Resposta imune mediada por linfócitos T CD8+ na infecção experimental pelo Trypanosoma cruzi
Arquivos
Data
2008
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Embora esteja bem estabelecido, há mais de quinze anos, que linfócitos T
CD8+ restritos por moléculas de MHC-Ia são importantes no controle da parasitemia
e sobrevivência de camundongos infectados com Trypanosoma cruzi, pouco se
sabia da especificidade desses linfócitos. A ausência de epítopos bem definidos
impedia o estudo detalhado da cinética da resposta imune e dos mecanismos de
controle da imunodominância. Assim, o objetivo inicial desta tese foi a identificação
de epítopos reconhecidos pelos linfócitos T CD8+ ativados durante a infecção pelo
T. cruzi. Uma vez definidos esses epítopos, estudamos a cinética de ativação
dessas células e alguns dos parâmetros que a controlavam. Por fim, estudamos os
possíveis mecanismos de imunodominância durante a resposta imune.
Durante a infecção experimental com a cepa Y de T. cruzi, nós observamos
que os epítopos VNHRFTLV, IYNVGQVSI ou TEWETGQI foram reconhecidos por
camundongos infectados C57BL/6, BALB/c ou B10.A, respectivamente. Esses
epítopos, expressos por membros da família das trans-sialidases, geraram forte
resposta imune medida pela citotoxicidade in vivo ou pela produção de IFN-γ ex vivo
(Elispot). A cinética da ativação desses linfócitos T CD8+ se iniciou no pico da
parasitemia e dependeu da carga parasitária. Ou seja, quanto mais tempo a
parasitemia demorou para atingir o pico, mais lenta foi a aparição das células
específicas. Uma vez que a resposta chegou ao máximo, essa se manteve alta por
meses e só então começou a declinar lentamente. O fenótipo dos linfócitos T CD8+
específicos de memória é CD62Llow, característico de células de memória efetoras.
Tanto a cinética, quanto o fenótipo dessas células diferiram dos achados observados
para vírus, bactérias e outros protozoários intracelulares. Em animais previamente
vacinados com DNA plasmidial ou proteínas recombinantes, uma significativa
aceleração da resposta imune específica foi observada, a qual correlacionou com a
imunidade protetora.
A fim de estudarmos os fatores que contribuíram para ativação dessas
células, nós utilizamos camundongos geneticamente deficientes. Observamos que a
deficiência para a produção de IL-12 e Interferon tipo I não causou nenhuma
redução na geração das células citotóxicas específicas. O mesmo foi observado em
animais deficientes para os receptores do tipo Toll 2, 4 ou 9. Por outro lado, animais
geneticamente deficientes que não expressavam as moléculas de MHC-II ou CD4
apresentaram significativa redução na resposta específica de linfócitos T citotóxicos.
O estudo dos mecanismos que controlam a imundominância da resposta
imune mediada por linfócitos T CD8+ específicos foi feito, inicialmente, pela
comparação da resposta imune em camundongos C57BL/6. Observamos que a
resposta imune para o epítopo VNHRFTLV foi imunodominante sobre os demais
epítopos restritos pelo MHC-Ia H-2Kb
. A fim de determinar se essa imunodominância
poderia ser exercida sobre epítopos restritos por MHC-Ia H-2Kk
(TEWETGQI) ou H-
2Kd
(IYNVGQVSI), comparamos as respostas imunes em camundongos infectados
homozigotos e heterozigotos. No caso do epítopo VNHRFTLV, nós observamos que
a resposta imune se manteve alta em ambas as linhagens de camundongo. Já no
caso dos dois outros epítopos restritos por MHC-Ia, H-2Kk
ou H-2Kd
, observamos
uma significativa redução na resposta imune dos animais heterozigotos em relação
aos homozigotos. Essa competição não foi dependente do tempo ou da dose de
parasitas inoculados. Também não foi observada quando os animais foram
imunizados com adenovírus recombinantes contendo esses mesmos epítopos. O
mais importante foi observar que a imunodominância pode ser evitada quando duas
cepas de parasitas, contendo epítopos imundominantes distintos, foram utilizadas
simultaneamente para infecção, sugerindo que há uma competição pelas células
apresentadoras de antígenos durante o “priming”. Esse mecanismo de
imundominância reduz a magnitude e o repertório da resposta imune e pode ser um
mecanismo sofisticado de escape do parasita para evitar a eliminação completa
pelos mecanismos efetores do hospedeiro.
Although it is well established for more than 15 years that CD8+ lymphocytes restricted by MHC-Ia molecules are important for the control of the parasitemia and survival during rodent infection with Trypanosoma cruzi, little was known about the specificity of these lymphocytes. The lack of well defined epitopes hindered the detailed study of the kinetic of the immune response and the mechanisms of control of the immunodominance. Therefore, the initial objective of this thesis, was to identify epitopes recognized by CD8+ T lymphocytes activated during the T. cruzi infection. Once we defined these epitopes, we studied the kinetics of activation of these cells and some of the parameters that controlled it. Finally, it was possible to study the mechanisms of immunodominance during the immune response. During the experimental infection with Y strain of T. cruzi, we observed that the epitopes VNHRFTLV, IYNVGQVSI or TEWETGQI were recognized by C57BL/6, BALB/c or B10.A infected mice, respectively. These epitopes, expressed by members of the trans-sialidase family of surface proteins, generated strong immune response as measured by the in vivo cytotoxicity or by the production of interferon-γ (Elispot). The kinetics of the activation of these CD8+ T cells initiated on the peak of parasitemia and depended on the parasite load. The longer delayed the parasitemia to reach its peak, slower was the appearance of these specific T cells. When the immune response reached the maximum, it was kept high for months and then, it declined slowly. The phenotype of memory CD8+ T lymphocytes was CD62LLow characteristic of effector memory cells. The kinetic one and phenotype of these cells had differed from the findings observed for other intracellular viruses, bacteria and protozoan parasites. In animals previously vaccinated with plasmidial DNA or recombinant proteins, a significant acceleration of the specific immune response was observed that correlated with the protective immunity. To study the factors that contributed for the activation of these cells, we used genetically deficient mice. We observed that deficiency for production of IL-12 and Interferon type I did not cause any reduction in the specific cytotoxic response. The same was observed in animals deficient for the Toll-like receptors 2, 4 or 9. On the other hand, genetically deficient animals that did not express MHC-II or CD4 molecules had significant reduction in the cytotoxic response mediated by CD8+ T cells. Finally, we described that following infection of mice with T. cruzi, an immunodominant CD8+ T cell immune response was developed directed to the epitope VNHRFTLV. To determine whether this immunodominance was exerted over other non H-2Kb -restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2Kb , H-2Kk (TEWETGQI), or H-2Kd (IYNVGQVSI)- restricted CD8+ T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2Kb -restricted immunodominant epitope. In contrast, H-2Kk or H-2Kd -restricted immune responses were significantly impaired in heterozygote infected mice when compared to homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi antigens. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8+ T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.
Although it is well established for more than 15 years that CD8+ lymphocytes restricted by MHC-Ia molecules are important for the control of the parasitemia and survival during rodent infection with Trypanosoma cruzi, little was known about the specificity of these lymphocytes. The lack of well defined epitopes hindered the detailed study of the kinetic of the immune response and the mechanisms of control of the immunodominance. Therefore, the initial objective of this thesis, was to identify epitopes recognized by CD8+ T lymphocytes activated during the T. cruzi infection. Once we defined these epitopes, we studied the kinetics of activation of these cells and some of the parameters that controlled it. Finally, it was possible to study the mechanisms of immunodominance during the immune response. During the experimental infection with Y strain of T. cruzi, we observed that the epitopes VNHRFTLV, IYNVGQVSI or TEWETGQI were recognized by C57BL/6, BALB/c or B10.A infected mice, respectively. These epitopes, expressed by members of the trans-sialidase family of surface proteins, generated strong immune response as measured by the in vivo cytotoxicity or by the production of interferon-γ (Elispot). The kinetics of the activation of these CD8+ T cells initiated on the peak of parasitemia and depended on the parasite load. The longer delayed the parasitemia to reach its peak, slower was the appearance of these specific T cells. When the immune response reached the maximum, it was kept high for months and then, it declined slowly. The phenotype of memory CD8+ T lymphocytes was CD62LLow characteristic of effector memory cells. The kinetic one and phenotype of these cells had differed from the findings observed for other intracellular viruses, bacteria and protozoan parasites. In animals previously vaccinated with plasmidial DNA or recombinant proteins, a significant acceleration of the specific immune response was observed that correlated with the protective immunity. To study the factors that contributed for the activation of these cells, we used genetically deficient mice. We observed that deficiency for production of IL-12 and Interferon type I did not cause any reduction in the specific cytotoxic response. The same was observed in animals deficient for the Toll-like receptors 2, 4 or 9. On the other hand, genetically deficient animals that did not express MHC-II or CD4 molecules had significant reduction in the cytotoxic response mediated by CD8+ T cells. Finally, we described that following infection of mice with T. cruzi, an immunodominant CD8+ T cell immune response was developed directed to the epitope VNHRFTLV. To determine whether this immunodominance was exerted over other non H-2Kb -restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2Kb , H-2Kk (TEWETGQI), or H-2Kd (IYNVGQVSI)- restricted CD8+ T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2Kb -restricted immunodominant epitope. In contrast, H-2Kk or H-2Kd -restricted immune responses were significantly impaired in heterozygote infected mice when compared to homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi antigens. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8+ T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.
Descrição
Citação
Tzelepis, Fanny. Resposta imune mediada por linfócitos T CD8+ na infecção experimental pelo Trypanosoma cruzi. 2008 97 f. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2008.