RESUMO
Brucella lumazine synthase (BLS) is a homodecameric protein that activates dendritic cells via toll like receptor 4, inducing the secretion of pro-inflammatory cytokines and chemokines. We have previously shown that BLS has a therapeutic effect in B16 melanoma-bearing mice only when administered at early stages of tumor growth. In this work, we study the mechanisms underlying the therapeutic effect of BLS, by analyzing the tumor microenvironment. Administration of BLS at early stages of tumor growth induces high levels of serum IFN-γ, as well as an increment of hematopoietic immune cells within the tumor. Moreover, BLS-treatment increases the ratio of effector to regulatory cells. However, all treated mice eventually succumb to the tumors. Therefore, we combined BLS administration with anti-PD-1 treatment. Combined treatment increases the outcome of both monotherapies. In conclusion, we show that the absence of the therapeutic effect at late stages of tumor growth correlates with low levels of serum IFN-γ and lower infiltration of immune cells in the tumor, both of which are essential to delay tumor growth. Furthermore, the combined treatment of BLS and PD-1 blockade shows that BLS could be exploited as an essential immunomodulator in combination therapy with an immune checkpoint blockade to treat skin cancer.
Assuntos
Interferon gama/genética , Melanoma Experimental/tratamento farmacológico , Receptor de Morte Celular Programada 1/genética , Receptor 4 Toll-Like/genética , Animais , Quimiocinas/genética , Citocinas/genética , Células Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Fatores Imunológicos/farmacologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Receptor 4 Toll-Like/agonistas , Microambiente Tumoral/efeitos dos fármacosRESUMO
Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responses.
Assuntos
Brucella , Flagelina , Complexos Multienzimáticos , Proteínas Recombinantes de Fusão , Salmonella typhimurium , Animais , Brucella/enzimologia , Brucella/genética , Brucella/imunologia , Células CACO-2 , Feminino , Flagelina/biossíntese , Flagelina/genética , Flagelina/imunologia , Humanos , Imunidade Humoral , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Salmonella typhimurium/metabolismo , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/imunologiaRESUMO
The multiple display of protein domains on polymeric scaffolds is an emerging technology for many applications. BLS is a highly immunogenic protein that has an oligomeric structure formed by a 17.2 kDa subunit arranged as a dimer of pentamers. Here we describe the production as well as the structural, functional, and immunological properties of a 9 kDa double-stranded RNA-binding domain (RBD3) fused to the structure of BLS. We demonstrate that the BLS and RBD3 modules are stably and independently folded in the structure of the chimera and form a decameric structure of 255 kDa as the native BLS oligomers. The polymeric display of RBD3 in the structure of BLS increases the dsRNA binding strength of this domain both in vitro and in vivo, and also enhances its immunogenicity to the point that it breaks the tolerance of mice to the RBD3 self-antigen. Our results underscore the BLS display strategy as a powerful tool for biotechnological and therapeutic applications.
Assuntos
Complexos Multienzimáticos/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sítios de Ligação , Brucella/enzimologia , Dicroísmo Circular , Clonagem Molecular , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Desnaturação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , TermodinâmicaRESUMO
The T-cell repertoire is shaped by the positive and negative selection of immature CD4(+) CD8(+) double positive (DP) thymocytes. Positive selection of DP T cells to the CD4(+) CD8(-) and CD4(-) CD8(+) simple positive (SP) lineages is a multistep process which involves cellular interactions between thymocytes and stromal cells. Mutant nackt (nkt/nkt) mice have been shown to have a deficiency in the CD4(+) CD8(-) T-cell subset both in the thymus and in the periphery. The present report suggests that nkt/nkt mice present alterations in early steps of positive selection because they show decreases in the percentages of CD69(+) and CD5(+) cells within the DP subset. Experiments involving bone marrow transfer and thymic chimeras demonstrate that the thymic epithelium of nkt/nkt mice is involved in the alterations registered during positive selection and dictates the ultimate fate of CD4(+) SP cells.
Assuntos
Antígenos CD4 , Camundongos Mutantes/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Alopecia/genética , Animais , Antígenos CD , Antígenos de Diferenciação de Linfócitos T , Antígenos CD5 , Antígenos CD8 , Imunidade Celular/imunologia , Lectinas Tipo C , CamundongosRESUMO
Mouse mammary tumour virus (MMTV) is a type B retrovirus that causes mammary tumours in susceptible mice. MMTV encodes a superantigen (SAg) that has the property of stimulating T-cell populations expressing a particular variable region of the T-cell receptor (TCR) beta chain (Vbeta) and needs to be presented in the context of major histocompatibility complex (MHC) class II molecules. Previously, we described two exogenous MMTV, MMTV BALB14, which encodes a superantigen that induces the deletion of Vbeta14+ Tcells, and MMTV BALB2, which encodes a SAg that induces the deletion of Vbeta2+ Tcells. We now describe their biological activity: the deletions involve both CD4+ and CD8+ populations, are progressive and can be detected in blood, lymph nodes and spleen. Such deletions reflect, at least in part, those occurring during intrathymic development. Both BALB2 and BALB14 viral variants are capable of inducing a strong increase of Vbeta-specific T cells in BALB/c mice (I-A+, I-E+). However, when injected into the footpad, their initial stimulatory capacity differs in that the presence of MHC I-E molecules is essential only for the stimulation of Vbeta2+ T cells. Both viral variants are able to induce deletion even in the absence of the I-E molecule in which case, however, deletion appears later and is less pronounced. Both exogenous MMTVs induce, at the end of a year, 30-35% of pregnancy-dependent mammary adenocarcinomas.
Assuntos
Neoplasias Mamárias Experimentais/etiologia , Vírus do Tumor Mamário do Camundongo/imunologia , Vírus do Tumor Mamário do Camundongo/patogenicidade , Infecções por Retroviridae/etiologia , Superantígenos , Infecções Tumorais por Vírus/etiologia , Adenocarcinoma/etiologia , Adenocarcinoma/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Variação Genética , Neoplasias Mamárias Experimentais/imunologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Gravidez , Complicações Neoplásicas na Gravidez/etiologia , Complicações Neoplásicas na Gravidez/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Infecções por Retroviridae/imunologia , Superantígenos/genética , Subpopulações de Linfócitos T/imunologia , Infecções Tumorais por Vírus/imunologiaRESUMO
A number of milk-borne exogenous mouse mammary tumor viruses (MMTV) infect mice shortly after birth and, when expressed, produce superantigens. Herein we describe the biological effects of new variants of exogenous MMTV: one of them (BALB14) present in BALB/c mice and showing a low ability to induce mammary tumors, and the other (MMTV-7) being the result of recombination between the BALB14 and the Mtv-7 endogenous provirus. The recombinant virus which has the SAg-specificity of Mtv-7 was amplified in BALB/c mice this fact correlating with a high incidence of mammary tumors. The role of strong SAgs in the mechanism by which the recombinant virus increases its title in a susceptible host is discussed. The results obtained suggest that the presence of non-productive endogenous proviruses--considered as conferring a selective advantage to the mouse population by protecting it from infection with exogenous MMTV--should also be advantageous to the pathogen by increasing its variability, thus broadening the host range and allowing the expansion of highly tumorigenic variants.
Assuntos
Neoplasias Mamárias Experimentais/imunologia , Vírus do Tumor Mamário do Camundongo/imunologia , Infecções por Retroviridae/imunologia , Superantígenos , Infecções Tumorais por Vírus/imunologia , Animais , Testes de Carcinogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Recombinação GenéticaRESUMO
Hosts and their pathogens have co-evolved for millions of years, developing multiple and intimate interactions. Vertebrates have evolved a very complex immune system which pathogens have often been able to circumvent, in some cases even managing to appropriate some of its components for their own purpose. Among the pathogens which do use components of the immune system to survive and propagate, those coding for the expression of superantigens (SAgs) are now under intense scrutiny. Investigations concerning one of these pathogens, the mouse mammary tumor virus (MMTV), led to the understanding of how the expression of such components is a critical step in their life cycle. A number of milk-borne exogenous MMTV infect mice shortly after birth and, when expressed, produce superantigens. Herein, we describe the biological effects of new variants of MMTV. Two of these, BALB14 and BALB2 encoding SAgs with V beta 14+ and V beta 2+ specificities, respectively, were present in BALB/c mice of our colony (BALB/cT); a third variant, termed MMTV LA, originated in (BALB/cTxAKR)F1 mice from recombination between BALB 14 and Mtv-7 endogenous provirus. The recombinant LA virus induces the deletion of V beta 6+ and V beta 8.1+ T cells as a consequence of the acquisition of SAg hypervariable coding region of Mtv-7. The SAg encoded by MMTV LA strongly stimulates cognate T cells in vivo leading to a very effective amplification of lymphoid cells in BALB/c mice, correlating with a high incidence of mammary tumors. These results suggest that the presence of non-productive endogenous proviruses--generally considered to confer a selective advantage to the host by protecting it from infection with exogenous MMTVs encoding cross-reactive SAgs--could also be advantageous for the pathogen by increasing its variability, thus broadening the host range and allowing the expansion of highly tumorigenic variants.