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. and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon , 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. 65-kDa heat-shock protein (pcDNA3-Hsp65) imparted protection against (12). However, clarifying the specific mechanisms by which B cells induce a protective immune response after DNA immunization is an important step in the development of more-effective DNA vaccines. Here, we investigated the mechanisms by which B cells modulate memory T cells in the pcDNA3-Hsp65 vaccinated mouse model. Our results SCH-503034 showed that a B-cell subpopulation expressing IL-10 mRNA downregulated the expression of proinflammatory cytokines, thus increasing the percentages of CD4+ and CD8+memory T cells in the spleen after DNA immunization. Material and Methods Mice Male 6-8-week-old C57BL/6 wild-type (WT) and B-cell-deficient (BKO; chain?/-) mice were obtained from Jackson Laboratories (USA) and maintained under specific-pathogen-free conditions in the animal house of the Departamento de Imunologia, Faculdade SCH-503034 de Medicina de Ribeir?o Preto, Universidade de S?o Paulo. The mice had access to water and sterile food … WT mice displayed reduced proinflammatory cytokine mRNAs in the spleen When the transcriptional profiles of the proinflammatory cytokines in the mouse spleens were evaluated 30 days after immunization, we found that DNA-Hsp65 immunization increased the mRNA levels of IFN- and IL-12 compared with empty-vector immunization in both the WT and BKO mice (Figure 2A and B, respectively). It is noteworthy that transcripts of IFN- and IL-12 were virtually undetectable in the cells of mice immunized with the empty vector. The WT mice showed lower mRNA expression of IFN- and IL-12 after DNA-Hsp65 immunization than the BKO mice. In contrast, no significant difference in IL-10 mRNA expression was observed between the WT and BKO groups when the mice were immunized with DNA-Hsp65. IL-10 mRNA expression was only elevated in the WT group immunized with the empty pcDNA3 vector (Figure 2C). These data indicate a role for B cells in the regulation of proinflammatory cytokine production in the mouse spleen. Figure 2 Relative expression of cytokine mRNAs in the spleens of wild-type (WT) and B-cell knockout (BKO) mice 30 days after immunization. C57BL/6 WT and BKO mice were injected intramuscularly on three occasions, at 15-day intervals, with 100 g pcDNA3 … DNA-Hsp65 immunization induced IL-10 mRNA expression by B cells To clarify the possible mechanisms by which B cells modulate the formation of memory T cells and regulate proinflammatory cytokine expression, the mRNA expression of IFN-, IL-12, and IL-10 was measured in B Rabbit Polyclonal to p300 cells purified from mouse spleen cells 30 days after immunization. The splenic B cells from mice immunized with DNA-Hsp65 or empty vector showed similar levels of IFN- and IL-12 mRNA expression (Figure 3A and B, respectively). However, B cells from the DNA-Hsp65-immunized mice displayed higher levels of IL-10 mRNA than the B cells from the empty-vector-immunized mice. This suggests that DNA-Hsp65 immunization activates a subpopulation of B cells that produces IL-10. Figure 3 Relative expression of cytokine mRNAs in purified B cells from wild-type (WT) mouse spleens 30 days after immunization. C57BL/6 WT mice were injected intramuscularly on three occasions, at 15-day intervals, with 100 g pcDNA3 encoding … Discussion Our results suggest that the presence of B cells is necessary to support the formation of memory after DNA immunization. Memory T cells develop after the evolution of the adaptive immune response. This protective response begins after the recognition of the antigen presented by professional APCs to na?ve T lymphocytes, which triggers their proliferation and differentiation into effector T cells. After antigen clearance, the immune response is downregulated and most activated lymphocytes undergo apoptosis. The pool of remaining lymphocytes then differentiates into long-lived memory T cells (15). A previous study showed that as well as presenting antigens, B cells also costimulate T cells through their interaction with CD40 and CD40L on the T-cell surface, enhancing T-cell activation SCH-503034 (16). Additional costimulation by their engagement with CD28 induces greater T-cell survival in the effector phase of the immune response, by promoting an increase in antiapoptotic molecules in the activated T cells. This event allows a larger number of the available cells to differentiate into.