs better classified in the two databases were docked into the three-dimensional (3D) structure of MHC (class I and II) complex to verify the calculated binding affinity. The binding interactions regarding the molecular recognition process by T-cells were also exploited through the MHC:ligand:T-cell complexes. RESULTS: Regarding the Kunitz-type sequence, four motifs were identified as potentially epitopes for MHC-I and three motifs were found for MHC-II. But, those motifs were classified as moderately immunogenic. Final remarks: The combined computer-aided protocol has significantly reduced the number of potential epitopes to be considered for further analysis and could be useful to identify immunogenic fragments (high, moderate and low) in protein pharmaceutics before in vitro/in vivo experimentation.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Desenho Assistido por Computador , Epitopos de Linfócito T/efeitos dos fármacos , Simulação de Acoplamento Molecular , Peptídeos/química , Peptídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Antineoplásicos Fitogênicos/metabolismo , Bases de Dados de Proteínas , Epitopos de Linfócito T/imunologia , Humanos , Proteínas Recombinantes/metabolismoRESUMO
The magnitude of Th1 cells response to vaccination is a critical factor in determining protection from clinical disease. Our previous in vitro studies suggested that exposure to the nicotine component of cigarette smoke skews the differentiation of both human and mouse dendritic cell (DC) precursors into atypical DCs (DCs differentiated ex vivo in the presence of nicotine) lacking parameters essential for the development of Th1-mediated immunity. In this study, we determined the causal relationship between nicotine-induced DC alterations and host response to vaccines. We show that animals exposed to nicotine failed to develop and maintain Ag-specific effector memory Th1 cells and Ab production to protein-based vaccine formulated with Th1 adjuvants. Accordingly, both prophylactic and therapeutic vaccines failed to protect and cure the nicotine-exposed mice from disease. More importantly, we demonstrate the nicotine-induced defects in the biological activities of in vivo DCs as an underlying mechanism. Indeed, i.v. administration of DCs differentiated in the presence of nicotine preferentially promoted the development of Ag-specific IL-4-producing effector cells in the challenged mice. In addition, DC subsets isolated from mice exposed to nicotine produced significantly less cytokines in response to Th1 adjuvants and inadequately supported the development of Ag-specific Th1 cells. Collectively, our studies suggest that nicotine-induced defects in the DC system compromises vaccine efficacy in smokers.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Nicotina/toxicidade , Vacinação/efeitos adversos , Animais , Formação de Anticorpos/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/patologia , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Humanos , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/transplante , Vacinação/métodosRESUMO
The final pathway of beta cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill beta cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8+ T cells from HLA-A2+ patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide-specific CD8+ T cells killed human beta cells in vitro. Critically, at high glucose concentration, beta cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human beta cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing beta cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining beta cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Epitopos de Linfócito T/imunologia , Glucose/metabolismo , Células Secretoras de Insulina/patologia , Insulina/imunologia , Precursores de Proteínas/imunologia , Adolescente , Adulto , Epitopos de Linfócito T/efeitos dos fármacos , Feminino , Glucose/farmacologia , Humanos , Insulina/metabolismo , Células K562 , Masculino , Fenótipo , Precursores de Proteínas/metabolismo , Sinais Direcionadores de ProteínasRESUMO
Tregitopes (T regulatory epitopes) are IgG-derived peptides with high affinity to major histocompatibility complex class II (MHCII), that are known to promote tolerance by activating T regulatory cell (Treg) activity. Here we characterized the effect of IgG Tregitopes in a well-established murine model of allergic asthma, demonstrating in vivo antigen-specific tolerance via adoptive transfer of Tregitope-and-allergen-activated Tregs. Asthma is a heterogeneous chronic inflammatory condition affecting the airways and impacting over 300 million individuals worldwide. Treatment is suppressive, and no current therapy addresses immune regulation in severely affected asthmatics. Although high dose intra-venous immunoglobulin (IVIg) is not commonly used in the asthma clinic setting, it has been shown to improve severe asthma in children and in adults. In our laboratory, we previously demonstrated that IVIg abrogates airway hyperresponsiveness (AHR) in a murine model of asthma and induces suppressive antigen-specific T-regulatory cells. We hypothesized that IgG-derived Tregitopes would modulate allergic airway disease by inducing highly suppressive antigen-specific Tregs capable of diminishing T effector cell responses and establishing antigen-specific tolerance. Using ovalbumin (OVA-) and ragweed-driven murine models of allergic airway disease, we characterized the immunoregulatory properties of Tregitopes and performed Treg adoptive transfer to OVA- and ragweed-allergic mice to test for allergen specificity. Treatment with Tregitopes attenuated allergen-induced airway hyperresponsiveness and lung inflammation. We demonstrated that Tregitopes induce highly suppressive allergen-specific Tregs. The tolerogenic action of IgG Tregitopes in our model is very similar to that of IVIg, so we foresee that IgG Tregitopes could potentially replace steroid-based treatment and can offer a synthetic alternative to IVIg in a range of inflammatory and allergic conditions.
Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Epitopos de Linfócito T/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/farmacologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Pulmão/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Transferência Adotiva , Animais , Animais Geneticamente Modificados , Antígenos de Plantas , Asma/imunologia , Asma/metabolismo , Asma/fisiopatologia , Broncoconstrição/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/fisiopatologia , Camundongos Endogâmicos C57BL , Ovalbumina , Extratos Vegetais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/transplanteRESUMO
BACKGROUND: Sterile protection against malaria, most likely mediated by parasite-specific CD8+ T cells, has been achieved by attenuated sporozoite vaccination of animals as well as malaria-naïve and malaria-exposed subjects. The circumsporozoite protein (CSP)-based vaccine, RTS,S, shows low efficacy partly due to limited CD8+ T cell induction, and inclusion of such epitopes could improve RTS,S. This study assessed 8-10mer CSP peptide epitopes, present in predicted or previously positive P. falciparum 3D7 CSP 15mer overlapping peptide pools, for their ability to induce CD8+ T cell IFN-γ responses in natural malaria-exposed subjects. METHODS: Cryopreserved PBMCs from nine HLA-typed subjects were stimulated with 23 8-10mer CSP peptides from the 3D7 parasite in IFN-É£ ELISpot assays. The CD8+ T cell specificity of IFN-γ responses was confirmed in ELISpot assays using CD8+ T cell-enriched PBMC fractions after CD4+ cell depletion. RESULTS: Ten of 23 peptide epitopes elicited responses in whole PBMCs from five of the nine subjects. Four peptides tested positive in CD8+ T cell-enriched PBMCs from two previously positive responders and one new subject. All four immunodominant peptides are restricted by globally common HLA supertypes (A02, A03, B07) and mapped to regions of the CSP antigen with limited or no reported polymorphism. Association of these peptide-specific responses with anti-malarial protection remains to be confirmed. CONCLUSIONS: The relatively conserved nature of the four identified epitopes and their binding to globally common HLA supertypes makes them good candidates for inclusion in potential multi-epitope malaria vaccines.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Epitopos de Linfócito T/química , Epitopos de Linfócito T/efeitos dos fármacos , Interferon gama/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologiaRESUMO
Recombinant DNA technology has, in the last decades, contributed to a vast expansion of the use of protein drugs as pharmaceutical agents. However, such biological drugs can lead to the formation of anti-drug antibodies (ADAs) that may result in adverse effects, including allergic reactions and compromised therapeutic efficacy. Production of ADAs is most often associated with activation of CD4 T cell responses resulting from proteolysis of the biotherapeutic and loading of drug-specific peptides into major histocompatibility complex (MHC) class II on professional antigen-presenting cells. Recently, readouts from MHC-associated peptide proteomics (MAPPs) assays have been shown to correlate with the presence of CD4 T cell epitopes. However, the limited sensitivity of MAPPs challenges its use as an immunogenicity biomarker. In this work, MAPPs data was used to construct an artificial neural network (ANN) model for MHC class II antigen presentation. Using Infliximab and Rituximab as showcase stories, the model demonstrated an unprecedented performance for predicting MAPPs and CD4 T cell epitopes in the context of protein-drug immunogenicity, complementing results from MAPPs assays and outperforming conventional prediction models trained on binding affinity data.
Assuntos
Antirreumáticos/farmacologia , Antígenos de Histocompatibilidade Classe II/imunologia , Infliximab/farmacologia , Redes Neurais de Computação , Rituximab/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Humanos , Espectrometria de Massas , Peptídeos/imunologia , Ligação Proteica , ProteômicaRESUMO
The prevalence of respiratory illness caused by the novel SARS-CoV-2 virus associated with multiple organ failures is spreading rapidly because of its contagious human-to-human transmission and inadequate globalhealth care systems. Pharmaceutical repurposing, an effective drug development technique using existing drugs, could shorten development time and reduce costs compared to those of de novo drug discovery. We carried out virtual screening of antiviral compounds targeting the spike glycoprotein (S), main protease (Mpro), and the SARS-CoV-2 receptor binding domain (RBD)-angiotensin-converting enzyme 2 (ACE2) complex of SARS-CoV-2. PC786, an antiviral polymerase inhibitor, showed enhanced binding affinity to all the targets. Furthermore, the postfusion conformation of the trimeric S protein RBD with ACE2 revealed conformational changes associated with PC786 drug binding. Exploiting immunoinformatics to identify T cell and B cell epitopes could guide future experimental studies with a higher probability of discovering appropriate vaccine candidates with fewer experiments and higher reliability.
Assuntos
Antivirais/farmacologia , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Cisteína Endopeptidases/química , Desenho de Fármacos , Pandemias/prevenção & controle , Peptidil Dipeptidase A/química , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/química , Proteínas não Estruturais Virais/química , Enzima de Conversão de Angiotensina 2 , Benzamidas , Benzazepinas , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/metabolismo , Sítios de Ligação , COVID-19 , Proteases 3C de Coronavírus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Avaliação Pré-Clínica de Medicamentos , Epitopos de Linfócito B/efeitos dos fármacos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Humanos , Simulação de Acoplamento Molecular , Peptidil Dipeptidase A/imunologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Compostos de Espiro/farmacologia , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismoRESUMO
Celiac disease (CD) is a T helper 1-driven autoimmune permanent enteropathy, triggered in susceptible individuals by the ingestion of gluten, the alcohol-soluble protein fraction of some cereals, such as wheat, rye, and barley. The only available treatment for CD is the life-long withdrawal of gluten-containing foods from the diet. Complying with gluten-free diet is difficult and affects the quality of life. Therefore, alternative therapies are being investigated. In this paper, we review a new therapeutic strategy for CD, relying upon peptides that are analogs of gliadin T-cell epitopes that show the ability to down-modulate the immune response pathogenic of CD. These peptides have been obtained artificially by amino acids substitution of gliadin T-cell stimulatory sequences and an immunomodulatory sequence has been identified in the alcohol-soluble protein fraction of cultivars of durum wheat.
Assuntos
Doença Celíaca/imunologia , Doença Celíaca/terapia , Epitopos de Linfócito T/química , Gliadina/química , Ativação Linfocitária/imunologia , Peptídeos , Doença Celíaca/fisiopatologia , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Gliadina/genética , Gliadina/imunologia , Humanos , Imunoterapia , Peptídeos/química , Peptídeos/imunologia , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Linfócitos T/imunologia , Triticum/químicaRESUMO
Cytolytic T lymphocytes (CTL) play an important role in defense against viral infections. Following clonal expansion and effector functions, a vast majority of the antigen-specific CTL undergoes programmed cell death to maintain homeostasis. We have shown earlier that melanoma epitope-specific CTL are quite sensitive to activation-induced cell death (AICD) even on the secondary encounter of the antigen. Excessive sensitivity of viral antigen-specific CTL to AICD, however, would be counterproductive. It might be argued that although CTL for a "self" epitope might be more prone to AICD for maintaining self-tolerance, viral antigen-specific CTL are likely to be less sensitive to AICD. We show here that influenza matrix protein-derived MP(58-66) epitope-specific CTL, activated in vitro and bearing a memory phenotype, are just as sensitive to AICD. The AICD in these CTL is not blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone or by soluble Ig-Fc chimeras of the death receptors [Fas, TNF receptor (TNF-R), TRAIL-RI, TRAIL-RII]. However, the MP(58-66)-specific CTL can be rescued from AICD by the c-jun-N-terminal kinase (JNK) inhibitor SP600125. These results have implications for immunotherapeutic intervention in rescuing viral epitope-specific CTL from AICD.
Assuntos
Antracenos/farmacologia , Epitopos de Linfócito T/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Linfócitos T Citotóxicos/imunologia , Proteínas da Matriz Viral/imunologia , Apoptose/efeitos dos fármacos , Biomarcadores/análise , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Epitopos de Linfócito T/efeitos dos fármacos , Humanos , Vírus da Influenza A/imunologia , Fenótipo , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
Dengue virus (DENV) has emerged as a rapidly spreading epidemic throughout the tropical and subtropical regions around the globe. No suitable drug has been designed yet to fight against DENV, therefore, the need for safe and effective antiviral drug has become imperative. The envelope protein of DENV is responsible for mediating the fusion process between viral and host membranes. This work reports an in silico approach to target B and T cell epitopes for dengue envelope protein inhibition. A conserved region "QHGTI" in B and T cell epitopes of dengue envelope glycoprotein was confirmed to be valid for targeting by visualizing its interactions with the host cell membrane TIM-1 protein which acts as a receptor for serotype 2 and 3. A reverse pharmacophore mapping approach was used to generate a seven featured pharmacophore model on the basis of predicted epitope. This pharmacophore model as a 3D query was used to virtually screen a chemical compounds dataset "Chembridge". A total of 1010 compounds mapped on the developed pharmacophore model. These retrieved hits were subjected to filtering via Lipinski's rule of five, as a result 442 molecules were shortlisted for further assessment using molecular docking. Finally, 14 hits of different structural properties having interactions with the active site residues of dengue envelope glycoprotein were selected as lead candidates. These structurally diverse lead candidates have strong likelihood to act as further starting structures in the development of novel and potential drugs for the treatment of dengue fever.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Desenho de Fármacos , Epitopos de Linfócito B/efeitos dos fármacos , Epitopos de Linfócito T/efeitos dos fármacos , Simulação de Acoplamento Molecular , Proteínas do Envelope Viral/antagonistas & inibidores , Antivirais/química , Humanos , Testes de Sensibilidade Microbiana , Proteínas do Envelope Viral/imunologiaRESUMO
Interleukin (IL)-10 secretion by tumor cells was demonstrated to be one of the mechanisms by which tumor cells can escape immunological recognition and destruction. In dendritic cells (DCs), which are currently used for vaccination therapies for malignant diseases, IL-10 inhibits IL-12 production and induces a state of antigen-specific anergy in CD4- and CD8-positive T cells. We therefore analyzed the effects of different activation stimuli including lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpha, and CD40 ligation on IL-10 mediated inhibition of DC development and stimulatory capacity. In our study, the addition of IL-10 to the cultures containing granulocyte/macrophage-colony stimulating factor and IL-4 with or without LPS completely inhibited the generation of DCs from peripheral blood monocytes. These cells remained CD14 positive and expressed high levels of IL-10 receptor (IL-10R), suggesting that IL-10 mediates its effects by up-regulating the IL-10R. In contrast, the simultaneous incubation of monocytes with IL-10 and TNF-alpha or soluble CD40 ligand (sCD40L) resulted in the generation of CD83-positive DCs, induction of nuclear localized RelB, and inhibition of IL-10R up-regulation. DCs grown in the presence of IL-10 and TNF-alpha or sCD40L elicited efficient CTL responses against viral and tumor-associated peptide antigens, which, however, were reduced as compared with DC cultures generated without IL-10. IL-10 decreased the production of IL-6 and the expression of IL-12 in the presence of TNF-alpha or sCD40L, but it had no effect on IL-15, IL-18, and TNF-alpha secretion. Our results show that TNF-alpha or CD40 ligation can antagonize the IL-10-mediated inhibition on DC function, suggesting that depending on activation stimuli, the presence of IL-10 does not necessarily result in T-cell anergy.
Assuntos
Células Dendríticas/imunologia , Interleucina-10/antagonistas & inibidores , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/farmacologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Antígenos CD , Ligante de CD40 , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Quimiocinas/biossíntese , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imunoglobulinas/biossíntese , Imunoglobulinas/imunologia , Interleucina-10/imunologia , Interleucina-10/farmacologia , Receptores de Lipopolissacarídeos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Interleucina/biossíntese , Receptores de Interleucina-10 , Solubilidade , Linfócitos T/efeitos dos fármacos , Fator de Transcrição RelB , Fatores de Transcrição/biossíntese , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , Antígeno CD83RESUMO
HER-3 expression has been reported to act as an important oncoprotein in head and neck squamous cell carcinoma. This protein is known to control tumor proliferation and acquisition of resistance by tumor cells towards EGFR inhibitors, therefore, development of a HER-3-targeted therapy is desirable. In this study, we found that HER-3 expression on tumor cells was increased after EGFR inhibition. To establish a novel therapeutic approach for HER-3-positive head and neck carcinoma, we identified a HER-3 helper epitope that could elicit effective helper T cell responses to the naturally processed HER-3-derived epitope presented in a HER-3 expressing tumors. This epitope induced potent cytolytic activity of CD4 T cells against such tumor cells. Moreover, pan HER-family tyrosine kinase inhibitor augmented the responses of HER-3-reactive CD4 T cells via upregulation of HLA-DR protein on the surface of tumor cells. Our results supports the validity of CD4 T cell-dependent HER-3-targeted therapy combined with a broad inhibitor of HER-family.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-3/metabolismo , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Epitopos de Linfócito T/efeitos dos fármacos , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Receptores ErbB/metabolismo , Antígenos HLA-DR/metabolismo , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Relapsing experimental autoimmune encephalomyelitis (R-EAE) is a Th1-mediated central nervous system (CNS) autoimmune disease with pathology similar to that of relapsing-remitting multiple sclerosis. Among recent therapeutic approaches to prevent or treat relapsing disease is the strategic blockade of the CD154-CD40 ligand pair interactions. We have previously shown that CD154 blockade at the peak of acute disease can, in the short term, inhibit spontaneous disease relapse and this is at least partly associated with the inhibition of T cell effector function and blockade of inflammatory cell recruitment to and/or retention in the CNS. However, little is understood about the long-term effects of CD154 blockade in the inhibition of immune responses to encephalitogenic antigens. Here we demonstrate that transient anti-CD154 blockade of CD154-CD40 interactions at the peak of acute phase of R-EAE resulted in significant long-term inhibition (by >80%) of clinical relapses and that clinical disease in those mice that did relapse was reduced in duration and severity compared to control antibody-treated mice. Additionally, we show that this strategy permanently inhibits DTH responses of T cells specific for relapse-associated encephalitogenic epitopes. Thus, transient CD154 blockade during ongoing disease has a long-term therapeutic efficacy in preventing disease relapses.