Fontoura I C, Trombone A P F, Almeida L P, Lorenzi J C C, Rossetti R A M, Malardo T, Padilha E, Schluchting W, Silva R L L, Gembre A F, Fiuza J E C, Silva C L, Panunto-Castelo A, Coelho-Castelo A A M
Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
Universidade Sagrado Coração, Bauru, SP, Brasil.
Braz J Med Biol Res. 2015 Dec;48(12):1095-100. doi: 10.1590/1414-431X20154409. Epub 2015 Sep 18.
In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.
在DNA疫苗中，将感兴趣的基因克隆到细菌质粒中，该质粒经过改造可在真核细胞中长时间诱导蛋白质产生。先前的研究表明，用编码麻风分枝杆菌65 kDa热休克蛋白的裸质粒DNA片段（pcDNA3-Hsp65）对BALB/c小鼠进行肌肉内免疫可诱导对结核分枝杆菌攻击的保护作用。免疫后保护性免疫反应的一个关键阶段是记忆T细胞的产生。此前，我们已经表明B细胞捕获质粒DNA-Hsp65，从而在小鼠结核分枝杆菌攻击后调节CD8+记忆T细胞的形成。因此，阐明B细胞在DNA免疫后如何作为保护性免疫反应的一部分发挥作用对于开发更有效的疫苗很重要。本研究的目的是研究DNA-Hsp65免疫后B细胞调节记忆T细胞的机制。以15天的间隔给C57BL/6和BKO小鼠每只注射100μg裸pcDNA-Hsp65，共注射三次。免疫30天后，用流式细胞术测量效应记忆T（TEM）细胞（CD4+和CD8+/CD44high/CD62Llow）和记忆CD8+T细胞（CD8+/CD44high/CD62Llow/CD127+）的百分比。还通过实时定量PCR对全脾细胞和纯化的B细胞（CD43-）中的干扰素γ、白细胞介素12（IL-12）和IL-10 mRNA进行定量。我们的数据表明，表达IL-10的B细胞亚群下调了脾脏中促炎细胞因子的表达，增加了CD4+TEM细胞和CD8+TEM/CD127+细胞的存活。
