RESUMO
Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation.
Assuntos
Imunidade Adaptativa , Herpesvirus Galináceo 2/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Doença de Marek/imunologia , Peptídeos/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Alelos , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Sítios de Ligação , Linhagem Celular , Galinhas , Cristalografia por Raios X , Regulação da Expressão Gênica , HIV-1/imunologia , Haplótipos , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Doença de Marek/virologia , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Fbxo7 is an unusual F box protein that augments D-type cyclin complex formation with Cdk6, but not Cdk4 or Cdk2, and its over-expression has been demonstrated to transform immortalised fibroblasts in a Cdk6-dependent manner. Here we present new evidence in vitro and in vivo on the oncogenic potential of this regulatory protein in primary haematopoietic stem and progenitor cells (HSPCs). Increasing Fbxo7 expression in HSPCs suppressed their colony forming ability in vitro, specifically decreasing CD11b (Mac1) expression, and these effects were dependent on an intact p53 pathway. Furthermore, increased Fbxo7 levels enhanced the proliferative capacity of p53 null HSPCs when they were grown in reduced concentrations of stem cell factor. Finally, irradiated mice reconstituted with p53 null, but not wild-type, HSPCs expressing Fbxo7 showed a statistically significant increase in the incidence of T cell lymphoma in vivo. These data argue that Fbxo7 negatively regulates the proliferation and differentiation of HSPCs in a p53-dependent manner, and that in the absence of p53, Fbxo7 expression can promote T cell lymphomagenesis.
Assuntos
Proteínas F-Box/metabolismo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Linfoma/patologia , Proteínas Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Diferenciação Celular/genética , Divisão Celular/genética , Proliferação de Células , Deleção de Genes , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Fator de Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6, enhances the level of cyclin D-Cdk6 complexes, and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here, we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which, unexpectedly, it was unable to do despite high levels of Cdk6. Instead, reduction of Fbxo7 expression increased proliferation, decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27, and increased levels of S phase cyclins and Cdk2 activity. Also, Fbxo7 protein levels correlated inversely with those of CD43, suggesting direct regulation of its expression and, therefore, of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle, indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however, decreased expression of Cdk6 also enhanced levels of CD43, indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations, consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells.
Assuntos
Diferenciação Celular , Proliferação de Células , Proteínas F-Box/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Linfócitos B/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Linhagem Celular , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ciclinas/biossíntese , Fase G1/genética , Fase G1/fisiologia , Humanos , Leucossialina/biossíntese , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genéticaRESUMO
F-box proteins are the substrate-recognition components of the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases. Here we report a structural relationship between Fbxo7, a component of the SCF(Fbxo7) E3 ligase, and the proteasome inhibitor PI31. SCF(Fbxo7) is known to catalyze the ubiquitination of hepatoma-up-regulated protein (HURP) and the inhibitor of apoptosis (IAP) protein but also functions as an activator of cyclin D-Cdk6 complexes. We identify PI31 as an Fbxo7.Skp1 binding partner and show that this interaction requires an N-terminal domain present in both proteins that we term the FP (Fbxo7/PI31) domain. The crystal structure of the PI31 FP domain reveals a novel alpha/beta-fold. Biophysical and mutational analyses are used to map regions of the PI31 FP domain mediating homodimerization and required for heterodimerization with Fbxo7.Skp1. Equivalent mutations in Fbxo7 ablate interaction with PI31 and also block Fbxo7 homodimerization. Knockdown of Fbxo7 does not affect PI31 levels arguing against PI31 being a substrate for SCF(Fbxo7). We present a model for FP domain-mediated dimerization of SCF(Fbxo7) and PI31.
Assuntos
Proteínas F-Box/química , Proteínas F-Box/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Dimerização , Humanos , Células Jurkat , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma , Mapeamento de Interação de Proteínas , Alinhamento de SequênciaRESUMO
Some B cell lymphomas lack important costimulatory properties that could prevent them from being used as cell based vaccines. Infection of A20 B lymphoma cells with a replication-defective adenovirus encoding murine (m) CD40L, but not mIL-2, produces an antigen presentation phenotype with upregulation of MHC Class I/II, induction of B7-1/2 molecules and production of MIL-12 and MIP-1alpha. Subcutaneous vaccination with irradiated Ad-mCD40L-infected- or Ad-mIL-2-infected-A20 cells generated A20-specific CD8+ T cell responses and cross reactive A20 Ig antibodies. Only vaccination with Ad-mCD40L-infected A20 cells produced a significant delay in tumor growth and long-term survival (p = 0.0039). Stronger protective immunity to A20 challenge was generated by intravenous priming with A20 cells infected with Ad-mCD40L, Ad-mIL-2 or their combination followed by a boost immunization with A20 cells activated with syngeneic fibroblasts expressing CD40L. Compared to Ad-LacZ-infected A20 priming, the combination priming was most effective followed by Ad-mCD40L and Ad-mIL-2 (p = 0.0027, p = 0.0027, p = 0.0163 respectively). Significant A20-specific CD8+ T cell-mediated cytotoxicity was only demonstrated in splenocytes from these groups of vaccinated animals. By contrast, ELISPOT assay of splenocytes from all A20 prime/boosted vaccinated groups demonstrated increases in gamma-interferon release by T cells elicited by in vitro stimulation either with A20 cells or another syngeneic 2PK-3 lymphoma, indicating the presence of cross reactive immunity. Similarly anti-A20 immunoglobulin antibodies generated after vaccination were not necessarily A20 idiotype-specific. Direct therapy of pre-established tumors was achieved with the combination of Ad-mCD40L and Ad-mIL-2 given at Days 4 and 8 at the tumor site with a significant long-term survival of 85% of tumor-bearing mice (p = 0.0001). Our study strongly supports the use of Ad-CD40L and Ad-IL-2 combination therapy for the treatment of patients with B cell lymphoma.
Assuntos
Apresentação de Antígeno , Ligante de CD40/genética , Ligante de CD40/imunologia , Vacinas Anticâncer , Imunoterapia , Interleucina-2/genética , Interleucina-2/imunologia , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Adenoviridae , Animais , Formação de Anticorpos , Feminino , Fibroblastos , Humanos , Imunidade Celular , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Baço/citologia , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Clearer evidence for immune surveillance in malignancy and the identification of many new tumour-associated antigens (TAAs) have driven novel vaccine and antibody-targeted responses for therapy in cancer. The exploitation of active immunisation may be particularly favourable for TAA where tolerance is incomplete but passive immunisation may offer an additional strategy where the immune repertoire is affected by either tolerance or immune suppression. This review will consider how to utilise both active and passive types of therapy delivered by T cells in the context of the failure of tumour-specific immunity by presenting cancer patients. This article will outline the progress, problems and prospects of several different vaccine and antibody-targeted approaches for immunotherapy of cancer where proof of principle pre-clinical studies have been or will soon be translated into the clinic. Two examples of vaccination-based therapies where both T cell- and antibody-mediated anti-tumour responses are likely to be relevant and two examples of oncofoetal antigen-specific antibody-directed T cell therapies are described in the following sections: (1) therapeutic vaccination against human papillomavirus (HPV) antigens in cervical neoplasia; (2) B cell lymphoma vaccines including against immunoglobulin idiotype; (3) oncofoetal antigens as tumour targets for redirecting T cells with antibody strategies.