RESUMO
TRIM28 is a component of heterochromatin complexes whose function in the immune system is unknown. By studying mice with conditional T cell-specific deletion of TRIM28 (CKO mice), we found that TRIM28 was phosphorylated after stimulation via the T cell antigen receptor (TCR) and was involved in the global regulation of CD4(+) T cells. The CKO mice had a spontaneous autoimmune phenotype that was due in part to early lymphopenia associated with a defect in the production of interleukin 2 (IL-2) as well as incomplete cell-cycle progression of their T cells. In addition, CKO T cells showed derepression of the cytokine TGF-ß3, which resulted in an altered cytokine balance; this caused the accumulation of autoreactive cells of the T(H)17 subset of helper T cells and of Foxp3(+) T cells. Notably, CKO Foxp3(+) T cells were unable to prevent the autoimmune phenotype in vivo. Our results show critical roles for TRIM28 in both T cell activation and T cell tolerance.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Ciclo Celular/imunologia , Interleucina-2/imunologia , Proteínas Nucleares/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Repressoras/imunologia , Fator de Crescimento Transformador beta3/imunologia , Animais , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/citologia , DNA/química , DNA/genética , Fatores de Transcrição Forkhead/imunologia , Humanos , Inflamação/imunologia , Interleucina-2/sangue , Células Jurkat , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Organismos Livres de Patógenos Específicos , Células Th17/imunologia , Fator de Crescimento Transformador beta3/biossíntese , Proteína 28 com Motivo TripartidoRESUMO
Anaphylaxis is a life-threatening immediate hypersensitivity reaction triggered by antigen capture by immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcvarepsilonRI) on mast cells. However, the regulatory mechanism of mast cell activation is not completely understood. Here we identify an immunoglobulin-like receptor, Allergin-1, that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain, and show it was preferentially expressed on mast cells. Mouse Allergin-1 recruited the tyrosine phosphatases SHP-1 and SHP-2 and the inositol phosphatase SHIP. Coligation of Allergin-1 and FcvarepsilonRI suppressed IgE-mediated degranulation of bone marrow-derived cultured mast cells. Moreover, mice deficient in Allergin-1 developed enhanced passive systemic and cutaneous anaphylaxis. Thus, Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice.
Assuntos
Degranulação Celular , Hipersensibilidade Imediata/imunologia , Mastócitos/metabolismo , Anafilaxia Cutânea Passiva/imunologia , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Animais , Células da Medula Óssea/patologia , Células Cultivadas , Humanos , Hipersensibilidade Imediata/genética , Hipersensibilidade Imediata/metabolismo , Imunoglobulina E/metabolismo , Inositol Polifosfato 5-Fosfatases , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Agregação de Receptores/imunologia , Receptores de IgE/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/imunologiaRESUMO
House dust mite (HDM) allergens are leading causes of allergic asthma characterized by Th2 responses. The lung-resident CD11b+ dendritic cells (DCs) play a key role in Th2 cell development in HDM-induced allergic asthma. However, the regulatory mechanism of HDM-induced CD11b+ DC activation remains incompletely understood. In this study, we demonstrate that mice deficient in an inhibitory immunoreceptor, Allergin-1, showed exacerbated HDM-induced airway eosinophilia and serum IgE elevation. By using bone marrow-chimeric mice that were sensitized with adoptively transferred HDM-stimulated wild-type or Allergin-1-deficient CD11b+ bone marrow-derived cultured DCs (BMDCs), followed by challenge with HDM, we show that Allergin-1 on the BMDCs suppressed HDM-induced allergic airway inflammation. We also show that Allergin-1 suppressed HDM-induced PGE2 production from CD11b+ BMDCs by inhibiting Syk tyrosine kinase activation through recruitment of SHP-1, subsequently leading to negative regulation of Th2 responses. These results suggest that Allergin-1 plays an important role in regulation of HDM-induced allergic airway inflammation.
Assuntos
Asma/imunologia , Hipersensibilidade/imunologia , Pneumonia/imunologia , Pyroglyphidae/imunologia , Receptores Imunológicos/imunologia , Animais , Células Dendríticas/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Although allergic contact dermatitis (ACD) is the most common T cell-mediated inflammatory responses against an allergen in the skin, the pathogenesis of ACD remains incompletely understood. In the sensitization phase in ACD, hapten-bearing dermal dendritic cells (DCs) play a pivotal role in the transport of an antigen to the lymph nodes (LNs), where they present the antigen to naïve T cells. Here we report that Allergin-1, an inhibitory immunoreceptor containing immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic region, is highly expressed on dermal DCs. Mice deficient in Allergin-1 exhibited exacerbated fluorescein isothiocyanate (FITC)-induced type 2 contact hypersensitivity (CHS) such as ear swelling and skin eosinophilia. Allergin-1-deficient mice also showed larger numbers of CD4+ T cells and FITC-bearing DCs and greater expressions of type 2 cytokines, including IL-5, IL-10 and IL-13, in the draining LNs than did wild type mice. In sharp contrast, Allergin-1-deficient mice showed comparable level of type 1 CHS induced by 2,4-dinitrofluorobenzene (DNFB). These results suggest that Allergin-1 on dermal DC inhibits type 2, but not type 1, immune responses in the sensitization phase of CHS.
Assuntos
Células Dendríticas/metabolismo , Dermatite de Contato/metabolismo , Fluoresceína-5-Isotiocianato/química , Receptores Imunológicos/fisiologia , Pele/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Células Dendríticas/citologia , Dinitrofluorbenzeno/química , Feminino , Hipersensibilidade Imediata , Interleucina-10/biossíntese , Interleucina-13/biossíntese , Interleucina-5/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Receptores Imunológicos/metabolismoRESUMO
Mast cells (MCs) play a critical role in oral allergen-induced anaphylaxis. However, the contribution of basophils to the anaphylaxis remains unclear. The inhibitory immunoreceptor Allergin-1 is highly expressed on MCs and basophils and inhibits FcεRI-mediated signaling in MCs. Here, we show that Allergin-1-deficient (Milr1-/-) mice developed more severe hypothermia, a higher mortality rate and a greater incidence of diarrhea than did wild-type (WT) mice in an oral ovalbumin (OVA)-induced food allergy model. MC-deficient Mas-TRECK mice, which had been reconstituted with either WT or Milr1-/- bone marrow-derived cultured MCs, did not develop hypothermia in this food allergy model. On the other hand, depletion of basophils by injection of anti-CD200R3 antibody rescued Milr1-/- mice from lethal hypothermia but not from diarrhea. In vitro analyses demonstrated that Allergin-1 inhibits IgE-dependent activation of both human and mouse basophils. Thus, Allergin-1 on basophils selectively suppresses oral allergen-induced anaphylaxis.
Assuntos
Anafilaxia/imunologia , Basófilos/imunologia , Receptores Imunológicos/imunologia , Animais , Modelos Animais de Doenças , Feminino , Hipersensibilidade Alimentar/imunologia , Imunoglobulina E/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores Imunológicos/administração & dosagem , Receptores Imunológicos/deficiênciaRESUMO
Although airway hyperresponsiveness (AHR) is a prominent feature of asthma, how it is regulated remains incompletely understood. Allergin-1, an inhibitory immunoglobulin-like receptor containing an immunoreceptor tyrosine-based inhibitory motif (ITIM), is expressed on human and mouse mast cells (MCs) and inhibits high-affinity receptor for IgE (FcεRI)-mediated signaling. Using MC-deficient KitW-sh/W-sh mice and Mas-TRECK mice, which carries a diphtheria toxin (DT)-induced MC deletion system based on il4 enhancer elements, we demonstrate here that MCs are involved in the induction of house dust mite (HDM)-induced AHR. Further, we show that MCs deficient in Allergin-1 exacerbated HDM-induced AHR, but had no effect on airway inflammation. In vitro analysis demonstrated that Allergin-1 inhibited anti-HDM allergen antibody-dependent HDM allergen-mediated degranulation by MCs. Thus, Allergin-1 on MCs plays an important role in the regulation of HDM-induced AHR.
Assuntos
Mastócitos/imunologia , Pyroglyphidae/imunologia , Receptores Imunológicos/imunologia , Hipersensibilidade Respiratória/imunologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Hipersensibilidade Respiratória/patologiaRESUMO
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not only a well-established immunotherapy for hematologic malignancies, but is potentially useful for treating solid tumors refractory to available therapies. However, application of allo-HSCT to solid tumors is limited, despite the beneficial antitumor effects, by the risk of graft-versus-host disease (GVHD). CD4+ T cells have been implicated in several aspects of GVHD, and suppress antitumor CD8+ T-cell responses. In the present study, we investigated clinically applicable allo-HSCT protocols designed to maximize antitumor effects while reducing the risk of GVHD. We used a mouse model of allo-HSCT with s.c. tumors. We found that myeloablative conditioning was associated with better inhibition of tumor growth but with severe acute GVHD. Early treatment with anti-CD4 mAb substantially ameliorated GVHD while preserving antitumor effects, leading to improved survival in myeloablative allo-HSCT. Late treatment with anti-CD4 mAb also ameliorated GVHD to some extent. Donor lymphocyte infusion in GVHD mice treated with anti-CD4 mAb further suppressed tumor growth without exacerbating GVHD. Collectively, our results suggest that myeloablative allo-HSCT followed by anti-CD4 mAb treatment and donor lymphocyte infusion could be a potent and safe immunotherapy for patients with cancers refractory to available therapies.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígenos CD4/imunologia , Neoplasias do Colo/terapia , Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Hematopoéticas/mortalidade , Transfusão de Linfócitos/métodos , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/imunologia , Imunoterapia Adotiva/métodos , Camundongos , Análise de Sobrevida , Condicionamento Pré-Transplante , Transplante Homólogo/mortalidade , Resultado do TratamentoRESUMO
TLR2 recognizes cell wall components of Staphylococcus aureus, which colonizes >90% of atopic eczematous skin lesions. The regulatory mechanisms of TLR2 signaling in the skin remain unclear. Allergin-1, an inhibitory immunoglobulin-like receptor containing an ITIM, is expressed on mast cells (MCs) and inhibits IgE-mediated anaphylaxis in mice. Here, we show that Allergin-1 inhibits TLR2-mediated activation of, and inflammatory cytokine production by, MCs in vitro Compared with wild-type mice, Allergin-1-deficient mice showed enhanced ear swelling with enhanced collagen deposition and greater Ly6G+ neutrophil recruitment after intra-dermal injection of Pam2CSK4 into pinnae. Using Mas-TRECK mice, which is an MC deletion system based on il4 enhancer elements, we also demonstrated that Allergin-1 on MCs is responsible for the Pam2CSK4-induced ear swelling. These results suggest that Allergin-1 on skin MCs suppresses TLR2-induced dermatitis.
Assuntos
Dermatite/imunologia , Mastócitos/imunologia , Receptores Imunológicos/imunologia , Receptor 2 Toll-Like/antagonistas & inibidores , Animais , Dermatite/patologia , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Imunológicos/deficiência , Receptor 2 Toll-Like/imunologiaRESUMO
Programmed cell death-1 (PD-1) is an inhibitory coreceptor for T lymphocytes that provides feedback inhibition of T cell activation. Although PD-1's expression on T cells is known to be activation dependent, the factors that determine the timing, intensity, and duration of PD-1 expression in immune reactions are not fully understood. To address this question, we performed a fine mapping analysis of a conserved 5'-flanking region of the PD-1 gene and identified a putative IFN stimulation response element, which was responsible for PD-1 transcription in the 2B4.11 T cell line. Consistent with this finding, activation by IFN-α enhanced both the induction and maintenance of PD-1 expression on TCR-engaged primary mouse T cells through an association IFN-responsive factor 9 (IRF9) to the IFN stimulation response element. Furthermore, PD-1 expression on Ag-specific CD8(+) T cells was augmented by IFN-α in vivo. We propose that strong innate inflammatory responses promote primary T cell activation and their differentiation into effector cells, but also cause an attenuated T cell response in sustained immune reactions, at least partially through type I IFN-mediated PD-1 transcription. Based on this idea, we demonstrate that IFN-α administration in combination with PD-1 blockade in tumor-bearing mice effectively augments the antitumor immunity, and we propose this as a novel and rational approach for cancer immunotherapy.
Assuntos
Antígenos de Superfície/genética , Proteínas Reguladoras de Apoptose/genética , Imunidade Celular , Interferon-alfa/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcrição Gênica/imunologia , Animais , Antígenos de Superfície/biossíntese , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/biossíntese , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Imunidade Celular/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/fisiologia , Interferon-alfa/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor de Morte Celular Programada 1 , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Elementos Reguladores de Transcrição/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
INTRODUCTION: Neutrophils are crucial to antimicrobial defense, but excessive neutrophilic inflammation elicits immune pathology. Currently, no effective treatment exists to curb neutrophil activation. However, neutrophils express a variety of inhibitory receptors which may represent potential therapeutic targets to limit neutrophilic inflammation. Indeed, we previously showed that the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) regulates neutrophilic airway inflammation and inhibits neutrophil extracellular trap formation. The inhibitory receptor Allergin-1 is expressed by myeloid cells and B cells. Allergin-1 suppresses mast cell and basophil activation, but a potential regulatory role on neutrophils remains unexplored. We aimed to demonstrate the regulation of neutrophils by Allergin-1. METHODS: We examine Allergin-1 isoform expression on human neutrophils during homeostatic (healthy donors) and chronic inflammatory (systemic lupus erythematosus patients) conditions in comparison to other circulating leukocytes by flow cytometry. To reveal a potential role for Allergin-1 in regulating neutrophilic inflammation, we experimentally infect wild-type (WT) and Allergin-1-deficient mice with a respiratory syncytial virus (RSV) and monitor disease severity and examine cellular airway infiltrate. Flow cytometry was used to confirm Allergin-1 expression by airway-infiltrated neutrophils in RSV infection-induced bronchiolitis patients. RESULTS: Only the short 1 (S1) isoform, but not the long (L) or S2 isoform could be detected on blood leukocytes, with the exception of nonclassical monocytes, which exclusively express the S2 isoform. Allergin-1 expression levels did not vary significantly between healthy individuals and patients with the systemic inflammatory disease on any interrogated cell type. Airway-infiltrated neutrophils of pediatric RSV bronchiolitis patients were found to express Allergin-1S1. However, Allergin-1-deficient mice experimentally infected with RSV did not show exacerbated disease or increased neutrophil airway infiltration compared to WT littermates. CONCLUSION: Allergin-1 isoform expression is unaffected by chronic inflammatory conditions. In stark contrast to fellow inhibitory receptor LAIR-1, Allergin-1 does not regulate neutrophilic inflammation in a mouse model of RSV bronchiolitis.
Assuntos
Bronquiolite , Inflamação , Receptores Imunológicos , Infecções por Vírus Respiratório Sincicial , Animais , Criança , Humanos , Camundongos , Inflamação/genética , Inflamação/metabolismo , Neutrófilos , Isoformas de Proteínas/genética , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sinciciais RespiratóriosRESUMO
Following the discovery that hydroxylated derivative 3 (Fig. 1) was one of the oxidative metabolites of the original lead 1, it was found that hydroxylated compound 4 possesses higher in vitro anti-HIV potency than the corresponding non-hydroxylated compound 2. Structural hybridation of 4 with the orally available analog 5 resulted in another orally-available spirodiketopiperazine CCR5 antagonist 6a that possesses more favorable pharmaceutical profile for use as a drug candidate.
Assuntos
Fármacos Anti-HIV/química , Antagonistas dos Receptores CCR5 , Dicetopiperazinas/química , Compostos de Espiro/química , Administração Oral , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacocinética , Linhagem Celular Tumoral , Dicetopiperazinas/síntese química , Dicetopiperazinas/farmacocinética , Dicetopiperazinas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Ratos , Receptores CCR5/metabolismo , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , EstereoisomerismoRESUMO
Based on the original spirodiketopiperazine design framework, further optimization of an orally available CCR5 antagonist was undertaken. Structural hybridization of the hydroxylated analog 4 derived from one of the oxidative metabolites and the new orally available non-hydroxylated benzoic acid analog 5 resulted in another potent orally available CCR5 antagonist 6a as a clinical candidate. Full details of a structure-activity relationship (SAR) study and ADME properties are presented.
Assuntos
Fármacos Anti-HIV/química , Benzoatos/química , Antagonistas dos Receptores CCR5 , Dicetopiperazinas/química , Administração Oral , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacocinética , Benzoatos/síntese química , Benzoatos/farmacocinética , Dicetopiperazinas/síntese química , Dicetopiperazinas/farmacocinética , Cães , Avaliação Pré-Clínica de Medicamentos , Cobaias , Haplorrinos , Humanos , Coelhos , Ratos , Receptores CCR5/metabolismo , Relação Estrutura-AtividadeRESUMO
Accumulating evidence suggests that PD-1, an immuno-inhibitory receptor expressed on activated T cells, regulates peripheral T cell tolerance. In particular, PD-1 is involved in the induction and/or maintenance of T cells' intrinsic unresponsiveness to previously encountered Ags, although the mechanism is yet to be determined. We used a simple experimental model to dissect the mechanism for anergy establishment, in which 2C TCR transgenic rag2(-/-) PD-1(+/+) mice were anergized by a single injection of a cognate peptide. Interestingly, 2C rag2(-/-) PD-1(-/-) mice were totally resistant to anergy induction by the same treatment; thus, PD-1 was responsible for anergy induction. Furthermore, PD-1 expression was induced within 24 h of the initial Ag exposure. The establishment of anergy was associated with a marked down-regulation of IL-2 from the CD8(+) T cells. In fact, IL-2 blockade resulted in anergy even in 2C rag2(-/-)PD-1(-/-) T cells. Furthermore, the complementation of the IL-2 signal in 2C rag2(-/-) PD-1(+/+) mice reversed the anergy induction. We propose that CD8(+) T cell anergy is induced by a reduction of cell-autonomous IL-2 synthesis, which is caused by the quick expression of PD-1 in response to Ag stimulation and the subsequent stimulation of this receptor by its ligands on surrounding cells.
Assuntos
Antígenos de Diferenciação/imunologia , Linfócitos T CD8-Positivos/imunologia , Anergia Clonal , Interleucina-2/antagonistas & inibidores , Animais , Antígenos/imunologia , Antígenos de Diferenciação/genética , Regulação da Expressão Gênica/imunologia , Interleucina-2/biossíntese , Interleucina-2/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptor de Morte Celular Programada 1RESUMO
The expansion of intratumoral stem-like/progenitor exhausted CD8+ T (Tstem/Tpex) cells provides a potential approach to improve the therapeutic efficacy of immune checkpoint blockade (ICB). Thus, here we demonstrate a strategy to facilitate Tstem/Tpex cell expansion by combining an alarmin high-mobility group nucleosome binding domain 1 (HMGN1) peptide with programmed death-ligand 1 (PD-L1) blockade. The antitumor effects of HMGN1, anti-PD-L1, and their combined treatment were monitored in the B16F10, LLC, Colon26, or EO771 tumor-bearing mice. The comprehensive immunologic analyses, such as high-dimensional flow cytometry, transcriptome analysis, and single-cell RNA-sequencing (scRNA-seq), were used to investigate the cellular and molecular mechanisms of antitumor immune responses after treatments. We identified the immunostimulatory domain (EPKRR SARLS AKPPA KVEAK PKK) on HMGN1 and synthesized this domain as a therapeutic peptide (minP1). Combined treatment with minP1 and PD-L1 blockade induced durable tumor regression in tumor-bearing mice. minP1 increased the number of intratumoral mature DCs enriched in immunoregulatory molecules (mregDC) and enhanced their MHC class I antigen-presenting program. minP1 also synergized with PD-L1 blockade in augmenting intratumoral Tstem/Tpex cell number. Analysis of our scRNA-seq dataset by CellPhonDB suggested potential interactions between mregDCs and Tstem/Tpex cells in tumors. Our results indicate that HMGN1 peptide (minP1) serves as an immunoadjuvant to promote effective anti-PD-L1 immunotherapy with increased Tstem/Tpex cells in tumors.
Assuntos
Alarminas/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Proteína HMGN1/uso terapêutico , Neoplasias/terapia , Animais , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Feminino , Proteína HMGN1/genética , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/imunologiaRESUMO
Spirodiketopiperazine-based CCR5 antagonists, showing improved pharmacokinetic profiles without reduction in antagonist activity, were designed and synthesized. We also demonstrate the anti-HIV activity of a representative compound 12, as measured in a p24 assay.
Assuntos
Fármacos Anti-HIV/farmacocinética , Antagonistas dos Receptores CCR5 , Piperazinas/farmacocinética , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Proteína do Núcleo p24 do HIV/metabolismo , Humanos , Microssomos Hepáticos/metabolismo , Piperazinas/síntese química , Piperazinas/química , Ratos , Receptores CCR5/metabolismoRESUMO
Using the previously reported novel spirodiketopiperazine scaffold, the design and synthesis of orally available CCR5 antagonists was undertaken. Compounds possessing a carboxylic acid function in the appropriate position showed improved oral exposure (AUC) relative to the initial chemical leads without reduction in the antagonist activity. The optimized compound 40 was found to show potent anti-HIV activity. Full details of structure-activity relationship (SAR) study are presented.
Assuntos
Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/farmacocinética , Antagonistas dos Receptores CCR5 , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Piperazinas/farmacologia , Piperazinas/farmacocinética , Administração Oral , Animais , Fármacos Anti-HIV/química , Disponibilidade Biológica , Células CACO-2 , Humanos , Piperazinas/química , Ratos , Receptores CCR5/metabolismoRESUMO
BACKGROUND: Transient depletion of CD4+ T cells results in tumor suppression and survival benefit in murine models; however, the tumor progression and recurrence still occur over more long-term monitoring of mice. Thus, we explored an additional strategy to enhance endogenous immune responses by an alarmin, high mobility group nucleosome binding protein 1 (HMGN1). METHODS: The anti-tumor effects of HMGN1, anti-CD4 depleting antibody, and their combined treatment were monitored in the Colon26 or the B16F10 subcutaneous murine models. The tumor-infiltrating CD8+ T cell proliferation, differentiation, exhaustion, and its gene expression were determined by flow cytometry, transcriptome analysis, and quantitative real-time PCR. RESULTS: Our results show that a systemic administration of low doses of HMGN1 with an anti-CD4 depleting antibody (HMGN1/αCD4) promoted expansion of CD8+ T cell populations (e.g. CD137+ PD-1+ and CD44hi PD-1+), recruited CCR7+ migratory dendritic cells to the tumor, and reduced co-inhibitory molecules (e.g. PD-1, LAG-3, and TIM-3) to counteract CD8+ T cell exhaustion. CONCLUSION: The HMGN1/αCD4 treatment expanded effector CD8+ T cells and prolonged their anti-tumor activities by rescuing them from exhaustion, thus resulting in tumor regression and even rejection in long-term monitored mice.
Assuntos
Anticorpos/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteína HMGN1/uso terapêutico , Neoplasias/terapia , Animais , Linhagem Celular Tumoral , Feminino , Proteína HMGN1/genética , Imunoterapia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Proteínas Recombinantes/uso terapêuticoRESUMO
Aplaviroc (GW873140) binds specifically to human cellular CC chemokine receptor 5 (CCR5) and demonstrates potent anti-human immunodeficiency virus activity in vitro in the subnanomolar range. In vitro studies show that aplaviroc selectively inhibits the binding of a particular monoclonal antibody, 45531, to CCR5. Based on this observation, a flow cytometry-based assay was developed to determine percentage CCR5 receptor occupancy (RO). CCR5 receptor occupancy was aplaviroc concentration-dependent and related to anti-human immunodeficiency virus activity in vitro. In the clinical setting, CCR5 receptor occupancy in peripheral blood was >98% in all subjects within 2 to 3 hours of dosing, which is consistent with the peak plasma concentrations of drug. Longitudinal analysis in the drug washout period revealed the time to 50% CCR5 receptor occupancy averaged >100 hours, in both human immunodeficiency virus-positive and human immunodeficiency virus-negative subjects, substantially longer than the plasma pharmacokinetic half-life of 3 hours. The duration of CCR5 receptor occupancy appeared to be dose-dependent and associated with antiviral activity as measured by plasma human immunodeficiency virus RNA nadir following 10 days of multiple dose administration. These data demonstrate that the analysis of CCR5 receptor occupancy, in addition to conventional plasma-based pharmacokinetic measures, provides an informative tool to assist in evaluating the pharmacodynamic and antiviral effects of cellular CC chemokine receptor antagonists.
Assuntos
Benzoatos/farmacologia , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Piperazinas/farmacologia , Receptores CCR5/efeitos dos fármacos , Compostos de Espiro/farmacologia , Adulto , Anticorpos Monoclonais/metabolismo , Benzoatos/administração & dosagem , Benzoatos/farmacocinética , Dicetopiperazinas , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Citometria de Fluxo , Inibidores da Fusão de HIV/administração & dosagem , Inibidores da Fusão de HIV/farmacocinética , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Piperazinas/administração & dosagem , Piperazinas/farmacocinética , Ligação Proteica/efeitos dos fármacos , RNA Viral/sangue , RNA Viral/efeitos dos fármacos , Receptores CCR5/metabolismo , Compostos de Espiro/administração & dosagem , Compostos de Espiro/farmacocinética , Fatores de Tempo , Adulto JovemRESUMO
Thymus-derived forkhead box p3(+) naturally occurring regulatory T cells (nTreg) are thought to circulate throughout the body to maintain peripheral immunological self-tolerance through interactions with dendritic cells (DCs), resulting in regulation of conventional T cells. However, the chemokine receptors, which are putatively involved in the in vivo migration of nTreg, have not been fully established. Here, we demonstrated that lymph node nTreg preferentially migrated to the paracortical area of lymph nodes after adoptive transfer, where they were observed to make contact frequently with CD8alpha(+) DCs and CD8alpha(-) CD11b(-) DCs. This migration of nTreg to the paracortical areas was impaired severely when cells were prepared from CCR7-deficient mice. However, to some extent, CCR7-independent migration of nTreg in such CCR7-deficient mice was also observed, but this occurred mainly in the medullary high endothelial venules. Taken together, these data provide the evidence that CCR7 mediates nTreg migration to the paracortical areas of lymph nodes under steady-state conditions; however, CCR7-independent migration also takes place in the medulla.