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Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation responsible for impaired hepatic metabolism and muscle weakness. To date, there is no curative treatment for GSDIII. We previously reported that 2 distinct dual AAV vectors encoding for GDE were needed to correct liver and muscle in a GSDIII mouse model. Here, we evaluated the efficacy of rapamycin in combination with AAV gene therapy. Simultaneous treatment with rapamycin and a potentially novel dual AAV vector expressing GDE in the liver and muscle resulted in a synergic effect demonstrated at biochemical and functional levels. Transcriptomic analysis confirmed synergy and suggested a putative mechanism based on the correction of lysosomal impairment. In GSDIII mice livers, dual AAV gene therapy combined with rapamycin reduced the effect of the immune response to AAV observed in this disease model. These data provide proof of concept of an approach exploiting the combination of gene therapy and rapamycin to improve efficacy and safety and to support clinical translation.
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Dependovirus , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos , Fígado , Sirolimo , Animais , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Dependovirus/genética , Terapia Genética/métodos , Camundongos , Fígado/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Músculo Esquelético/metabolismo , Fenótipo , Sistema da Enzima Desramificadora do Glicogênio/genética , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Humanos , MasculinoRESUMO
One of the major goals of in vivo gene transfer is to achieve long-term expression of therapeutic transgenes in terminally differentiated cells. The extensive clinical experience and the recent approval of Luxturna® (Spark Therapeutics, now Roche) and Zolgensma® (AveXis, now Novartis) place vectors derived from adeno-associated viruses (AAV) among the best options for gene transfer in multiple tissues. Despite these successes, limitations remain to the application of this therapeutic modality in a wider population. AAV was originally identified as a promising virus to derive gene therapy vectors because, despite infecting humans, it was not associated with any evident disease. Thee large proportion of AAV infections in the human population is now revealing as a limitation because after exposure to wild-type AAV, anti-AAV antibodies develops and may neutralize the vectors derived from the virus. Injection of AAV in humans is generally well-tolerated although the immune system can activate after the recognition of AAV vectors capsid and genome. The formation of high-titer neutralizing antibodies to AAV after the first injection precludes vector re-administration. Thus, both pre-existing and post-treatment humoral responses to AAV vectors greatly limit a wider application of this gene transfer modality. Different methods were suggested to overcome this limitation. The extensive preclinical data available and the large clinical experience in the control of AAV vectors immunogenicity are key to clinical translation and to demonstrate the safety and efficacy of these methods and ultimately bring a curative treatment to patients.
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Vetores Genéticos , Imunidade Humoral , Anticorpos Neutralizantes , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , HumanosRESUMO
Neuromuscular disorders are a large group of rare pathologies characterised by skeletal muscle atrophy and weakness, with the common involvement of respiratory and/or cardiac muscles. These diseases lead to life-long motor deficiencies and specific organ failures, and are, in their worst-case scenarios, life threatening. Amongst other causes, they can be genetically inherited through mutations in more than 500 different genes. In the last 20 years, specific pharmacological treatments have been approved for human usage. However, these "à-la-carte" therapies cover only a very small portion of the clinical needs and are often partially efficient in alleviating the symptoms of the disease, even less so in curing it. Recombinant adeno-associated virus vector-mediated gene transfer is a more general strategy that could be adapted for a large majority of these diseases and has proved very efficient in rescuing the symptoms in many neuropathological animal models. On this solid ground, several clinical trials are currently being conducted with the whole-body delivery of the therapeutic vectors. This review recapitulates the state-of-the-art tools for neuron and muscle-targeted gene therapy, and summarises the main findings of the spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD) and X-linked myotubular myopathy (XLMTM) trials. Despite promising efficacy results, serious adverse events of various severities were observed in these trials. Possible leads for second-generation products are also discussed.
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Recombinant adeno-associated virus (rAAV) vectors are one of the most promising in vivo gene delivery tools. Several features make rAAV vectors an ideal platform for gene transfer. However, the high homology with the parental wild-type virus, which often infects humans, poses limitations in terms of immune responses associated with this vector platform. Both humoral and cell-mediated immunity to wild-type AAV have been documented in healthy donors, and, at least in the case of anti-AAV antibodies, have been shown to have a potentially high impact on the outcome of gene transfer. While several factors can contribute to the overall immunogenicity of rAAV vectors, vector design and the total vector dose appear to be responsible of immune-mediated toxicities. While preclinical models have been less than ideal in predicting the outcome of gene transfer in humans, the current preclinical body of evidence clearly demonstrates that rAAV vectors can trigger both innate and adaptive immune responses. Data gathered from clinical trials offers key learnings on the immunogenicity of AAV vectors, highlighting challenges as well as the potential strategies that could help unlock the full therapeutic potential of in vivo gene transfer.
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Adenoviridae/genética , Linfócitos B/imunologia , Terapia Genética/tendências , Vetores Genéticos/genética , Linfócitos T/imunologia , Ensaios Clínicos como Assunto , Humanos , Imunidade , Ativação LinfocitáriaRESUMO
Foxp3+ regulatory T cells (Tregs) play a major role in acquired immune tolerance to allogenic transplants. Their suppressive activity is thought to require T cell receptor (TCR)-driven antigen recognition; little, however, is known about the fraction of Tregs able to recognize alloantigens within this T cell subset primarily educated against self-antigens. Performing transfer experiments of Tregs or conventional T cells (Tconv) into both lymphoreplete and lymphopenic mice, we observed a similarly high proportion of cells signaling through their TCR and proliferating in allogenic hosts. Furthermore, using an in vivo proliferation assay with limited T cell numbers infused into lymphopenic mice, we found that the overall frequency of alloreactive Tregs was similar if not higher to that of alloreactive Tconv. Overall our study highlights a noticeably high level of alloreactive Foxp3+ regulatory T cells accounting for their predominant role in transplantation tolerance.
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Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/imunologia , Isoantígenos/imunologia , Animais , Proliferação de Células/fisiologia , Tolerância Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante/imunologiaRESUMO
A key aspect to consider for vaccinal protection is the induction of a local line of defense consisting of nonrecirculating tissue-resident memory T cells (TRM), in parallel to the generation of systemic memory CD8+ T cell responses. The potential to induce TRM has now been demonstrated for a number of pathogens and viral vectors. This potential, however, has never been tested for recombinant adeno-associated virus (rAAV) vectors, which are weakly inflammatory and poor transducer of dendritic cells. Using a model rAAV2/1-based vaccine, we determined that a single intradermal immunization with rAAV2/1 vectors in mice induces fully functional TRM at the local site of immunization. The optimal differentiation of rAAV-induced transgene-specific skin TRM was dependent on local transgene expression and additional CD4+ T cell help. Transgene expression in dendritic cells, however, appeared to be dispensable for the priming of transgene-specific skin TRM, suggesting that this process solely depends on the cross-presentation of transgene products. Overall, this study provides needed information to properly assess rAAV vectors as T cell-inducing vaccine carriers.IMPORTANCE rAAVs display numerous characteristics that could make them extremely attractive as vaccine carriers, including an excellent safety profile in humans and great flexibility regarding serotypes and choice of target tissue. Studies addressing the ability of rAAV to induce protective T cell responses, however, are scarce. Notably, the potential to induce a tissue-resident memory T cell response has never been described for rAAV vectors, strongly limiting further interest for their use as vaccine carriers. Using a model rAAV2/1 vaccine delivered to the skin, our study demonstrated that rAAV vectors can induce bona fide skin resident TRM and provides additional clues regarding the cellular mechanisms underlying this process. These results will help widen the field of rAAV applications.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Parvovirinae/imunologia , Animais , Células Dendríticas/imunologia , Dependovirus , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Parvovirinae/genética , Pele/citologia , Pele/imunologia , Transgenes/genética , Transgenes/imunologia , Vacinação , Vacinas Virais/imunologiaRESUMO
Recombinant adeno-associated viral (rAAV) vectors exhibit interesting properties as vaccine carriers for their ability to induce long-lasting antibody responses. However, rAAV-based vaccines have been suggested to trigger functionally impaired long-term memory CD8+ T cell responses, in part due to poor dendritic cell (DC) transduction. Such results, albeit limited to intramuscular immunization, undermined the use of rAAV as vaccine vehicles against intracellular pathogens. We report here that intradermal immunization with a model rAAV2/1-based vaccine drives the development of bona fide long-term memory CD8+ T cell responses. The intradermal route of immunization and the presence of potent major histocompatibility complex (MHC) class II responses showed synergistic effects on the overall quantity and quality of systemic long-term effector memory transgene-specific CD8+ T cells being generated against the transgene. Of key interest, we found that the induction of memory cytotoxic T lymphocytes (CTLs) following intradermal immunization was solely dependent on the cross-presentation of skin-expressed transgene products, which appeared highly enhanced as compared to muscle-expressed transgene products. Overall our results highlight key tissue-specific differences in transgene presentation pathway requirements of importance for the design of rAAV-based T cell-inducing vaccines.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Dependovirus/genética , Animais , Linfócitos T CD4-Positivos/metabolismo , Feminino , Citometria de Fluxo , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0157822.].
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Natural regulatory T (Treg) cells interfere with multiple functions, which are crucial for the development of strong anti-tumour responses. In a model of 4T1 mammary carcinoma, depletion of CD25+Tregs results in tumour regression in Balb/c mice, but the mechanisms underlying this process are not fully understood. Here, we show that partial Treg depletion leads to the generation of a particular effector CD8 T cell subset expressing CD11c and low level of PD-1 in tumour draining lymph nodes. These cells have the capacity to migrate into the tumour, to kill DCs, and to locally regulate the anti-tumour response. These events are concordant with a substantial increase in CD11b+ resident dendritic cells (DCs) subsets in draining lymph nodes followed by CD8+ DCs. These results indicate that Treg depletion leads to tumour regression by unmasking an increase of DC subsets as a part of a program that optimizes the microenvironment by orchestrating the activation, amplification, and migration of high numbers of fully differentiated CD8+CD11c+PD1lo effector T cells to the tumour sites. They also indicate that a critical pattern of DC subsets correlates with the evolution of the anti-tumour response and provide a template for Treg depletion and DC-based therapy.
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UNLABELLED: Cancer onset and progression involves the accumulation of multiple oncogenic hits, which are thought to dominate or bypass the physiologic regulatory mechanisms in tissue development and homeostasis. We demonstrate in T-cell acute lymphoblastic leukemia (T-ALL) that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent T-cell receptor (TCR) signaling has antileukemic properties and enforces a molecular program resembling thymic negative selection, a major developmental event in normal T-cell development. Using mouse models of T-ALL, we show that induction of TCR signaling by high-affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3ε chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse- or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of patients with TCR-expressing T-ALL and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. SIGNIFICANCE: T-ALLs are aggressive malignant lymphoid proliferations of T-cell precursors characterized by high relapse rates and poor prognosis, calling for the search for novel therapeutic options. Here, we report that the lineage-specific TCR/CD3 developmental checkpoint controlling cell death in normal T-cell progenitors remains switchable to induce massive tumor cell apoptosis in T-ALL and is amenable to preclinical therapeutic intervention. Cancer Discov; 6(9); 972-85. ©2016 AACR.See related commentary by Lemonnier and Mak, p. 946This article is highlighted in the In This Issue feature, p. 932.
Assuntos
Leucemia de Células T/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Complexo CD3/imunologia , Complexo CD3/metabolismo , Seleção Clonal Mediada por Antígeno , Modelos Animais de Doenças , Feminino , Humanos , Imunofenotipagem , Leucemia de Células T/tratamento farmacológico , Leucemia de Células T/genética , Leucemia de Células T/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Antitransgene CD8(+) T-cell responses are an important hurdle after recombinant adeno-associated virus (rAAV) vector-mediated gene transfer. Indeed, depending on the mutational genotype of the host, transgene amino-acid sequences of foreign origin can elicit deleterious cellular and humoral responses. We compared here two different major histocompatibility complex (MHC) class I epitopes of an engineered ovalbumin transgene delivered in muscle tissue by rAAV1 vector and found very different strength of CD8 responses, muscle destruction being correlated with the course of the immunodominant response. We further demonstrate that robust CD8(+) T-cell priming can occur through the cross-presentation pathway but requires the presence of either a strong MHC class II epitope or antibodies to the transgene product. Finally, manipulating transgene subcellular localization, we found that provided we avoid transgene expression in antigen presenting cells, the poorly accessible cytosolic form of ovalbumin transgene lacking strong MHC II epitope, evades CD8(+) T-cell priming and remains permanently expressed in muscle with no immune cell infiltration. Our results demonstrate that the intrinsic immunogenicity of transgenes delivered with rAAV vector in muscle can be manipulated in a rational manner to avoid adverse immune responses.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Músculo Esquelético/imunologia , Transgenes , Animais , Epitopos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismoRESUMO
Extrathymically induced Foxp3⺠regulatory T (Treg) cells contribute to the pool of Treg cells and are implicated in the maintenance of immune tolerance at environmental interfaces. The impact of T-cell senescence on their generation and function is, however, poorly characterized. We report here that steady-state induction of Foxp3 is impaired in aged T cells in vivo. In vitro assays further revealed that this defective generation of Treg cells was independent from the strength of TCR stimulation and arose before T-cell proliferation. Importantly, they also revealed that this impairment of Foxp3 induction is unrelated to known age-related T-cell defects, such as IL-2 secretion impairment, accumulation of activated T-cell populations, or narrowing of the T-cell repertoire. Finally, a loss of extrathymic induction of Foxp3 and tolerance to minor-mismatched skin graft were observed in aged mice treated by nondepleting anti-CD4 antibody. The T-cell intrinsic impairment of Treg-cell generation revealed here highlights age as a key factor to be considered in immune tolerance induction.
Assuntos
Envelhecimento/imunologia , Senescência Celular/imunologia , Transplante de Pele , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Bloqueadores/administração & dosagem , Antígenos CD4/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Timo/imunologia , Tolerância ao Transplante/efeitos dos fármacosRESUMO
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.
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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.
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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
Foxp3(+) Treg are crucial for the maintenance of self-tolerance and have been shown to control CD8(+) T-cell effector functions. In addition, Treg are thought to control the priming of CD8(+) T cells, which recognize the same antigens as Treg. Taking advantage of our model of peripheral tolerance induction to influenza hemagglutinin (HA) after HA gene transfer, we found that HA-specific Treg suppress antigen-linked CTL responses through early blockade of CD8(+) T-cell expansion. Confronted with their cognate antigen, Treg expand more rapidly than CD8(+) T cells and are highly suppressive only during the initial stages of immune priming. They nullify HA-specific CD8(+) T-cell responses, local inflammatory responses and rejection of HA transduced cells. When HA gene transfer is performed with extensive tissue inflammation, HA-specific Treg are less effective but still reduce the frequency of newly primed HA-specific CD8(+) T cells and the ensuing frequency of memory CD8(+) T cells. Our results demonstrate that Treg control CTL priming in an antigen-specific manner at the level of T-cell expansion, highlighting how self-reactive Treg could prevent the induction of autoimmune responses through selective blockade of autoreactive T-cell proliferation.
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Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Feminino , Citometria de Fluxo , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígenos H-2/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Antígeno de Histocompatibilidade H-2D , Memória Imunológica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismoRESUMO
Exon-skipping AAV1-U7-associated therapy is a promising treatment for Duchenne muscular dystrophy (DMD). We have shown earlier that the newly rescued dystrophin protein is stably expressed for months in mice and dogs, and does not induce immune rejection of transduced fibers. In this study, we used the dystrophic mdx mouse as a preclinical model to characterize the immune response to the adeno-associated virus 1 (AAV1) vector, and tested the feasibility of administering multiple AAV1 injections to extend the treatment to several muscles. We found that re-injections of AAV1 vector are compromised as early as 3 days after the first injection, coincident with a rapid increase in AAV1-specific immunoglobulin M (IgM) and IgG in the serum. Adoptive transfer of immune sera confirmed the rapid appearance of an AAV1 neutralization activity, and experiments with immunoglobulin-deficient (microKO) mice proved that antibodies (Abs) are the only effectors responsible for AAV1-U7 elimination. It is important to note, however, that the AAV2 vector still generated an adverse immune response in microKO mice. By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, we found that a mere 5 days of immunomodulation treatment was sufficient to totally abrogate the formation of anti-AAV1 Abs and to allow for the correction of muscular dystrophy in multiple muscles, provided the treatment was administered during each challenge.
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Dependovirus/genética , Vetores Genéticos/genética , Distrofia Muscular de Duchenne/terapia , Abatacepte , Animais , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Antígenos CD40/imunologia , Imunofluorescência , Terapia Genética/métodos , Imunoconjugados/farmacologia , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Distrofia Muscular de Duchenne/genética , Transdução Genética/métodosRESUMO
Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.
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Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/análise , Tolerância Imunológica , Ativação Linfocitária , Linfócitos T Reguladores/imunologia , Animais , Antígenos/genética , Antígenos/imunologia , Antígenos/farmacologia , Antígeno CD11c/análise , Antígenos CD4/análise , Hemaglutininas/genética , Hemaglutininas/imunologia , Hemaglutininas/farmacologia , Inflamação/imunologia , Subunidade alfa de Receptor de Interleucina-2/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
The inducible heat shock protein Hsp70 has been described as a tumour antigen being frequently overexpressed in tumours of various histologic origins, with a role in tumourigenicity, as a critical event in tumour progression. A strategy to enhance the immune response to an antigen is the identification of multiple epitopes and the induction of a polyspecific response. Applied to tumour vaccination, such a polyspecific response should lead to a more robust antitumour efficacy. The long peptide Hsp70380-402 encompasses three nonamer peptides with a high affinity for HLA-A *0201. In a previous paper, we have shown that two of these nonamer peptides, p391 and p393, can raise CTL to recognize tumour cells overexpressing Hsp70. In the present paper, we demonstrate that the third nonamer peptide, p380, is a new epitope efficient in raising an antitumour immune response. The p380-402 polypeptide was able to induce an immune response against each of the three constituent epitopes both in vivo in HLA-A *0201 transgenic mice and in vitro with human PBMC. This polypeptide therefore constitutes an interesting candidate for the induction of multiple HLA-A *0201-restricted anti-Hsp70 antitumour CTL responses.
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Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Animais , Antígenos de Neoplasias/química , Western Blotting , Células COS , Chlorocebus aethiops , Epitopos de Linfócito T/química , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Proteínas de Choque Térmico HSP70/química , Humanos , Camundongos , Camundongos Transgênicos , Peptídeos/química , Peptídeos/imunologia , TransfecçãoRESUMO
A major impediment to gene replacement therapy is immune elimination of genetically modified cells. In principle, this can be dealt with by inducing a strong, specific, and enduring tolerance through engraftment of transgene-modified autologous bone marrow (BM). Because usual myeloablation and/or immunosuppression are risk factors in most pathologies, we assessed the potential of monospecific CD4(+)CD25(+) regulatory T cells (Tregs) to engraft minor-mismatched BM without preconditioning. We found that as few as 5 x 10(4) Tregs directed to the male DBY protein promote the engraftment of foreign male BM into sex-mismatched female hosts, establishing sustained chimerism in all hematopoeitic compartments. We achieved concomitantly strong tolerance to all foreign antigens expressed in the BM, likely occurring through induction of anergy and/or deletion of antidonor T cells. Chimerism was obtained in thymectomized mice too, underlining the major role of peripheral tolerance mechanisms in our system. This allowed us to engraft gene-modified tissues while preserving full immunocompetence to third-party antigens. Our results demonstrate that very few donor-specific Tregs are effective as the sole conditioning to induce mixed molecular chimerism and long-term tolerance to multiple foreign antigens.
Assuntos
Transplante de Medula Óssea/imunologia , Linfócitos T Reguladores/imunologia , Condicionamento Pré-Transplante/métodos , Animais , Sequência de Bases , Quimera/imunologia , RNA Helicases DEAD-box , Feminino , Fatores de Transcrição Forkhead/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Tolerância Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Proteínas/genética , Proteínas/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transplante de Pele/imunologia , Doadores de Tecidos , Transplante HomólogoRESUMO
Thymus-derived regulatory T cells (Tregs) expressing CD4, CD25, and the transcription factor Foxp3 play major roles in preventing autoimmunity. The Treg population is enriched in T cells expressing high-avidity self-reactive T cell receptors, and thymic epithelial cells expressing self-antigens (Ag) have been implicated in their induction and/or selection. However, the thymic selection events leading to Treg lineage commitment remain unclear. We followed the thymic development of self-Ag-specific Tregs in double-transgenic mice coexpressing a neo-self-Ag, hemagglutinin (HA) under the control of a neural tissue-specific promoter, and a transgenic class II-restricted T cell antigen receptor specific for HA111-119. Our data show that the promiscuous expression of the HA transgene in thymic epithelial cells is involved in the selective induction and/or expansion of HA-specific Foxp3(+) Treg thymic precursors as early as the double-positive stage.