Terapia gênica visando à neutralização da interleucina-10 in vivo como uma nova alternativa imunoterapêutica para o melanoma murino B16F10-Nex2
Arquivos
Data
2009
Autores
Marchi, Luis 
Orientadores
Rodrigues, Elaine Guadelupe
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Citocinas do tipo 2 estão presentes em fases mais avançadas do
desenvolvimento de diversos tumores humanos, funcionando em alguns
casos como fatores prognósticos de neoplasias. O desenvolvimento do
melanoma murino B16F10 está relacionado a um aumento na produção de
IL-10 por células NK e linfócitos T CD4+
e uma diminuição acentuada na
produção de IFN- por essas células, nas fases mais avançadas da
progressão tumoral.
Embora o modelo singeneico do melanoma murino B16F10-Nex2
apresente baixa imunogenicidade in vivo, uma pequena porcentagem de
camundongos C57Bl/6 são naturalmente resistentes ao desenvolvimento
subcutâneo desse tumor, mesmo após mais de uma inoculação das células
tumorais. Animais naturalmente resistentes produziram maiores
concentrações de IFN- e animais susceptíveis ao tumor produziram
maiores concentrações de interleucinas anti-inflamatórias, IL-10 e IL-6.
Confirmando o papel imunoregulador negativo da IL-10 na resposta
imune protetora natural desse modelo, camundongos geneticamente
deficientes em IL-10 (IL-10KO) foram mais resistentes à implantação
subcutânea de células B16F10-Nex2. Animais que resistiram à progressão
tumoral produziram uma resposta protetora com perfil de citocinas do tipo 1
(IFN- e IL-12), e a proteção foi dependente de IFN- .
Macrófagos e células dendríticas de animais IL-10KO após estímulo in
vitro com antígenos do melanoma B16F10-Nex2 apresentaram maior
produção de citocinas pró-inflamatórias (IFN- , TNF- e IL-12) e uma maior
expressão de marcadores de ativação celular (MHCII, CD40, CD80 e CD86).
Com a transferência adotiva de células dendríticas IL-10KO, mas não IL-10
competentes, associadas a um lisado de células B16F10-Nex2, animais
C57Bl/6 foram protegidos contra o desenvolvimento tumoral, sugerindo que
células dendríticas que não expressam IL-10 sejam responsáveis pela
indução da resposta protetora natural mais eficiente observada em animais
IL-10KO.
Tendo em vista o papel imunossupressor da IL-10 na resposta
protetora induzida pelo melanoma B16F10-Nex2, foi construído um vetor
plasmidial eucariótico contendo o mini-gene referente à porção extracelular
do receptor da IL-10 murina, a ser utilizado em protocolos de terapia gênica
para neutralização da IL-10 in vivo pela expressão de um receptor decoy da
interleucina.
Animais tratados com o plasmídeo recombinante apresentaram
aumento na sobrevida, em um efeito dependente de IFN- e potencializado
pela associação de uma terapia gênica adjuvante para produção de IL-12.
Células dendríticas de animais C57Bl/6 foram transfectadas com esse
plasmídeo recombinante, associadas a antígenos tumorais e transferidas
adotivamente à animais C57Bl/6, que foram subsequentemente desafiados
com células tumorais subcutaneamente. Essas células, que tiveram o IL-10
neutralizado pelo receptor decoy, induziram resposta protetora
significativamente mais eficiente que as células controle transfectadas com
o plasmídeo vazio, sugerindo que essas células estejam envolvidas in vivo
na indução da resposta protetora observada após a imunização com o
plasmídeo recombinante e neutralização da IL-10 sistêmica.
Concluímos que a terapia gênica desenvolvida foi eficaz na
neutralização da IL-10 sistêmica, podendo ser aplicada como uma nova
alternativa imunoterapêutica antitumoral.
Type 2 cytokines are increased in late phases of several human tumors, and in some cases are considered prognostic factors for these neoplasias. Late development phases of murine melanoma B16F10 correlate with an increased production of IL-10 by NK cells and CD4+ T lymphocytes, and also with a decreased production of IFN- by these cells. Syngeneic murine melanoma B16F10-Nex2 shows low immunogenicity in vivo, however, a small percentage of C57Bl/6 mice are naturally resistant to subcutaneous tumor development, even after several tumor cell inoculations. Naturally resistant animals produced higher concentrations of IFN- and tumor susceptible animals produced higher concentrations of anti-inflammatory cytokines, IL-10 and IL-6. The immunoregulatory role of IL-10 on the natural protective immune response induced in this model was confirmed by the increased resistance to tumor development observed in IL-10 genetically-deficient mice (IL- 10KO) subcutaneously inoculated with B16F10-Nex2 cells. A type 1 immune reponse (IFN- and IL-12) was induced in resistant IL-10KO animals, and the protection was IFN- -dependent. IL-10KO macrophages and dendritic cells stimulated in vitro with B16F10-Nex2 melanoma antigens secreted higher concentrations of proinflammatory cytokines (IFN- , TNF- e IL-12), and expressed an increased amount of surface activation markers (MHCII, CD40, CD80 e CD86). Adoptive transfer of IL-10KO dendritic cells, but not IL-10-competent cells, in association with tumor antigens, induced a protective response in C57Bl/6 mice, suggesting that IL-10-negative dendritic cells induce the efficient protective response observed in IL-10KO animals. As IL-10 showed a clear immunosuppressive role on the natural protective response induced by B16F10-Nex2 cells, we constructed an eukaryotic plasmidial vector carrying the murine IL-10 receptor extracellular portion gene, to be used in gene therapy protocols for IL-10 neutralization in vivo by the expression of a decoy receptor. Recombinant plasmid-treated animals showed an IFN- -dependent increased survival, and this protective effect was augmented by the association with an adjuvant IL-12 gene therapy Dendritic cells from C57Bl/6 animals were transfected with recombinant plasmid, adoptively transferred to C57Bl/6 mice, and treated animals were challenged subcutaneously with B16F10-Nex2 tumor cells. Interleukin-10-neutralized dendritic cells induced a significantly increased survival, as compared to IL-10-containing dendritic cells, suggesting that dendritic cells induce the protective response observed in vivo after recombinant plasmid immunization and systemic IL-10 neutralization. We concluded that gene therapy using a plasmid expressing the IL-10 receptor extracellular region was effective on systemic IL-10 neutralization, and this tool could be used as a novel antitumor immunotherapeutic alternative.
Type 2 cytokines are increased in late phases of several human tumors, and in some cases are considered prognostic factors for these neoplasias. Late development phases of murine melanoma B16F10 correlate with an increased production of IL-10 by NK cells and CD4+ T lymphocytes, and also with a decreased production of IFN- by these cells. Syngeneic murine melanoma B16F10-Nex2 shows low immunogenicity in vivo, however, a small percentage of C57Bl/6 mice are naturally resistant to subcutaneous tumor development, even after several tumor cell inoculations. Naturally resistant animals produced higher concentrations of IFN- and tumor susceptible animals produced higher concentrations of anti-inflammatory cytokines, IL-10 and IL-6. The immunoregulatory role of IL-10 on the natural protective immune response induced in this model was confirmed by the increased resistance to tumor development observed in IL-10 genetically-deficient mice (IL- 10KO) subcutaneously inoculated with B16F10-Nex2 cells. A type 1 immune reponse (IFN- and IL-12) was induced in resistant IL-10KO animals, and the protection was IFN- -dependent. IL-10KO macrophages and dendritic cells stimulated in vitro with B16F10-Nex2 melanoma antigens secreted higher concentrations of proinflammatory cytokines (IFN- , TNF- e IL-12), and expressed an increased amount of surface activation markers (MHCII, CD40, CD80 e CD86). Adoptive transfer of IL-10KO dendritic cells, but not IL-10-competent cells, in association with tumor antigens, induced a protective response in C57Bl/6 mice, suggesting that IL-10-negative dendritic cells induce the efficient protective response observed in IL-10KO animals. As IL-10 showed a clear immunosuppressive role on the natural protective response induced by B16F10-Nex2 cells, we constructed an eukaryotic plasmidial vector carrying the murine IL-10 receptor extracellular portion gene, to be used in gene therapy protocols for IL-10 neutralization in vivo by the expression of a decoy receptor. Recombinant plasmid-treated animals showed an IFN- -dependent increased survival, and this protective effect was augmented by the association with an adjuvant IL-12 gene therapy Dendritic cells from C57Bl/6 animals were transfected with recombinant plasmid, adoptively transferred to C57Bl/6 mice, and treated animals were challenged subcutaneously with B16F10-Nex2 tumor cells. Interleukin-10-neutralized dendritic cells induced a significantly increased survival, as compared to IL-10-containing dendritic cells, suggesting that dendritic cells induce the protective response observed in vivo after recombinant plasmid immunization and systemic IL-10 neutralization. We concluded that gene therapy using a plasmid expressing the IL-10 receptor extracellular region was effective on systemic IL-10 neutralization, and this tool could be used as a novel antitumor immunotherapeutic alternative.
Descrição
Citação
MARCHI, Luis. Terapia gênica visando à neutralização da interleucina-10 in vivo como uma nova alternativa imunoterapêutica para o melanoma murino B16F10-Nex2. 2009. 160 f. Dissertação (Mestrado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2009.