RESUMO
A primary goal in transplantation medicine is the induction of a tolerogenic environment for prevention of transplant rejection without the need for long-term pharmacological immunosuppression. Generation of alloantigen-specific regulatory T cells (Tregs) by transduction with chimeric antigen receptors (CARs) is a promising strategy to achieve this goal. This publication reports the preclinical characterization of Tregs (TR101) transduced with a human leukocyte antigen (HLA)-A*02 CAR lentiviral vector (TX200) designated to induce immunosuppression of allograft-specific effector T cells in HLA-A*02-negative recipients of HLA-A*02-positive transplants. In vitro results demonstrated specificity, immunosuppressive function, and safety of TX200-TR101. In NOD scid gamma (NSG) mice, TX200-TR101 prevented graft-versus-host disease (GvHD) in a xenogeneic GvHD model and TX200-TR101 Tregs localized to human HLA-A*02-positive skin transplants in a transplant model. TX200-TR101 persisted over the entire duration of a 3-month study in humanized HLA-A*02 NSG mice and remained stable, without switching to a proinflammatory phenotype. Concomitant tacrolimus did not impair TX200-TR101 Treg survival or their ability to inhibit peripheral blood mononuclear cell (PBMC) engraftment. These data demonstrate that TX200-TR101 is specific, stable, efficacious, and safe in preclinical models, and provide the basis for a first-in-human study.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Órgãos , Receptores de Antígenos Quiméricos , Camundongos , Animais , Humanos , Linfócitos T Reguladores , Leucócitos Mononucleares/transplante , Antígenos HLA-ARESUMO
The outcome of organ transplantation is largely dictated by selection of a well-matched donor, which results in less chance of graft rejection. An allogeneic immune response is the main immunological barrier for successful organ transplantation. Donor and recipient human leukocyte antigen (HLA) mismatching diminishes outcomes after solid organ transplantation. The current evaluation of HLA incompatibility does not provide information on the immunogenicity of individual HLA mismatches and impact of non-HLA-related alloantigens, especially in vivo. Here we demonstrate a new method for analysis of alloimmune responsiveness between donor and recipient in vivo by introducing a humanized mouse model. Using molecular, cellular, and genomic analyses, we demonstrated that a recipient's personalized humanized mouse provided the most sensitive assessment of allogeneic responsiveness to potential donors. In our study, HLA typing provided a better recipient-donor match for one donor among two related donors. In contrast, assessment of an allogeneic response by mixed lymphocyte reaction (MLR) was indistinguishable between these donors. We determined that, in the recipient's humanized mouse model, the donor selected by HLA typing induced the strongest allogeneic response with markedly increased allograft rejection markers, including activated cytotoxic Granzyme B-expressing CD8+ T cells. Moreover, the same donor induced stronger upregulation of genes involved in the allograft rejection pathway as determined by transcriptome analysis of isolated human CD45+cells. Thus, the humanized mouse model determined the lowest degree of recipient-donor alloimmune response, allowing for better selection of donor and minimized immunological risk of allograft rejection in organ transplantation. In addition, this approach could be used to evaluate the level of alloresponse in allogeneic cell-based therapies that include cell products derived from pluripotent embryonic stem cells or adult stem cells, both undifferentiated and differentiated, all of which will produce allogeneic immune responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/prevenção & controle , Antígenos HLA/imunologia , Teste de Histocompatibilidade , Histocompatibilidade , Leucócitos Mononucleares/transplante , Transplante de Órgãos , Baço/imunologia , Tolerância ao Transplante , Animais , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Bases de Dados Genéticas , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto , Antígenos HLA/genética , Humanos , Isoanticorpos/metabolismo , Leucócitos Mononucleares/imunologia , Teste de Cultura Mista de Linfócitos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Órgãos/efeitos adversos , Fenótipo , Valor Preditivo dos Testes , Baço/metabolismo , Transcriptoma , Transplante HomólogoRESUMO
OBJECTIVE AND METHODS: This study investigated the potential for protective effects of human umbilical cord blood mononuclear cells (UCB-MCs) genetically modified with the VEGF and GNDF genes on contusion spinal cord injury (SCI) in rats. An adenoviral vector was constructed for targeted delivery of VEGF and GDNF to UCB-MCs. Using a rat contusion SCI model we examined the efficacy of the construct on tissue sparing, glial scar severity, the extent of axonal regeneration, recovery of motor function, and analyzed the expression of the recombinant genes VEGF and GNDF in vitro and in vivo. RESULTS: Transplantation of UCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF at the site of SCI induced tissue sparing, behavioral recovery and axonal regeneration comparing to the other constructs tested. The adenovirus encoding VEGF and GDNF for transduction of UCB-MCs was shown to be an effective and stable vehicle for these cells in vivo following the transplantation into the contused spinal cord. CONCLUSION: Our results show that a gene delivery using UCB-MCs-expressing VEGF and GNDF genes improved both structural and functional parameters after SCI. Further histological and behavioral studies, especially at later time points, in animals with SCI after transplantation of genetically modified UCB-MCs (overexpressing VEGF and GDNF genes) will provide additional insight into therapeutic potential of such cells.
Assuntos
Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Leucócitos Mononucleares/transplante , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/terapia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Axônios/fisiologia , Cicatriz , Modelos Animais de Doenças , Feminino , Sangue Fetal/citologia , Técnicas de Transferência de Genes , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Leucócitos Mononucleares/citologia , Masculino , Atividade Motora/fisiologia , Neuroglia/patologia , RNA Mensageiro/biossíntese , Distribuição Aleatória , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/fisiologia , Medula Espinal , Traumatismos da Medula Espinal/fisiopatologia , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Graft-versus-host disease (GVHD) remains a frequently occurring and difficult-to-treat complication in human allogeneic stem cell transplantation. Murine transplantation models are often used to study and understand the complex pathogenesis of GVHD and to explore new treatment strategies. Although GVHD kinetics may differ in murine and human models, adequate models are essential for identification of the crucial factors responsible for the major pathology in GVHD. We present a detailed description of the specific histological features of a graft-versus-host-induced fibrotic response in xenogeneic RAG2(-/-)γc(-/-) mice after total body irradiation and injection with human peripheral blood mononuclear cells. We describe the full morphological features of this reaction, including a detailed analysis of the specific tissue infiltration patterns of the human peripheral blood mononuclear cells. Our data show the development of fibrosis, predominantly near blood vessels, and reveal different cell populations and specific cell migration patterns in the affected organs. The combination of immunohistochemical cell characterization and mRNA expression analysis of both human (donor)- and murine (host)-derived cytokines reveals an interaction between host tissues and donor-derived cells in an entangled cytokine profile, in which both donor- and host-derived cytokines contribute to the formation of fibrosis.