RESUMO
Hematopoietic stem cells (HSCs) are precursor cells that give rise to blood, immune and tissue-resident progeny in humans. Their position at the starting point of hematopoiesis offers a unique therapeutic opportunity to treat certain hematologic diseases by implementing corrective changes that are subsequently directed through to multiple cell lineages. Attempts to exploit HSCs clinically have evolved over recent decades, from initial approaches that focused on transplantation of healthy donor allogeneic HSCs to treat rare inherited monogenic hematologic disorders, to more contemporary genetic modification of autologous HSCs offering the promise of benefits to a wider range of diseases. We are on the cusp of an exciting new era as the transformative potential of HSC gene therapy to offer durable delivery of gene-corrected cells to a range of tissues and organs, including the central nervous system, is beginning to be realized. This article reviews the rationale for targeting HSCs, the approaches that have been used to date for delivering therapeutic genes to these cells, and the latest technological breakthroughs in manufacturing and vector design. The challenges faced by the biotechnology cell and gene therapy sector in the commercialization of HSC gene therapy are also discussed.
Assuntos
Doenças Hematológicas , Transplante de Células-Tronco Hematopoéticas , Humanos , Células-Tronco Hematopoéticas , Terapia Genética , HematopoeseRESUMO
Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a curative treatment for hematologic malignancies, relies on donor cytotoxic T lymphocyte (CTL)-mediated graft-versus-leukemia (GVL) effect. Major complications of HSCT are graft-versus-host disease (GVHD) that targets specific tissues and tumor relapses. However, the mechanisms dictating the anatomical features of GVHD and GVL remain unclear. Here, we show that after HSCT, CTLs exhibited different killing activity in distinct tissues, being highest in the liver and lowest in lymph nodes. Differences were imposed by the microenvironment, partly through differential PD-1 ligand expression, which was strongly elevated in lymph nodes. Two-photon imaging revealed that PD-1 blockade restored CTL sensitivity to antigen and killing in lymph nodes. Weak CTL activity in lymph nodes promoted local tumor escape but could be reversed by anti-PD-1 treatment. Our results uncover a mechanism generating an anatomical segregation of CTL activity that might dictate sites of GVHD and create niches for tumor escape.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Tumor/imunologia , Transplante de Células-Tronco Hematopoéticas , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/imunologia , Aloenxertos , Animais , Feminino , Citometria de Fluxo , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
The inflammasome is activated in response to a variety of pathogens and has an important role in shaping adaptive immunity, yet the spatiotemporal orchestration of inflammasome activation in vivo and the mechanisms by which it promotes an effective immune response are not fully understood. Using an in vivo reporter to visualize inflammasome assembly, we establish the distribution, kinetics and propagation of the inflammasome response to a local viral infection. We show that modified vaccinia Ankara virus induces inflammasome activation in subcapsular sinus (SCS) macrophages, which is immediately followed by cell death and release of extracellular ASC specks. This transient inflammasome signaling in the lymph node generates a robust influx of inflammatory cells and mobilizes T cells from the circulation to increase the magnitude of T cell responses. We propose that after infection, SCS macrophages deliver a burst response of inflammasome activity and cell death that translates into the broadening of T cell responses, identifying an important aspect of inflammasome-driven vaccination strategies.
Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Inflamassomos/imunologia , Linfonodos/imunologia , Macrófagos/imunologia , Infecções por Poxviridae/imunologia , Linfócitos T/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Adaptadoras de Sinalização CARD , Citometria de Fluxo , Camundongos , Vaccinia virus/imunologiaRESUMO
DC vaccines have been used to induce tumour-specific cytotoxic T cells . However, this approach to cancer immunotherapy has had limited success. To be successful, injected DCs need to migrate to the LNs where they can stimulate effector T cells . We and others have previously demonstrated by MRI that tumour antigen-pulsed-DCs labelled ex vivo with superparamagnetic iron oxide nanoparticles migrated to the draining LNs and are capable of activating antigen-specific T cells . The results from our study demonstrated that ex vivo superparamagnetic iron oxide nanoparticles-labelled and OVA-pulsed DCs prime cytotoxic CD8(+) T-cell responses to protect against a B16-OVA tumour challenge. In the clinic, a possible noninvasive surrogate marker for efficacy of DC vaccination is to image the specific migration and accumulation of T cells following DC vaccination.
Assuntos
Células Dendríticas/imunologia , Linfócitos T Citotóxicos/imunologia , Tecnécio Tc 99m Exametazima , Vacinação , Animais , Camundongos , Camundongos Endogâmicos BALB C , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios XRESUMO
Modification of allogeneic dendritic cells (DCs) through drug treatment results in DCs with in vitro hallmarks of tolerogenicity. Despite these observations, using murine MHC-mismatched skin and heart transplant models, donor-derived drug-modified DCs not only failed to induce tolerance but also accelerated graft rejection. The latter was inhibited by injecting the recipient with anti-CD8 Ab, which removed both CD8(+) T cells and CD8(+) DCs. The discrepancy between in vitro and in vivo data could be explained, partly, by the presentation of drug-modified donor DC MHC alloantigens by recipient APCs and activation of recipient T cells with indirect allospecificity, leading to the induction of alloantibodies. Furthermore, allogeneic MHC molecules expressed by drug-treated DCs were rapidly processed and presented in peptide form by recipient APCs in vivo within hours of DC injection. Using TCR-transgenic T cells, Ag presentation of injected OVA-pulsed DCs was detectable for ≤ 3 d, whereas indirect presentation of MHC alloantigen by recipient APCs led to activation of T cells within 14 h and was partially inhibited by reducing the numbers of CD8(+) DCs in vivo. In support of this observation when mice lacking CD8(+) DCs were pretreated with drug-modified DCs prior to transplantation, skin graft rejection kinetics were similar to those in non-DC-treated controls. Of interest, when the same mice were treated with anti-CD40L blockade plus drug-modified DCs, skin graft survival was prolonged, suggesting endogenous DCs were responsible for T cell priming. Altogether, these findings highlight the risks and limitations of negative vaccination using alloantigen-bearing "tolerogenic" DCs.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Forkhead/imunologia , Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Isoantígenos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/imunologia , Transplante de Pele/imunologia , Doadores de Tecidos , TransplanteRESUMO
Chronic graft-versus-host disease (cGVHD) is characterised by a complex etiology of both alloimmune- and autoimmune-mediated disease progression and pathology, and is consequently difficult to control. The therapeutic potential of regulatory T (Treg) cells for cGVHD is currently being investigated; however, the relative ability of Treg cells with defined antigen specificities for auto- and alloantigen to prevent disease has not been previously examined. In this study, we show that donor-derived Treg-cell lines generated with self-MHC H-2(b) specificity or specificity for BALB/c H-2(d) alloantigen presented via the direct or indirect pathways are able to mediate an equal protection against cGVHD immune pathology in a disease model associated with recipient B-cell-driven humoral autoimmunity and glomerulonephritis. Mechanistically, autospecific Treg cells prevented disease induction by blocking donor T-cell engraftment whereas allospecific Treg cells permitted long-term engraftment of donor T cells. Donor T cells, while unresponsive to auto- and recipient alloantigens, retained the capacity to respond to third party alloantigens on ex vivo stimulation. These findings indicate that allospecific Treg cells may therefore be more clinically relevant as a cell therapy for cGVHD in the context of haplo-identical hematopoietic transplantation, as they allow persistence of donor T cells capable of responding to foreign antigens whilst preventing cGVHD-mediated autoimmunity.
Assuntos
Autoimunidade , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Antígenos H-2/imunologia , Isoantígenos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Doença Crônica , Modelos Animais de Doenças , Feminino , Glomerulonefrite/imunologia , Glomerulonefrite/prevenção & controle , Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas , Imunidade Humoral , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Reguladores/transplante , Transplante HomólogoRESUMO
Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4(+) T cells but, most importantly, they primed cytotoxic CD8(+) T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Dextranos/metabolismo , Melanoma Experimental/imunologia , Coloração e Rotulagem , Vacinação , Animais , Bioensaio , Células da Medula Óssea/citologia , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Meios de Contraste/metabolismo , Células Dendríticas/citologia , Células Dendríticas/ultraestrutura , Linfonodos/metabolismo , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Linfócitos T/citologiaRESUMO
Graft rejection by the immune system is a major cause of transplant failure. Lifelong immunosuppression decreases the incidence of graft rejection; however, nonspecific immunosuppression results in increased susceptibly to infection and cancer. Regulatory T cells (T(regs)), which suppress the activation of the immune system and induce tolerance, are currently under evaluation for use in clinical transplantation. Ex vivo expanded polyclonal T(regs) that are introduced into transplant recipients alter the balance of T effector cells to T(regs); however, experimental data suggest that alloantigen-specific T(regs) would be more effective at preventing graft rejection. We have developed a method to enrich alloantigen-specific human T(regs) based on the coexpression of activation markers, CD69 and CD71. These T(regs) could be readily expanded in vitro and demonstrated potent antigen-specific suppression. In a humanized mouse model of alloimmune-mediated injury of human skin grafts, alloantigen-specific T(regs) resulted in a significant reduction in clinically relevant indicators of dermal tissue injury when compared with polyclonal T(regs), restoring a histology comparable to healthy skin. This method of human allospecific T(reg) selection should be scalable to the clinic. The improved in vivo efficacy of alloantigen-specific T(regs) over polyclonal T(regs) shown here suggests that generating "customized" T(regs) with defined anti-donor allospecificities may improve current practice in clinical immunotherapy.
Assuntos
Isoantígenos/imunologia , Transplante de Pele/imunologia , Linfócitos T Reguladores/imunologia , HumanosRESUMO
In order to optimise and improve the efficacy of transfection mediated by dendrimers, it is essential to fully understand the mechanisms of cell entry and intracellular trafficking by these complexes. Previously, we have shown that gene delivery by dendrimers is dependent from cholesterol and membrane rafts. The inhibition of transfection by treatment with filipin III suggested that gene delivery might be occurring by a caveolin-dependent pathway. We therefore investigated the internalisation and transfection properties of dendriplexes using cell lines (HeLa and HepG2) that express few caveolae. We show that, in contrast to other cells, cholesterol depletion does not affect the ability of dendriplexes to transfect these cells. Inhibition of clathrin-independent, phagocytic and macropinocytic pathways also failed to inhibit transfection of these cells and endothelial cells. However, overexpression of caveolin 1 resulted in an increased rate of dendriplex uptake into HeLa, HepG2 and endothelial cells, and increased transfection efficiency. Furthermore, in endothelial cells, confocal microscopy demonstrated colocalisation of dendriplexes and caveolin 1. These data highlight that dendriplexes may use different internalisation pathways in different cells, and that caveolae form a preferential route for gene delivery by this non-viral vector.
Assuntos
Caveolina 1/metabolismo , Dendrímeros/química , Transfecção/métodos , Androstadienos/farmacologia , Caveolina 1/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Clorpromazina/farmacologia , Colesterol/química , Cromonas/farmacologia , Citocalasina D/farmacologia , Dendrímeros/farmacocinética , Dendrímeros/farmacologia , Células Endoteliais , Células HeLa , Humanos , Morfolinas/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , WortmaninaRESUMO
PURPOSE: Proinflammatory cytokines are integral components of the allogeneic response to a corneal transplant and contribute to the pathogenesis of graft failure that results from irreversible damage to donor corneal endothelium. As yet, the mechanism and effectors of tissue damage during graft rejection remain unidentified. In the current study, the synergistic apoptotic effect of sustained proinflammatory cytokine insult was investigated in excised cornea and in transformed and primary corneal endothelial cells. METHODS: Apoptosis was assessed by tissue- and flow cytometry-based TUNEL staining. Downstream signaling events of cytokine stimulation and subsequent activation status of endothelium were studied by RT-PCR and Western blot analysis. Cellular production of NO was examined by the Griess reaction. RESULTS: Prolonged exposure (48 hours) of corneal endothelium to IL-1, IFNgamma, and TNF (100 ng/mL each) resulted in induction of apoptosis. Synergy in induction of apoptosis was found after exposure to cytokine combinations. Cytokine-mediated cytotoxicity was correlated with high and sustained (up to 36 hours) endothelial activation (specifically through NF-kappaB, p38, and STAT-1), upregulation of inducible nitric oxide synthase (iNOS), and elevated de novo production of NO. Pharmacologic inhibition of iNOS elicited complete cytoprotection from inflammatory cytokine insult. CONCLUSIONS: The specific release of proinflammatory cytokines from alloreactive infiltrating cells, in combination with the inflamed environment of a corneal allograft, results in apoptosis in the corneal endothelium. This effect is mediated by the de novo generation of NO and sustained activation of NF-kappaB, p38, and STAT-1. Inflammatory cytokine-induced apoptosis presents a new target for the development of interventions to prevent or attenuate endothelial injury in graft rejection.