To analyse the role of CD4+ T subsets in this protection, we took two approaches. First, we compared CD4+ T-cell activation and TCR Vβ diversity from draining LN at 1 week post-infection

with La alone versus La infection following pre-infection with Lb for 8 weeks (short-term). We focused on IFN-γ production in Vβ8, Vβ4 and Vβ6 (because of their relatively high frequencies) and used Vβ7 as an example of low-frequency types (Figure 1). Compared to La infection HSP assay alone, pre-infection with Lb increased IFN-γ production from total CD4+ T cells, as well as from Vβ6- and Vβ8-bearing CD4+ T cells (Figure 3c). Second, we compared CD4+ T-cell activation and TCR Vβ diversity from draining LN and the spleen at 1 week post-infection with La versus Lb parasites in mice that were pre-infected with Lb for 24 weeks (long-term). As shown in Figure 4(a), the secondary infection with Lb (the Lb/Lb group) consistently showed higher IFN-γ but lower IL-17 production from draining LN CD4+ T cells than did the La counterparts (the Lb/La group). For the tested Vβ-bearing CD4+ T-cell subsets (Vβ4, 6, 7, and 8), the Lb/Lb groups

displayed 2.1- to 9-fold higher frequencies of IFN-γ-producing cells in draining LNs. It was evident in Figure 4(b) that the Lb/Lb groups showed high frequencies of IFN-γ-producing cells in the tested T-cell subsets. Likewise, the similar trends were observed for cells obtained from the spleen (Figure 4c,d). Collectively, our results indicate SAR245409 mouse that repeated exposures to Lb parasites (the Lb/Lb groups) preferentially stimulate the expansion of IFN-γ-producing cells among multiple Vβ-bearing CD4+ T-cell subsets and that such responses contribute to the protection against a secondary infection with La parasites. To further characterize CD4+ T-cell activation during the primary and secondary infections, we collected draining LN cells at 4 weeks post-infection with La or Lb and stimulated cells briefly (6 h) with PMA/ionomycin, The ex vivo production of intracellular cytokines (IFN-γ, IL-10, IL-17, IL-2 and TNF-α) in CD4+ CD44+ T cells was

analysed by FACS. As shown in Figure 5(a), CD4+ CD44+ T cells from Lb-infected Quinapyramine mice contained higher frequencies of IFN-γ-producing cells, but lower frequencies of IL-10- and IL-17-producing cells than did the counterparts from La-infected mice. On average, the ratios of IFN-γ- vs. IL-10-producing cells in Lb-, La- and noninfected mice were 4.7, 2.0 and 1.7, respectively. The frequencies of IL-2- and TNF-α-producing CD4+ CD44+ T cells were comparable in two infection models. Therefore, CD4+ T cells derived from Lb-infected mice were highly activated with a strong Th1 phenotype. Next, we designed a cross-stimulation experiment, in which draining LN cells from La- or Lb-infected mice were restimulated in vitro with La or Lb antigens, and vice versa.