RESUMO
Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human OTC cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTCSpf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic.
RESUMO
Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8+ T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25+ T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration.
Assuntos
Dependovirus/imunologia , Terapia Genética , Vetores Genéticos/imunologia , Imunossupressores/administração & dosagem , Sirolimo/administração & dosagem , Animais , Avaliação Pré-Clínica de Medicamentos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas , Linfócitos T/efeitos dos fármacosRESUMO
Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa-/-) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa-/- mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector-mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease.
Assuntos
Dependovirus/genética , Doença de Depósito de Glicogênio Tipo II/terapia , Fígado/metabolismo , Animais , Terapia Genética , Vetores Genéticos , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/fisiologiaRESUMO
Use of adeno-associated viral (AAV) vectors for liver-directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)-dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV-mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short-term treatment with a single dose of torin 1 significantly increased vector-mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid-α-glucosidase in nonhuman primates. CONCLUSION: We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver-directed gene therapy using AAV vectors and showed in a proof-of-principle study how this virus-host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252-265).
Assuntos
Autofagia/genética , Dependovirus/genética , Terapia Genética/métodos , Hepatócitos/citologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Transdução GenéticaRESUMO
Results from clinical trials of liver gene transfer for hemophilia demonstrate the potential of the adeno-associated virus (AAV) vector platform. However, to achieve therapeutic transgene expression, in some cases high vector doses are required, which are associated with a higher risk of triggering anti-capsid cytotoxic T-cell responses. Additionally, anti-AAV preexisting immunity can prevent liver transduction even at low neutralizing antibody (NAb) titers. Here, we describe the use of exosome-associated AAV (exo-AAV) vectors as a robust liver gene delivery system that allows the therapeutic vector dose to be decreased while protecting from preexisting humoral immunity to the capsid. The in vivo efficiency of liver targeting of standard AAV8 or AAV5 and exo-AAV8 or exo-AAV5 vectors expressing human coagulation factor IX (hF.IX) was evaluated. A significant enhancement of transduction efficiency was observed, and in hemophilia B mice treated with 4 × 1010 vector genomes per kilogram of exo-AAV8 vectors, a staggering â¼1 log increase in hF.IX transgene expression was observed, leading to superior correction of clotting time. Enhanced liver expression was also associated with an increase in the frequency of regulatory T cells in lymph nodes. The efficiency of exo- and standard AAV8 vectors in evading preexisting NAbs to the capsid was then evaluated in a passive immunization mouse model and in human sera. Exo-AAV8 gene delivery allowed for efficient transduction even in the presence of moderate NAb titers, thus potentially extending the proportion of subjects eligible for liver gene transfer. Exo-AAV vectors therefore represent a platform to improve the safety and efficacy of liver-directed gene transfer.
RESUMO
Since the early days of gene therapy, muscle has been one the most studied tissue targets for the correction of enzyme deficiencies and myopathies. Several preclinical and clinical studies have been conducted using adeno-associated virus (AAV) vectors. Exciting progress has been made in the gene delivery technologies, from the identification of novel AAV serotypes to the development of novel vector delivery techniques. In parallel, significant knowledge has been generated on the host immune system and its interaction with both the vector and the transgene at the muscle level. In particular, the role of underlying muscle inflammation, characteristic of several diseases affecting the muscle, has been defined in terms of its potential detrimental impact on gene transfer with AAV vectors. At the same time, feedback immunomodulatory mechanisms peculiar of skeletal muscle involving resident regulatory T cells have been identified, which seem to play an important role in maintaining, at least to some extent, muscle homeostasis during inflammation and regenerative processes. Devising strategies to tip this balance towards unresponsiveness may represent an avenue to improve the safety and efficacy of muscle gene transfer with AAV vectors.
Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Músculo Esquelético/imunologia , Transgenes/imunologia , Animais , Dependovirus/imunologia , Terapia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Tolerância Imunológica/genética , Injeções Intramusculares , Camundongos , Músculo Esquelético/virologia , Doenças Neuromusculares/terapiaRESUMO
The immunogenic properties of recombinant adeno-associated virus (rAAV) gene transfer vectors remain incompletely characterized in spite of their usage as gene therapy vectors or as vaccines. Molecular interactions between rAAV and various types of antigen-presenting cells (APCs), as well as the impact of these interactions on transgene or capsid-specific immunization remain unclear. We herein show that binding motifs recognized by the capsid and which determine the vector tissue tropism are also critical for key immune activation processes. Using rAAV capsid serotype 1 (rAAV1) vectors which primary receptors on target cells are α2,3 and α2,6 N-linked sialic acids, we show that sialic acid-dependent binding of rAAV1 on APCs is essential to trigger CD4(+) T-cell responses by increasing rAAV1 uptake and contributing to antigenic presentation of both the capsid and transgene product although this involves different APCs. In addition, the nanoparticulate structure of the vector in itself appears to be sufficient to trigger mobilization and activation of some APCs. Therefore, combinations of structural and of serotype-specific cell-targeting properties of rAAV1 determine its complex immunogenicity. These findings may be useful to guide a selection of rAAV variants depending on the intended level of immunogenicity for either gene therapy or vaccination applications.
Assuntos
Dependovirus/genética , Nanopartículas/química , Sorogrupo , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Capsídeo/imunologia , Dependovirus/imunologia , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Injeções Intramusculares , Lectinas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nanosferas/química , Nanosferas/virologia , Análise de Componente Principal , Transgenes , Vacinas Sintéticas/virologiaRESUMO
The generation of large amounts of induced CD4+ CD25+ Foxp3+ regulatory T (iTreg) cells is of great interest for several immunotherapy applications, therefore a better understanding of signals controlling iTreg cell differentiation and expansion is required. There is evidence that oxidative metabolism may regulate several key signalling pathways in T cells. This prompted us to investigate the effects of oxygenation on iTreg cell generation by comparing the effects of atmospheric (21%) or of low (5%) O2 concentrations on the phenotype of bead-stimulated murine splenic CD4+ T cells from Foxp3-KI-GFP T-cell receptor transgenic mice. The production of intracellular reactive oxygen species was shown to play a major role in the generation of iTreg cells, a process characterized by increased levels of Sirt1, PTEN and Glut1 on the committed cells, independently of the level of oxygenation. The suppressive function of iTreg cells generated either in atmospheric or low oxygen levels was equivalent. However, greater yields of iTreg cells were obtained under low oxygenation, resulting from a higher proliferative rate of the committed Treg cells and higher levels of Foxp3, suggesting a better stability of the differentiation process. Higher expression of Glut1 detected on iTreg cells generated under hypoxic culture conditions provides a likely explanation for the enhanced proliferation of these cells as compared to those cultured under ambient oxygen. Such results have important implications for understanding Treg cell homeostasis and developing in vitro protocols for the generation of Treg cells from naive T lymphocytes.
RESUMO
Gene transfer efficacy is limited by unwanted immunization against transgene products. In some models, immunization may be avoided by regulating transgene expression with mir142.3p target sequences. Yet, it is unclear if such a strategy controls T-cell responses following recombinant adeno-associated viral vector (rAAV)-mediated gene transfer, particularly in muscle. In mice, intramuscular rAAV1 gene delivery of a tagged human sarcoglycan muscle protein is robustly immunogenic and leads to muscle destruction. In this model, the simple insertion of mir142.3p-target sequences in the transgene expression cassette modifies the outcome of gene transfer, providing high and persistent levels of muscle transduction in C57Bl/6 mice. Such regulated vector fails to prime specific CD4 and CD8 T cells; although, transgene tolerance seems to result from ignorance and could be broken by a robust antigenic challenge. While effective in normal mice, the mir142.3p-regulated transgene remains immunogenic in sarcoglycan-deficient dystrophic mice. In these mice, transgene expression is only prolonged but does not persist as effector CD4 and CD8 T-cell responses develop. Thus, using a mir142.3p-regulated transgene can improve rAAV muscle gene transfer results, but the level of efficacy depends on the context of application. In normal muscle, this strategy is sufficient to prevent immunization and functions even more effectively than tissue-specific promoters. In dystrophic models, additional strategies are required to fully control T-cell responses.
Assuntos
Dependovirus/genética , Expressão Gênica , Tolerância Imunológica/genética , MicroRNAs/genética , Músculo Esquelético/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Distrofia Muscular Animal/imunologia , Distrofia Muscular Animal/metabolismo , Sarcoglicanas/genética , Sarcoglicanas/metabolismo , TransgenesRESUMO
The administration of recombinant adeno-associated viral vectors (rAAV) for gene transfer induces strong humoral responses through mechanisms that remain incompletely characterized. To investigate the links between innate and adaptive immune responses to the vector, rAAVs were injected intravenously into mice deficient in cell-intrinsic components of innate responses (Toll-like receptors (TLRs), type-1 interferon (IFN) or inflammasome signaling molecules) and AAV-specific antibodies were measured. Of all molecules tested, only MyD88 was critically needed to mount immunoglobulin G (IgG) responses since MyD88(-/-) mice failed to develop high levels of AAV-specific IgG2 and IgG3, regardless of capsid serotype injected. None of the TLRs tested was essential here, but TLR9 ensured a Th1-biased antibody responses. Indeed, capsid-specific Th1 cells were induced upon injection of rAAV1, as directly confirmed with an epitope-tagged capsid, and the priming and development of these Th1 cells required T cell-extrinsic MyD88. Cell transfer experiments showed that autonomous MyD88 signaling in B cells, but not T cells, was sufficient to produce Th1-dependent IgGs. Therefore, rAAV triggers innate responses, at least via B cells, controlling the development of capsid-specific Th1-driven antibodies. MyD88 emerges as a critical and pivotal regulator of both T- and B-cell adaptive immunity against AAV.
Assuntos
Adenoviridae/imunologia , Anticorpos/imunologia , Anticorpos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Células Th1/imunologia , Animais , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: It is generally accepted that all transplants are not rejected in the same fashion. However, the extrinsic and intrinsic factors that control the recognition and rejection of a particular allograft by the host are not well characterized. METHODS: We compared the mechanisms underlying the response with donor antigens by T cells activated after transplantation of fully allogeneic skin and corneal grafts in mice. RESULTS: In corneal-transplanted mice, the CD4+ T-cell response was exclusively mediated by T cells recognizing minor antigens in an indirect fashion and producing low levels of interleukin-2. In contrast, skin grafts elicited both direct and indirect CD4+ T-cell responses primarily directed to major histocompatibility complex antigens and characterized by high interleukin-2 levels. Although CD8+ T-cells producing gamma interferon were activated directly in both skin- and corneal-grafted mice, only CD8+ T cells from skin-transplanted mice mounted a cytotoxic response. Next, we investigated whether failure of corneal transplants to induce a CD4+ direct alloresponse is due to their poor immunogenicity or due to the site of placement (eye). We observed that corneas transplanted under the skin and splenocytes transplanted in the eye were both capable of inducing direct CD4+ T-cell alloreactivity. CONCLUSIONS: This shows that failure of orthotopic corneal allotransplants to elicit a CD4+ T-cell direct alloresponse is associated with the combination of two factors, their low immunogenicity and the immune-privileged properties of the eye.
Assuntos
Transplante de Córnea/imunologia , Rejeição de Enxerto/imunologia , Transplante de Pele/imunologia , Animais , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Antígenos de Histocompatibilidade/imunologia , Imunidade Inata/imunologia , Ativação Linfocitária/imunologia , Camundongos , Transplante Homólogo/imunologiaRESUMO
We investigated whether Toll-like receptor ligands (TLR-Ls) can bypass the requirement for CD4(+) T-cell help in the induction of fully efficient memory CD8(+) T cells (cytotoxic T lymphocytes [CTLs]). "Helpless" CTLs were induced by a synthetic CD8(+) T-cell epitope administered with TLR3-L and TLR9-L, but not with TLR2/6-L, TLR4-L, or TLR7-L. The up-regulation of MHC-I and costimulatory molecules by dendritic cells following TLR stimulation was not sufficient for the priming of "helpless" CTLs, which depended essentially on the induction of a strong IFN-alpha/beta response. The "helpless" CTLs induced by TLR-Ls differentiated into fully functional memory CTLs able to proliferate as well as their "helped" counterparts upon challenge, in the absence of CD4(+) T-cell help.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Memória Imunológica , Receptor 3 Toll-Like/fisiologia , Animais , Linfócitos T CD4-Positivos , Proliferação de Células , Apresentação Cruzada , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/farmacologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos , Receptor 3 Toll-Like/imunologia , Regulação para Cima/efeitos dos fármacosRESUMO
Controlling the cross-presentation of exogenous Ags to CD8+ T cells represents a major step for designing new vaccination strategies. Whereas several recombinant pseudo-viral particles have been used as delivery systems for triggering potent CTL responses to heterologous exogenous Ags, the adjuvant properties of virus-like particles (VLPs) themselves were little questioned. Here, we analyzed the contribution of the porcine parvovirus (PPV)-VLPs to the induction of protective cellular responses to exogenous Ags carried by an independent delivery system. Microspheres, which are known to transfer exogenous Ags into the MHC class I pathway, were chosen for delivering the immunodominant OVA(257-264) CD8+ T cell epitope (B-OVAp). This delivery system fulfills the requirements in terms of cross-presentation, but fails to induce cross-priming of specific CD8+ T cells. Coinjection of PPV-VLPs with B-OVAp results in the priming of potent CTL responses and type 1-biased immunity in a CD4- and CD40-independent manner, as efficiently as the recombinant PPV-VLPs carrying the same epitope (PPV-OVAp). Furthermore, vaccination with PPV-VLPs and B-OVAp was fully efficient to protect mice against the development of OVA-bearing melanoma. These findings indicate that PPV-VLPs act not only as a delivery system but also as a strong adjuvant when independently provided with exogenous Ag. Thus, dissociation between delivery system and adjuvant would provide a more flexible and reliable system to induce potent and protective CTL.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos Imunodominantes/administração & dosagem , Animais , Antígenos CD40/genética , Antígenos CD40/metabolismo , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Proteínas do Ovo/administração & dosagem , Proteínas do Ovo/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Fatores Imunológicos/administração & dosagem , Técnicas In Vitro , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microesferas , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Parvovirus Suíno/imunologia , Fragmentos de Peptídeos , Transdução de Sinais , Linfócitos T Citotóxicos/imunologiaRESUMO
Neonatal cytotoxic T cell responses have only been elicited to date with immunogens or delivery systems inducing potent direct APC activation. To define the minimal activation requirements for the induction of neonatal CD8(+) cytotoxic responses, we used synthetic microspheres (MS) coated with a single CD8(+) T cell peptide from lymphocytic choriomeningitis virus (LCMV) or HIV-1. Unexpectedly, a single injection of peptide-conjugated MS without added adjuvant induced CD4-dependent Ag-specific neonatal murine cytotoxic responses with adult-like CTL precursor frequency, avidity for Ag, and frequency of IFN-gamma-secreting CD8(+) splenocytes. Neonatal CD8(+) T cell responses to MS-LCMV were elicited within 2 wk of a single immunization and, upon challenge, provided similar protection from viral replication as adult CTLs, demonstrating their in vivo competence. As previously reported, peptide-coated MS elicited no detectable activation of adult CD11c(+) dendritic cells (DC). In contrast, CTL responses were associated with a partial activation of neonatal CD11c(+) DC, reflected by the up-regulation of CD80 and CD86 expression but no concurrent changes in MHC class II or CD40 expression. However, this partial activation of neonatal DC was not sufficient to circumvent the requirement for CD4(+) T cell help. The effective induction of neonatal CD8(+) T cell responses by this minimal Ag delivery system demonstrates that neonatal CD11c(+) DC may mature sufficiently to stimulate naive CD8(+) neonatal T cells, even in the absence of strong maturation signals.
Assuntos
Antígeno CD11c/imunologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Microesferas , Animais , Animais Recém-Nascidos , Antígenos Virais/administração & dosagem , Antígenos Virais/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/virologia , Epitopos de Linfócito T/imunologia , HIV-1/imunologia , Imunização , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Peptídeos/imunologiaRESUMO
Murine mixed hematopoietic chimerism can be achieved following nonmyeloablative conditioning with cyclophosphamide, T cell-depleting monoclonal antibodies, and thymic irradiation. Donor lymphocyte infusions (DLIs) 35 days after bone marrow transplantation (BMT) convert mixed to full donor chimerism and mediate graft-versus-lymphoma effects without graft-versus-host disease. We evaluated the role of T-cell subsets in DLIs in converting mixed to full donor chimerism in a fully major histocompatibility complex-mismatched strain combination. Whereas DLIs administered on day 35 converted 100% of mixed chimeras to full donor chimerism, conversion was less frequent when either CD4 or CD8 cells were depleted, indicating that both subsets contribute to the conversion. Surprisingly, administration of CD8-depleted DLIs led to complete loss of donor chimerism in a high proportion (54%) of recipients compared with CD4-plus CD8-depleted DLIs (15%) or CD4-depleted DLIs (0%) (P <.05). DLIs administered at early time points after BMT (eg, day 21) also precipitated rejection of donor marrow by recipient alphabeta T cells, in association with donor CD4 cell expansion and high production of interleukin 2 (IL-2), IL-4, and interferon-gamma. Thus, DLIs can paradoxically induce marrow rejection by residual host alphabeta T cells. These results have implications for the timing of and use of subset depletion of DLIs in recipients of nonmyeloablative transplants.
Assuntos
Transplante de Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/imunologia , Reação Enxerto-Hospedeiro/imunologia , Linfócitos T/imunologia , Condicionamento Pré-Transplante/métodos , Animais , Sobrevivência de Enxerto/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Subpopulações de Linfócitos T/imunologia , Fatores de Tempo , Quimeras de Transplante , Transplante Homólogo/imunologiaRESUMO
In this study, we measured direct and indirect T-cell alloresponses mediated by CD4(+) and CD8(+) T cells in three mouse transplantation models: skin, cornea, and retina. We show that the contribution of direct and indirect antigen recognition pathways to the alloresponse to fully allogeneic grafts varies depending upon the nature of the tissue/organ transplanted. The implications of this finding for understanding the cellular mechanisms by which rejection is mediated in different transplant models are discussed.
Assuntos
Rejeição de Enxerto/imunologia , Isoantígenos/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/fisiologia , Transplante de Córnea , Interferon gama/biossíntese , Interleucina-2/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Pele/imunologia , Transplante HomólogoRESUMO
The development of antiviral vaccines has almost exclusively been based on live attenuated vaccines up until now. However, the efficacy of HBsAg particles as an antiHBV vaccine has clearly demonstrated that protective antiviral immunity can be achieved by other strategies. Virus-like particles formed by structural proteins were proven to be highly immunogenic and capable of inducing protective immunity against various viral infections in preclinical studies. Clinical trials using virus-like particles confirmed their safety and immunogenicity. Moreover, chimeric virus-like particles carrying foreign peptidic sequences were shown to elicit potent B- and T-cell responses. Virus-like particles formed by a fusion protein between the HBsAg and the circumsporozoïte surface protein are safe and immunogenic in volunteers and induce a partial protection against natural Plasmodium falciparum infection.
Assuntos
Sistemas de Liberação de Medicamentos , Vacinas Virais/administração & dosagem , Vírus/imunologia , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Feminino , Humanos , Imunidade nas Mucosas/imunologia , Papillomaviridae/imunologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Vacinas Sintéticas/imunologiaRESUMO
This study aimed to determine the effects of anti-CD154 on T cell cytokine profiles and ocular chemokine gene expression after high-risk corneal transplantation and to specifically determine if CD154 blockade is associated with a switch from a Th1 to a Th2 alloimmune response. Mice were used as recipients of syngeneic or multiple minor H or MHC antigen-mismatched corneal grafts. Recipient beds were neovascularized (high-risk). Hosts were randomized to receive either anti-CD154 antibody or control immunoglobulin (Ig) perioperatively. Two weeks after corneal transplantation, allospecific delayed-type hypersensitivity (DTH) was evaluated. Frequencies of interferon-gamma (IFN-gamma)-, interleukin-2 (IL-2)-, IL-4-, and IL-5-secreting T cells in the hosts were measured by enzyme-linked immunospot (ELISPOT) assay. Ocular chemokine gene expression in anti-CD154-treated and control hamster Ig-treated groups was determined using a multiprobe ribonuclease protection assay (RPA). Leukocyte infiltration of corneal grafts was evaluated microscopically. Anti-CD154-treated mice did not exhibit allospecific DTH. The frequencies of Th1 cytokine-producing but not Th2 cytokine-producing T cells were significantly reduced in anti-CD154-treated hosts. Postoperative mRNA levels of RANTES and macrophage inflammatory protein-1beta (MIP-1beta) in anti-CD154-treated eyes were substantially suppressed compared with hamster Ig-treated controls. Leukocyte infiltration was profoundly suppressed in grafts of anti-CD154-treated hosts. These data demonstrate that blockade of the CD40-CD154 costimulatory pathway after corneal transplantation inhibits Th1-mediated responses but does not induce a switch to a Th2-specific response. In addition, anti-CD154 therapy suppresses ocular chemokine gene expression and leukocytic infiltration into allografts.