major-vaccinated animals, again at values just above the limit of detection. Together, these results confirm that Lm/CpG accelerates

the natural immune response to L. major in the C57BL/6 mice, eliminating the “silent” phase of infection. This is dominated by the production of IL-6, IL-12, TNF-α, and IFN-γ. IL-10, TGF-β, IL-4, and FK506 IL-23 are produced at very low levels. Importantly, Table 1 also shows that parasites are required to initiate a vigorous immune response; immunization with CpG DNA alone did not induce a significant, sustained increase in cytokine secretion. L. major killing is driven by IFN-γ in the resistant C57BL/6 mice 14. One of the specific features of Lm/CpG vaccination is the early increase in IL-6 secretion, which has been linked to Th17 development 15. We studied the expression of both cytokines at wk 0 (prior to vaccination) and wk 2, 6, and 10, to span all the immunological phases of live vaccination. Figure 1 shows the absolute number of dermal CD4+IL-17+ and CD4+IFN-γ+ T cells in both L. major (A) and Lm/CpG-vaccinated mice (B). At wk 2, CD4+ T cells from Lm/CpG-vaccinated mice mostly expressed IL-17, although

IFN-γ+ cells were also detectable; expression of both cytokines decreased in these mice thereafter. Although CD4+IL-17+ T cells could be transiently detected in L. major-vaccinated BYL719 cost animals at wk 2, their numbers were significantly lower (by 2.5-fold). At wk 6, CD4+IFN-γ+ T cells were revealed as the main population present in the skin of L. major-vaccinated animals. At this time point, few CD4+ T cells could be found in the skin of Lm/CpG-vaccinated animals, since resolution

of infection had already taken place. Figure 1C represents representative flow cytometry plots from vaccinated mice at wk 2 and 6, and it shows that IFN-γ and ILı7 are not expressed by the same cell populations. It also shows that PDK4 CpG DNA alone does not produce a sustained adaptive immune response. Together, these results demonstrate that Lm/CpG (i) modifies the natural immune response against L. major infection by increasing Th17 cells during the “silent” phase and (ii) the combination of parasites and CpG DNA is required for Th17 expansion. To confirm that Lm/CpG-induced IL-6 causes Th17 expansion, we neutralized IL-6 during the first 2 wk following vaccination by treatment with anti-IL-6 receptor (anti-IL-6R), as described previously 11. Neutralization of the cytokine for longer periods of time did not change the outcome of the experiment (data not shown). Control mice were inoculated with the same dose of an isotype control (rat IgG). We then analyzed the number of CD4+ T cells expressing IL-17 and IFN-γ in the ears of vaccinated mice during the “silent” and acute phase (wk 1, 2, 3, and 6 post vaccination). Neutralization of IL-6 provoked a remarkable decrease in IL-17 expression at wk 1 and 2 in Lm/CpG-vaccinated mice (Fig.