RESUMO
Antibodies, specific to murine DEC205, can be used to target antigens to dendritic cells. The immunodominant domain of human type XVII collagen, hNC16A, was fused to this antibody (DEC-hNC16A) and was administered as expression plasmid by gene gun transfection with the aim of inducing tolerance to human type XVII collagen in a skin transplantation model. Mice transfected with DEC-hNC16A were challenged with skin grafts from transgenic mice engineered to express human type XVII collagen. Graft survival was either prolonged or grafts were accepted infinitely (33% and 16%, respectively) upon treatment with DEC-hNC16A while 100% of grafts were rejected in untreated controls. Graft acceptance was associated with the absence of a CD4+ infiltrate and a dense CD8+ T-cell infiltrate and was not strictly dependent on antibody production. Our results show that DEC-hNC16A targets dendritic cells in vivo leading to prolonged survival of transgenic skin grafts. This indicates that DEC205-targeting may be used for the induction of tolerance to skin antigens, which would increase the chances of successful skin gene therapy of epidermolysis bullosa patients.
Assuntos
Autoantígenos/imunologia , Tolerância Imunológica/imunologia , Células de Langerhans/imunologia , Células de Langerhans/patologia , Colágenos não Fibrilares/imunologia , Transplante de Pele/imunologia , Transplante de Pele/patologia , Animais , Autoantígenos/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Terapia Genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Colágenos não Fibrilares/genética , Estrutura Terciária de Proteína , Transfecção , Colágeno Tipo XVIIRESUMO
HAX1 was originally described as HS1-associated protein with a suggested function in receptor-mediated apoptotic and proliferative responses of lymphoid cells. Recent publications refer to a complex and multifunctional role of this protein. To investigate the in vivo function of HAX1 (HS1-associated protein X1) in B cells, we generated a Hax1-deficient mouse strain. Targeted deletion of Hax1 resulted in premature death around the age of 12 wk accompanied by a severe reduction of lymphocytes in spleen, thymus and bone marrow. In the bone marrow, all B-cell populations were lost comparably. In the spleen, B220(+) cells were reduced by almost 70%. However, as investigated by adoptive transfer experiments, this impairment is not exclusively B-cell intrinsic and we hypothesize that a HAX1-deficient environment cannot sufficiently provide the essential factors for proper lymphocyte development, trafficking and survival. Hax1(-/-) B cells show a significantly reduced expression of CXCR4, which might have an influence on the observed defects in B-cell development.
Assuntos
Linfócitos B/imunologia , Movimento Celular/imunologia , Linfopoese/imunologia , Proteínas/imunologia , Animais , Linfócitos B/metabolismo , Medula Óssea/imunologia , Medula Óssea/metabolismo , Movimento Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Peptídeos e Proteínas de Sinalização Intracelular , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Linfopoese/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas/genética , Proteínas/metabolismo , Receptores CXCR4/biossíntese , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Baço/imunologia , Baço/metabolismo , Timo/imunologia , Timo/metabolismoRESUMO
Immune recognition and rejection of tissues expressing transfected genes is a major complication of gene replacement therapy for inherited genetic disorders. Owing to the high immunogenicity of human bullous pemphigoid antigen 2 (hBPAG2), the induction and maintenance of tolerance to this neo-antigen is essential to deliver the gene product to patients with epidermolysis bullosa junctionalis. In a skin grafting mouse model, we used gene gun transfection with a construct encoding hNC16A, the immunodominant domain of hBPAG2, to induce antigen-specific immune tolerance. Eighty percent of wild-type mice transfected with hNC16A showed long-term survival of skin grafts expressing hBPAG2. Tolerance was stable and transferable by T cells but not by B cells of tolerant mice to naive hosts. A dense Foxp3(+) regulatory T-cell (T(reg)) infiltrate was noticed in grafts of tolerant mice and depletion of these cells resulted in a loss of tolerance. Taken together, we show that long-lasting hBPAG2-specific tolerance was induced with gene gun delivery of hNC16A through a T(reg)-dependent mechanism. This is of relevance to patients undergoing gene therapy and has broader implications for the treatment of antigen-specific autoimmune diseases.
Assuntos
Autoantígenos/genética , Terapia Genética/métodos , Rejeição de Enxerto/prevenção & controle , Tolerância Imunológica/genética , Colágenos não Fibrilares/genética , Penfigoide Bolhoso/genética , Penfigoide Bolhoso/terapia , Administração Cutânea , Transferência Adotiva , Animais , Autoantígenos/imunologia , Membrana Basal/imunologia , Terapia Combinada , Feminino , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Humanos , Tolerância Imunológica/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Colágenos não Fibrilares/imunologia , Penfigoide Bolhoso/imunologia , Transplante de Pele/imunologia , Linfócitos T Reguladores/imunologia , Transfecção/métodos , Colágeno Tipo XVIIRESUMO
BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFkappaB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1), accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1) dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1). The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM) and Rap1-GTP-interacting adaptor molecule (RIAM). This alters actin dynamics and renders focal adhesion kinase (FAK) constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1(-/-) mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naïve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer.