RESUMO
Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies1. Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies2-4. Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2+ breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Feminino , Humanos , Camundongos , Receptor ErbB-2/genéticaRESUMO
BLT (bone marrow-liver-thymus) humanized mice, which reconstitute a functional human immune system, develop prototypic human virus-specific CD8+ T cell responses following infection with human immunodeficiency virus type 1 (HIV-1). We explored the utility of the BLT model for HIV-1 vaccine development by immunizing BLT mice against the conserved viral Gag protein, utilizing a rapid prime-boost protocol of poly(lactic-co-glycolic) acid microparticles and a replication-defective herpes simplex virus (HSV) recombinant vector. After HIV-1 challenge, the mice developed broad, proteome-wide gamma interferon-positive (IFN-γ+) T cell responses against HIV-1 that reached magnitudes equivalent to what is observed in HIV-1-infected individuals. The functionality of these responses was underscored by the consistent emergence of escape mutations in multiple CD8+ T cell epitopes during the course of infection. Although prechallenge vaccine-induced responses were largely undetectable, the Gag immunization increased both the magnitude and the kinetics of anamnestic Gag-specific T cell responses following HIV-1 infection, and the magnitude of these postchallenge Gag-specific responses was inversely correlated with acute HIV-1 viremia. Indeed, Gag immunization was associated with a modest but significant 0.5-log reduction in HIV-1 viral load when analyzed across four experimental groups of BLT mice. Notably, the HSV vector induced elevated plasma concentrations of polarizing cytokines and chemotactic factors, including interleukin-12p70 (IL-12p70) and MIP-1α, which were positively correlated with the magnitude of Gag-specific responses. Overall, these results support the ability of BLT mice to recapitulate human pathogen-specific T cell responses and to respond to immunization; however, additional improvements to the model are required to develop a robust system for testing HIV-1 vaccine efficacy.IMPORTANCE Advances in the development of humanized mice have raised the possibility of a small-animal model for preclinical testing of an HIV-1 vaccine. Here, we describe the capacity of BLT humanized mice to mount broadly directed HIV-1-specific human T cell responses that are functionally active, as indicated by the rapid emergence of viral escape mutations. Although immunization of BLT mice with the conserved viral Gag protein did not result in detectable prechallenge responses, it did increase the magnitude and kinetics of postchallenge Gag-specific T cell responses, which was associated with a modest but significant reduction in acute HIV-1 viremia. Additionally, the BLT model revealed immunization-associated increases in the plasma concentrations of immunomodulatory cytokines and chemokines that correlated with more robust T cell responses. These data support the potential utility of the BLT humanized mouse for HIV-1 vaccine development but suggest that additional improvements to the model are warranted.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Viremia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Doença Aguda , Animais , Evolução Biológica , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Infecções por HIV/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunização , Camundongos , Camundongos Transgênicos , Linfócitos T/metabolismo , Carga ViralRESUMO
A large and diverse array of chemoattractants control leukocyte trafficking, but how these apparently redundant signals collaborate in vivo is still largely unknown. We previously demonstrated an absolute requirement for the lipid chemoattractant leukotriene B(4) (LTB(4)) and its receptor BLT1 for neutrophil recruitment into the joint in autoantibody-induced arthritis. We now demonstrate that BLT1 is required for neutrophils to deliver IL-1 into the joint to initiate arthritis. IL-1-expressing neutrophils amplify arthritis through the production of neutrophil-active chemokines from synovial tissue cells. CCR1 and CXCR2, two neutrophil chemokine receptors, operate nonredundantly to sequentially control the later phase of neutrophil recruitment into the joint and mediate all neutrophil chemokine activity in the model. Thus, we have uncovered a complex sequential relationship involving unique contributions from the lipid mediator LTB(4), the cytokine IL-1, and CCR1 and CXCR2 chemokine ligands that are all absolutely required for effective neutrophil recruitment into the joint.
Assuntos
Artrite/imunologia , Quimiocinas/imunologia , Interleucina-1alfa/imunologia , Interleucina-1beta/imunologia , Leucotrieno B4/imunologia , Neutrófilos/imunologia , Animais , Artrite/genética , Artrite/patologia , Células Cultivadas , Quimiocinas/biossíntese , Modelos Animais de Doenças , Suscetibilidade a Doenças , Interleucina-1alfa/deficiência , Interleucina-1beta/biossíntese , Interleucina-1beta/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR1/imunologia , Receptores de Interleucina-8B/imunologia , Receptores de Leucotrienos/deficiência , Receptores de Leucotrienos/imunologia , Líquido Sinovial/imunologiaRESUMO
After host entry through mucosal surfaces, human immunodeficiency virus-1 (HIV-1) disseminates to lymphoid tissues to establish a generalized infection of the immune system. The mechanisms by which this virus spreads among permissive target cells locally during the early stages of transmission and systemically during subsequent dissemination are not known. In vitro studies suggest that the formation of virological synapses during stable contacts between infected and uninfected T cells greatly increases the efficiency of viral transfer. It is unclear, however, whether T-cell contacts are sufficiently stable in vivo to allow for functional synapse formation under the conditions of perpetual cell motility in epithelial and lymphoid tissues. Here, using multiphoton intravital microscopy, we examine the dynamic behaviour of HIV-infected T cells in the lymph nodes of humanized mice. We find that most productively infected T cells migrate robustly, resulting in their even distribution throughout the lymph node cortex. A subset of infected cells formed multinucleated syncytia through HIV envelope-dependent cell fusion. Both uncoordinated motility of syncytia and adhesion to CD4(+) lymph node cells led to the formation of long membrane tethers, increasing cell lengths to up to ten times that of migrating uninfected T cells. Blocking the egress of migratory T cells from the lymph nodes into efferent lymph vessels, and thus interrupting T-cell recirculation, limited HIV dissemination and strongly reduced plasma viraemia. Thus, we have found that HIV-infected T cells are motile, form syncytia and establish tethering interactions that may facilitate cell-to-cell transmission through virological synapses. Migration of T cells in lymph nodes therefore spreads infection locally, whereas their recirculation through tissues is important for efficient systemic viral spread, suggesting new molecular targets to antagonize HIV infection.
Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV/imunologia , Animais , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Células Gigantes , Infecções por HIV/transmissão , Humanos , Linfonodos/virologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos TransgênicosRESUMO
BACKGROUND: Induction of endogenous regulatory T (Treg) cells represents an exciting new potential modality for treating allergic diseases, such as asthma. Treg cells have been implicated in the regulation of asthma, but the anatomic location in which they exert their regulatory function and the mechanisms controlling the migration necessary for their suppressive function in asthma are not known. Understanding these aspects of Treg cell biology will be important for harnessing their power in the clinic. OBJECTIVE: We sought to determine the anatomic location at which Treg cells exert their regulatory function in the sensitization and effector phases of allergic asthma and to determine the chemokine receptors that control the migration of Treg cells to these sites in vivo in both mice and human subjects. METHODS: The clinical efficacy and anatomic location of adoptively transferred chemokine receptor-deficient CD4(+)CD25(+) forkhead box protein 3-positive Treg cells was determined in the sensitization and effector phases of allergic airway inflammation in mice. The chemokine receptor expression profile was determined on Treg cells recruited into the human airway after bronchoscopic segmental allergen challenge of asthmatic patients. RESULTS: We show that CCR7, but not CCR4, is required on Treg cells to suppress allergic airway inflammation during the sensitization phase. In contrast, CCR4, but not CCR7, is required on Treg cells to suppress allergic airway inflammation during the effector phase. Consistent with our murine studies, human subjects with allergic asthma had an increase in CCR4-expressing functional Treg cells in the lungs after segmental allergen challenge. CONCLUSION: The location of Treg cell function differs during allergic sensitization and allergen-induced recall responses in the lung, and this differential localization is critically dependent on differential chemokine function.
Assuntos
Asma/imunologia , Asma/metabolismo , Quimiocinas/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Alérgenos/imunologia , Animais , Asma/genética , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Movimento Celular/imunologia , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Imunização , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Receptores CCR4/deficiência , Receptores CCR4/genética , Receptores CCR7/deficiência , Receptores CCR7/genéticaRESUMO
Humanized mice historically have not been good models of human humoral immunity induced by either infection or immunization. However, newer versions of humanized mice generated in severely immunodeficient mice with a targeted disruption of the IL2Rγc gene have recently been reported to produce antigen-specific class-switched human antibodies, with some demonstrating neutralizing activities. Here we review the growing ability of humanized mice to support the study of human humoral immune responses, discussing the current and future potential of these models as well as their current limitations.
Assuntos
Imunidade Humoral , Animais , Humanos , Camundongos , Camundongos SCID , Modelos AnimaisRESUMO
Lung transplantation remains the only effective therapy for patients with end-stage pulmonary diseases. Unfortunately, acute rejection of the lung remains a frequent complication and is an important cause of morbidity and mortality. The induction of transplant tolerance is thought to be dependent, in part, on the balance between allograft effector mechanisms mediated by effector T lymphocytes (Teff), and regulatory mechanisms mediated by FOXP3(+) regulatory T cells (Treg). In this study, we explored an approach to tip the balance in favor of regulatory mechanisms by modulating chemokine activity. We demonstrate in an adoptive transfer model of lung rejection that CXCR3-deficient CD8(+) Teff have impaired migration into the lungs compared with wild-type Teff, which results in a dramatic reduction in fatal pulmonary inflammation. The lungs of surviving mice contained tolerized CXCR3-deficient Teff, as well as a large increase in Treg. We confirmed that Treg were needed for tolerance and that their ability to induce tolerance was dependent on their numbers in the lung relative to the numbers of Teff. These data suggest that transplantation tolerance can be achieved by reducing the recruitment of some, but not necessarily all, CD8(+) Teff into the target organ and suggest a novel approach to achieve transplant tolerance.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Rejeição de Enxerto/prevenção & controle , Transplante de Pulmão/imunologia , Receptores CXCR3/imunologia , Tolerância ao Transplante/imunologia , Animais , Rejeição de Enxerto/imunologia , Inflamação/prevenção & controle , Transplante de Pulmão/efeitos adversos , Camundongos , Linfócitos T Reguladores/imunologiaRESUMO
Neutrophil recruitment into tissue plays an important role in host defense and disease pathogenesis, including the inflammatory arthritides. A multitude of diverse chemoattractants have been implicated in neutrophil recruitment, suggesting that they have overlapping functions in mediating this critical biological response. However, here we demonstrate a unique, non-redundant role for the leukotriene B4 receptor BLT1 in mediating neutrophil recruitment into the joint in the K/BxN mouse model of inflammatory arthritis. We demonstrate that neutrophil expression of BLT1 was absolutely required for arthritis generation and chemokine production in this model, and that specific BLT1 inhibition reversed established disease. Adoptive transfer of wild-type (WT) neutrophils restored arthritis and chemokine production in BLT1(-/-) mice. Surprisingly, the primary effect of the transferred WT neutrophils into BLT1(-/-) mice was to promote the entry of endogenous BLT1(-/-) neutrophils into the joints of these mice. However, continued joint inflammation was dependent on the presence of WT neutrophils, indicating an ongoing specific requirement for BLT1-activated neutrophils in mediating BLT1(-/-) neutrophil recruitment by other chemoattractants. These experiments demonstrate that neutrophil BLT1 functions in a novel and essential non-cell-autonomous manner to enable the recruitment of additional neutrophils not expressing this receptor, thereby amplifying the inflammatory response in autoantibody-induced arthritis.
Assuntos
Artrite Experimental/imunologia , Leucotrieno B4/metabolismo , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Receptores do Leucotrieno B4/fisiologia , Receptores Purinérgicos P2/fisiologia , Animais , Artrite Experimental/patologia , Autoanticorpos/administração & dosagem , Movimento Celular/imunologia , Células Cultivadas , Quimiocinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Neutrófilos/metabolismo , Receptores do Leucotrieno B4/metabolismo , Receptores Purinérgicos P2/metabolismo , Membrana Sinovial/metabolismoRESUMO
Leukotriene B4 is a lipid mediator that recently has been shown to have potent chemotactic activity for effector T lymphocytes mediated through its receptor, BLT1. Here, we developed a novel murine model of acute lung rejection to demonstrate that BLT1 controls effector CD8+ T cell trafficking into the lung and that disruption of BLT1 signaling in CD8+ T cells reduces lung inflammation and mortality in the model. In addition, we used BLT1-deficient mice and a BLT1 antagonist in two tracheal transplant models of lung transplantation to demonstrate the importance of BLT1 for the recruitment of T cells into tracheal allografts. We also show that BLT1-mediated CD8+ T cell recruitment plays an important role in the development of airway fibroproliferation and obliteration. Finally, in human studies of lung transplant recipients, we found that BLT1 is up-regulated on T lymphocytes isolated from the airways of patients with obliterative bronchiolitis. These data demonstrate that BLT1 contributes to the development of lung rejection and obliterative bronchiolitis by mediating effector T lymphocyte trafficking into the lung. This is the first report that describes a pathologic role for BLT1-mediated T lymphocyte recruitment in disease and identifies BLT1 as a potential therapeutic target after lung transplantation.
Assuntos
Bronquiolite Obliterante/etiologia , Linfócitos T CD8-Positivos/fisiologia , Rejeição de Enxerto/etiologia , Transplante de Pulmão/efeitos adversos , Receptores do Leucotrieno B4/fisiologia , Animais , Sequência de Bases , Bronquiolite Obliterante/imunologia , Bronquiolite Obliterante/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Colágeno/metabolismo , Modelos Animais de Doenças , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Transplante de Pulmão/imunologia , Transplante de Pulmão/patologia , Transplante de Pulmão/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Receptores do Leucotrieno B4/deficiência , Receptores do Leucotrieno B4/genética , Traqueia/transplanteRESUMO
STAT6-mediated chemokine production in the lung is required for Th2 lymphocyte and eosinophil homing into the airways in allergic pulmonary inflammation, and thus is a potential therapeutic target in asthma. However, the critical cellular source of STAT6-mediated chemokine production has not been defined. In this study, we demonstrate that STAT6 in bone marrow-derived myeloid cells was sufficient for the production of CCL17, CCL22, CCL11, and CCL24 and for Th2 lymphocyte and eosinophil recruitment into the allergic airway. In contrast, STAT6 in airway-lining cells did not mediate chemokine production or support cellular recruitment. Selective depletion of CD11b(+) myeloid cells in the lung identified these cells as the critical cellular source for the chemokines CCL17 and CCL22. These data reveal that CD11b(+) myeloid cells in the lung help orchestrate the adaptive immune response in asthma, in part, through the production of STAT6-inducible chemokines and the recruitment of Th2 lymphocytes into the airway.
Assuntos
Antígeno CD11b/biossíntese , Quimiotaxia de Leucócito/imunologia , Pulmão/imunologia , Células Mieloides/imunologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Fator de Transcrição STAT6/fisiologia , Células Th2/imunologia , Animais , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Células Cultivadas , Quimiocina CCL17/biossíntese , Quimiocina CCL22/biossíntese , Quimiocina CCL24/biossíntese , Modelos Animais de Doenças , Imunidade Inata , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/metabolismo , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Hipersensibilidade Respiratória/metabolismo , Fator de Transcrição STAT6/deficiência , Fator de Transcrição STAT6/genética , Células Th2/patologia , Células Th2/transplanteRESUMO
The generation of humanized BLT mice by the cotransplantation of human fetal thymus and liver tissues and CD34(+) fetal liver cells into nonobese diabetic/severe combined immunodeficiency mice allows for the long-term reconstitution of a functional human immune system, with human T cells, B cells, dendritic cells, and monocytes/macrophages repopulating mouse tissues. Here, we show that humanized BLT mice sustained high-level disseminated human immunodeficiency virus (HIV) infection, resulting in CD4(+) T-cell depletion and generalized immune activation. Following infection, HIV-specific humoral responses were present in all mice by 3 months, and HIV-specific CD4(+) and CD8(+) T-cell responses were detected in the majority of mice tested after 9 weeks of infection. Despite robust HIV-specific responses, however, viral loads remained elevated in infected BLT mice, raising the possibility that these responses are dysfunctional. The increased T-cell expression of the negative costimulator PD-1 recently has been postulated to contribute to T-cell dysfunction in chronic HIV infection. As seen in human infection, both CD4(+) and CD8(+) T cells demonstrated increased PD-1 expression in HIV-infected BLT mice, and PD-1 levels in these cells correlated positively with viral load and inversely with CD4(+) cell levels. The ability of humanized BLT mice to generate both cellular and humoral immune responses to HIV will allow the further investigation of human HIV-specific immune responses in vivo and suggests that these mice are able to provide a platform to assess candidate HIV vaccines and other immunotherapeutic strategies.
Assuntos
Formação de Anticorpos , Modelos Animais de Doenças , Infecções por HIV/imunologia , HIV/imunologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , Imunidade Celular , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Receptor de Morte Celular Programada 1RESUMO
Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy.
Assuntos
Anticorpos Biespecíficos , Mieloma Múltiplo , Animais , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD28 , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Receptores de Antígenos de Linfócitos T , Linfócitos TRESUMO
Allogeneic hematopoietic chimerism leading to central tolerance has significant therapeutic potential. Realization of that potential has been impeded by the need for myeloablative conditioning of the host and development of graft-versus-host disease (GVHD). To surmount these impediments, we have adapted a costimulation blockade-based protocol developed for solid organ transplantation for use in stem cell transplantation. The protocol combines donor-specific transfusion (DST) with anti-CD154 mAb. When applied to stem cell transplantation, administration of DST, anti-CD154 mAb, and allogeneic bone marrow leads to hematopoietic chimerism and central tolerance with no myeloablation and no GVHD. Tolerance in this system results from deletion of both peripheral host alloreactive CD8+ T cells and nascent intrathymic alloreactive CD8+ T cells. In the absence of large numbers of host alloreactive CD8+ T cells, the transfusion that precedes transplantation need not be of donor origin, suggesting that both allospecific and non-allospecific mechanisms regulate engraftment. Agents that interfere with peripheral transplantation tolerance impair establishment of chimerism. We conclude that robust allogeneic hematopoietic chimerism and central tolerance can be established in the absence of host myeloablative conditioning using a peripheral transplantation tolerance protocol.
Assuntos
Transplante de Medula Óssea/imunologia , Hematopoese/imunologia , Quimeras de Transplante/imunologia , Condicionamento Pré-Transplante , Tolerância ao Transplante/imunologia , Animais , Transfusão de Sangue , Ligante de CD40/fisiologia , Feminino , Doença Enxerto-Hospedeiro/prevenção & controle , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Receptores de Interleucina-2/antagonistas & inibidores , Transplante HomólogoRESUMO
Progressive Multifocal Leukoencephalopathy (PML) is an often fatal disease caused by the reactivation of the JC virus (JCV). Better understanding of viral-host interactions has been hampered by the lack of an animal model. Engrafting NOD/SCID/IL-2-Rg (null) mice with human lymphocytes and thymus, we generated a novel animal model for JCV infection. Mice were inoculated with either a PML isolate, JCV Mad-4, or with JCV CY, found in the kidney and urine of healthy individuals. While mice remained asymptomatic following inoculation, JCV DNA was occasionally detected in both the blood and the urine compartments. Mice generated both humoral and cellular immune responses against JCV. Expressions of immune exhaustion marker, PD-1, on lymphocytes were consistent with response to infection. Using this model we present the first in vivo demonstration of virological and immunological differences between JCV Mad-4 and CY. This model may prove valuable for studying JCV host immune responses.
Assuntos
Vírus JC/imunologia , Leucoencefalopatia Multifocal Progressiva/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos de Diferenciação/metabolismo , Antígenos Virais/imunologia , Células COS , Proteínas do Capsídeo/imunologia , Chlorocebus aethiops , DNA Viral/sangue , DNA Viral/urina , Modelos Animais de Doenças , Humanos , Imunidade Celular , Imunidade Humoral , Vírus JC/genética , Rim/virologia , Leucoencefalopatia Multifocal Progressiva/metabolismo , Leucoencefalopatia Multifocal Progressiva/virologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptor de Morte Celular Programada 1RESUMO
An estimated 34 million people are living with HIV worldwide (UNAIDS, 2012), with the number of infected persons rising every year. Increases in HIV prevalence have resulted not only from new infections, but also from increases in the survival of HIV-infected persons produced by effective anti-retroviral therapies. Augmentation of anti-viral immune responses may be able to further increase the survival of HIV-infected persons. One strategy to augment these responses is to reinvigorate exhausted anti-HIV immune cells present in chronically infected persons. The PD-1-PD-L1 pathway has been implicated in the exhaustion of virus-specific T cells during chronic HIV infection. Inhibition of PD-1 signaling using blocking anti-PD-1 antibodies has been shown to reduce simian immunodeficiency virus (SIV) loads in monkeys. We now show that PD-1 blockade can improve control of HIV replication in vivo in an animal model. BLT (Bone marrow-Liver-Thymus) humanized mice chronically infected with HIV-1 were treated with an anti-PD-1 antibody over a 10-day period. The PD-1 blockade resulted in a very significant 45-fold reduction in HIV viral loads in humanized mice with high CD8(+) T cell expression of PD-1, compared to controls at 4 weeks post-treatment. The anti-PD-1 antibody treatment also resulted in a significant increase in CD8(+) T cells. PD-1 blockade did not affect T cell expression of other inhibitory receptors co-expressed with PD-1, including CD244, CD160 and LAG-3, and did not appear to affect virus-specific humoral immune responses. These data demonstrate that inhibiting PD-1 signaling can reduce HIV viral loads in vivo in the humanized BLT mouse model, suggesting that blockade of the PD-1-PD-L1 pathway may have therapeutic potential in the treatment of patients already infected with the AIDS virus.
Assuntos
Anticorpos Bloqueadores/farmacologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Carga Viral/efeitos dos fármacos , Animais , Western Blotting , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Subunidade gama Comum de Receptores de Interleucina/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Receptor de Morte Celular Programada 1/imunologiaRESUMO
The development of mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT (bone marrow, liver, thymus) mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exist regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. We infected humanized BLT mice with HIV-1 as a model pathogen and characterized HIV-1-specific immune responses and viral evolution during the acute phase of infection. HIV-1-specific CD8(+) T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics, and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication and restricted the same CD8(+) T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen-specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the use of a small-animal model for human pathogens.
Assuntos
Linfócitos T CD8-Positivos/imunologia , HIV-1/imunologia , Animais , Infecções por HIV/imunologia , Antígenos HLA-B/metabolismo , Humanos , Imunidade Celular/imunologia , CamundongosRESUMO
The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4⺠T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.
Assuntos
Aptâmeros de Nucleotídeos/uso terapêutico , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Colo do Útero/efeitos dos fármacos , Genes gag , Genes vif , Infecções por HIV/prevenção & controle , Macrófagos/efeitos dos fármacos , RNA Interferente Pequeno/uso terapêutico , Receptores CCR5/genética , Quimeras de Transplante/virologia , Vagina/efeitos dos fármacos , Administração Intravaginal , Animais , Aptâmeros de Nucleotídeos/administração & dosagem , Sequência de Bases , Antígenos CD4/genética , Linfócitos T CD4-Positivos/imunologia , Polaridade Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Colo do Útero/virologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Infecções por HIV/transmissão , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , RNA Interferente Pequeno/administração & dosagem , Especificidade da Espécie , Quimeras de Transplante/imunologia , Vagina/virologiaRESUMO
Receptors involved in innate immunity to fungal pathogens have not been fully elucidated. We show that the Caenorhabditis elegans receptors CED-1 and C03F11.3, and their mammalian orthologues, the scavenger receptors SCARF1 and CD36, mediate host defense against two prototypic fungal pathogens, Cryptococcus neoformans and Candida albicans. CED-1 and C03F11.1 mediated antimicrobial peptide production and were necessary for nematode survival after C. neoformans infection. SCARF1 and CD36 mediated cytokine production and were required for macrophage binding to C. neoformans, and control of the infection in mice. Binding of these pathogens to SCARF1 and CD36 was beta-glucan dependent. Thus, CED-1/SCARF1 and C03F11.3/CD36 are beta-glucan binding receptors and define an evolutionarily conserved pathway for the innate sensing of fungal pathogens.
Assuntos
Caenorhabditis elegans/microbiologia , Candida albicans/imunologia , Sequência Conservada , Cryptococcus neoformans/imunologia , Evolução Molecular , Imunidade Inata , Receptores Depuradores/imunologia , Animais , Antígenos CD36/deficiência , Antígenos CD36/imunologia , Caenorhabditis elegans/imunologia , Proteínas de Caenorhabditis elegans/imunologia , Candida albicans/citologia , Candidíase/imunologia , Candidíase/microbiologia , Adesão Celular , Criptococose/imunologia , Criptococose/microbiologia , Cryptococcus neoformans/citologia , Citocinas/biossíntese , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas de Membrana/imunologia , Camundongos , RNA Interferente Pequeno/metabolismo , Análise de Sobrevida , Receptor 2 Toll-Like/metabolismoRESUMO
Lung transplantation remains the only effective therapy for patients with end-stage lung disease, but survival is limited by the development of obliterative bronchiolitis (OB). The chemokine receptor CXCR3 and two of its ligands, CXCL9 and CXCL10, have been identified as important mediators of OB. However, the relative contribution of CXCL9 and CXCL10 to the development of OB and the mechanism of regulation of these chemokines has not been well defined. In this study, we demonstrate that CXCL9 and CXCL10 are up-regulated in unique patterns following tracheal transplantation in mice. In these experiments, CXCL9 expression peaked 7 days posttransplant, while CXCL10 expression peaked at 1 day and then again 7 days posttransplant. Expression of CXCL10 was also up-regulated in a novel murine model of lung ischemia, and in bronchoalveolar lavage fluid taken from human lungs 24 h after lung transplantation. In further analysis, we found that 3 h after transplantation CXCL10 is donor tissue derived and not dependent on IFN-gamma or STAT1, while 24 h after transplantation CXCL10 is from recipient tissue and regulated by IFN-gamma and STAT1. Expression of both CXCL9 and CXCL10 7 days posttransplant is regulated by IFN-gamma and STAT1. Finally, we demonstrate that deletion of CXCR3 in recipients reduces airway obliteration. However, deletion of either CXCL9 or CXCL10 did not affect airway obliteration. These data show that in this murine model of obliterative bronchiolitis, these chemokines are differentially regulated following transplantation, and that deletion of either chemokine alone does not affect the development of airway obliteration.